Promotion of Endometriosis in Mice by Polychlorinated Dibenzo-p-Dioxins,Dibenzofurans, and BiphenylsKrista L. Johnson,' Audrey M. Cummings,2 and Linda S. Birnbaurn2lCurriculum in Toxicology, University of North Carolina, Chapel Hill, NC 27599-7270; 2U.S. Environmental Protection Agency, NationalHealth and Environmental Effects Research Laboratory, Environmental Toxicology Division, Research Triangle Park, NC 27711

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evaluating the structural relevance ofPHAHs7,8-CDD to the proliferation of endometriosis.etsof other Therefore, a selection of PHAHs with vary-

Is micewere ing degrees of affinity for the Ah receptor3, or 10 pg were selected. In addition to 2,3,7,8-TCDD,iCE1s four other PHAHs were administered:,or10pg 3,3',4,4',5,-pentachlorobiphenyl (PCB 126),DIkga10 2,2',4,4',5,5'-hexachlorobiphenyl (PCB

Three weeks 153), 1,3,6,8-tetrachlorodibenzo-p-dioxin:bodyliver, (1,3,6,8-TCDD), and 2,3,4,7,8-pen-sians, tter- tachlorodibenzofuran (4-PeCDF). The

isurement of hypothesis of this study is that induction ofl a4#-i endometriosis by PHAHs may be Ah recep-statistically tor-mediated and will be influenced by struc-eitherPCB tural differences among these chemicals.

ally observed 2,3,7,8-TCDD and the two additional diox-rodue wno in-like compounds (PCB 126 and 4-PeCDF)

1 by 2,3,7,8- (19) should evoke increased proliferation ofof this study endometriotic lesions, while the nondioxin-an-promoted like chemicals (PCB 153 and 1,3,6,8-* I an amatic TCDD) (19) should not induce increasedIs lx uo-p- endometriotic growth.

Materials and Methods

Polyhalogenated aromatic hydrocarbons(PHAHs) are a group of environmental cont-aminants that include the polychlorinateddibenzo-p-dioxins (PCDDs), dibenzofurans(PCDFs), diphenylethers (PCDEs), biphenyls(PCBs), and naphthalenes (PCNs) (1,2).PHAHs evoke a broad range of toxic andbiochemical responses (3,4). One commonadverse effect is reproductive toxicity, includ-ing reduced fertility, decreased litter size,diminished uterine weight, and altered ovari-an functioning in several species (5-7).Recent studies showed an effect onendometriosis in several species followingexposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (2,3,7,8-TCDD) (8,9). Endometriosisis the growth of endometrial tissue outsidethe uterus, often causing infertility and pain(10). A major characteristic of endometriosisis the presence of lesions and hemorrhagiccysts in the peritoneum (11). Proliferation ofendometrial lesions is estrogen dependent(12) and often associated with immune dys-function (13) and it is possibly caused bycompounds such as the most potent PHAH,2,3,7,8-TCDD (14). 2,3,7,8-TCDD in-duced an increase in the prevalence and sever-ity of endometriosis in rhesus monkeys (8)and provoked an increase in the growth ofsurgically induced endometriotic lesions inboth rats and mice (9) following subchronic

exposure. Because humans are exposed to abroad range of PHAHs, identification of theeffects of additional PHAHs on endometrio-sis is warranted.

Endometriosis is difficult to diagnosis,thus estimates of prevalence vary widely.Many researchers suggest that the preva-lence of endometriosis is increasing in thegeneral population (15). While a 10%prevalence rate in the general populationhas often been accepted (10), estimates areas high as 60-80% among women withinfertility or pain in Belgium (16).Researchers suggest that the high incidencerate of endometriosis in Belgian womencoincides with elevated concentrations ofpersistent organochlorine environmentalcontaminants in this country (17). Forexample, elevated blood levels of PCBswere discovered in Belgian women whosuffer from endometriosis (17). A recentstudy in Israel has also demonstrated anassociation between endometriosis and ele-vated 2,3,7,8-TCDD levels (18).

The mechanism of action by whichdioxin induces increased proliferation ofendometriosis in recent animal studies (8,9)has not yet been determined. Objectives ofthis investigation were to determine ifPHAH-promotion of endometriosis may bearyl hydrocarbon (Ah) receptor mediated by

Chemicals. Both dioxin-like and nondioxin-like chemicals were used in addition to 2,3,7,8-TCDD (Radian Corp., Austin, TX) (199.Dioxin-like chemicals used were PCB 126(Ultra Scientific Chemical Co., NorthKingstown, RI) and 4-PeCDF (Accustandard,New Haven, CT), and nondioxin-like chemi-cals used were PCB 153 (Accustandard) and1,3,6,8-TCDD (Accustandard.) All chemi-cals had purities greater than 98%. No2,3,7,8-TCDD-like contaminants were pre-sent in the other chemicals.

Address correspondence to L.S. Birnbaum, U.S.Environmental Protection Agency, National Healthand Environmental Effects Research Laboratory,Environmental Toxicology Division, MD-66, RoomL-318, Research Triangle Park, NC 27711 USA.The research described in this article has been fund-ed in part by the U.S. Environmental ProtectionAgency with the University of North Carolina,Chapel Hill. The manuscript has been reviewed inaccordance with EPA policy and approved for pub-lication; however, it does not necessarily reflect theviews of the agency. Mention of trade names orcommercial products does not constitute endorse-ment or use recommendation.The authors are grateful for the technical assistanceof Michael DeVito, Janet Diliberto, Joan Metcalf,David Ross, Vicki Richardson, Joe Jackson, FrancesMcQuaid, Dennis House, Chris Hurst, and MichaelSantostefano. In addition, the authors would like tothank Michael Santostefano, Barbara Abbott, andMichael DeVito for reviewing ihis manuscript.Received 16 September 1996; accepted 2 April 1997.

Volume 105, Number 7, July 1997 * Environmental Health Perspectives

-.

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750

Articles * Structure-activity relationships of HAHs and endometriosis

Animals. Previous studies using rodentmodels focused on the effects of 2,3,7,8-TCDD exposure on the promotion ofendometriosis in rats and mice (9). Becausemice showed a greater degree of endometriot-ic severity (9), appeared to lack overtendocrine disruption at the doses studied (9),and demonstrated greater immunosensitivity(20) following exposure to 2,3,7,8-TCDDthan did rats, the mouse model was chosenfor this study. Female B6C3F1 mice wereobtained from Charles River BreedingLaboratories (Raleigh, NC) at 70 days of age,randomly assigned to a treatment group, andacclimated for 1 week prior to dosing. Micewere maintained at the National Health andEnvironmental Effects Research Laboratoryof the U.S. Environmental Protection Agency(EPA). Animal care and treatment were con-ducted according to established guidelines.All animals were housed in an environmentwith controlled humidity (40-50%), 12:12-hr light:dark cyde, and constant temperature(20-24°C). Animals received food (Prolabrat, mouse, hamster 3000, Agway, Syracuse,NY) and water ad libitum

Treatment. Chemical doses wereassigned based on published toxic equiva-lency factor (TEF) values (3) for all chemi-cals and solubility considerations for PCB153 and 1,3,6,8-TCDD. Experiment 1included doses of 0 (corn oil; SigmaChemical Co., St. Louis, MO), 1, 3, and10 pg 2,3,7,8-TCDD/kg bw with 10 ani-mals per group. Experiment 2 includeddoses of 0, 3, and 30 mg PCB 153/kg bwand 100, 300, and 1000 pg PCB 126/kgbw with 10-12 animals per group.Experiment 3 included doses of 0, 10, 30,and 100 pg 4-PeCDF/kg bw and 2 and 20mg 1,3,6,8-TCDD/kg bw with 10-12 ani-mals per group. Dosing was administeredvia oral gavage a total of five times, with 3-week intervals between each dosing asdescribed (9). All animals were dosed witha corn oil dosing vehicle at 10 ml/kg bw.

Surgial technique. Because mice have aclosed reproductive tract with a bursa-enclosed ovary and an estrous cycle insteadof a menstrual cycle, they do not developendometriosis naturally (12). Endometriosismust be induced in these animals throughsurgical methods performed during theweek of the second dosing. The two majorsteps of the surgical process as described byVernon and Wilson (21) in a rat model andextended to mice by Cummings andMetcalf (12) were 1) ablation of the leftuterine horn with longitudinal bisection toexpose the epithelial cells and 2) suturing ofuterine segments onto alternating mesen-teric blood vessels in the peritoneal cavity.

Necropsy. At the conclusion of 16weeks, the animals were euthanized by

carbon dioxide asphyxiation followed byexanguination via cardiac puncture.Necropsies were performed in a randomorder to prevent bias during the measuringof lesion diameters. Lesions, ovaries, uter-ine horn, liver, and thymus were extractedand weighed. Lesions, ovaries, and uterinehorns were fixed for histology, while liverand thymus were frozen on dry ice andstored at -70°C.EROD assay. Ethoxyresorufin and

resorufin were purchased from MolecularProbes (Eugene, OR). Microsomal proteinswere prepared (22) and quantified (23)using bovine serum albumin (BSA) as astandard. The reaction buffer contained0.1 M KPO4, 5 mM Mg2SO4, and 2 mgBSA/ml at pH 7.5. Liver microsomes werediluted in 0.1 M KPO4 (100 pl) to providereaction linearity, added to 0.1 M KPO4buffer containing 1.5 nM ethoxyresorufin,and preincubated for 2 min at 37°C. Theproduction of resorufin was started by theaddition of 100 pl of ,-NADPH (5mg/ml) and monitored spectrofluorimetri-cally as described (22). A logl0 transforma-tion was utilized to normalize ethoxyre-sorufin O-deethylase (EROD) data.

Histopathology. Microscopic examina-tion of endometriotic lesions and ovarieswas performed to characterize histologicalchanges associated with exposure to halo-genated aromatic hydrocarbons (HAHs).Animals were selected for histopathologyrandomly to obtain an unbiased representa-tive sample for review. Histology was pre-pared by Experimental PathologyLaboratories Inc. (Research Triangle Park,NC) and pathology was performed by John

Seely at PATHCO (Research Triangle Park,NC). Endometriotic lesions were examinedfor the presence of inflammation and lumi-nal exudate or transudate, while ovaries wereexamined for both the presence of primary,growing, and antral follicles and the pres-ence of corpora lutea (both active andregressing). Active corpora lutea weredefined as newly formed corpora lutea, aswell as those that became increasinglyeosinophilic and those that appeared foamy.Regressive corpora lutea were characterizedby degeneration, necrosis, and fibrous tissueproliferation.

Statistical analysis. Primary statisticalanalysis of endometriotic lesion diametersand secondary analyses of lesion, ovarian,uterine, and thymus weights from all chem-ical treatment groups were performed usingthe Dunnett's test and a level of probabilityof statistical significance of p<0.05. Meanswith standard deviations (SD) were deter-mined for all dose groups for body, liver,ovarian, uterine, and lesion weights andlesion diameters. For EROD activity, thestatistical intergroup comparisons weredetermined using a one-way analysis of vari-ance (ANOVA) followed by Fisher's pro-tected least significant difference (PLSD).The levels of probability of statistical signif-icance for EROD data are p<0.01.

ResultsEROD activity is a marker for CYPlAl-dependent enzyme induction by 2,3,7,8-TCDD and related compounds (19). Inthis study, constitutive EROD activity incontrol animals for three separate experi-ments were similar (Table 1). A statistically

Table 1. Mean parameter values and standard deviations

Dose EROD activity Ovarian(per kilogram (pmoles/min/ Lesion diameter Lesion weight weight8

Chemical body weight) mg protein) (mm) (mg) (mg)

Control 1 0 pg 114 ± 40 5.46 ± 1.27 22.50 ± 13.0 8.51 ± 0.812,3,7,8-TCDD 1 pg 201 ± 100 7.27 ± 1.42* 109.20 ± 63.60* 9.11 ± 2.18

3 pg 657 ± 378** 6.96 ± 0.77* 94.50 ± 43.30* 9.30 ± 1.4510 pg 844 ± 386** 6.12 ± 0.76 61.70 ± 26.70* 7.03 ± 2.05

Control 2 0 mg 71 ± 27 5.71 ± 1.43 71.30 ± 51.50 8.38 ± 2.98PCB 153 3mg 116±51 5.74±0.90 59.50±26.20 10.22±1.14

30 mg 91 ± 32 5.84 ± 0.52 64.10 ± 18.90 13.49 ± 10.57PCB 126 100 pg 805 ± 408** 6.25 ± 1.03 65.70 ± 28.90 10.68 ± 1.97

300 pg 1576 ± 420** 6.83 ± 1.46 101.00 ± 55.80 9.91 ± 1.561000 pg 1827 ± 439** 6.6-1 ± 1.75 91.90 ± 57.90 9.14 ± 2.25

Control 3 0 mg 110 ± 34 6.05 ± 0.86 71.00 ± 31.70 10.78 ± 1.811,3,6,8-TCDD 2 mg 84 ± 23 5.98 ± 0.91 63.30 ± 27.80 9.17 ± 1.72

20 mg 159 ± 49 5.96 ± 0.82 61.00 ± 29.40 10.38 ± 1.564-PeCDF 10 pg 676 ± 293** 6.05 ± 0.54 70.00 ± 22.00 10.42 ± 2.38

30 pg 1303 ± 527** 6.26 ± 1.27 84.20 ± 46.40 9.58 ± 1.72100 pg 1516 ± 782** 7.32 ± 1.29* 114.30 ± 58.50 9.08 ± 1.09

Abbreviations: 2,3,7,8-TCDD, 2,3,7,8-tetrachlorodibenzo-p-dioxin; PCB 153, 2,4,5,2',4',5'-hexachloro-biphenyl; PCB 126, 3,4,5,3',4'-pentachlorobiphenyl; 4-PeCDF, 2,3,4,7,8-pentachlorodibenzofuran, 1,3,6,8-TCDD, 1,3,6,8-tetrachlorodibenzo-p-dioxin.aRight ovary of intact uterine horn.*p<0.05; **p<0.01.

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Articles - Johnson et al.

significant (p<0.01) dose-dependentincrease in EROD activity in mice treatedwith increasing doses of 2,3,7,8-TCDD,PCB 126, or 4-PeCDF was observed; incontrast, mice treated with 1,3,6,8-TCDDor PCB 153 had EROD activities similarto control groups (Table 1).

Previous studies in a rodent model for2,3,7,8-TCDD-promoted endometriosisfocused on lesion diameter as an indicatorof 2,3,7,8-TCDD-induced responses (9).Examination of lesion diameter in threeseparate control groups in this study indi-cated similar values with no statistical dif-ferences (Table 1). Treatment of animalswith 1 or 3 pg 2,3,7,8-TCDD/kg bwresulted in a statistically significant increasein lesion diameter (p<0.05). Although thediameter was increased relative to controlsat 10 pg 2,3,7,8-TCDD/kg bw, it was notstatistically significant (Table 1). Anincrease in lesion diameter with dose wasobserved in animals treated with 4-PeCDF,although a statistically significant increasewas only observed in animals treated with100 pig 4-PeCDF/kg bw (Table 1).Animals treated with PCB 126 resulted inan apparent increase in lesion diameter;however, this increase was not statisticallysignificant compared to control animals(Table 1). Analysis of lesion diameter val-ues for animals treated with PCB 153 or1,3,6,8-TCDD resulted in lesion diametervalues similar to control animals in all dosegroups (Table 1).

Endometriotic lesion weight was used asan additional marker to examine HAH-pro-moted endometriosis. Examination oflesion weights in three separate controlgroups revealed high variability. Lesionweights for control animals in experiment 1were significantly different from controlanimals in experiments 2 and 3, which maycontribute to the variability in resultsamong dose and chemical groups (Table 1).Treatment of animals with 1, 3, or 10 pg2,3,7,8-TCDD/kg bw resulted in a dose-dependent decrease in endometriotic lesionweight. However, endometriotic lesionweights in all dose groups of 2,3,7,8-TCDD-treated mice were significantly ele-vated compared to control animals inexperiment 1 (Table 1). Lesion weights ofanimals treated with 1 or 3 pg 2,3,7,8&TCDD/kg bw also appear elevated whencompared to control animals of experiments2 or 3. Elevated endometriotic lesionweights were not observed in the lowesttreatment groups for 4-PeCDF or PCB 126when compared to controls, although anapparent, but nonsignificant dose-depen-dent increase in lesion weight was observedin animals treated with 4-PeCDF/kg bw or100 or 300 pig PCB 126/kg bw. Lesion

weight values for the highest dose of PCB126 (1000 pg/kg bw) decreased nonsignifi-cantly from 300 pg/kg bw. High variabilitywas present in all treatment groups, espe-cially in lesion weight data of animals treat-ed with PCB 126 or 4-PeCDF. Analysis oflesion weights in mice treated with PCB153 or 1,3,6,8-TCDD resulted inendometriotic lesion weights similar to con-trol animals.

Another tissue examined in this study toassess the effects of HAH exposure onendometriosis was the ovary. Because bodyweight values did not vary significantly acrosschemical dasses or doses, similar results wereobserved between crude ovarian weights(Table 1) and ovarian weight/bw ratios (datanot shown). Ovarian weights from ablateduterine horns appeared slightly lower than theovarian weights from intact uterine horns.However, trends in both sets of ovarianweights are consistent within most dosegroups. Therefore, the crude ovarian weightsfrom intact uterine horns (Table 1) were usedas markers to describe the dose-dependenteffects of administered compounds on ovari-an weights. Although no significant differ-ences in ovarian weight were found in com-parisons of treated to control animals, possi-bly due to high data variability, examinationof ovarian weights from animals treated with1 or 3 pg 2,3,7,8-TCDD/kg bw revealed atrend toward an increase in ovarian weights,which was followed by an apparent decreasein ovarian weight in animals treated with 10pg 2,3,7,8-TCDD/kg bw (Table 1). Animalstreated with PCB 153 exhibited an apparentbut nonsignificant increase in ovarian weightwith increasing dose (Table 1). In contrast,mice treated with PCB 126 or 4-PeCDF

showed an apparent decrease in ovarianweight with increasing dose (Table 1).Furthermore, analysis of ovarian weightsfrom mice treated with 1,3,6,8-TCDDshowed ovarian weights similar to controlanimals at all dose levels (Table 1). A micro-scopic examination of endometriotic lesionsand ovarian tissue was used to characterizehistological changes associated with exposure.In all endometrial lesions examined, endome-trial epithelium, endometrial glands withstroma, and the myometrium were present,but these structures varied in thickness andprominence, without relation to treatment.The presence of luminal exudate or transu-date was more severe in lesions characterizedas nonstandard than in those characterized asstandard, but no significant dose-dependentdistribution of severity was noticed becauseincidence rates of nonstandard lesions, suchas discolored or hardened lesions, were con-sistent across dose groups and chemical dass-es. Inflammation of the endometriotic uter-ine segments was consistent across all animalsand, in most instances, appeared acute (asso-ciated with polymorphonuclear cells.)Examination of ovarian tissue revealed theabsence of active corpora lutea in animalsonly from the 10 pg 2,3,7,8-TCDD (in twoof three animals examined), 100 pg PCB 126(one of three animals), and 1000 pig PCB126/kg bw (two of three animals) treatmentgroups. In addition, animals with the highestnumber of regressive corpora lutea comparedto total corpora lutea (both active and regres-sive) were animals treated with 10 pg 2,3,7,8-TCDD (53.3%), 100 pg PCB 126 (60%), or1000 pig PCB 126/kg bw (75%). In contrast,only 26.7% of the total corpora lutea in con-trol animals, was characterized as regressive.

Table 2. Mean parameter values and standard deviations

Dose Uterine(per kilogram horn weighta Thymus weight Liver weight

Chemical body weight) (g) (g) (g)Control 1 0 pg 0.069 ± 0.016 0.031 ± 0.012 1.247 ± 0.1072,3,7,8-TCDD 1 pg 0.060 ± 0.020 0.035 ± 0.014 1.359 ± 0.148

3 pg 0.074 ± 0.029 0.048 ± 0.022 1.323 ± 0.11510 pg 0.058 ± 0.020 0.031 ± 0.013 1.581 ± 0.159

Control 2 0 mg 0.050 ± 0.023 0.033 ± 0.004 1.238 ± 0.140PCB 153 3 mg 0.057 ± 0.019 0.035 ± 0.010 1.291 ± 0.166

30 mg 0.058 ± 0.020 0.042 ± 0.011 1.306 ± 0.146PCB 126 100 pg 0.049 ± 0.008 0.043 ± 0.012 1.410 ± 0.160

300 pg 0.058 ± 0.015 0.031 ± 0.012 1.367 ± 0.1511000 pg 0.053 ± 0.011 0.028 ± 0.008 1.521 ± 0.128

Control 3 0 mg 0.061 ± 0.017 0.036 ± 0.005 1.329 ± 0.0811,3,6,8-TCDD 2 mg 0.053 ± 0.014 0.039 ± 0.009 1.197 ± 0.087

20 mg 0.061 ± 0.016 0.041 ± 0.007 1.300 ± 0.1824-PeCDF 10 pg 0.063 ± 0.015 0.036 ± 0.007 1.359 ± 0.131

30 pg 0.060 ± 0.029 0.027 ± 0.008* 1.440 ± 0.310*100 pg 0.052 ± 0.023 0.026 ± 0.006* 1.467 ± 0.211*

Abbreviations: 2,3,7,8-TCDD, 2,3,7,8-tetrachlorodibenzo-p-dioxin; PCB 153, 2,4,5,2',4',5'-hexachloro-biphenyl; PCB 126, 3,4,5,3',4'-pentachlorobiphenyl; 4-PeCDF, 2,3,4,7,8-pentachlorodibenzofuran, 1,3,6,8-TCDD, 1,3,6,8-tetrachlorodibenzo-p-dioxin.alntact uterine horn.*p<0.05.

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Articles - Structure-activity relationships of HAHs and endometriosis

The absence of active corpora lutea and thehigh percentages of regressive corpora lutea inthese dose groups indicate either a direct orindirect chemically induced atrophic effect onthe ovaries in these animals.

The final parameters measured in thestudy were uterine, thymus, and liverweights. Uterine weights of all treated ani-mals were not statistically different from val-ues of control animals, and analysis of uter-ine weights for three separate control experi-ments revealed no statistical differences(Table 2). There was an apparent nonsignif-icant increase in uterine weights of micetreated with 3 pg 2,3,7,8-TCDD/kg bw,which was followed by an apparent decreasein uterine weights in the highest dose group,10 pg 2,3,7,8-TCDD/kg bw. These effectson uterine weights were similar to thoseobserved in animals treated with PCB 126.A trend toward a slight decrease in uterineweights with increasing dose of 4-PeCDFwas observed. Treatment of animals with alldoses of PCB 153 resulted in a slight, non-significant dose-dependent increase in uter-ine weights as compared to controls.However, all animals treated with 1,3,6,8-TCDD had uterine weights similar to con-trol animals. In contrast, a dose-dependentdecrease in thymus weights with increasingdose of 4-PeCDF or PCB 126 was observed(Table 2). Thymus weights of animals treat-ed with 30 or 100 pg 4-PeCDF/kg bw weresignificantly less than control thymusweights (p<0.05), while the suggesteddecrease in thymus weights in animals treat-ed with PCB 126 was nonsignificant.Finally, treatment of animals with 2,3,7,8-TCDD, PCB 126, or 4-PeCDF resulted inan apparent increase in liver weight withincreasing dose, although this was only sig-nificant in animals treated with the middleand high doses of 4-PeCDF (Table 2). Incontrast, exposure of animals to PCB 153 or1,3,6,8-TCDD resulted in liver weights inall dose groups similar to controls.

DiscussionThe promotion of endometriosis only byPHAHs with high binding affinity to theAh receptor is consistent with the hypothe-sis that PHAH promotion of endometriosisis Ah receptor mediated. Due to variabilityin these results from study limitations suchas individual animal variability, bioassayinsensitivity, and gross invasiveness of thesurgical procedures, conclusions about themechanism of action by which HAHs pro-mote endometriosis are only preliminary.Follow-up studies should attempt to refinethe surgical model to more accurately assessthe effects ofHAHs on endometriosis.

As in previous studies that focused onlesion diameter as the primary endpoint to

assess the influence of 2,3,7,8-TCDD onthe promotion of surgically inducedendometriosis (9), this study evaluated theeffects of 2,3,7,8-TCDD, PCB 126, PCB153, 1,3,6,8-TCDD, and 4-PeCDF onendometriotic lesion diameter (Table 1).The mechanism by which PHAHs such as2,3,7,8-TCDD, PCB 126, or 4-PeCDFincrease lesion diameter is unknown, but itappears to be Ah receptor mediated, as istrue for all other well-characterized 2,3,7,8-TCDD-induced responses (24).

Structure binding relationships (SBRs),based on affinity of ligand binding for theAh receptor, are often indirectly assessed byinduction of arylhydrocarbon hydroxylase(AHH), EROD, and other enzyme activities,and are described by structure-activity rela-tionships (SARs). Previous studies showed acorrelation between endpoints, such asCYPlAl induction, and affinity for the Ahreceptor expressed through SARs (25). Forexample, in this study 2,3,7,8-TCDD, PCB126, and 4-PeCDF, the congeners thatincreased endometriotic lesion diameters(statistically significant only in animals treat-ed with 2,3,7,8-TCDD or 4-PeCDF), havethe strongest binding affinities for the Ahreceptor (1,26,27). In contrast, compoundssuch as 1,3,6,8-TCDD and PCB 153, whichdid not induce any increases in endometriot-ic lesion diameter, have weak affinitiestowards the Ah receptor (27,28).

Endometriotic lesion weights were alsomeasured for secondary analysis of changesin endometriotic lesion sizes due to HAHexposure (Table 1). Increases in lesionweight, like increases in lesion diameter,occured in animals exposed to chemicalswith the highest affinities for the Ah recep-tor (statistically significant increasesinduced by 2,3,7,8-TCDD and nonsignifi-cant increases induced by PCB 126 and 4-PeCDF) and not in animals exposed tochemicals with significantly lower bindingaffinities (PCB 153 and 1,3,6,8-TCDD).This supports the hypothesis that promo-tion of endometriosis by HAHs may be Ahreceptor mediated.

This study also revealed changes inovarian weight and differences in ovarianhistopathological evaluation based onchemical and dose (Table 1). As with otherendpoints, chemicals with greater bindingaffinities for the Ah receptor evoked greatertoxic responses such as decreases in ovarianweights. Ovarian histological examinationof animals treated with 1 or 3 pg 2,3,7,8-TCDD/kg bw, PCB 153, 1,3,6,8-TCDD,or 4-PeCDF was consistent with ovarianweights for these animals and indicated nopresence of ovarian atrophy. The absence ofcorpora lutea and the high percentages ofregressive corpora lutea in animals treated

with 10 pg 2,3,7,8-TCDD, or 100 or 1000pg PCB 126/kg bw supports the hypothesisthat ovarian atrophy may have occurred inanimals treated with high doses of chemi-cals with strong binding affinities for the Ahreceptor. The lack of ovarian atrophyobserved in the 4-PeCDF-treated animalsmay be due to the low concentration of 4-PeCDF available to extrahepatic tissues dueto its sequestration in the liver (29). Still,the results of the histological evaluations arepreliminary because only a small, randomlyselected representative sample from eachdose group was examined.

Ovarian atrophy at high doses of2,3,7,8-TCDD and PCB 126 is consistentwith the results of decreased lesion diame-ters and weights observed in animalsadministered high doses of these com-pounds compared to the resulting lesiondiameters and weights in animals adminis-tered lower doses. High doses of PHAHsmay cause antiestrogenic effects, leading toeither indirect or direct ovarian toxicity oratrophy (30). The resulting decrease in thepromotion of endometriosis (observable inthe variability in lesion diameter and lesionweight values at the high dose levels of2,3,7,8-TCDD and PCB 126) is consistentwith the requirement for estrogen to pro-mote lesion growth (31). Thus, Ah recep-tor binding may correlate with ovarianatrophy, as well as lesion diameter andlesion weight. Future studies examiningendometrial promotion should use lowerdose levels of 2,3,7,8-TCDD and PCB 126to avoid adverse effects on the ovary.

Several additional endpoints were mea-sured in this study to assess the adverseeffects of PEHIAH exposure. Resulting antie-strogenic effects from HAH exposure couldalso be mediated by an indirect route via adecrease in the concentration of circulatingestrogens (32). This can also lead to adecrease in uterine weights (33). Analysis ofuterine weights in this study revealed a corre-lation between changes in uterine weightsand binding affinities for the Ah receptor.Chemicals that bind strongly to the Ahreceptor (2,3,7,8-TCDD, PCB 126, and 4-PeCDF) caused decreases in uterine weightsat high doses, while chemicals that bindweakly to the Ah receptor (PCB 153 and1,3,6,8-TCDD) evoked no apparentdecreases in uterine weights when comparedto controls. Also, even though changes inuterine weights were not statistically signifi-cant, they appeared to correlate with changesin ovarian weights and ovarian histopatho-logical evaluations, supporting the theory ofovarian atrophy in animals treated with thehighest doses of 2,3,7,8-TCDD or PCB126. Therefore, a relationship appears toexist between structure-activity and binding

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Articles * Johnson et al.

relationships and antiestrogenic effects, suchas decreases in uterine weights.

Another significant effect often associat-ed with 2,3,7,8-TCDD exposure is thymicatrophy, shown by previous studies to beAh receptor mediated (25). Originally, thisstudy design was for levels of chemicalexposure to produce no overt toxicity.However, analysis of thymic atrophy in thisstudy revealed decreases in thymus weightsonly at high doses of chemicals with highbinding affinities for the Ah receptor(2,3,7,8-TCDD, PCB 126, and 4-PeCDF);this was only statistically significant in ani-mals treated with either of the two highestdoses of 4-PeCDF. Therefore, the mecha-nism of thymic atrophy correlates with thestructural relationships of PHAHs observedin mice with endometriosis.

Hepatotoxicity is a common responsein mice following exposure to PHAHs (32)and often results in increased liver weights(34). In this study, nonsignificant increasedliver weights with increasing dose wereapparent only in animals treated with2,3,7,8-TCDD or PCB 126, while statisti-cally significant increases were apparent inanimals treated with 4-PeCDF. A greaterhepatotoxic response may have beenobserved in animals treated with 4-PeCDFthan in animals treated with 2,3,7,8-TCDD or PCB 126 because 4-PeCDF issequestered in the liver (29). Because thesechemicals bind with great affinity to the Ahreceptor, increases in liver weight andhepatotoxicity appear to correlate withbinding affinities for the Ah receptor.

In addition to induction of toxicresponses such as thymic atrophy and hepa-totoxicity, PHAHs have been identified asmicrosomal monooxygenase inducers (35),frequently inducing cytochrome P450isozymes (36). CYP4501A1 enzyme induc-tion is often measured by AHH or ERODactivity. Increased enzyme activity coincideswith increased gene expression and PHAHexposure (37). Therefore, EROD activitywas measured in this study as an indicator ofPHAH-induced effects and to interpretinformation about endometrial growth.Based solely on analysis of EROD activities(Table 1) for animals treated with PCB 126versus animals treated with 2,3,7,8-TCDD,lesion size of animals treated with the 100pg PCB 126/kg bw should be comparable tolesions in animals treated with 10 pg2,3,7,8-TCDD/kg bw. Thus, lesion diame-ters of animals treated with PCB 126 shouldhave decreased with increasing dose as didlesion diameters in animals treated with thehighest dose of 2,3,7,8-TCDD when com-pared to lower doses. This effect is mostprobably the result of ovarian atrophy asseen histologically. Even though the EROD

values of animals treated with the highestdose of 4-PeCDF were comparable toEROD values in animals treated with 300pg PCB 126/kg bw, no ovarian atrophy wasobserved in these animals, possibly becauseof the lack of availability of this chemical toextrahepatic tissue due to its extensivesequestration in the liver (29). Thus, lesiondiameters continued to increase because noovarian atrophy occurred and circulatingestrogen levels were probably normal.

In conclusion, this study was an analysisof the influence of the structural relevanceof PHAHs on the proliferation of surgicallyinduced endometriotic lesions in B6C3F1female mice. Analysis of all the parametersmeasured, especially lesion diameter, sug-gests that the mechanism of PHAH-pro-moted endometriosis may be Ah receptormediated, with structure-activity and bind-ing relationships influencing the degree ofendometriotic proliferation.

Some endpoints in this study, such asendometriotic lesion diameter, did not corre-late directly with dose level or hepaticEROD induction because of influences ofadditional responses such as hormonal inter-actions and possible antiestrogenic effects.Chemicals exerting antiestrogenic effects athigh doses can induce ovarian atrophy,which in turn may decrease circulating estro-gen levels and proliferation of endometriosis.In general, the responses in lesion diameter,lesion weight, ovarian weight, uterineweight, and thymus weight correlate withstructure binding relationships of the admin-istered PHAHs. Specifically, analysis of theprimary endpoint measured, endometrioticlesion diameter, demonstrates a dose-depen-dent increase in size following administrationof chemicals with strong binding affinitiesfor the Ah receptor, when the effects of ovar-ian atrophy on lesion diameter are con-trolled. Therefore, the results of this studyprovide preliminary support for the hypothe-sis that PHAH promotion of endometriosismay be Ah receptor mediated.

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International Conference Mapping Environmental Risksand Risk Comparison

October 21 th - 24th, 1997 Amsterdam, The Netherlands

The conference Risk 97 will be held October 21-24, 1997, in Amsterdam and is organized by RIVM, incooperation with various international organizations. The central theme of the conference will be the relevanceof geographical maps for the discussion on and the management of risks. Risk concepts were developed invarious, widely differing fields of science and policy. Within the environmental sciences, differences inapproach can also be distinguished, e.g., between radiological risks, risks of carcinogenic compounds, andecotoxicological risks. Whereas the assessment of radiological risks for humans is largely regulated, themethods for assessing risks of toxic compounds for humans and the ecosystem are still developing. However,in all these disciplines the application of geographical information and transferring estimated risk values intomaps are emerging. Although the aims of risk assessments in each of these fields are similar, the terminologyand the methods differ widely, which creates profound difficulties in policy making and negotiations with theparties involved, such as industries and the public. The conference aims to bring these subjects together toadvance mutual understanding and the technology of risk mapping.

If you are interested in attending the conference, contact:RISK 97 Conference Secretariat

GEOPLAN, Emmastraat 28, 1075 HV AmsterdamFax +31 20 - 664 63 06

E-mail: risk97.rivm@geoplan.nlhttp://www.risk97.rivm.nl/

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