[CANCER RESEARCH (SUPPL.) 54, 1929s-1933s, April 1, 1994]

Inflammation, Chromosomal Instability, and Cancer: The Schistosomiasis Model1

Miriam P. Rosin,2 Wagida A. Anwar, and Amanda J. Ward

Epidemiology Division, British Columbia Cancer Agency, Vancouver, British Columbia, Canada [M. P. R., A. J. W.], and Department of Community, Environmental andOccupational Medicine, Ain Shams University, Cairo, Egypt ¡W.A. A.j

Abstract

Evidence is accumulating in support of a role for reactive oxygenspecies in the etiology of cancer. Inflammatory cells, such as neutrophils,macrophages, and eosinophils, are an important endogenous source ofoxygen radicals. Stimulation of these cells by tumor promoters or byforeign bodies (parasites, bacteria, etc.) causes the release of reactiveoxygen species. Laboratory studies have shown that genetic damage andneoplastic transformation are induced in vitro in cells cocultured withactivated inflammatory cells. We have recently begun to study the role ofinflammatory reactions in inducing genetic damage in a human population. This paper describes our initial studies of Egyptian patients infectedwith Schistosoma haematobium. This infection induces chronic inflammation and irritation in the urinary bladder and is associated with increasedcancer at this site. We describe a recently completed population study thatshows that infected individuals have elevated levels of genetic damage intheir bladders, as measured by the exfoliated cell micronucleus test.Treatment that kills the parasite also reduces the micronucleus frequencies. We also explore the hypothesis that altered sensitivity of clones ofcells in these patients to reactive oxygen species could be a force thatdrives the development of neoplasia by facilitating clonal expansion. Evidence is presented for the possible involvement of loci on chromosome 11in controlling the level of chromosomal breakage caused by oxidativedamage. We have shown that bladder carcinoma cells are sensitive tomicronucleus induction by promoter-activated neutrophils and that they

can be protected from this damage by insertion of a normal chromosome11. Further work is in progress to define the source of chromosomalbreakage in schistosomiasis patients and to begin to develop an understanding of the host factors protecting bladder cells in these individualsfrom genetic damage.

Introduction

One of the hallmark activities of tumor promoters in animals is theirability to recruit inflammatory cells to an application site and stimulate a respiratory burst in these cells. The cells release ROS3 such as

Superoxide anión and hydrogen peroxide, as well as lipid oxidationproducts (1, 2). This activity may be critical to tumor promotion.There is a strong concordance between the capacity of tumor promoters to stimulate inflammatory cells to release ROS and their capacityto promote tumors (3). Furthermore, blocking the respiratory burst inanimal models with agents such as retinoids, steroids, or antioxidantsleads to the inhibition of tumor promotion (3).

Little is known about the forces driving tumor promotion andprogression in humans. However, there is some suggestion that inflammatory processes may again play a role. Clinical studies havedocumented an association between inflammation and cancer fordecades (2, 4—7).As shown in Table 1, this association has been

observed in many different tissues. The source of the inflammation isvaried: in some cases it is not known, while in others viral, bacterial,or parasitic infections are implicated. However, in each case the

1 Presented at the 4th International Conference on Anticarcinogenesis & Radiation

Protection, April 18-23, 1993, Baltimore, MD. Supported by grants from the NationalCancer Institute of Canada and the Natural Science and Engineering Research Council ofCanada (M. P. R.).

- To whom requests for reprints should be addressed, at Division of Epidemiology,

Biometry, and Occupational Oncology, British Columbia Cancer Agency, 600 W. 10thAve., Vancouver, BC, V5Z 4E6 Canada.

1 The abbreviations used are: ROS, reactive oxygen species; MN, micronucleus; TPA,12-O-tetradecanoyl-phorbol-13-ace(ate; O^-MedG, O''-methyldeoxyguanosine; hpf, high-

power field; PMN. polymorphonuclear leukocyte; X/XO, xanthine/xanthine oxidase.

inflammation is longstanding (chronic) and the cancer develops at thesite of the inflammation. It is not known whether transient, recurrentinflammatory reactions are also associated with increased cancer risk.Nor is it known to what extent chemical and physical agents are acting

in humans to increase tumorigenesis by stimulating inflammatoryreactions. Many irritants thought to increase cancer development bystimulation of proliferation may also act by aggravating inflammationin the tissue and vice versa (4, 6, 8). It is often difficult to separate thetwo events since they commonly occur concurrently.

Interest in the role of inflammation in carcinogenesis has beenrevived recently. This renewed attention is largely due toaccumulating evidence that stimulated inflammatory cells arecapable of inducing genotoxic effects, such as DNA strand breaks(9, 10), sister chromatid exchanges (11), and mutation (12, 13), andof promoting neoplastic transformation (14, 15) in nearby cells.Evidence suggests that these effects may be oxidant mediated.DNA base modifications in cells cocultured with stimulatedinflammatory cells (i.e., activated with TPA to produce arespiratory burst) are characteristic of hydroxyl radical injury (16).Oxidized DNA bases are also induced in vivo in mice receiving atopical exposure to TPA. Wei and Frenkel (17) reported theinduction of 8-hydroxyl-2'-deoxyguanosine, thymidine glycol, and5-hydroxymethyl-2'-deoxyuridine in epidermal DNA of such mice

and attribute this induction to TPA-induced infiltration and

activation of inflammatory cells. The path by which this damage isinduced in a cell is still unresolved. One possibility is that thehydrogen peroxide produced by activated inflammatory cells isreacting with chromatin-bound metal ions and generating hydroxyradicals via a Fenton-type reaction. An alternative explanation is

that Superoxide anions interact with nitric oxide (another product ofstimulated inflammatory cells) and form a peroxynitrite radical thatdecomposes to hydroxyl radical and damages the DNA (18-20).

There are two other mechanisms by which inflammatory cells mayincrease genotoxic damage in a tissue. Inflammatory cells have beenshown to participate in the metabolic activation of procarcinogens toDNA-damaging species (7, 21, 22). Neutrophils activate aromatic

amines, aflatoxins, estrogens, phenols, and polycyclic aromatic hydrocarbons by oxidant-dependent mechanisms (7). Finally, several

studies implicate inflammatory cells in the formation of carcinogenicnitrosamines (23). Leaf et al. (24) reported that immunostimulatedrats are capable of nitrosating morpholine, probably from reactantsarising from nitric oxide production by macrophages.

The main goal of our current research is to develop a betterunderstanding of promotion in humans by focusing on the role ofinflammation and dysregulated cell proliferation in cancer development. Our initial focus is on inflammatory reactions. We have approached this goal by devising in vivo and in vitro model systems. Thein vivo approach has focused on studies of early cellular changes inthe bladders of Egyptian patients infected with Schistosoma haematobium. This parasite infection induces chronic inflammation andirritation in the urinary bladder and is associated with increased cancerat this site (25-28). The in vitro studies are aimed at identifying host

factors that elevate sensitivity of bladder cells to oxidative stress. Thispaper will describe our experience to date with these models.

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INFLAMMATION AND ELEVATED CHROMOSOMAL INSTABILITY

Table I //(i,'/i cancer risk in individuals with chronic inflammation

Adapted from Refs. 2 and 4-7.

Cancer sue Source of inflammation in tissue

Colon

Bladder

SpleenLiver

Biliary tract

Gall bladderLungStomach

Esophagus

Skin

.1. japontcurn infectionUlcerative colitisCrohn's disease

.V.liacinalobiutn infectionCatheterized patients with chronic cystitisRecurrent cystitis.V.mansitni infectionHepatitis B virusHepatic cirrhosisCionorchis sincsis infectionOpisioihorchi* mvrrini infectionChronic cholecystitisTuberculosislli'licohtictcr infection

Atrophie gastritisBarrett's esophagus

Reflux esophagitisChronic skin ulcers

Schistosomiasi* Model

Schistosomiasis is a common parasitic disease that ranks secondonly to malaria as a global cause of morbidity and mortality (29). Itoccurs in 74 tropical and subtropical countries and affects about 10%of the world's population (27, 28). The prevalence of this infection

remains high despite the availability of efficacious antischistosomaldrugs. Individuals become infected with the parasite at an early age byexposure to the free-swimming larval stage (termed cercariae) in

contaminated water. Even when treated, they will often becomereinfected, since they return to the same water source. Thus, theinfection is long term with the progressive development of pathological changes, often throughout a lifetime.

Three major species infect man. In each case the infection isassociated with an increase in cancer. Schistosoma munsoni infectionsare associated with the development of follicular lymphoma of thespleen (27, 30), Schistosoma japonicum infections with colon cancer(31), and 5. haemalobiiim infections with cancer of the urinary bladder (25-28). Although the evidence supporting the first two associa

tions is somewhat limited, the involvement of 5. haematobiiim infection in bladder cancer is more strongly supported. Elevated numbersof bladder carcinomas occur in geographic areas that are endemic forthe parasite. In these regions, the majority of bladder cancers willoccur in patients with schistosoma! cystitis. Finally, carcinoma ofschistosome-infected bladders have clinical features that are distinc

tive from those observed in bladder carcinomas in populations not atrisk for schistosomal infection. The cancers develop 1 to 2 decadesearlier and are predominantly squamous rather than transitional cellcarcinoma (for reviews see Refs. 26 and 32).

Various hypotheses have been proposed to explain why S. haema-

tohiiun infections predispose to bladder cancer (26, 32). It has beensuggested that environmental carcinogens such as cigarette smoke orpesticides (many infected individuals are farmers) could play a role inthe development of cancer by interacting synergistically with theinfection (26, 33). Another more favored suggestion implicates urinary nitrosamines as initiating agents. In support of this hypothesis aredata showing that W-nitroso compounds are elevated in the urine of

infected individuals to levels greater than those observed in uninfectedindividuals (34—37).These ^/-nitroso compounds may come fromexogenous sources (e.g., diet) or, alternatively, be formed endog-

enously through the action of bacteria or stimulated inflammatorycells, both commonly present in the bladder of these patients (36, 37).Further support for the involvement of nitrosamines in schistosomalbladder cancers comes from a recent study by Badawi et al. (37).These workers reported the presence of O''-MedG in bladder tissue

DNA of Egyptian subjects with bladder carcinoma and suffering fromschistosomal infections. O6-MedG was detected more frequently in

the bladder DNA of these patients than in normal bladder tissue ofEuropean origin (93% of Schistosomiasis samples compared with 33%of normal bladders) and at significantly higher levels. The formationand persistence of O''-MedG in DNA is correlated with the carcino

genic and mutagenic action of nitrosamines.Another factor that may play a major role in bladder carcinogenesis

in Schistosomiasis patients is the presence of continuous physicalirritation and inflammation produced by schistosomal eggs in thebladder mucosa. The adult S. Imematobium worms inhabit the veins ofthe perivesical plexus, where the female lays eggs. Some eggs passthrough the bladder mucosa and are excreted in the urine. Other eggsare trapped in the tissue. A chronic inflammatory reaction is initiated,with the invasion of histiocytes and other inflammatory cells into thebladder, the formation of granulomas, and eventually fibrosis. Inaddition, the eruption of the eggs through the mucosa stimulatesreparative urothelial hyperplasia and cell turnover.

Several workers have proposed multistep and multifactorial modelsfor bladder cancer development in Schistosomiasis patients (26, 32,36). These models attribute the initiation of carcinogenesis to lowdoses of nitrosamines or some other environmental carcinogen, withthe infection supplying the proliferative stimulus to drive the expansion of clones of initiated cells. Although animal studies with thisparasite infection have been difficult to perform, data obtained in astudy by Hicks et al. (38) support such a model. These workersstudied the effect of S. haematobiiim infections and low initiatingdoses of the bladder carcinogen jV-butyl-jV-(4-hydroxybutyl)nitro-

samine on the development of urothelial neoplasia in the baboon.Their data showed that, although proliferative and inflammatorychanges occurred in infected animals without carcinogen exposure,the nitrosamine treatment was necessary before early neoplasticchanges (carcinoma in situ) became apparent. The carcinogen treatment alone was too low to produce changes in the tissue. Thisexperiment was terminated after 2'/2 years, while the animals were still

relatively immature, so the long-term effects of the treatments are not

known. Also left unresolved is the mechanism by which the infectionwas contributing to the carcinogenic process. It is not known whetherthe inflammation itself was important or whether the infection wasworking through its effect on proliferation.

Involvement of Inflammation in Genetic Damage inSchistosomiasis Patients

Schistosomiasis is endemic in Egypt, particularly in rural communities. In 1990 we began working in a small village in the Fayyum, anoasis some 65 miles southwest of Cairo. This region is extensivelyirrigated by canals from the Nile and represents a major farming andfishing region in Egypt. Approximately 40% of the 10,000 inhabitantsof the village of Shakshouk are infected with the parasite.

The question that we posed was whether or not Schistosomiasispatients would have elevated levels of chromosome damage in theirbladders, and if so, whether these levels would be associated with thepresence of the infection (39). Our study design involved collectingexfoliated urothelial cells from the urine of Schistosomiasis patientsbefore and after treatment with an antischistosomal drug. MN frequencies were determined in these cells and used as an estimate ofgenetic damage occurring in the bladder. Micronuclei are formed bydamage to chromosomes or to the spindle apparatus in the basal cellsof the tissue epithelium. When these cells divide, chromosomal fragments (or entire chromosomes lacking an attachment to the spindleapparatus) lag behind and are excluded from the main nuclei in thedaughter cells. These fragments form their own membranes andappear as Feulgen-positive bodies in the cytoplasm of the daughter

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INFLAMMATION AND ELEVATED CHROMOSOMAL INSTABILITY

UJO

o 2

S

IoÅ’U

A. INFECTED

2

B. TREATED

2

1

Lft

C. CONTROLS

Fig. 1. Micronuclei frequencies in exfoliated cells collected from 37 schistosomiasispatients before (A ) and after (fl) treatment with praziquantcl. C. MN frequencies ofvillage controls.

cells. The daughter cells mature and are exfoliated. The exfoliated cellmicronucleus test has been shown to be a sensitive technique forquantifying the level of chromosomal breakage in tissues of individuals exposed to carcinogenic agents (40, 41).

Fig. \A is a histogram showing MN frequencies in exfoliated cellscollected by centrifugaron of urine samples from 37 male patientswith 5. haematohium infection. For comparison, Fig. 1C showsurothelial MN frequencies of 32 villagers, matched for age, sex, andsocioeconomic status but free of infection. Infection status is confirmed by analyzing urine from each individual for eggs and hema-

turia on at least two sampling occasions. A wide range of MNfrequencies is present in the infected group (range, 0-2.67%); how

ever, the frequencies are significantly elevated above those observedin the control group (Table 2, P < 0.0001). Fig. Ißshows the MNfrequencies of each infected patient in a second urine sample taken 2months after treatment with praziquantel. A significant decrease inMN frequencies is observed in response to the treatment (Fig. Iß.P < 0.0001). Posttreatment MN values did not differ significantlyfrom those observed in the control population (Table 2. P = 0.35).

These data show that schistosomiasis patients have elevated levelsof genetic change in their bladders and suggest that this changerequires the presence of the infection. When the parasite is killed. MNfrequencies return to levels observed in control populations. In orderto obtain more information concerning the source of the geneticdamage in these patients we evaluated the same specimens for inflammatory cells and bacteria. WBC and bacterial counts were estimated microscopically (X400 magnification) in preparations previously scored for MN frequencies. A determination was made of thenumber of eosinophils, PMNs, and bacterial cells per hpf.

The majority of urine samples from infected patients containedlarge numbers of eosinophils (35% of samples had >30/hpf). PMNs(44% of samples had 6-20/hpf), and bacteria (68% of samples had>50/hpf). A significant reduction in the number of eosinophils (P =0.0001) and PMNs (P = 0.01) occurred after treatment (comparisonsof pre- and posttreatment values with paired I test), resulting in levels

that were no longer significantly different from those observed in

uninfected controls (comparison of posttreatment and control valueswith x2 test: for eosinophils. P = 0.20; for PMNs, P = 0.06). In

contrast, the bacterial infection remained approximately unchanged inpatients before and after treatment (/-" = 0.44). staying at levels

significantly higher than those observed in controls (P < 0.001). Thisapproach provides only a crude estimate of bacterial infection; however, the data suggest that secondary bacterial infections are notresponsible for the elevated MN frequencies in patients with schistosoma! infection. These bacterial infections have been proposed as onesource for the /V-nitroso compounds found in the urine of infectedindividuals (34-37).

Although the data support a role of inflammatory cells in thegenetic damage, further studies are required to show that the relationship is causal or to determine the mechanism by which these cellswould be inducing the damage. A major problem in these studies is indistinguishing between effects due to altered cell proliferation inresponse to injury and effects resulting from the action of inflammatory cells. Elevated epithelial proliferation will also increase geneticdamage (for reviews see Refs. 4, 6, and 40). Furthermore, the inflammatory cells will themselves play a role in mediating proliferativechanges in the infected bladder via the generation of eicosanoids andother factors. We are currently extending this research to better defineinteractions among chromosomal breakage, proliferation, and oxida-

tive damage by using a battery of biomarkers for these events (forreviews see Refs. 3, 7, 40, and 42).

Is Altered Sensitivity to Oxidative Stress a Driving Force forAccumulation of Genetic Change?

Reactive oxygen species are continuously produced during normalcell metabolism as well as generated in response to an array ofxenobiotics. A complex defense system has evolved to deal with thesespecies. This system includes low molecular weight antioxidants anddetoxifying enzymes (supcroxide dismutase, catalase, and peroxi-

dases) in addition to a repair process that prevents fixation of DNAdamage resulting from radical attack. When the radical load increasesbeyond the capacity of these defenses (such as in the bladder ofschistosomiasis patients) or the system becomes defective, a situationknown as "oxidative stress" (43) or a "prooxidant state" (44) is

created and genetic damage occurs. One of the interests of thislaboratory is the identification of host factors that increase sensitivityto oxidative damage and, more specifically, the products of activatedinflammatory cells.

Recent studies suggest that loci on chromosome 11 may be involved in the control of oxidative DNA damage in cells. Parshad et al.(45) reported an abnormally high frequency of chromatid breaks andgaps when human tumors are X-irradiated during the G-, phase of the

cell cycle. This effect occurs in tumors of different tissue origin and/orhistopathology. Insertion of normal chromosome 11 into these tumorcell lines results in a reduction of radiation-induced damage to the

level observed in normal cells. These workers attribute this effect toa restoration of a defective DNA repair process and suggest that this

Table 2 MN frequencies nf bladder cells in ScUstOSOmiasis patients"

SubjectsSchistosomiasis

patientsBefore treatmentAfter treatment

ControlsNo.

ofpatients37

3732%

of cells withMN(mean)0.82r' 3. PMNs > 5. and bacteria > 3 per high-power field (X4(X) magnification).

Indicated comparison significantly different at /' < O.IMHH.

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INFLAMMATION AND ELEVATED CHROMOSOMAL INSTABILITY

Table 3 MN induction in response to oxidative stress in bladder carcinoma cell lineswith and without a chromosome 11 insert0

CelllineA1698

A1698 + dcr(ll)%

of MN inuntreatedcultures3.3

±0.44.0 ±0.3%

of MN induced with differenttreatments'"TPA-activaled

PMNs5.5

±0.70.5 ±0.6X/XO(1)8.0

±1.10.4 ±0.4X/XO

(2)9.6

±0.12.6 ±0.7

" Original study described by Ward et ai (48). Cultures were kindly provided by Dr.

O. Pereira-Smith (Baylor College of Medicine. Houston. TX).h MN frequencies in untreated cultures have been subtracted to provide induced values:

treatments were coincubatcd with human neutrophils stimulated with TPA (10 ng/ml) orexposure to xanthine (174 mg/ml) plus xanthine oxidase (doses 1 and 2, 1.2 X 10~6 and1.8 X 10~6 UIHK/i I. respectively). Data represent results of 3 replicate experiments for

treatments and 12 for untreated controls. Parent vs. hybrid. P < 0.01 for all treatments butnonsignificant for untreated cultures.

defect is a critical early event in tumorigenesis (46). It is possible thatthe same mechanism is affecting the capacity of bladder cells to dealwith DNA damage by ROS. Chromosome 11 alteration is a commonoccurrence in tumors of the urinary bladder (47).

In order to test this hypothesis we assayed two cell cultures forsensitivity to oxidative stress: A1698, a bladder carcinoma culture(termed parent), and A1698 + derll, a cell line created from A1698by microcell-mediated transfer of a normal chromosome 11 (termed

hybrid). The cultures were exposed for l h to X/XO (a treatmentwhich produces a mixture of Superoxide and hydrogen peroxide) orcoincubated with TPA-activated human neutrophils. Micronucleus

frequencies were significantly lower in the hybrid cultures containingthe chromosome insert (Table 3).

We have yet to determine the mechanism underlying this protection. However, the altered sensitivity does not appear to be due to adifference in the level of single-strand DNA breakage and its rejoining. In a recent study, we assayed parent and hybrid cells for single-strand DNA breakage after X/XO treatment using the single-cell gel

electrophoresis assay (alkaline comet assay). This assay analyzesindividual cells within a population and provides data concerning theheterogeneity of DNA breakage and repair. There was no significantdifference between parent and hybrid cultures in either the initialamount of single-strand DNA breakage at treatment (P > 0.1) or after

20 min of repair (P > 0.1) (48).It is not yet known whether the phenomenon described above will

be restricted to the cell culture being used or be more universal innature. However, it is possible that alterations such as this will play amajor role in driving the progression of a tissue from a normal to aneoplastic state. Clones of cells with an inherited alteration in thecapacity to repair oxidative DNA damage would show an elevation ingenetic instability and an increased probability of acquiring the multiple alterations to specific oncogenes or suppressor genes required fortumor development (40). Schistosomiasis patients would appear to bean appropriate model system in which to test such an hypothesis.

Conclusion

In this paper we have summarized some of the first evidencesupporting an association between chronic inflammation in humansand an elevated level of genetic damage in the inflamed site. Furtherwork is necessary to prove that this damage is actually due to theinflammation and, if so, to define the mechanism by which theinflammation is acting to produce the damage. We have stressed thepossibility that ROS are involved. However, we have indicated alternatives that need to be considered. The inflammatory cells could alsobe increasing genetic damage through nitrosamine formation, bybioactivation of carcinogens, and/or through enhanced stimulation ofcell proliferation.

In summary, we have described a human model system that is veryamenable to studies of the role of inflammation in cancer. Further

expansion of these studies should involve the use of the biomarkerscurrently available for oxidative damage, genetic change, and cellproliferation to test these hypotheses.

Acknowledgments

The authors would like to acknowledge the expert technical assistance ofMs. C. Michelsen.

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