[CANCER RESEARCH 60, 5414–5419, October 1, 2000]

Ovarian Tumors in Rats Induced by Chronic 2,3,7,8-Tetrachlorodibenzo-p-Dioxin Treatment

Barbara J. Davis,1 Elizabeth A. McCurdy, Brian D. Miller, George W. Lucier, and Angelika M. TritscherLaboratory of Women’s Health [B. J. D., E. A. M.] and Laboratory of Computational Biology and Risk Analysis [B. D. M., G. W. L.], National Institute of Environmental HealthSciences, Research Triangle Park, North Carolina 27709, and Nestle Research Center, CH-1000 Lausanne 26, Switzerland [A. M. T.]

ABSTRACT

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is a multispecies repro-ductive toxicant, and it has been recently classified by IARC as a knownhuman carcinogen. Here, we report that TCDD promotes the developmentof ovarian tumors in an initiation-promotion model in female SpragueDawley rats. Rats were initiated with diethylnitrosamine (DEN) or vehicleat 70 days of age. Starting 2 or 18 weeks after initiation, rats were exposedbiweekly to TCDD at a daily average dose of 125 ng/kg/day for 14, 30, or60 weeks continuously or for 30 weeks plus withdrawal periods of 16 or 30weeks. Fifteen of 76 (20%) rats initiated with DEN and promoted withTCDD for various lengths of time developed ovarian sex cord-stromaltumors of Sertoli cell type, whereas no ovarian tumors developed in 86rats used as vehicle controls or that received DEN alone or TCDD alone.The highest tumor incidence occurred in 6 of 14 rats (43%) after 60 weeksof continuous TCDD after DEN initiation. One of six rats developed atumor by 30 weeks of exposure. Because most effects of TCDD can beattributed to its activation of the aryl hydrocarbon receptor (AhR), thepresence and localization of AhR was determined in the rat ovary and inthe ovarian tumors by reverse transcription-PCR, immunohistochemistry,and in situ hybridization. AhR was localized to oocytes, granulosa andthecal cells of growing follicles, surface epithelial cells, and epithelial cellslining single tubules in ovaries from adult control Sprague Dawley rats.Neoplastic cells in the ovarian tumors were also positive for both AhRmessage and protein. These results indicate that the ability of TCDD tocause ovarian tumors is dependent on initiation, length of promotion, andage of the animal when exposed and evaluated. The tumor type induced byTCDD in this experimental system is the same histological subtype as thatreported from an early study of youngsters exposed during an industrialaccident in Seveso, Italy.

INTRODUCTION

The polyhalogenated aromatic hydrocarbon TCDD2 is a ubiquitousenvironmental contaminant that produces a spectrum of biochemicaland adverse biological effects in people and in a wide variety ofexperimental animal models, including reproductive, developmental,and carcinogenic effects (1, 2). TCDD has recently been classified asa known human carcinogen (3). Whereas TCDD is a multisite car-cinogen in rodents, the most commonly studied model is liver tumorinduction, particularly through initiation and promotion studies (4, 5).Using an initiation and promotion model, Lucieret al. (6) demon-strated that the ovary needs to be intact to observe the hepatocarci-nogenic and proliferative action of TCDD in female rats. This findingsuggested that the tumor-promoting effect of TCDD may be mediatedeither by direct modulation of ovarian function or through indirectendocrine modulation. Antiestrogenic activity has been ascribed toTCDD (7) through studies showing TCDD inhibits the uterotropicaction of estrogen (8), induces uterine atrophy, and causes reproduc-

tive failure in mice (9). TCDD also decreases expression of ER in theliver (10) and uterus (11).

TCDD may act directly on the ovary because the AhR message hasbeen identified in rodent and primate ovaries by RT-PCR (12), and thegeneral scientific consensus is that most, if not all, of the effects ofTCDD are mediated by initial binding to this transcription factor (13,14). Moreover, the binding affinity of polycyclic aromatic hydrocar-bons to the AhR correlates with the ability of these chemicals to causeovarian toxicity and ovarian tumors in a strain and species-dependentmanner in mice (15). Thus, it is possible that TCDD elicits directeffects on the ovaries through the AhR that, in turn, modulateshormone levels, ER, or other ER-mediated pathways. Given thecentral role of the ovary and ovarian hormones in TCDD carcinoge-nicity in the liver, the purpose of this study was to determine theovarian pathology within the framework of an initiation-promotionmodel and determine whether AhR is a factor in the ovarian pa-thology.

MATERIALS AND METHODS

Chemicals. TCDD dosing solutions were obtained from Radian Corp.(Morrisville, NC). Concentration and purity were controlled by gas chroma-tography. All other chemicals were obtained from Sigma Chemical Co. (St.Louis, MO), unless specified in the text. All PCR reagents were from Promega(Madison, WI).

Animals and Experimental Design. The study design has been describedelsewhere (16). Briefly, at 70 days of age, female Sprague Dawley rats wereinitiated with a single dose of DEN i.p. at 175 mg/kg in saline vehicle (1 ml/kgbody weight) or saline only. Two or 18 weeks after initiation, promotion wasstarted with biweekly oral gavage of TCDD in corn oil at a dose of 1750 ng/kg,equivalent to 125 ng/kg/day. Controls received corn oil. The study includedfour treatment categories: (a) groups dosed continuously with TCDD orvehicle for 14 weeks, 30 weeks, or 60 weeks, beginning 2 weeks afterinitiation; (b) groups dosed continuously with TCDD for 30 weeks, followedby 16 or 30 weeks of treatment with corn oil alone; (c) groups dosed withTCDD for 14 or 30 weeks beginning 18 weeks after initiation; and (d) groupsdosed with TCDD for 30 weeks, followed by 16 weeks of dosing with corn oilonly beginning 18 weeks after initiation. Necropsies were performed 1 weekafter the last treatment. All time points included control animals receiving thevehicles saline and corn oil only and animals initiated only.

Serum was collected from cardiac puncture at necropsy. Serum levels ofestradiol, progesterone, and testosterone were analyzed by RIA (DiagnosticProducts Inc., Los Angeles, CA) from serum stored at270°C. A completehistomorphological examination was done on ovaries, uterus, cervix, vagina,and all grossly characterized masses of the reproductive tract at all time pointsof the study. Rats were categorized into stages of the estrous cycle by vaginalhistology and correlated with uterine and ovarian histology.

Cell proliferation rates were determined by BrdUrd incorporation by miniosmotic pumps (Alzet model 2 ml, 10 ml/hr; Alza Corp., Palo Alto, CA) filledwith 30 mg/ml BrdUrd implanted into each rat 7 days before necropsy. NuclearBrdUrd incorporation was detected by immunohistochemistry using 1:50 di-lution of mouse-anti-BrdUrd antibody (Becton Dickinson, San Jose, CA). Cellproliferation rates were generated by computer-assisted image analysis using aMicroimage Video system and NIH image (v5.8). Positive (brown) nuclei andtotal nuclei were counted from at least five,340 fields per slide from fourseparate ovaries with a diagnosis of hyperplasia and three ovaries with adiagnosis of tumor.

Received 1/3/00; accepted 8/2/00.The costs of publication of this article were defrayed in part by the payment of page

charges. This article must therefore be hereby markedadvertisementin accordance with18 U.S.C. Section 1734 solely to indicate this fact.

1 To whom requests for reprints should be addressed, at National Institute of Envi-ronmental Health Sciences, P.O. Box 12233, MD B3-06, Research Triangle Park, NC27709. Phone: (919) 541-2764; Fax: (919) 541-7666; E-mail:Davis1@NIEHS.NIH.GOV.

2 The abbreviations used are: TCDD, 2,3,7,8-tetrachlorodibenzo-p-dioxin; ER, estro-gen receptor; AhR, aryl hydrocarbon receptor; RT-PCR, reverse transcription-PCR;BrdUrd, bromodoxyuridine; DEN, diethylnitrosamine.

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AhR Studies. RT-PCR was performed on total RNA isolated from a singlefrozen rat ovary using TRI Reagent (Molecular Research Center, Inc., Cincin-nati, OH). Primer pairs for RT-PCR were selected from the rat AhR (GenBankaccession no. U09000), nucleotides 1399–1422 (59-tgcggggctcgaaagaagaca-gag-39) for reverse primer and nucleotides 1073–1096 (59-ggaggtgggtccagtc-caatgcac-39) for forward primer and made by Bioserve Biotechnologies (Lau-rel, MD). RT-PCR was performed on 800 ng of total RNA. The PCR reactionwas 35 cycles of 30 s at 94°C, 30 s annealing at 60°C, and 30 s at 72°C,followed by a 4-min extension at 72°C. Restriction enzyme digest using FokI and BamHI (Promega) confirmed the AhR product.

In situ hybridization was performed using a digoxigenin-labeled probegenerated from a 1.8-kb cDNA fragment of the mouse amino terminal DNAsequence subcloned into pBluescript II plasmid (Stratagene, La Jolla, CA),which was kindly provided by C. Bradfield (17) and used previously forin situhybridization studies (18). The mouse sequence has nearly 100% homology tothe rat AhR (19). The plasmid was linearized withXhoI or XbaI (Stratagene),then labeled with digoxigenin using an Ambion transcription kit (Austin, TX).Riboprobes were then reduced to 150–200 bp by alkaline hydrolysis and usedat a final concentration of 1.0 mg/ml. The hybridization was performed onformalin-fixed, paraffin-embedded sections of mouse and rat liver (as con-trols), rat ovary, and ovarian tumor using methods described previously (20).

Immunolocalization of AhR was done on normal Sprague Dawley rat ovaryand ovarian tumors using a standard avidin-biotin antiperoxidase detectionsystem (Vector Laboratories, Burlingame, CA). TCDD-treated rat liver servedas a positive control. AhR antibodies were generated from rabbits immunizedwith a synthetic peptide corresponding to amino acids 371–399 of the humanAhR plus a COOH-terminal cysteine conjugated to maleimide-activated ImjectKLH (Pierce Chemical Co., Rockford, IL). The specificity of the immunopu-rified antibody (Immunopure Plus Protein A IgG purification kit; PierceChemical Co.) as well as the cross-reactivity with rat AhR was confirmed byWestern blot analysis (data not shown) on normal rat liver. AhR was detectedin 5-mm sections of fixed tissues following standard protocols. Positive stain-ing was visualized with 3,39-diaminobenzidine tetrachloride (Sigma Fast DABtablet set; Sigma Chemical Co.) using 0.05% Toluidine Blue as counterstain(Fisher Scientific, Fair Lawn, NJ).

Statistical Analyses.Serum hormones were analyzed by ANOVA after logtransformation of data (JMP; SAS Institute, Inc., Cary, NC). Tumor incidencedata were analyzed by Fisher’s exact test.

RESULTS

Ovarian tumors developed in 15 of 76 (20%) rats initiated withDEN and promoted with TCDD, whereas no ovarian tumors devel-oped in the 86 rats that served as vehicle controls or received DENalone or TCDD alone (Table 1). The highest tumor incidence occurredin DEN-initiated rats treated with TCDD continuously for 60 weeks (6of 14 rats). The tumor incidence in this group was statistically differ-ent (P, 0.05) from the DEN-initiated concurrent control group (0 of11). The earliest time point of occurrence of this tumor was after 30weeks of continuous TCDD treatment, with one of six animals de-veloping this tumor (17% incidence). Tumors were also in rats ingroup II that were continuously dosed for 30 weeks with TCDD, butthen kept for an additional 15 or 30 weeks on vehicle alone. In groups

Fig. 1. Sprague Dawley rat ovaries from the 60 week,DEN/TCDD treatment group.A, ovarian tumor character-istic of Sertoli cell tumor (large arrow), with remnantovarian follicle (small arrow); H&E.Bar, 200 mm. B,typical tubular pattern of ovarian Sertoli cell tumor athigher magnification; H&E.Bar, 50mm. C, higher mag-nification of B demonstrating cell morphology. Thelargearrow points to a mitotic figure and thesmall arrowpoints to intracytoplasmic granules.Bar, 25 mm. D, nu-clear BrdUrd incorporation in ovarian Sertoli cell tumorwith typical heterogeneousbrown staining of nuclei (ar-rows); hematoxylin counterstain.E, small tumor develop-ing adjacent to follicles, both demonstrating marked stain-ing for BrdUrd.Bar, 200mm. F, higher magnification offollicle showing both typical granulosa cell morphologyand aberrant Sertoli cell morphology with remnant ofoocyte still within the follicle.Bar, 50 mm.

Table 1 Incidence of ovarian tumors by TCDDa

GroupTime

(weeks)

Tumor incidence

Vehicle initiatedb DEN initiatedb

Control TCDD Control TCDD

I. 2 weeks after initiation 14 0/6 0/6 0/6 0/6Continuous dose 30 0/6 0/5 0/6 1/6c

60 0/6 0/6 0/11 6/14c,d

II. 2 weeks after initiationContinuous dose (30 weeks)- 30–16 0/6 0/6 0/6 1/6c

Discontinue (16/30 weeks) 30–30 0/6 0/9 0/11 1/11c

III. 18 weeks after initiation:Continuous dose 14 1/6c

30 1/15c

IV. 18 weeks after initiation:Continuous dose (30 weeks)-Discontinue (16 weeks) 30–16 4/12c

a Data represent the number of female Sprague Dawley rats with ovarian tumors pertotal number rats at study end. Rats were treated biweekly with TCDD at a daily averagedose of 125 ng/kg/day for up to 61 weeks. Control rats received corn oil.

b Rats were treated at 10 weeks of age with a single dose of 175 mg DEN/kg(DEN-initiated) or saline (noninitiated).

c Significantly different from control (P, 0.05) by Fisher’s exact test.d One rat had bilateral tumors.

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III and IV in which rats were exposed to TCDD beginning 18 weeksafter initiation, 1 of 6 rats was identified with an ovarian tumor afteronly 15 weeks of TCDD treatment. After 30 weeks, 1 of 15 ratsdeveloped an ovarian tumor. Four of 12 (33%) rats developed ovariantumors when dosed for 30 weeks and then kept for an additional 17weeks on vehicle only.

All tumors were diagnosed as ovarian sex cord-stromal tumors ofSertoli cell pattern. The microscopic pattern of the tumors was char-acterized by expansile nodules of densely packed, varying sizedtubules (resembling seminiferous tubules) separated by a fine fibro-vascular stroma (Fig. 1,A–C). Tubules were lined by one to threedisorganized layers of elongated or polyhedral cells with abundantpale eosinophilic wispy cytoplasm and basally located nuclei. At leastone-fourth of the cells in a tubule contained large clear intracytoplas-mic vacuoles, and other cells contained numerous round, 1–2-mmdiameter, bright eosinophilic intracytoplasmic granules (Fig. 1C).Nuclei were vesicular with a single small nucleolus. Benign tumorswere diagnosed microscopically based on size (1-mm diameter orgreater), distinct separation from ovarian stroma into nodular masseswith partial or complete encapsulation, and compression of surround-ing parenchyma.

Whereas most tumors were benign, ovarian Sertoli cell carcinomaswere diagnosed in four rats in the 60-week continuous exposuregroup. Diagnosis of carcinoma was based on size, complete efface-ment of ovarian tissue, lack of a capsule, marked anaplasia, cellularand nuclear atypia, and pleomorphism of the tumor cells, although nometastasis occurred. Two of the carcinomas had been identified atnecropsy. The largest tumor was located in the right ovary andmeasured 2.43 1.43 1.4 cm, and the second, also in the right ovary,measured 6 mm in diameter. One rat in the 60-week continuousTCDD exposure group had bilateral benign ovarian Sertoli cell tu-mors, whereas all other rats had unilateral ovarian tumors with con-tralateral ovarian atrophy or interstitial cell hyperplasia. Hyperplasticlesions were characterized by poorly demarcated, unencapsulated,

1-mm or less areas of Sertoli cell-like tubular proliferations. Hyper-plastic Sertoli cell-like foci appeared concurrently with tumors, butdid not precede tumor formation temporally (data not shown).

Ovaries from four rats with hyperplastic lesions and three rats withbenign tumors were stained for BrdUrd incorporation as an indicatorof cell proliferation (Fig. 1,D–F). Cells were stained for nuclearBrdUrd in hyperplastic foci in a random pattern with a mean of4.75 6 1 per 62 cells (7.6% proliferation rate). Neoplastic cellsstained for nuclear BrdUrd in a much higher frequency, and in sometubules all cells incorporated BrdUrd. The mean number of positivenuclei was 306 2.6 per 53 cells (78% proliferation rate).

The presence of ovarian AhR message was confirmed by RT-PCR(Fig. 2). AhR protein and message were localized by immunohisto-chemistry andin situ hybridization in a hyperplastic lesion, twobenign tumors, and one malignant tumor. Faint nuclear and cytoplas-mic immunopositive staining and intense staining for message waspresent in the large hyperplastic and neoplastic pleomorphic cellslining tubules (Fig. 3). Additionally, AhR message and protein werelocalized to the nucleus and cytoplasm in oocytes, and granulosa andthecal cells of primordial and growing follicles in ovaries from acontrol rat, a noninitiated animal treated with TCDD for 60 weeks,and in an ovary from a DEN-initiated rat with 60 weeks of TCDDtreatment that also contained a benign tumor (Fig. 4). Minimal to nostaining for receptor was seen in granulosa cells or thecal cells ofmature, antral follicles.

AhR protein and message were also localized the surface epithelialcells. An intensedark brownnuclear staining was found in the surfaceepithelial cells and in Sertoli-like stellate cells lining single tubules ofabout 250mm diameter presumed to be cross-sections of aberrant

Fig. 2. Expression of AhR in normal ovaries from young adult Sprague Dawley rats.Top, expected 349-bp PCR product;bottom, confirmed AhR product by restrictionenzyme digest using Fok I andBamHI (both) orBamHI.

Fig. 3. AhR localization in ovarian Sertoli cell tumor.A, AhR protein localization byimmunohistochemistry.Brownstaining is positive signal localized to many nuclei (arrow)compared with other nuclei (arrowhead).Bar, 60mm. B, negative control forA. C, AhRmessage localization by nonisotopicin situ hybridization.Blue-purplestaining is positivesignal (arrow), compared with cells not staining (arrowhead); nuclear fastred counter-stain.Bar, 60 mm. D, negative control forC.

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atretic follicles (Fig. 4C). These cells also had faint cytoplasmicstaining. Such tubules may have represented precursor lesions giventhat similar structures, but surrounding an oocyte, were found withintumors (see Fig. 1,E andF).

No significant treatment-related changes in serum estradiol andprogesterone levels or ratios of estradiol:progesterone were detectedafter data were log transformed and analyzed by ANOVA. No sig-nificant differences were detected when rats with tumors were com-pared with appropriate controls at 60 weeks. Testosterone levels werebelow the detection levels of the assay in all cases.

DISCUSSION

This is the first study to demonstrate that TCDD promotes thegrowth of ovarian sex cord-stromal tumors of the Sertoli cell variantin Sprague Dawley rats initiated with DEN. The experimental evi-dence supporting the tumor promotional effect of TCDD in this designinclude the observations that: (a) ovarian tumors were confined to theDEN/TCDD groups; (b) the highest incidence of tumors were found

in rats with the longest promotion (60 weeks); and (c) more tumorshad features characteristic of malignancy in this group. In contrast, noovarian tumors developed in rats used as vehicle controls or thatreceived DEN alone or TCDD alone. The time course analysis sug-gests that continuous exposure to TCDD (60 weeks) is most effectivein promoting tumors. However, the occurrence of tumors in groups inwhich rats were older when promotion was started suggests that, inaddition to the length of TCDD exposure, the development of ovariantumors can be influenced by the length of time after DEN initiation,and/or by the age of the animal at the time of exposure. Suchobservations for the induction of ovarian cancer are consistent withthose previously determined for liver tumors (21). Additionally, cellproliferation rates were dramatically increased in the ovarian tumorscompared with hyperplastic foci and normal tissue, suggesting thatTCDD promotes proliferation of preneoplastic cells, leading to tumorssimilar to that seen in promotion of liver foci (22).

The primary effect of TCDD on the adult female rat reproductivetract appears limited to the ovary, because no other significant lesions

Fig. 4. AhR localization in ovaries.A, immunohis-tochemistry for AhR in an ovary that also contained atumor. AhR protein localized to surface epithelial cellsand oocyte and granulosa cells of primary follicle(arrow) compared with primordial follicle (arrow-head).Bar, 60mm. B, light positive staining for AhRprotein localized to surface epithelial cells and manygranulosa cells of a growing follicle (arrow). *, oo-cyte; bar, 60mm. C, intense staining for AhR proteinin Sertoli-like cells within a degenerative follicle (ar-row). Surface epithelial cells also stain positive.Bar,50 mm. D, negative control for immunohistochemistryfor A–C. Thearrow points to follicle. *, oocyte;bar,60mm. E, AhR message localization by nonisotopicinsitu hybridization.Blue-purplestaining is a positivesignal in granulosa cells, thecal cells, and oocyte offollicles (arrows) and in surface epithelial cells; nu-clear fastred counterstain. *, oocyte;bar, 60 mm. F,negative control forE. *, oocyte;bar, 60 mm.

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were found that could not be attributed to age or weight (data notshown). Given the specificity of effect in this study, TCDD appears toact directly on the ovary to promote tumor development. Previousstudies have found that TCDD acts as an endocrine disrupter. Forexample, a single treatment of TCDD suppressed estradiol productionby the ovary in immature rats stimulated with ovulatory doses ofgonadotropins (23). Additionally, in primates, chronic administrationof TCDD decreased serum estrogen and progesterone levels (24).Although changes in serum hormone levels may have occurredacutely but undetected in our study, there were no significant differ-ences in serum hormone levels between the various treatment groupsat any time point. Thus, it is likely that the tumors were promoted byTCDD directly acting on the ovary. The resulting ovarian changesmay then secondarily impact hormonal balances. Indeed, Sertoli celltumors in women may manifest clinically as hyperestrogenism (25).

Sertoli cell tumors belong to the class of sex cord-stromal tumorsthat includes the histological subtypes of granulosa cell tumors, the-comas, Sertoli cell tumors, and stromal tumors (26). Sertoli celltumors or Sertoliform-like tumors occur as a rare spontaneous tumorin about 2% of Sprague Dawley rats (154 of 7748 rats) at 130 weeksof age (27). These tumors have also been induced byN-ethyl-N-nitrosourea but not by DEN at equivalent doses used in this study (28).Thus, the rarity of this tumor in rats and the initiation/promotionmodel used in this study are further support that TCDD promotes thedevelopment of Sertoli cell tumors in female rats.

The histomorphological observations of our study suggest that theovarian Sertoli cell tumors are derived from granulosa cells, becausea number of tumors contained both granulosa cells and Sertoli cells,and in some cases both cell phenotypes surrounded an oocyte. Duringdevelopment of the gonad, Sertoli cells and granulosa cells arise fromthe same progenitor sex cord cell while their function and fate becomedifferentially determined in each sex. In the ovary, granulosa cellshave a finite life span and die through apoptosis either during follic-ular development or after differentiating to a luteal cell after ovula-tion. In the testes, Sertoli cells normally have an indefinite life span asthey support spermatogenesis. We propose that promotion by TCDDaltered a fundamental apoptotic program of the DEN-initiated gran-ulosa cell, allowing for survival and transdifferentiation to the male-patterned Sertoli cell. Indeed, TCDD has been shown to inhibitapoptosis in initiated hepatocytesin vivo (29) and in vitro (30).Moreover, this inhibition of apoptosis of selected subpopulations ofhepatocytes has been proposed as critical mechanistic step in livertumor promotion in these models. The observed tumor promotion inthe rat ovary could follow a similar mechanism of action.

A dysmorphogenesis or transdifferentiation of follicular growth andatresia characterized by follicles containing remnants of oocytes sur-rounded by Sertoli cells has been described in the ovaries of micedeficient in both estrogen receptorsa andb (31). As TCDD has beenshown to down-regulate ER in the liver and uterus (10, 11), it ispossible that TCDD interacts and down-regulates ER in the granulosacells as it promotes tumor growth. Because most effects of TCDD canbe attributed to its activation of the AhR, the presence and localizationof AhR in undifferentiated granulosa and thecal cells, which are of sexcord-stromal lineage, and within the neoplastic cells in the ovariantumors in this rodent study suggests that TCDD may directly promotethe survival and growth of initiated cells through receptor-mediatedevents. The role of the AhR and its activation in mediating TCDDtoxicity and potentially its carcinogenicity is complex because TCDDmay mediate some toxic and carcinogenic effects independently ofactivation of the AhR (32). Nonetheless, the identification of the AhRmessage and protein within TCDD-promoted ovarian tumors coupledwith the well-documented role of AhR in TCDD-mediated effectsprovide strong evidence that this receptor pathway may be involved in

ovarian tumorigenesis. Moreover, the identification of the AhR mes-sage and protein in cells of immature follicles but not in differentiatedcells suggests this receptor may have a physiological role in follicularcell function as well. However, additional studies of the role of AhRin ovarian function and in tumorigenesis is needed because the anti-body used for the immunolocalization studies was a polyclonal pep-tide antibody that can cross-react with other unidentified proteins3 andbecause a systematic evaluation of expression in all tumors and in allovarian cell types under various hormonal and cycle conditions hasyet to be done.

An IARC working group concluded that TCDD seems to actsimilarly in rodents and people, and several epidemiological studieshave shown an association between high dioxin exposure and adverseeffects, including increased overall cancer risk in people (3). Resultsfrom two epidemiological studies further support a link betweenovarian cancers in women and the ovarian cancers produced experi-mentally through TCDD promotion in the rat. The first study inves-tigated cancer incidence in 20,000 young, 0–19-year-old, Seveso,Italy, residents within the first 10 years after exposure to TCDD in anindustrial accident (33). One ovarian androblastoma and one ovariantumor of germ cell origin were reported based on hospital dischargerecords. Sertoli cell tumors are also known as “androblastomas” or“arrhenoblastomas” and occur occasionally in women (25). However,no ovarian tumors were expected or found in the aged-match refer-ence population, and the relative risk for ovarian tumor developmentwas infinity in the TCDD-exposed group. Because no direct exposuremeasurements were available from the persons developing the tumors,the true exposure of the tumor-developing individuals is unknown.The second study investigated cancer incidence in a cohort of 334women exposed to high levels of dioxins as well as hexachlorocyl-cohexane during the manufacturing of different herbicides and insec-ticides (34). An increased incidence of ovarian cancer (4 cases ob-servedversus2.6 cases expected; 95% CI, 0.41–3.96) was reported.However, no information is available on the exact types of tumors inthis cohort.4 It is possible that the tumors occurred by random chancebecause there is a low number of cases and these tumor types canoccur in young children as well as women. However, it may also bepossible that the occurrence of ovarian tumors in these exposedhuman populations is truly significant. Given that our experimentalstudies produced ovarian tumors through TCDD promotion, that theAhR is present in follicular cells and tumors that are common betweenspecies, and that common ovarian tumor types are now reported inrodents and women exposed to TCDD, it is possible that exposure toTCDD and activation of the receptor may increase the risk for thedevelopment of ovarian cancer in women as is the case in the exper-imental model.

ACKNOWLEDGMENTS

We thank Dr. Joseph Haseman for review and statistical analysis and Dr.Nigel Walker for review.

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- Dioxin Treatmentp2,3,7,8-Tetrachlorodibenzo-Ovarian Tumors in Rats Induced by Chronic

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