THE HEALTH IMPACT OF NANOPARTICLES

N A N O P A T H O L O G Y

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THE HEALTH IMPACT OF NANOPARTICLES

N A N O P A T H O L O G Y

ANTONIETTA M GATTIUniversity of Modena & Reggio Emilia, Italy

STEFANO MONTANARILaboratory Nanodiagnostics, Italy

British Library Cataloguing-in-Publication DataA catalogue record for this book is available from the British Library.

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Copyright © 2008 by Pan Stanford Publishing Pte. Ltd.

NANOPATHOLOGYThe Health Impact of Nanoparticles

Rhaimie - Nanopathology.pmd 10/3/2007, 5:24 PM1

This book is dedicated to those who are neither young nor old enough to know everything, but know, as we do,

that they don’t know anything.

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vii

Acknowledgements

All this research would not have come into fruition if it had not been

financed by the European Project NANOPATHOLOGY. Word of thanks

to the independence of the referees and the European Commission for its

farsightedness. It was due to this initiative that people could now be

properly treated and the cause of their contamination could be identified

and eradicated.

Prof. James Kirkpatrick, Prof. William Bonfield, Prof. Peter Revell

and Dr. Diana Boraschi were the first to understand what we meant and

to support and encourage us.

We are indebted to Dr. Andrea Gambarelli, Dr. Roberta Salvatori,

Dr. Federico Capitani, Dr. Daniela Tossini, Dr. Gianluca Sighinolfi for

their technical help.

Special thanks to Miss Lavinia Nitu, our efficient and patient

secretary.

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ix

Preface

Nanotechnologies can represent a real innovation for human society

and life. The possibility of “bottom-up” construction makes man look

like God, but the wise man knows that every progress can have a

negative side and too often, when he realizes that, disasters have already

occurred.

The primary raison d’être of this book is to help society avoid the

repeat of the mistakes made by the Curies and their followers when they

discovered radioactivity and started, on the wings of enthusiasm, to use it

on people affected by a number of diseases, and did that without being

able to anticipate the harmful side effects of those therapies.

A new trend has spread all over Europe, meetings after meetings

dedicated to radiotherapy were organized, and people have even started

to wear necklaces with beads of Cobalt core.

It was only a few decades later, after having paid a high price

in terms of deaths, that the side effects of radioactivity became

evident, but now we can use radioactive materials in a safe way daily,

taking advantage from this phenomenon and the technologies it had

generated.

The new frontier opened by nanotechnology especially in medicine

looks extremely exciting. In the future we might see nanodevices

equipped with nanomotors inserted in the blood vessels and driven to

areas damaged by an infarction to destroy the thrombus or the atheroma

and restore circulation, or toward the pneumothorax area to seal the

lesion. Or even devices that act like guardians to check the onset of

inflammations in precancerous areas.

All these may look like a dream, but, it is a dream fast becoming a

reality and, before, it becomes real, it is crucial that we verify how

organisms, tissues, cells react to the presence of nanoparticles, i.e.

foreign bodies whose behaviour is still largely unknown.

Nanopathology

x

Recent European research projects (Nanosafe1, Nanosafe2,

Nanoderm, Nanopathology) have explored the possible risks of

nanoparticles on human health, and their results are controversial. Some

assert the safety of nanoparticles through in-vitro tests, others are more

doubtful and less optimistic, while a few scientists have already

presented clinical evidences of the presence of nanoparticles in

pathological tissues [A.M. Gatti, 2005]. Besides other pieces of evidence,

unintentionally released, nanosized particles were found in soldiers who

served in former Yugoslavia during the Balkans War (1993-97). It is

widely known that the explosion of Depleted Uranium bombs can

develop a temperature exceeding 3000°C [Annual Report, 1978]. The

magnitude of this combustion is capable of vaporizing everything. As

soon as the vaporized materials cool down, nanosized particles are

created and are scattered in the environment. The inhalation or ingestion

of those mainly metallic particles by humans and animals can bring

about pathological effects [Nemmar A. et al., 2002]. Warfare is not the

only one to be blamed for the formation of nanoparticles as a pollutant.

Car engines, industry, incineration and high-temperature procedures in

general are just a few examples of particulate pollutants producers. It is

easy to conclude that in more than one instance, the environment is

already contaminated by nanosized particles.

It has been proved that 100-nm-sized particles when inhaled can

bypass the lung barrier in 60 seconds and reach the liver in 60 minutes.

It may not be possible to eliminate nanoparticles from the

environment, but, awareness of their possible adverse effects on human

health is important. More research are needed to determine the safest

procedures to handle them.

This book intends to help society change its mind set as in the words

of John Steinbeck, Nobel Prize Winner, 1964,

“The ability to think differently today than yesterday is

what separates the wise from the stubborn.”

Preface xi

Bibliography

Annual Technical report of the Air Force Armament Laboratory. (1978). Armament

development and test Center, Eglin Air Force Base, Florida, USA. Project n°

06CD0101 (From October 1977 to October 1978). Gatti, A.M. Handbook (2005).

Risk assessment of micro and nanoparticles and the human health, Handbook of

nanostructured biomaterials and their applications. Ed. by American Scientific

Publisher USA, cap. 12, pp. 347-369.

Nemmar A., Hoet P.H.M., Vanquickenborne B., Dinsdale D., Thomeer M., Hoylaerts

M.F., Vanbilloen H., Mortelmans L., Nemery B. (2002). Passage of inhaled

particles in to the blood circulation in humans, Circulation, 105 (4), pp. 411-417.

Authors

Antonietta M Gatti

University of Modena & Reggio Emilia, Italy

Stefano Montanari

Laboratory Nanodiagnostics, Italy

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xiii

Contents

Acknowledgements vii

Preface ix

1. How the Whole Thing Began or the Logic Path Towards a

Discovery 1

1.1 Introduction...................................................................................... 6

1.2 Bibliography .................................................................................... 10

2. In-vitro and in-vivo Biological Behaviour of Micro and Nanoparticles 11

2.1 Introduction...................................................................................... 11

2.2 Nanoparticles and medical devices .................................................. 13

2.2.1 Dentistry ................................................................................ 13

2.2.2 Orthopaedics.......................................................................... 14

2.2.3 Nanostructured surfaces ........................................................ 16

2.2.4 Drug delivery......................................................................... 17

2.2.5 Nanomedicine........................................................................ 19

2.3 The results of the nanopathology project ......................................... 22

2.3.1 In-vivo experiments ............................................................... 28

2.4 Bibliography .................................................................................... 33

3. Clinical Cases: Lung, Blood, Liver, Kidney, Digestive System,

Vessels, Sperm 39

3.1 Introduction...................................................................................... 39

3.2 Lung ................................................................................................. 42

3.3 Blood................................................................................................ 70

3.4 Liver................................................................................................. 80

3.5 Kidney and adrenal gland ................................................................ 95

3.6 Digestive system .............................................................................. 102

3.7 Vessels ............................................................................................. 114

3.8 Sperm ............................................................................................... 115

3.9 A few considerations on reproduction ............................................. 121

3.10 Bibliography .................................................................................... 131

4. Six “Detective Stories” 135

4.1 The 1st case....................................................................................... 135

4.2 The 2nd

case...................................................................................... 142

4.3 The 3rd

case ...................................................................................... 145

4.4 The 4th

case ...................................................................................... 148

Nanopathology xiv

4.5 The 5th

case ...................................................................................... 152

4.6 The 6th

case ...................................................................................... 155

5. War and Nanoparticles 161

5.1 Civilians living around a firing ground ............................................ 198

5.2 A few reflections.............................................................................. 199

5.3 Bibliography .................................................................................... 200

6. Nanoparticles in the Environment and Working Places 203

6.1 Introduction...................................................................................... 203

6.2 Chimneysweeps: a historical case .................................................... 205

6.3 Welding metals ................................................................................ 207

6.4 Toner................................................................................................ 210

6.5 The particulate active filter or FAP.................................................. 212

6.6 The environment around a foundry.................................................. 215

6.7 The environment around a power plant............................................ 220

6.8 The case of a ship............................................................................. 227

6.9 Incinerators ...................................................................................... 228

6.10 Tobacco smoke ................................................................................ 231

6.11 Bibliography .................................................................................... 237

7. Nanoparticles in Food, Cosmetics and Other Products 239

7.1 Bibliography .................................................................................... 249

8. New York 9/11 251

8.1 Bibliography .................................................................................... 263

9. The Future and Prevention Criteria 265

9.1 The state of the art............................................................................ 265

9.2 What is next?.................................................................................... 267

9.3 The future......................................................................................... 280

9.4 A few reflections.............................................................................. 281

9.5 Bibliography .................................................................................... 284

Appendix 287

Index 291

1

Chapter 1

How the Whole Thing Began or the Logic Path Towards a Discovery

___________

ack in 1990, the hospital of Monza, a branch of the University of

Milan - Italy, sent to the Laboratory of Biomaterials of the

University of Modena a vena cava filter (Montanari, 2000) which

had two broken legs and that had been explanted surgically from a 62-

year-old female patient (Emanuelli, et al., 1995) (Gatti, et al., 2006). The

surgeon who had first implanted and then removed the filter just wanted

to know why the device had failed and solving his problem was not

particularly hard, as it was due to a caudal migration with the two legs

trapped in a collateral vein. The filter moved downward, while the two

legs could not follow the movement, broke. One of the consequences was

that the fracture surfaces remained exposed to the blood and interacted

with its components. What remained of the filter and the broken legs was

observed under a Scanning Electron Microscope equipped with an X-ray

microprobe (Energy Dispersive Spectroscope or EDS).

This analysis [A highly energetic electron beam is aimed at the

sample, and that yields a number of by-products, among which X-rays.

Each chemical element has characteristic energies/wavelengths which

can be detected using a solid state energy dispersive spectrometer

detector] (See Appendix) on this surface, where the legs had broken,

revealed a relatively high concentration of Chlorine, Silicon, Phosphorus

and Magnesium; all elements that did not belong either to the filter’s

alloy - an AISI 316L stainless steel, whose composition is Chromium

18%, Nickel 14%, Molybdenum 3%, Carbon below 0.03% and Iron to

balance - or to the blood, at least not in such quantities. At that time, we

could not find any reasonable explanation to what we had seen.

%

Nanopathology 2

A couple of years later, the same hospital sent another broken vena

cava filter to the Laboratory. This time the device was a temporary one

(Bovyn, et al., 1997) removed from a 58-year-old male 23 days after

having been implanted. Also that filter had failed, having lost one of its

legs which was then retrieved from the patient’s right inferior interlobar

renal vein by means of a transvenous noose catheter. The scanning-

electron-microscope observation of the fracture showed a few inorganic

deposits that could not belong to the product’s composition. As the EDS

analysis proved that the filter’s alloy was composed of Cobalt,

Chromium, Nickel, Molybdenum, Silicon and Iron (alloy called Phynox),

while Calcium and Aluminium were found in one of the deposits,

Chlorine, Silicon, Potassium, Calcium, Sulphur, Aluminium, Sodium,

Titanium and Magnesium in another, and single particles of Aluminium

and Calcium-Sulphur were also detected. Those deposits had obviously

formed in vivo, since the specimen had been carefully washed in a

Potassium oxide solution, and cleaned in a Hydrogen peroxide ultrasonic

bath, a treatment that destroys biofilm and leaves only insoluble

precipitates strictly adhered to the surface. Some of the materials

detected can be found as trace elements in ionic form or bound to organic

molecules in the human organism, but what we saw was particles and the

quantities we were confronted with were comparatively high. Again, no

satisfactory explanation could be offered then to that phenomenon.

In December 1997 a 62-year-old patient (Ballestri, et al., 2001)

(Gatti, et al., 2002) was admitted to the University hospital of Modena,

where the Laboratory of Biomaterials was then located. He was affected

by acute renal failure, hepato-splenomegaly and a mild haemolytic

anaemia. In addition to that, for the last eight years he had suffered from

a slight increase of the body temperature, a phenomenon that manifested

itself in the late afternoon and remitted to a normal temperature in a

matter of a few hours, without recurring to any medication. That fever

followed periodic cycles, each of which lasted a few months. Among the

symptoms the patient suffered from there was also a constant tiredness.

Blood and urine culture had been consistently negative and, despite

numerous hospitalizations, no aetiology could be determined for such

collection of symptoms.

How the Whole Thing Began or the Logic Path Towards a Discovery 3

So, a percutaneous echo-assisted biopsy was performed in the liver

and the lower pole of the left kidney, revealing non-caseating

granulomatous areas with Langhans-type giant cells in the kidney, while

the liver showed a mild fibrosis of the portal tracts filled with

mononuclear infiltrates and with the presence of epithelioid giant cells.

Scattered polarized light-blue particles either in the kidney and the liver

were shown by polarized-light microscopy observation, often inside

giant cells located in granulomatous areas, suggesting a foreign-body

multi-system granulomatosis. No yeasts or alcohol-acid resistant bacteria

could be identified. Clinical and immunological tests excluded

tuberculosis, mycotic infection, protozoa infestation, granulomatous

vasculitis or sarcoidosis, and the patient’s history was negative for

exposure to chemicals, drug abuse or arteriographic procedures.

That was indeed a very hard problem to solve through the traditional

means of investigation. So, the samples were given to our Laboratory

where we studied the debris by means of a scanning electron microscope

(SEM XL40 by Philips) and an environmental scanning electron

microscope (ESEM XL50 by Fei Company) equipped with an EDS. Both

in the liver and kidney specimens, we could identify solid particles

whose electronic density was higher than the biological background’s.

Their size ranged 6-20 microns in the liver and was smaller than 6

microns in the kidney, and the EDS elemental analyses, identical in both

types of specimens, revealed that they were made of Aluminium, Silicon,

Oxygen, Sodium, and Potassium or Barium, a composition that

suggested that of a ceramic material (and was compatible with feldspars,

non-fibrous silicates that are regular components of porcelain). In order

to understand the origin of such debris, a specific anamnestic study of the

patient was carried out, looking for an internal source of these debris like

prostheses, implant, etc.

What we learned from the patient, was that nine years before, two

ceramic bridges had been placed in his left upper and right lower dental

arches. That caused immediately a marked discomfort, an uncontrolled

lachrymation of the left eye, and a homolateral earache that could not be

solved by the antibiotic treatments the patient had undergone. In addition

to that, the patient became a bruxer.

Nanopathology 4

The prostheses had been milled a little later to try and solve the

problem of malocclusion and bruxism, so leaving a highly worn

prosthetic surface.

So, we started to suspect that the source of the debris found in the

patient’s tissues could be those prostheses and had both removed. They

were cross-sectioned and subjected to an Rx microanalysis that showed

the same elemental composition as the foreign bodies discovered in the

tissues. Ceramic particles smaller or as large as 40 microns were found

in the patient’s stools before the prostheses were removed, but none

one month after. So the patient underwent a 6-month therapy of

methylprednisolone and that induced the remission of the fever along

with the reversal of hepato-splenomegaly. Inflammation, haemolysis

and cholestasis disappeared, while the renal function recovered. After

therapy, no steroids were administered for another six month and

the clinical picture worsened. A recourse to methylprednisolone and

cyclophosphamide, an immunosuppressive therapy, lead to a complete

remission. Liver and kidney biopsies performed three years after the first

hospitalization showed a marked reduction of the granulomatosis.

Now, the question was: how could that debris, whose origin was hard

to call into question, have reached the liver and the kidneys? In our

opinion, the hypothesis that those particles had been absorbed by the

gastrointestinal system looked the likeliest, but no literature existed to

support what we suspected. Debris of different sizes had been detected in

the stools, the same kind of foreign bodies had been found in the liver

and the kidneys, but those in the kidneys were smaller than those in the

liver. That could suggest that the particles, undoubtedly present in the

bowels, which were absolutely healthy in our patient, had passed through

the intestinal mucosa and had been cleared by the liver before entering

the general circulation and, from there, the kidneys.

Such passage was not described in the then current handbooks of

Physiology and sounded hard to believe.

Professor Peter Revell of the Free Royal Hospital of London gave us

then four bioptic samples of liver granulomatosis whose origin had been

declared to be unknown, but viruses, bacteria and parasites had been

ruled out as possible causes. In three of them we found evident traces of

How the Whole Thing Began or the Logic Path Towards a Discovery 5

surprisingly enough but undoubtedly environmental dust, while in one

we detected particles of a metal that had been used for therapeutic

purposes. Gold nanoparticles had been injected in the patient’s knee joint

to treat an arthrosis and it was that Gold that we found in the liver tissue.

That made us understand that solid environmental pollution could be

suspected as being able to penetrate the body and settle in the liver. How,

it was far from clear, but the most reasonable ways of entry seemed us to

be the digestive system – for which we had already a piece of evidence

with the case of the dental prosthesis - and, perhaps - but it was just a

guess - the respiratory system.

In the meantime, we had received more samples both from our

University Hospital and the Istituto Tumori (Cancer Institute) of Milan –

Italy, and they regarded cases of colon cancer and Crohn’s disease. In all

instances, inorganic dust was present in considerable concentrations and

variety. In one case we could even detect 15 different chemical elemental

compositions in the same cluster in less than 1 cm2 of tissue.

Thanks to our experience in caval filtration and the new availability

of filters that could be explanted even after a long time, we started to

take into consideration the blood clots that were regularly found stuck to

the device when it was removed from the patient and the thrombi that the

device had trapped. If inorganic dust, especially composed of chemical

elements that should not be present in the organism, had been detected,

that would have meant, without any possible doubt, that that dust had an

exogenous origin. As a matter of fact, as will be illustrated in detail later

in this book, all samples showed that presence.

Finding once unexpected inorganic particulate had become a daily

experience, but we could say very little or nothing at all about its way of

entrance.

So, we started to look at some pathological lung sample we got from

our University and the University of Siena (Italy), and among them there

were also some cases of pneumothorax. Then, we studied cases of cystic

fibrosis, sarcoidosis and of different forms of lung cancer.

Inorganic micro- and nanodust was to be found with a great

frequency.

We decided, then, to present a project to the European Community, a

project we called “Nanopathology”, by that neologism we had created

Nanopathology 6

meaning the pathologies due to micro- and nanodust, and by that project

intending to study if what we had observed for a few years had a

scientific basis.

1.1 Introduction

Man has only recently landed on what to him was a new planet: Nano.

But Nature has always been there. Just to give one example, Nature uses

a “nanocode” called genome to preserve all the information about the

species each individual, be it a man, a virus or any other living being,

belongs to, and that code is extremely complex, sophisticated and

efficient. Genome, in its turn, gives all the necessary instructions, many

of which we are not aware of, to build the (nanosized) proteins necessary

to live, and does that at a nanoscale.

We are accustomed to interacting with objects the size we can handle

and/or see, and have also been accustomed for a relatively long time to

investigating and trying to understand how atoms and molecules behave;

but Nano is something different from both worlds. Nanoscale systems

are too big to be considered molecules, but definitely too small to be

understood in terms of macro, and attempting to extrapolate their

behaviour starting either from the smaller or from the larger would lead

us astray. Neither quantum nor classic physics can be fully applied.

Of this sort of “mesoworld” we know very little, but what has

immediately become visible is that the properties of those objects are

extremely interesting from a scientific point of view and, in addition to

that, offer many prospective possibilities to be exploited to our benefit.

Right for that latter reason, an enormous throng of scientists, technicians,

industrialists and financiers flung themselves, and are still doing with

growing enthusiasm and hopes, headlong into that field, the way pioneers

did in the past when a new land was discovered or gold was

unexpectedly found somewhere.

More than a couple of decades ago, in 1986, Kim Eric Drexler, an

American engineer, published Engines of Creation, a book describing

nanomachines capable of reproducing both themselves and virtually any

material object, extending life duration, curing diseases, in short,

How the Whole Thing Began or the Logic Path Towards a Discovery 7

working all sorts of wonders and, at the same time, reducing pollution.

Many scientists ridiculed such outlook, and nevertheless the book

exerted a strong influence on many people. And many people means

good business. Now, after more than twenty years have elapsed, we are

still far from what Drexler we do not know how rightfully anticipated,

but it is a matter of fact that nanotechnology is finding more and more

applications and the limit seems to be imagination.

According to Forbes, (web ref. 1) now the most common applications

of nanotechnology are in sports goods, ski wax, tennis racquets and

tennis balls being the top three, but some medical applications are

already in use and many more are being attempted, cosmetics,

photocatalists and computer chips are already available and what are

called “smart” surfaces, by that meaning hydrophobic, self-cleaning,

anti-bacteria, anti-mould treatments are best sellers. Just as a brief

observation, the adjective “smart” sounds very à-la Drexler.

So, after having landed in what if not a promised is at least a

promising land, it would be fool not to advance and explore it with the

purpose of exploiting it. But like any unknown territory, also this can

hide unexpected dangers and, for that reason, using prudence and

exercising some patience may look advisable. Who carried out

exhaustive studies about the impact those technology and, in particular,

handling nanodust may have on human and animal organisms? How can

we know how long those entities will take to interact with environment

and organisms, if ever they will interact in a perceptible way? Is there

any long enough experience in that field? No satisfactory answer can be

given to those questions.

What we can do is extrapolate.

Micro- and, much more rarely, nano-scale particles have always been

produced by a number of natural sources: belching volcanoes, forest

fires, rock erosion, and airborne desert and beach sand, but the greatest

quantity of such dust, particularly the smaller, comes from man. Much of

it is the undesired by-product of combustion, a process that has started to

be used on a large, industrial scale only a couple of centuries ago, i.e.

roughly 1/10,000 of the time man has spent on the Earth. Generating heat

at temperatures higher than that of burning wood has always been

difficult and, when the concept of selling value was introduced,

Nanopathology 8

expensive. For those reasons, materials such as, for example, glass have

been rather precious for a long time. But when we started to exploit fossil

combustibles, heat has grown cheaper and cheaper and easy to come by,

and now very high temperatures can be attained without any difficulty

and at a low cost. All that creates dust, and, as a rule of thumb, the higher

the temperature, the tinier the dust.

So, foundries, cement plants, incinerators and internal-combustion

engines, among an indefinite number of other sources, produce particles,

and their quantity is constantly on the increase, particularly as regards

nanoparticles, since industrial filters are not efficient enough to trap them

and the increase in temperature makes microparticles rarer and

nanoparticles more common.

Particulate matter is released in the atmosphere and behaves in a way

that is very similar, or all but identical in the case of nanosize, to gas.

Some of those particles, especially the small ones, tend to form clusters

and, for that reason, making a clear distinction between micro and nano

is not always possible and often not even meaningful. However, in any

case the structure of a nanoparticle remains the same.

The dust we deal with is inorganic, most of the times crystalline,

insoluble in water or other common, natural solvents, and non

biodegradable. Being so small, pressure and thermal gradients and wind

carry it virtually everywhere, and when it eventually falls to the ground,

a gentle breath of wind can lift it up again, thus restarting the cycle. That

way, those particles can travel very long distances and stay with us

forever.

Besides, when we look at those inorganic particles from the pollution

point of view, we must not forget that they are often the carriers of

organic pollutants like, for example and among many others, dioxins,

furans or polycyclic aromatic hydrocarbons. And we must also remember

that in a number of environmental conditions, even inside biological

tissues, those particles tend to coalesce and, for that reason,

differentiating micro and nanoparticles is not always possible and in

some instances could be meaningless. Another important point is that

spherical particles generated by combustion, especially the larger ones,

are often hollow and very fragile, so, when they break, they create

How the Whole Thing Began or the Logic Path Towards a Discovery 9

fragments which are obviously smaller and, as a consequence,

microparticles can create nanoparticles.

From the compositional point of view, the nanoparticles we deal with

are very often alloys fortuitously created by the fact that the chemical

elements that make them up were present in the materials burnt and

combined to form an alloy that in most cases is not to be found in any

metallurgy handbook. So, their elemental chemical composition can be a

telltale indication that allows to identify the source.

But there are also cases when those particles come from wear and

friction of machines or industrial working, and heat is not involved in

their formation. In that case, their composition is the same as that of the

original material.

An important issue regards mass and volume. If, for the sake of

simplification, we assume that particles are spherical (which is often the

case with the particles we deal with, when created at high temperature),

we can easily calculate their volume and their mass, which depends,

obviously, on the matter that object is made of. Let’s suppose that we

have a sphere whose diameter is 10 µm, i.e. that of a coarse particle. In

that case, its volume is 4πr3/3 = 523.3 µm

3. Now, let us suppose that we

wish to make spheres with a diameter of 2.5 µm, i.e. ¼ of the one of the

former ball. In that case, the volume of each sphere will be 8.0593 mµ3,

i.e.64 times as small. That means that, using the same amount of matter

necessary to make a particle whose diameter is 10 µm, we can create 64

particles whose diameter is 4 times as small. And, continuing, we can

create 1,000 particles with a diameter of 1 µm or 1,000,000 with a

diameter of 0.1 µm. This is particularly important if we look at this

simple geometric fact with the nanopathologist’s eye, since the same

amount of matter, if burnt at different temperatures, can give origin to

particles whose effects on the organism is very different [see also

Chapter 6 – Introduction]

On that dust we collected some experience and thanks to that we can

try and extrapolate about nanotechnological dust and its biological

behaviour.

Small particles are classified in a number of different ways, according

to the classifier and his discipline. Physicists, chemists and biologists do

not seem to be always agreed. We are not much interested in academic

Nanopathology 10

classifications and we do not really know where micro ends and nano

starts in the organism. It is highly probable that a threshold exists below

which natural, physiological barriers are ineffective and even one below

which cells do not oppose any resistance to the entrance of foreign

bodies. Our studies do not allow us to quantify that barrier nor to say if

and, in case, to what extent that threshold is influenced by size, shape,

surface/volume ratio, chemistry, state of aggregation or other factors.

Ours is a work in progress.

1.2 Bibliography

Ballestri, M., Baraldi, A., Gatti, A. M., Furci, L., Bagni, A., Loria, P., Rapanà, R. M.,

Carulli, N. and Albertazzi, A., (2001), Liver and Kidney Foreign Bodies

Granulomatosis in a Patient with Malocclusion, Bruxism and Worn Dental

Prosthesis – Gastroenterology,121:1234-38

Bovyn, G., Gory, P., Reynaud, P. and Ricco, J. B., (1997), The Tempofilter: A

multicenter study of a new temporary caval filter implantable for up to six weeks –

Ann Vasc Surg;11:520-25

Emanuelli, G., Gatti, A. M., Cigada, A. and Brunella, M. F., (1995), Physico-chemical

observations on a failed Greenfield vena cava filter – J Cardiovasc Surg;36:121-5

Gatti, A. M. and Montanari, S., (2006), Retrieval Analysis of Clinical Explanted Vena

Cava Filters – J Biomed Mat Res Part B: Appl Biomater 77B:307-314

Gatti, A. M., Ballestri, M. and Bagni, A., (2002), Granulomatosis associated to porcelain

wear debris – American Journal of Dentistry, Vol. 15, No. 6

Montanari, S., (2000), Malattia tromboembolica e filtri cavali - Ed. C. Rabbia, G.

Emanuelli – 90-140 Minerva Medica – Turin

Ref.web 1

[http://www.forbes.com/investmentnewsletters/2005/01/12/cz_jw_0112soapbox.html]

11

Chapter 2

In-vitro and in-vivo Biological Behaviour of Micro and Nanoparticles

___________

2.1 Introduction

he 21st century opened with a revolution due to emerging

technologies called nanotechnologies, which are growing

exponentially and even if we know that they must have a physical

limit, their outlook seems to have no end.

“Nano” is a word of Greek origin meaning “very small”, and

nanotechnologies are characterized by smaller and smaller technologies,

i.e. they deal with items ranging in magnitude between 10-9

and 10-8

m.

The first definition was given by Norio Taniguchi, of the Tokyo Science

University, in 1971: “Nano-technology is the production technology to

get the extra high accuracy and ultrafine dimensions, i.e. the preciseness

and fineness of the order of 1 nm (10-9

m in length)”. This definition has

been updated by the Europäische Akademie in 2003:

“Nanotechnology deals with functional systems based on the use of

subunits with specific size-dependent properties of the individual sub-

units,” in order to include not only the production of nanoparticles, but

also the production of systems or processes based on the highest possible

miniaturization.

Nanotechnologies refer to the technological field concerning the

controlled manufacturing of functional nanosystems or the creation of

nanostructures, which results in the production of entities with at least

one dimension of 100 nm-length scale.

7

Nanopathology 12

To give an idea of the smallness of these things, we can compare

them with some biological objects (see Fig. 2.1). A human hair can range

between 10 and 60 microns in width, human red cells have a diameter of

5-7 microns, while, at the cell order, ATPsynthase measures 10 nm and a

DNA molecule ranges between 0.5 nm and 2 nm.

Fig. 2.1 Scale of dimensions.

The synthesis of such small entities, never seen in the world, as the

ones we include in the definition of nano and their (sometimes

involuntary) dissemination can represent a new pollution and new stimuli

for the animal and human organisms strongly influencing their health.

Nobody knows what the impact will be of these new technologies on

our society and what side effects are to be expected. It is a novel situation

and it is imperative to carry out researches to investigate if and how

nanoparticles can represent a possible risk to the environment and who

lives in that environment.

But our aim is to investigate:

- what is considered “nano” for the human body, namely,

- what is its reaction against a discrete (not continuous) stimulus,

- what is the threshold size below which tissues or cells do not react

in a known way since the sensors do not “see” or recognize the stimulus.

Nanopathology will try to give an answer.

In-vitro and in-vivo Biological Behaviour of Micro and Nanoparticles 13

Applications in the medical field are already possible and a brief

overview of the most promising ones is given below.

2.2 Nanoparticles and medical devices

Applications of nanotechnologies are being experimented in a number of

specific fields of medical interest, but dentistry, by the creation of

nanocomposites for dental restorations, is the discipline where they are

already commonly available.

2.2.1 Dentistry

At present, dental restorations employ amalgams, composites, and Gold

or ceramic inlays.

There is a real need to dispose of mouldable, more resistant materials,

especially for posterior teeth where mastication load is particularly high,

capable of replacing amalgam, long suspected to be toxic. Though there

are no final scientific demonstrations to support the thesis of its toxicity,

a few diseases, and some lethal ones at that, have been associated to

the use of amalgams, in particular, amiotrophic lateral sclerosis and

Alzheimer’s disease. Amalgam is, in fact, the most common, and

inescapable for its carriers, source of human exposure to Mercury, and a

chronic low-level exposure to Mercury should be considered as a factor

in Alzheimer’s disease or in other diseases of the central nervous system,

including multiple sclerosis and Parkinson’s disease (Wenstrup et al.,

1990) (Saxe et al., 1999).

Some dental materials such as composites, dental filling resins

employing nanoparticles made, for instance, of Zirconia (Zirconium

oxide), are already available on the market, but other, more sophisticated,

materials are being developed. Organo-functionalized nanospheres,

containing X-ray absorbing elements such as Zirconium, combined with

liquid crystal monomers produce, in fact, composites with minimal

polymerization shrinkage and improve mechanical properties (Mitra et

al., 2003), (Mayer et al., 1998), (Williams et al., 2003).

Nanopathology 14

The improvement of these properties is only partial, since the wear

due to mastication cycles is not decreased and the risk of wear debris

ingestion continues (Ballestri et al., 2001).

Until a few years ago, this event did not cause any alarm, since it was

believed that that debris was gotten rid of with the faeces like all the

non degradable, small things people, and babies in particular, ingest

involuntarily. Unfortunately, it is not so.

It is growing clearer and clearer that biocompatible materials can

lose their biocompatibilty when they pass from a bulk form to a

fragmented one. The nanosized form is responsible for until-now

hardly suspected direct interactions with single cells, and those

interactions can induce unknown, unexpected reactions (Peters et al.,

2004), (Lucarelli, et al 2004). Toothpastes with either Silver or Gold or

hydroxypatite nanoparticles and used as nanofillers are already on

the market, as well as the bristles of some toothbrushes coated with

nanoSilver (see Chapter 7).

2.2.2 Orthopaedics

Besides dental implantology, another field of application of

nanotechnology is orthopaedics.

Orthopaedic devices, especially hip and knee joint prostheses,

present two main problems that show over time: wear of the matching

surfaces in the joint and loosening of the stem in the femoral channel.

The former causes the failure of the device because of the granulomatous

reaction induced by the wear debris, the loss of the three-dimensional

movement due to the loss of sphericity of the head, and, finally,

the rupture of the acetabulum with the ensuing migration of the

head.

To overcome these problems, new nanocomposite-based, wear-

resistant and self-lubricating PVD (plasma vapour deposition) coatings

(Stueber et al. 2002) are being studied. The wear of the hip joint

prostheses and the health problem due to the production of wear debris

induce researchers to develop new coatings more resistant to wear and,

In-vitro and in-vivo Biological Behaviour of Micro and Nanoparticles 15

possibly, self-lubricating. A possible solution can be the development

of a metastable hard matrix incorporating homogenously distributed

nanoclusters of MoS2. The use of cross-linked polyethylene does not

seem to solve the problems, also because wear produces sub-micronic

particles that can trigger a tissue reaction and be widely disseminated in

the tissues because of their ultrafine size (Urban et al., 2000), (Revell

et al., 1997).

The creation of nanosized-morphology surfaces enhances

biocompatibility and is supposed to ameliorate the interaction between

the device and specific cellular components. In biomaterial research, it

has been found that even though a bulk material may be well tolerated by

the body, finely divided particles of the same material can often lead to

severe, even carcinogenic, complications. Differences in particle size

influence histological reactions and cytokine production. Macrophage

response to particulate debris appears, for example, to be dependent on

particle size, composition, and dose as given by surface area ratio.

Several studies have also been carried out to determine the relationship

between cell and particle size with respect to cytotoxicity. (Goodman

et al., 1990), (Shanbagh et al., 1994), (Tamura, 2003).

Carbon nanofibres have been proposed as a possible, new

orthopaedic implant material because of their unique mechanical,

electrical and cytocompatibility properties. These fibres have a size close

to hydroxyapatite (Calcium phosphate) crystals and collagen fibres

found in the bone. Moreover, “in vitro” studies provided evidence that

nanophase Carbon fibres enhance osteoblast function over conventional

Carbon fibres and current orthopaedic implant materials (such as

titanium). To determine the influence of Carbon nanofibre wear debris on

osteoblast viability, direct contact toxicity studies were performed. The

results proved that nanophase Carbon fibres were less detrimental to

osteoblast viability than larger-diameter, conventional Carbon fibres. In

other words, they provide evidence of the promise of nanophase

materials in improving orthopaedic implant efficiency (Price et al.,

2004).

Nanopathology 16

2.2.3 Nanostructured surfaces

Implantable medical devices are bound to employ biocompatible

materials, i.e. materials that must be not only accepted by the biological

environment, but also promote an appropriate and favourable biological

interaction. The basic concept is that biological activity depends on the

structure of the matching matter and, at the same time, much of the

matter properties depends on size (Dobson et al., 1999).

So the biocompatibility of a material must have an “appropriate” cell

adhesion, that is preceded by a specific protein adsorption from the

extracellular matrix. The adsorption of a specific monolayer of proteins

will be of the utmost importance for the success of an implant. In fact,

once the proper protein has been adsorbed by the implanted surface, the

appropriate cells need to adhere and proliferate. The target of achieving

proper cell adhesion and proliferation is every biomaterial engineer’s

challenge. (Whiteside et al., 1991).

There are many cellular processes which are triggered by the type of

protein adsorbed, its conformation and its biological activity. If the

presence of a certain protein is requested to guarantee a “proper”

interaction of an implant with the biological environment, it may be

possible to manipulate the implant surface in order to induce in advance

that situation. The so-called “biomimetic” surfaces base their activity in

the human body upon this concept and, typically, biomimetics aims at

copying nature’s way of organizing and ordering complex molecules at

surface, to inhibit non-specific surface reactions and, finally, to create

“smart surfaces” that undergo rapid shifts in properties with small

external change (Castner et al., 2002).

The ability to design such a system is greatly supported by

biotechnologies and nanotechnologies. Examples of that are the

molecular self–assembly as a strategy for nanofabrication that involves

designing molecules and supramolecular entities so that shape-

complementarity causes them to aggregate into desired structures. Just as

an example, the Self-Assembly Monolayers (SAMs) are considerably

important in developing arterial prostheses, where it is absolutely

essential that the interior of the walls gets quickly coated by endothelial

cells. The efforts to attach heparin molecules to the surface of vascular

In-vitro and in-vivo Biological Behaviour of Micro and Nanoparticles 17

catheters in order to avoid blood clotting was not particularly successful,

especially because thrombosis cannot be reduced to a simple surface

problem, but the new strategy of SAMs can certainly improve and

stabilize these surfaces, and help to achieve better results. (Zhang et al.,

2002).

Nanotechnologies can create functionalised nanosurfaces attaching

polipetides or dendrimers (repeatedly branched molecules) that can

increase the biocompatibility of the implantable devices. Suitable

candidates for dendrimers synthesis and study include monomers

currently used for the preparation of medical-grade linear polymers,

and natural metabolites. For example, monomers from synthetic

polymers such as polyethylene-glycol, polytrimethylene-carbonate and

natural amino-acids represent classes of monomers suitable for use.

Incorporation of one or more of these building blocks in a dendritic

structure provides new opportunities to create well-defined polymers for

tissue engineering applications. The versatility of dendrimer technology

provides also unlimited possibilities in the synthesis of novel

nanomaterials and unprecedented control over these systems (Eppel

et al., 2002), (Bellingham et al., 2003), (Carnahan et al., 2001), (Balogh

et al., 1999).

2.2.4 Drug delivery

Other emerging and especially promising applications are those in the

drug-delivery field and gene therapy. Those are made possible by the

versatility of nanotechnologies and, particularly, by the extremely

reduced size of those devices, equal to that of their targets. In the targeted

delivery of drugs there are attempts to use dendrimers because drug

molecules can be loaded both in the interior of the dendrimers and

attached to the surface groups in order to control the rate of drug’s

release into the body. ( Kilts, 2003), (Orive et al., 2003), (Buxton, 2003).

One of the most promising application of nanoparticles is their use in

pharmaceuticals as a drug-releasing support for neural diseases, capable

of negotiating the blood-brain barrier. The existence of this barrier is

in fact a severe limitation for the delivery of potentially useful drugs

such as cytostatics and central nervous system in-vitro active agents.

Nanopathology 18

Nevertheless, it has been demonstrated that different drugs bound to

nanoparticles can be transported across the blood-brain barrier and

achieve pharmacological effects in the brain (i.e. brain tumour treatment)

(Li et al., 2003), (Lockman et al., 2002), (Koziara et al., 2003), (Rantioa

et al., 2004), (Schroeder et al., 1998).

Nanosized materials can be the substrate where viruses or DNA

molecules can be encapsulated or sorted. The interaction of these

biomolecules with the nanoparticle, nanotube or nanosized surface can

serve to detect specific proteins or viruses and can guarantee that the

molecules are carried to the specific target. The integration of biological

molecules with Carbon nanotubes (i.e. fullerene molecules with a

cylindrical or toroidal shape) has potential applications in gene- and

drug-delivery technology and enables the use of hybrid systems like

biomolecular sensors. In addition, peptide functionalized nanoparticles

were created as a new class of therapeutic and diagnostic agents with the

purpose of specifically recognizing and binding the characteristic

molecular signature of cancer and other pathological conditions. (Salem

et al, 2003), (Zheng et al, 2003), (Sando et al,2003), (Chakrabarti et al.,

2003), (Lehmann-Horn et al., 2003), (Ai et al. 2003), (Gao et al., 2003).

But, besides its visible advantages, the possibility to interact with the

smallest components of the human body makes nanoparticles potentially

dangerous, and verifying the results of this interaction is an awkward

matter (Pereira et al., 1999), (Saebo, 2004).

Many researchers and organizations (web ref. 1), (Tran et al., 2005),

(web ref. 2), (Greenpeace Environment Trust Report, 2003), (US EPA

Report, 2005), (SCHENIR Report, 2005), (web ref. 3), (Proceedings of

St. Gallen, 2006), (web ref. 4), (web ref. 5), (web ref. 6) are busy

discussing the possible risks related to these new technologies and the

possibility to manipulate in such a small-scale way the cell response.

They can actually enter the caveolar openings in cell membranes and

the perturbations produced can cause the death of the cell. Nanoparticles

have also a high surface reactivity and can interact with enzymes or

proteins, disturbing biological processes and causing impairments to

structural or metabolic processes. (Schler et al., 2003), (Colvin, 2003),

(Warheit, 2004), (Borm et al., 2004), (Brook et al., 2004), (Maynard et

al., 2004), (Hett, 2004).

In-vitro and in-vivo Biological Behaviour of Micro and Nanoparticles 19

2.2.5 Nanomedicine

Nanomedicine (Editorial, 2003), (web ref. 7), (web ref. 8) is a new field

of research that deals with all the applications of nanoparticles or

nanodevices for medical uses.

It refers to highly specific medical intervention at the molecular scale

that involves the use of engineered nanodevices and nanostructures to

monitor, repair, construct and control the human biological system. The

most elementary of nanomedical devices will be used in the diagnosis of

illness. A more advanced use of nanotechnology might involve, for

example, implanted devices to dispense drugs or hormones as needed in

people with chronic imbalance or deficiency states. Lastly, the most

advanced nanomedicine involves the use of nano-robots as miniature

surgeon. Such machines might repair damaged cells or even get inside

cells and replace or assist damaged intracellular structures (heart

defibrillators and pacemakers are likely to be replaced by more

sophisticated devices that can act on the individual cells). And in a

possibly not so far future, nanomachines might replicate themselves or

correct genetic deficiencies by replacing DNA molecules.

Nanospheres are already employed in humans, though only

experimentally, in diagnostics. Inhalable nanoparticles can sense, for

example, the local ventilatory status, releasing drugs at appropriate local

dosage in response to physiological stimuli and lipid-based nanoparticles

are being considered for site-specific delivery of antifungal therapy

to the pulmonary epithelium. Moreover, it was observed that highly

lymphotrophic super-paramagnetic nanoparticles (monocrystalline Iron

oxide) can easily gain access to lymph-nodes by means of interstitial-

lymphatic transport in patients affected by prostate cancer. Their

presence in lymph-nodes can be detected by MRI (Magnetic Resonance

Imaging) and their concentration can indicate a metastasis (Harishingani

et al., 2003), (Babes et al., 1999).

Scientists have also shown that Iron oxide nanoparticles as small as a

virus can outline not only brain tumours under MRI, but also other

cerebral lesions caused by multiple sclerosis, stroke and neurological

disorders, that may otherwise go unnoticed. Iron oxide nanoparticles can

Nanopathology 20

be delivered across the blood-brain barrier and stay in the brain lesions at

least for days. For this reason, Iron oxide has some advantages over

Gadolinium, which must be administered just before surgery and which

doesn’t enter cells. Advances in magnetic resonance imaging using Iron

oxide are showing to be particularly promising to improve diagnoses also

in liver and neck tumours (Mack et al., 2002), (Schultz et al., 1999).

The same nanoparticles can be used also for cancer therapy. Once

delivered in the tumour they are involved in a field of radiofrequency

waves that induce a heating of the Iron particles and of the cells

that produces the death of the cells and the necrosis of the tumour.

(Brigge et al., 2002), (Fahlvik et al., 1990), (Boomemain et al., 2001),

(Hogemann et al., 2001), (Moghimi et al., 2001), (Allport et al., 2001),

(Wunderbaldinger, 2001), (Wilhelm et al., 2002), (Moore et al., 2001).

This new possible therapy is under evaluation with in-vitro and

in-vivo experiments. The given references can be the base for a

deepening of the matter.

From our point of view, we look with a critical eye such an invasive

diagnosis/therapy that releases intentionally not biodegradable particles

inside the organism, particles which are inevitably sensed as foreign

bodies. The reaction against that unusual, unexpected invasion is not the

one produced against, for example, foreign proteins: we do not have

specific enzymes, specific antibodies and there is no anaphylactic shock.

Those particles represent something unknown to the human body, but

when they are inside the blood circulation they are immediately sensed

and interact with the blood’s components and they do it physically,

chemically and biologically. This interaction is the key-point of

modifications that can have systemic effects, not immediately the way it

happens with foreign proteins, but after a more or less long while.

Nanoparticles have a size that is roughly the same as the tri-dimensional

size of proteins. The attachment of a nanoparticle to a protein (for

physical adhesion, for electrostatic attraction, etc.) can cause a stretching

of chemical bonds, a stress of the protein’s morphology, and the

denaturation of the proteinic structure, causing the non recognition of the

compound as something rightfully belonging to the organism. That can

be the foundation of an immunological disease.

In-vitro and in-vivo Biological Behaviour of Micro and Nanoparticles 21

But what is very critical, is the fact that after the imaging or the

therapy have been performed, these foreign bodies cannot be disposed of,

remain inside the body as foreign particulate matter, and their long- term

effects are mostly unknown. Something about those effects is described

in the following paragraph.

Also the application of quantum dots in Medicine may be seen as a

possible cause of unknown effects.

Quantum dots (QDs) are semiconductive crystals that do not exist in

nature, with dimensions ranging from 1 to 10 nm, and that can be

fluorescent. They can emit light throughout the visible spectrum from the

infrared to the ultraviolet. What makes our dots unique is that their

luminescence can be tuned to any wavelength over a broad spectral range

and be stable under ambient conditions (Bruchez Jr M et al., 1998),

(Bruchez Jr M, 2005). (web ref. 9).

Examples of QDs are Cadmium selenide, Lead selenide, and Lead

Sulphur. Cadmium selenide can be the core of a QD with an outer shell

of Zinc.

Their peculiarity is the fluorescence emitted after having been

excited. That fluorescence is more perceptible and more prolonged

as to that of the normal fluorescent agents used in Medicine

(fluorescin, etc.)

They can be combined with antibodies in an easy way and can

be driven inside a cell. Their fluorescence can be detected and be

used to verify its state. Studies to use them in vivo seem to demonstrate

their capacity to be attracted by tumour cells and show their borders

in a body.

But the main problem that remains beyond the technological beauty

of these items is “what is the fate of them after use inside the body?”

They can enter easily in the cells but they are inorganic matter masked by

organic one.

Nanopathology 22

2.3 The results of the nanopathology project

In 2001, we coined a new word: “nanopathology”, meaning by that the

collection of pathologies due to micro- and nanoparticles. At that time,

this was a void concept, since a comparatively small number of

pathologies were recognized worldwide as triggered by particulate

matter. Silica particles and asbestos fibres are recognized to be able to

induce lung diseases as silicosis and asbestosis. Inhalation of dust

containing particles of quartz or fibres of a silicate is known to trigger

lethal pathologies of the lungs. It is a well-known fact that the inhalation

of mine dust or of cigarette smoke are risk factors for the onset of lung

cancer. But a possible correlation with nanoscaled particulate matter was

completely unknown.

In 2001, submicronic matter was recognized to be dispersed in the

environment, though very few studies existed, though none of them was

exhaustive, on its behaviour, but nothing had been done for nanosized

particles.

No evidence identified nanodust as responsible for pathologies. Just

micronic and, in very few instances, submicronic particles were

considered to be the cause of pathologies (e.g. pneumoconioses).

Now that nanotechnologies are a rapidly growing discipline, society

is scared by possible side effects of the production, manipulation and use

of nanoparticles, and nanosafety has become by necessity a priority.

Many researchers are investigating the toxicity of nanoparticles towards

cells, while our project has already gone further by investigating their in-

vivo interaction and the impact they have to humans.

Among the most abundant air pollutants in urban areas is particulate

matter with a mean diameter ≤10 µm (also called PM10 by

environmental toxicologists, defined as particulate matter with an

aerodynamic size of 10 µm or smaller). Over the years, it has become

clear that particles with a very low size (especially ≤100 nm) are more

significant health-wise than larger particles, since they have been shown

to be capable of inducing far more severe effects. (Oberdörster, 2001).

The surface/size-ratio increases exponentially with the decreasing of

particle sizes, leading to enhanced surface reactivity. This enhanced

In-vitro and in-vivo Biological Behaviour of Micro and Nanoparticles 23

surface reactivity might lead to greater biological activity per given mass,

compared to larger particles, which, in turn, might have effects, for

example, on the internalization of particles into tissues, cells and

organelles, or on the induction of oxidative stress (Oberdörster et al.,

2005). By definition, particles with a size below 100 nm are called nano-

scaled particles (short: nanoparticles) or ultrafine particles by

toxicologists.

Because of the minute size of nanoparticles, the internalization into

the body’s tissues appears to be extremely easy. This was shown by

experiments in human volunteers with radioactive-labelled Carbon

nanoparticles (i.e. `Technegaz`) that were shown to pass rapidly into the

systemic circulation after inhalation. Radioactivity could already be

detected in the blood one minute after inhalation (Nemmar A. et al.,

2002). Furthermore, animal studies revealed that inhaled nanoparticles

were relocated into the liver (Oberdörster G. et al., 2002) and the brain

(Oberdörster G. et al., 2004). Thus, nanoparticles seem to be able to

circumvent the tight blood-brain-barrier and possibly cross the blood-

placenta barrier (Reichrtova E. et al., 1998), (Kaiglova A. et al., 2001).

Moreover, it has been suggested, and we offered further evidence, that

nanoparticles are involved in thrombus formation (Nemmar A. et al.

2002), (Gatti A.M. et al., 2004) and today we know that particulate air

pollution is associated with enhanced mortality from respiratory and

cardiovascular diseases (Pope C.A., 2000).

As the sources of internalized nanoparticles (food, air, etc.) and the

location of particle detection are generally far apart, a distribution via the

blood stream must have occurred. Thus, endothelial cells, which line the

inner surface of blood vessels, will have direct contact with the particles.

Those cells are important in inflammation mechanisms and wound

healing. Upon pro-inflammatory stimulation of the endothelium,

adhesion molecules are expressed on the cell surface, thus mediating

leukocyte attachment (e.g. E-selectin and intercellular adhesion mole-

cule-1/ICAM-1). Besides, endothelial cells can release cytokines, such as

interleukin-8 (IL-8, a key factor in neutrophil chemotaxis). Thus, these

features contribute to the pro-inflammatory endothelial phenotype that

permits the transmigration of leukocytes from the blood into the

perivascular space (Cook-Mills J.M. et al., 2005).

Nanopathology 24

The activation of IL-8, E-selectin and ICAM-1 is regulated by the

same transcription factors NF-κB (nuclear factor-κB) and AP-1 (acti-

vator protein-1) (Montgomery K.F. et al., 1991), (Roebuck K.A., et al.,

1995), (Mukaida N. et al., 1994).

The in-vitro tests we carried out within the scope of the European

project considered the interaction of 5 different engineered nanoparticles:

Titanium oxide: titania (TiO2), Silicon oxide: silica (SiO2),

Polyvynilchoride (PVC), Cobalt and Nickel with cells (human

endothelial, gut epithelial, liver epithelial and monocytic cells) and

organs (in-vivo tests with rats). Those nanoparticles were examined with

respect to the cellular internalisation and their influence on the cell

viability, proliferative activity, and the pro-inflammatory endothelial

phenotype. Titanium oxide (titania) was selected since it is employed in

many beauty and sun-screen creams and in wall paints, Silicon oxide for

its use in chewing gum and coatings, PVC because it is used in a bulk

form for medical devices, Cobalt because of its toxicity and Nickel

because it is allergenic1.

One of the main characteristics of nanoparticles is their clustering,

namely their capability to form microsized aggregates that provoke their

sinking in the medium. So, a real interaction with cells is not allowed if

proper procedures to disperse them in the medium are not adopted. Some

authors claim good non-cytotoxic results, but they did not verify if a real

cell-nano interaction was set-up.

The nanoparticles mentioned were added to macrophages/monocytes,

gut and liver epithelial cell culture medium and the cytotoxicity assays

verified the cell survival, the production of inflammatory and defence

mediators as the cytokine production, and the modulation of TLR

expression. These are parameters that can give an idea if the presence of

nanoparticles weakened the “healthy” state of the cells .

The cytotoxicity studies carried out by Dr. D. Boraschi (CNR Pisa –

Italy) showed that Cobalt nanoparticles induced a lesser expression of

1 SiO2 and TiO2 particles were produced by flame spray pyrolysis. The size range of SiO2

was 4-40 nm with 14 nm mean particle size; of TiO2 20-160 nm (mean size 70 nm),

of Co particles (Sigma) 50-200 nm (mean size 120 nm), of Ni was about 62 nm

(Nanoamor), of PVC (polyvinylcloride without phthalate) 100 nm.

In-vitro and in-vivo Biological Behaviour of Micro and Nanoparticles 25

TLR4, TLR7, TLR8, TLR5 TLR10 and CD14. The other materials have

different behaviours (Lucarelli M. et al., 2004).

The tests verified that:

1- Nano-particles do not affect cell survival or proliferation. Only Co

nano-particles are toxic at >100 mg/106 cells

2- Co nano-particles could have an M2 biasing inhibiting effect of

TLR4/CD14-mediated responses. It was noted a down-regulation

of TLR4 and CD14 expression, inhibition of reactivity to LPS in

macrophages.

3- Nano-particles, in particular ZrO2, induce increase expression of viral

TLR. There is a possible impact on cell reactivity to viral infections

4- SiO2 nano-particles have M1 biasing effect on macrophages. There is

the induction of M1 markers in naive cells, amplification in M1 cells

5- Nano-particles, in particular TiO2, amplify IL-18 activity (role in

initiation of autoimmunity?). That means that there is a differential

expression of IL-18 regulatory molecules in macrophages and

hepatocytes.

So there is not an evident and significant cytotoxic effects at subtoxic

concentration, but an impairment of cellular viability was observed at

different levels (i.e. decrease of cell number, protein expression of the

proliferation marker Ki67, and the metabolic activity).

According to studies carried out by J. Kirkpatrick (University of

Mainz – Germany) a pro-inflammatory effect in HDMEC (Human

Dermal Microvascular Endothelial Cells) occurred after exposure to

SiO2, Co, and Ni particles and was apparent by an enhanced release of

IL-8. Only higher particle concentrations (25 and 50 µg/ml) induced this

increase in IL-8 release. The E-selectin protein expression was enhanced

by high amounts of Co-particles whereas Ni-particles induced no protein

expression of E-selectin. In contrast to the particles, divalent Co and Ni

ions induced the expression of all pro-inflammatory markers tested (i.e.

IL-8, E-selectin, ICAM-1) (Peters K. et al., 2004).

That means that the normal cell defences are weakened in presence of

nanosized foreign bodies

In order to evaluate and quantify of pro- and anti-angiogenic

characteristics of the nanoparticles we selected another model system

Nanopathology 26

that leads to the formation of in-vitro capillaries within a three-dimen-

sional extracellular matrix (fibrin and type I collagen).

The addition of the different particles (TiO2, SiO2, Ni, Co, PVC) into

the three-dimensional matrix revealed that only the presence of Co

particles led to changes in the in-vitro capillary phenotype. A number of

abortive sprouts were formed and the developing sprouts were not so

pronounced as in the positive control. However, cell viability appeared

unaffected. Software-supported quantification of these Co-particle-

induced changes revealed a significant reduction in the degree of

angiogenesis in vitro.

Also the tests employing the co-cultivation of endothelial cells

together with the monocytic cell line U937 in the angiogenesis model

system verified the hypothesis according to which the addition of PMA-

stimulated U937 cells to the culture could lead to a distinct decrease of

the length of in-vitro-capillaries and also to a reduction of endothelial

cell number.

In detail the tests with endothelial cells verified:

a- The nanoparticles investigated do not affect cell survival or

proliferation. Only Co nanoparticles are toxic at >100 mg/106 cells. They

inhibit the expression of LPS receptors TLR4 and CD14 and induce a

down-regulation of mRNA expression for TLR4 and CD14 (the two

receptor chains that recognise bacterial lipopolysaccharide and activate

macrophage defence functions).

An interesting result is that Co nanoparticles impair macrophages

activation by bacterial LPS, in fact a negligible cytokine production

was detected. These results mean that in presence of these metallic

nanoparticles the macrophages become unable to mount appropriate

defences to bacterial challenge. That means that danger exists of

increased susceptibility to infections. These very interesting results were

not obtained with Ni nanoparticles since, due to their forming micrometric

aggregates and sinking in the medium, they did not interact with the cells.

b- The Cobalt nanoparticles with endothelial cells showed an

angiogenic behaviour.

Our study has shown that human endothelial cells possess a large

capability to internalize nanoparticles. All nanoparticles tested were

taken up by the endothelial cells and to a major extent into vacuoles.

In-vitro and in-vivo Biological Behaviour of Micro and Nanoparticles 27

The Cobalt nanoparticles showed an angiogenic behaviour, that can

trigger a carcinogenic reaction.

The results induced to put forward the hypothesis that the observed

pro-inflammatory activation after Co particle exposure may be attributed

to a release of divalent Co ions by the particles [Cobalt nanoparticles

inside the cell corrode and release Cobalt ions], since the exposure of en-

dothelial cells to these ions leads to the impaired endothelial viability and

pro-inflammatory stimulation. This contrasts with the effects of the Ni

particles. Here, the suggestion of a Ni ion release by the particles

resulting in an induced pro-inflammatory stimulation is not congruent

with the pro-inflammatory effects induced by the respective ions, since

Ni ions induced both an increase in the release of IL-8 and the protein

expression of endothelial cells adhesion molecules (i.e. E-selectin and

ICAM-1), whereas Ni particles induced only an increased IL-8 release

and the expression of adhesion molecules was not initiated. This

indicates an activation mechanism for the Ni-particles that deviates from

the Ni-ion-induced activation that is shown to occur via a cooperation of

the above-mentioned transcription factors NF-κB and AP-1. Since

oxidative stress is also a relevant aspect in the mechanisms of (Ni-)

particulate matter-induced effects (Koziara et. al., 2003) this mechanism

of differential activation of pro-inflammatory gene promoters might

play a role. Thus, it can be suggested that the Ni-ion release by the

nanoparticles remains under the critical limit for pro-inflammatory

activation but further Ni nanoparticle induced effects (possibly oxidative

stress) are responsible for the enhanced IL-8 release. Also the tests with

the co-cultures (macrophage-endothelial cells) indicate a deep interaction

of Co nanoparticles with the cells. The addition of PMA-stimulated

U937 cells to the culture led to a distinct decrease in the length of in-

vitro capillaries and also to a reduction of endothelial cell number. In

addition to that, the number of U937 colonies within the 3-dimensional

extracellular matrix rose.

These in-vitro results indicate some mechanisms of actions of

nanoparticles with cells and explain the in-vivo results and the clinical

evidence.

Nanopathology 28

2.3.1 In-vivo experiments

The 5 different materials mentioned above (Cobalt, Nickel, silica, titania,

PVC) were implanted in rats in two forms (bulk samples as discs, and

nanoparticles).

That experiment was performed in order to verify if the size of these

debris is an important factor in triggering pathological reaction.

Each rat (see Fig. 2.3.1) was implanted in the dorsal muscle with two

similar implants: an amount of 5 cc of nanoparticles and a 10-mm

diameter, 0.8 mm height disc of the same chemical composition. For

each group of materials, 2 rats were sacrificed after 6 months (short

term), and 3 rats were sacrificed after 12 months (long term). In addition,

5 control rats were implanted with a reference material. Two of them

were sacrificed after 6 months and 3 of them were sacrificed after 12

months. Five rats were fed by gavage with a mixture of nanoparticles

(50% Ni and 50% ZrO2 with two different ranges) (about 0.030 – 60 mg

by day): 2 were sampled after 6 months and 3 were sampled after 12

months, in order to see if the small-size particles can negotiate the bowel

barrier and contribute to the pathogenesis of some diseases. The animals

were sacrificed after 6 and 12 months.

After the short- and long-term implantation, samples were explanted

and the gross necropsy of the animals performed. All samples were

formalin-fixed and paraffin-embedded, microtome-sliced and Hematoxylin-

Eosin-stained. The sections were analyzed histopathologically and under

ESEM in order to verify the interface of the bulk and nanosized materials

after the interaction.

Our animal studies verified that the metallic nanoparticles were

cancerogenic, while the bulk material induced just a fibrotic capsule with

a granulomatosic reaction.

In-vitro and in-vivo Biological Behaviour of Micro and Nanoparticles 29

Fig. 2.3.1 Cobalt and Nickel nanoparticles induced rhabdomyosarcoma in the rats’ back.

The metallic disks induced only fibrotic capsules. (Courtesy of Biomatech-France)

The other materials (PVC, silica, and titania) developed only a

fibrotic capsule or a granulomatous tissue. The reason could be the

agglomeration of the nanoparticles that transforms them to micrometric

debris. In this case, the body reacts to the size of particulate matter, as the

materials are chemically inert.

A summary of macroscopic and microscopic findings are presented in

Tab. 2.3.1.

Nanopathology 30

Tab. 2.3.1 Synopsis of the histological findings.

Material 6-month period 12-month period

TiO2: NP Granuloma Granuloma

TiO2: B Inflammation Inflammation

SiO2: NP Inflammation Inflammation

SiO2: B Inflammation Inflammation

PVC: NP Granuloma Granuloma + fibroblastic

proliferation

PVC: B Inflammation Inflammation

Co: NP Preneoplasia Neoplasia*

Co: B Inflammation +

granuloma Preneoplasia*

Ni: NP Neoplasia #

Ni:B Neoplasia #

Note: B= bulk, NP= Nanoparticles; * Animals were euthanized after 8 months. # No

analysis - animals were euthanized after 6 months.

TiO2 – In detail: a thin, fibrous, moderately capillarized pocket

surrounding the bulk material implanted subcutaneously was observed in

all cases, but no granulomas were present.

SiO2 – In detail: in all cases, an adherent thin fibrous pocket

surrounding the bulk material implanted subcutaneously could be seen,

like what happened with TiO2, without any evidence of local intolerance.

The inflammatory infiltrate consisted mainly of lymphocytes and

monocytes, but not of mast cells.

At the site of the intramuscular implantation, neither particles nor

macroscopic abnormality could be seen. Histological examination of the

specimens with implanted particles revealed inflammatory infiltrates

which were localised subcutaneously and intramuscularly. An increased

numbers of mast cells, typically localized perivascularly, could be

detected in most cases.

PVC – Also in all these cases, a thin fibrous pocket around the bulk

material implanted subcutaneously was observed, without any visible,

local lesion. And also in these cases, the bulk material itself did not

reveal anything abnormal. Histological evaluation of implanted PVC

In-vitro and in-vivo Biological Behaviour of Micro and Nanoparticles 31

bulk showed discrete chronic inflammatory infiltrates. No particles could

be observed.

The sites implanted intramuscularly with nanoparticles did not show

anything macroscopically remarkable. In 8 cases out of 10, particles were

easily visible in the subcutaneous tissue. On histology, all cases of

implanted PVC nanoparticles revealed intramuscular foreign body-type

granuloma. Macrophages and multinucleated foreign-body giant cells

could be seen around amorphous, unstained polygonal material that did

not exhibit birefringence under polarized light. However, the particles

revealed intense cherry-red colour by staining with the Oil Red-reaction.

A slight perigranulomatous fibrosis was also observed. An interesting

find is the observation of foci of fibroblastic proliferation in three

animals. The cells were spindle-shaped and showed some nuclear

pleomorphism. On immuno-histochemistry, they showed an enhanced

nuclear expression of PCNA (Proliferating Cell Nuclear Antigen).

Cobalt – Clinical examination revealed the formation of nodules in

three animals occurred within the 6-month observation period. Then, all

remaining animals implanted with Co developed nodules at the sites

where nanoparticles had been implanted. One animal of the 12-month

observation group died 8 months after implantation. So, for ethical

reasons because of the development of these handicapping tumors, all

remaining animals were sacrificed at 8 months. In three out of the four

cases belonging to the 6-month group and in six cases of the 8-month

group, nodules developed at the site where nanoparticles had been

implanted.

Through histological observation of the specimens with implanted

nanoparticles, malignant, intramuscular, mesenchymal tumors at the

original implantation site was demonstrated in one out of four animals of

the 6-month observation period and in five out of six cases of the 8-

month period. Particles were observed in three of six animals belonging

to the 8-month group.

No such tumors were not observed around the bulk material, where

a discrete inflammatory infiltrate composed of mononuclear cells and

lymphocytes was present. In the subcutaneous area, a discrete fibrosis

had developed, but not granulomas.

Nanopathology 32

In two of the four animals of the 6-month observation period (at the

nanoparticulate site) and in two of the six animals of the 8-month

observation period, a capsule with fibroblastic proliferations was visible.

The cells showed an increased pleomorphism of the nuclei and mitotic

rate. In addition, they displayed strong expression of the proliferation

marker PCNA. These lesions are classified as pre-neoplasia.

Grouped together, these results show the presence of a malignant

mesenchymal tumor in five of the six cases of implanted Co nanoparticle

sites and two further cases (one around bulk material and one at the

nanoparticulate site) with pre-neoplasia.

Nickel – Three of the animals from the Ni-implanted group died

respectively 4, 4.5 and 5.5 months after implantation. Because of such a

high mortality rate and the development of tumors, we decided to

sacrifice all the remaining animals belonging to that group at 6 months.

Both subcutaneous and intramuscular implantation sites had developed

visible nodules in all cases and in all cases necrosis was detected inside

the nodule associated with deposits and exudate seen in the implanted

subcutaneous pocket.

Both Ni bulk and nanoparticles of implanted Ni showed a malignant

mesenchymal tumor surrounded by a fibrous capsule localized

subcutaneously and intramuscularly. In general, abundant areas of central

necrosis, dystrophic calcification and ossification were also observed. In

contrast with that, tumors in tissue specimens implanted with bulk

material showed a central cystic component, suggestive of the implant

site. In addition to that, necrosis looked less important, and ossification

was more rarely seen. Particles could not be observed histologically.

The findings indicate a malignant mesenchymal tumor, which is

classified on the basis of the morphology and the immuno-histochemical

marker profile as rhabdomyosarcoma.

The results obtained, though preliminary and in need to be confirmed

by further experiments, show that size is a decisive factor in determining

the compatibility of a material with the organism. As to materials, metals

seem to be much more reactive in a pathogenic way then ceramics and

polymers. These results must be reminded for all the following chapters.

In-vitro and in-vivo Biological Behaviour of Micro and Nanoparticles 33

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39

Chapter 3

Clinical Cases: Lung, Blood, Liver, Kidney, Digestive System,

Vessels, Sperm ___________

3.1 Introduction

his chapter contains a number of clinical cases studied on bioptic or

autoptic samples. No homogeneity or statistical meaning is to be

expected or looked for here, as this is just the start of a collection of

cases that will hopefully keep coming and being added to the ones

illustrated here. Anyhow, what is described in this part of the book

represents answers given to patients, or their relatives or doctors when

the patients had died, regarding the exposure those patients had

undergone. In many instances, we had no advice to give as to a possible

treatment, but when somebody knows he is dying, knowing why can be

important to him. In any case, the experience we are collecting can be

useful to prevent the repetition of similar cases or to find an explanation

to cryptogenic diseases.

It was hard, when not impossible at all, to guess how particles

actually behave once they have entered the organism. So, one of the

problems was to select the organs that make up a favourite mark for

those particles, if such a thing as favourite organs exists.

Inflammatory pathologies of unknown origin had already proved to

be a very interesting source of knowledge, but in our then limited

experience we had seen that dust might possibly be blamed for other

forms of adverse reactions like, for example, the formation of blood

7

Nanopathology 40

thrombi or - but in that case the thing was far more complicated - for

some neurological diseases or pathologies whose classification is

controversial like chronic fatigue.

We studied more than 600 aggregate cases of vein thrombosis,

chronic fatigue, lymphomas, solid cancers of various organs, etc.

Reference tissues were obtained from cadavers of young, presumably

healthy, subjects, died in car accidents.

All the pathological specimens we had the chance to examine showed

the presence of inorganic, non biocompatible and non biodegradable

debris, ranging 10 nm - 10 microns. Some of those debris (10-100 nm)

were also photographed inside cellular nuclei.

The observation and study of the pathological samples we came

across and are described here showed a few interesting data. In the cases

of biological reactions typical of some diseases like cryptogenic

granulomatosis, sarcoidosis and Crohn’s disease, foreign bodies were

always present inside the granulomatous tissue. In some cases, the

chemical composition of the particulate detected made it possible to

identify the kind of exposure the patient had undergone. Micro and

nanoparticles were always present in cancerous tissues, but it must be

emphasized that this constant presence was due to a pre-selection of the

cases we observed. As a matter of fact, in a number of patients suffering

from cancer we had the chance to check outside the scope of this project,

no particles were found. This is only natural, as predisposing and causing

factors exist, such as genetics, radiations, exposure to organic solvents

and many other pollutants. In the cases investigated, mainly concerning

primary cancers, the inorganic foreign bodies were concentrated at the

interface between cancer and healthy tissue. This specific location, never

discovered before, seems to be meaningful in understanding the

mechanism regulating the onset of cancerogenicity. This specific

location could also explain why in some cases we can’t find anything:

The planar section we analyzed was part of a bulk, tri-dimensional

sample and it is likely that the interface was not included in that

particular section.

Once, we could not use the tissue harvested from one of the “healthy”

cadavers we used as reference, as it contained Calcium carbonate. It

turned out that that boy had been drug addicted and the mineral was used

Clinical Cases: Lung, Blood, Liver, Kidney, Digestive System, Vessels, Sperm 41

to blend the drug. After that, considering the difficulties that carrying out

an anamnestic study implies with relatives or friends about the health

state of somebody who has died, our attention was addressed to people

we had reasons enough to consider “non contaminated”.

After the 3-year project sponsored by the European Community we

carried out, the reference samples, i.e. “the standards”, are represented by

the internal organs of feti of induced abortions. Their tissues are “clean”,

since we believed they had not been exposed to environmental pollution.

That was an extreme choice to obtain reference samples with “zero

contamination”, but that choice reserved some surprise.

In fact, the study mentioned above involved the verification of animal

and human malformed feti. In a few instances, dust was detected in their

tissues, and that showed that nano-contamination can be shared between

mother and child through the umbilical circulation.

Fig. 3.1 Schematic view of the entrance ways of particles in the organism.

Nanopathology 42

Fig. 3.1 serves to understand the paragraphs that follow, as it

describes graphically how particles, be they inhaled or ingested or, as a

matter of fact, introduced in any other way into the organism, reach any

tissue. The lung is the first structure to be interested, followed by the

blood, the liver and the kidney. After that, the digestive system has been

treated, but the contaminations found in the liver and the kidney could

have originated from inhalation as well as from ingestion. Sperm closes

the chapter. We did not describe anything regarding the brain, as we

could examine only two cases, in both of which particulate matter could

be seen.

All the images below are electron-microscope picture of pathological

tissues. They are presented together with their relative chemical EDS

spectrum (see Appendix). All spectra show the peaks of Carbon and

Oxygen that belong to the biological substrate, to the Carbon support

where the specimen is laid, but may as well be part of the spot aimed at,

which is in all cases an inorganic particle. We cannot be certain if

Carbon and Oxygen are real components of the particle or just an

artifact. For that reason, Carbon and Oxygen are not listed in the

elements detected.

3.2 Lung

We may say that we inaugurate our life with an inspiration of air. In fact,

neither our entire body nor any of the cells that make it up could survive

without Oxygen, since much of our metabolism is based upon a series of

oxidations.

That is the reason why the inhalation of air, a blend of gases where

Oxygen represents about one fifth, is necessary. But the air we breath is

not so pure as we would desire it to be. Among many other pollutants,

it contains all the by-products of the combustion processes related to

fires, heating, and industrial and automotive processes. So it contains

gases like Carbon dioxide and Carbon monoxide, furans, dioxins,

hydrocarbons, etc. And it contains dust which for size and chemical

composition is different from the natural one.

Clinical Cases: Lung, Blood, Liver, Kidney, Digestive System, Vessels, Sperm 43

Medical Geology is a new discipline of investigation that studies the

impact of natural dust on human health. (Centeno, 2005).

Who, for instance, lives close to a volcano has a higher probability to

suffer from lung diseases, since, even if that volcano is not active, the

erosion of lava streams by rain, winds and temperature changes pollutes

the environment in a constant way. But the industrial and/or combustive

processes release dusts with chemical compositions that have never been

present on this planet and, therefore, are completely unknown to the

human body and its metabolism.

Because of its morphology and size, inhaled dust can interfere

physically with the passage of Oxygen through the alveoli, since it

creates a hard-to-negotiate barrier to the exchanges of the gases (Oxygen

against Carbon dioxide). But dust, perceived as a foreign body in the

alveoli, can also create a different local problem consisting in an

inflammatory reaction which can degenerate.

For those very well-known reasons, many environment-protection

agencies, among which that of the European Community, set limits to the

concentration of particulate pollution in the atmosphere. Nowadays those

limits are based on the mass of PM10 in a fixed volume, but a more

scientific determination, like number of particles evaluated according to

the reciprocal of their size is certainly hoped for. (Armaroli, N. et al.,

2003), (L.M. Brown et al., 2000), (J. Ruuskanen et al., 2001), (S. Ebelt

et al. 2001), (R.M. Harrison et al., 2000). The European Community is

preparing a new regulation based on PM2.5, unfortunately still

considering just mass.

For these reasons, we started to investigate samples of lungs affected

from pathologies with different levels of seriousness. Tab. 3.2.1 reports

the list of the pathologies investigated and all the images that follow are

related to these cases.

As can be seen, there are some cases seemingly not related in a direct

way to lung pathologies. For instance: Hodgkin’s and non-Hodgkin’s

lymphoma, skin pesudolymphoma, Wegener’s granulomatosis,

nephrocalcinosis, and Gulf War Syndrome. All these pathologies are

multi-organ-diseases and we analyzed many samples coming from the

same subject, included the lung, even if that was not the organ that

looked mainly interested by the pathology.

Nanopathology 44

We selected the particles shown according to peculiarity of

morphology, size or chemistry.

The lungs, as well as the mouth, the stomach or the colon have a

surface that has been exposed for a long time to pollution, so we find

much dust that remained trapped for a certain period of time. In the case

of a half-mm2 sample of the mouth mucosa we found as many as 600

sub-micronic particles of 10 different chemical compositions.

Tab. 3.2.1 List of the investigated samples of the lung.

Pathology No.

Fibrosis 8

Silicosis 1

Granulomatosis 3

Wegener’s granulomatosis 1

Pulmonary Emphysema 1

Sarcoidosis 3

Gulf War Syndrome 2

Pneumothorax 14

Pleural Coniosis 1

Nephrocalcinosis 1

Racemose ossification 1

Hodgkin’s Lymphoma 1

Non-Hodgkin’s Lymphoma 1

Skin Pseudo-lymphoma 1

Pleural Mesothelioma 32

Cancer 17

Not diagnosed 4

Reference 20

Total 112

First of all, a basic concept must be clear: most gross particles remain

on the inner surface of the alveoli, while the smaller ones, and the more

so the smaller they are, can negotiate that anatomical barrier and enter

the bloody stream.

Fig. 3.2.1a shows the surface of a lung alveolus with large particles of

surely combustive origin. That can be inferred by their high porosity,

something typical of combustion processes. Fig. 3.2.1b, instead, shows

the section of a blood vessel with red cells in its lumen stuck together,

Clinical Cases: Lung, Blood, Liver, Kidney, Digestive System, Vessels, Sperm 45

and white particles with a size smaller than 2 µm. They look whiter than

the biologic matter around them because of their atomic density, higher

than the biological surroundings’. In this context they can express their

possible non-biocompatibility by a thrombogenic effect and thus induce

the formation of a thrombus. Pulmonary thromboembolism may be the

consequence of that phenomenon in the venous circulation, while stroke

and infarction may be what the phenomenon triggers in the arteries.

What we documented in the blood vessels suggests also another

possible effect. Particles are much harder than any blood components

and of the tissue making up the vessels. They are transported by the flow,

so they can hit and “scratch” the endothelium, causing a vasculitis. As a

matter of fact, many of the pathologies investigated present also that

particular symptom.

Fig. 3.2.1a – Fig. 3.2.1b ESEM images of 2 types of particles. The former is deposited on

the surface of an alveolus, while the latter is trapped inside a blood vessel.1

Much literature exists describing an increase of lethal episodes in

cardiopaths in relation with an increase of PM2.5 in the atmosphere, and

the fact becomes easy to explain in the light of our observations (Courter

1 This photo was published in Handbook of Nanostructured Biomaterials and Their Applications, Volume 2, Gatti A.M., Montanari S. “Risk assessment of micro and nanoparticles and the human health”, cap. 12, 347-369 ed American Scientific Publisher USA 2005.

Nanopathology 46

L. A. et al., 2007), (Schwarze, P. E. et al., 2006), (Newby, J. A. et al.,

2006).

For a long time, silica and silicates particles have been known as the

responsible to a form of pneumoconiosis called silicosis, while asbestos

has been blamed as the cause of mesothelioma (asbestos can be

considered nanoparticle, since one of its dimensions is in the nanosize).

Pleural Malignant Mesothelioma, a malignant tumour that spreads

from the parietal pleura in the chest cavity and sometimes involves the

lung, is a kind of tumour strictly correlated with inflammatory reactions

and is due to environmental contamination.

Though not the only one, asbestos exposure is the most common risk

factor (involving more or less 80 percent of mesothelioma patients), but

other promoting factors are known, including chronic lung infections,

tuberculous pleuritis, radiation and exposure to the simian virus 40

(SV40) or other mineral fibres.

Of that highly malignant tumour very little is known to date about its

molecular and metabolic features and awareness is very recently growing

about the relevance of cell-to-cell signalling involved in the modulation

of its cell growth and differentiation, e.g. the presence of NOX and other

signal molecules (Soini et al., 2000) recently found. Of course it is

impossible to see gaseous compounds like NOx, COx, SOx, etc., so only

their effects are detectable, like the production of cytokines or others

compounds of the cell down to gene expression.

Our studies started from a pragmatic vision: if particulate matter

originating from environmental pollution is to be blamed for some

human diseases, we must find it where the biological tissue shows its

main lesions.

Fig. 3.2.2 shows asbestos fibres (a silicatic compound containing,

according to their species, Calcium, Silicon, Magnesium and Iron) inside

a pathological human lung tissue affected by mesothelioma. Around a

fibre, formations called “pearls of asbestos” can be seen. Those are

precipitates of Iron protein induced by the chemical reaction of the

silicatic fibre with the extracellular matrix, rich in Iron. The periodicity

of those pearls is due to the diffusion of ions that rules the phenomenon.

This aspect is well known among biomaterialists expert of bioactive

glasses (a compound of Silicon-Sodium-Calcium and Phosphorus),

Clinical Cases: Lung, Blood, Liver, Kidney, Digestive System, Vessels, Sperm 47

materials commonly used for bone defect repair. (Gatti, et al, 1998) In

fact, these materials are not inert in the human organism, but degrade

releasing ions that create a concentration gradient activating diffusive

processes. In this specific case, the glass releases immediately Sodium

ions and there is a diffusion from the core toward the surface of the glass

of the Phosphorus and Calcium ions, leaving a matrix rich in Silicon (a

Silicon hydrated gel). The surface grows now rich in Calcium and

Phosphorus that precipitate as Calcium phosphate, the matrix for the

colonization of the osteocytes. This way, new bone is rapidly generated.

Fig. 3.2.2 ESEM Images of asbestos fibres with its chemical composition, found inside

a pleural mesothelioma. “Pearls of asbestos” (arrow) are visible(marker 5 µm), (marker

10 µm).2

Pleural mesothelioma has been described in literature for a long time

as related to the inhalation of asbestos nanoparticles with their peculiar

form of needles. But it also a fact that some patients who developed that

2 This photo was published in Handbook of Nanostructured Biomaterials and Their Applications, Volume 2, Gatti A.M., Montanari S. “Risk assessment of micro and nanoparticles and the human health”, cap. 12, 347-369, ed American Scientific Publisher USA 2005.

Nanopathology 48

disease were never exposed to that pollutant. It is a well-known

fact that there is a correlation between environmental pollution and

some diseases, so we looked for environmental contaminants in the

pathological lung tissues.

Fig. 3.2.3 shows another example of asbestos fibres embedded in the

lung tissue. Iron protein pearls are clearly visible.

Fig. 3.2.3 Asbestos fibre in a pleural mesothelioma. (marker 10 µm).

Asbestos is evident in the latter picture, with the typical pearls of Iron

protein. It is important to observe the pointed shape of asbestos crystals,

a shape that makes penetration into tissue easier as compared to other,

bulkier shapes.

Racemose ossification, the disease related to Fig. 3.2.4 is a, rare,

diffuse pulmonary ossification of unknown origin in which mature bone

is found in the pulmonary parenchyma. It affects in general middle-aged

males and is asymptomatic. In the vast majority of cases, the condition is

discovered incidentally at autopsy. The arrow in Fig. 3.2.4 shows a

particle of Iron embedded in the new “bone”. (Wells et al. 1943), (Fried

et al. 1992) (Ikeda et al. 1998), (Trejo et al. 2002).

Clinical Cases: Lung, Blood, Liver, Kidney, Digestive System, Vessels, Sperm 49

Fig. 3.2.4 Image of a racemose ossification. The arrow indicates a particle entrapped in

the part of lung that modified its chemical content into a Calcium-phosphate (marker

50 µm).

Fig. 3.2.5 shows a case of round atelectasis (Stark, 1982) (Menzies

et al., 1987). That disease is a rare lung pseudotumour that in most cases

is associated with asbestos-related pleural disease but can result from

a variety of other chronic pleural diseases like, for example, an acute

pleuropulmonary illness caused by Legionella pneumophila. The disease

presents often as an asymptomatic radiographic finding associated with

chronic pleural disease, usually related to asbestos exposure. In the case

we studied, we found silicatic particles containing Silicon-Aluminium-

Magnesium-Iron-Sodium-Phosphorus-Sulphur-Chlorine-Potassium-

Calcium-Titanium-Iron inside the round-shaped whiter areas. (Stark P.,

1982), (Menzies R et al., 1987).

Nanopathology 50

Fig. 3.2.5 Image of a lung sample affected by round atelectasis. Inside the lung fibrotic

tissue with chronic inflammation we found “round” areas full of silicatic nanoparticles.

Like all sarcoidoses, lung sarcoidosis is characterized by the presence

of small granulomas. In the case of the lung, granulomas can appear on

the walls of the alveoli or on those of the bronchioles. Its origin is still

unknown, but most scientists think it is caused by a disorder of the

immune system (Heffner DK, 2007). The case to which Fig. 3.2.6 refers

shows Mercury-Sulphur nanoparticles in the reaction cutaneous

lymphoid infiltrate. Granulomas can contain foreign bodies. In this case

the nanoparticles detected are chemically toxic.

Fig. 3.2.6 Lung sarcoidosis. Inside a non necrotizing dermal granulomas, chemically toxic

nanoparticles of Mercury-Sulphur-Silicon-Chlorine were found. (marker 5 µm).

Clinical Cases: Lung, Blood, Liver, Kidney, Digestive System, Vessels, Sperm 51

Fig. 3.2.7 concerns a case affected from tubercolo-silicosis. At optical

microscopic observation, the lung parenchyma showed silicotic nodules

with concentric bands of hyalinized collagen surrounded by fibroblasts

and histiocytes with a blackish pigment .

We report images of wide lung calcific areas (white dots). Their

chemical analyses reveal that that is not a normal calcification but the

biological tissue turned into Calcium phosphate. This tissue is hard, not

elastic and can compromise the elasticity of the lung and of breathing.

Fig. 3.2.7 Low-magnification image of tubercolo-silicosis with wide Calcium phosphate

areas (marker 300 µm).

Fig. 3.2.8 High-magnification image of tubercolo-silicosis. At such magnification, Silicon-

Titanium-Aluminium-Iron particles become visible. (marker 50 µm).

Nanopathology 52

At higher magnification (Fig. 3.2.8), it can be seen that the white dots

are micro and nanoparticles of a compound made of Titanium-Silicon-

Aluminium-Iron-Magnesium-Sodium-Chorine-Potassium-Calcium-

Sulphur-Phosphorus. It is hard to tell the origin of that pollution, but

wide areas of the lung are interested by these foreign bodies. The lung

tissue is a composite material with altered morphology and physical

properties.

Fig 3.2.9 Image of a squamocellular carcinoma sample containing Tungsten–based particles

(marker 20 µm).

The patient whom the biopsy relative to a squamocellular carcinoma

(Fig. 3.2.9 and Fig. 3.2.10) was taken from worked at the milling and

rough-hewing of metal bushings for oil-pressure pumps where mineral

oils derived from hydrocarbons were used. Tungsten-Aluminium-Iron-

Titanium nanoparticles were present in his pathological tissue. At

histopathological observation, the squamocellular carcinoma was hardly

differentiated, and differentiation areas were in an adeno-squamous

direction. Wide necrotic foci and marked atypias were also present.

Clinical Cases: Lung, Blood, Liver, Kidney, Digestive System, Vessels, Sperm 53

Fig. 3.2.10 Squamous cellular carcinoma with Titanium-Silicon-Aluminium-Phosphorus-

Chlorine-Potassium-Iron particles (marker 20 µm).

Fig. 3.2.11 Iron nanoparticles found in the lung of a soldier suffering from Gulf War

Syndrome (marker 5 µm).

Nanopathology 54

Fig. 3.2.12 Solitary Gold-Silver-Copper particle found in the lung of a soldier suffering

from Gulf War Syndrome (marker 10 µm).

The images of Fig. 3.2.11 and 3.2.12 show nanoparticulate matter of

different compositions in the lung tissue of a veteran from the First Gulf

War, suffering from the so-called “Gulf War Syndrome”

The samples come from an officer active in the Canadian Intelligence

who died in 1999 as a victim of the so-called Gulf War, during which he

served as a mine clearer. Natural Uranium (90%) and depleted Uranium

(9-10%) were found in his urine by another laboratory when the patient

was still alive. No such element, though, was found in particulate form in

our investigations. In the samples studied we found nanoparticles of

Titanium compounds and of Iron, along with particulate matter of Silver

and precious alloys like Gold-Copper. Finding such small particles inside

that patient’s lungs was strange indeed, as he had lived most of his life

lived in the Canadian countryside, and had never worked in a

nanotechnology laboratory where particles that size are common. He

grew ill after a 6-month mission in Iraq and, in fact, had been exposed to

the war pollution created by bombing. (See Chapter 5: War and

nanoparticles)

In another case, this time of a patient affected from the Gitelman’s

syndrome, we found many different foreign bodies in the same sample.

Gitelman’s syndrome is a rare, often asymptomatic, autosomal-

recessive disorder affecting the renal tubules, causing them to pass

Sodium, Magnesium, Potassium and Chloride into the urine. The patient,

Clinical Cases: Lung, Blood, Liver, Kidney, Digestive System, Vessels, Sperm 55

suffering from chronic interstitial pneumonia with allergic fibrosing

alveolitis, had been in touch with depleted Uranium. Inactivating

mutation in the SLC12A3 gene was found in the patient, which is

compatible with a diagnosis of Gitelman’s syndrome. (O’Shaughnessy

KM et al., 2004), (Naesens M. et al., 2004).

Nanoparticles of a rather complex composition are visible in the

tissue. The different chemical composition of the particles found

indicates an origin from different sources.

Fig. 3.2.13 Low-magnification image of a lung tissue affected by Gitelman’s Syndrome.

Arrows show Silicon-based pollution (marker 300 µm).

The first image (Fig. 3.2.13) shows a common silicate particle

(normal dust) and the second one (Fig. 3.2.14) is a round-shaped particle

of a compound of Cerium-Lanthanum-Neodymium. In our experience it

can have been originated by smoking, as in some tobacco leaves we

found the presence of this strange environmental, weakly radioactive

composition. The third image (Fig. 3.2.15) is related to the presence

of another unusual compound containing Copper-Chlorine-Silicon-

Aluminium-Sulphur-Phosphorus-Iron.

Nanopathology 56

Fig. 3.2.14 Images of a single Cerium-Neodymium-Iron particle in a lung sample affected

by Gitelman’s Syndrome (marker 20 µm).

Fig. 3.2.15 Images of a lung sample affected by Gitelman’s Syndrome. Note the nano-scale

Chlorine-Copper-Silicon-Sulphur particulate matter (marker 2 µm).

The case shown in Fig. 3.2.16 concerns a 42-year-old woman who

died of malignant pleural mesothelioma without having been exposed to

asbestos. In fact, no such mineral could be found in her pathological

samples, but we detected Iron nanoparticles and fibres of Aluminium-

Silicon-Iron, whose long-shaped morphology is similar to asbestos. The

only anamnestic detail of this patient is that she underwent a five-year

chemotherapy treatment because of a breast cancer, before falling ill of

mesothelioma (Melato M et al., 2001). Another case of the same disease

(Fig. 3.2.17) shows the presence of Iron micro and nanoparticulate.

Clinical Cases: Lung, Blood, Liver, Kidney, Digestive System, Vessels, Sperm 57

Fig. 3.2.16 Images of malignant pleural mesothelioma’s sample. A fibre of Aluminium

silicate is clearly visible (marker 20 µm).

Fig. 3.2.17 Images of malignant pleural mesothelioma’s sample with Iron micro and

nanoparticles. (marker 10 µm).

Nanopathology 58

Fig. 3.2.18 Iron-based particles in the sample of a patient with a multi-visceral

granulomatosis (marker 100 µm).

Fig. 3.2.18 concerns the case of a patient for whom no clear diagnosis

was issued. He suffered from a multi-visceral granulomatosis and what

we found in the lung and spleen samples were wide areas where Iron-

Silicon-Aluminium-Magnesium-Sulphur-Phosphorus were present. The

presence of Silicon, Aluminium and Magnesium with Iron induces to

think that Iron is not endogenous but comes from outside the body.

The case of Fig. 3.2.19 is about a young lady of 22 who suffered from

talcosis and pneumothorax. The histological study revealed the presence

of blebs, fibrosis, lymphocytic phlogosis and giant cells with particles.

The particles we detected were composed mainly of Copper (Fig. 3.2.20)

and of Silicon-Magnesium (talc).

Clinical Cases: Lung, Blood, Liver, Kidney, Digestive System, Vessels, Sperm 59

Fig. 3.2.19 Talc particles contained cells in a pneumothorax (talcosis) (marker 10 µm).

Fig. 3.2.20 Solitary Copper particle in a pneumothorax area (marker 10 µm).

The round-shaped Copper particle shows that the patient had been

exposed to a toxic material coming from a combustion occurred at about

1,100°C, since the melting temperature of Copper is 1,083°C. That

element has been known for a long time to be toxic both in vitro and in

vivo causing tissue necrosis. If the particles are located outside of the

lung, in touch with the pleura, they can induce a necrosis, a rupture and

the onset of a pneumothorax.

Nanopathology 60

Fig. 3.2.21 Case of pneumotorax with the presence of a Titanium needle (marker 10 µm).

Fig. 3.2.22 Case of pneumotorax with the presence of macrophages full of Iron-Silicon-

Aluminium-Calcium particles (marker 20 µm).

In a 65-year-old workman a pneumoconiosis was diagnosed. The

histology showed blebs, fibrosis, lymphocytic inflammation and giant

cells with particles we showed to be composed of Iron-Silicon-Sulphur-

Calcium-Aluminium (Fig. 3.2.22). The pricking needles of Titanium

(Fig. 3.2.21) can be responsible of the pneumotorax, but the damage

created by the Iron particles may have also contributed.

Clinical Cases: Lung, Blood, Liver, Kidney, Digestive System, Vessels, Sperm 61

Fig. 3.2.23 Cells full of Iron-Silicon-Aluminium-Phosphorus-Sulphur-Calcium-Titanium-

Chromium particles surrounding a pneumothorax area (marker 20 µm).

Fig. 3.2.24 Particular of Fig. 3.2.24 (marker 10 µm).

A further case of pneumothorax, this time of a 42-year-old solderer

came to our observation. Coniotic materials in giant cells (at different

magnifications) were easily visible, composed of Iron-Silicon-Titanium-

Calcium-Aluminium-Phosphorus-Sulphur-Chromium (Fig. 3.2.23 and

Fig. 3.2.24). White, round shaped particles are clearly visible inside the

cells. This evidence is compatible with a working-place exposure.

Nanopathology 62

Fig. 3.2.25 Area around pneumothorax. Many silicatic particles are visible (marker

20 µm).

Fig. 3.2.26 Particular of the lung sample around the area of the pneumothorax above.

Cerium-Iron particles are contained in cells (marker 10 µm).

A businessman of 42 suffered from a pneumothorax. Blebs with

fibrosis, chronic lymphocytic infiltration and giant cells containing

nanoparticles whose main component was Silicon were present. (Fig.

3.2.25). Also in this case, in the sample of the lung lesion, the tissue was

full of micro and nanosized foreign bodies. Rather peculiar is the

probably weakly radioactive debris containing Cerium-Iron-Aluminium-

Silicon-Phosphorus and Sulphur (Fig. 3.2.26).

Clinical Cases: Lung, Blood, Liver, Kidney, Digestive System, Vessels, Sperm 63

Fig. 3.2.27 Image of a lung sample in the area of the pneumothorax. Silicon-Aluminium

based particles are visible (marker 100 µm).

Fig. 3.2.28 Particular of Fig. 3.2.27. At higher magnification, numerous needle-shaped

Silicon-Aluminium based particles can be seen (marker 10 µm).

A case similar to the one illustrated in Fig. 3.2.25 is shown in

Fig. 3.2.27. In this instance the patient was a 71-year-old workman. The

coniotic nodule and the giant cells contained particles whose principal

component was a composition of Silicon-Aluminium-Potassium-

Titanium-Iron-Magnesium-Sodium-Phosphorus-Sulphur, i.e. a ceramic

dust. Fig. 3.2.28 regards the same patient as above, but in this portion

of the lung the particles had the same composition but a different

morphology.

Nanopathology 64

Fig. 3.2.29 Cluster of Silver nanoparticles in a lung sample affected by allergic pneumonia

(marker 5 µm).

The picture above (Fig. 3.2.29) shows a case of chronic, allergic

pneumonia of exogenous origin with lung interstitial fibrosis and

secondary leiomyomatosis, a rare, benign tumour that generally develops

in the uterus, but can affect organs with smooth muscle cells. In this case,

clusters of Silver nanoparticles were clearly visible in the pathological

tissue.

Fig. 3.2.30 Unusual morphology of pulmonary cells (adenocarcinoma) containing Iron-

Sulphur-based particles (marker 50 µm).

Clinical Cases: Lung, Blood, Liver, Kidney, Digestive System, Vessels, Sperm 65

Fig. 3.2.31 Same sample as the one of Fig. 3.2.29. Normal red cells are inside the ellipse,

while lighter red cells are indicated by arrows (marker 20 µm).

Fig. 3.2.32 Same sample as the one of Fig. 3.2.29 and Fig. 3.2.30. What look like Iron

phosphate precipitates on a needle-shaped Iron particle (marker 10 µm).

Figs. 3.2.30-31 and 32 show different types of particles found in a

small lung sample affected by adenocarcinoma. The first image show

round-shaped organic bodies with Iron nanoparticles inside. The second

one shows metallic particles (Chromium-Iron-Nickel) embedded in red

cells (lighter in colour and indicated by arrows), while the third one is an

Iron needle with endogenous Iron-phosphatic crystals. A surgical metal

clip was detected at histology. It could be responsible for the debris

found.

A few cases of pleural mesothelioma are shown below.

Nanopathology 66

Fig. 3.2.33 Images of Iron-Sulphur and Iron-Zinc-Sulphur-Silicon-Phosphorus

nanoparticles of in a lung tissue affected by mesothelioma (markers 10 µm and 20 µm

respectively).

In the same case of mesothelioma, two different types of

nanoparticles were detected. One was made of Iron-Sulphur, while the

other was composed of Iron, Zinc, Silicon, Sulphur and Phosphorus

(Fig. 3.2.33).

Among the particles found in the case illustrated in Fig. 3.2.34 and

Fig. 3.2.35, we focused our attention to some debris with unusual

compositions. The former contained Uranium-Phosphorus-Potassium

particles, (a radioactive composition) the latter contained Tungsten,

Tantalum, Niobium, Phosphorus and Sulphur.

Clinical Cases: Lung, Blood, Liver, Kidney, Digestive System, Vessels, Sperm 67

Fig. 3.2.34 Pleural mesothelioma with particles containing Uranium (marker 50 µm).

Fig. 3.2.35 Lung tissue affected by mesothelioma with Tungsten-Tantalum nanoparticles

(marker 20 µm).

Nanopathology 68

Fig. 3.2.36 The same sample of a tissue affected by mesothelioma are shown at different

magnifications (marker 200, 50 and 5 µm respectively).

Clinical Cases: Lung, Blood, Liver, Kidney, Digestive System, Vessels, Sperm 69

This case of mesothelioma is rather peculiar. In fact, macrophages

were found in the tissue full of small, round-shaped particles of

Aluminium-Magnesium. Fig. 3.2.36 shows part of the tissue at

increasing magnifications. The second image shows a blood vessel

surrounded by whiter cells. They are full of sub-micronic globular

entities containing Aluminium.

The patient had worked for a long time in a mechanic industry and he

was exposed to vapours exhaled by what is called “Aluminothermy

process”. Also this case confirms that what was found inside is the

expression of the environmental exposure the patient underwent. The

inorganic matter we inhale can remain trapped in the lung at different

tissue levels. In the case of needle-shaped particles, once inside the lung,

because of the respiratory acts, they are pushed toward the more external

part of the organ, i.e. the pleura. When they are there, they have the time

to express their toxicity and induce a tissue or cell reaction. The

biocompatibility of a material is also a function of time: a short time

interaction is less detrimental then a long term contact.

But in the analysis of the possible damage due to environmental

pollution we need to take into account other phenomena that were

observed by the School of Leuven (Belgium).

The main work is due to Dr. Nemmar who had some 100 nm-sized

Technetium-radiolabelled Carbon nanoparticles inhaled by 5 volunteers.

The experiment showed that those particles negotiate the lung barrier in

60 seconds and their dissemination in internal organs, the liver in this

particular case was the organ observed, occurs in 60 minutes. (Nemmar

et al., 2002)

That simple experiment simulated what probably happens every day.

It is well-known that 100nm-sized particles are released, of course

unintentionally, by car engines, industrial fumes, incinerators, etc. and

the paper mentioned offers an explanation to what we find in many

tissues other than hepatic (Tonomori et al., 2004). The next paragraphs

show that contamination can be very deep, and no organ is free from

danger. It is only a matter of size, chemical composition, affinity for

some function of the organ and chemistry of the particle.

Nanopathology 70

3.3 Blood

The presence of the inorganic particles found in the internal organs was

explained putting forward the hypothesis that they are carried by the

blood circulation.

In order to demonstrate this presence inside the blood, we wanted to

take pictures of circulating particles, but also of possible problems

induced by their interaction with the blood components. To this aim, we

tackled the problem from three different points of view.

Firstly we looked for inorganic debris in the blood of clinically

healthy people living in polluted areas.

Then, we checked thrombi formed in-vivo, either in people at risk of

pulmonary thromboembolism and in patients who had a myocardial

infarction. In the former case, we analyzed venous clots trapped by caval

filters, and in the latter the thrombi removed from the coronary arteries

by means of a new procedure making use of a thrombectomy equipment.

And finally we analyzed samples of patients suffering from vasculitis,

a cryptogenic inflammation of the blood vessels.

The list of Tab. 3.3.1 shows the pathologies considered.

Fig. 3.3.1 shows the image of red cells smeared on an acetate sheet.

Small particles are visible, but it is hard to tell if they are immersed in the

plasma, attached to the cell or embedded inside it.

Clinical Cases: Lung, Blood, Liver, Kidney, Digestive System, Vessels, Sperm 71

Tab. 3.3.1 List of the pathologies analysed.

Pathologies No.

Thromboembolic disease 65

Aneurism 1

Idiopathic Thrombocytosis 1

Diabetes 1

Parassitosis 2

Cerebral angioma 1

Hemangioma - Angiolipoma 1

non-Hodgkin’s Lymphoma 1

Hodgkin’s Lymphoma 1

Lymphadenopathy 2

Not diagnosed cases 6

Reference samples 2

Total 84

This analysis itself is simple to perform and particles are easy to

detect, but that is not enough to allow us to say that the presence of

foreign bodies means that a pathology is starting.

More interesting proved the study of thrombi formed in-vivo, since

particles found inside them can mean that they acted as nucleating

agents, or their non biocompatibility triggered the coagulation cascade.

Fig. 3.3.1 Image of an Antimony–Cobalt nanoparticle and red cells (marker 5 µm).

Nanopathology 72

In order to demonstrate the interaction particle-blood in vivo, we took

patients treated with caval filters. Those are medical devices that are

implanted in the inferior vena cava either as a preventive means before

surgery in patients who were deemed as being at risk of developing a

deep-vein thrombosis (DVT) with a consequent pulmonary embolism

(PE), or to prevent PE episodes in potentially relapsing patients. In those

cases, a metallic structure is inserted in the inferior vena cava and left

there to capture the thrombi formed in the lower limbs and pelvis.

Fig. 3.3.2 Image of a vena cava filter by ALN (France).

The analyses of explanted temporary vena cava filters (ALN Implants

Chirurgicaux, - France) (Fig. 3.3.2) offered the possibility to analyse the

thrombus entrapped, without influencing the system. Those filters,

usually implanted in the lumen of the inferior vena cava, are the most

common mechanical device for the prevention of pulmonary thrombo-

embolism and their use is growing more and more widespread, especially

in the USA.

The observation of the filters explanted allowed to analyze the small

thrombi captured in the human venous circulation. (Gatti, A. M. et al.,

2006) (Montanari S., 2000). The vena-cava filters examined are

composed of nine AISI-316L stainless steel, 0.3-mm diameter, wires

Clinical Cases: Lung, Blood, Liver, Kidney, Digestive System, Vessels, Sperm 73

gathered together inside an ogival capsule made of the same material,

from where they branch out, forming a conical skeleton. Each of the

shorter 6 of the 9 prongs that make up the device has a distal hook that

penetrates the venous wall and serves to keep the filter anchored in place,

while the longer three are meant as stabilizers to keep the device lined up

with the vascular axis, and have no hooks. One of the long prongs has a

small ring at its distal end. The overall height of the device is 5 cm and,

being it elastic, its diameter adjusts itself to the section of the vessel up to

32 mm.

In two different studies, we analyzed respectively 14 and 20

temporary filters explanted from patients who had had this device

implanted for different reasons and for different periods (from 18 to 384

days). The main indications were:

1. Anti-thrombosis prophylaxis in bone fractures or implant of hip

joint prosthesis or cancer;

2. Deep Vein Thrombosis (DVT);

3. Protection in loco-regional thrombolysis treatment.

The particles found in the thrombi trapped in the filters were

characterized with a size ranging from 10 down to 0.1 micron and for a

wide variety of chemical compositions.

Some of the compositions found are listed below. The first element

considered is that expressed in the spectrum with the highest peak:

- Si;

- Si; Mg;

- Cr, Fe, Si, S, Na;

- S, O, Ba ,Na;

- Fe, Sb, S, P, Si, Na;

- Sb, S;

- W, O, S, P, Fe;

- Bi ,S, Cl, Si, Na Ca;

- Pb, Si, Al ,Cr, Fe, Mg, Ca;

- Ag, Si, Mg, S, Bi, Cl, O, Ca, Cu;

- Co, S, Ag, Ca, Cl, P, W, Al, Zn, Fe;

- S, Ba, Ca, Cl, Na;

- Fe, Cr, O, S, Ca, Cl, Cu;

- S, Cl, Ca, Bi, P, Na, Mg;

Nanopathology 74

- Ag, O, S, Ca, P, Si, Mg, Ni;

- Au, Cl, S, O, Ca, Ag, Na, Si, Mg, Cu, Fe.

It is interesting to see how all the thrombi come to our observation

had foreign bodies trapped.

One case was particularly interesting from the nanopathological point

of view, as the thrombus contained micro- and nano-sized foreign bodies

composed of as many as 6 different chemical compositions (Co-W, Al-

Si, Si, Fe, Ag-S, Fe-Cr-Ni). Fig. 3.3.3 and 3.3.4 show particles of Barium

Sulphate and Silver dispersed in the thrombus. (Gatti, A. M. et al., 2006)

Fig. 3.3.3 Image of red cells in a thrombus trapping nanoparticles of Barium-Sulphate

(marker 10 µm).

Fig. 3.3.4 Image of a particle composed of Silver and Calcium found in a thrombus (marker

10 µm).

Clinical Cases: Lung, Blood, Liver, Kidney, Digestive System, Vessels, Sperm 75

Fig. 3.3.5 Sulphur-Barium-Sodium-Aluminium-Silicon-Phosphorus-Calcium-Iron particles

in an fibrotic tissue adherent to a caval filter (marker 10 µm).

The presence and quantity of thrombotic material found in the filters

did not seem to depend on the time the device had stayed implanted in

the organism, but on the extent of contamination of the blood, i.e. how

many particles per volume unit were present. That means that the higher

the patient’s exposure was, the greater was the concentration in the blood

and the higher was their probability to induce a thrombotic reaction. It

has been demonstrated that foreign bodies the size of 100 nanometres,

once inhaled, can leave the alveoli and enter the blood circulation in 60

seconds. (Nemmar et al. 2002). In Fig. 3.3.5 Sulphur-Barium-Sodium-

Aluminium-Silicon-Phosphorus-Calcium-Iron nanoparticles are shown

trapped in the fibrotic tissue adherent to a caval filter.

Nanopathology 76

Fig. 3.3.6 Cluster of nanoparticles composed of Titanium-Sulphur–Barium-Calcium-

Chlorine (marker 10 µm).

Fig. 3.3.6 and Fig. 3.3.7 show metallic debris found in thrombi

trapped by two caval filters. The former is a cluster of Titanium-

Sulphur–Barium-Calcium-Chlorine nanodebris, while the latter is a

single Iron-Chromium-Nickel needle-shaped particle.

Fig. 3.3.7 Image of stainless-steel debris found inside a thrombus (marker 20 µm).

Clinical Cases: Lung, Blood, Liver, Kidney, Digestive System, Vessels, Sperm 77

In the cases we had the chance to observe, particles much larger than

100-nanometer were detected in the blood. Whether they are the cause of

the thromboses or they are just mechanically trapped by the blood during

its pathologic coagulation may be discussed, but it is not unreasonable to

suppose that a foreign body can be thrombogenic, especially, as has

always been the case in this study, when those foreign bodies are neither

biodegradable nor biocompatible, and being not biocompatible means, in

the vast majority of cases, being also thrombogenic.

Recently it was demonstrated that when the environmental

concentration of PM2.5 increases, the mortality rate of cardiopaths, or

patients suffering from cardiovascular diseases increases as well. The

images shown can confirm these data.

The size and composition of the debris found is rather typical of

industrial pollution, in particular of high-temperature processes involving

inorganic materials, such as occurs in a foundry, in a cement plant, in an

incinerator or in engine fumes.

All patients were considered to be at risk of developing a deep-vein

thrombosis (DVT) or, in some cases, were already diagnosed to be

affected by that disease. This is one of reasons that make us suspect that

the thrombi detected or, at least, part of them, embolized from the DVT

foci. Some of them, though, could have been originated by inorganic

particulate circulating in the blood and become actually thrombogenic

once the organism has been stimulated by particular, well-known,

conditions like, for example, surgery, bone fractures, cancer or a

relatively long stay in bed.

A number of the many pulmonary embolism episodes observed

worldwide are classified as idiopathic, since no focus can be identified

nor an explanation regarding their origin is possible according to the so-

called Virchow’s Triad, that states that pathological clotting of the blood

in the vessels occurs when the blood flow is somehow obstructed or

slowed down, when the vascular endothelium is damaged or when the

chemistry of the blood is disturbed or incompetent. In many instances, it

is a combination of the three factors that is responsible for the

thrombosis, as one single factor may not always be enough.

The presence of particulate matter in the blood, added to the Triad as

a fourth factor, may explain in part or totally the not infrequent cases

Nanopathology 78

when none of those classical three causes exist, yet a pulmonary

thromboembolism occurs.

That particulate air pollutants are responsible for or, at least,

have some form of relationship with the onset of cardiovascular and

pulmonary diseases has been amply demonstrated (Peters A. et al.,

1997), (Samet J. M. et al., 2000) and is supported by clinical evidence,

though much remains still to be explained as to the patho-mechanisms

they follow. At present, for instance, we are not in a position to say if the

blood reacts to the presence of clusters of nanoparticles and of isolated

microparticles in a different way.

Also analyzing the material that occluded a coronary vessel we found

foreign bodies.

Myocardial infarction is caused by an acute obstruction of the

coronary lumen and in many cases that obstruction is caused by a

thrombus or, at least, contains thrombotic material together with

atheromatous plaque. A few years ago, a mechanical technique aimed at

aspiring that material grew popular in interventional cardiology. (Jun-

ichi K. et al., 2002), (Andrew, M. W. et al., 2006). We had the chance to

investigate on a sample taken from a myocardial infarction thanks to the

technique of mechanical coronary thrombectomy and found micro and

nanoparticulate in that thrombus as well.

Fig. 3.3.8 and the ones that follow (3.3.9 and 3.3.10) show inorganic

particulate inside coronary thrombi. Such unexpected finding may rouse

Fig. 3.3.8 Coronary thrombus with an agglomeration of many nanoparticles with one

microparticle of a Titanium-Bismuth-Sulphur-Phosphorus compound (marker 10 µm).

Clinical Cases: Lung, Blood, Liver, Kidney, Digestive System, Vessels, Sperm 79

the suspicions that those foreign bodies can have triggered the

pathological coagulation. This could explain why a higher cardiogenic

mortality is reported when PM2.5 concentration in the atmosphere grows

higher (Pope, C. A. et al., 2006), (Pope, C. A. et al., 2006).

Fig. 3.3.9 Small Platinum-Tungsten-Copper-Sodium debris found in a coronary thrombus

(marker 10 µm).

Fig. 3.3.10 Cluster of nanoparticles composed of Antimony-Cobalt Sulphur-Sodium

embedded in a fibrin reticulum (marker 10 µm).

Nanopathology 80

3.4 Liver

Granulomatous hepatitis is a lesion found in a wide spectrum of liver

diseases (Denk, H. et al., 1994). TBC and sarcoidosis infection have been

most frequently incriminated, while other causes are represented by viral

infections, immunological disorders, drug-induced injuries, Crohn’s

disease and foreign-body reaction (Anderson, D. S. et al., 1998).

A minor percentage of cases, about 10%, remains of unknown

etiology and is indicated as GLUS (Granulomatous Lesions of Unknown

Significance), (Brinker, H., 1990)

From the histological point of view, such lesions are usually non

caseating, with a few multinucleate giant cells and some surrounding

chronic inflammatory infiltrate. Medical literature analyses local effects

of wear from joint replacements, identifying the debris as the main result

of in situ degradation of implants (Jacobs, J. J. et al., 1998). In addition

to obvious local effects, particulate debris created by an implanted device

has been reported as present in lymph nodes (Albores-Saavedra, J. et al.,

1994), (Bos, I. et al., 1990) and in distant organs, but literature on the

subject is rather scanty (Urban, R. M. et al., 2000).

As a general rule, the larger particles thus generated cause a fibrous

or giant-cell reaction, while the smaller ones prompt a macrophage

phagocytosis reaction. Chapter 2 demonstrates that nanoparticles behave

in a different way and do not induce “normal” inflammatory reactions.

Our first case of liver granulomatosis is the one described in Chapter

1. In this later series, we analyzed a group of patients with phlogosis

(13 cases) and “granulomatous phlogosis” (resulted from liver biopsies)

(29 cases), since they are related to tissue inflammations also caused by

foreign bodies . The relevant data are shown in Tab. 3.4.1.

In order to have a reference, not shown in the table, five specimens of

liver taken from presumably healthy young people dead in car accidents

were observed in the same way under ESEM together with ten more

samples from feti. Those specimens were in all cases free from any

particulate contamination.

Clinical Cases: Lung, Blood, Liver, Kidney, Digestive System, Vessels, Sperm 81

Tab. 3.4.1 List of the pathologies investigated with the relevant number of cases.

Pathology No.

Phlogosis 13

Fibrosis 2

Granulomatosis 29

Wegener’s granulomatosis 1

Steatosis 7

Hepatitis 6

Siderosis 3

Haemorrhage 3

Wilson’s disease 1

Cancer 28

Metastasis 17

Hodgkin’s Lymphoma 3

non-Hodgkin’s Lymphoma 1

Gulf War Syndrome 1

Nephrocalcinosis 1

Not diagnosed cases 23

Reference samples 15

Total 154

All these cases were checked by a histopathologist. Just three of the

cases chosen showed the presence of foreign material inside the

granulomas, whose nature remained undetermined to him. In any case,

he ruled out any infectious origin according to the results obtained with

Pas, Grocott and Zihl-Nielsen reactions.

Once again, the observations under ESEM proved essential in the

identification of materials responsible for granulomatous reactions, as the

presence of micro- and nano-debris in all samples except the references

was consistently found. The particles detected were different in size (in

general <20 µm) and in chemistry. As all the cases selected are

meaningful in demonstrating the hypothesis mentioned above, some of

them are discussed singularly because of their clinical history. Their

anamnestic records confirmed the exposure we found in the tissue.

Case No. 1: 61-year old female with a clinical history of rheumatoid

arthritis; increase of GOT, GPT and gamma-GT. Use of corticosteroids

and Gold salts injected directly in her joints. Histologically, the liver

Nanopathology 82

biopsy showed a mild chronic phlogosis and a small granuloma in the

proximity of a portal tract, with black material inside.

At ESEM analysis, particles trapped in a degenerated tissue

containing Gold nanoparticles were identified. (Fig. 3.4.1). The oddity of

such finding got us to analyse more in detail different areas of the same

specimen. The EDS spectrum (Fig. 3.4.2) showed that Gold alone was

not accompanied by any other element, the spherical, homogenous

morphology of the particles as small as 50-nm excluded a dental origin.

The study of the clinical record revealed that years before falling ill of

liver granulomatosis, the patient had suffered from a knee arthrosis and

had been subjected to a colloidal Gold therapy. We could not find any

other reasonable explanation to the presence of that form of pure Gold in

the liver, where it had behaved like a foreign body, but a migration from

the joints, where it had been injected.

Fig. 3.4.1 Gold nanoparticles in a liver granulomatosis (marker 50 µm).

Clinical Cases: Lung, Blood, Liver, Kidney, Digestive System, Vessels, Sperm 83

Fig. 3.4.2 Cluster of Gold nanoparticles in a liver granuloma (marker 2 µm).

Case No. 2: A 25-year-old female was affected by a hepatic

echinococcosis. At ESEM observation, small debris of Barium-sulphate

could be detected in the section. The patient’s clinical file revealed that

she suffered from digestive disorders, which suggested the possibility

that she had undergone a gastroscopy with BaSO4 used as contrast

medium. The fact was confirmed by the patient herself who also reported

an allergic reaction from that chemical.

Case No. 3: A 63-year-old male suffered from indefinite symptoms,

and a constant feeling of fatigue. Liver biopsy showed the presence of a

lympho-monocytic infiltrate, areas of macrosteatosis and an epithelioid

granuloma lined with a lympho-monocitic halo. At ESEM, degenerated

areas of the liver showed numerous particles of Calcium-sulphate

(gypsum) and fillo-silicate (Fig. 3.4.3). Such chemistry suggested the

possibility that the patient lived or worked in a particularly polluted

environment. As a matter of fact, the clinical record reported that he was

a mason.

Nanopathology 84

Fig. 3.4.3 Gypsum and fillo-silicate micro and nanoparticles in an epithelioid granuloma.3

Case No. 4: A 65-year-old African male was operated on for a spleen

cancer. Surgery revealed that his liver was compromised and a biopsy of

that organ was taken, which showed a large granuloma containing

necrotic tissue. The ESEM observation in that tissue revealed the

presence small spherical Calcium-phosphate particles with a narrow

distribution of diameters (2-7µm) (Fig. 3.4.4). The hypothesis we

advanced, a hypothesis never confirmed because of the patient’s death

and the consequent stop of all investigations, was that those findings

were of biological origin, perhaps a parasite.

3 This photo was published in Biomateriali, vol 23, issue 11, Gatti A.M., Rivasi F. “Biocompatibility of micro- and nanoparticles Part I in liver and kidney”, June 2002, p. 2381-2387.

Clinical Cases: Lung, Blood, Liver, Kidney, Digestive System, Vessels, Sperm 85

Fig. 3.4.4 400-µm granuloma with fibrous capsule (low-magnification). (marker 200 µm).

Fig. 3.4.5 Same sample as above. Higher magnification shows Calcium phosphate

nanospherules (marker 5 µm).

The same morphology, size range and composition as those seen in

this case was found in a number of other biological tissues (Fig. 3.4.5).

Still today we do not understand if the reaction is a sort of unusual

calcification (why spheres? Why Sodium and Magnesium?) or it is due

to some other mechanism.

Case No. 5: The only symptom of a 27-year-old male with a history

of drug abuse was fever. Liver echography showed multiple nodules of a

diameter never exceeding 2 cm. The liver samples we had the chance to

Nanopathology 86

check showed the presence of foreign bodies with different chemical

composition (silicates). Such finding was probably due to the powder

used to mix the drug.

Case No. 6: The liver biopsy of a 62-year-old male with a clinical

history of peritoneal dialysis showed granulomas in correspondence with

the glissonian membrane and focal hepatic fibrosis. The spectrum of the

debris seen in the pathological tissue reveals the presence of Carbon,

Oxygen, Silicon, Sulphur, Calcium and Titanium. Such debris might be

correlated with a possible pollution of the dialysis solutions or to the

pharmaceutics taken in a chronic way.

Diagnostics uses either light transmission microscopy with a

maximum of up to 400 magnifications or molecular biology, that

analyses the sequence of the aminoacidic bases, whose size is

nanometric. Between this two terms there is a wide interval spanning

about 2-3 orders of magnitude where information about particular

pathologies, not otherwise explained, may be lost. And such information

may be important or even essential to understand the problem.

As mentioned above, no less than 10% of all cases of liver

granulomatosis remains unexplained as to their origin, and that brought

us to face this problem under the different point of view of our technique.

The use of a novel technique, a technique typical of physicists,

represented by the use of Environmental Scanning Electron Microscopy

(ESEM) that allows the analysis of samples under different gradients of

pressure, without any physical and chemical pre-treatment demonstrated

that within this gap evidence can be found essential to issue a correct

diagnosis.

In fact, just as an example, the case of the patient with a fever and

renal failure of unexplained aetiology described in Chapter 1, whose

renal and liver biopsies were subjected to ESEM examination, showed a

spectrum of elemental composition compatible with the porcelain his

dental prosthesis was made of. This could explain the aetiology of his

granulomatous hepatic lesions and was fundamental to set an efficacious

therapy.

In the same way, the X-ray microanalyses carried out on the

particulate matter of case No. 3, gave information on the particles’

Clinical Cases: Lung, Blood, Liver, Kidney, Digestive System, Vessels, Sperm 87

chemistry and these data could be related, at least in part, to the clinical,

environmental and employment history of that patients. A likely

identification of the cause of the disease is certainly useful for the

therapy, and, which is of extreme importance, to set prevention

measures.

On the basis of these observations, we can assume that defining “non

toxic” what are just chemically inert (or hardly reactive) particles is

erroneous. Below a critical size, still hard to establish, particles, whatever

they are made of, are not biocompatible and may interact with biologic

tissues just because of their presence, and they may be responsible of

chronic inflammatory reactions that can lead to more severe forms of

disease. Nano-scaled particles can induce other unknown phenomena not

yet described in books or papers.

This first part concerning the pathologies of the liver has been

dedicated to cases of granulomatosis because such reaction by tissues

against foreign bodies has been often described in literature. Local

granulomatosis around the head of hip joint prostheses due to the wear

debris of the matching surfaces (head against acetabulum) is a far from

rare complication in orthopaedics. So, there is no problem in accepting

the existence of a correlation between presence of foreign bodies and

biological reaction. More difficult to accept are the following images.

They show the presence of dust that induced more severe reactions, like

cancer.

For a better understanding of the subject, we are presenting a genetic

disease involving the metabolism of endogenous Iron, in order to show

the difference between a genetic and an acquired disease. The genetic

disease we will deal with is siderosis.

Siderosis is a pathological build up of Iron in the organism. The lungs

are the most interested organs, but the liver and the urinary system can be

also affected. An Iron overload is known to impair the immune response

of the liver, and cause hepatic fibrosis and cirrhosis. There are different

opinions about the relative risk of developing hepato-cellular carcinoma

in patients with siderosis as compared with patients with hepatic fibrosis

and cirrhosis and the mechanism of liver carcinogenesis in genetic

hemochromatosis is still to be discovered. The main association with

genetic hemochromatosis is with a defect in gene HFE which helps

Nanopathology 88

regulate the amount of Iron absorbed from food. In the case below

(Fig. 3.4.6), precipitates of Iron in the form of numerous nanoparticles

are clearly visible inside the cytoplasmatic area. The picture shows some

nuclei surrounded by Iron precipitates (whiter).

Fig. 3.4.6 Image of a liver affected by siderosis. Globular Iron precipitates in the cytoplasm

can be seen (marker 20 µm).

In two cases of liver cancer, we detected particles inside the nucleus

of viable, “healthy” cells (Fig. 3.4.7). The mechanism of entry of those

particles is still to be explained.

Fig. 3.4.7 ESEM image of a liver cancerous tissue with a viable cell containing

nanoparticles in the nucleus (marker 10 µm).

Clinical Cases: Lung, Blood, Liver, Kidney, Digestive System, Vessels, Sperm 89

Fig. 3.4.8 Silicon-Aluminium-Phosphorus-Sulphur-Iron particles in a non clearly diagnosed

liver pathology (marker 20 µm).

This sample of Fig. 3.4.8 was taken from a cylinder of hepatic tissue

that showed a partially changed architecture. Portal spaces looked

slightly dilated, with inflammatory infiltrate. No steatosis, no Iron or

biliary accumulations were visible. Kupfer cells were not evident.

Some hepatocytes looked glycogenated. Foreign-body giant cells

with a marked lymphocyte halo were present at one of the edge of

the cylinder. Silicon-Aluminium-Phosphorus-Sulphur-Iron particles were

disseminated inside the area and could be detected in the tissue.

Fig. 3.4.9 Chlorine-Potassium-Sulphur-Nickel crystal in an apparently empty cavity

(marker 20 µm).

Nanopathology 90

Fig. 3.4.9 is a cylinder of liver tissue with a preserved lobular

architecture. A small quantity of inflammatory, lymphocyte infiltrate is

visible in the portal spaces. No Iron or bile, but some small Iron

accumulation were present. Kupfer cells were not evident. At an edge of

the sample, some giant cells mingled with a thick lymphocyte infiltrate

were visible. At ultrastructural observation, the sample revealed the

presence of Chlorine-Potassium-Sulphur-Nickel particles. The edged

morphology defines it as a crystal. The void space around it may mean

that it is toxic.

Fig. 3.4.10 Two images of a neoplastic sample of the liver cancer where Sulphur-based

debris are visible (marker 10 µm).

Clinical Cases: Lung, Blood, Liver, Kidney, Digestive System, Vessels, Sperm 91

The images of Fig. 3.4.10 show a wide dissemination of Sulphur-

Phosphorus-Barium-Calcium-Potassium-Iron-Sodium-Copper micro and

nanoparticles inside the hepatic tissue. The same chemistry was

homogeneously found throughout all the sample. The patient suffered

from a colon adeno-carcinoma, but his liver was already interested by

metastases. The portal space was larger and infiltrated by lymphocytes

and the metastatic tissue was interested by fibrosis, phlogosis and

formation of ductal-like structures. The particles of the bottom image of

Fig. 3.4.10 are attached to the surface of red cells.

Another pathology of unknown origin is lung fibrosis. Among the

cases examined, in one of them we found Lead-Iron nanoparticles in the

lung and in the liver. Fig. 3.4.11 presents a preserved liver architecture

with a lymphocyte infiltration, without neoplasm, and without Iron

accumulation. No mycetes were present, but chemically toxic foreign

bodies of Lead and Iron at nano-scale level.

Fig. 3.4.11 The image shows a hepatic tissue affected by a mild fibrosis. Lead-Iron-based

nanoparticles can be easily seen (marker 20 µm).

After a cholecystectomy, the patient developed a diabetes and later a

pancreas neoplasm. The liver presented a fibrosis, where we found the

toxic Lead-Iron-based nanoparticles.

Nanopathology 92

Fig. 3.4.12 Zinc-Sulphur-Phosphorus particles in a liver. The same particles were found in

the lung cancer of the patient (marker 20 µm).

Fig. 3.4.13 Liver cancer sample with two Zinc nanoparticles inside a cell nucleus (marker

5 µm).

Figs. 3.4.12 and 3.4.13 show a particular of the liver with small

steatosic modifications. Zinc nanoparticles are clearly visible inside a

cell nucleus. Fig. 3.4.13 shows that there is no efficient physiological

barrier at cellular level for nano-scaled particles.

Clinical Cases: Lung, Blood, Liver, Kidney, Digestive System, Vessels, Sperm 93

Fig. 3.4.14 The image shows a particular of a neoplastic tissue of a cholangiocarcinoma full

of Barium-Sulphate micro and nanodebris (marker 20 µm).

Fig. 3.4.15 Same hepatic tissue as that in Fig. 3.4.14, interested by cancer with round-

shaped particles of a Magnesium-Aluminium-Barium compound (marker 20 µm).

Fig. 3.4.14 and Fig. 3.4.15 are about the same case of

cholangiocarcinoma. A particle inside a cell nucleus is visible in Fig.

3.4.15. Particles gather and accumulate in preferential locations and

finding those is essential to investigations. Another important point is the

influence locations could have.

Nanopathology 94

Fig. 3.4.16 Liver with metastases from colon adenocarcinoma (marker 50 µm).

Fig. 3.4.16 shows a cancerous metastasis characterized by the

formation of fibrosis and gland-like structures. The sample is full of

nanoparticles composed of Barium-Sulphur-Iron. We did not have the

possibility to check if the same particulate was present in what was

called primary cancer located in the colon.

Fig. 3.4.17 Image of a small particle of a Aluminium-Phosphorus-Chlorine-Sulphur-

Calcium-Iron-Lead-Strontium compound (marker 50 µm).

Clinical Cases: Lung, Blood, Liver, Kidney, Digestive System, Vessels, Sperm 95

Fig. 3.4.17 shows a small particle of a strange Aluminium-Phosphorus-

Barium-Chlorine-Sulphur-Calcium-Iron-Lead-Strontium compound found

in the liver of a patient affected by the so-called Gulf-War syndrome.

That alloy does not exist in any metallurgy handbook. Every time we

find such strange chemical compositions, not having any application, we

have reasons to believe they are created by uncontrolled combustions:

Outside the chimney of an incineration plant or during the explosion of a

weapon against a target or when an airplane crashes or explodes inside a

building as happened in New York in 2001, a matter vaporization occurs

with the creation of a new pollution whose composition changes from

point to point. So, a coupling of elements is obtained that are not

matched in any intentionally made alloy.

3.5 Kidney and adrenal gland

From a certain point of view, the kidney tissue behaves like a mechanical

filter, the way other organs, the liver, for example, do. The difference

between kidney and liver seen as mechanical filters is that the former can

trap tinier objects.

Also for this organ we analyzed samples from subjects suffering from

different pathologies, as reported in Tab. 3.5.1.

Tab. 3.5.1 List of the renal diseases examined.

Pathology No.

Granulomatosis 2

Wegener’s granulomatosis 1

Glomerulo-nephritis 1

Gulf War Syndrome 3

Neu-Laxova Syndrome 2

Cancer 2

Nephrocalcinosis 1

Kidney failure (IRA) 1

Reference samples 3

Total 16

Nanopathology 96

The first case concerns a granulomatose reaction. Granuloma is a

typical reaction of animal tissues to the presence of a foreign body that

can be either organic (bacteria, parasites, etc) or inorganic (dust, etc).

Sometimes, a fibrotic capsule is created in the attempt to isolate the

exogenous inorganic presence from the biological context. Fig. 3.5.1

shows this type of reaction. When the foreign body is isolated that way,

it can stay there for years without inducing particularly severe symptoms.

An example may be the debris of a bullet that can remain sequestered in

the tissues.

Fig. 3.5.1 Fibrous capsule in a kidney grown around a silicate particle (marker 20 µm).

Clinical Cases: Lung, Blood, Liver, Kidney, Digestive System, Vessels, Sperm 97

The image above shows the presence of a microsized debris of a

silicate, i.e. a ceramic material, in the kidney. That debris can be “regular

household” dust, but can as well be a material coming from sources like,

for instance, ingested food. Chapter 1 showed the presence of dental

prosthetic debris in a granuloma of the liver and the kidneys with the

larger particles in the liver and the smaller in the kidneys. So, finding

silicate debris present in the composition of chewing gums in those

organs should not be surprising. As a matter of fact, chewing gums sold

on the Italian market contain silicates under particular form, as the

producer claims they can remove food remains from between the teeth

when no water is available to brush them. During mastication, the gum

matrix releases this particulate matter which gets swallowed and, thanks

to its small size, can pass through the bowel barrier and be filtered by

this tissue.

Fig. 3.5.2 with two spectra is about a patient suffering from acute

renal failure (ARF), i.e. a loss of kidney function occurring very rapidly.

The immediate result is a retention of urea and creatinine and non-

Nitrogenous waste products that are normally excreted by the kidney.

According to the severity and duration of the renal dysfunction, this

accumulation is accompanied by a number of metabolic disturbances,

such as metabolic acidosis, hyperkalaemia, changes in body fluid

balance, and adverse effects on many other organ systems. The condition

can be characterized by oliguria or anuria. The patient died of multiorgan

failure after sessions of plasmapheresis and continuous arterovenous

filtration. The histologic aspect was that of a tubular necrosis. Slight

lymph-monocyte infiltrate in the interstitium

Nanopathology 98

Fig. 3.5.2 Silicate and Tin foreign body in a renal tissue (marker 50 µm).

Fig. 3.5.3 Right adrenal gland with adenoma containing Iodine-Copper-Sulphur particles

(marker 20 µm).

Clinical Cases: Lung, Blood, Liver, Kidney, Digestive System, Vessels, Sperm 99

The patient to what Fig. 3.5.3 refers was a career soldier in the

coastguard corps who had been on duty in Albania 2000-2001. In 2003 a

Crohn’s syndrome was diagnosed, from adrenal-gland adenoma. In this

case of Crohn’s syndrome, strange particles of Iodine-Copper-Sulphur

were found. Copper is a toxic metal, but no literature could be found

about the biocompatibility of this material in the adrenal gland.

Fig. 3.5.4 Chromium-Iron nanoparticles in a kidney, most probably a stainless steel (marker

5 µm).

Nanopathology 100

Fig. 3.5.5 Image of a particular of globular calcification in a kidney (marker 100 µm and

50 µm respectively).

Fig. 3.5.4 and Fig. 3.5.5 refer to a patient died of multi-organ

failure. Fig. 3.5.4. shows the presence of what looks like stainless steel

nanoparticles, while Fig. 3.5.5 shows globular entities, in fact Calcium-

Phosphate precipitates, formed inside the kidney. The calcification

occurred modifies the physical properties of this tissue, like, for instance

its elasticity. As a consequence of this irreversible phenomenon, the

tissue grew harder and lost much of its functionality.

Clinical Cases: Lung, Blood, Liver, Kidney, Digestive System, Vessels, Sperm 101

Figs. 3.5.6a and 3.5.6b and spectrum. The images show a calcification (Calcium-

Phosphorus-Magnesium-Chlorine-Sodium) formed by single spherules (markers 200 µm

and 5 µm respectively).

The patient the sample of Fig. 3.5.6 comes from suffered from a

multi-organ failure with no clear diagnosis about its aetiology. He

presented many different particles disseminated in all organs, but in the

kidneys we found these Calcium-phosphate particles with a narrow range

of size distribution (1–10 micron). The difference with the previous case

is not only in the morphology but also in the composition. The spheres

are also composed of Sodium and Magnesium. The case looks like that

of the liver granuloma of Fig. 3.4.4, full of spherules very similar to

these.

Even after consulting one of the greatest experts of Calcium-

phosphates, Dr. Rachel LeGeros of the University of New York, we

cannot tell if it has an organic or an inorganic origin, nor if it is

endogenous or exogenous.

Nanopathology 102

The series of images shown demonstrates that environmental

pollution can be trapped also in the kidneys. It is possible that the

smallest nanoparticles can escape through the mesh of this filter,

go through the renal tubules, the ureters and the bladder and are

drained through the urethra. Our studies on urine could not confirm

any possible hypothesis, since the salt crystallization masks any other

presence.

3.6 Digestive system

Our studies on the digestive system showed that every inorganic, non

biodegradable, particulate matter can remain sequestered at different

levels along the pathway between the mouth and the anus.

This particulate pollution can be intentionally or unintentionally

contained in the food, or in drugs as excipient, or can derive from the

wear debris of dental materials. Part remains stuck in the oral mucosa

and cases of “Burning Mouth Disease”, ameloblastoma, cancer examined

confirmed the local entrapment.

We present an example of a very small bioptic sample taken from the

oral mucosa of a subject affected by a Burning Mouth Disease (Fig.

3.6.1). The patient was cured with tranquilizers and antidepressant drugs.

In the one-by-one millimetre specimen we detected more than 600

particles, the debris from a dental prosthesis were found. During

restoration works, the dentist drilled the old Gold-Platinum alloy

prosthetic material without the protection of a dam and polluted the

whole mouth cavity, but specially the mucosa, with the drilling debris.

The mucosa responded with an inflammatory, highly disseminated,

reaction.

Clinical Cases: Lung, Blood, Liver, Kidney, Digestive System, Vessels, Sperm 103

Fig. 3.6.1 Oral mucosa (marker 500 µm).

Our study of stomach cancer is just at an initial stage.

The few cases, but particularly the too few samples we had available

do not allow to put forward any hypothesis. Another important point as

to the stomach cases is that we have never had the possibility to select in

the explanted cancerous mass the interface between cancer and healthy

tissue. From our point of view, that is the most significant position where

inorganic pollution concentrates. The only meaningful oddity we came

across was the finding of titania (Titanium dioxide) nanoparticles in a

cancer tissue of a stomach of a 75-year old patient. Titania nanoparticles

are something that is being employed more and more frequently in

sunscreen creams and in paints as an application of nanotechnology, but

whose use is rather recent. So, it is strange to find high-technology

material in the stomach of an old person. A possible, though not

demonstrated, explanation is that he may have eaten them mixed with

some food. That and all the following images witness that those particles

can remain entrapped inside the tissues and are not entirely eliminated

through defecation.

More than 50% of the 400 samples of food examined show a

contamination coming either from environmental sources or from the

wear of the machines used to work it (See Chapter 7).

Nanopathology 104

In the case of pathologies of the most distal tract of the digestive

system, we analyzed different levels of seriousness of disease (Tab.

3.6.1): from mildly inflamed tissues to very severe illnesses like cancer.

Tab. 3.6.1 List of the pathologies examined.

Pathology No.

Phlogosis 2

Crohn’s disease 3

Ulcerous rectocolitis 2

Granulomatosis 2

non-Hodgkin’s Lymphoma 1

Cancer 30

Mesothelioma 1

Metastasis from hepatic carcinoma 1

Reference samples 3

Total 45

One of the cases of Crohn’s disease we examined is particularly

interesting. A 27-year-old patient had taken a antistaminic tablets since

the age of 7. In his inflamed tissue we found silicate particles which,

according to the drug’s producer, were part of the excipient used in those

tablets.

The next image (Fig. 3.6.2) gives an idea of what our colon is: a

receptor of everything is introduced through the mouth. In this case, a

large, curled debris of what is probably Teflon is adherent to the mucosa

wall. The polymeric material is easily identified because of the presence

of Fluorine in the Carbon-Oxygen spectrum. Observing the morphology

of the debris, a suggestive hypothesis can be proposed: When food is

cooked in an anti-adherent pan with a Teflon coating and is stirred or

turned with a non appropriate tool like a knife or fork, the possibility

exists of scratching the surface with the ensuing detachment of the

Teflon layer with its typical folds.

The deposition of such large debris on the mucosa surface can lead to

the following consequences: They behave as mechanical barriers

obstructing the adsorption of nutritive components. They are foreign

bodies that the tissue tries to eliminate with spasms (probably), pain,

inflammatory reaction, granuloma or fibrotic capsule formation, etc. But

Clinical Cases: Lung, Blood, Liver, Kidney, Digestive System, Vessels, Sperm 105

what may look strange is that also this macroscopic debris was not

eliminated with defecation.

The images below witness the presence of inorganic foreign bodies in

the digestive tract wall. In a case we found 14 chemically different

particles in a 2-mm square sample. In this situation it is particularly

difficult to find a correlation between the particles, their origin and the

disease. That is likely to be the result of multiple “insults”, all concurring

to trigger a reaction.

Fig. 3.6.2 Bulk sample of a colon mucosa where a folded Teflon debris (arrow) is deposited.

Nanopathology 106

Fig. 3.6.3 Lead-Chlorine-Aluminium nanoparticles in a patient suffering from Crohn’s

disease (marker 10 µm).

The sample illustrated in Fig. 3.6.3, containing Lead-Chlorine-

Aluminium nanoparticles, comes from a patient affected by Crohn’s

disease, a pathology characterized by granulomatous reactions that,

in the case shown, were treated with Asacol (a non steroidal anti-

inflammatory drug). Inside the tissue we found also toxic particles

containing Lead.

Fig. 3.6.4 Colon microvilli with Aluminium-Copper micro and nanoparticles (marker

100 µm).

Clinical Cases: Lung, Blood, Liver, Kidney, Digestive System, Vessels, Sperm 107

In the colon cancer shown in Fig. 3.6.4 we found, among other

particles, Aluminium-Copper micro and nanoparticulate. In many

pathologic instances, the colon contains particles presenting different

shape, size and composition. It is likely that the reaction is mainly due to

the presence of foreign bodies without a particular influence due to their

chemistry.

Fig. 3.6.5 Probably zirconia nanoparticles in a cancerous tissue (marker 10 µm).

Fig. 3.6.6 Tin-Calcium-Silicon, Aluminium-Magnesium-Sodium-Phosphorus-Sulphur-Iron

particles sequestered in a colon reaction tissue (marker 100 µm).

Nanopathology 108

The images of Figs. 3.6.5 and 3.6.6 present another case of colon

cancer with particles of what is probably zirconia (Zirconium dioxide)

and a strange compound whose complex composition can be seen in the

spectrum attached. It is difficult to guess both the origin and the

biological properties of these particles.

Fig. 3.6.7 A radioactive micro-sized particle in a peritoneal mesothelioma (marker 20 µm).

Fig. 3.6.8 Cluster of Lead-Silicon-Titanium-Chromium-Iron-Aluminium-Magnesium

nanoparticles (marker 10 µm).

Clinical Cases: Lung, Blood, Liver, Kidney, Digestive System, Vessels, Sperm 109

Fig. 3.6.9 Round-shaped Silicon-Silver-based particle (marker 5 µm).

The images of Figs. 3.6.7, 3.6.8 and 3.6.9 show different samples

coming from a case of peritoneal mesothelioma. We analyzed more than

that 10 samples from different areas compromised by the disease and

there we found numerous different micro and nanosized particles. What

we found most frequently were stainless steel particles (non shown), but

we could also identify toxic Lead particles (Fig. 3.6.8), Silver-Silicon

particles and some ceramic radioactive material like that shown in Fig.

3.6.7. That material contained Titanium, Zirconium, Cerium but also

Uranium, Thorium, Niobium and Yttrium. Industrial ceramic material

could be the origin of such particulate.

Fig. 3.6.10 Particle aggregate of a salt in a sigma adenocarcinoma.

Nanopathology 110

Beside unusual chemical compositions detected in the intestine, we

found also common products. In this case (Fig. 3.6.10) a Sodium-

Chlorine particle is shown. The presence of a familiar composition like

that is intriguing, because those are the elements composing soluble salts.

So we wondered why such a compound could be present in the intestinal

tissue in an evidently insoluble form. In an medium like urine, salts like

Sodium chloride or Sodium chlorate solidify only during the sample

preparation and at that time they crystallize forming dendrites. In this

case we suppose they were already aggregated in an insoluble way

before fixation of the sample. We are not in a position to tell if such a

particle, with its composition and its being insoluble, therefore not a

source of ions, is biocompatible.

Fig. 3.6.11 Particular of a colon mucosa affected by cancer with sub-micronic stainless steel

particles (marker 10 µm).4

4 This photo was published in Biomateriali vol.25, A.M. Gatti “Biocompatibility of micro- and nano-particles in the colon (part II)”, 385-392, Feb 2004.

Clinical Cases: Lung, Blood, Liver, Kidney, Digestive System, Vessels, Sperm 111

Fig. 3.6.12 Higher magnification of Fig. 3.6.11.

The case Fig. 3.6.11 and Fig. 3.6.12 refers to is rather singular as

the patient, suffering from a colon cancer, was a 19-year-old girl. Her

pathologic specimens were full of stainless steel particulate matter.

Fig. 3.6.13 Cluster of Silver-Molybdenum nanoparticles in a cancerous colon mucosa.

The previous images are referred to colon cancer samples. In the

same specimen we found metals like Silver-Molybdenum (Fig. 3.6.13) or

Silver alone (Fig. 3.6.14). If we were somewhat surprised at finding

those metals in the colon, the analysis of some food containing Silver

(see Chapter 7) offered us a possible explanation for those presences.

Nanopathology 112

Fig. 3.6.14 Cluster of Silver nanoparticles deposited on the colon mucosa in a cancer case.

Red cells are clearly visible at the right, lower corner.

A case of an amateur long-distance runner is shown below (Fig.

3.6.15). During his sports activity, a period of about 20 years, the subject

had taken very large quantities of food integrators and stimulant drugs

without any medical supervision. At the age of 50 a colon cancer was

diagnosed. In this case, we were not in a position to blame either diet or

drugs for his disease, since, because of the variety of substances

introduced, many of which escaped from the subject’s memory, no

traceability study was possible.

(a) (b)

Figs. 3.6.15a and 3.6.15b5 Silver nanoparticles inside the colon mucosa (marker 20 and

2 µm).

5 This photo was published in Biomateriali vol.25, A.M. Gatti “Biocompatibility of micro- and nano-particles in the colon (part II)”, 385-392, Feb 2004.

Clinical Cases: Lung, Blood, Liver, Kidney, Digestive System, Vessels, Sperm 113

Fig. 3.6.16 Silicon–Magnesium particle, i.e. talc, trapped in a colon cancer.

The colon cancer of Fig. 3.6.16 contained talc particles. That mineral

is generally ingested only as excipient in tablets.

The samples above show particulate matter in a wide variety of

morphologies, sizes and chemical compositions. Today’s world has

grown particularly complex and taking into account all the variables that

make it up and can interfere with living organisms is all but impossible.

If we limit ourselves to food and consider it under the nanopathological

point of view, we have to consider that:

a- an overwhelming majority of primary products grow in polluted

environments;

b- cereals and flours are generally stored in silos, many of which are

old and release debris that is inevitably added to food;

c- manufacturing adds further contaminants like the wear debris of

crushing and grinding tools;

d- polluted ingredients can be added to clean ones, thus giving origin

to a contaminated final product;

e - animals fed with polluted food can retain contamination, and that

contamination is transferred to man when he eats those animals which

are never checked under this point of view.

Nanopathology 114

3.7 Vessels

As demonstrated, environmental pollution can contaminate the blood, but

also the vessel structure can be interested by those entities. Those

pollutants are mainly metallic or ceramic and they are obviously harder

than the red and white cells, the platelets, and the intima, the inner lining

of the blood vessels. Because of that, when carried by the blood flow,

they can graze those structures and damage them, for example scratch the

vessel walls. A vessel damaged that way might be more prone to start

thrombosis or host atheromasic deposits or calcifications. We saw that

the particles carried by the blood can cross the vessel’s wall, perhaps

pushed by the blood pressure or through other mechanisms we still do

not know. During that passage, they can be trapped by the wall. It is only

reasonable to accept the fact that they can interact with that tissue.

Fig. 3.7.1 shows a silicatic particle that, probably because of its

arrow-shaped morphology, scraped the endothelium and remained stuck

in the vascular wall. Fig. 3.7.2 represents another example of entrapment

of debris.

Fig. 3.7.1 Silicatic particle in a vessel wall.

Clinical Cases: Lung, Blood, Liver, Kidney, Digestive System, Vessels, Sperm 115

Fig. 3.7.2 Silver-Chlorine particle trapped in an inflammatory aourtic aneurism case.

In Chapter 4, the case of a girl affected by vasculitis will be discussed

extensively where Tungsten carbide nanoparticles were found. It is

certain that they were present in the blood where they induced the

disease, but also extravasated, i.e. they passed through the vessel wall

and went into the muscles where they caused a different reaction.

3.8 Sperm

Our first analysis of a sperm sample was made as a consequence of a

telephone call from a mother, whose son had died after his return from a

peace-keeping mission in the Balkans in 2001. Her son had decided to

join the Italian mission after the end of the Balkan war, had left perfectly

healthy and after a three-month mission had come home ill. His

conditions were underestimated and misdiagnosed by the doctors who

took care of him and he died of a Hodgkin’s lymphoma. Before

undergoing chemotherapy, he donated his sperm in order to use it for a

future fertilization in the hope he could recover from his disease.

After his death, his parents were informed that the sperm should have

been destroyed, but his mother asked to get it and gave it to us to be

examined.

That first analyses gave important results (see Tab. 3.8.1), since we

found foreign bodies there. At the beginning, since it was our first match

with sperm, finding foreign bodies in a fluid we believed to be protected

Nanopathology 116

enough surprised us, but, continuing our experience, we noticed that the

fact is far from uncommon.

Tab. 3.8.1 List of the sperm samples analyzed with the relevant patient’s pathologies.

Pathology No.

Non-Hodgkin’s Lymphoma 3

Hodgkin’s Lymphoma 1

Lymphoma 1

Testicle cancer 1

Gulf War Syndrome 3

Blood disorder 2

Non diagnosed cases 2

Reference samples 5

Total 18

The following images show some examples of the debris and their

chemical compositions. The study involved mainly the sperm of veterans

from Iraq and former Yugoslavia (see Chapter 5) given to us because of

the fear of contaminations suspected be responsible for the children

malformation sometimes observed among American veterans. At

present, the epidemiological studies tend to deny any correlation among

the “working site exposure” of the fathers and these pathologies, but it is

true that the protocols of the US Army suggest to avoid procreation

within a year after the mission or longer than that.

Another case dealt with a soldier who had some health problems after

having returned from the Balkans and asked to have his sperm analyzed

and there we found metallic particles of different compositions, mainly

of Tungsten-Cobalt-Copper-Titanium-Sodium-Aluminium-Phosphorus-

Sulphur-Chlorine-Potassium-Calcium. The round-shape of most particles

was the obvious consequence of fortuitous combustions, a fact confirmed

by the great quantity of different elements present in the composition.

Clinical Cases: Lung, Blood, Liver, Kidney, Digestive System, Vessels, Sperm 117

Fig. 3.8.1 Tungsten-Cobalt-Copper-Titanium nanoparticle in a sperm sample. (marker

20 µm).

The soldier, to whom Fig. 3.8.1 refers, and his wife wished to have a

child, but were afraid something wrong could happen as a consequence

of the pollution we had detected and, they asked for our help. So, with

the help of two young doctors, we tried a sort of decontamination,

separating the spermatozoa from the plasma, and we saw that the

particles remained in the plasma. Having seen that, we suggested to recur

to artificial fertilization with the “clean” spermatozoa and the result was

a couple of healthy twins.

Fig. 3.8.2 A viable spermatozoon is close to a Lead-Chromium particle (marker 20 µm).

Nanopathology 118

Fig. 3.8.2 shows a viable spermatozoon close to two Lead-Chromium

(two toxic elements) particles. The obvious questions are: “What is the

fate of this spermatozoon in contact with a toxic material? What is the

fate of this particle when the sperm is donated during a sexual

intercourse? What will the answer be of the vaginal mucosa and the

uterus when a toxic particle remains in close contact with them? Is

Burning Semen Disease (see Chapter 5) the response for this presence?

All that is something really new, very little has been done in that field

and we do not have the answers. We can formulate only hypotheses, and

those hypotheses may show the way the research should follow.

Another case we came across and illustrated in Fig. 3.8.3 is that of a

soldier whose sperm was polluted by clusters of nanoparticles composed

mainly of Lead and Iron.

Fig. 3.8.3 Cluster of Lead-Chromium-Iron-Titanium-Silicon-Aluminium-Magnesium-

Sodium-Chlorine-Calcium nanoparticles (marker 10 µm).

Clinical Cases: Lung, Blood, Liver, Kidney, Digestive System, Vessels, Sperm 119

Fig. 3.8.4 Image of an Iron particle together with a viable spermatozoon (marker 20 µm).

Both pictures (Fig. 3.8.4 and Fig. 3.8.5) show foreign bodies in a drop

of sperm from two different subjects.

Fig. 3.8.5 Image of a spherical zirconia particle in the sperm (marker 20 µm).

Nanopathology 120

Fig. 3.8.6 Cluster of Lead-Chromium-Chlorine nanoparticles (marker 20 µm).

Fig. 3.8.7 Image of a viable spermatozoon with a spherical Antimony-Cobalt-Sulphur-

Sodium nanoparticle (marker 20 µm).

Clinical Cases: Lung, Blood, Liver, Kidney, Digestive System, Vessels, Sperm 121

Figs. 3.8.6 and Fig. 3.8.7 come from the same patient and show

viable spermatozoa in contact with toxic foreign bodies. This finding

interested us for two reasons: the first because the so-called physiological

barriers prove to be inefficient against such form of assault, the second

because it may represent a key to understand some medical mysteries of

our age. For instance, the so-called “industrial sterility” that affects some

workers in certain industries could be explained if we accept that the

contamination in some working places, represented by a toxic pollution,

reaches the testicles, pollutes the sperm and causes the death of

spermatozoa or reduces their vitality. This kind of contamination could

be verified also in the female partners suffering from Burning Semen

Disease.

In the case of contaminated sperm, the simplest prevention measure

that can be adopted to avoid possible problems to the female partner is

the use of a condom

3.9 A few considerations on reproduction

We leave in an age whose sexual customs are somewhat looser than in

the past, and meeting occasional partners without knowing anything

about their health is common enough. Knowing that we do not know

what we should and that ignorance may lead to grievous consequences

should induce to adopt more prudent approaches.

What we found in the sperm has other important consequences.

What happens if an egg is fertilized by contaminated sperm? What

happens to a fertilized egg that develops in a medium where particles of

toxic material are present?

All those questions remain without direct answers, but we had a

chance to observe some of the possible consequences. When we

analyzed malformed feti (both human and animal), we discovered

something that had escaped consideration until that moment.

We received two malformed lambs whose mothers had been grazing

close to an interforce military firing ground in Sardinia, and samples of

the internal organs of three feti affected by Neu-Laxova syndrome from

Malta. The peculiarity of the latter was that they were born, along with

Nanopathology 122

three more, in a period of twenty moths, something really surprising,

since that syndrome is extremely rare and Malta counts no more than

400,000 inhabitants.

The animal cases presented two different macroscopic kinds of

malformations: the first lamb had no developed brain nor eyes, in whose

place sat its ears. The second had the extrusion of the intestine. Both

survived just few hours.

The three cases examined out of six feti affected by Neu-Laxova

syndrome were sent to us by the University of Malta for the search of

possible genotoxic material. Despite its extreme rarity, six cases were

reported in Malta, a country with a population of 400,000 inhabitants, in

the space of just 18 months.

Neu-Laxova syndrome is a rare, incompatible with life, syndrome of

unknown origin characterized by ichthyosis (scaly skin), intrauterine

growth retardation, microcephaly, short neck, central nervous system

abnormalities, hypoplastic or atelectasia of the lungs, deformities of the

limbs, oedema, polyhydramnios (an abnormally high level of amniotic

fluid), and short umbilical cord.

As a matter of fact, inorganic particulate matter was clearly visible in

all samples we examined.

At the same time, we analyzed some internal organs of fetus from

induced abortions and in all cases they appeared free from any

particulate contamination.

Clinical Cases: Lung, Blood, Liver, Kidney, Digestive System, Vessels, Sperm 123

Fig. 3.9.1 Morphology of four different tissues: brain, thymus gland, spleen and kidney of

a fetus whose delivery was artificially induced (markers: A = 20 µm, B = 50 µm, C =

100 µm, D = 100 µm).

Fig. 3.9.1 shows different tissues belonging to a healthy fetus from an

induced abortion. The tissues appear free of any foreign body. They are

composed of only Carbon and Oxygen. (Hydrogen and Nitrogen are part

of the composition of the natural tissues, but it is impossible to see them

with our equipment).

All the images that follow regard samples taken from malformed feti.

Surprisingly enough, since those beings had never lived in the

environment we all share, particulate of exogenous origin could be

detected in their tissues. The compositions of some of those particles

were particularly strange and, in a few cases, certainly toxic.

Nanopathology 124

Fig. 3.9.2 Antimony particle inside the baby’s liver affected by Neu-Laxova syndrome

(marker 20 µm).

Fig. 3.9.3 Strontium-Sulphur-Iron-Aluminium particle inside a baby’s liver affected by

Neu-Laxova syndrome (marker 20 µm).

Fig. 3.9.2 and Fig. 3.9.3 regard the same case.

Clinical Cases: Lung, Blood, Liver, Kidney, Digestive System, Vessels, Sperm 125

Fig. 3.9.4 Lead-Bismuth particle surrounded by red cells in a liver sample of a case of

Neu-Laxova syndrome (marker 10 µm).

Fig. 3.9.5 Antimony particle inside a baby’s kidney affected by Neu-Laxova syndrome.

The subject is the same as the one of Fig. 3.9.4 (marker 10 µm).

Fig. 3.9.4 and Fig. 3.9.5 belong to the same subject. In the three cases

we had the chance to examine, the particles found showed different

compositions but Antimony was always present.

Nanopathology 126

Fig. 3.9.6 Zinc particle in a Neu-Laxova’s syndrome liver (marker 10 µm).

Fig. 3.9.7 Antimony particles inside a baby’s kidney affected by Neu-Laxova syndrome

(marker 20 µm).

The pictures above (Figs. 3.9.6 and 3.9.7) belong to the same case.

All the three cases of malformed feti showed the presence of micro

and nanoparticles inside the liver and the kidneys. The spectral analyses

of all the particles found in the small samples received revealed that they

are contaminated with metallic foreign bodies, sometimes toxic, like the

compounds of Antimony, Strontium or Lead. This presence is really

peculiar, if we consider that feti are never directly exposed to the

environment and its pollution. According to what we found, those

specimens present different levels of pollution as far as concentration is

concerned, but they all share the presence of Antimony compounds. A

Clinical Cases: Lung, Blood, Liver, Kidney, Digestive System, Vessels, Sperm 127

study aimed at searching the origin of this contaminant investigating

parents’ life and environment they lived in is in progress.

Fig. 3.9.8 Iron-Zirconium-Silicon-Aluminium-Magnesium-Zinc-Copper-Chromium-Calcium-

Antimony particle in the liver of a malformed fetus (marker 10 µm).

Fig. 3.9.9 Lung tissue from a malformed fetus. Clustered Nickel-Chromium nanoparticulate

is present (marker 20 µm).

Nanopathology 128

Among the oddities found in the lung pathologic tissues, we want to

show also an indirect exposure. The direct exposure to pollution regarded

a pregnant woman, but that pollution moved to the fetus through the

placental blood circulation. The pulmonary tissue of a 20-week fetus is

shown in Fig. 3.9.8 and Fig. 3.9.9. The artificial abortion was due to the

discovery of a 2-cm vertebral fission. Clusters of micro and nanoparticles

were visible in the lung tissue, composed of Nickel-Chromium. This

debris is chemically toxic. The particle shown in Fig. 3.9.8 is rather

large, about the same size of a red cell, and its composition is very

complex. Also in the case of this fetus, Antimony was present.

The next is not a human case, but a veterinary one (Fig. 3.9.10 and

Fig. 3.9.11). It regards the malformed fetus of a lamb died immediately

after delivery. Its mother grazed in the close vicinity of a firing ground in

Sardinia where grass was contaminated by the dust coming from the

explosions consequent to military experiments and drills.

Fig. 3.9.10 Antimony-Sulphur particle found in a malformed lamb’s brain (marker 10 µm).

Also in the cases of the malformed lambs we found a wide

dissemination of nanoparticles in all its organs, including the brain and

testicles. As in the human cases, Antimony was a constant presence.

Clinical Cases: Lung, Blood, Liver, Kidney, Digestive System, Vessels, Sperm 129

Fig. 3.9.11 Antimony-Cobalt-Sulphur nanoparticle in the liver tissue of the malformed lamb

of Fig. 3.9.10 (marker 20 µm).

It was somewhat odd to find recurrent chemical compositions shared

by either the human feti of Malta and the animal ones in Sardinia. But

Antimony, the element in question, could also be found in combination

with Cobalt in the sperm of a soldier who worked in a firing ground in

Sardinia and in a Canadian soldier who served during the First Gulf War.

This series of coincidences urged us to start a project on fetal

malformation which is in progress.

Nanopathology 130

Tab. 3.9.1 Flow-chart of nanoparticle dissemination with the entrance points.

Note: The number 1 in the insets means a primary “door” of entrance of the particles inside the human body.

All the images shown in the previous pages suggest a particular

dissemination of the nanoparticles once inhaled or ingested in the human

body. Their persistence in the tissues can induce possible pathological

reactions.

The Tab. 3.9.1 is an attempt to summarize the achieved experience.

Further studies are necessary to confirm the findings.

Clinical Cases: Lung, Blood, Liver, Kidney, Digestive System, Vessels, Sperm 131

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135

Chapter 4

Six “Detective Stories” ___________

he technique we developed allowed us to set-up a new investigative

method to assess the patient’s actual exposure to micro and

nanopollution.

The identification of the particle chemistry found in the pathological

tissues, along with their size and shape, induced us to investigate on the

patient’s life and look for possible sources of that particular

contamination, the real possibilities of his exposure and how that

pollution contamination may have occurred. In some cases we saw that

the exposure continued to be possible, so, some suggestions were given

to the patient to avoid it.

That kind of work had us discover unexpected ways of contamination

and, quite often, unknown ways of dissemination of the particles inside

the human body.

Six of such cases are briefly described below.

The 1st Case

The first case is related to a 25-year old non smoking girl.

She was admitted to the hospital because of vascular problems

affecting her hands and an altered electrical conduction of her peripheral

nerves.

The first diagnosis she received was sclerodermia, but later, after

the neuro-electrical test results, a diagnosis of ischemic miopathy was

advanced. The steroid therapy undertaken proved to be ineffective.

7

Nanopathology

136

A biopsy of the left-leg muscles (Fig. 4.1) was performed and specific

tests on the fresh tissue revealed an endothelial vasculitis, diagnosis

confirmed by Harvard University researchers.

Another histological section was sent to us to be processed and

analyzed with our technique. What we saw among other particulate were

submicronic particles of Carbon-Tungsten outside a blood vessel.

Fig. 4.1 Image of Carbon-Tungsten nanoparticles inside the muscle biopsy (marker 20 µm).

The way we do in similar cases, because of the presence of such

unusual foreign bodies which we took as a sort of marker, we started to

investigate on the patient’s occupational life that had taken place all

along in a small hairdresser’s shop.

We checked a great variety of the products used there to see if

Tungsten was present somewhere, but no trace of that metal was to be

found. So, we analyzed the dust from the plaster of the shop premises,

the indoor particulate pollution in the air, the dust inside the hairdryers

(the electrical resistances of the hairdryers can be made of Tungsten) and

the smoke of the cigarettes smoked by the only smoker who worked

there. Again, no trace of Tungsten was to be found.

Special attention was dedicated to parents’ story, also considering the

fact that the patient’s father had died of a lung cancer. That diagnosis had

been issued by observing bronchial mucus smeared on a glass slide, and

we look for it in the hospital’s archive. Had we found Tungsten there, we

could have identified a pollutant shared by both father and daughter and

that would have put us on an easier track.

Six “Detective Stories” 137

It must be said that biological samples on glass are not ideal for our

analyses, since glass issues signals that can interfere with those issued by

the particles.

In that particular specimen we found a few metallic particles, but no

Tungsten. So, that hypothesis of domestic pollution had to be ruled out

and we had to restart the investigation, which we did in the patient’s

house.

The indoor particulate contamination in the air was analyzed together

with the wall plaster, the cigarettes smoked by her mother and the food

more frequently eaten by the patient. But not even there any trace of

Tungsten was found.

We had to find a source of Tungsten if we wanted to solve the case.

That metal or its carbide (Carbon-Tungsten particles were what we had

found in the girl’s biopsies) are rather common in metallurgical

industries, but the patient did not seem to be likely to be or to have been

involved in such a contamination, as she had lived all her life in a flat

downtown, far from industrial sites.

In the course of our many interviews, the girl told us that two years

ago she had had a bleeding from the endometrium which did not respond

to any pharmaceutical treatment and that was solved surgically. Before

the operation, she had also suffered from two bleeding episodes from the

anus, and in both cases she was treated at the Emergency Room of the

local Hospital.

During the surgical operation, a small biological sample of

endometrium (Fig. 4.2) was taken and processed for the usual

histological tests. We analyzed the archived sample and found small

particles of Iron-Chromium-Nickel (stainless steel) there. The presence

of very small particles ranging from 0.5 to 8 µm in such an anatomical

site seemed very strange. Their presence could have been the cause of

the bleeding, since those are bodies foreign to the human organism and,

in contact with the mucosa, can trigger an inflammatory reaction and, in

some instances, a localized necrosis. The inflammation can also reach

deeper into the tissues. That could explain the strange episodes of

bleeding from the anus which could depend on an acute inflammation of

the internal tissue in close vicinity of the vagina or the uterus.

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138

Fig. 4.2 Image of a sample of endometrium with stainless steel nanoparticles (marker

20 µm).

Such an event reminded us of the Burning Semen Disease suffered by

the wives of the veterans after their coming back from the war missions

in Iraq or Afghanistan during the 1st and 2

nd Gulf War (see Cap. 5)

This findings had us redirect the investigations toward the patient’s

partner and we discovered that he had suffered from a testicle

cancer, operated on four years ago. So, we thought that analyzing his

pathological tissues could give us some important clues.

At the time of the disease, the partner worked in a tile factory as a

clerk, but his desk was located in a large room, close to a micronizing

tool used to ground the clay before it was mixed with water, and to a

special machine used to cut the edge of the tiles. The grinding and

cutting tools were made respectively of stainless steel and Tungsten

carbide.

The partner told us that during the period he was employed there, the

man who worked in direct contact with the latter machine had died of a

lung cancer.

The analyses on the four surgical samples we got revealed the

massive presence of micron-sized particles of silicates (tiles) and debris

of stainless steel and Tungsten.

The finding of this material inside a testicle had us put forward a

mechanism of dissemination and translocation: The partner had been

exposed to the materials making up the tiles and to the wear debris

coming from two different tool machines. Following a mechanism that

Six “Detective Stories” 139

we cannot yet fully explain but that we have observed in many instances,

we think that those debris were entrapped in a testicle (we had no

samples from the contralateral one, so, nothing about it may be said)

after having been inhaled, and the metallic nanoscaled particles (stainless

steel and Tungsten) extravasated and contaminated the sperm which was

donated to our patient during sexual intercourse. The presence of non

biocompatible, not biodegradable particles in the girl’s vaginal mucosa

caused pain, inflammation and bleeding as occurs in the veterans’ wives.

The surgery that followed the bleeding episodes exposed a

compromised tissue, without any effective defense against sperm

contamination. As a matter of fact, the symptoms referred to the sclerosis

started a few months after the surgical operation.

The probable reasons why Tungsten, and not stainless steel, particles

were so widely disseminated were because of their size, much smaller

than that of the stainless steel particulate, or because of a physical-

chemical interaction withy the blood components, or both. Such

dissemination in the blood circulation had been the cause of the

vasculitis.

The particles extravasated and came into contact with the muscle

fibres, causing a fibrosis, which is the normal biological reaction to the

presence of non biocompatible foreign bodies.

Of course, we did not have the possibility to check but a few of the

girl’s tissues, but, as far as we saw, the particle dissemination seemed to

be ubiquitous. So, we suggested to have protected sex in order to avoid

further exposure, but the physical elimination of every single particle

from the organism looked impossible, at least to our experience and for

the time being.

In the case described, Tungsten acted as a marker and the search of its

specific source of pollution allowed us to identify the kind of exposure

and the gross mechanism of dissemination inside the organism. The

following images show the Tungsten particles found in the spermatic

vessel (Fig. 4.3), the stainless steel debris (Fig. 4.4) and the particles of

silicatic material from the ceramic tiles (Fig. 4.5, 4.6 and its spectrum).

Nanopathology

140

Fig. 4.3 Image of a Tungsten particle found inside the spermatic vessel (marker 50 µm).

Fig. 4.4 Image of a stainless steel particle (Iron-Chromium and Nickel) found in the

cancerous tissue of the testicle (marker 20 µm).

Six “Detective Stories” 141

Fig. 4.5 Image of the part of the resected cancerous tissue of the testicle with the

spectrum of the normal biological tissue (marker 1.000 µm).

Fig. 4.6 shows the “dust” found in the resected tissue of the testicle

cancer. The samples contained many different particles, mainly silicatic

debris coming from the dust of the tiles (white particles indicated by

arrows). They are composed of Silicon-Aluminium-Calcium. Normal

biological tissue contains only Carbon-Oxygen-Sodium-Phosphorus-

Sulphur-Chlorine. We do not know if the cancer was induced by the

metallic particles or by the enormous pollution.

Fig. 4.6 Image of an agglomerate of silicatic nanoparticles trapped in a calcific tissue

(marker 50 µm).

Nanopathology

142

The 2nd

Case

A professor of Mathematics started to betray some problems in

articulating the sound “r” during his lectures at the University. After

different neurological examinations, a diagnosis of Lateral Amyotrophic

Sclerosis was issued, a diagnosis he did not believe in and refused to

accept.

Thus, the professor started to search the Internet about that disease

and learnt that the one of the first, more common symptoms is a slight

claudication and this, among other symptoms he had read about, was not

something he suffered from.

In the enormous quantity of not always reliable information he found

in the Internet, he read that dental amalgam, because of its content of

Mercury, could be somehow related to the disease he had been

diagnosed.

So, the professor asked Dr Gatti her opinion as a biomaterial expert

and a teacher of Dental Materials at the University of Modena and

Reggio Emilia.

Actually, he had either amalgam restorations and dental prostheses in

his mouth and hardly in two teeth the amalgam did not look corroded. (It

is only in the course of corrosion that amalgam releases Mercury, Silver,

Tin and Copper ions which can be toxic, locally in the oral mucosa and

in a systemic way after ingestion).

But the observation of the mouth revealed an unusual situation: The

surface of the tongue was not symmetrical and the right half was not as

flat as the left one, but was corrugated and wavy. The surface of the

mucosa seemed to have a larger extension than the substrate, and that

created a sort of gibbosity, not adherent to the tongue base. In addition to

that, we noticed a discoloration in the internal cheek mucosa at the level

of the intersection of the teeth. We also observed that in the right part of

the mouth some porcelain prostheses had been implanted.

Having seen all that, we thought that if there is a wear phenomenon,

debris must be released, and those debris can be entrapped by the nearby

mucosa and induce a fibrosis. The tongue mucosa has a great aptitude to

absorb, so a passage of some fine particles through it cannot be

surprising and their entrapping occurs at the level of the underlying

Six “Detective Stories” 143

tissue called corium. That formation is a thick network of fibrous

connective tissue strictly connected with numerous elastic fibres,

forming the septa between the muscular bundles of the tongue. It

contains the branches of many nerves and vessels that feed the papillae.

The tissue can react to a foreign body giving origin to a fibrosis.

A thickening and the following contraction of that layer could explain

the humped morphology of the mucosa surface and the loss of agility of

the tongue. It is also reasonable to think that all that exerts a compression

on the underlying muscle and nerves. But, in any case, a collection of

foreign bodies can give origin to a fibrosis, and a localized fibrosis could

be responsible for the problem in articulating the sound “r”, because a

fibrotic tissue is thick, less elastic than normal and does not allow fine

movement.

The first step we had to take was to demonstrate the presence of that

fibrosis with the inclusion of prosthetic debris in a biopsy of the internal

side of the cheek. As a matter of fact, the tissue presented a fibrosis and

some porcelain debris were present there.

The second step was to demonstrate a similar presence in a biopsy of

the right, ipsilateral side of the tongue. The hope was that if a fibrotic

reaction due to foreign bodies was present, probably the removal of this

tissue or a cut made on the tongue could interrupt the phenomenon and

its worsening. We thought that stopping the fibrosis’ progress could

probably prevent further damage to nerves and blood vessels. In fact, the

shrinkage of a fibrotic tissue can make the vessels occlude and stop of

the supply of nutritious elements and Oxygen. The afferent and deferent

nervous terminations can simultaneously stop the propagation of the

electrical signal, thus causing the opening of the electrical circuit.

While the histopathologist confirmed the presence of a fibrosis in the

bioptic sample, we saw some strange precipitates of Zinc there.

It is easy to tell the difference between exogenous particles and

endogenous precipitates: The EDS signal is high in the case of “stones”,

since the atomic structure is compact, while, in the case of precipitates,

the inorganic element (Zinc in this case) is bound to proteins, forming

insoluble metalloproteins. The density of these precipitates is very low

and also the signal they issue is low (proteins contain Carbon, Oxygen,

Nanopathology

144

Hydrogen and Nitrogen, all very light elements). Also the morphology is

characteristic.

In our case, Zinc ions must have been absorbed by the tongue mucosa

and in the sub-mucosa the interaction with proteins created insoluble

precipitates. The sub-mucosa is reach of nerves and nervous terminations

that can be compromised by the presence of toxic ions. Also the

formations of metalloproteic precipitates can interfere with the cellular

metabolism according to different mechanisms: protein depletion, protein

immobilization or a change in the local pH. A further possibility is the

entrance of ions within the cell where they can precipitate, forming

aggregates that disturb the intracellular metabolism. The sequestration of

proteins, namely the formation of insoluble precipitates, can interrupt

cascade reactions that cause “alternative metabolic solutions”.

It was not only Zinc what we detected, but a wide variety of particles

ranging 0.8 – 2 µm. Gold particles were there, and this was compatible

with the bridges the patient carried. 0.8 µm particles of Silicon,

Aluminium, Calcium, Magnesium, Potassium and Iron were also present,

and they could certainly be blamed for the fibrosis, the roughness of the

tongue surface and the discolouration of the mucosa.

Fig. 4.7 Image of nanoparticles of Phosphorus-Zinc-Magnesium-Aluminium-Silicon-

Calcium-Iron found in the biopsy of the mouth mucosa (marker 20 µm).

But what puzzled us most was the presence of Zinc (Fig. 4.7). That is

a metallic element commonly used as oxide because of its antibacterial

activity, but its presence is unexpected inside the oral mucosa and an

explanation for such a presence must be given.

Six “Detective Stories” 145

As usual, we questioned the patient about his diet, the use of chewing

gum, drugs, etc., but no clue was to be found there.

The truth emerged unexpectedly when we asked the patient about his

visits to the dentist’s. The patient had been advised to use a mouthwash

and he had used it in an obsessive way, many times a day, for more than

3 years. The label of the product reported the presence of a Zinc salt as a

bactericide, but the solution contained also ethylic alcohol, a substance

that increases the permeability of the tongue epithelium, thus making the

entrance of the Zinc salt easier. That salt hydrolyzed, Zinc formed a

metalloprotein and that metalloprotein precipitated. Its presence was the

likely cause of the partial necrosis of the biological structures there

present.

Zinc is a well-known bactericide and is toxic to cells. Therefore, it

could interact with the myelin sheath of the nerves and damage it.

When we realized that, the patient had already quitted using the

mouthwash and the damage to his nervous system had grown

irreversible; so, all that was of no practical use to him and he died in six

months.

If our hypothesis is correct, this is a case where very simple

precautionary measures could have avoided the gravest of consequences.

The 3rd

Case

The manager of a very important company consulted our laboratory for a

case of dermatitis afflicting his trunk, asking to check the biopsy of the

derma interested by the affection.

The observation of the very small sample showed the presence of

numerous particles of stainless steel (Fe-Cr-Ni).

Such a finding looked strange, because neither at home nor at the

patient’s working place stainless steel particulate should have been

present.

He lived an apparent healthy life in a “normally” polluted big city. He

had been a smoker in the past but in the last 10 years he had never lit a

cigarette.

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146

The patient told us that in the course of a yearly check up he had been

subjected to a chest X-ray examination that revealed light spots in his

lungs and, because of that, some biopsies of those spots had been

performed. The response was that the areas were calcified.

We took those bioptic samples, observed them with our usual

method, and found that stainless steel was present there in the form of

small micrometric and submicronic particles.

Having been found in the lungs, we supposed as a probable

hypothesis that those pollutants had been inhaled; some of them had

stayed in the lungs where they had triggered an inflammation with a

consequent calcification, and others had been disseminated throughout

the organism because of a size small enough to allow the passage

through the lung barrier and enter the blood stream.

Their presence in the skin (Fig. 4.8) could be explained with an

attempt of the organism to get rid of them, but they were halted by the

derma which is very compact and hard to cross, and there they caused the

irritation the patients complained of.

Such a hypothesis must be demonstrated, and to do that we had to

find out the origin of that particular pollution and the way our patient had

been exposed to it.

Questioning him, we got to know that he had suffered from nasal

congestion for more than twenty years and for that reason, to try and

breath better, he had been operated on his nasal septum one year ago. But

for the previous twenty years, he had put drops in his nose to decrease

the congestion. During all that period, he had used only two types of

herbal drugs, one coming from Italy and one from Switzerland, and we

checked both products from bottles the patient still conserved, even if he

did not use them any more after the successful surgery he had undergone.

What we saw was that only one of the two oily liquids contained

particles, and they were the same size and composition as those we had

detected in the pathological tissue.

That could be explained by guessing that the herbs used by that

particular laboratory were ground with a stainless steel tool that, because

of the obvious wearing of the machine, released particles to the product.

We wondered why the presence of metal particles did not induce a

more serious disease like, for example, a cancer?

Six “Detective Stories” 147

A possible explanation is that the quantity of particles was relatively

small and the oil used as a vehicle protected them from corrosion, thus

preventing a release of Iron, Chromium and Nickel ions. Besides, that oil

was organic and the tissues had an interaction with something they

recognized and did not see the inorganic bodies below. So, all that

occurred was a comparatively mild inflammation with a consequent

calcification.

In that case, the exposure had been interrupted and nothing else could

be done as a preventive or therapeutic measure.

Fig. 4.8 Image of three particles of stainless steel embedded in a biopsy of derma (marker

20 µm).

Fig. 4.9 Image of a small stainless steel particle found in a lung biopsy (marker 20 µm).

Nanopathology

148

Fig. 4.10 Image of a stainless steel particle found inside the nose drops used by the

patient (marker 20 µm).

In Fig. 4.9, a single stainless-steel particles found in the patient’s lung

is visible. Fig. 4.10 and Fig. 4.11 show the contamination of Iron-based

particles detected after filtration in two different nasal drops used by the

patient.

Fig. 4.11 Image of the metallic contamination of the nose drops used by the patient, and

deposited on a paper filter (marker 200 µm).

The 4th

Case

In this case, the patient was a 45-year-old lady suffering from a

peritoneal mesothelioma.

Six “Detective Stories” 149

She had been operated on and some bioptic samples of her

peritoneum were sent to us to be analyzed.

There we found many debris singularly disseminated in the specimen:

metals like, for example, stainless steel were present, but one particular

chemical composition attracted our attention.

The EDS spectrum indicated that a number of submicronic particles

were a silicate containing, among other elements, Uranium and Thorium.

In that very period we were busy with specimens coming from Italian

and French veterans back from former Yugoslavia, and Uranium was

what we were looking for, but without any result: No trace of Uranium

was to be found in those pathological tissues.

Now we were confronted with the case of a lady who had never been

in a war theatre nor, to her knowledge, in the vicinity of a source of

radioactivity, but had spent her whole life in a quiet Italian town working

as a clerk in an office, yet she had Uranium and Thorium in her body.

So, we started a hunt to identify the origin of such an unusual and

unexpected particulate.

Being a silicate, we immediately suspected that the origin of the

pollution found could be an area about 20 kilometres south of the place

where the patient lived, where numerous ceramic factories are located.

As to Uranium, it is used to give a yellow colour to enamels and

Thorium is used as well. The study of the winds, though, seemed to rule

out such a hypothesis or, at least, to make it very unlikely.

Fig. 4.12 Image of a particular debris containing radioactive elements found in the

peritoneal mesothelioma (marker 20 µm).

Nanopathology

150

Fig. 4.12 shows a particle that contains Titanium-Zirconium-

Calcium-Iron-Aluminum-Yttrium-Niobium-Uranium-Thorium-Cerium-

Manganese-Copper. That can be originated by tiles industries that use a

silicatic or Zirconium-based earth for the tiles.

Fig. 4.13 Image of a particle found on a leaf surface. Its composition includes radioactive

elements (marker 20 µm).

Fig. 4.13 shows a particle composed of Silicon-Phosphorus-

Aluminium-Uranium-Thorium-Lanthanum-Cerium-Neodymium-Iron-

Potassium-Calcium. That composition is typical of a type of earth used

for ceramic tiles. Uranium and Thorium are likely to have been added as

components of some glazes.

Since those particles had been found in the peritoneum, i.e.

anatomically close to the digestive system, we assumed as a likely,

though not exclusive, hypothesis that ingestion could have been the way

they were introduced into the organism. According to that assumption,

we began to search the food the patient ate more frequently, despite the

fact that what was in the peritoneum could have entered long ago with a

food, if food was indeed the Trojan horse, that had been used in the past

and then forgotten. Nevertheless, we tried.

All the family composed by a husband and a girl, both healthy, was

involved in that sort of treasure hunting.

The patient told us that both her parents had died of liver cancer,

without any doubt something worth investigating, but we could not find

Six “Detective Stories” 151

their biopsies and, of course, the histopathological records did not

contain anything that could help us.

Questioning the family about their preferences in the matter of food,

we got to know that the lady preferred vegetables to meat, unlike her

husband and daughter, and she was particularly fond of a kind of wild

chicory that her parents had been bringing to her at least once a week

from a village in the hills, about 30 kilometres away from town, where

she had moved at the age of 22 when she married. That chicory had been

a usual dish to her since her childhood and had never ceased to be.

Chicory was what we had to check and we went to the small valley

were it had always been picked.

It was winter time and no chicory could be found in that place nor

other edible vegetables, but, looking for possible clues of pollution, we

saw a plume of smoke in the distance and we drove there, four or five

kilometres away as the crow flies. There we saw a group of small

ceramic factories whose existence was unknown to us.

Then, we went back to the valley and picked some oak leaves still

hanging from a few trees that we brought to the laboratory and observed

under ESEM. On those leaves we detected particles containing silicatic

compounds with Uranium and Thorium.

According to what we discovered, the lady and her parents had been

eating something they believed to be very safe but which, in fact,

contained toxic (and slightly radioactive) particulate.

Now, a final piece was missing to the solution of the puzzle: why had

our patient contracted a peritoneal mesothelioma, while her parents had

died of liver cancer (granting the fact that the aetiology was the same)?

Twenty three years ago, our patient had spent her honeymoon in the

North African desert and, when a guest at some Bedouins’, she had

drunk tea which gave her a typhoid fever. Intestinal problems lasted a

long time and, probably, that inflammatory condition altered the

permeability of her intestinal mucosa which became more negotiable to

particles.

If we accept that hypothesis, had she had a healthy gut, she would

have contracted not a mesothelioma but a liver cancer, the way her

parents did.

Nanopathology

152

The 5th

Case

A couple of somewhat unusual patients, two lady doctors working in the

neonatal department of a big hospital, came to our laboratory, asking us

if we could help solve their case.

Four doctors, including the chief of the department, and two nurses

suffered from different diseases: cancer, sarcoidosis, and two missing

diagnoses, as the operators developed symptoms that could not be

referred to any known disease.

One of the two ladies suffered from a lung sarcoidosis and inside the

granulomatous formations typical of that disease we found micro and

nanoparticles of stainless steel (Fig. 4.14) and silicates (Fig. 4.15).

Fig. 4.14 Image of a small stainless-steel particle (indicated by arrow) found in a lung

biopsy performed to confirm the diagnosis of sarcoidosis (marker 20 µm).

Six “Detective Stories” 153

Fig. 4.15 Biopsy of a nodule containing Silicon-Aluminium particles (marker 10 µm).

Fig. 4.16 Bacterium (inside the circle) containing Aluminium particles collected by

gravity on a Carbon sensor (marker 10 µm).

The second case was different. The doctor-patient contracted a

bacterial infection in the ring finger of her left hand, an infection that

proved resistant to the specific antibiotic administered. Those bacteria

had been detected within the Intensive Care Unit of the department,

where the patient was usually busy (Fig. 4.16).

In a short time, the patient had started to suffer from fatigue, than

from acute episodes of sometimes monolateral and sometimes bilateral

pain at the lower limbs followed by the formation of nodules that were

surgically removed as many as 18 times without any advantage to the

patient, since the pathology continued its progress. Our investigations in

Nanopathology

154

those nodules discovered the presence of stainless steel and Aluminium

particulate. A section of those nodules was sent to the Harvard School of

Medicine in Boston and the diagnosis issued was of a vasculitis.

Our hypothesis was that phenomena of extravasation of those

particles, running in the circulatory system, had been occurring, and that

could explain the acute pain. Then, the presence of foreign bodies in the

muscular tissue could have reasonably induced a biological reaction that

drove to the formation of the nodules.

We had to look for the source of pollution that could have caused the

pathologies in doctors who had been living what for doctors is a normal

life.

So, we decided to sample the air in two adjacent rooms where the

doctors work more frequently and we did so by means of an air pump

that worked for one hour. The dust was collected on cellulose filters

where particles smaller than 2.5 µm were detected.

We were told that just outside the building where the department was

located, another building had been standing and that building had been

very slowly pulled down raising great clouds of dust. The still standing

broken stainless-steel reinforcement had been left on place for a long

time.

The pollution created was aspirated by the air conditioning system set

on the roof of the department. Every room was equipped with a filtration

system that can only stop particles larger than 2.5 micron, while smaller

particles can pass easily and their presence inside the rooms was

concentrated because of a recirculation system which created an

accumulation of fine pollutants.

It is only natural that everybody who works in such environmental

conditions is exposed to the effects of that sort of pollution.

The main difference between the two cases was the much more

regular presence of the second doctor in the Intensive Care Unit, where a

bacterial contamination had been found, and those bacteria were, in turn,

contaminated with Aluminium and had served as a carrier. Aluminium

particulate was inside the bacteria we found and that bulky presence

changed their morphology. It was probably for that change in shape that

the antibiotics administered did not have any effect. In the first case, the

inhalation of foreign bodies had induced a granulomatosic reaction,

Six “Detective Stories” 155

while in the a second one the infective carrier of the pollution was

predominant.

To the same way of entrance, two different pathologies had ensued.

The cases proposed and the solution found should have us reflect.

The solution of the twin cases had been possible because we started

from looking for and finding the real exposure the patient had undergone.

Every patient has a personal history and epidemiological studies are not

always helpful or are not meaningful at all if the number of cases is

very restricted. So, a single-patient oriented, “customized” investigation

should be carried out in such circumstances. That is certainly far from

easy, expensive and time consuming, all things our society does not look

willing to accept, but we do not see any other way to deal with patients

like the ones we were confronted with.

Far from easy, expensive and time consuming but not useless. The

knowledge gained from such cases is of the utmost usefulness to prepare

prevention guide lines and, in a very pragmatic way, prevention means

saving money.

In the case in point, we got a further proof that any uncontrolled

release of nanoparticles in the working places must be avoided. Masks,

specific filtration means and effective systems to abate acute dust

pollution, especially when it is the nanometric size, must be studied and

readily adopted. When dust is released in the environment, everybody

must take proper care. How, it is the scientist’s task to say.

The 6th

Case

The case of a dentist who had developed a lung sarcoidosis showed the

existence of a close correlation between his disease and the powder he

had used to bleach his patients’ teeth.

In 20 years he had used about 300 kilograms of that fine powder, a

part of which he had certainly inhaled and whose concentration in his

organism had probably exceeded his tolerable threshold concentration.

That dentist, male, 52 years old, never been a smoker, gradually

developed shortness of breath during exercise, cough and retrosternal

pain, which prompted for investigation of lung and heart function.

Nanopathology

156

Plain X-ray chest film showed signs of pulmonary fibrosis. A

subsequent CT scan confirmed that fibrosis and showed the presence of

several micro-nodules. All other diagnostic tests were normal.

Sarcoidosis was suspected and a bronchoscopy was performed,

during which a biopsy of the bronchial mucosa and bronchoalveolar

lavage were carried out.

Optical microscope observation of the biopsy revealed chronic

inflammation of the bronchial mucosa, with edema and eosinophilic

granulocytes.

Bronchoalveolar lavage showed an increase in total cell number and

CD4/CD8 lymphocytes ratio. A diagnosis of sarcoidosis was established

and a steroid therapy was started.

An investigation was performed in order to determine whether the

disease could be traced to an occupational environment agent.

The activity of dental surgeons is associated with several possible

hazardous exposures (such as X-ray, dental material components, blood-

borne pathogens and so on). Among these, aerosols of biological material

and inorganic substances are specifically responsible for airborne

exposure.

The instruments used during dental surgical procedures may produce

intense heat (electrocautery, laser) and such procedures usually generate

fumes containing biological material (even partially unburnt).

High-speed, air-driven dental handpieces, ultrasonic scalers, the

polishing of composite and ceramic restorations and the use of the

milling cutter on metallic prostheses disperse a large amount of aerosol

and spatter including fine particulate matter.

During the polishing and whitening of natural teeth, chemical

compounds (composed by particles of Sodium di-carbonate and

tricalcium phosphate) are sprayed on the tooth surface detaching part of

its enamel and dispersing particles in the environment, thus exposing

both patient and operator to risk of inhalation.

For example, a standard hygiene procedure involves the use of

prophylactic material and ultrasonic and/or manual instrumentation. The

prophylaxis phase can be substituted by an artificial bicarbonate aerosol

and that was what our subject had been doing. Artificial cleaning

aerosols are formed by a Mini-Clean device (Castellini, Bologna, Italy)

Six “Detective Stories” 157

with air pressure set to 6 to 7 atmospheres and water flow to 1

atmosphere. Each individual run lasts 20-30 minutes for each patient.

It has been reported that 95% of the particles measure less than 5

micrometers and are mainly concentrated within 2 meters of the patient

where they can be easily inhaled by dental clinicians.

For this reason, the dentist always protected himself with a face mask

[3M ESPE 1942 F.B. (1800 NL) resistant molded face] whose level of

efficiency was 90-92%.

To test our hypothesis that sarcoidosis could be in this case related to

a possible environmental agent, a further examination of biopsy

specimens was performed. So, sections of the lung biopsy were sent to us

to be analyzed.

The label on the product used by our subject declared that the powder

contained particles of Sodium carbonate (Fig. 4.17) and tri-Calcium

phosphate (Fig. 4.18).

Fig. 4.17 Sodium-carbonate particle of and its EDS spectrum (marker 20 µm).

Nanopathology

158

Fig. 4.18 Tri-Calcium phosphate particles and their spectrum (marker 20 µm).

Fig. 4.19 Silica particles: a component non declared by the Manufacturer (marker

50 µm).

Fig. 4.19 shows other silicatic particles found in the polishing

material. Those particles were not declared in the product label.

Considering the patient’s long exposure to dust, a hypothesis was

advanced that the particulate matter could originate from the wear of

materials used during the abrasion and polishing operations.

It was hypothesized that the material used for dental cleaning

procedures could have a chemical composition similar to that of the

Six “Detective Stories” 159

debris found. A sample was observed under ESEM and EDS analyzed.

One of the spectra obtained proved similar to that of the debris found in

the lung. (Fig. 4.19).

In the case we described, the diagnosis of sarcoidosis was issued

according to standard criteria (ATS 1999).

Additional information about the possible origin of the disease was

provided by ESEM with EDS of biopsy specimens (Fig. 4.20 and Fig.

4.21), which demonstrated that the material found inside the sarcoidotic

granulomas was identical to that found in the powder the dental surgeon

had used in large quantities for several years. More Calcium-phopshatic

particles were found in the bioptic specimens. Fig. 4.22 shows spherical

particles composed by Calcium-phosphate with Sodium and Magnesium

ranging from 0.5 to 2.5 µm. The prolonged inhalation of soluble tri-

Calcium-phosphate and Sodium-carbonate pollution provided a large

supply of Calcium, Phosphorus and Sodium ions. So, a logical

correlation can be established between the inhaled pollution and the

formation of those Calcium-phosphate entities.

Fig. 4.20 Particular of the lung biopsy (marker 200 µm).

Nanopathology

160

Fig. 4.21 Silica particle found in the lung biopsy (marker 20 µm).

Fig. 4.22 Spherules of Calcium-phosphate (marker 10 µm).

Such result cannot be considered a conclusive proof, but it does

however induce a strong suspicion that the disease could have been

initiated by the material used by the surgeon.

161

Chapter 5

War and Nanoparticles ___________

fter the 1

st Gulf War many soldiers (mainly American, British and

French) became ill. After the Balkan War also Italian and in our

direct knowledge also Spanish and French soldiers, in the course

or soon after their peace-keeping missions, developed strange collections

of diseases, called later with the name of the site where that condition

seemed to have had its origin.

Then, after the 2nd

Gulf War, other soldiers became ill.

Medicine can neither explain the collective, simultaneous presence of

all those symptoms nor cure them.

Also the epidemiological studies carried out on that problem are not

conclusive, also because they involve a relatively small number of cases,

in a limited time.

A new vision is now presented with some specific pieces of evidence

that clarify the impact of the new war technologies with human life.

It is a well-known fact that every kind of combustion generates

particles, and explosions can rightfully be classified as combustions.

Since the invention of gunpowder, probably in the middle of the

thirteenth century, explosions have been increasingly used in warfare and

have produced as a consequence clouds of dust, but it is only relatively

recently that blasting bombs have started to be widely used.

To our knowledge, the first connection between explosion and micro

and nanoparticles dates back to the late Seventies of the last century,

when the Air Force Laboratory, Armament Development and Test

Center, of Eglin Air Base, Florida, issued a report about the results of a

few experiments carried out on depleted-Uranium weapons which

included the Air Force GAU-8, the Army XM 774 and M735E1 and the

PHALANX gun systems (Technical Report, 1978).

$

Nanopathology 162

Depleted Uranium (DU) is a by-product of the enriching process of

natural Uranium meant to be used in nuclear reactors, when the fissile

and more radioactive isotopes are removed. Another, though less

common, source of DU is reprocessed spent reactor fuel.

DU is not so radioactive as natural Uranium and definitely less than

enriched Uranium (the specific activity [the activity in becquerels (Bq)

per unit mass i.e. one radioactive decay takes place per second] of DU is

about 15 Bq per mg compared with 24.5 Bq per mg for natural uranium),

but some not negligible radioactivity remains, and, in any case, DU is

toxic. Once natural Uranium has been deprived of much of its precious

235 isotope or enriched Uranium is not usable any more, DU must be

disposed of, and doing that is rather expensive. So, whoever faces that

problem is more than willing to rid himself of that metal in a quite

inexpensive way.

In the late 1940s, the US and the then existing USSR started their

respective nuclear programs and, in a short time, began to store huge

quantities of DU in stockpiles, in the hope that future technologies would

allow to further extract the 235 isotope, but the expectations of both

armies were disappointed.

In the 1970s, the USSR developed a particular armor plating for their

tanks that US ammunitions could not pierce, so the Americans began to

test materials dense and penetrating enough, and found that the best

compromise between efficiency and cost was DU, of which there was an

enormous availability.

Uranium, be it natural, enriched or depleted, is an excellent penetrator

and, in addition to that, is pyrophoric, i.e. gets oxidized and ignites

spontaneously upon impact with a solid target in the presence of Oxygen,

and the temperature that combustion reaches exceeds 3,000°C.

The study mentioned above and completed between October 1977

and October 1978 at the Ford Farm Firing Range, Aberdeen Proving

Ground, MD, placed a particular emphasis on size (just out of curiosity,

they describe the formation of ultrafine particles less than 0.1 µm in

diameter as an “unexpected phenomenon”), structure and stability of the

particles resulting from the impact against multiple-armor-plate targets of

penetrators consisting of 3.5 kg DU containing 0.75% Titanium by

weight.

War and Nanoparticles 163

The airborne particles were collected on a double-stick cellophane

tape which was cut in small portions, and those portions were placed on

Carbon-coated Aluminum stubs, coated with 100%-Gold and observed

under SEM.

The soil samples were sieved and treated in order to select and

concentrate particles in the respirable range and observed as described

above.

The elemental composition of the particles observed down to the size

of 0.5 µm in diameter was identified with an X-Ray energy spectrometer.

As could be expected, a large number of particles in a broad range of

sizes (< 50 µm - > 1 µm) resulted from the explosions, and most of them

were either spherical or ellipsoidal in shape, thus showing that the heat

generated was enough to melt Uranium and its oxides. On page 6, the

document reads: “Fragments produced are ignited spontaneously by a

combination of shock and friction heating at impact. Combustion of

fragments in air is exothermic and self-sustaining. Flash temperature

reached during impact of depleted Uranium penetrators with armor plate

have been shown to fall in the range of 5500 to 5600°F (…) Test results

show further a nearly constant (3037 to 3093°C) impact flash

temperature over the entire range of impact velocities from 4010 to 5560

feet per second. Such temperatures (…) are sufficient to initiate

combustion of the numerous particles produced.” And further on: “

Examination of numerous particles revealed several distinct

morphologies. The great majority of airborne particles exhibited a rugose

or convoluted structure which frequently appeared, at surface level, to

consist of large, interconnecting concave plates (…). Each plate, formed

independently from the solidification of molten material, was observed to

radiate from a common origin or focal point upon the surface. At or near

the junction of adjacent plates considerable overlapping and fusion

occurred, resulting in distinct but irregularly delineated boundary edges.

Higher magnification revealed numerous imperfections in the overall

crystalline structure along the surface, thereby serving to further

subdivide each major plate. (…) some rough particles exhibited a more

uniform surface morphology, devoid of major plate divisions. The

convoluted surface, although similar to that previously described,

consisted of deeper, more numerous folds and extensive dimpling.

Nanopathology 164

Fissures and pore-like invaginations were frequently observed and

progressively developed into deep fractures, presumably as a

consequence of thermal expansion or through collisions with other

objects. These fractures, which traversed irregular courses along the

convoluted folds, were the eventual cause of extensive particle breakup

(…). Internal morphology was frequently revealed by examination of

fractured particles, particularly those in which large portions had become

detached. Both solid and hollow particles were observed, the latter

clearly demonstrated by the presence of hemispherical fragments (…).

Although wall thickness varied greatly, the inner surface morphology of

hollow particles consistently resembled that of the outer surface. (…)

Prior to weathering, the surfaces of most airborne particles were covered

to a varying extent by immense numbers of nearly spherical, ultrafine

particles less than 0.1 µm in diameter (…) Identity of these particulates

as pure or alloyed Uranium was confirmed by X-ray analysis.

“An example of the uniform dispersal of ultrafine particulates on the

surface of a depleted Uranium/iron particle is demonstrated (…) Any

further accumulation, however, generally resulted in extensive

coagulation and hence the formation of billowing aggregates (…).

“Although generally found in association with larger particles, the

ultrafine particulates were also detected in the free state directly upon the

surface of the collective tape. At low magnification these particulates

appeared as large concentric masses often reaching several hundred

micrometers in diameter (…). At greatly increased magnification (…),

these masses were revealed as consisting of vast numbers of small

aggregates and long angular chains. The ultrafine spherical particles

comprising these aggregates measured approximately 0.01 to 0.1 µm in

diameter. (…)

“Morphology of particles removed from soil samples was quite unlike

that of their airborne counterparts. Far greater numbers of irregularly

shaped fragments were present, presumably the result of interaction and

fusion with sand and other materials within the soil (…). Spherical

particles, although quite numerous, generally lacked the convoluted

surface morphology so apparent in airborne samples. Their surfaces were

consistently smoother and frequently speckled with knobby blebs (…).

War and Nanoparticles 165

Some of these blebs were clearly continuous with the surrounding

surface whereas others appeared nearly detached.

“The relative fragility of these Uranium particles was clearly evident

following brief exposure at the laboratory to ultrasound (…). Although

sonification lasted no longer than 15 seconds, particles showed extensive

fracturing and in many instances compete disintegration.

“(…)

“The elemental composition of individual particles was qualitatively

determined by energy dispersive X-ray spectroscopy. Depleted Uranium

particles frequently contained iron, aluminum, silicon, magnesium,

potassium, titanium, and tungsten as a result of contamination during

impaction and settling.”

Unfortunately, a document of such importance was never published

to the scientific community and is still very hard, if not impossible at all,

to come by, in spite of what is declared in the preface: “This report has

been reviewed by the Information Office (OI) and is releasable to the

National Technical Information Service (NTIS). At NTIS, it will be

available to the general public, including foreign nations.”

It was only in the 1990s, starting from the first Gulf War, that DU

weapons were largely and regularly employed in warfare, and it was

soon apparent that both soldiers and civilians involved in that conflict

showed unexpected health problems whose nature was unclear. The

collection of symptoms they suffered from was called “Gulf Syndrome”

since they did not fit in any already classified pathology and its origin

was attributed to various causes, from the radioactivity and toxicity of

Uranium to the improper use of vaccines and other drugs, but that was

done without any convincing scientific evidence. Much of it was due to

an emotional wave caused by the fact that some people, especially

journalists, had discovered that the Allied Forces had used bombs

containing DU, and Uranium, with all that that name recalls, looked an

excellent scapegoat and also the spectre of the diseases induced by the

radioactivity of the A-bomb resurfaced.

Uranium is dangerous if it is inhaled, ingested or its debris are

incorporated in biological tissue. It is chemically toxic and its

radioactivity can induce lethal effects in biological tissues. The effects of

the radiations emitted by Uranium can be deterministic or stochastic.

Nanopathology 166

That means that in the former case, if a threshold dose is reached, the

probability to contract a medullar aplasia or cutaneous lesions or sterility

is equal to 1; in the latter case, a malignant disease originates in a

fortuitous way without a critical dose having been administered.

Not much later, another undeclared war broke out, that time in what

remained of Yugoslavia, and DU weapons were used there as well. Like

in Iraq, also there soldiers and civilians showed symptoms of disease,

mainly, but far from exclusively, of oncologic nature, and those

symptoms were classified under the common expression of “Balkan

Syndrome”. A few mistakes were made in the epidemiologic studies that

were implemented in order to try and quantify the actual extent of what

someone described as a disaster and others as something of no

importance when non existing at all, and that delayed and then slackened

the scientific investigations. In any case, it was a matter of fact that the

collection of symptoms was not consistent with any already known

pathology and that alone was enough to stimulate our curiosity.

Stress may be blamed for disorders like fatigue, shortness of breath,

headache, sleep disturbance, forgetfulness and impaired concentration,

but cancer, diseases of the genitourinary system (GC Gray et al., 1996),

diseases of the blood and the haematopoietic organs, and some forms of

mental disorders can probably be ascribed to other causes. But also now,

long after the end of the war, final reports are delivered from the

Departments of the Veterans Affairs and new suspicions of associations

with known diseases, (but of unknown origin) are rising; among others,

for instance, with the Lou Gehrig’s disease (web ref. 1, 2001), and

unexpected deaths (web ref. 2, 2002)

Studies on cohorts of soldiers who had served in different wars had

already shown the existence of previously unknown collections of

symptoms related to the particular conflict they had taken part in.

Already in the XIX century, after the US civil war, the so-called Da

Costa Syndrome was described (JM Da Costa, 1871). Then, after the

first World War, the so-called effort Syndrome was observed, while

psyconeurosis was reported as a consequence of the participation in the

second World War. Anxiety, neurosis, panic disorders, mitral-valve

prolapse and chronic fatigue Syndrome were seen in soldiers employed

in the Korean conflict, and a post-Vietnam Syndrome was described in

War and Nanoparticles 167

veterans from the long war fought in Vietnam, but in that conflict an

important contribution was most probably given by chemically toxic

agent as Agent Orange, i.e. dioxin.

The Syndrome suffered by the soldiers who were engaged in the first

Gulf War included fatigue, headache, muscle and joint pain, diarrhoea,

frequent fevers, skin rashes, shortness of breath, chest pain, sleep

disturbance, irritability and depression, but also, in some cases, increased

birth defects among the veterans’ children. In the UK that condition was

called SSIDC, by that acronym meaning Symptoms and Signs of Ill-

Defined Conditions.

The symptoms suffered by the veterans from the Balkans were

somewhat different from those of the ones who took part in the Gulf

War, but, in that latter case, some veterans had died in a very short time.

Then, between the end of the 1990s and the beginning of the new

century, the claims that radioactivity was to be blamed grew louder and

louder. To verify the truthfulness of that claim a specific Committee

from the U.N. Environment Protection Agency checked the radioactivity

in ex-Yugoslavia on January 2001. After 340 samplings in 11 sites, only

a few of them resulted to have a high level of radioactivity apparently

linked to DU ammunitions (web ref. 3).

Though not overwhelming, evidence that DU ammunitions was not

only used (something NATO had then already admitted) but a certain

radioactivity was still present was there. Circular red lines are still

present in the ground in many areas, delimiting an area around a DU

bullet non exploded entrapped in the soil. Nevertheless, a few Italian

soldiers who were not deployed in those zones reported some of the

symptoms listed above, so the probability that radioactivity could be the

cause of the diseases grew much weaker.

Another group of people identified the cause of the Syndrome in the

toxicity of multiple vaccinations that part of the soldiers were subjected

to in a short period of time, but also for this claim there was no scientific

evidence strong enough, though that factor may not be ruled out.

The multifarious and often unexplained symptoms shown by a

comparatively high number of veterans of the Desert Storm Conflict

induced many researchers to call it a Syndrome, commonly referred to as

the Gulf War’s. As a matter of fact, the collection of symptoms was not

Nanopathology 168

consistent with any already known pathology. Also the delay in showing

up and the duration of the symptoms was something hard to explain still

today.

The studies on soldiers who had served in different wars previously

mentioned had already shown symptoms related to that particular

conflict:

1 - after the US civil war: DaCosta Syndrome

2 - I World War: so-called Effort Syndrome

3 - II World War: Psyconeurosis

4 - Korean conflict: anxiety, neurosis, panic disorders, mitral valve

prolapse and chronic fatigue Syndrome

5 - Vietnam war: post-Vietnam Syndrome (In the course of that war,

an important contribution was given by chemically toxic agent as Agent

Orange [dioxin])

6 - Gulf War Syndrome: fatigue, headache, muscle and joint pain,

diarrhoea, frequent fevers, skin rashes, shortness of breath, chest pain,

sleep disturbance, irritability and depression, but also (in some cases)

increased birth defects among the veterans’ children. In the UK that

condition was called SSIDC, i.e. Symptoms and Signs of Ill-Defined

Conditions.

There is a similarity of symptoms as fatigue, headache, shortness of

breath, forgetfulness, sleep disturbance, impaired concentration, but they

can found also in adult population under psychological stress. It is

singular the symptom of diarrhoea.

The variety of the pathologies and their intensity, that in some cases

led to death, seem to have different aetiologies. That is the conclusion of

many researchers even if many veterans have expressed concern that

their unexplained illnesses may result from their experience in the war.

Also after the end of the war in what was Yugoslavia, the soldiers

engaged there denounced a collection of symptoms that has been called

the Balkans Syndrome.

The symptoms were different from those of the Gulf War, but, in this

case, some veterans died in a very short period. After the first casualties,

somebody tried to explain why young and healthy (before the war)

soldiers died and to find a possible correlation among pathologies like

the various forms of cancer and their presence in the war theatre.

War and Nanoparticles 169

Some people, especially journalists, discovered that the allied forces

used bombs containing Depleted Uranium, and Uranium looked an

excellent scapegoat.

Uranium is a high density, metallic element that, in its natural

form, consists of three radioisotopes: 238

U, 235

U, 234

U.(2). It decays

spontaneously emitting alpha, beta and gamma radiations. This

peculiarity was sufficient to suspect that some soldiers were highly

exposed to its radioactivity due to the enormous quantity of DU

bombs used in the conflict and developed cancers and others diseases.

The correlation radiation = cancer was already been demonstrated

in people who lived in Nagasaki and Hiroshima during the second

World war.

Uranium is dangerous principally (Grandolfo et al., 2003) if it is

inhaled, ingested or debris are incorporated in biological tissue. It is

chemically toxic and its radioactivity can induce lethal effect in the

biological tissues.

The rumours of weapons containing radioactive materials dropped

in the Balkans were verified by a specific Committee from the

United Nations for the Environmental Protection Agency on January

2001. After 340 samplings in 11 sites, only three sites resulted to have a

high level of radioactivity apparently linked to the DU ammunition,

for instance Hadzici, a small village, 60 km far from Sarajevo.

(web ref. 3).

So there is evidence that DU ammunition were used and a certain

radioactivity is still present, but some ill Italian soldiers were not

deployed in those zones, so the radioactivity cannot be the cause of the

diseases.

Nanopathology 170

Another cause was identified in the toxicity of multiple vaccinations

in a short period of time, but also for this cause there is no scientific

evidence.

The Italian Commission (web ref. 4) considered 44 cases reported in

Tab. 5.1 and the first conclusion was that there was no relationship with

their permanence in the war theatre. One year later the Commission

elaborated again the data and reached a different conclusion. They found

that there was a slightly higher incidence of Hodgkin’s lymphoma and

Acute Lymphatic Leukaemia among the military people deployed in ex-

Yugoslavia or in close contact with DU ammunition in fire grounds.

Specific observations were carried out in soldiers exposed to high-

temperature combustion processes involving the blast of Depleted

Uranium weapons. Our original aim was to find Uranium nanoparticles

in the bioptic samples we received, but what we found instead of

Uranium was metal dust coming from target and bomb volatilized by the

heat generated by Uranium and re-condensed under nanoparticle form.

Inhaled and ingested nanoparticles can negotiate either the alveolar and

the digestive system walls, migrate to the blood and be carried virtually

to any organ. Sperm samples belonging to dead soldiers who were active

in war territories were referred to our laboratory and we developed a

novel technique to be able to test them. In all cases, nanoparticles were

present, while nothing was found in similar samples coming from

healthy subjects we used as reference. [See chapters 3 and 5]

Having found dust dispersed in the sperm, we checked if malformed

human and animal feti contained that particular form of pollution.

War and Nanoparticles 171

Tab. 5.1 Cases examined by the Mandelli Commission in Italy.

Pathology N. of Cases

Hodgkin’s lymphoma 12

non-Hodgkin’s lymphoma 8

Acute lymphatic leukaemia 2

Thyroid Carcinoma 3

Colon or rectum cancer 4

Larynx and Pharynx cancer 1+1

Lung and Bronchus cancer 1+1

Kidney cancer 1

Stomach cancer 1

In her capacity of coordinator of the European project on

nanopathology and consultant of the 2nd Italian Governmental

Commission, Dr Gatti had the opportunity to check a small cohort of 52

soldiers (for a total of 59 samples, because from autopsy we had more

samples for 1 subject) with our technique. The aim was to look for

Depleted Uranium inside the pathological tissues of the sick veterans.

Some of the Italian soldiers had already died before getting a clear

diagnosis, while others had developed symptoms typical of well-known

diseases (for instance, Parkinson’s or Alzheimer’s). All declared to suffer

from a common symptom: chronic fatigue, a symptom usually

complained for also by the New York fire fighters who took part in the

rescue operations of 9/11. So we analyzed autoptic specimens, biopsies,

surgical samples or “fresh” specimens as blood or sperm. (See Tab. 5.2)

In eleven of them, colon cancer had been diagnosed and the rest

suffered from non-Hodgkin’s lymphoma. In all the small specimens

inorganic particulate could be detected and its chemistry verified. The

samples were very small, and there is the possibility that further debris

included Uranium could be entrapped in other organs and tissues, but

what we found is meaningful of a certain contamination and it responds

to a logic behavior of the bombing: it creates a new “special”

environmental pollution.

Nanopathology 172

Tab. 5.2 Type of samples analyzed.

Sample No. of Cases

Bone marrow 12

Liver 6

Lymph node 8

Thyroid 3

Bowel 3

Larynx and Pharynx 2

Lung 5

Kidney 3

Spleen 3

Stomach 2

Bladder 1

Sperm 6

Gonads 2

Brain 1

Skin 2

Total 59

In none of the cases we checked could we find trace of DU debris,

either micro- or nano-sized. In many of them, instead, we detected the

presence of nanoparticles, often showing very strange elemental

chemical compositions, different from case to case. The explanation to

that phenomenon is relatively easy: As described above, when a bomb

goes off, the explosion induces a great quantity of heat and involves a

large number and variety of objects and materials. Those objects and

materials volatilize and most of the molecules they are made of break

into chemical elements that recombine, often under the form of an alloy,

as soon as the atmosphere cools down. Since all that matter is cast far

away from the spot where the explosion had taken place, it finds an

environment cold enough to allow it to recombine very quickly, in a

matter of seconds. Because of the variety of the materials that take part in

the explosion and the fact that the soldiers we checked had been

deployed in a comparatively vast territory, the alloys we found were

rather different from each other.

That finding agrees with the deployment of the soldiers. They did not

go in the same bombed places and at the same time.

War and Nanoparticles 173

Like in many other, not war-related, cases we could study,

nanoparticles were scattered in many different tissues, including red cells

(an excellent carrier), brain and gonads.

Fig. 5.1 Image of red cells spread on a plastic sheet, of a contaminated spouse, observed

under ESEM, with a white particle of Antimony –Cobalt attached (marker 5 µm).

The first of the cases we checked was particularly interesting and was

related to the 1st Gulf War (web ref. 5).

A Canadian officer with a history of amateur marathon runner and,

also because of that, enjoying an excellent physical condition, was

repatriated after a six-month stint on a wheel-chair, despite the fact that

he had never been injured, but for three days he had been highly exposed

to bombing pollution.

The symptoms since February 1991 were loss of motor control,

chronic fatigue, respiratory difficulties, chest pain, insomnia, short-term

memory loss, testicle pain, aching bones, diarrhea, and depression.

After his return he survived for 8 years. During that time he

developed symptoms specific of different pathologies, included

Parkinson’s and Alzheimer’s diseases.

Nanopathology 174

Among the unusual aspects of this Syndrome there was the change of

his eye colour, originally brown, but after 2 years turned to greyish, then

to intense blue. Then, just before his death, they turned to grey again. His

widow did not find any doctor who could give an explanation to this

strange phenomenon.

Besides his difficulty in walking, he had problems with his sexual

life, as, after sexual intercourse, his wife felt a smart burning sensation in

her vagina, which started to bleed and, in a short time, developed sores

which did not respond to any therapy. That particular condition, later on

reported by other partners of veterans when we interviewed them, was

called “Burning Semen Disease” (see also Chapter 3).

Blaming a particular chemical composition for that disease does not

seem possible, but the cases we had the chance to study so far are still

few and deeper investigations on a sufficient number of cases are

necessary to be able to say something conclusive.

The analyses were carried out on 4 different types of samples: the

lung (Fig. 5.2), the spleen (Fig. 5.3), the liver (Fig. 5.4) and the kidney

(Fig. 5.5).

In the liver we found Cobalt and Antimony nanoparticles. The

presence of Cobalt debris attracted our attention and pushed out our

fantasy, beyond the border of the present knowledge.

Cobalt is blue (Fig. 5.4). The patient’s eye became blue and all his

body contained Cobalt nanoparticles. All his body contained metallic,

toxic nanoparticles, finely disseminated. To explain the colour eye

mystery we can remind the symptoms of a genetic disease called

Wilson’s Syndrome. In this case the lack of a protein does not allow to

metabolize Copper that precipitates, mainly in the liver. One of the

symptoms considered for the diagnosis is a yellow circle around the iris.

Copper is brown-yellow in colour. Can this comparison be sustainable?

For now there is no possibility of epidemiological studies, since that was

a unique case. The particles we found in the liver were present in the

blood circulation and reached also the eye microcirculation. We found

also nanoparticles of Mercury-Selenium in the kidney. A Canadian

doctor performed analyses on his urine and he found traces of Uranium,

but in the samples we analyzed the amount of toxic particles was

sufficient to explain the symptoms.

War and Nanoparticles 175

This case was deeply analyzed since we had sections from autoptic

samples of different organs, and we could understand that the

dissemination is total, and some of the particles accordingly to their

chemistry can be trapped selectively in some organs. The disease and the

symptoms are related to this wide contamination.

Fig. 5.2 ESEM image of a solitary spherical nanoparticle of Bismuth-Chlorine-Sodium

found in the lung (marker 20 µm).

Fig. 5.3 ESEM image of a post mortem section with spherical nanoparticles of

Antimony-Chlorine found in the spleen (marker 20 µm).

Nanopathology 176

Fig. 5.4 ESEM image of a post-mortem sample of the liver. Nanoparticles of Cobalt were

found (marker 10 µm).

Fig. 5.5 ESEM image of a post-mortem sample of the kidneys. Nanoparticles of toxic

Mercury-Selenium were found (marker 20 µm).

Especially in the kidney the particles were found as singlets

(Fig. 5.5). This fine dissemination is peculiar. Until now it is impossible

to reproduce that phenomenon in an in-vitro simulation test for the

capacity of clustering of engineered nanoparticles.

We analyzed also soldiers not directly involved in the war, but who

served in peace-keeping missions in bombed sites.

The Italian soldiers’ samples we analyzed were provided by the

patients themselves or by their families. We present a selection of some

results. It is possible that if we had the opportunity to analyze other

tissues we could have found more or less debris with further

War and Nanoparticles 177

compositions, since a selective capture from an organ of specific

chemistry is possible. The soldiers we analyzed served in the Balkans in

different periods and in different areas, so the difference in compositions

of the pollution is a logic consequence. Many slept and/or worked in

destroyed buildings, other visited polluted areas or were busy destroying

weapons. It is a common procedure, after the destruction of enemy

ammunition collected inside a hole dug in the ground, to verify directly

on the explosion site, on the smoking ashes, if everything had been

actually destroyed. That is a dangerous procedure: the risk exists of

inhaling the pollution generated by the explosion. That habit was kept

also during the 2nd

Gulf War. That means that soldiers slept close to

primitive open-air incinerators, no more than holes in the ground where

they dispose of daily wastes of food, but also many other kinds of

unwanted materials, including captured weapons.

The first 3 cases (Fig. 5.6, Fig. 5.7, and Fig. 5.8) show images of

pollution inside biopsies of lungs and bone marrow of Italian soldiers.

Fig. 5.6 Image of micro and nanoparticles of compounds containing Iron-Silicon-

Phosphorus-Aluminum-Sulphur-Chlorine-Sodium-Potassium (marker 20 µm).

Nanopathology 178

Fig. 5.7 Image of nanoparticles of stainless steel inside the bone marrow (marker 50 µm).

Fig. 5.8 Image of spherical particles of Iron-Manganese-Chromium-Silicon found in a

bone marrow biopsy (marker 5 µm).

Figs. 5.7 and 5.8 show different particles found in a bone marrow

sample. They are metallic debris both irregularly and, in most cases,

round-shaped; their origin was from combustive processes at a

temperature higher than 900°C.

War and Nanoparticles 179

Fig. 5.9 Image of a cluster of nanoparticles of Iron found inside the liver (marker 10 µm).

Fig. 5.10 Image of particles of a compound of Zirconium-Sulphur-Chlorine-Calcium

found in the liver (marker 10 µm).

Figs. 5.9 and 5.10 are related to the same soldier who died after

having contracted a lymphoma. His organs were contaminated mainly by

Iron-based particles, found mainly embedded in the bone marrow. Also

Zirconium-based particles where found in the spleen biopsies after the

organ was removed.

Nanopathology 180

Fig. 5.11 Image of a debris of Antimony found in the lung (marker 20 µm).

Fig. 5.12 Image of a particle of Tungsten found in the lung (marker 20 µm).

Figs. 5.11 and 5.12 are related to the case of a soldier who had a lung

neoformation removed. Solitary particles of Antimony and Tungsten

were found inside. Those are recognized as chemically toxic materials.

War and Nanoparticles 181

Fig. 5.13 Image of a cluster of Cobalt particles inside a bladder cancerous tissue (marker

10 µm).

Fig. 5.14 Image of round-shaped particles inside a bladder cancer (marker 2 µm).

Figs. 5.13 and 5.14 show particles found in the same sample of

bladder cancer. They belong to a mine clearer who had served for 25

years in the Army and had been present at very numerous explosions for

the elimination of ammunition. For that reason, he was exposed to the

pollution created by that kind of combustion. Inside his pathological

tissue we found nanoscaled particles of Cobalt, Tungsten, stainless steel,

etc.

Nanopathology 182

Fig. 5.15 Image of a solitary particle of stainless steel surrounded by red cells (marker

50 µm).

The picture above (Fig. 5.15) is relative to a rhino-laryngeal

neoformation in a soldier affected by non-Hodgkin’s lymphoma.

Fig. 5.16 Image of a wide dissemination of debris of stainless steel inside the bone

marrow in an (marker 50 µm).

The sample of Fig. 5.16 is related to an acute mieloid leukaemia a

veteran from the Balkan War suffered from.

War and Nanoparticles 183

Fig. 5.17 Iron-Zinc micro and nanodebris inside a calcific tissue found in the liver

(marker 20 µm).

The soldier whose liver biopsy was observed in Fig. 5.17 suffered

from pancytopenia. The tissue contained a wide variety of particles with

different compositions. Calcium was detected together with Iron, Zinc,

Magnesium, Silicon, Phosphorus and Sulphur.

Fig. 5.18 Image of an Antimony-Cobalt debris found inside the sperm of a soldier who

served in a fire ground (marker 10 µm).

Hodgkin’s lymphoma was the disease diagnosed to the soldier,

now died, whose sperm we examined. It contained 800-micron sized

Antimony-Cobalt particles (Fig. 5.18). The patient was never deployed in

the Balkans but served in a firing ground in Sardinia.

Nanopathology 184

Fig. 5.19 Image of Iron micro and nanoparticles disseminated in a brain tissue affected by

glioblastoma (marker 20 µm).

Fig. 5.19 shows Iron-Silicon-based nanoparticles finely disseminated

in a soldier’s brain affected by glioblastoma. The presence of Silicon

witnesses that the particles are of exogenous origin.

Fig. 5.20 Image of a solitary debris of Lead-Chlorine-Antimony found in a

ganglioneuroma (marker 20 µm).

Fig. 5.20 and Fig. 5.21 present two solitary particles disseminated in a

ganglioneuroma (Antimony-Lead-Chlorine) and in an adrenal gland

(Mercury-Silver). The elements composing those particles are recognized

as toxic.

War and Nanoparticles 185

Fig. 5.21 Image of a Mercury-Silver-Calcium-Phosphorus singlet particle found in an

adrenal gland. (marker 20 µm).

Fig. 5.22 Image of two Strontium-Sulphur-Calcium-Sodium debris found in an adrenal

gland (marker 20 µm).

Nanopathology 186

Fig. 5.23 Image of three Copper-Iodine-Sulphur particles found in an adrenal gland.

(marker 20 µm).

Figs. 5.21, 5.22 and 5.23 show three different types of particles

found in a removed adrenal gland. They contain chemically very oddly

composed toxic materials. The pathology that affected the patient who

served in a peace-keeping mission in the Balkans was adrenal adenoma.

Fig. 5.24 Image of a 5-micron sized particle of a silicate containing also radioactive

materials like Uranium-Thorium-Yttrium-Cerium-Neodimium (marker 10 µm).

War and Nanoparticles 187

Fig. 5.25 Image of micron- and nano-sized (white arrow) debris of a silicatic

composition. The debris on the left is composed of nanoparticles found in a bone marrow

sample (marker 2 µm).

Figs. 5.24 and 5.25 show mostly nanometric debris found in the same

bone marrow biopsy that contains mainly ceramic materials, some of

them radioactive (Fig. 5.24). The patient had served in Bosnia and

developed a thyroid carcinoma with lymph-nodal metastases, and acute

leukemia. In that biopsy, we found ceramic debris containing, among

other materials, radioactive elements like Uranium, Thorium and

Yttrium. We do not know when and where the soldier was exposed, but

these materials may come from the pollution of buildings, perhaps

containing tile with radioactive glazes. After the buildings were

destroyed, a part of them was aerosolized.

Among the samples we received, there were some coming from a few

French soldiers who had taken part in the peace-keeping missions in the

Balkans.

One of those soldiers had served for six months as a clerk in an office

based in Kosovo and had developed a Hodgkin’s lymphoma because of

which he eventually died. In the biopsies we got, we found nanoparticles

composed of alloys containing Silver and Gold, among other elements

like Iron, Chromium, etc. (Fig. 5.26).

No such alloy is to be found in any metallurgy manual or, at least to

our knowledge, in any commercially available product. As a matter of

fact, Gold is very often used for its anti-corrosive properties and is never

Nanopathology 188

mixed with stainless steel, which is what the rest of the alloy looked like.

So, as happens in many cases, we guessed that that strange composition

could be the result of a combustion and the consequent interaction of the

elements occasionally present. Explosions are obviously very common in

war theatres, and explosion is a form of combustion. But the subject we

were dealing with had never been involved in battlefield actions or was

close to actual war theatres.

Fig. 5.26 Image of the cluster of nano-debris composed of Silver-Gold-Chlorine-Iodine-

Chromium,-Iron- Gold,-Magnesium-Silicon-Sulphur (marker 5 µm).

In the course of our investigations, though, we were informed of a

kind of exposure our subject might have undergone.

He had slept and eaten for two months in a truck, and that truck had

been previously used in Iraq, from where it had been moved to Kosovo

without having been cleaned.

Of course, we did not have any chance to get samples of the dust

contained inside the truck, but it is only natural that such a condition can

rouse suspicion about the possibility that that dust may have polluted the

people who had stayed there for a relatively long time. Long enough, in

any case, to get contaminated. The sleeping bags could have been

particularly interesting to check, as dust trapped there would have kept

the soldiers closely exposed to particles for many hours a day.

The small size of the particles found and their composition made their

origin clear: combustion at high temperature. But we have no answer to

the question about their origin, Balkans or Iraq.

War and Nanoparticles 189

One of the questions we wondered about was: Can only Depleted

Uranium and Tungsten bombs create nanoparticles, or is the temperature

developed by collections of more conventional weapons enough to make

them?

Thus, we carried out a simple experiment with the Italian

Governmental Commission Dr Gatti worked with.

Since the Italian soldiers stationed in Baghdad confiscated all kinds

of small armaments and bombs, put them in large holes and had them

blow, we had gravimetric passive sensors set at distances of 100, 200 and

300 meters from the hole and exposed their surface just a few seconds

before the explosion. Then let them exposed to the environment for 30

minutes and analyzed them to check for the presence of nanopollution.

In all samples we found a great quantity of nanosized dust and, in

addition to that, we detected there particles with the same, unusual

composition we had already found in the bioptic specimens of soldiers

we had had the possibility to observe. That was a simple demonstration

that very fine pollution can be generated also by “low-technology”

weapons. The next images show the pollution created during the firing of

a pool of ammunitions.

Fig. 5.27 and Fig. 5.28 show particles with their chemical

composition found 100 m away from the explosion, while Fig. 5.29 and

Fig. 5.30 represent the dust collected 200 m away.

Nanopathology 190

Fig. 5.27 Image of dust created 100 m away from the firing of a pool of weapons in

Baghdad. The big 20 micron-sized particle is a cluster of nanoparticles (marker 5 µm).

Fig. 5.28 Image of a 700 nm-sized round-shaped particle of Iron-Aluminium-Silicon

found at 100 m from the explosion (marker 10 µm).

War and Nanoparticles 191

Fig. 5.29 Low-magnification image of the dust collected at 200 m from the explosion

(marker 200 µm).

Fig. 5.30 Image of a spherical Zirconium-Sulphur-Sodium particle found 200 m away

from the site of explosion (marker 20 µm).

Nanopathology 192

Fig. 5.31 Image of another group of particles found 200 m away from the explosion site.

(marker 20 µm).

Fig. 5.31 shows debris collected on the passive sensor 200 m away

from the explosion site.

Micro and nanoparticles were found in all the three points of measure

(100, 200, 300 m). Some showed strange chemical compositions like that

of Fig. 5.31 and Fig. 5.32. The first spectrum shows the presence of Lead

together with Chromium, Iron, Silicon, Aluminium, Calcium. That

composition is the result of an occasional melting. This alloy is not be

found in any metallurgy handbook and is the occasional result of

elements present when high-temperature combustions occur. The particle

of Fig. 5.32 shows a round-shaped morphology formed by other smaller

“balls” composed of Iron, Silicon, Sulphur, Aluminium, Sodium and

Calcium.

War and Nanoparticles 193

Fig. 5.32 Image of a composite round-shaped particle composed of Iron, Silicon, Sulphur,

Aluminium, Sodium and Calcium from the explosion occurred 200 m away (marker

10 µm).

We had the opportunity to analyze the oil used in the engine of trucks

that had worked for 2 years in Iraq. That oil contained dust of different

compositions (Fig. 5.33 and Fig. 5.34). Some of the particles detected

presented the same morphologies and chemical compositions as we

found in some pathological tissues. Zirconium, one of the rather unusual

elements detected in the oil, was also present in the specimens of two

soldiers.

Fig. 5.33 Image of a round-shaped, hollow particle of Iron found inside the engine oil

used in the truck in Baghdad (marker 5 µm).

Nanopathology 194

Fig. 5.34 Image of a Zirconium bar found inside the engine oil used in the trucks in

Baghdad (marker 10 µm).

So, we observed something that probably did not need any particular

demonstration, but just common sense, i.e. that explosions cause a new

pollution contaminating the environment. For how long? Are those

particles somehow degradable? In our opinion, seen their composition,

we can hardly believe that they, or, at least, the vast majority of them,

can “disappear”, even after a long time. And that persistence applies both

to the environment and to the organism.

Once they have been released in the atmosphere, they float in the air

and can cover long distances, carried by the wind. When they eventually

fall to the ground - and rain and snow can be the occasional carriers of

particles - a gust of wind is enough to raise them again and have the

circle restart.

As to the organism, we have demonstrated that particles, no matter

how they are introduced, can escape most physiological barriers and

settle virtually everywhere. Their presence in the lymph nodes, where

some physiological waste collects, looks almost natural, but their being

in the bone marrow, in the brain and in the sperm is somewhat less

expected.

Now, having observed all that evidence, one may ask whether their

presence in the pathological tissues is actually related to the pathology?

Is their chemistry a crucial, or even just an important, factor? And do all

War and Nanoparticles 195

subjects polluted in the same way develop identical symptoms and

disease?

We try and answer those questions checking places where explosions

occur on a regular basis, i.e. in firing grounds.

Italy has a few of them, but one in particular attracted our attention

because of the rumors and the actual complaints we heard from the

people living in its vicinity.

The firing ground of Salto di Quirra is located in the middle of the

east coast of Sardinia, close to a village called Quirra, along a beach of

“uncontaminated” beauty. There are no industries worth mentioning in a

radius of many tens of kilometers and traffic is scarce. The village has no

more than 150 inhabitants.

The firing ground specializes in testing missile aiming systems (web

ref. 6) and is used not only by different Italian services but also by

private companies producing weapons and foreign armies.

A few soldiers serving there had died in the same short period of time

of Hodgkin’s disease. Among those soldiers we could analyze the case of

a 22-year old boy whose Hodgkin’s lymphoma had not been diagnosed

in time and he had died.

In a supra-clavicular neoformation we found particles of Antimony

and Iron-Copper, while in his sperm we found round-shaped particles,

about 1-micron-large particles of Antimony-Cobalt and Iron-Chromium-

Sulphur. Their spherical shape and chemical composition witnessed a

high-temperature combustive origin.

Some of the inhabitants of the village had developed forms of cancer

and the activities of the firing ground had been blamed by them as the

responsible of the diseases, and in another village of the inland called

Escalaplano, twelve malformed babies were born, but that was limited to

the period between 1988 and 1990.

By request of the authority responsible for public health of that

district, we analyzed pathological samples (see Tab.5.3) from ten of the

inhabitants of the village of Quirra, located closer to the firing ground

than Escalaplano.

Nanopathology 196

Tab. 5.3 List of the pathologies and the examined samples.

Pathology Examined sample

Melanoma Skin

Non-Hodking’s lymphoma Lymph node

Chronic Myeloid leukemia Aspiration biopsy

Breast cancer Breast

Adenocarcinoma Uterus

Thyroid carcinoma Lymph node

Lobular carcinoma Breast

In order to verify if this pollution was related to the activities of the

firing ground, a specific investigation was carried out inside the area with

the permission of the military authority and under their supervision.

At the time of the investigations, no activities were planned, so, we

collected on adhesive disks the dust already present in that environment,

touching the areas behind the missile launching pads on the ground and

on the special metallic devices that adsorb the fire.

We collected samples also from a pool under a platform where

special, big engines for rockets were tested (Fig. 5.35). The water of the

pool served to absorb the heat of the exhausts.

Fig. 5.35 Particle found attached to the wall of the pool. It is composed of Lead-

Phosphorus-Iron-Copper-Silicon-Aluminium-Sodium-Calcium (marker 10 µm).

War and Nanoparticles 197

The samples collected there contained dust with different

composition, but one attracted our attention since it contained Lead. A

specific research verified that the engine used a solid red propellant that

contains Lead. The sample was not analyzed in our laboratory, but by

RIS (Crime Scene Investigative Institute) of Parma (Italy) which gave us

the result shown in Fig. 5.36.

:

Fig. 5.36 Image of a particle of solid fuel with its spectrum. It is composed of Carbon-

Oxygen-Lead and Aluminium (marker 20 µm).

Lead was important for our study, since some patients showed its

presence in their pathological tissues.

Nanopathology 198

5.1 Civilians living around a firing ground

In some cases the patients living around the firing ground and who

developed a disease presented metallic particles, some of them Lead-

based. That presence is puzzling for three reasons: Firstly, the village

where those people live is very small, with little traffic car and without

any industry. Secondly, this contaminant is nanosized and its production

needs a very high temperature. Finally, Lead is bound to elements like

Titanium, Chromium, Iron, Silicon, Aluminium and such an alloy does

not exist in any metallurgical handbook and can only be the result of

an occasional meeting of all those elements, which is typical of a

combustion where some presences are accidental.

For these reasons, we think that there is a certain correlation

among some activities of the firing ground (rocket engines or weapon

elimination), the chemistry of the pollution generated and some cases of

the patients examined. The activities of launching missiles are controlled

and the population is alerted when a launch is planned, but the bench

tests of the rocket engines might be considered a non risky activity, so,

no precaution is taken.

There are also other specific activities we had no opportunity to

investigate, that probably were responsible for the creation of the

Antimony–Cobalt pollution we found in the samples of the soldiers.

Fig. 5.1.1 Image of a cluster of nanoparticles found in a lymph node affected from non-

Hodgkin’s lymphoma (marker 10 µm).

War and Nanoparticles 199

In the technical report of 1978, Dr. Joe A. Framer declares that

“Depleted Uranium as the most desirable candidate is based primarily

upon on its 1-high density 2- pyrophoricity 3-metallurgcalproperty, 4-

availability and 5- relatively low cost.” But after the test, he says that

“additional research is necessary to better define the physical and

chemical nature of the fragmentary depleted Uranium generated as a

consequence of its military use as weaponry. Foremost attention should

be focused on its potential for dissemination within the environment and

entry into biological systems, particularly that of man”

In addition to that, “Such specific knowledge is required for

determination of potential safety hazards associated with the respiration

and deposition of Depleted Uranium aerosol within the lungs. Particles in

the 0.1 – to 0.5 micron size range are of great concern because of high

efficiency for deposition in the lungs. This range has been defined

appropriately as the respirable size range.”

Back in 1978, nobody talked about nanoparticles and

nanopathologies, but the concept is clear.

The concern for the environment and humans caused by the effects of

explosions was already expressed almost three decades ago, but nothing

was made to control or to avoid the problem. Now we extend the

concept: All the particulate matter generated by high-temperature

combustive processes is greatly aggressive to environment and human

and animal health.

5.2 A few reflections

We are all aware that the new technologies we invented or what we call,

without much insight, progress have always a more or less important

negative side.

In this case we see that bombing creates a new environmental

pollution, sometimes nanoscaled, but always with a chemical

composition that can be very dangerous for human and animal life.

The new wars create very small bullets, impossible to see and for now

undetectable by soldiers since they have no sensors for them. But

nanotechnologies can help find the right solution to develop equipment

Nanopathology 200

sensitive enough. Through them we can develop sensors for

nanoparticles, filters to capture them, masks to avoid their inhalation,

personal protections for contamination. But for now no technology is

available or even thinkable for an environmental remediation. When the

environment (ground, vegetables, water) is contaminated, it is practically

impossible to clean it.

The effects of such contamination will be paid not only by the

populations living there, where that contamination had actually occurred,

but by the neighbouring countries as well, perhaps not directly involved

in the war but, unfortunately, pollution does not stop at frontier stations.

Wind carries particles far from their origin, but so does water through

torrents and rivers that flow into the sea. Fish eat particles along with

food and are contaminated, thus becoming a contamination carrier to

animals they are food for.

Our planet is too small to stand all that. So, before starting another

war, governments ought to be aware of the hardly soluble problems they

are triggering, last but not least among them that of the money that will

be spent in what is going to be the unsuccessful attempt to reclaim a so

polluted territory.

5.3 Bibliography

Da Costa, J. M. (1871) On irritable heart: a Clinical study of a form of functional cardiac

disorders and its consequences Am. J. Med. Sc., 61, 17-52

Grandolfo, M., Mele, A., Ferrigno, L., Nuccetelli, C., Risica, S and Tosti, M. E. (2003).

Uranio impoverito e linfomi di Hodgkin nei soldati italiani in Bosnia e Kossovo:

una possibile associazione?, Not Ist Super Sanità; 16 (7/8)

Gray, G. C., Coate, B. D., Anderson, C. M., Kang, H. K., Berg, S. W., Wignall, F. S.,

Knoke, J. D. and Barrett-Connor E. (1996). The postwar hospitalization experience

of U.S. veterans of the Persian Gulf War, N Engl J Med. 1996 Nov

14;335(20):1505-1513.

Technical report of the Air Force Armament Laboratory – Armament development and

test Center, Eglin Air Force Base, Florida, USA, From October 1977 to October

1978, Project n° 06CD0101

Web Ref. 1: www.gulfwarvets.com/gehrig.htm,

Web Ref. 2: www.gulfwarvets.com/study.htm, The Washington Post Company 4 March

2002, by Suzanne Gamboa

War and Nanoparticles 201

Web Ref. 3: http://balkans.unep.ch

Web Ref. 4: www.uranioimpoverito.it/mandelli.htm

Web Ref. 5 (http://www.umrc.ney/riordon.aspx)

Web Ref. 6: www.perdasdefogu.it/MILITARE.htm

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203

Chapter 6

Nanoparticles in the Environment and Working Places ___________

6.1 Introduction

ollution taken in a broad sense depends both on the source and

the environment. Restricting the subject to particles generated at

high temperature, their composition depends mostly on what is

being burnt and, in part, on the physical conditions at which that

combustion takes place. The environment where particulate matter is

issued affects in a determinant way its concentration and scattering. It is

only natural that, in general, a windy place will suffer less from

concentrated dust pollution than a place where the calm of the wind is a

predominant condition and, even more so, an indoor environment will be

very critical in comparison with the open air, because of stagnation. Also

atmospheric pressure and inversion phenomena influence diffusion in the

environment of that dust.

The traceability method we use takes all those factors into

consideration. A few examples are described in this chapter where

traceability has been of the utmost importance to understand some

phenomena otherwise difficult to make out. In some of those examples, a

chemical element has been taken as the marker and the guiding thread

that led us to the solution of some cases.

There are instances where the technical attempt of solving a problem

linked to pollution leads involuntarily to the creation of different,

unexpected problems that, in some circumstances, can be more serious

than the original ones. Filters used in Diesel cars (FAP, see Chap. 6.5) to

capture the particulate matter produced by the combustion of gasoil are

3

Nanopathology 204

just one possible example and are briefly taken into consideration as the

producers of a novel form of pollution. Another example is that of

incinerators, meant to solve the problem of getting rid of wastes but

causing the formation of pollutants far more dangerous than what was to

be eliminated.

Distribution of particulate pollutant is of the utmost importance and

may be expressed in terms of mass, volume, surface area or number.

Already back in 1998, toxicological studies on rat models showed that

nanoparticles are much more toxic than coarser ones made of the same

components (K. Donaldson et al., 1998) and even earlier, in 1995,

ultrafine particles were demonstrated to be able to penetrate in the

pulmonary interstitium, thus causing cardiovascular diseases (A. Seaton

et al., 1995), something that larger particles are unable to do.

As already explained in Chapter 1.1, nanoparticles are dominant in

the particle number count and are significant in contributing to surface

area, but mean little in terms of mass.

Tab. 6.1 shows this concept.

Tab. 6.1 List of the ratio size-number and surface of nanoparticles.

Diameter (in µµµµm) Relative number Relative surface

10 1 1 1 10

3 10

2

0.1 106 10

4

0.01 109 10

6

Nevertheless, it is a well-known fact that size is a critical parameter

when the point of view is that of human health, as the smaller the

particles, the easier they can penetrate the organism and the deeper they

can reach. It is another fact that the larger the surface area of those

particles, the more chemically reactive they become. And, finally, count

is also determinant, since it represents the number of impacts the

organism may undergo. So, evaluating particulate pollution in terms of

weight alone has very little significance, at least when health is the issue.

Condensation of non volatile materials from supersaturated hot

combustion gases is a process responsible for the formation of airborne

nanoparticles, the size of about 1-2 nm. Those particles can grow very

Nanoparticles in the Environment and Working Places 205

rapidly in size by condensation of low-volatility materials. It must be

remembered that inorganic, microsized particles formed at high

temperature are often fragile and can break into much smaller, often

nanosized, particles. In that case, the number of actual particles is

increased and so is their surface area, while their mass stays constant.

The following examples are presenting places and situation were

nanopollution is already unintentionally released in the environment.

This knowledge can be very useful since, being aware of the problem, we

can study precautionary measures, preventive procedures and counter-

measures.

6.2 Chimneysweeps: a historical case

“Ramazini [sic, correct spelling is Ramazzini] has written a book De

Morbis Artificium. The colic of Poictou is a well known distemper; and

every body is acquainted with the disorders to which painters, plumbers,

glaziers, and the workers in white lead, are liable: but there is a disease

as peculiar to a certain set of people, which has not, at least to my

knowledge, been publicly noticed; I mean the chimney-sweepers’ cancer.

It is a disease which always makes its first attack on, and its first

appearance in, the inferior part of the scrotum; where it produces a

superficial, painful, ragged, ill-looking sore, with hard and rising edges:

the trade call it the soot-wart. I never saw it under the age of puberty,

which is, I suppose, one reason why it is generally taken, both by patient

and surgeon, for venereal; and being treated with mercurials, is thereby

soon and much exasperated. In no great length of time, it pervades the

skin, dartos, and membranes of the scrotum, nod seizes the testicle,

which it enlarges, hardens, and renders truly and thoroughly

distempered; from whence it makes its way up the spermatic process into

the abdomen, most frequently indurating and spoiling the inguinal

glands: when arrived within the abdomen, it affects some of the viscera,

and then very soon becomes painfully destructive.”

That is how Dr Percivall Pott (1819) [The Chirurgical Works of

Percivall Pott, F. R. S. Surgeon to ST. Bartholomew’s Hospital with his

last corrections. To which are added, a short account of the life of the

Nanopathology 206

Author, a Method of curing the Hydrocele by injection, and occasional

notes and observations by Sir James Earle, F. R. S. Surgeon

Extraordinary to the King, &C. First American, from the last London

edition. In two volumes. Vol. I. Philadelphia: Published by James

Webster, No. 24, South Eight Street. William Brown, Printer. 1819] in

one of his numerous texts on this subject, starts to describe a sort of

cancer that had affected chimneysweeps for centuries and for which no

therapy had been found.

The chimneys built in North Europe especially in the XVIII century

had a very small diameter and followed long and tortuous courses. So,

people with a very slight build and a nimble body, and scantily dressed to

reduce their bulk, were needed to clean them and, in fact, children were

usually employed to do that job. And, already in their early adolescence,

a non negligible number of those young workers contracted scrotum

cancer.

What Dr Pott did was to perform a surgical ablation of the tissues

involved, but, unfortunately, to no avail, thus inflicting further sufferings

and pain to his patients.

Already in 1775 Pott had realized that soot was responsible to the

disease, in this way admitting that some “poison” could induce a cancer.

The practical problem was solved in a very pragmatic way by the

Danish government that obliged all chimneysweeps to take daily baths,

thus avoiding a too long exposure of the skin (children worked all but

naked) to the soot that smeared their body. Great Britain followed later

with a law that forbade the employment of children under eight years of

age and imposed weekly baths (G. Paganetto. 2001)

The same type of disease was observed at the first half of the 20th

century in mule spinners in cotton textile factories, where they were

exposed to shale oil, which was used as a lubricant for cotton spindles.

Between 1920 and 1943, 824 out of the 1,303 skin carcinomas identified

in textile-industry workers affected the scrotum (S.A. Henry 1943).

In both cases, for the onset of the pathology polycyclic aromatic

hydrocarbons (PAHs), particularly some 3-, 4- and 5-ring PAHs such as

benz(a)pyrene and dibenz(a,h)anthracene were blamed.

Two things are particularly noteworthy: An environmental agent was

recognized as cancerogenic, and effective laws were passed on the sole

Nanoparticles in the Environment and Working Places 207

basis of medical observations without waiting for a complete theoretical

explanation of the physical, chemical and biological mechanisms

involved, the three of them then completely missing.

6.3 Welding metals

Welding is used to join different metal parts by having them coalesce,

and that is done by melting them. Many different energy sources can be

used to carry out that process, and, among them are gas flame, electric

arc, laser light, electron beam, friction, and ultrasound. All those

methods generate particles that scatter in the environment. Their shape is

generally spherical and their size depends mostly on the temperature at

which the procedure has occurred (Fig. 6.3.1). Most of those particles are

composed by a thin, crystalline skin, are hollow inside and are very

fragile.

Fig. 6.3.1 Image of a hollow particle obtained by laser welding (marker 20 µm).

Nanopathology 208

Even a sight collision can break them up and the fragments thus

originated become particles in their turn. Those particles are obviously

irregular in shape and smaller then the objects they come from, growing

that way more numerous and more aggressive. Many of them are likely

to be nanometric in size, so a single microparticle may give birth to a

high number of nanoparticles (Fig. 6.3.2 and Fig. 6.3.3).

It is only natural that welding is more critical from the point of view

of the health impact it may have, when it happens in a closed space, and

among the effects the dust produced can cause is its being trapped in the

welder’s clothes. Besides rather uncommon circumstances, those clothes

are eventually handled, often to be washed, by the welder’s wife or, in

any case, by somebody who is not the welder himself and it is not rare

the case when that person, never directly involved in the workman’s job,

falls ill with the same occupational diseases that typically affect welders.

Fig. 6.3.2 Image of an agglomeration of spherical particle of Iron-Copper-Zinc-Silicon-

Sulphur-Calcium found on the ground around a welding site (marker 5 µm).

Nanoparticles in the Environment and Working Places 209

Fig. 6.3.3 Image of spherical Zinc debris collected by air filtration during a flame

welding (marker 10 µm).

Some of the particle have a submicronic size and, if inhaled, can pass

through the lung barrier and enter the blood circulation, causing a

chemico-physical pollution. Also the microsized particles that have been

blocked in the alveoli because of their large size and the weakness of the

aggregation bounds can degrade along the time. Or, better, the material

that induced their aggregation degrades, releasing all the nanosized

debris, that, being so small, have a high probability to pass through the

lung barrier. That means that pollution can have a delayed effect,

practically impossible to predict.

In the case of these workers, it is mandatory that they use an

effectively protective mask when at work; that they wear overalls that are

never brought back home but are washed at the working place; that they

do not eat in the working place; that they change their cloth when they

eat.

Many working places are potential sources of pollution. So, a

dedicated risk analysis is necessary, along with the proper education of

every single worker. In most cases, the protective measures necessary

are simple enough and even inexpensive.

Nanopathology 210

6.4 Toner

Nowadays printer toner is widely used in offices and even in household

environments. No particular attention is usually reserved to it, but when

there are many printers gathered together in the same room, black dust is

often visible on the furniture and on the machines themselves. We had

the chance of checking a few toners made by different producers and

saw that the Carbon granules contain metal micro and nanoparticulate

whose composition depends on the effect the manufacturer wants

to obtain. Fig. 6.4.1, Fig. 6.4.2 and Fig. 6.4.3 show Carbon-based

spherules containing respectively Iron, Titanium-Tin-Antimony-Silicon

and Titanium-Strontium-Sulphur nanoparticles.

Fig. 6.4.1 Image of dust of toner (Olivetti) composed of tiny Iron particles contained in

larger Carbon-Oxygen particles (marker 5 µm).

Toner can be dangerous particularly when the cartridge is being

changed, and especially so if that operation is made in room without any

proper air aspiration. Repeated exposure, like the one undergone by

technicians assigned to service of photocopiers, may be particularly risky

because of accumulation of this kind of dust.

Nanoparticles in the Environment and Working Places 211

Fig. 6.4.2 Small Carbon-Oxygen particles contaminated by Titanium-Tin-Antimony-

Silicon (marker 5 µm).

Fig. 6.4.3 Coloured toner containing Titanium-Strontium-Sulphur nanoparticles (marker

10 µm).

Nanopathology 212

6.5 The particulate active filter or FAP

Because of a more favourable taxation as to gasoline engines come into

force decades ago, Diesel cars have grown very popular in Europe. One

of their drawbacks, though, is the great quantity of particles they give

out, a major problem in European cities where pollution has already gone

beyond any sustainable limit, and PM10 may not exceed by law a certain

mass per cubic metre (now, in 2007, the limit is 40 µg/m3), a mass limit

that will become smaller and smaller along with the approval of new

rules.

In the year 2000, a filter invented the year before, meant to capture

that dust, started to equip some French cars. Owing to its capability of

having particles coalesce, Cerium oxide contained in a small tank is let in

the filter, so that the dust becomes coarse enough to be detained. In order

to avoid obstruction and keep the filter clean, every 300-400 kilometres

the system breaks up what has been captured and emits gases and

ultrafine, mostly Carbon-based, particles from the tailpipe. Those

particles contain inorganic nanodust and are much finer than the

relatively big ones produced originally by the combustion in the engine.

That “cleaning” operation occurs when the car is running at high enough

speed for a comparatively long time, so, presumably, not in urban areas,

but nevertheless, from the nanopathological point of view, the inorganic

dust let free is far more dangerous than the one that comes out of cars

that are not equipped with that device and, because of its size, it can stay

suspended in the atmosphere a long time and travel very long distances.

Another problem is Cerium, an element belonging to lanthanides

whose toxicity has not been exhaustively investigated but which is not

negligible, which is excreted from the organism rather slowly when in

ionic form (but, as explained all over this book, particles behave in a

different way) and which we start finding under particulate form in urban

surroundings. Fig. 6.5.1 shows a Cerium particle detected in the

environmental pollution of Mantua, an Italian 48,000-inhabitants town.

In the case of the Cerium particles of Fig. 6.5.2, they contain also smaller

Platinum particulate that may be used as a marker indicating a particular

source of pollution.

Nanoparticles in the Environment and Working Places 213

Fig. 6.5.1 Image of a Cerium particle found on a trafficked street in Mantua (Italy)

(marker 2 µm).

Fig. 6.5.2 Platinum nanoparticles contained in a larger Cerium particle (marker 500 nm).

Such pollution was unknown until a few years ago. In fact, we

performed a similar analysis on a trafficked street of two North-Italy

streets, but the dust we found, in spite of its complexity, did not contain

any Cerium.

Nanopathology 214

One of the elements we found was Silver in particle form, maybe

coming from the coating of low-cost catalytic mufflers (Fig. 6.5.3).

During the periodic ignition and killing procedures, the muffler is

subjected to heating and cooling cycles that cause the detachment of the

Silver coating and the expulsion of the debris in the environment.

Environmental pollution can depend on current technology and,

therefore, can change along with progress. An example of that can be

that of Sapporo (Japan) where snow tyres with steel spikes where largely

used. The wear debris of those spikes was related to an increase of lung

cancer incidence and, out of prudence, those particular tyres were

forbidden. Epidemiologic studies will tell if that measure was indeed

effective.

Fig. 6.5.3 Micro and nanoparticles of a Silver compound released by cars in a trafficked

street (marker 20 µm).

From Lavoisier’s principle’s point of view (the total mass of an

isolated system is unchanged by interaction of its parts), it is obvious that

no inorganic pollutants is disposed of, particles are made smaller and, as

a consequence, more penetrating and, in addition to that, a new element,

in this case Cerium, is introduced into the mass of pollutants.

What is surprising is that this aspect of the problem has never been

taken into consideration and cars equipped with those devices are

allowed to circulate when car traffic is forbidden by municipal

authorities because pollution limits have been exceeded.

Nanoparticles in the Environment and Working Places 215

If more sensible, more adherent to science, regulations were enforced,

devices like that would have never been proposed by industry.

6.6 The environment around a foundry

In January 2006 we had the chance to check the territory around a huge

steelworks at the outskirts of Udine, a medium-sized town in North Italy

(95,000 inhabitants).

As it often happens in similar circumstances, the people leaving in a

radius of a few kilometres from the plant had complained for years about

the poor quality of the air, the ubiquitous presence of dust that made it

impossible to hang out the washing and forced them to keep their

windows constantly closed, and the fact that fruit trees and greens

withered and died. An unusually high rate of cancer was also unofficially

reported, but, not unlikely what happens in most similar cases, no

epidemiologic evidence (or, to be sure, serious research) existed to

support that widespread persuasion.

Fig. 6.6.1 Image of micro and nanoparticles of an Iron-based compound found in the

vicinity of a foundry (marker 5 µm).

Fig. 6.6.1 and Fig. 6.6.2 show some examples of round-shaped

pollutants found in the environment.

Nanopathology 216

Fig. 6.6.2 Image of an Iron-Manganese particle (marker 10 µm).

The environment around the factory was full of the material worked

inside and dispersed in the environment through the chimneys. Dross

was also heaped outside, that contributed to disperse particles in the

environment (Fig. 6.6.3).

Fig. 6.6.3 Images of the two morphologies of the Iron-based particles released in the

environment. The first is hollow but filled with smaller spherical debris, the second one is

an aggregation of very small debris on an Iron sphere (marker 5 µm).

Nanoparticles in the Environment and Working Places 217

So, the municipality of Udine together with those of three

neighbouring small towns interested by the same problem asked us to

carry out an investigation limited to ten specimens sampled inside and in

the vicinity of the plant.

Unfortunately, the person in charge of the factory did not allow us to

enter the premises where metals were actually worked nor to take

samples from the dross that were heaped outside the buildings. So, we

had to be content with what we could gather in the air, on some

vegetables and a few standing objects within about 800 meters from the

area of the plant.

What we found was a huge quantity of micro- and nanoparticles

ranging from 0.1 to 20 µm, in many cases spherical in shape and with an

elemental chemical composition mainly based on Iron, but containing

also other elements.

Failing a way prescribed by law to characterize particles and in

presence of an undeniable pollution, car traffic is generally the source

blamed.

In this particular case of Udine, the spherical shape prevalent in the

particles, the fact that those spheres were hollow, the nanometric size of

most of them and the unusually high content of Iron, in that case

considered as a marker, made it evident that the origin could not be

traffic exhausts. Non spherical, nanometric particulate matter was also

present, and also in that case, due to its size and composition where Iron

was again predominant, blaming cars did not look reasonable. Besides, in

the case in point, traffic was not particularly heavy and no other industry,

big enough to justify such a degree of pollution, stood in the territory. It

must also be considered how fragile hollow particles are and how the

breaking fragments inevitably released in the environment are nothing

else but irregularly shaped nanoparticles.

That the particles detected were formed at high temperature is further

proved by their composition. Just as an example, in one sample we saw

the presence of 12 elements (C, Fe, O, Zn, Si, Ca, Mn, S, Al, Mg, Cl,

Cr), while in another one, the elements were as many as 14 (Fe, O, Ca,

Si, C, Al, Mn, Mg, Cr, P, K, S, Ti, Zn). In both cases- but similar

conditions are present in the majority of the particles examined - those

elements are alloyed and the alloy is formed in a fortuitous way,

Nanopathology 218

according to the elements present in the crucible that vaporize and

subsequently combine as soon as they go up and find a cooler

environment, a fact, this, shared by all similar cases like, for example,

those that pass in the course of explosions.

A comparatively small number of particles were also detected, whose

origin was most probably different from the former. Calcium, Potassium

and Magnesium are typically found in the soil and it is only natural that

some particles thus composed are airborne and are detected among the

others. And it is as likely that traffic, however scarce, is responsible for a

fraction of the pollution present in that territory, though with a particulate

which is not the same as the one generated by the steelworks.

All samples were taken in places where dust had the time to

accumulate, with the exception of a sample, which was obtained sucking

air for 45 minutes in a place 200 meters away from the factory. Also in

that case a great quantity of Iron-based micro and nanoparticles were

filtered, most of which were spherical and clustered like those harvested

elsewhere.

Fig. 6.6.4 shows a fully contaminated acarus found on a leaf picked

about one kilometer away from a foundry. Also its mouth (Fig. 6.6.5), a

sort of open door to its organism, is surrounded by the metallic debris.

Unfortunately, only the first part of the investigation could take place.

What should have followed to come to a meaningful and much more

useful conclusion, i.e. a research on biopsies of people suffering from

diseases likely to be due to particulate pollution, could not be carried out

because of pressures that convinced the municipalities not to pursue the

matter further.

Nanoparticles in the Environment and Working Places 219

Fig. 6.6.4 Image of an acarus found on a leaf. It is widely contaminated by the Iron-based

particles (marker 500 µm).

Fig. 6.6.5 Image of the mouth of the acarus at higher magnification with the chemical

composition of the homogenous pollution (marker 100 µm).

Nanopathology 220

6.7 The environment around a power plant

The Italian river Po flows into the Adriatic Sea forming a delta, and that

delta was transformed into a regional park. Between 1970 and 1984, an

electric power station was built on a small island called Polesine

Camerini situated in those wetlands, and the combustible it used was

heavy oil at a yearly rate of 3 million tons.

The not many people living in that territory started soon to complain

about occasional oil fallouts and, in a relatively short time, about some

health problems that they ascribed to the pollution generated by the

power station.

Their suspicion was impossible to prove, as no epidemiological study

had ever been carried out and the population was too small to justify

such an undertaking. In addition to that, epidemiology is often tainted by

mistakes in what should be the population taken as a reference, as in

many cases that population is living in conditions that are not the ones

where contamination is absent.

In 2005, together with other experts in fields different from ours, we

started to investigate the problem on behalf of the cognizant criminal court.

Our idea was to cover the whole course the possible particulate

pollutants, if any were present, had followed.

We had, then, the possibility to check a certain number of samples

that were suspected to contain micro and nanoparticles with the obvious

aim of discovering if the power plant could be blamed for the pollution

of the park it was accused of. The samples had been collected years

before, when the plant was fully operational, since at the time of the trial

it was closed.

The first specimen we took into consideration was a cotton singlet

which had been washed and then hung to dry in the open. After a short

time, yellowish oily drops had fallen on that garment, leaving easily

visible spots.

Different varieties of oil had been used in the station, according to

their content of Sulphur, and we analyzed all of them according to our

method. Then, we checked the different ashes deriving from combustion.

Lichens are considered excellent biological indicators and we check

numerous samples of those vegetables. We also received dried salad

Nanoparticles in the Environment and Working Places 221

leaves picked in the territory, some of them grown outside and some

inside greenhouses. Those grown indoor did not carry particulate

pollutants, unlike those grown outside.

Another kind of specimens we checked were the particles captured by

the filters of the devices used by the local agency for the protection of the

environment.

The following images (Fig. 6.7.1) show the content of the oil

polluted by Lead, Iron-Sulphur, Barium-Sulphur-Strontium and Silicon-

Aluminium-Sulphur-Iron and some Vanadium-Sulphur compounds. Part

of them remains in the ashes, but part is released through the chimneys to

the environment, with a chemical composition that a combination of

these elements.

Fig. 6.7.1 Image of a drop of oily fuel. The oil contains Carbon-based material and also

different particulate matter, for each of which the relative spectrum is shown (marker 100 µm).

Nanopathology 222

After having been burnt, the elements contained in the oil may

recombine in particulate form. In addition to the spectra shown, small

quantities of elements such as Vanadium, Calcium and Bismuth were

also present and, as a consequence, were found in the ash (Fig. 6.7.2 and

Fig. 6.7.3).

Fig. 6.7.2 Image of the ash with its chemical composition. It contains mainly Barium

sulphates, Vanadium-Calcium-Iron-Nickel-Strontium-Magnesium-Silicon (marker 400 µm).

Fig. 6.7.3 Image of a particular of the ash. The particle is round-shaped and is composed

of Iron-Sulphur-Vanadium-Magnesium-Aluminium-Silicon (marker 20 µm).

Nanoparticles in the Environment and Working Places 223

The flying ash is particularly light and is easily carried by the wind.

For that reason, it can be found in the environment, including vegetables

like, for example, lichens (Fig. 6.7.4).

Fig. 6.7.4 Particulate pollution in dry lichens (marker 10 µm).

In the dry lichens we analyzed, we found a great number of particles.

Most of them were spherical, as is typical of a combustive origin. The

spectra of Fig. 6.7.4 shown two different types of particles: the former

contains mainly Sulphur and Iron with a ratio similar to that found in the

oil, along with other elements in a much smaller quantity. The latter

contains a mixture of many elements like, for instance, Silicon, Lead,

Titanium, Iron, Zinc, etc.

Nanopathology 224

Fig. 6.7.5 Round-shaped hollow debris made of Iron, Aluminium, etc. found in a lichen

(marker 5 µm).

Fig. 6.7.6 Sulphur-Iron-based nanoparticle cluster found in a lichen (marker 2 µm).

Fig. 6.7.5, Fig. 6.7.6 and Fig. 6.7.7 show single and composite round-

shaped particles found over the lichen surface. Though they have

different chemical compositions, they contain a combination of the same

elements present in the oil.

Nanoparticles in the Environment and Working Places 225

Fig. 6.7.7 Round-shape particle containing, among other elements, Vanadium (marker

10 µm).

The lichens examined contained particulate whose composition

recalls that of the elements found in the oil. The same elements have

been combined in a different way as a consequence of combustion. It is

important to remember that the power plant was placed in a regional

park, close to the sea and rather far from any other pollution source.

Besides lichens, we analyzed also some lettuce on which we found

Sulphur and Iron in the same ratio as in the oil and in the particles

detected on the lichens. It is only natural that the lettuce grown in

greenhouses, which we examined as well, was free from particulate

contamination (Fig. 6.7.8).

Nanopathology 226

Fig. 6.7.8 Spherical particles on a lettuce leaf, containing Sulphur-Iron and Sulphur-

Barium (marker 20 µm).

On the basis on the evidence described, along with that of other

experts, the criminal court condemned to damages the property of the

plant and sentenced its president and its manager to imprisonment

(suspended sentences).

On behalf of the criminal court, we checked about thirty cases of

cancers suspected to be related to the power-plant’s pollution. Not all the

specimens we received were particularly useful for our kind of

investigation, since they were not taken from the interface between

cancerous and healthy tissue. In the case of pathological lymph nodes,

particles are inside their structure and, for that reason, we did not meet

any problem. In ten of those samples, we found pollution compatible

with the combustion of the oil previously checked.

In Fig. 6.7.9 particles are shown with the same Iron-Sulphur ratio

detected in oil, lichens and lettuce.

Nanoparticles in the Environment and Working Places 227

Fig. 6.7.9 Debris detected in a supra-clavicular lymph-node in a patient living in the

power-plant’s area, suffering from Hodgkin’s lymphoma (marker 20 µm).

The same kind of Iron-Sulphur particles were found in dust fallen on

car bodies. That dust induced corrosion of the metal parts. The obvious

question is: If those particles are aggressive enough to corrode a car, can

they be accepted by the organism?

6.8 The case of a ship

A ship is a sort of maze made up of an unbelievable number of rooms.

Some of them are enormous, some are very small. Some received an

abundant supply of air directly from the atmosphere, some, and those are

the majority, are ventilated only through air which is forced there

through a complex network of pipes.

One of our investigations concerned a ship under construction and we

had thus the possibility to observe men at work, the particulate pollution

they produced and how those particles behaved in such an environment.

Samples were taken close to welders busy in a huge shed placed in

the dock yard. As could be expected, the vast majority of the particles

were made of Iron and were spherical. A very similar type of particles

we found in the air inside the ship, where workmen were doing the same

job as their colleagues in the shed, and those particles had been captured

by the bulk insulators and the varnish used to paint the walls of the

Nanopathology 228

rooms. Obviously, those particles may be slowly released in the scarcely

ventilated room and be a continuous source of pollution.

6.9 Incinerators

We would have much liked to study the pollution generated by

incinerators, but that proved all but impossible. The reason is not a

technical one, but is due to the huge economical interests involved in the

building and running of those systems used to dispose of urban and

industrial wastes, interests that involve powerful lobbies of builders,

businessmen and politicians that oppose any form of serious control.

Those lobbies describe incineration as a way to have waste disappear, but

such a claim is in jarring contrast with the law of conservation of matter,

that states that the mass of a closed system of substances will remain

constant, regardless of the processes acting inside the system, provided

no nuclear reactions take place. That means that matter may change

form, but cannot be created or destroyed, a notion that was already

familiar to Greek philosophers of the fifth century B.C. and was

experimentally demonstrated by Antoine Lavoisier in 1786.

It is hard to think that burning tons and tons of matter every day,

whatever that matter is made of, produces just steam, Carbon monoxide,

Nitrogen oxides and little more. Besides organic pollutants as dioxins,

furans, polychlorinated biphenyls (PCB), polycyclic organic matter

(POM) and a very long list of further pollutants, many of which probably

unknown, non existent in the original load but the result of chemical

transformations, mainly oxidations, and depending on what is being

burnt, incinerators process virtually all sorts of materials and many

components of those materials are of inorganic origin. Since chemical

elements cannot be transformed, at least in the working conditions of an

incinerator, into other elements and experience proves that combustion,

when occurring in the presence of inorganic materials, generates

inorganic particles, it is evident that those particles must be taken into

consideration when dealing with the fumes coming from waste

incineration. Those particles can either form directly where combustion

occurs or farther than there, in the latter circumstance beyond the

Nanoparticles in the Environment and Working Places 229

filtration system any incinerator is equipped with. In the former case, it

must be kept in mind that the majority (not in mass but in number) of the

particles generated are most probably nanometric in size and escape any

possibility of capture by the filters used, hardly mattering what filters are

made of. In the latter case, what passes through the filter has not yet

condensed into particulate and, for that reason, the filter is hardly

effective. In both cases, even in the event the filter has trapped all the

nanodust generated upstream, something that waits to be proved in an

indisputable way, what the fate of that material is remains dubious and

the subject is always dealt with in an ambiguous and far from exhaustive

way. As a matter of fact, that dust is somehow, sooner or later,

introduced into the environment. As an example, activated charcoal may

be cited. Because of the high surface area of its granules, activated

charcoal is sometimes used as a filter, but as soon as it grows no more

usable because it has already captured all it could, it is thrown into the

incinerator the way any waste is, thus introducing the dust it had trapped

into the cycle again.

In a way, incineration as a means to dispose of waste is a sort of naïve

conjuring trick. It is a fact that most of what is burnt is not visible any

more, but Lavoisier teaches us that nothing has actually disappeared, but

must have been transformed into something else. And that something

else is heavier than the rubbish we did not want to see. The reason for

that is that burning means oxidizing, and, in the case in point, oxidizing

means involving atmospheric Oxygen in the reaction, i.e. adding mass.

But more mass is being added, as, depending on the different techniques

used, chemicals like, for example, bicarbonate, lime and ammonia, are

employed for various reasons. Water is also generously used to cool parts

of the plant and that water is poured in streams at a higher than natural

temperature, thus influencing negatively the habitat of plants and

animals. It is only natural that also those substances contribute to

increase the mass that results from the combustion the original waste

undergoes. So, if we consider all the addenda, what comes out of the

process weights more or less twice as much as what we wanted to get

rid of.

Setting aside organic pollutants, in most cases much more aggressive

than the original rubbish, and setting also aside what are called secondary

Nanopathology 230

particles, i.e. those that form in the atmosphere from gaseous pollutants,

particularly Sulphur dioxide, Nitrogen oxides, ammonia, and volatile

organic compounds and that can be the carriers of other particulate,

incineration produces a wide variety of particles, presumably often in the

form of alloys whose composition is actually impossible to prognosticate

because of the almost infinite assortment of materials entered into

combustion. So, guessing what their toxicity will be is impossible. All

we can say is that thinking that dust is not formed by incineration is a

preposterous notion and one of the many dangers is that particles, even if

microsized, are very likely to break up into a higher quantity of

nanoparticulate.

We had the chance of analyzing some of the ash produced by an

incinerator and scattered on the ground for a radius of many hundred

metres and we also collected dust from vegetables grown in the vicinity

of one of such plants, but we were never allowed to carry out more

systematic investigations.

Biomass like dead trees, tree branches, yard clippings, left-over crops,

wood chips, bark and sawdust from lumber mills is used in some

circumstances as a fuel to produce energy. Apart from the generation of

pollutants like, for instance, dioxins because of the presence of Chlorine

in the wood, such process is responsible for the release in the atmosphere

of inorganic particulate, since inorganic molecules and metallic elements

are contained in vegetables.

In spite of the fact that vegetable biomass used for that purpose

should not contain chemical additives, in fact, in the vast majority of

cases, those vegetables contain pesticides and evident traces of chemical

fertilizers. In other, almost as numerous cases, the timber used is the

industrial production reject of furniture, frames or other wooden objects,

containing all the chemicals used to enhance the characteristics of those

materials, chemicals that end up, in chemically and physically changed

forms, often as more toxic substances, in the fumes given off by through

the chimneys.

In many circumstances, because actual biomass coming from

comparatively short distances is not enough to fuel this kind of plants,

they are gradually transformed into waste incinerators.

Also in this case, we have never been allowed to investigate.

Nanoparticles in the Environment and Working Places 231

6.10 Tobacco smoke

About 15 billion cigarettes are smoked every day in the world, but,

though the most popular, cigarettes are not the only system to inhale

burnt tobacco, and pipes, cigars and less common systems are currently

used.

Smoking tobacco is a concentrate of particulate atmospheric pollution

because its leaves are relatively large and dust falls on their surface. In

order to be suited for consumption, those leaves must be desiccated and

that procedure makes them loose an enormous fraction of their weight.

So, dust, being not volatile, concentrates on the final product.

Smokers inhale smoke on a voluntary basis, and that smoke,

produced at a temperature of about 800°C, contains more then 4,000

known pollutants, among which are Carbon monoxide, Nitrogen oxides,

Hydrogen cyanide, ammonia, and other toxic irritants such as acrolein

and formaldehyde. Inorganic particulate is more difficult to define

chemically as it depends on fortuitous, variable circumstances like the

place where the plant has grown and dried and what was actually present

in the atmosphere during those periods of time.

Everybody, particularly a non-smoker, is aware of the fact that

tobacco smoke does not interfere exclusively with smokers but reaches

also, though in a more diluted way, the respiratory system of whom is

unwilling to breath burnt tobacco. That way, cigarettes, cigars and pipes

become the producers of environmental pollution which is particularly

perceptible and aggressive in confined rooms, and that pollution contains

suspended particles.

The following pictures show the particles of what we found in some

of the cigarette tobacco we observed and their relative elemental

spectrum.

Nanopathology 232

Fig. 6.10.1 Biological morphology of a tobacco leaf with its spectrum (marker

1,000 µm).

The elemental composition shown in Fig. 6.10.1 is the one typical of

tobacco leaves. The elements shown (Potassium, Carbon, Oxygen,

Calcium, Chlorine, Magnesium, Phosphorus and Sulphur) in the

spectrum are homogeneous throughout the whole leaf and are to be

subtracted from the following spectra.

A few pictures of particulate pollution found in cigarettes from

Sarajevo, Baghdad and the US are shown below. Some of the particles

are nanosized, while others are microsized but equally interesting as they

contain typical war pollutants, some of which are radioactive. Those

cigarettes had been produced in 2003, but we do not know when that

tobacco had been grown.

Fig. 6.10.2, Fig. 6.10.3 and Fig. 6.10.4 show the pollution on the

tobacco leaf strips in cigarettes (Drina King Size and Aura Light) from

Sarajevo after the war, in 2000. Fig. 6.10.5 represents the war pollution

in a cigarette from Baghdad, (Sumer Star King Size) in 2004.

Nanoparticles in the Environment and Working Places 233

Fig. 6.10.2 Different debris found in a cigarette from Sarajevo (marker 100 µm).

The heavy metals present in the particles are notoriously toxic and

their penetration may be enhanced by the hot surface of burning tobacco.

Fig. 6.10.3 Drina King Size Cigarette from Sarajevo (after the Balkan war). The particles

found contain Uranium and Thorium (marker 20 µm).

Nanopathology 234

Fig. 6.10.4 Aura Light Cigarette from Sarajevo with very unusual particulate:

Lanthanum, Praseodymium, Neodymium, Cerium and Samarium are present in its

composition (marker 20 µm).

Fig. 6.10.5 Sumer Star King Size Cigarette from Baghdad with particles containing

Uranium and Thorium (marker 50 µm).

Nanoparticles in the Environment and Working Places 235

Fig. 6.10.6 Cigarette from the US (Marlboro) polluted by particles containing rare earths

like Lanthanum-Cerium-Neodymium (marker 10 µm).

Fig. 6.10.7 Cigarette from the US (Marlboro) polluted by particles containing rare earths

and Lead (marker 10 µm).

The Fig. 6.10.6, Fig. 6.10.7 and Fig. 6.10.8 show the environmental

pollution on an American tobacco leaf. During smoking this pollution is

burnt and inhaled.

Nanopathology 236

Fig. 6.10.8 Cigarette from the US (Marlboro) In this case, the particles polluting the

leaves contained Gold, among other inorganic pollutants.

Nowadays world is vastly polluted by anthropic activities and sooner

or later we will realize that we have no other choice left but face the

problem instead of clumsily try to hide it the way most politicians are

doing. A political action, common to all countries, is inescapable and the

sooner the better. But, beyond politics, the problem is also a

technological one, as, in many circumstances, no available technology

can clean where we have made a mess and something must be contrived.

Our work is meant to contribute to solve this problem, showing the

presence of a very particular, infinitesimally small but highly dangerous

pollution inside Man and animals in the deepest parts of their body, and

that presence is mainly caused by Man himself. As soon as this

contamination exceeds a certain threshold now impossible to tell, and do

it in a continuous and stable way, our species is doomed to extinction.

Let us call all that catastrophism, if we like it better, but let us get up and

do something.

Nanoparticles in the Environment and Working Places 237

6.11 Bibliography

Donaldson, K. and McNee, W. (1998). The mechanism of lung injury caused by PM10 In

Issues of environmental science and technology, Ed. R. E. Hester and R. M.

Harrison, no. 10, pp- 21-32. The Royal Society of Chemistry

Henry, S. A. (1943). Cancer of the Scrotum in Relation of Occupation”, 16, Oxford

University Press,

Paganetto, G. (2001). Percorsi storici di cancerogenesi chimica. Basell Poliolefine Italia.

Ferrara

Seaton, A., McNee, W., Donaldson, K. and Godden, D. (1995). Particulate air pollution

and acute health effects. Lancet 345, 176-178

The Chirurgical Works of Percivall Pott, F. R. S. Surgeon to ST. Bartholomew’s Hospital

with his last corrections. To which are added, a short account of the life of the

Author, a Method of curing the Hydrocele by injection, and occasional notes and

observations by Sir James Earle, F. R. S. Surgeon Extraordinary to the King, &C.

First American, from the last London edition. In two volumes. Vol. I. Philadelphia:

Published by James Webster, No. 24, South Eight Street. William Brown, Printer.

1819. (http://www.mindfully.org/Health/Chirurgical-Works-Percivall-Potts.htm.)

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239

Chapter 7

Nanoparticles in Food, Cosmetics and Other Products ___________

anotechnological items are already on the market and we interact

with them everyday even if we are not informed and we are

not aware. Label on the product rarely contains the definition

“nanotechnological” or the notice “contains nanoparticles” or something

of the kind, because industries are afraid of a negative perception people

may have and the a-priori rejection of a nanoproduct.

Singular is the case occurred in Germany involving “Magic Nano”, a

protective glass and bathroom sealant in the form of a spray, which

claimed to have antibacterial activity (web ref. 1).

Within a few days, almost 100 people were hospitalized for

pulmonary problems, some of them edema. The producer Kleinmann

GmbH, a multinational industry, assured that it did not contain

nanoparticles or other nano-things, as confirmed also by Rene Zimmer of

the Federal Institute for Risk Assessment (BfR) in Berlin (web ref. 2),

(web ref. 3), (web ref. 4), (web ref. 5) and that the toxicity was due to the

propellant. No matter what the origin of the problem was, the product

had to be withdrawn in a hurry. We do not know what the truth is, but

propellants are contained in many sprays and there is no information

about other similar cases. In any case, people rightly or wrongly,

perceived that “nano” is suspicious. And that event made also the

scientists ill at ease.

L’Oreal, the French cosmetics company, invented liposomes,

nanostructures capable of crossing the derma and penetrate deeply. They

are investing a lot in these technologies and the hope is that they are

putting money also in safety, because beauty is certainly something

1

Nanopathology

240

pleasant that may be rightfully pursued, but safety is far more important.

So, biocompatibility of the nanostructures they add (web ref. 6) and their

long-term reactions must be ascertained beyond any possibility of doubt.

Matter at nanoscale level can hide surprises.

For instance, Aluminium is stable in bulk shape, but explosive

at nano-levels. Carbon nanostructures are not soluble and not

biodegradable. So, taking advantage of that characteristics, they are used

for the construction of golf balls, tennis rackets and skis. From our point

of view, these items can be considered safe, since the nanomaterials used

are trapped in the solid structure and cannot be released.

“Nanotea” by a Chinese company will increase tenfold the amount of

Selenium adsorbed with green tea through nanocapsule engineered to

bypass the acid environment of stomach and dissolve in the bowel.

Canova Activa oil contains nanocapsule-delivered chemicals in

rapeseed cooking oil that will stop cholesterol entering the bloody

stream.

Slim Shake chocolate is a powdered drink that uses nanotechnology

to cluster cocoa cells.

Among their all but infinite applications, nanotechnologies can be

applied also to create sensors for food. In fact, there is a project using

that technology to detect bacteria inside meat, doping 60-nm sized silica

nanoparticles with molecules of fluorescent Ru(bpy) dye. The scientists

of the University of Florida attached antibodies for antigens that are

present on the surface of the bacteria to nanoparticles. That can work for

Escherichia Coli bacteria present in mincemeat and the test takes only 20

min.

Nanotechnological devices have started to be used in washing-

machines, air conditioners and freezers made by two industries: Samsung

and Daewoo. (web ref. 7), (web ref. 8), (web ref. 9)

Samsung inserted a device that releases “Silver Nano” ions (Ag+)

that bind themselves with the fabric at a molecular level. “This draws out

impurities and bacteria from the clothes, leaving them completely

sterilized and totally fresh”. “Silver Nano employs the safe and sanitizing

power of silver to eradicate airborne bacteria and germs”.

Nanoparticles in Food, Cosmetics and Other Products

241

Obviously, something like nano-sized silver ions (Ag+) is a nonsense.

Does the device release nanoparticles or does it release ions? The

difference is enormous.

When the advertisement of this product line appeared for the first

time, it publicized Silver nanoparticles and nanotechnology. The toxic

activity of Silver nanoparticles is well-known (see Chapter 9) and is

being taken advantage of by Monsanto that use it in pesticides to control

the Colorado potato beetle. FDA say they do not have the authority to

regulate pesticides and that is an EPA’s job. EPA say that FDA has

responsibility for requiring a label, because potato is a food (web ref.

10), (web ref. 11).

What is occurring is that nanotechnologies, creating new products,

create at the same time new situations that are not regulated. So, when

Samsung changed their advertisement from just Silver nano into Silver

nano ions, it is possible that they did so as soon as they knew of the

possibility that the material used in the device is a pesticide.

Daewoo uses Silver particles mixed with a plastic resin. “Using Nano

Poly Technology in “Pulsator” & “Tub U” in Washing Machines many

hurtful bacteria in clothes shall be sterilized perfectly”. That is what they

claim (web ref. 9).

All that is very exciting, but we cannot exclude that there may be a

risk that we inhale Silver nanoparticles directly from our clothes and/or

dispersed in our house from air conditioners. And no studies have been

undertaken about a potential skin contamination. But further hypotheses

can be put forward. The waste water from the washing machine is

drained to the water treatment plant through the sewer system, but at

present there are no filters that can capture nanoparticles. So, there is a

real possibility that they reach the sea, through the rivers, and are

ingested by fish. The end of the story is that we can eat a material

released by our own washing machine.

That waste water can also pollute the environment, contaminating

through irrigation the grass eaten by animals as well as the vegetables

that are our food.

So, we should not be too surprised by what we found in a hamburger

bought in a shop: a cluster of Silver nanoparticles (Fig. 7.1).

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242

Fig. 7.1 Image of a cluster of Silver nanoparticles contained in the meat of a hamburger

(marker 20 µm).

We do not the actual origin of that Silver, but our hypothesis is not

unreasonable.

After some time from this finding we had the possibility to analyze

dried hay in a small Italian farm where a case of mad cow had been

reported. The hay contained the environmental pollution we usually find

in that area, but we detected also numerous Silver particles, though in a

composition containing also Magnesium, Aluminium and Silicon (Fig.

7.2). We supposed that they could come from a pesticide, but we had no

possibility to check.

Fig. 7.2 Image of small Silver particles on the surface of a hay blade (marker 50 µm).

Nanoparticles in Food, Cosmetics and Other Products

243

Silver or Silver oxide nanoparticles are already present in socks and

T-shirts, as the aversion for body smell induces industries to make yarn

with bactericidal properties.

Fig. 7.3 shows a thread coated with an already scratched Silver layer.

What is the appearance of this layer going to be after many washings? If

the metal coating is no more there, we will have good reasons to think

that probably some fish, clams and other beings have been contaminated.

Fig. 7.3 Image of a thread of a T-shirt with Silver-oxide nanoparticles (top marker 20 µm;

bottom marker 2 µm).

There are famous examples of “translocation” of environmental

pollution.

The people of Minamata City in Japan suffered from the toxicity of

Mercury eaten along with contaminated fish (web ref. 12). That Mercury

was ingested in particulate form and not as ion, but the corrosion the

particles underwent in the body had Mercury ions released and their

toxicity induced the pathology.

Nanopathology

244

Another example not yet entirely investigated is related to the Balkan

war. A refinery in Pančevo was bombed for three days and a 800-m

column of soot was created that moved also toward the American

headquarters (web ref. 13).

Airplanes took off and bombed the cloud with Silver iodide in the

attempt to make it precipitate with the rain. Unfortunately, that operation

caused the river Danube to be contaminated by this tiny, not

biodegradable pollution as well as the Black Sea, where the Danube

flows. Having fish polluted by Silver-iodide particles is a logic

probability, but nobody seems to show any particular interest in that and

no proper prevention measures are taken, except a control for Uranium

and a few other heavy metals on game and mushrooms imported to Italy

from former Yugoslavia.

Heavy-metal pollution is something we detected in 140 samples of

bread or biscuits coming from 10 different countries in three continents.

40% of them were contaminated by environmental or industrial

particulate pollution.

Nanoparticles in Food, Cosmetics and Other Products

245

Fig. 7.4 Image of Tungsten-Cobalt micro and nanoparticles detected in non-industrial

bread (marker 1000 µm, image above and 20 µm, image below).

Fig. 7.4 shows a metallic contamination in a bread sample. Because

of the particle shape, we may say that that is probably due to the wear of

some crushing or milling tools, probably from the anti-wear metal used

as coating of those machines.

In “nanotechnological” food, non biodegradable nanoparticles can be

added intentionally, for commercial, technological or aesthetic reasons.

One clear example is represented by a chocolate bar called Mars

where Titanium dioxide nanoparticles are added to avoid the separation

of cocoa and cocoa butter due to aging and changes of temperature.

Inside our body, there is no way to degrade titania nanoparticles. They

are insoluble, non biodegradable and can remain as foreign bodies inside

the digestive system or migrate elsewhere. There is no evidence that they

are eliminated with defecation and, to our knowledge, no studies have

been undertaken on the subject.

Nanopathology

246

Fig. 7.5 Image of Iron-Chromium nanoparticles in a chocolate (Mars) (marker 10 µm).

Fig. 7.5 shows nanoparticles of a type of stainless steel that were

probably unintentionally released in the chocolate during a specific

industrial manufacturing process.

Fig. 7.6 shows titania nanoparticles contained in a chewing gum.

It contains also silicates and silica micro particles added to remove

food debris from the teeth.

Fig. 7.6 Image of a cluster of titania nanoparticles in a chewing gum (marker 2 µm).

Nanoparticles in Food, Cosmetics and Other Products

247

But nanoparticles can be present also in toothpastes and even as

coatings of toothbrushes (head and bristles). Nano-UP, a Japanese

company, has a product line composed of toothbrushes (Fig. 7.7) and

toothpastes where Gold and Silver as coating and nanoparticles

respectively are used. Their toothpaste contains Silver and few coarse

Gold debris.

Fig. 7.7 Image of a toothbrush bristle with Silver nanoparticles (marker 100 µm).

It is only obvious that the presence of unwanted, toxic, not

biodegradable contaminants (pesticides, dioxin, etc.) in food decreases

its nutritive value and has a negative impact on human health. Among

the numerous contaminants that can be present, heavy metals make up a

well-known group.

Recently, with the diffusion of nanotechnology, the problem of

toxicity related to nanoparticle size (and not just to its chemical

composition) has started to be investigated (Donaldson, K. et al., 2004),

(Kipen, H. M. et al., 2005). Indeed, we can say that some in-vivo studies

suggest that nanoparticles are not inherently benign (Hillyer, J. F. et al.,

2001), and that they affect the biological behaviour at the cellular, sub-

cellular and protein levels (Donaldson, K. et al., 2001), (Zhao, Z. et al.,

2006)

The possible presence of metals as salt has been investigated in food

(Charbonneau, J. E. 2001), (Szefer, P. et al., 2003).

Nanopathology

248

So far, inorganic pollutants not as soluble salts (i.e. ions) but under

particulate form has not been investigated in depth. The previous

chapters proved that exogenous, inorganic, micro- and nanoparticles can

be found in biological and, more specifically, human pathological

tissues, in tissues affected by pathologies of unknown origin like Crohn’s

disease (a granulomatous disease of the colon) or various forms of cancer

(e.g. colon, stomach cancer).

Food could be an important carrier for “dust” since, in many cases,

row materials grow in polluted environments, more and more

contaminated by industrial wastes.

Nanotechnological food can be very useful, but also very dangerous

for human and animal life.

The information gathered will be used to set up prevention program.

Then we should study food “from the fork to the farm” and detect what

the sources polluting it are. The study should also include animal feed.

As our method allows to work on archived biological samples, “mad

cow” disease could and should be part of the study, in order to verify if

cattle were actually fed with exhausted lubricants mixed with animal

flour as is sometimes reported. A focused study could be carried out on

the poultry food in order to verify if there is a nano-contamination. If so,

the infected animals must be verified for this contamination and the

nanoparticle-virus interaction should be evaluated.

It is desirable to analyze as basic research ice carrots in the Antarctica

to check if environmental nanopollution was already present in the past

and to what extent. Hiroshima and Nagasaki destruction and Chernobyl

accident with its Cesium dust could be particularly interesting.

Then, again as basic research, it would be desirable to know how

bacteria and parasites behave and develop after having interacted with

nanodust.

It would be of great interest to study how viruses adhere to

nanoparticles and how (and if) they change, for example in

pathogenicity, after having interacted.

Nanodust seems to be teratogenic and this is an issue that certainly

deserves to be deeply investigated.

Nanoparticles in Food, Cosmetics and Other Products

249

We should also develop traps and filters to capture nanodust and

sensors to detect and measure it. Without any doubt, this will prove very

useful to nanotechnology industries to protect workers.

Then, it would be very useful to develop a network of infrastructures

to monitor nanodust in the environment, capable of alerting people in

case of danger (see, for example, what was not available at Chernobyl).

Setting up courses and schools to educate scientists and technicians in

this new, particular field of environmental science is of the greatest

importance and an urgent necessity to nanotechnology companies

producing or working with engineered nanoparticles.

Special attention must be paid to security, since nanoparticles can be

inexpensive and silent bullets to the cell nucleus and could be used as

such.

Further research directions can be the study of the so-called industrial

sterility, malformed feti in specific industrial areas, rare diseases and new

pathologies investigated, but not yet understood. Epidemiological studies

with homogenous cohorts of patients should be carried out, where,

starting from the analysis of the embedded particulate matter, exposure is

verified and the source of pollution is identified.

The results of our research have been readily applied by the Italian

senatorial commission dealing with the problem of pathologies

contracted by peacekeeping troops in former Yugoslavia. A non

negligible number of them were in the vicinity of the explosion of high-

temperature rounds (e.g. depleted Uranium or Tungsten weaponry) or

were involved in the destruction of weapons which was carried out by

heaping them up in large holes dug in the ground and having them burn.

Some of those soldiers were also close to burning oil wells. In all those

cases, they inhaled particulates produced be the combustion of large

quantities of matter and may also have ingested them. Part of those

soldiers developed diseases that can be classified as nanopathologies.

7.1 Bibliography

Charbonneau, J. E. (2001), Investigation of corrosion and container integrity in metal

food containers using scanning electron microscopy-X-ray microanalysis,

Scanning, May-Jun;23(3):198-203.

Nanopathology

250

Charbonneau, J. E. (2001), Investigation of foreign substances in food, Scanning, Jan-

Feb;23(1):51-7.Donaldson, K., Stone, V., Tran, C. L., Kreyling, W., and Borm, P.

J. (2004b), Nanotoxicology. Occup.Environ.Med. 61, 727-728.

Donaldson, K., Stone, V., Clouter, A., Renwick, L., and MacNee, W. (2001a). Ultrafine

particles. Occup.Environ.Med. 58, 211-6, 199

Hillyer, J. F. and Albrecht, R. M. (2001), Gastrointestinal persorption and tissue

distribution of differently sized colloidal gold nanoparticles, Journal of

Pharmaceutical Sciences 90(12):1927-1936

Kipen, H. M. and Laskin, D. L. (2005), Smaller is not always better: nanotechnology

yields nanotoxicology. Am J Physiol Lung Cell Mol Physiol, Nov;289(5):L696-7.

Szefer, P., Domagała-Wieloszewska, M., Warzocha, J., Garbacik-Wesołowska, A. and

Ciesielski, T. (2003), Distribution and relationships of mercury, lead, cadmium,

copper and zinc in perch (Perca fluviatilis) from the Pomeranian Bay and Szczecin

Lagoon, southern Baltic, Food Chemistry 81, 73-83.

Zhao, Z., Hyun, J. S., Satsu, H., Kakuta, S. and Shimizu, M. (2006), Oral exposure to

cadmium chloride triggers an acute inflammatory response in the intestines of

mice, initiated by the over-expression of tissue macrophage inflammatory protein-2

mRNA, Toxicol Lett. Jul 1;164(2):144-54. Epub 2006 Jan 18.

Web Ref. 1:

www.washingtonpost.com/wp-dyn/content/article/2006/04/05/AR2006040502149.html

Web Ref. 2:

http://www.bfr.bund.de/cms5w/sixcms/detail.php/7750

Web Ref. 3:

http://www.quackwatch.org/01QuackeryRelatedTopics/PhonyAds/silverad.html

Web Ref. 4:

http://www.fda.gov/bbs/topics/ANSWERS/ANS00971.html

Web Ref. 5:

www.smalltimes.com/Articles/Article_Display.cfm?ARTICLE_ID=270664&p=109

Web Ref. 6:

www.wired.com/medtech/health/news/2005/09/68683)

Web Ref. 7:

www.samsung.com/in/products/washingmachine/thesamsungwashingmachinea

dvantage/index.htm)

Web Ref. 8:

www.samsung.com/ph/presscenter/samsunginthephilippines/productnews_20040608_00

00058341.asp)

Web Ref. 9:

www.daewoo-electronics.de/eu/products/living_washing_glos.asp

Web Ref. 10:

(www.biotech-info.net/seeds.html,

Web Ref. 11:

(www.lawbc.com/fda.html).

Web Ref. 12:

(www1.umn.edu/ships/ethics/minamata.htm)

Web Ref. 13:

(www.ieer.org/reports/bombing/index.html)

251

Chapter 8

New York 9/11 ___________

n 9 September 2002, Dr Gatti sent the following message to

the Herald Tribune, as an answer to a specific request by the

editor to remind the anniversary of the Towers’ collapse with a

100-word message.

“I was in London, at Harrod’s, when I watched the Twin Towers

collapse.

The immediate slaughter was something everybody could understand,

but there was something more sadly sophisticated I thought of. All people

who survived the disaster inhaled and ingested particles of dust which

got stored in the tissues of their respiratory and digestive tracts. There

they will induce more or less slow, but often deadly, reactions, what a

European research started relatively recently calls a ‘nanopathology’.

(An innovative technique makes it possible to detect and diagnose them.)

As a scientist involved in the research, I know that New York is bound to

suffer more mourning that will pass unrecognized.”

Probably somebody remembered that letter, since a little less than one

year later we received a phone call from New York, and the person who

called was a lady who had been heavily involved in the event of 9/11.

She was one of those people who had their working place inside one of

the Twin Towers and survived the attack; but she inhaled a huge quantity

of dust and, because of that and the respiratory problems that followed,

she spent two months inside an Oxygen tent. She called to know

something about the theory of Nanopathology mentioned in the letter and

about the possible existence of remedies. So, we started to exchange

messages.

2

Nanopathology 252

After two years, she developed an angioma in her brain, she was

operated on and eventually recovered after a long convalescence, during

which she followed what looked a strange detoxification program (Web.

Ref. 1)

The Council of Down Manhattan had accepted the proposal made by

the Foundation for the Advancement of Science and Education located in

Los Angeles to apply a particular procedure aimed at detoxifying a

certain number of workers employed by the Council and exposed to

dust: firemen, sanity workers etc. That detoxification consisted of a 3-

week treatment based on physical exercise, sauna and a sort of vitamin

cocktail. Dr Gatti was invited to take part in meetings where medical

doctors and patients presented their data and discussed their experiences

and, in case, their progress. At the beginning, she was sceptical, but,

before rejecting any theories or hypotheses, knowing them well enough

is necessary, in order to be able to find out their possible weak points, if

any. So, every judgement was left suspended.

From what is a traditional scientific point of view, such a treatment

looked indeed much of a nonsense and a very poor remedy. Moreover,

we are accustomed to believing that the higher the cost, the more

effective the result is, and, in that case, the cost of the treatment proposed

was in fact very low.

The particulate pollution generated by the collapse was extremely

fine, in our opinion especially the one formed in the part of the buildings

crossed by the aircrafts that had “disappeared” there. In that particular

point, the temperature had been very high, high enough to have the

metallic structure and the airplanes vaporize and it was particularly so in

the vicinity of the blast, and that blast involved a great variety of

different things, from glass to plastics, from steel to computers.

Everything was inside disappeared from sight. But, in fact, as every

student knows, nothing had disappeared: everything had been sublimed

and transformed into something else: an aerosol whose composition

depended on the elements present in the reaction matter. That new

pollution thus generated had a huge variety of chemical compositions

that did not exist before.

The Environmental Protection Agency (EPA) workers looked for

asbestos in the pipelines of the air conditioning systems three years after

New York 9/11

253

the collapse to demonstrate a possible correlation between the diseases

become manifest and the presence of such pollution. But blaming just

asbestos is a limited way of facing the problem which is much bigger and

with more culprits than just that.

As a consequence of the collapse of the buildings, another form of

pollution was created and that was the one originated from the

pulverization of concrete, whose particles were larger in size than the

ones generated by the parts of the buildings where the planes had

entered.

The constant, persistent cough of the firemen is a demonstration of

the inhalation and entrapment of those micro-sized particles that, because

of their larger size, had not penetrated deep enough to reach the alveoli

but had remained in touch of the bronchial walls where the ciliary

activity tried, not so successfully, to get rid of them.

One of the subjects belonging to the group had started his work

September 11, 2001, but grew seriously ill next May. After such a

comparatively long time, that person developed chronic fatigue, that did

not allow him to stand up from his bed without exhausting all his

energies. A number of other, apparently not homogeneous, symptoms

like blurred vision, nightmares, depression, etc. affected him and, for all

those reasons he was unable to work. For him, like for all the other

patients, no current drug was effective against those symptoms and some

of the subjects even suffered from the side effects those drugs produce.

So, this person entered the group and underwent the “detoxification“

treatment after which he recovered and we lost his track.

During the meetings held at the place where doctors and patients

gathered and carried out their program, and thanks to long discussions

with the patients, Dr Gatti got to know interesting details about the onset

of those people’s diseases, exposure, latency period, symptoms, their

getting better and their personal feelings. In particular, a sentence of a

survivor attracted her attention: while he talked about his impressions

during the detoxification treatment, he told of his surprise when he

observed the dramatic change in colour of his own sweat which had

turned to a rich brown. None of us had ever heard of such a phenomenon.

Sweat’s main functions are regulation of body temperature and

elimination of some waste products and it was probably those products

Nanopathology 254

that had caused that reaction. As we work on inorganic debris, we

thought it natural to check if that liquid contained them and, in case,

what could their size and composition be. Had we found what we wanted

to look for in those people’s sweat, the next question was: are those

things, expelled from the organism, responsible for the partial recovery

experienced by the majority of those subjects participating in the

programme?

If the particulate matter inevitably inhaled, dispersed in the blood

circulation and in the internal organs, can induce a body reaction with

those unusual collections of symptoms, its complete, or even only partial,

elimination can eliminate or even just decrease the biological reaction.

So, we proposed to analyze the sweat of those patients and some of

them collected it during their saunas following a protocol simple enough

not to be a bother, but strict enough not to yield polluted samples. We

asked the patients to clean their skin scrubbing it with alcohol, not to use

soap, creams or other potential contaminants. They were provided with a

sterile vial where they collected the sweat drops during the sauna, mainly

from the breast and the arms, and not from the armpits.

The analyses on the sweat we got were very interesting. They

revealed that Nature is not unkind and a possibility exists to get rid of so

unusual a pollutant from the pores of the skin. When the body

temperature rises and sweat is produced, the pores open and a certain

amount of liquid is thus eliminated. In that liquid we actually found the

debris we were looking for.

The following images show the particles we found in the fluids

examined.

We present below a series of unusual chemical compositions found in

the particles detected in the sweat, that could be related to the collapse.

New York 9/11

255

Fig. 8.1 Particles of n. 1 rescue-worker’s sweat containing a particle composed of

Copper-Zinc-Gold-Chlorine-Potassium (marker 20 µm).

Fig. 8.2 Particle from n. 2 worker’s sweat containing Zinc-Lead-Iron-Chlorine-Silicon-

Calcium (marker 5 µm).

Fig. 8.1 and Fig. 8.2 show small particles found in two different

workers that present two different peculiarities. The first chemical

compound (Copper-Gold-Zinc) contains Gold and Copper, typical of a

commercial golden alloy, but in commercial alloys Zinc is never present.

Such a composition is not used in any known material. The second one

contains again Zinc but now that element is bound to Phosphorus-Lead-

Iron-Chlorine-Calcium-Silicon. The oddity is represented by the hard-to-

explain presence of Phosphorus. This can be the result of an occasional

combustion: a residual of the melting of pipelines of water-gas, window

structures, etc.

Nanopathology 256

Fig. 8.3 Particle from n. 3 rescue-worker’s sweat containing Copper-Silver-Gold-Zinc

(marker 20 µm).

Fig. 8.4 Particle from n. 4 worker’s sweat containing Iron (marker 10 µm).

Fig. 8.3 shows an 8-micron sized particle of Copper-Gold-Silver-

Zinc. It is strange to find golden compounds in the sweat of two different

workers who shared only one comparable exposure. The presence of

Gold and Silver as parts of computers, furnishings, jewels kept inside

safes as well as Copper in electrical wires is understandable. Zinc is

another element whose presence can be explained as it may derive from a

coating (zincing), but is never used to form golden alloy. That means

that this type of particles can be originated by an accidental, local,

uncontrolled combustion.

Fig. 8.4 presents a particle containing a mixture of elements

coming from two different origins. Chlorine-Sodium is the elemental

composition of a salt commonly and predominantly present in the sweat,

New York 9/11

257

so of biological origin. Iron-Sulphur, instead, with the specific

morphology observed (a sharp-cornered debris) is of exogenous origin.

The particle appears immersed in the sweat and surrounded by other

nanosized debris (Fig. 8.5).

Fig. 8.5 Image of a Sodium chloride crystal trapping other nanometric particles

containing Iron-Barium-Sulphur-Magnesium-Aluminium-Silicon-Calcium-Potassium

(marker 20 µm).

Thanks to a lucky coincidence, we could get the dust deposited on an

object on display in a shop that was located inside one of the Towers that

its owner found completely covered with dust just after the collapse, and

on a fireman’s helmet. Unfortunately, we did not have the possibility to

analyze the flying ashes that hovered over what is today called Ground

Zero and flew toward the other boroughs of New York. Those are likely

to preserve the memory of the two airplanes and the parts of the

skyscrapers where they stroke.

The following images show the dust collected on the firemen’s

helmet used for 3 months and put inside a bag together with his overalls.

Neither helmet nor overalls were cleaned. After 3 years from the

collapse, when we formulated the hypothesis that the dust on the helmet

represented the exposure the subject had suffered and caused the disease

he suffered from, he asked us to check and try to explain why his weight

had increased and why his breath had grown short. The helmet was very

dusty, and the main composition was concrete (Fig. 8.8). In that dust we

found some particles with compositions similar to those we had found in

Nanopathology 258

his sweat samples. Much of our attention was focused on golden alloys,

not common in the normal pollution, and on debris composed of

numerous elements, since this is one of the clues left by a random

combustion.

Fig. 8.6 Image of micro and nanosized debris containing Gold-Silver-Copper-Calcium-

Silicon (marker 50 µm).

Fig. 8.7 Image of particles of the dust collected on the helmet. They contain Sulphur-

Copper-Silicon-Calcium-Chlorine (marker 20 µm).

New York 9/11

259

Fig. 8.6 and Fig. 8.7 show particles detected in the dust found on the

rescue worker’s helmet. They have different morphology and size. The

bigger and sharp-edged ones are concrete, while the smaller have

different origins. Some of them present a matrix where very small

particles are embedded. The first contains noble metals that were also

present in the sweat. That should not look strange, but what is just

obvious to find in such circumstance.

When the disaster occurred, the atmospheric temperature was still hot

and many air conditioners were working. (In New York, air conditioners

are kept on even if there is no actual need of them.) For that reason, the

dust produced, much of which was very thin and behaved more or less

like a gas, entered the pipes. That transferred and probably even

concentrated that kind of pollution indoors. A problem that is somehow

similar to that is due to the rubble and remains of the collapse moved

elsewhere and disposed of in sorts of landfills. Those too were the

obvious origin of dust. Since some of those particles are still nested

somewhere, are actually impossible to get rid of and can have only been

diluted in the atmosphere, it is only natural that they can still have an

impact on the people who live in the territory.

Fig. 8.8 Debris of the concrete found at Ground Zero with the spectrum (marker 100 µm).

Nanopathology 260

Fig. 8.9 Image of round-shaped Iron-Sodium-Silicon-Sulphur-Chlorine-Calcium debris.

(marker 20 µm).

Fig. 8.10 Image of composite debris found on the overalls of the fireman. It includes

nanosized, round-shaped Lead-Iron-Sodium-Aluminium-Silicon-Phosphorus-Chlorine-

Calcium particles (marker 5 µm).

Fig. 8.9 and Fig. 8.10 show particles of compounds of Iron and Lead:

similar debris were found also inside the sweat specimens. There is a

correlation between the chemical compositions of the particles found in

the sweat and those found in the pollution.

New York 9/11

261

Fig. 8.11 Image of a round, submicronic Chlorine-Cerium-Sodium-Magnesium-

Aluminium-Silicon-Sulphur-Calcium-Iron particle (marker 20 µm).

Fig. 8.11 shows a 500-nanosized spherical particle with a strange

composition: Chlorine-Cerium-Sodium-Magnesium-Aluminium-Silicon-

Sulphur-Calcium-Iron, found on a firefighter’s fatigue dress.

Fig. 8.12 Image of New York debris. The whiter particle is composed of Gold-Copper-

Nickel-Zinc-Sodium-Aluminium-Silicon-Chlorine-Potassium-Calcium-Iron (marker 10 µm).

Nanopathology 262

Fig. 8.13 Image of a round-shaped particle containing Calcium-Lanthanum-Cerium-

Neodymium-Sodium-Magnesium-Aluminium-Silicon-Sulphur-Potassium-Iron. (marker

10 µm).

Fig. 8.12 and Fig. 8.13 show particles detected on the object found

intact below the ruins, inside a shop of sanitary fixtures by the owner. He

collected that as a memento of his destroyed activity and life. Also the

chemistry of this dust containing Gold-Copper, but also Zinc, Iron,

Nickel was related to that found in the sweat samples. Once again, it is a

random combination of elements that are not related to the melting of a

single material or object, but to the presence of many other objects in

the same crucible. In many of these spectra there is the presence of

Sodium-Magnesium-Aluminium-Silicon-Calcium-Iron, i.e. is the basic

composition of a ceramic glass material. It has been observed that during

the melting in some parts of the Towers, there was the formation of glass

debris entrapping other materials. The composition of the image of

Fig. 8.13, a spherical debris, is rather peculiar. Its spectrum contains

Sodium-Magnesium-Aluminium-Silicon-Calcium-Iron, but that spectrum

could be due to the part that surrounds it. Certainly, it contains earth

elements like Cerium-Lanthanum-Neodymium, and Sulphur. In our

experience, though not exclusively, the earth elements’ triplet signal may

be related to an environmental contamination of tobacco smoke. A more

accurate investigation about the history of the object, of the shop and of

the owner (was he a smoker?) could explain much about those presences.

In any case, nothing can be completely destroyed.

New York 9/11

263

Most of the dust we analyzed was related to concrete. In this chapter

we presented the more meaningful or unusual debris to try and

demonstrate if a passage from inside the organism to outside is possible.

We considered those strange compositions as traceability markers and

the conclusion we came to was that a passage looks possible. Can this

finding help to devise an efficient detoxification method? There is a

possibility, but a limited one, since, probably, the passage is allowed

only to the dust trapped in the fat. For the one remained in the blood or in

other tissues there seems to be no way to be exteriorized. Let us hope

that scientists and technologists are smart enough to find a solution.

8.1 Bibliography

Web. Ref. 1 http://www.nyc.gov/html/doh/html/wtc/index.html

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265

Chapter 9

The Future and Prevention Criteria ___________

9.1 The state of the art

an has always lived with nanoparticles: proteins and colloids,

when observed according to their size, may be so classified, but

proteins and colloids are organic compounds and, at least in most

cases, can be tolerated by man or even used for his metabolism.

Inorganic micro and nanoparticles are constantly generated by nature:

belching volcanoes throw out huge quantities of minerals in the form of

tiny specks of dust, fires produce particulate, sand is blown by the wind

in the atmosphere and part of the products of rock erosion caused by the

weather ends up in the air.

So, man has been confronted all along with that kind of pollution, but

very little can be said of its interactions with his organism, since, with

rare exceptions, that has always been considered as an inescapable

condition, almost a sort of background noise, wherein man is bound to

live. Therefore, little or no attention at all has been paid by medicine to

that phenomenon that in most cases was not seen as a problem.

One of the characteristics that distinguishes man from all other

animals is his unique way of using the planet where he lives. Man is the

only animal who can lit a fire, and this now apparently very simple and

harmless action has been the first source of non natural pollution, as all

combustions produce particulate matter whose composition is different

from the one of the materials it derives from. For millenniums, though,

fire was very sparingly used, and in the vast majority of applications just

to warm oneself or cook food, and the pollution generated represented

a comparatively trifling problem in a planet inhabited by a scanty

population. Energy has not been in great demand for the largest part of

0

Nanopathology

266

human presence on the Earth: Muscles, be they offered by animals or by

man, have been the primary form of energy for a very long time, only

much later boosted by natural sources like wind and flowing water, and it

was only in Europe, in the second half of the eighteenth century, with the

so-called First Industrial Revolution, that combustion started to be used

on a relatively large scale, and that was done to heat water meant to

produce steam exploited to drive machinery. A few decades later, with

the Second Industrial Revolution, coal became quantitatively the most

important source of energy, thus supplanting wood and peat as a fuel and

transforming water-driven mills into plants whose output was strictly

dependent on man’s decisions. Thus, man started to impinge noticeably

upon nature by producing through combustion particulate matter that,

when inorganic, could not be degraded.

Nowadays the sources of anthropic micro and nanoparticulate are all

but endless. Heat has become very easy to come by and very cheap, and

higher and higher temperatures can be reached with no technical

difficulties. This allows us to produce energy and materials we

commonly and often absently use in our every-day life without

considering that one of the by-products is inevitably dust, and the higher

the temperature, the tinier the particles. And what we produce nowadays

is particles much smaller than and very different from those produced by

nature.

One of the most obvious and visible responsible for this kind of

pollution is certainly car traffic. And not only with its exhaust fumes, but

also with the wear of the tyres and the brakes. Heating systems, when

using fossil fuels, are also polluters, and so are all plants that have

recourse to combustion: incinerators, cement works (often used, more or

less legally, as incinerators as well), power plants, foundries and many

factories in general. But great quantities of heat can also be generated by

the explosion of weapons, especially when they are high-technology ones

like those based on depleted uranium or tungsten, and much heat and

dust was generated in New York when the World Trade Center

collapsed.

Besides unintentionally produced, noxious nanodust, there is dust

of the same size we manufacture and whose properties, for which we

keep finding new applications, we exploit more and more frequently.

The Future and Prevention Criteria

267

Engineered nanoparticles are already used in a number of commercially

available products, while scientists and technicians are busy

experimenting new possibilities for their use.

Huge amounts of money and human resources are being invested in

nanotechnologies and great expectations, both technical and economical,

are placed in the indeed extraordinary properties of nanosized matter.

If, on one hand, one can share this enthusiasm, some prudence is

necessary if we don’t want to run the risk of being sorely disappointed.

There are a few applications, mainly in food and in medicine, that require

the introduction of nanoparticles in the organism, and that is done

without having carried out a sufficient experimentation and without

having allowed enough time to elapse to observe and evaluate the

consequences of such use. In too many cases it is taken for granted that

our organism can get rid of those particles, but no scientific evidence

does really exist to confirm such an assumption. As far as we have been

the possibility to see, we could not detect any biological mechanism of

elimination for inorganic particles once they have been trapped in a

tissue, and evidence is being collected that shows that those foreign

bodies are far from harmless. In our opinion, this should be regarded as

an invitation not to act in a hurry in the hope of outrunning one’s

competitors.

9.2 What is next?

Nanotechnologies, creating new materials and structures not far from an

atomic level, at least from the dimensional point of view, will represent a

societal revolution. But the other side of the coin inherent in every

progress may be not so rosy and it is impellent and mandatory to find it

out before the side effects are the cause of phenomena that may be hard

to tackle or even be or become irreversible.

The synthesis of new matter and the discovery of its incredible

properties excite not just the researchers involved but also businessmen

and a market always searching for new possibilities of gain.

The previous chapters showed pieces of evidence that are hard, when

not impossible at all, to deny.

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Everybody remembers how GMOs (Genetically Modified

Organisms) flopped partly because of a generally bad perception but,

above all, because research organizations and media were not wise

enough and lied on the possible effects those organisms could induce on

human bodies. So, now, great care is taken on the news released by

scientists, and this, in some cases awkward, prudence may cause a bad

perception of nanotechnologies. Booklets are prepared for the instruction

of scientists in order to alert them of the danger and teach them how to

present the subject to the public opinion. Specific European projects

(web ref. 1) of communication in the specific field are being financed

and a sort of censorship has been set up by the European Community in

order to prevent the release of news that may be unfavourably interpreted

by the public.

But if, from one side, there is a need to control information in order to

protect the investments and avoid that huge amounts of money go

wasted, from another, certainly more important, point of view, we need

to set up all the possible preventive measures we are capable of in order

to avoid risks for the workers, the end-users and the environment.

It is a matter of fact that a number of nanotechnological products are

distributed on the market without informing the consumer of that

particular origin and something like that cannot be accepted. The fear of

a flop surrounding these new technologies due to a possible bad,

emotional, reaction of the public, induces the producers perhaps not to

lie, but to issue only partial information. When, within 3-4 years, no

adverse side effects are reported, it is very likely that they will not only

reveal but even advertise the nanotechnological origin of their product.

But not to run risks in the event of something having gone wrong and

their products having caused some sort of damage to their users,

producers have no interest in furthering the research on nanotoxicity so

to be able to maintain that there is no evidence to blame their goods. One

of the dangers inherent in that kind of attitude is that the full

understanding of what is a scientific problem involving health be delayed

and a possible wide dissemination of the diseases originated by a misuse

of nanotechnology, deriving from the globalization of the market and the

dissemination of the products worldwide, go unrecognized even by the

cleverest epidemiologist. In such a scenario, what will be registered will

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be nothing more than a “normal” increase of different types of cancer,

leukaemia, lymphoma, etc. of “hard-to-explain” origin.

Another problem is the attitude of some scientists, hopefully a

minority, but in some instances an important one, at least from the

acquired prestige point of view, to yield to the allurements of money or

power. The way it happened some decades ago with tobacco, those

scientists agree to write papers that are then published even by serious

journals, and in those papers the essence of science gets twisted and, in

the most favourable of hypotheses, truth is concealed and facts are

passed over in silence. People who obtained money for their research or

people who invest their money to develop nanotechnological products,

will hardly look for side effects. So, all that is used as an alibi based on a

mistaken feeling of authority to escape from responsibilities and, in our

case, the risk is that the use of nanoproducts and its relevant exposure do

not enter among the parameters examined and are not considered in the

statistical evaluations.

What is written above must not be seen as a rejection of

nanotechnology but just a sort of warning intended to make it stronger,

more effective and much less open to attack.

A discipline that comes often into the picture is epidemiology.

Research in that field requires huge numbers of cases and a long time,

and, for that reason, a research whose conclusions are presented today

was actually started ten or fifteen years ago when the conditions, due to

the rapid evolution of pollution, were not comparable with the present

ones. If, as an example, we take into consideration a population exposed

to the pollution originating from an incinerator built according to the

most modern technology, we must know that the particulate matter it

eventually produces as a final result of its processes is probably much

finer than that produced by the older plants. So, the modern particulate’s

behaviour in the environment is much more similar to a gas than the old

one’s and limiting the epidemiologic evaluation to areas were gross

particles are likely to fall to the ground becomes meaningless and

deceptive. And equally meaningless and deceptive is considering just

respiratory, oncologic and cardiovascular diseases, neglecting all those

belonging to nanopathologies, including, for example and among others,

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Parkinson’s and Alzheimer’s diseases, diabetes, fetal malformations, etc.

On top of that, many studies are published only if they are “reassuring”

and some of them draw conclusions that should be rejected by any

serious epidemiologist or, in any case, looked at with suspicion. It is not

exceptional to find articles maintaining that increasing pollution has no

effect on the morbidity of diseases notoriously due to pollution, and even

taking for granted that that had been written in good faith, something like

that, in contrast with science and common sense, should be considered as

a false negative, waiting for more and stronger evidence.

We must keep in mind that nanoparticles behave in a very peculiar

way, unsuspected until not long ago, and probably much is still to be

learnt and psychologically accepted. Nanoparticles are sets of atoms, but,

because of their size, they have properties that are not ruled by the

atomic-physics laws. And, in more than a way, their adverse effects are

different from those of the chemicals containing the same elements but in

a form that is not that of nanoparticulate.

At certain threshold concentrations, nanoparticles have been found to

be toxic (Brayner et al.,2006), and capable of inducing oxidative stress

(RDS) to cells (Nell, A. et al., 2006), but under that critical concentration

they can present new, maybe long-term, toxic aspects never investigated

or suspected before. (Hansen, T. et al., 2006)

This reveals the particular physico-chemical aspect of inorganic

nanoparticles. In fact, in addition to the particle chemistry, their

inorganic nature and particulate structure makes them respond in a

special way in a biological environment. They have greater densities

(and greater inertia, in a world small enough to be dominated by

Brownian motion), high electron densities (the response to light is very

different and this is the basis of the remote photoablation), they have

strong redox potentials, they have a marked thrombogenic activity, etc.

Some of them have another peculiarity: they are not soluble and not

biodegradable, so, when they penetrate the organism, since it is likely

that we have no elimination mechanisms made available by nature, they

stay there forever.

An example of such a behaviour can be found in silicosis. Inhaled

silica microparticles are attacked and incorporated by macrophages that

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try to digest them but, because of their absolute non biodegradability,

they do not succeed. So, when those macrophages die, they are

“digested” by the organism the way all macrophages are. Not so their

content which is indestructible and is released again in the biological

environment to be, in case, attacked by another macrophage. This

continuous, repetitive behaviour causes a state of disease. Single

nanoparticles represent a very small stimulus to cell, too small to be

recognized by the membrane sensors and activate the normal cell

defences (the activation of the macrophagic reaction), but they disturb

the cellular metabolism.

So, the presence of inorganic nanoparticles in biological media, even

without being directly toxic to the cell, may cause environmental stress.

Cells are very sensitive to signals from the environment. For

example, cellular reproduction is regulated by complex exterior signals

and synchronized with the neighbouring cells. In the presence of a stress

(i.e. a non expected modification of the environment), the cell stops

temporarily its reproduction cycle until it re-adjusts to the new situation.

The responsible for that are the Stress-Activated Proteins (SAP), kinases

which transmit the alarm signal to the cell. If this process does not

function properly, erroneous cellular duplication may be induced, which

might result into tumour cells.

In addition to that, nanoparticles can be a reservoir of perturbing or

toxic cations which are slowly released and induce noxious effects in

tissues. Furthermore, nanoparticles may modify ternary structure of

proteins, changing their functionality. In addition to that, they can be not

recognized as self, and give an immunological disease.

Prion-related pathologies as Creutzfeldt-Jacob’s and amyloidosis as

Alzheimer’s are classified as conformational diseases where misfolded

proteins induce nearby proteins to change their conformation. Moreover,

the biodistribution of the molecule attached to a nanoparticle can be very

different from the monomeric form. For example, compared to free

chemotherapeutic molecules, delivery to cancerous tissues may be

favourably biased by several mechanisms, including particle size (Tang

Z. et al., 2001) and by attaching targeting ligands to the surface of the

particles.

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All this is enough to understand that conventional tests to assess the

cyto- and eco-toxicity are not designed nor suited to study nanoparticles–

cell interaction and that the study of acute toxicity may not be as relevant

as other slower processes, where inorganic contamination progressively

induces stress and damages tissues.

There are no homogenous and certain results about the in-vitro and

in-vivo toxicity of nanoparticles, so floods of words are being written to

debate the problem. Failing data, writing has the effect to remove the fear

of the unknown.

Many articles, reports, strategic documents, risk assessments, contain

recommendations. An article by Dr. Maynard of the Woodrow Wilson

International Center for scholars in Washington (Maynard, A. D. et al.,

2006) is a series of good intentions mixed with fundamental, theoretical

rules of risk assessment and management. The most used words are “we

must develop”, and, in particular, what we must develop is instruments,

tests, validations of tests, and models to predict toxicity. That sounds

rather premature since, without knowing the laws ruling the nanoworld,

it is impossible to predict anything meaningful. The incredible properties

we verified in some nanoparticles witness that nanoworld presents rules

that can be either wonderful or terrifying.

All the reports of different associations and organizations underline

the “must” task (Research Needs on Nanoparticles, 2005), (web ref. 2),

(web ref. 3)

A far from negligible quantity of money is dedicated to research,

much of it by the European Community and the US. But now there is a

risk: nanotechnologists who had new institutes built, money and honours

as pioneers may not want to kill “the goose that lays the golden egg”, so

it may be not so likely that they declare the existence of a possible risk.

The death of Richard Smalley, co-discoverer of the so-called “bucky-

balls” and Nobel Prize laureate, occurred October 31, 2005, could be a

sign of danger not to be ignored, as he died of lung cancer and

leukaemia, simultaneously.

There is a possibility that such an unusual combination of diseases be

the expression of the exposure to the nanoparticles he himself had

created and that possibility should be considered among the risks related

to handling nanoparticles.

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A hypothesis that can be put forward is that part of the inhaled

particulate matter forming clusters may have remained trapped in the

lungs where, because of its non biodegradability and non

biocompatibility, it may have induced a cancer. The nanoparticles that

did not form clusters or with a size small enough, for example, below

100nm, may have passed rapidly, before having the time to cluster,

through the lung barrier and gone into the bloody stream. There, they

may have created bounds with some proteins or cells specific of this

environment, so they were not filtered by organs but went to the bone

marrow.

We do not know for certain if the cell reaction is due to the particle’s

chemistry or its physical structure or only because nanoparticles

are foreign bodies interfering with the normal cell metabolism, but a

reaction is possible at the deeper levels.

What is sure is that this approach leads to a “customized medicine” to

cure diseased people rather than diseases as a sort of abstract entities,

since each individual has necessarily undergone his own exposure to

pollutants. This implies more time and more money dedicated to patients

and this, in its turn, means that only affluent people will be able to afford

it. If we want to avoid such a situation, the only possible solution is

primary prevention, that set of measures aimed at removing the causes of

a disease, rather than diagnose it precociously or cure it when it has

already grown manifest. At present, no pharmaceuticals show any real

activity against the effects the interaction between non biodegradable and

non biocompatible nanoparticles and organism has, and the most they

can do is a simple palliation, and that in case they are correctly

prescribed, something that means a correct diagnosis has been issued.

From our point of view, we have already a basic knowledge

to understand what the possible risks are and how and where they

can be expressed. Identifying risks does not mean at all rejecting

nanotechnologies on principle and the business hoped for as some

scientists and entrepreneurs claim and fear, but means the intention to

drive their development safely in order to exploit their great potential in

the best and most lasting way. Nanotechnology-based industries must

have taken precautions and probably check their staff from the health

point of view, but the question is: How? And how much do they really

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want to know? If, for example, one of their employees develops a cancer,

how willing are they to investigate on a possible correlation between

working place exposure and disease? Cancer morbidity is growing more

and more common in industrialized areas, and children are not spared by

that trend, too often hastily dismissed as an inescapable fatality caused

by wrong parental genes. One of the likely causes, almost certainly not

the sole but nevertheless not negligible, is particulate pollution and there

is no reason to believe that engineered particles differ under this special

aspect from unintentionally produced ones. But there are also diseases

other than cancer that must be kept under control, and among them

troubles like chronic fatigue, insomnia, loss of memory and irritability

that are often attributed to stress and treated accordingly but that may

have nanoparticles as cause.

For all these reasons, referable to the prudence of common sense, we

suggest to apply the precautionary principle before an intentional,

uncontrolled, systematic, massive release of engineered nanoparticles in

the environment occurs.

Something we will be bound to face soon is the nanoscaled pollution

unintentionally released in some specific working places and, later, in the

environment.

If it can be easy or, at least, feasible to check the release of

engineered nanoparticles in the place where they are produced or

worked, it is extremely complicated and expensive, if not sometimes

impossible, to control them when they are already disseminated in the

environment.

The need to be competitive in the market has already induced

industries to develop a fair number of nanotechnological products. It is

the case of Samsung and Daewoo that constructed washing machines

with the NanoSilver technology. Inside the machines there is a tablet

releasing Silver nanoparticles (Silver oxide is well-known for its

bactericide activity). So, their presence in the fabric guarantees no odours

since the bacteria responsible for the problem are killed. Those

manufacturers claim also a lesser use of detergents. The use of molecule

of Silver oxide as a bactericide is regulated in “Private area and public

health area disinfectants and other biocidal products” under the Directive

98/8/EC (web ref. 4) but in the case of Silver oxide in nanoparticulate

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form, its properties are due mainly to their being nanoscaled, so, the

reference to the disinfectants regulation is improper and should not be

considered valid. It may be curious to observe that after the news was

spread that the US EPA (Registration Review Schedule: Antimicrobial

Pesticides of October, 4, 2006) had declared that Silver Oxide (AgO) is

a pesticide, the Italian advertisements for those products changed and

they claim a release only of Silver ions and not nanoparticles. A direct

measure of this presence in the washing water could solve the mystery.

With a similar technology, Samsung make also air conditioners and

refrigerators (web ref. 5), (web ref. 6), (web ref. 7). From our point of

view, the clothes, having come out of washing full of “free”

nanoparticles, are very close to people who wear them and, as a

consequence of such a proximity, those people can inhale them. But

those particles can also be disseminated in the house and outside and the

waste water, not filtered efficiently at nanolevel, can disperse the

nanoparticles in the rivers and in the sea, providing unwanted “food” to

clams, fish, etc.

It is somehow absurd that the scientists that discovered the nano

phenomena be still busy assessing their potential toxicity, that the

funding agencies be just starting broadly funding the assessments of risks

and funding research, while industry is already prepared to release

massively and in an uncontrolled way biocide nanoparticles. Such

nanoparticles, despite being used as a substitute of bleach to disinfect

water for a long time, should be banned from therapeutic use because

their mechanism of action is still unknown, because, in contrast with

common antibiotic agents, nanoparticles may persist indefinitely active

since they are not degraded during their bacteria killing function and one

has to bear in mind that attacking the microbe world means to attack the

life substrate itself, and, for example, it has been found that biocide

Al2O3 nanoparticles reduce root growth in different crops, due to the

perturbation of the soil composition (Yang, L. et al., 2005).

Of course, releasing nanoparticles through air conditioners inside a

close ambient is a nonsense. We fear to inhale dust from incinerators,

from cement plants, from diesel engines, etc. and allow industries to

install a source of indoor pollution with nanotechnological particles in

the house. This pollution can be inhaled by children or pregnant women,

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which is something that must be taken into very serious consideration

and not dealt with so superficially as we are doing now.

The case of refrigerators releasing bactericide nanoparticles is

somewhat different. Only when its door is open the Silver pollution is

released in the domestic environment, but that pollution is likely to

contaminate food. Thus, the same device can potentially give two

different pathologies, the former starting from inhalation, the latter from

ingestion.

Special attention must be paid to what happened with asbestos. It was

used in brakes, buildings and innumerable further applications, from

where it was expected not to be released, but actually it was. The next

observation was that it accumulates in the lung, but as it did not produce

acute toxicity, it was happily overlooked until many years later with a

burst of lung and brain (where they may have entered through the

olfactory nerves) diseases related to cancer.

The results obtained through our research should put scientists,

technicians and politicians on the alert and, maybe, induce them to revise

their way of understanding the impact of environmental pollution upon

our health. We should start by considering a simple natural law, stating

that nothing can be created and nothing destroyed, but everything can

just be transformed. It is a matter of fact that all forms of combustion

change matter and produce micro- and nanoparticulate whose size

depends mainly on the temperature reached in the crucible, and that most

of that particulate, which, in many instances, is neither biodegradable nor

biocompatible, gets dispersed in the environment. So, the air, the soil,

water and vegetables are polluted and both humans and animals are the

victims of that condition through the inhalation of air and the ingestion of

food. The situation may grow worse to humans who eat animals whose

flesh is polluted, and nowadays that pollution may be imported from

places that are very far from where those humans live. In our century, it

is particularly hard to set boundaries to everything and pollution is no

exception.

What the majority of the systems aimed at getting rid of wastes do is

reduce them to a very small size, sometimes incomparably smaller than

the original, and disperse them. As already pointed out, the problem is

that the smaller the size of that dust, the more penetrating and aggressive

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to the organism it is, and making it smaller and smaller does not seem

particularly wise.

One of the most promising results of our research is the possibility it

offers to trace back the source of pollution and the kind of exposure the

subject underwent, by comparing what we detect in his pathological

specimens with the environment where that subject lives or work, or the

food he eats. So, it will not be too difficult to understand whether the

strategy of having wastes “disappear” by making them small enough to

become hard to detect or we had better resort to different solutions.

To summarize, there are some pathologies strictly related to the

environmental pollution; so, in order to understand them it is necessary

to know and characterize that.

The following Tab. 9.1 summarizes the parameters we need to know

to explain these pathologies.

Tab. 9.1 Scheme of the factors that can trigger a disease.

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As a result of our research, but much is still ignored, being a foreign

body seems to be the most important among the factors inducing

pathologies. No matter what particles are made of, they are not

recognized as compatible with the environment they have entered and

that lack of compatibility is mainly due to their being a physical entity.

This is something that could be somewhat hard to classical toxicologists,

accustomed to reasoning rather in terms of ions or molecules and their

chemical noxiousness.

Size is also of the utmost importance, since, as often mentioned, the

smaller particles are, the worse their effect on organisms is. Clustering in

a tissue is a common phenomenon and, under certain aspects, for

instance in inducing the formation of granulation tissue, clustered

particles may behave like larger entities, but the sum of their surface

areas is in any case much larger than the one of bigger particles and the

result is an enhanced reactivity.

As just mentioned, surface area also influences pathogenicity and, in

particular, its ratio with volume, as the higher it is, the higher reactivity

particles have.

Another factor worth considering is particle shape. According to our

experience, still in need of being confirmed by more observations, a

needle-like shape like that of asbestos fibres is more capable of

penetrating tissues than a bulkier one.

Concentration is also a critical factor: unless particle penetrate cell

nuclei, in which case this consideration may lose much of its meaning, it

seems that a comparatively low concentration of particulate matter can

pass unnoticed and be tolerated by the host tissue that, on the contrary,

reacts when a threshold concentration is exceeded. That such

concentration exists is a fact hard to doubt of, but how to measure it is

now beyond our possibilities. We strongly suspect that that may depend

on a number of factors including size, shape, surface-area/volume,

chemical composition, tissue involved and its health condition, along

with the subject’s health and habits. If and how other chemicals or

pollutants in general interfere with that hypothetical threshold we do not

know, though it is reasonable to think so, and even to think that in some

cases a sort of synergy may occur.

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A phenomenon we have often noted is how intake velocity can

influence the onset of a disease. In some cases, that can be rather easily

explained: Let us take a non-smoker and a smoker and have them inhale

quickly the same amount of dust. A non-smoker is likely to be able to get

rid of it because of the better efficiency of his respiratory system and, in

particular, of his muco-ciliary cells. In other instances, a quick intake

may happen when a certain organ is especially prone to capture those

particular particles and the consequence is they are not more evenly

distributed throughout the organism.

As to radioactivity, we have no direct experience, since we never

found any trace of it in the cases of soldiers we had a chance to observe

and in the case of peritoneal mesothelioma described in Chapter 4 we did

not measure it. So, radioactivity has been listed as a likely factor but, as a

matter of fact, we have no direct experience to support its influence.

Though probably not so important as being a foreign body, chemistry

is certainly a weighty factor of pathogenicity. It is only obvious that a

particle made of Arsenic is more toxic than an Iron particle sharing the

same shape and size. Most of the particles we observed, though, are

alloys and their behaviour is notoriously not the same as the sum of each

of their elemental components. If a particle gets corroded in a tissue, the

substances resulting from that chemical phenomenon are something

different from the material composing the original entity and, in that

case, the resulting toxicity can be the one well-known by toxicologists. It

is reasonable to think that chemistry influences the possibility of a

nanoparticle to be bound, for example, by a protein and thus acquire a

different dynamics.

It should not be surprising that every individual reacts to nanodust in

a way that can be different from his neighbour’s. The same individual

variability occurs with exposure to bacteria, viruses, parasites and most

other aggressions.

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9.3 The future

As described in Chapter 5, we know from war experience that as soon

as the hot, volatilized materials generated by explosions cool down,

nanosized particles are created and scattered in the environment. The

inhalation or ingestion of those, mainly metallic, particles by humans and

animals can bring about pathological effects. But for the formation of

nanoparticles as pollutants, there is not only warfare to blame. Car

engines, industry, incineration and, in general, all high-temperature

procedures are just a few examples of particulate pollutants producers.

So, it is easy to guess that in more than one case, the environment is

already contaminated.

As far as we know now, it is not possible to reclaim the environment

from nanoparticles and even in the future, with better technologies than

those available today, that may be extremely difficult. In many cases, the

proposed environmental remediation obtained with techniques making

use of nanoparticles does not solve the problem but simply shifts it from

a variety of pollution to another, this time induced by the nanoparticles

employed, nanoparticles that in most cases are not biodegradable and

stay in the environment, perhaps not the same as the one that had been

“cleaned”, but still somewhere on this planet, forever. But, being aware

of their possible adverse effects on the human health, even now it is not

so hard to confine them in a laboratory and study the safest procedures to

handle them.

At present, we have sufficient knowledge to suggest some simple

criteria to try and avoid toxicological problems in a “not alerted”

population, criteria that should be adopted as a precautionary measure.

The following ten “golden rules” can be adopted immediately and at

trifling costs:

1 - Nanoparticles contained in any product should be declared in the

product’s label, so that consumers are free to make a conscious selection.

2 - A notice should be clearly visible in any rooms or sites where

nanoparticles are released (e.g. some new air conditioners).

3 - Do not disperse intentionally, non biodegradable nanoparticles in

the environment, specially indoors, and take all necessary measures not

to disperse them unintentionally.

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4 - Do not add non biodegradable nanoparticles in food.

5 - Do not introduce in any way (inhalation, ingestion, injection,

instillation, etc.) non biodegradable, non biocompatible nanoparticles in

living organisms.

6 - Do not put non biodegradable, non biocompatible nanoparticles in

contact with external or internal parts of living organisms.

7 - Do not have sexual intercourse with persons who were exposed to

micro- and nano-pollution. In case, the use of condoms is mandatory.

8 - Do not procreate if the male partner was exposed to pollution.

Assisted fertilization can help in such a situation, if only decreasing risk.

9 - Do not eat vegetables grown in a polluted area. Water can be

polluted as well.

10 - Do not smoke tobacco leaves grown in a polluted environment,

especially the one created by war. Weapon explosion can create a new,

mainly inorganic, form of pollution.

The criteria above are simple enough and should be applied

immediately for the sake of human and animal health, but more will

certainly be added as our knowledge advances. Those criteria give also

clear indications to the laboratories and companies busy in the

fabrication of new nanoproducts.

That does not mean that nanotecnologies must be sacked, it means

that certain products are safe, other that release nanoparticles not. We

need nanotechnologies to face the nanosized world and contrast that for

our safety and of our society.

9.4 A few reflections

“Memento, homo, quia pulvis es et in pulverem reverteris.” (Remember,

man, that you are dust and unto dust you shall return.) That is what

priests say when they put a pinch of ash on the head of the members of

their flock on Ash Wednesday. Is that sentence adapted from the Genesis

a prophecy?

Like a parasite, man has already destroyed a part of his habitat and

any being doing that is doomed to extinction. If he has done so in an

irreversible way is hard to say.

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Every day we can create forms of pollution that did not existed before

and whose impact on this planet where vegetables, animals and men

belong on an equal basis, has never been experimented. Can living

beings adapt themselves to the attack of those unexpected pollutants? In

many cases the question has no answer. What we can say is that Nature

rests on a delicate equilibrium and her times are long, much longer than

today’s Homo sapiens sapiens.

Nanopollution is a stimulus living beings had never been confronted

with before; at least, not to the present extent. We should not be mistaken

and think it does not have an effect or has a minor one just because those

particles are so small. What they do in comparison with the larger dust

generated by Nature or by less technological human activities, is change

their target. As an example, let us take 10-micron particles and 0.1-ones.

When inhaled, the former are likely to penetrate no farther than the

alveoli, and that just in case they can get that deep; the latter, instead,

reach the alveoli very easily, stay there no longer than few seconds and

enter quite easily the blood circulation to invade virtually all the

organism and be sequestered inside it by some tissue impossible to guess.

What we did in the last few years is create new forms of pollution as

side effects of novel, very sophisticated technologies. At the beginning,

we did not pay attention to it. We did not want to. And now that

pollution has already shown some of its possible interferences upon

human and animal organisms, but we are not entitled to say that we have

already seen everything. Much is likely to be still unknown and we shall

have to wait many years before being able to say something conclusive.

Let us take as an example asbestos. We can inhale it again and again and

in most cases nothing visible happens for a long time. But we know that

mesothelioma may take up to forty years after exposure before growing

manifest and we have not the least evidence allowing us to say that

something similar is not happening with nanodust. Nature does not come

to terms with what we do: she just lives according to her rules and is

blind and deaf to any protest. Once again, the way we have been doing

for millennia using our brain to develop more and more deadly weapons,

challenging Nature instead of living in harmony with her as all living

beings do, we are showing all our naïve arrogance and lack of wisdom.

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As it always happens, those who will suffer the severest

consequences of nanopollution belong to the weaker part of society:

children and old people. Children can be affected even before they are

born as may be seen reading what is reported in Chapter 3 on malformed

feti, while old people are obviously more prone to be affected because of

their generally poorer health condition and less efficient defence systems.

But, if we look at the problem from a social standpoint, we must admit

that less affluent people will suffer more or, better, will be the first to

suffer. In many cases, large factories and incineration plants are built

in the poorest quarters of cities and in already environmentally

compromised areas where the rich do not live in a sort of more or less

conscious attempt to escape disaster, a disaster, however, that in the long

run cannot be dodged. As already described, nanoparticles, the most

aggressive particulate, behave like gases and their distribution is

homogeneous over large areas, knowing no boundaries. In addition to

that, as far as we know, physiological known barriers are hardly

effective.

Anthropic pollution will probably cause an increase in sterility and

miscarriages may become more and more common. This is not

psychological terrorism: more than one medical association has already

alerted governments and international agencies about that danger, while

symposia, congresses, meetings on nanotoxicity are being held all over

the world, articles are being written, TV and radio programs are being

broadcast, but the truth is that no meaningful political decision has ever

been taken while we keep approaching a no-return point at increasing

speed. Politicians are not only short-sighted but are often in bad faith, as

what they keep trying to do is hiding the problem. And they do that by

hiring scientists available to produce literature fit for that aim, and by

calling terrorists all the others.

So, an honest, effective political action on a global scale is

mandatory, as we cannot afford to live the way we are doing, with a

small - small from the population size point of view - country like the

United States consuming a disproportionate amount of energy resources

and being responsible for not much less than half the production of

greenhouse gases. All that, while emergent countries like China and

India dream to adopt as behavioural models our most reprehensible

Nanopathology

284

habits in terms of respect for planet we live on. We are spoilt children

and are the model which billions of people aim at.

Nature does not care for money, political colours, philosophical ideas,

sociology or any other superstructure we created. Nature does not even

care too much for Man. Man is just another animal and if the new

equilibrium is not compatible with Man’s life, let Man be extinct.

Dinosaurs reigned on this planet for a very long time, far longer than we

Men have done. Yet, something happened that had the scale tip just a

little and those enormous animals simply disappeared. We are not sure,

but perhaps a meteorite hit the Earth creating a huge, long-lasting cloud

of very fine dust, and that dust became an impassable screen to some

solar radiations, causing the extinction of organisms that were at the base

of the food chain. And the same dust has certainly been inhaled by

animals and ingested along with vegetables so that only the more

resistant to that kind of unusual aggression survived.

That pollution may cause disasters is something even some historians

maintain. One of the factors they add to those which led to the fall of the

Roman empire is heavy-metal pollution in their aqueduct pipelines and

in their wine. And if we look at the tragedies of Hiroshima and Nagasaki

in the light of what we know now, we must accept that the two very

high-temperature explosions which destroyed the cities and had all sorts

of materials sublime must have created millions of tons of nanodust

whose existence is impossible to deny and is proved by the black rain

that fell about half an hour after the bombing. And if nanodust was

present, it must have behaved the way all nanodust does. So, it is

reasonable to think that part of the pathologies that affected the

inhabitants of those cities and the malformations so common in their

offspring is due to particulate matter entered in their tissues.

One of our present themes of research is aimed at finding out if that

hypothesis is correct and will be one of the subjects of our next book.

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287

Appendix

Our analyses are mainly made with an Environmental Scanning Electron

Microscope (ESEM-Quanta, FEI-Company, The Netherlands)

The reason why that instrument is called “Environmental” is because,

besides being able to work in high and medium vacuum, it can analyze

samples at room, i.e. environmental, conditions. Thanks to that feature, it

can easily accept biological specimens. (Report, 1996)

In addition to that, there are 3 major characteristics that make ESEM

excellent for our use:

Pure secondary-electron detection;

Compatibility with water;

High chamber pressure.

1- Secondary electrons come from the atoms of the sample that derive

from interactions with the primary electrons of the beam. Those electrons

are responsible for the best sample resolution at low energies. ESEM can

detect pure secondary electrons in a gaseous environment. In

conventional scanning electron microscopes (SEM), the electronic gun

must work at high-vacuum conditions because of the high voltage it

needs. Because of that, the equipment accepts only dried and clean

samples, since humidity and dirt are incompatible with high vacuum. In

addition to that, the samples must be electron-conductive and so they

must be coated inside a vacuum chamber through a sputtering process

with Carbon or with a Gold/Palladium alloy. In order to remove the

electrostatic charge created by the electron beam, their morphology must

be flat and simple enough, so as to allow the coating to be homogeneous.

2- The imaging process is directly affected by the ionization

characteristics of the gas inside the chamber. Different gases can be

employed. There is no difficulty in having water vapour ionise, and that

yields excellent imaging performance, so biological samples can be kept

fully hydrated virtually indefinitely.

Nanopathology 288

3- The lowest pressure necessary to keep water in a liquid state is

about 4.6 Torr. In a conventional low-vacuum scanning electron

microscope, the highest chamber pressure does not exceed 2 Torr. High

chamber pressure permits to study samples with high outgassing rate as

well as to perform dynamic experiments like re-crystallization, or to

observe living cells in the medium.

Our ESEM is equipped with a Tungsten filament, an ionisation

secondary-electron sensor, a back-scattered sensor, SW controlled

Peltier cooled specimen stage and an Energy Dispersive System (EDS by

EDAX, USA).

The electron beam issued by the ESEM hits the sample surface and

produces X-ray fluorescence from the atoms in its path. The energy of

each X-ray photon is typical of the element which generated it. The EDS

microanalysis system collects the X-rays, sorts and plots them by energy,

and allows to identify the elements that produced the peaks in this energy

distribution. The system can identify the elemental composition of the

materials imaged for all elements with an atomic number greater than

Boron. Most elements can be detected at concentrations of order 0.1%.

In general, when analyzed by the EDS, biological samples from

healthy tissues show just the peaks of Carbon and Oxygen, but some

specialized tissues reveal also a content of other elements like Calcium

and Phosphorus in the bone, Iron in the liver and Phosphorus in the

brain.

When their ion size is below the sensitivity threshold of the system,

some elements undoubtedly present in a tissue are not detectable with

this method. Iron bound to haemoglobin in the red cells, for example,

cannot be seen, while when it precipitates in the liver as it does in

patients affected by siderosis, detecting it becomes quite easy.

If the tissue to be observed has been fixed chemically, dehydrated and

then embedded in paraffin, it can show small peaks of elements like

Sulphur or Chlorine not belonging to the specimen, and that depends on

the chemicals present in the liquids used for the treatments. In this case,

the spectrum of the biological tissue is necessary as a reference and must

be subtracted from that of the foreign body contained in it.

Appendix 289

Sample Preparation

The biological sample must be properly prepared in order to detect

inorganic particulate it contains.

Biological samples can be divided into two groups:

fresh tissues (not chemically fixed), bulk or sections; and

chemically fixed and paraffined tissues.

a – Fresh tissues are: 1- the bulk samples from surgery, including

bioptic and autoptic specimens, 2 – the blood , 3 – the sperm, and 4 –

the living cells from in-vitro simulation.

As briefly described above, they can be observed in wet conditions,

keeping the sample at a temperature of 5°C degree and at a relative air

humidity of 90-95%. Since these samples did not undergo any additional

chemical treatment, no pollution is possible.

According to the kind of sample, different preparation methods have

been developed.

The samples obtained from surgery are usually bulk and, to compare

them with the histo-pathological sections on which diagnoses are

performed, they are frozen at –20°C and then cut into 20-micron slices.

Then, they are deposited on an acetate sheet and observed under

ESEM.

Acetate was selected since, under Energy Dispersive Spectroscopy, it

emits only minor signals for Carbon and Oxygen, ever-present elements

in any biological specimen. When the section is very thin, the contribute

from the acetate substrate is just added to that from the biological

specimen and does not affect the measure carried out on the inorganic

debris.

Since every fresh sample represents a potential a risk of infections

(HIV, hepatitis) for the operators, we decided to subject them to a half-

hour’s immersion in formalin before handling it.

Bulk samples may be subjected to a critical-point dehydrating process

through the exchange of water with Nitrogen.

The blood and the sperm are smeared on the acetate sheet and then

covered with another sheet of the same material. A few seconds later,

they are delicately turned and separated in order to obtain a cell

Nanopathology 290

monolayer. The sheet is glued with a Carbon disc on a stub and observed

immediately.

The living cells, not treated in any way, are put on a support and

observed under ESEM directly in the medium. As, because of the

medium surface, observing the cell morphology is impossible, humidity

is slightly and smoothly decreased, thus allowing a mild dehydration

without prejudice to the morphology.

Sometimes, fixation is performed (a few drops of 2,5%

glutaraldehyde) so the morphology is preserved and, what is most

important, the interaction between cells and nanoparticles is preserved as

well.

b – The following method allows the observation of old samples

preserved in archives. Those samples had already been fixed, dehydrated

and embedded in paraffin and, from those blocks, we cut 20-micron-

thick sections. The slices are then suspended in warm water and

deposited on the acetate sheet. To remove the paraffin, the samples are

covered with a few drops of xylol and 98%-alcohol and, a few seconds

after, the excess of liquid is slid along the sheet edge and absorbed in

blotting paper. The samples can be observed in different modalities: in

high and low vacuum; in secondary and in backscattered electron mode.

Bibliography

Report from Environmental Scanning Electron Microscopy: An Introduction to ESEM by

Philips Electron Optics, Eindhoven, The Netherlands 1996, Robert Johnson

Associates California.

291

Index

1st Gulf War, 161

2nd

Gulf War, 161

9/11, 251

A-bomb, 165

acarus, 218

acrolein, 231

Acute Lymphatic Leukaemia, 170

acute renal failure, 97

adenocarcinoma, 65

adenoma, 99

adrenal adenoma, 186

adrenal-gland, 99

Adriatic Sea, 220

Agent Orange, 167

air conditioners, 259

Air Force Laboratory, Armament

Development and Test Center,

of Eglin Air Base, Florida, 161

Allied Forces, 165

alloy, 9, 95

Aluminium, 2

alveoli, 43

alveolitis, 55

Alzheimer’s disease, 13

amalgam, 13

ameloblastoma, 102

amiotrophic lateral sclerosis, 13

ammonia, 229

amyloidosis, 271

angiogenic, 26

animal feed, 248

Antarctica, 248

antibodies, 21

Antimony, 125

anti-wear, 245

arteries, 45

arthrosis, 5

Asacol, 106

asbestosis, 22

ash, 230

atelectasis, 49

Baghdad, 189, 232

Balkans War, x

Barium, 3

beauty and sun-screen creams, 24

bicarbonate, 229

biocompatibility, 16

biocompatible, 16

biomass, 230

biscuits, 244

Bismuth, 222

Black Sea, 244

Blebs, 62

blood, 1

blood-brain barrier, 18

blood-placenta barrier, 23

blurred vision, 253

bombing, 54

bone, 47

bone marrow, 177

Bonfield, William, vii

Boraschi, D, 24

Boron, 288

Bosnia, 187

Nanopathology 292

bowels, 4

brain, 18

brakes, 266

bread, 244

bronchial mucus, 136

bronchoalveolar lavage, 156

Brownian motion, 270

bucky-balls, 272

Burning Mouth Disease, 102

Burning Semen Disease, 121

Cadmium, 21

calcification, 100

Calcium, 2

cancerogenic, 28

cancerogenicity, 40

Canova Activa, 240

Capitani, Federico, vii

car bodies, 227

Carbon, 1

Carbon monoxide, 42

cardiogenic mortality, 79

cardiovascular, 23

cardiovascular diseases, 77

caval filtration, 5

cell, 16

cellular duplication, 271

Cerium, 55

Cesium, 248

Chernobyl, 248

chewing gum, 24

children malformation, 116

chimney-sweepers’ cancer, 205

China, 283

Chlorine, 1, 76

chocolate, 245

cholangiocarcinoma, 93

cholestasis, 4

Chromium, 1

chronic fatigue, 40, 253

cigarette, 22, 231

cirrhosis, 87

clams, 275

clothes, 208

coal, 266

Cobalt, 2, 24

colloids, 265

colon, 5

combustion, 7

composites, 13

concrete, 259

coronary vessel, 78

cough, 155, 253

Council of Down Manhattan, 252

COx, 46

Creutzfeldt-Jacob, 271

criminal court, 220

Crohn’s disease, 5, 40

cryptogenic diseases, 39

customized medicine, 273

cystic fibrosis, 5

cytokine, 15

Da Costa Syndrome, 166

Daewoo, 240

Danube, 244

De Morbis Artificium, 205

defecation, 245

dendrimers, 17

dental filling resins, 13

dental prosthesis, 5

dental restorations, 13

Departments of the Veterans

Affairs, 166

depleted Uranium, x, 54

depression, 167, 253

Index 293

derma, 145

dermatitis, 145

Desert Storm Conflict, 167

detoxification, 252

diabetes, 91

digestive system, 5

dioxin, 8, 42, 167

DNA, 12

Drexler, Kim Eric, 6

drug, 85

EDAX, 288

edema, 239

Effort Syndrome, 168

enamel, 156

endometrium, 137

endothelial cells, 23

endothelium, 45

Energy Dispersive Spectroscope

(EDS), 1

energy, 266

engineered particles, 274

environment, 22

Environmental Scanning Electron

Microscopy, 86

enzymes, 18

EPA, 241

epidemiologic studies, 166

epidemiological studies, 249

epidemiology, 269

Escalaplano, 195

Escherichia Coli, 240

ESEM, 28

Europäische Akademie, 11

Europe, 266

European Commission, vii

European Community, 5, 43

excipient, 102

explosions, 161

exposure, 39, 253

eye, 174

faeces, 14

fat, 263

fatigue, 83

FDA, 241

Federal Institute for Risk

Assessment (BfR), 239

FEI-Company, 287

fertilizers, 230

feti, 41

fever, 85

fibrosis, 3

fibrotic capsule, 29

filters used in Diesel cars (FAP),

203

firemen, 252

firing ground, 121

First Industrial Revolution, 266

fish, 200

Fluorine, 104

food, 88, 248

Ford Farm Firing Range, Aberdeen

Proving Ground, MD, 162

foreign-body, 3, 20

forgetfulness, 168

formaldehyde, 231

fossil fuels, 266

Foundation for the Advancement of

Science and Education, 252

foundries, 266

Framer, Joe A, 199

Free Royal Hospital of London, 4

fullerene, 18

furan, 8, 42, 228

Nanopathology 294

Gadolinium, 20

Gambarelli, Andrea, vii

ganglioneuroma, 184

gastrointestinal system, 4

Gatti, 171

Germany, 239

giant cells, 58

Gitelman’s syndrome, 54

glass, 8

glioblastoma, 184

globalization, 268

GMOs (Genetically Modified

Organisms), 268

Gold, 5

gonads, 173

granulation tissue, 278

granuloma, 31

granulomatosic reaction, 28

granulomatosis, 3

granulomatous tissue, 29

Ground Zero, 257

Gulf War, 54

Gulf War Syndrome, 43, 95

Hadzici, 169

haemolysis, 4

haemolytic anaemia, 2

hamburger, 241

Harrod’s, 251

Harvard School of Medicine in

Boston, 154

headache, 167

heart, 19

heavy oil, 220

hepatic echinococcosis, 83

hepatitis, 289

hepato-splenomegaly, 2

Herald Tribune, 251

HIV, 289

Hodgkin’s lymphoma, 115

hydrocarbons, 42

Hydrogen, 2

hydroxyapatite, 14, 15

immune system, 50

immunological disease, 20

incinerators, 8

India, 283

industrial sterility, 121

infarction, 45

inflammation, 4

inflammatory pathologies, 39

Information Office (OI), 165

inhalable, 19

inhalation, 22

insomnia, 173

internalization, 23

interstitial pneumonia, 55

intestinal mucosa, 4, 151

Iodine, 99

Iraq, 54

Iron, 1

irritability, 167

Istituto Tumori (Cancer Institute)

of Milan, 5

Italian Commission, 170

Italian senatorial commission, 249

Kleinmann GmbH, 239

knee, 5

Korean conflict, 166

Kosovo, 188

landfills, 259

Lanthanum, 55

Lateral Amyotrophic Sclerosis, 142

Index 295

Lavoisier, 214

law of conservation of matter, 228

Lead, 21, 91

LeGeros, Rachel, 101

leiomyomatosis, 64

lettuce, 225

lichens, 223

lime, 229

liposomes, 239

London, 251

Los Angeles, 252

Lou Gehrig’s disease, 166

lung, 5

lung barrier, 209

lymph-nodal metastases, 187

lymph-nodes, 19

lymphocytes, 30

lymphomas, 40

macrophage, 15

mad cow disease, 248

Magic Nano, 239

Magnesium, 1, 2

malformed lambs, 121

Malta, 121

Manganese, 150

Mantua, 212

Mars, 245

masks, 155

mast cells, 30

Maynard, 272

medical devices, 16

Medical Geology, 43

medullar aplasia, 166, 169

Mercury, 13

mesenchymal tumors, 31

mesothelioma, 46

mesoworld, 6

metalloproteic precipitates, 144

metalloprotein, 145

metastasis, 19

mieloid leukaemia, 182

Minamata City, 243

miopathy, 135

Molybdenum, 1, 111

monocytes, 30

Monsanto, 241

mouth, 44

mouthwash, 145

muco-ciliary cells, 279

mule spinners, 206

multi-organ failure, 100

multi-organ-diseases, 43

multiple sclerosis, 13

muscles, 266

Nagasaki and Hiroshima, 169

Nano Poly Technology, 241

nanocomposites, 13

nanopollution, 135, 205

nanoproduct, 239

nanospheres, 19

Nanotea, 240

nanotechnologies, ix

nanotube, 18

nasal congestion, 146

National Technical Information

Service (NTIS), 165

NATO, 167

neck, 20

Nemery, Ben, 69

Neodymium, 55

nephrocalcinosis, 43

Neu-Laxova syndrome, 121

neurological, 19

New York, 171, 251

Nanopathology 296

Nickel, 1

nightmares, 253

Niobium, 66, 150

Nitu, Lavinia, vii

non-Hodgkin’s lymphoma, 43

NOX, 46

nucleating agents, 71

oil wells, 249

olfactory nerves, 276

osteoblast, 15

oxidative stress, 27, 270

Oxygen, 3

paints, 103

Palladium, 287

Pančevo, 244

pancreas, 91

pancytopenia, 183

Parkinson’s disease, 13

peace-keeping missions, 176

Percivall Pott, 205

peritoneum, 150

pesticides, 230

phagocytosis, 80

Phosphorus, 1

photoablation, 270

Phynox, 2

physiological barriers, 121

platelets, 114

Platinum, 102, 212

PM10, 22

PM2.5, 43

pneumoconioses, 22

pneumothorax, 5

Po, 220

Polesine Camerini, 220

pollutants, 8

polychlorinated biphenyls, 228

polycyclic aromatic hydrocarbons,

8, 206

polycyclic organic matter, 228

post-Vietnam Syndrome, 166

Potassium, 2

power station, 220

Praseodymium, 234

precautionary principle, 274

pre-neoplasia, 32

prevention, 87

primary electrons, 287

pro-inflammatory, 27

prostate, 19

protein, 16, 265

pseudotumour, 49

psyconeurosis, 166

pulmonary, 19

pulmonary intestitium, 204

pulmonary thromboembolism, 70

Pulsator, 241

PVC, 24

pyrophoric, 162

quantum dots, 21

quartz, 22

Racemose ossification, 48

Ramazzini, 205

rats, 28

red cells, 65

redox potentials, 270

renal failure, 2

respiratory, 23

respiratory system, 5

retrosternal pain, 155

Revell, Peter, 4

rhabdomyosarcoma, 32

Index 297

rheumatoid arthritis, 81

RIS (Crime Scene Investigative

Institute) of Parma (Italy), 197

risk factors, 22

Ru(bpy), 240

Salto di Quirra, 195

Salvatori, Roberta, vii

Samarium, 234

Samsung, 240

Sapporo, 214

Sarajevo, 169

sarcoidosis, 5

Sardinia, 121

sclerodermia, 135

sclerosis, 139

Second Industrial Revolution, 266

second World War, 166

secondary electrons, 287

secondary particles, 229

Selenium, 240

September 11, 253

Shake, Slim, 240

shale oil, 206

ship, 227

shortness of breath, 155

siderosis, 87

Sighinolfi, Gianluca, vii

Silicon, 1

silicosis, 22

Silver, 14

Silver Nano, 240

skin, 205

skin carcinomas, 206

skin pesudolymphoma, 43

sleep disturbance, 167

Smalley, Richard, 272

Sodium, 2

soldier, 99

SOx, 46

sperm, 42

spleen, 84

squamocellular carcinoma, 52

stainless steel, 72

steatosis, 89

steelworks, 215

stomach, 44

Stress-Activated Proteins (SAP),

271

stroke, 19

Strontium, 95

Sulphur, 2

sunscreen creams, 103

surface/size-ratio, 22

sweat, 254

Symptoms and Signs of Ill-Defined

Conditions, 167, 168

talc, 113

Taniguchi, Norio, 11

Tantalum, 66

tea, 240

teeth, 13

Teflon, 104

teratogenic, 248

testicle, 121, 205

Thorium, 109, 150

threshold, 10

thrombectomy, 70, 78

thrombi, 5

thrombus, 23

thyroid, 187

tiredness, 2

Titanium, 2

tobacco, 55, 231

Tokyo Science University, 11

Nanopathology 298

toner, 210

tongue, 142

toothbrushes, 247

toothpaste, 14, 247

Tossini, Daniela, vii

toxicity, 212

traceability, 203

Tub U, 241

tuberculous pleuritis, 46

Tungsten, 66

Twin Towers, 251

typhoid fever, 151

tyres, 266

U.N. Environment Agency, 169

U.N. Environment Protection

Agency, 167

Udine, 215

ultrafine, 23

umbilical circulation, 41

University of New York, 101

University of Siena (Italy), 5

Uranium, 54, 150

urine, 2

US, 272

USSR, 162

uterus, 118, 137

vaccines, 165

vagina, 137

vaginal mucosa, 118

Vanadium, 221

vein thrombosis, 40

vena cava filter, 1

veterans, 116

Vietnam, 167

Virchow’s Triad, 77

volcanoes, 265

wall paints, 24

Washing Machines, 241

wastes, 204

water, 266

wear, 14

Wegener’s granulomatosis, 43

welding, 207

white cells, 114

wind, 266

Woodrow Wilson International

Center, 272

World Trade Center, 266

X-ray, 288

Yttrium, 109, 150

Yugoslavia, x, 116

Zimmer, Rene, 239

Zinc, 21

Zirconia, 13

Zirconium, 13