Correlation between Particle Size, In VivoParticle Persistence, and Lung InjuryGunter Oberdorster,1 Juraj Ferin,1 and Bruce E. Lehnert2'University of Rochester, Department of Environmental Medicine, Rochester, New York; 2Los Alamos NationalLaboratory, Life Sciences Division, Los Alamos, New Mexico

Dosimetry parameters such as deposition, clearance, retention, and translocation and dissolution of inhaled particles in and to different lung com-partments may be important for the persistence of particles in the lung and may correlate with adverse pulmonary effects. We investigated suchcorrelations using a model involving TiO2 particles of two particle sizes (20 nm diameter, ultrafine; 250 nm diameter, fine) of the same crystallinestructure (anatase). A 12-week inhalation experiment in rats resulted in a similar mass deposition of the two particle types in the lower respiratorytract. The ultrafine particles elicited a persistently high inflammatory reaction in the lungs of the animals compared to the larger-sized particles. In thepostexposure period (up to 1 year) retention in the alveolar space per se was not different between fine and ultrafine TiO2. However, the followingdifferences between the particle types were noted: a significantly different total pulmonary retention, both quantitatively (significantly prolongedretention of the ultrafine TiO2) and qualitatively (increased translocation to the pulmonary interstitium and persistence there of the ultrafine TiO2);greater epithelial effects (Type II cell proliferation; occlusion of pores of Kohn) and the beginning of interstitial fibrotic foci with ultrafine TiO2; signifi-cant sustained impairment of alveolar macrophage function after ultrafine TiO2 exposure as measured by the clearance of test particles. A correla-tion between particle surface area and effects was observed. A comparison of the adverse reactions with dosimetric parameters of TiO2 in differentlung compartments in the postexposure period showed a correlation of the persistence of effects in both the alveolar and interstitial space with thepersistence of particles in the respective compartment. - Environ Health Perspect 102(Suppl 5):173-179 (1994)

Key words: ultrafine particles, dosimetry, retention, lung clearance, particle surface area, inflammation

IntroductionWhen evaluating exposure-dose-effectrelationships of inhaled particles the defini-tion and determination of the relevant doseis crucial (Figure 1). The initially depositeddose may not be a decisive parameter, sinceparticles probably clear at varying ratesfrom different lung compartments. In con-trast, the retained dose may be the moreimportant parameter. Conventionally andconveniently, doses usually are expressed interms of particle mass (gravimetric dose).However, when different types of particlesare compared, doses may be more appro-priately expressed as particle volume, parti-cle surface area, or numbers of particles,depending on the effect in question. Forexample, the retardation of alveolarmacrophage-mediated clearance due to par-ticle overload appears to be better corre-lated with phagocytized particle volumerather than mass ( ).

This paper was presented at the Workshop onBiopersistence of Respirable Synthetic Fibers andMinerals held 7-9 September 1992 in Lyon, France.

The authors thank Nancy Corson, R. Gelein,Pamela Mercer, and Kiem Nguyen for their excellenttechnical assistance in these studies.

These studies were supported by NIEHS grantsES04872 and ES01247.

Address correspondence to GOnter Oberd6rster,University of Rochester, Department of Environmen-tal Medicine, Rochester, NY 14642. Telephone (716)275-3804. Fax (716) 256-2631.

Exposure Ds I5ffect

Retention = Deposition - ClearanceDose retained Dose depited Arnount cleared

(Parameters: mass; volume; surface area; number)

Biopersistence

Physical/mechanical processes Chemical processes(Translocation; splitting; breaking) (Biodurability: dissolution; leaching)

Compartments: Alveolar; Interstitial; Intracellular; Extracellular. Lymph-node

Figure 1. Biopersistence and biodurability in relation to dose parameters for exposure-dose-effect relationships ofinhaled nonfibrous and fibrous particles.

The retained dose is a result of the should be reserved for chemical processesbiopersistence of a particulate compound,which is based on several clearance mecha-nisms. These can summarily be describedby physical/mechanical and chemicalprocesses. Although the terms retention,biopersistence, and biodurability often areused interchangeably, we suggest that"biopersistence" should refer to the in vivobehavior of a particle (fibrous or nonfi-brous) and that "retention" should reflect adosimetric term. The term "biodurability"

occuring in vivo and contributing to biop-ersistence. Biopersistence mechanisms canbe very different for different pulmonarycompartments (e.g., alveolar vs interstitial,intracellular vs extracellular). The issue ofbiopersistence and the correlation betweenparticle dose parameters and retention inindividual lung compartments have beenstudied here by performing inhalationstudies with TiO2 particles of two sizes andevaluating resulting specific effects. Both

Environmental Health Perspectives 173

OBERDORSTER ETAL.

particle types were TiO2 of submicronicparticle size with the crystalline structure ofanatase, one particle type with an average

particle diameter of about 20 nm (ultra-fine) and the other with an average diame-ter of about 250 nm (fine). Both are highlyinsoluble, so their biopersistence is mainlydue to physical/mechanical processes. Thisstudy was based in part on our previousobservation that the intratracheal instilla-tion of ultrafine particles led to a signifi-cantly greater pulmonary inflammatoryresponse than larger-sized particles (2,3).

MethodsMale Fischer 344 rats (bw 177 g ± 12 g)were exposed in whole body exposure

chambers to either ultrafine TiO2 (TiO2 -

D) at a concentration of 23.5 ± 2.9 mg/m3or fine TiO2 (TiO2-F), at 22.3 ± 4.2mg/m3. The exposure was for 6 hr/day, 5days/week for 12 weeks; there were 64 ani-mals per group. Control animals were

sham-exposed to filtered air. Uponaerosolization, both TiO2 particle typesformed agglomerates with mass medianaerodynamic diameters of 0.71 lpm (TiO2-D) and 0.78 pm (TiO2-F) and with geo-metric standard deviations of 1.9 forTiO2-D and 1.7 for TiO2-F. Since the aero-

dynamic diameters of the aerosols were

essentially the same for the two particletypes, the compartmental deposition in therespiratory tract of the animals was expectedto be very similar. After deposition in thelung these particle agglomerates appear to

disaggregate to smaller aggregates, as we

and others (4) have observed by electronmicroscopy of lung sections. At 4, 8, and 12weeks of the exposures, six animals pergroup were killed by an overdose of pento-barbital. Further animals were killed at 29and 64 weeks after cessation of exposure.

The following measurements during andafter exposure were performed: analysis ofTiO2 burdens in different compartments ofthe lung, i.e., lavagable versus nonlavagableparticle burdens, and particle content inregional lymph nodes. An extensive lunglavage was performed, and lavagable cellswere analyzed for macrophages, polymor-phonuclear cells (PMNs), lymphocytes, andother types. Furthermore, lysosomal andcytosolic enzymes and total protein were

determined in the lavage fluid, as additionalparameters of toxicity. Light and electronmicroscopy were performed after respectivepreparation of fixed lung sections to evalu-ate epithelial and interstitial responses.

An important parameter of alveolarmacrophage (AM) function, i.e., AM-mediated particle clearance, was deter-

Table 1. Lavagability of polystyrene particles from rat lungs by extensive lavage.

Day after 3.5-pm particles 10.3-pm particlesadministration % lavagable % nonlavagable % lavagable % nonlavagable

4103062132202

80807975757776

20202125252324

86858380807876

14151720202224

With inflammation (20-40% PMN)1 81 191 78 221 76 24

Radioactively labeled polystyrene particles of two sizes were administered by intratracheal instillation to differentiatebetween alveolar lung burden, interstitial lung burden, and total lung burden. Interstitial dose = total lung burden - alveo-lar burden - lymph node burden. (Particle dose 20-100 pg(. On average, 23% of alveolar burden was nonlavagable afterday 30. (Alveolar burden = 1.3 x lavagable amount(. This number was not influenced by inflammation induced by coin-stilling ultrafine TiO2 together with the polystyrene particles.

mined by measuring in vivo the retentionkinetics of 85Sr-labeled polystyrene test

particles (3M Company) following inhala-tion via tracheal tubes. For this purpose,

four rats of each group were anesthetizedwith halothane after 12 weeks of exposure

(end of exposure) and another four rats per

group after 7 months, and connected to a

respirator system by intratracheal tubes(inserted via the mouth), which allowedgroups of up to 16 rats to be exposed to thetest particles simultaneously without any

external fur contamination. An aerosol ofthe labeled particles was inhaled over a

period of 15 min. The retained activity of85Sr was determined on subsequent days

0

E

C(40

E

Total Lung

T =501 days

4 I - (20nm)

2 g/ T, =174 days -.

exposure (250 nm)

0 100 200 300 400Days

Lavaged Lung

3 exposure (

i\ 1

r0 s nn onrs arzn srhninon n n

(up to 200 days postexposure) by a doubledetector system collimated to the lungregion of the rats. Short- and long-termeffective clearance rates were determinedfrom the data and the respective biologicalretention halftimes were calculated.

Results of these measurements were

correlated with specific doses of theretained particles in individual lung com-

partments. To assess the burden of particlesin the alveolar space versus the interstitium,the excised lungs of the animals were

lavaged extensively 10 times with a volumeof 5.0 ml saline per wash cycle. Thisyielded an average of approximately1.1 to 1.2 x 107 total cells in the lung

1000 ~Lymph Nodes(Om)

200; I/O @o _ *0(250 nm)

0~~~~~~~~

0 100 200 300 400

Days

('4

0

oEt

uu zuu sDuu 4UUDays

0 100 200 300 400Days

Figure 2. TiO2 burdens in different lung compartments during and after 12 weeks of exposure of rats to fine and ultrafineTiO2 particles (mean ± SD; n= 4).

Environmental Health Perspectives

5 1 Lavage Pellet

4 i34 t4(2SOnm)

(20 in)

exposurs.0 6O ~~ ~ ~ -|.."@|@@u

174

PARTICLE PERSISTENCEAND LUNG INJURY

3501

Exposure:

6 1102D

T102-F */9 * more than 50% of

ok_Qio *11- .

total lung burden

8Weeks

41

3001

2501N

° 2001HO' 1501

100

50

64

0 -

0 Exposure: 0TiO2D 0

0-.0-,

10-- 00~~~~~~~~~o. 0

o0 A TiO2-F

o. -8

Weeks41 64

Figure 3. Estimated interstitial burden (left) and interstitial plus regional lymph node burden (right) during and after exposure of rats to ultrafine (TiO2-D) and fine (TiO2-F) particles.

washes. However, even an "extensive"lavage cannot completely deplete the alveo-lar space of macrophages and particles, andit cannot be assumed that the lavagableportion is equivalent to the retained dose inthe alveolar space. To determine the alveo-lar as well as the interstitial dose moreaccurately, a pilot study was performedwith instillation of very large (10.3 pm)and smaller (3.5 pm) polystyrene test parti-cles labeled with two radioactive tracers('4'Ce, 95Nb). Groups of animals werelavaged during the subsequent 202 daysand the lavagable and nonlavagable por-tions of the radioactively labeled test parti-cles were determined (5). It was assumedthat the larger particles would not betransferred to the interstitium and that,accordingly, the sum of lavagable andnonlavagable fractions would represent theburden in the alveolar space. Histologicalevaluations confirmed that none of thelarge test particles were in epithelial cells orthe interstitium, whereas a very few of thesmaller (3.5 pm) particles could occasion-ally be seen at those sites. On average,beyond day 30 after instillation, 22.9 ± 2.0percent of the total retained lung burdenwas not lavagable (Table 1). Thus, the alve-olar burden can be estimated from thelavagable fraction by multiplying this frac-tion by 1.3. Furthermore, the interstitialdose of other retained particulate materialcan be calculated by subtracting the alveo-lar burden and the lymph nodal burdenfrom the total lung burden.*

To demonstrate that these very differ-ent polystyrene particles are an accuratemodel of TiO2 interaction with AM, 500pg TiO2-F were coinstilled with the poly-

styrene particles; we found that, bothshortly after instillation and 24 hr later,there was no difference in the lavagable andnonlavagable fraction of TiO2 and poly-styrene particles. Furthermore, since a highinflammatory response expected with theultrafine TiO2 might affect the lavagabilityof the lung, we also determined the lavaga-bility of radioactively labeled test particlesin the presence of such inflammation bycoinstilling 500 and 1000 pg ultrafineTiO2 with radioactive polystyrene particles.The nonlavagable fraction of the poly-styrene particles was essentially the same asbefore, i.e., approximately 22%, demon-strating that the inflammatory response didnot influence alveolar lavagability. In con-trast, a larger fraction of the ultrafine TiO2,compared to the polystyrene particles, wasnonlavagable, indicating some transloca-tion of the ultrafine TiO2 to epithelial andinterstitial sites. We conclude from thispilot study that for subsequent determina-tions of the alveolar and interstitial burdensof particles, a nonlavagable fraction of23%(using our lavage technique) of the alveolarburden in rat lungs should be assumed(Table 1).

ResultsDosimetryThe total retained lung burden at the endof the 12-week exposure was 6.62 ± 1.22mg for TiO2-F and 5.22 ± 0.75 mg forTiO2-D (Figure 2). Assuming first-orderclearance kinetics, retention halftimes forthe ultrafine particles were approximately500 days and for the larger-sized particlesapproximately 170 days postexposure. Thelavagable amount (lavage pellet) postexpo-sure was generally similar in both particlegroups, indicating no large differences inthe overall clearance of the different parti-cle sizes from the alveolar compartment.

However, large differences were observedin the fraction remaining in the lung. Theultrafine TiO2 particles showed a signifi-cantly greater fraction being retained com-pared to the fine TiO2 particles. Asignificantly larger fraction of the ultrafineparticles was transferred to the regionallymph nodes, as compared with fine parti-cles, indicating a greater ability of theultrafine particles to enter interstitial spacesafter alveolar deposition (Figure 2).

The interstitial dose of the retainedTiO2 particles was determined (seeMethods). More than 50% of the totalretained lung burden of the ultrafine parti-cles could be attributed to interstitial local-ization at the two postexposure time pointsstudied (Figure 3). For the fine particles,this was also the case at the latest timepoint examined. The interstitial dosedecreased somewhat beyond week 41 forthe ultrafine particles, whereas it continuedto increase for the fine particles. This mayindicate a faster clearance rate of the ultra-fine particles from interstitial sites asopposed to the fine particles, perhaps, viathe bronchus-associated lymphatic tissue(BALT) onto the mucociliary escalator ofthe conducting airways (6). This, however,needs to be investigated further.

The clearance of both TiO2 particletypes from the alveolar space showed simi-lar kinetics as indicated in Figure 2 (lavagepellet). A more complete analysis of thedata is given in Table 2 by comparing theamounts of TiO2 in the alveolar and inter-stitial spaces 1 year after the end of expo-sure. In spite of the prolonged pulmonaryretention of ultrafine TiO2 (Figure 2), theoverall clearance of both particle typesfrom the alveolar space is virtually identi-cal. Within the 1-year period postexposure,93% of either particle type present atthe end of exposure in the alveolar spacewas removed from this compartment.

Volume 102, Supplement 5, October 1994

3500-

3000-

2500-

° 2000-

> 1500-

1000

500

*The term interstitial dose should not imply that all par-ticles of this compartment are in the pulmonary intersti-tium; some also may be in epithelial cells. Regardless,they are no longer in the alveolar space per se.

175

OBERDORSTER ETAL.

Table 2 Clearance of TiO2 particles from alveolar space during 1 year after cessation of 12-week exposure.

Interstitial spaceAlveolar space (incl. LN)

Time after Clearance Input Clearance to other sites (GI)exposure days pg pg % pg pg % (of Alveolar) %(of alveolar)

0 4192 10233915 93

365 277 2864

0 6202 412

365 4595743 93

1200

However, clearance pathways for the twoTiO2 particles evidently differed markedly.A large fraction (44%) of alveolar burdenof the ultrafine particles appeared in theinterstitial space, compared to only 13%for the fine particles. Most of the fine par-ticles presumably cleared to the GI tract,although fecal excretion of TiO2 particleswas not specifically evaluated in the presentstudy. Clearance to the GI tract was muchless for the ultrafine particles, indicatingprolonged clearance of these particles from

1841 44

788 13

50

80

the alveolar space via AM and up themucociliary escalator.

Ineflammatory ResponseThere was a progressive increase in totalcells lavaged from the TiO2-D exposed ani-mals during the 12-week exposure period(Figure 4), due to an increase of both AMand PMNs. The inflammatory response tothe TiO2-D persisted for a long period afterexposure. The numbers of these cell typeshad returned almost to normal levels 1 yearafter exposure. These effects were not (or

only modestly) observed with TiO2-F. Thelavagable protein (Figure 4), (as well ascytoplasmic and lysosomal enzymes-LDHand P-glucuronidase-data not shown),was also significantly elevated in the lavagefluid from animals exposed to the ultrafineparticles during and at the end of the expo-sure and at week 41 after the start of expo-sure. Taken together, these data show thegreater pulmonary inflammatory potencyof inhaled ultrafine TiO2 particles com-pared to fine TiO2 particles, whenlung doses are expressed in terms of particlemass (equal gravimetric doses). This con-firms earlier results obtained with intratra-cheally instilled ultrafine and fine TiO2particles (3).

However, when the inflammatoryinflux of PMNs was correlated with thesurface area of the retained particles (i.e.,lavagable particles) both ultrafine and fineparticle effects could be described by acommon dose-effect curve. This underlinesthe importance of considering differentdose parameters (e.g., mass vs surface area)when interpreting particle effects. Theseresults also confirmed earlier findings withinstilled particles (3).

Exposure1 Total Cells

- o.0\/ . \2-D/I \

° 2 Ti02'-FControl

8Weeks

co

0

*

z

0-

T41 64

Exposure' Lavage AM0

T TiQ2D*0 :

IT12-

t ~~~~Control

8Weeks

41

6)

C00

0

E

0'1E

CF4-,-

0L-

a.

64

0 41 64Weeks

Exposure: Lavage Protein

1--, TiO2 D L

Cont0.14-

u.u I I i I I

8Weeks

41 64

0.5

0.44

0.34

Figure 4. Lung lavage parameters during and after 12 weeks of exposure of rats to TiO2-D (ultrafine) and TiO2-F (fine) particles compared to sham exposed controls (mean ± SD; n= 8).

Environmental Health Perspectives

TiO2-D

T1i2-F

LN, lymph nodes.

to

0

xU)

0

40

30

20i

lot

u -_

40 Tto

0'- 30Q*

(nQ)C" 20-0

0o 100

50 T

4 nI

176

PARTICLE PERSISTENCEAND LUNG INJURY

Control>% ,o,102 -F

1.00 To02-Dp2N b

o ~~~~~~~b

(D 0.10-Caa

0.01Immediately 7 Monthsafter exposure after exposure

Figure 5. Pulmonary clearance rates of 'Sr labeled testparticles inhaled either immediately after 12 weeks ofexposure to TiO2 or 7 months later. (Mean and 95% confi-dence interval; a = significantly different from control; b =significantly different from TiO2-F).

Alveolar Macrophag ClearanceFunction

Subchronic inhalation of ultrafine and fineTiO2 particles resulted in a significantimpairment of AM-mediated test particleclearance (Figure 5). Control animals had aclearance rate of the test particles ofapproximately 1% per day, for the animalsexposed to the fine particles, the clearancerate was reduced to 0.6% per day; and forthose exposed to the ultrafine particles, itwas even further reduced, to 0.13% perday. Retention half-times were 66 days forthe control animals, 117 days for thoseexposed to fine particles, and 541 days forthose exposed to ultrafine particles. Sevenmonths after exposure, when the TiO2 bur-dens in the lung had decreased (Figure 2)the test particle clearance for animalsexposed to fine particles was no longerimpaired, whereas the animals exposed toultrafine particles still showed a signifi-cantly diminished clearance rate of 0.52%.

Since prolonged AM clearance functionin a particle overload situation may be cor-related with the volumetric burden of thephagocytized particles in the AM (1), dif-ferent dose parameters of the TiO2 particlesretained in the AM were correlated withthe impaired particle clearance. Neitheraverage gravimetric nor average volumetricburdens of both particle types correlatewell with the observed effect on AM clear-ance function (Table 3). With fine TiO2particles, there was an AM volumetric loadof 9%, greater than the 6% that Morrowhypothesized (1) to indicate a particleoverload condition with subsequent effecton particle clearance; indeed, prolongedclearance was seen in the present study.Exposure to ultrafine particles, however,resulted in much lower average volumetricburdens, which possibly indicated anincreased cytotoxicity of these particles.Expressing these doses as the retainedparticle surface area in the macrophages

shows that the effects on AM-mediatedclearance function of the two different par-ticle types can be expressed by a commondose-response curve (Figure 6). (Calcula-tion of the phagocytized volume is basedon an average rat AM volume of 1000 lim3[7] and of a TiO2 anatase density of3.8 g/cm3. The specific surface area hadbeen determined previously to be 50 m2/gfor ultrafine TiO2 and 6.4 m2/g for fineTiO2).

Lung MorphologyHistological evaluation of lung sections at41 weeks of the study showed early fibroticreactions in the ultrafine TiO2-exposedanimals and to a lesser degree also in theanimals exposed to fine TiO2. However, 1year after the exposure the early fibroticreactions had shown signs of regression andwere no longer as prominent (8). Type IIcell hyperplasia, especially in alveoli thatcontained aggregates of particle-laden AM,was also observed in the animals exposed toultrafine particles. The observation thatsuch epithelial response resulted inthe occlusion of the pores of Kohn mayhave important implications, since thesepores are thought to serve as interalveolarmigration pathways for AM (9-11).Interstitial inflammatory reactions, (i.e.,PMN increases), were also observed to ahigher degree at the end of the exposureperiod in the animals exposed to the ultra-fine particles.

DiscussionThis subchronic inhalation study with twosizes of TiO2 particles confirmed the previ-ous findings with these particles after intra-tracheal instillation. When the samegravimetric doses of ultrafine and fineTiO2 particles were delivered to the lung,ultrafine particles produced significantlygreater inflammation and interstitialtranslocation (2,3). The study also showsthat the pulmonary clearance of the ultra-fine particles was significantly slower, and

that this was due to an altered biopersis-tence in both the alveolar space and in thepulmonary interstitium. The overall reten-tion in the alveolar space was no differentfor fine and ultrafine TiO2 particles.However, compared to the fine TiO2, alarger fraction of the ultrafine TiO2 wastranslocated to the pulmonary interstitiumwhere it was retained for a longer time.Dissolution of TiO2 particles in lung tissuehas not been reported, and we can assumethat pulmonary clearance of these particlesis solely dependent on physical/mechanicalprocesses (Figure 1).

Several inhalation studies by differentlaboratories in the past have shown that alarge load of highly insoluble particles inthe alveolar space results in severe retarda-tion of AM-mediated particle clearance(12,13). This phenomenon was referred toas "particle overload," indicating an over-loading of the AM by phagocytized parti-cles with subsequent impairment of theirclearance function. Morrow (1) suggestedthat this is correlated with the phagocytizedparticle volume and that AM functionstarts to be impaired when on average 6%of the normal AM volume is filled byphagocytized particles. The load of AMwith fine TiO2 particles in our study didindeed reach the value of 9% (Table 3),which was associated with an almost dou-bling of the retention of inhaled test parti-cles. However, ultrafine particles onlyreached a phagocytized volume in the AMof 2.6%, yet they prolonged retention ofthe test particles by a factor of more than 4(Table 3). Even after 7 months, test parti-cle clearance continued to be retarded. Incontrast, correlating the prolonged reten-tion with the retained surface area of theparticles in AM showed that the data couldbe expressed with a dose-effect relationshipcommon to both particle types (Figure 6),independent of postexposure time.

Several points should be emphasized.One is that the overall retention of boththe fine and ultrafine particles did not dif-

Table 3. Particle dose parameters and effects on AM-mediated particle clearance.

Retained dose/l 0o AM AM-effectTime

postexposure, nl Particle no. Test particle retention,weeks pg (% of AM volume) cm2 xl 0- control = 1

Control 0, 29 0 0 (0) 0 0 1.0TiO2.F 0 340 90 (9) 21.9 10.9 1.8a

29 32.5 9 (0.9) 2.1 1.1 1.1TiO2-D 0 99.8 26 (2.6) 49.9 5420 8.2a

29 43.8 11 (1.1) 21.9 2380 2.0a

AM = alveolar macrophages. aSignificant change against controls p< 0.05.

Volume 102, Supplement 5, October 1994 177

OBERDORSTER ETAL.

_ . I I I

0 1*0 20 30 40 50

surface area of retained particles/1 06 AM

cm2Figure 6. Correlation between surface area of TiO2 particles phagocytized by AM and pulmonary retention half-time ofinhaled polystyrene test particles.

fer in the alveolar space (Table 1), althoughthe ultrafine particles were cleared via themucociliary escalator into the GI tract at a

significantly slower rate. This can beexplained by the effect on AM function as

demonstrated by the result of the test parti-cle clearance. A second point is that theretention in the alveolar space-more pre-

cisely the dose retained in AM expressed as

particle surface area-rather than the over-

all pulmonary retention of the TiO2 parti-cles, is important for the effect on AMclearance function. Third, the volumetricload of the AM is not a good predictor forthe delayed clearance effect when ultrafineparticles are involved. The surface area ofthe retained particles appears to be a betterdose determinant. Although our data are

limited at this point (four data pairs only,Figure 6), other studies have also demon-strated the importance of the dose parame-ter "surface area" for the adverse biologicaleffects of both fibrous and spherical parti-cles (14,15). This does not mean that AMvolume load may not be important for AMclearance function, in particular for largerparticles, as has been shown repeatedly(5,12,16). However, surface area may

become a significant factor when particlesize gets smaller, apparently gaining greaterimportance as dose parameter for particlesin the submicronic range.

The mechanism(s) underlying theretarded removal of AM-phagocytized par-

ticles from TiO2-D exposed lungsremain(s) to be elucidated. Green (9) andFerin (10) speculated that the pores ofKohn may serve as a shortcut by which AMmigrate from distal alveoli to more proxi-mal alveoli, positioned nearer the ciliatedairways, for subsequent transport up theconducting airways. It is therefore tempt-ing to speculate that the prolonged AM-mediated particle clearance observed withthe ultrafine particles was related to theType II cell hyperplastic response andocclusions of the pores of Kohn in affectedalveoli. These alveoli often contained largeaggregates of particle-filled AM, which, inturn, appeared subjectively to contain thevast bulk of the TiO2 present in the alveo-lar space compartment.

The dose parameter "surface area" ofthe particles also correlates better thanother dose parameters with the inflamma-tory PMN influx into the alveolar space.

This corroborates the earlier results withinstilled particles of several types includingultrafine and fine TiO2 rutile and anatase,and with ultrafine carbon black (3). In thepresent and earlier studies, the surface area

of the particles retained in the alveolarspace represented a better dose determinantthan surface area of the particles retained inthe lung in toto, which underscores the sig-nificance of determining particulate doselevels in individual lung compartments.

However, there may also be "interfer-ence" between lung compartments viareleased cellmediators affecting specificresponses, depending on the particle loadin each compartment. For example, theacute particle-induced inflammatory influxofPMN into the alveolar space was signifi-cantly reduced, when more than 50% of anacutely administered high dose of ultrafineTiO2 particles was rapidly translocated tothe interstitial space, thereby shiftinginflammatory events (e.g., release ofchemotactic factors) to the interstitium.This reduced PMN influx was notobserved with ultrafine carbon black parti-cles, which were not rapidly translocated tothe interstitium (3).

The observed early fibrotic reactions inthis study were more pronounced afterTiO2-D exposure than after TiO2-F. Thismay also correlate with the larger intersti-tial dose of the ultrafine particles, althoughit is conceivable that the particle burden inthe alveolar space also contributed to theseevents. It has been clearly demonstratedthat particles, including TiO2, will activateAM to release both proinflammatorycytokines and fibrogenic factors (17,18). Inthe present study it was observed thatfibroblast growth factors are released in thealveolar space, partly due to release by AM(data not shown). Since we could not assessthe fibrogenic activity occurring in theinterstitial space, we cannot differentiatebetween the contribution of alveolar andinterstitial burdens to the fibrogenicresponse. However, histological evidencefrom other studies points to the interstitialparticle load as being most important (19).

We conclude from these studies thatthe greater pulmonary effects of ultrafineparticles, compared to larger submicronicparticles, can be explained by their largerspecific surface area, the greater interstitialaccess, and their altered biopersistence,resulting in the increased retention ofultrafine particles. Pulmonary compart-mental doses thus should be consideredwhen evaluating effects (alveolar versusinterstitial versus total dose), and the intra-pulmonary kinetics and translocation ofparticles should be evaluated. We concludefurther that when evaluating the biopersis-tence and related dose-effect relationshipsof inhaled particles of largely different sizes,the particle surface area rather than themass of the retained particles appears to bethe most relevant dose parameter.

Environmental Health Perspectives

C\

H

-

c

0Ef-0c

5004

4004300-

200-

100-

A Control*TiO2FOTiO2D

---- - control (66d)-

178

PARTICLE PERSISTENCEAND LUNG INJURY

REFERENCES

1. Morrow PE. Possible mechanisms to explain dust overloading ofthe lungs. Fundam Appl Toxicol 10:369-384 (1988).

2. Ferin J, Oberd6rster G, Soderholm SC, Gelein R. Pulmonary tissueaccess of ultrafine particles. J Aerosol Med 4:57-68 (1991).

3. Oberdorster G, Ferin J, Gelein R, Soderholm SC, Finkelstein J.Role of the alveolar macrophage in lung injury: studies with ultra-fine particles. Environ Health Perspect 97:193-199 (1992).

4. Takenaka S, Dornh6fer-Takenaka H, Muhle H. Alveolar distribu-tion of fly ash and of titanium dioxide after long-term inhalationby Wistar rats. J Aerosol Sci 17:361-364 (1986).

5. Oberdorster G, Ferin J, Morrow P. Volumetric loading of alveolarmacrophages (AM): a possible basis for diminished AM-mediatedparticle clearance. Exp Lung Res 18:87-104 (1992).

6. Ferin J, Oberd6rster G. Translocation of particles from pulmonaryalveoli into the interstitium. J Aerosol Med, 5:179-187 (1992).

7. Dethloff LA, Lehnert BE. Pulmonary interstitial macrophages: iso-lation and flow cytometric comparisons with alveolar macrophagesand blood monocytes. J Leukocyte Biol 43:80-90 (1988).

8. Baggs R, Oberdorster G, Ferin J. Apparent regression of pulmonarylesions produced by inhaled titanium dioxide. Toxicologist 12:224(1992).

9. Green GM. Alveolobronchiolar transport mechanisms. Arch InternMed 131:109-114 (1973).

10. Ferin J. Pulmonary alveolar pores and alveolar macrophage-medi-ated particle clearance. Anat Rec 203:265-272 (1982).

11. Lehnert BE, Sebring RJ, Oberdorster G. Particle-cell relationshipsand cellular and anatomical changes in the lung following the depo-

sition of relatively high burdens of fine and ultra-fine titaniumdioxide. Am Rev Resp Dis 145:A800 (1992).

12. Muhle H, Creutzenberg 0, Beldmann B, Heinrich U,Mermelstein R. Dust overloading of lungs: investigations of variousmaterials, species differences and irreversibility of effects. J AerosolMed 3:S-111-S-128 (1990).

13. Mauderly JL, Jones RK, Griffith WC, Henderson RF, McClellanRO. Diesel exhaust is a pulmonary carcinogen in rats exposedchronically. Fundam AppI Toxicol 9:208-221 (1987).

14. Timbrell V, Ashcroft T, Goldstein B, Heyworth F, Meurman LO,Rendall REG, Reynolds JA, Shilkin KB, Whitaker D. Relationshipsbetween retained amphibole fibres and fibrosis in human lung tis-sue specimens. Ann Occup Hyg 32:(Suppl I), 323-340 (1990).

15. Oberdorster G, Yu CP. The carcinogenic potential of inhaled dieselexhaust: A particle effect? J Aerosol Sci 21:S397-S401 (1990).

16. Lehnert BE. Alveolar macrophages in a particle "overload" condi-tion. J Aerosol Med 2: S9-S39 (1990).

17. Driscoll K, Maurer JK. Cytokine and growth factor release by alve-olar macrophages: potential biomarkers of pulmonary toxicity.Toxicol Pathol. 19:398-405 (1991).

18. Piguet PF, Collart MA, Gran GE, Sappino A-P, Vassalli P.Requirement of tumor necrosis factor for development of silica-induced pulmonary fibrosis. Nature 344:245-247 (1990).

19. Adamson IYR, Letourneau HL, Bowden DH. Enhancedmacrophage-fibroblast interactions in the pulmonary interstitiumincreases fibrosis after silica injection to monocyte-depleted mice.Am J Pathol 134:411-418 (1989).

Volume 102, Supplement 5, October 1994 179