Estrogenic and antiestrogenic actions of PCBs in the female rat: In vitro and in vivo studies

Reproductive Toxicology, Vol.7, pp. 237-248, 1993 Printedin the U.S.A.All rightsreserved.

0890-6238/93 $6.00 + .00 Copyright© 1993PergamonPress Ltd.

• Original Contribution

ESTROGENIC A N D ANTIESTROGENIC ACTIONS OF PCBs IN THE FEMALE RAT: IN VITRO A N D IN VIVO STUDIES HEIKO T. JANSEN,* PAUL S. COOKE,* JOHN PORCELLI,* T S U E I - C H u LIU,'~ a n d LARRY G. HANSEN* *Department of Veterinary Biosciences, College of Veterinary Medicine, University of Illinois, Urbana, Illinois; and tDepartment of Medical Technology, National Yang Ming Medical College, Shih-Pai, Taipei, Taiwan, Republic of China Abstract m A series of studies were performed to examine the effects of various PCB congeners [3,4,3',4'TCB (PCB77), 2,5,2',5'-TCB (PCB52), 4' OH-2,4,6-TCB (OHPCB)] and a complex PCB mixture (Aroclor 1242; A1242) on the weights and patterns of cell proliferation of immature rat uteri in vivo and the effects of A1242 on gonadotropin release by dispersed anterior pituitary cells in vitro. Immature (20-day) rats were treated ip with corn oil (vehicle), PCBs, or 17/l-estradiol (E2) once or daily for two consecutive days. Except for PCB77, all PCBs and E2 produced significant increases in uterine weights ( P < 0.01) and increases in uterine 3H-thymidine labeling. PCB77 attenuated ( P < 0.01) the increase in uterine weight due to either A1242 or E2 treatment. Cultured anterior pituitary cells exposed to varying concentrations of A1242 (0.1 to 50 ppm) exhibited enhanced gonadotropin responses to gonadotropin-releasing hormone (GnRH) similar to that of E2. These results demonstrate that PCBs can produce either estrogenic o r antiestrogenic effects. Key Words: polychlorinatedbiphenyls;rat; pituitary; uterus; gonadotropins;estrogen;antiestrogen;female.

Certain PCB congeners, putative PCB metabolites, and complex PCB mixtures (<48% chlorine content) are estrogenic and bind competitively to estrogen receptors (8-10) leading to increases in uterine weight (6,9-11). These estrogenic effects of PCBs are potentially deleterious to a variety of reproductive organs, as shown by extensive earlier work indicating that exposure to naturally occurring or synthetic estrogens (e.g., coumestral, DDT, methoxychlor, and chlordecone) can lead to reproductive abnormalities (12,13). While the estrogenic PCBs may have direct effects on reproductive organs such as the ovary (5,14) and uterus (6,8-11), some of these reproductive effects may result from the actions of PCBs on the pituitary and/or hypothalamus. These effects could lead to changes in luteinizing hormone (LH) or follicle-stimulating hormone (FSH) secretion and thereby cause secondary changes in gonadal function. However, little information exists regarding possible direct influences of PCBs on pituitary function. The accumulation of certain PCBs within the pituitary (15) suggests that this may be a site of PCB action and this could explain some of the observed alterations in reproductive function. Both hypotha-

INTRODUCTION

Polychlorinated biphenyls (PCBs) belong to a large group of persistent environmental contaminants known to produce adverse reproductive effects in the female (I). For example, mice, rats, and monkeys treated with PCBs exhibit significantly longer estrous cycles and/or anovulation (2-4). Reductions in the percent of ova implanted and in the number of ovarian follicles have also been documented (2,5). Neonatal treatment with Aroclor 1221 (a PCB mixture containing 21% C1) induces precocious puberty, persistent vaginal estrus, and premature reproductive quiescence (6), and another PCB mixture, Aroclor 1254, produces complete reproductive failure when given to mink (7).

Presented in part at the 72nd annual meeting of the Endocrine Society, Atlanta, GA, June 1990 and at the 25th annual meeting of the Society for the Study of Reproduction, Raleigh, NC, July, 1992. Address correspondence to Heiko T. Jansen, Department of Veterinary Biosciences, University of Illinois, 2001 S. Lincoln, Urbana, IL 61801 Received 8 September 1992; Revision received 17 November 1992; Accepted 21 November 1992. 237

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lamic gonadotropin-releasing hormone (GnRH) and ovarian 17/3-estradiol (E2) are important in maintaining the proper pituitary gonadotropin secretion required for ovulation (16-20). Therefore, estrogenic PCBs could potentially have significant effects on pituitary gonadotropin production. In addition to the known estrogenic effects of PCBs, several PCB congeners bear structural similarities to 2,3,7,8-tetrachloro-dibenzo-dioxin (TCDD), which exhibits antiestrogenic activity in vitro and in vivo (21,22). The antiestrogenic effects of several PCBs in vitro have recently been described (23) but there are presently no data available indicating that these compounds are capable of producing antiestrogenic effects in vivo. To fully understand the potential reproductive effects of PCBs in vivo it is therefore necessary to assess both the estrogenic and antiestrogenic activity of individual PCB congeners. This information would be useful in making more accurate predictions of reproductive hazards as a result of exposure to the complex and variable composition of PCB mixtures present within the environment. In the present report, we describe the results of studies designed to examine the estrogenic and antiestrogenic effects of a complex PCB mixture and individual PCB congeners using cultured anterior pituitary cells and the immature rat uterine weight bioassay. The results demonstrate estrogen-like effects of A1242 on the normal physiologic responses of the pituitary in vitro and on uterine weight in vivo. Furthermore, individual PCB congeners can produce either estrogenic or antiestrogenic effects on the uterus.

METHODS Animals In vitro studies. Young female SpragueDawley rats (Harlan, Indianapolis, IN: 19 to 85 per experiment) weighing approximately 200 g were ovariectomized and killed 4 days later. Anterior pituitaries were then dissociated and cultured as described below. In vivo studies. Immature Sprague-Dawley rats (Harlan, Indianapolis, IN; 3 to 18 per treatment) were weaned at 20 days of age and injected ip with PCBs o r E2 dissolved in corn oil or the corn oil vehicle alone. Some animals were killed on day 21 while others received a second injection of the test substance on day 21 and were then killed on day 22.

Volume 7, Number 3, 1993

Chemicals Aroclor 1242 (A1242) was a gift from Monsanto (St. Louis, MO); 3,4,Y,4'-tetrachlorobiphenyl (PCB77), 2,5,2',5'-tetrachlorobiphenyl (PCB52), and 2,4,6-trichloro-4'-hydroxy-biphenyl (OHPCB)) were purchased from Ultra Scientific (North Kingston, RI). E2 w a s purchased from Sigma (St. Louis, M O ) . E2 stock solutions were made in 100% ethanol and diluted appropriately for in vitro experiments. For in vivo studies, E2 was dissolved in corn oil and administered at a dose of 1 t~g/day/rat in a volume of 0.1 mL. PCBs were diluted initially in acetone, dried, and then re-diluted in corn oil. Daily doses of PCBs (in 0.1 mL corn oil) administered per rat were: A1242 (80, 320, 640/.~g, 2.56 mg, and 8.0 mg), PCB77 (160/~g), PCB52 (640/~g), OHPCB (250 k~g). In vitro studies General. Anterior pituitaries from ovariectomized rats were excised and sliced. Pituitary fragments were then dissociated by collagenase and hyaluronidase following brief exposure to trypsin (24). Dispersed pituitary cells (4 to 5 × 105 cells/1.5 mL/culture) were cultured for 1 day in suspension as aggregates in siliconized Leighton tubes (a preliminary experiment indicated that A1242 interfered with cell attachment to culture dishes) at 37 °C under moist 5% CO2 and 95% air. Cell viability was determined by vital dye exclusion (nigrosin). The basic culture medium was medium 199 without phenol red (a weak estrogen (25)) supplemented with amino acids, vitamins, antibiotics, and dextrancharcoal-treated (to remove endogenous steroids) bovine-calf and fetal-calf sera (24,26). Test compounds (diluent, A1242 o r E2) were then added to cultures (n = 3 to 4 per treatment) and incubated for 24 to 48 h before the end of a 3-day preincubation period. Following the preincubation period, cells were washed and incubated with or without GnRH for 4 h in the presence of initial treatments. Eagle's minimum essential medium without phenol red and sera containing 25 mM HEPES was used for cell wash and incubation. After the 4 h incubation, medium was separated from cells by centrifugation (225 x g for 10 min), and stored at -20 °C. Cells were solubilized (24), centrifuged at 33,000 × g for 1 h, and the cytosols collected and stored at -20 °C. LH and FSH in both media and cytosols were measured by radioimmunoassay (26). Experiment 1: effects ofA1242 and E2 on medium and total L H and FSH. This experiment was designed to characterize the effects of E2 and various concentrations of A1242 on 1) basal and GnRH-

Estrogenic/AntiestrogenicPCBs • H. T. JANSENET AL. stimulated LH and FSH release, and 2) total (medium + cytosol) LH and FSH. Cultures were preincubated with diluent (0.5% ethanol), A1242 (0.1, 1.0, 10, or 50 ppm [0.34, 3.4, 34, or 170/xM]), or E2 (6 × 10-l° M) for 24 or 48 h. Cells were then washed and incubated with or without GnRH (1 nM) for 4 h. Medium and cellular LH and FSH were determined as described above.

Experiment 2: effects ofA1242 with or without E2 on GnRH-stimulated LH release. The objective of this experiment was to determine whether or not A1242 affects GnRH-stimulated LH release in the presence of Ez. Cells were preincubated for 48 h with A1242 (0, 0.1, 1.0, and 10 ppm [0, 0.34, 3.4, and 34/zM]) in the presence or absence of E2 (6 × 10-1°). Cell washes, GnRH treatment, and cell solubilization were as described for Experiment 1. Media and cytosols were assayed for LH. Experiment 3: effects of A1242 and E2 on GnRH dose-response. The objective of this experiment was to compare the effects of A1242 and E2 on LH release in response to increasing doses of GnRH. Cells were pretreated with diluent, A1242 (5 or 10 ppm [17 or 34 ~M]), or Ez (6 x 10-1° M) for 48 h. After washing, cells were exposed to increasing doses of GnRH (10 -jJ to 10-7 M) for 4 h. Media were then collected and assayed for LH. In vivo studies Single dose effects ofA1242 on uterine weight. Immature female rats (n = 5 to 6 per group) were injected ip once on day 20 with corn oil (control) or A1242 (0.64 rag, 2.56 mg, and 8 mg dissolved in 0.1 mL corn oil). Animals were killed on day 21 and the uteri excised, freed of connective tissue, blotted dry, and weighed. Effects of repeated doses ofA1242, individual PCB congeners, and E2 on uterine weight. Immature female rats (n = 11 to 14 per group) were injected with corn oil alone (control), E2 (1 /xg) or PCBs at doses of 80 and 320/xg (A1242), 160/~g (PCB77), 640/zg (PCB52), 250/zg (OHPCB) in corn oil on days 20 and 21. Animals were killed on day 22. The uteri were excised and weighed as described above. Effects of PCB77 coadministered with E2 or A1242 on uterine weight. Immature female rats (n = 3 to 18 per group) were given either corn oil, PCB77 (160/~g), A1242 (80 and 320/xg), E2 (1/xg) alone or PCB77 in combination with either A1242 or E2. Test compounds were administered on days 20 and 21

239

and rats were killed on day 22. The uteri were excised and weighed as described above.

Radioimmunoassays. LH in medium and cells from in vitro experiments was determined as previously described (24). Inter- and intra-assay coefficients of variation for three pooled sera were 3.9% and 6.6%, respectively. FSH in medium and cells was determined as previously described (26). The intra-assay coefficient of variation for three pooled sera was 2.8%. 3H-thymidine autoradiography. The technique used for autoradiography has been described in detail previously (27). Briefly, uteri were removed from rats 24 h after the second test injection. The tissue was cut into 2 mm pieces and incubated in Dulbecco's modified Eagle's medium containing 5 ttCi of 3H-thymidine (specific activity = 80 Ci/mmol; Amersham) per mL for 4 h. The tissues were then fixed, sectioned and processed for autoradiographic examination (27). Statistical analysis. Data were subjected to one- or two-way analysis of variance. Individual differences were determined using a post-hoc Student-Newman-Keuls test (28). RESULTS

In vitro studies Effects ofA1242 on medium and total LH and FSH in cultured pituitary cell aggregates Experiment 1: effects of increasing doses of A1242 medium LH and FSH (Figure 1). Pretreatment with 0.5% ethanol (diluent) alone for either 24 h or 48 h did not affect pituitary LH and FSH responses (data not shown). Pretreatment with E2 or A1242 (0.1 to 10 ppm) for 24 h had no significant effect on basal LH or FSH release. Similar effects were observed after 48 h pretreatment; however, with the addition of a higher dose of A1242 (50 ppm) a significant increase in basal LH (P < 0.01) and FSH (P < 0.05) release was observed. GnRH significantly increased (P < 0.01) medium LH and FSH in cells pretreated with diluent (24 h and 48 h). Pretreatment with E2 for 24 h and 48 h potentiated (P < 0.01) the GnRH-induced LH and FSH release. After a 24 h preincubation period, the GnRH-induced LH response was also potentiated (P < 0.01) by 0.1, 1.0, and 10 ppm A1242. However, after a 48 h preincubation period, only 10 ppm A1242 significantly (P < 0.01) potentiated the GnRH-induced LH release, whereas 50 ppm A1242 significantly (P < 0.01) reduced GnRH-induced LH release. GnRH-induced FSH release was potenti-

240

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Fig. 1. Effects of increasing doses of PCB (Aroclor 1242; A1242) on basal and GnRH-stimulated LH and FSH release by aggregate cultures of rat anterior pituitary cells. Anterior pituitary cell cultures (5 x 105 cells per tube) were washed three times with MEM containing 0.5% ethanol (Diluent; D), 6 x 10-j° M E2, or 0.1, 1.0, 10, or 50 ppm [0.34, 3.4, 34, or 170 /zM] A1242 after either 24 h or 48 h preincubation with the same test agents as described in Methods. Cultures were then incubated with either 0 (control) or 1 nM GnRH for 4 h. Each point represents the mean -+ SEM of 3 or 4 replicate cultures. NIH ovine LH-S20 was used as the standard for radioimmunoassay of LH and NIDDK-rFSH-RP2 as the standard for FSH. ** P < 0.01 vs. corresponding diluent value; * P < 0.05 vs. corresponding diluent value; + P < 0.01 vs. ( - ) GnRH.

ated ( P < 0.01) by A1242 only at 10 ppm after both 24 and 48 h preincubation periods.

L H ( P < 0.01) in the presence of G n R H and total F S H only in the absence of G n R H ( P < 0.05)

Total L H and F S H (Figure 2). Total (medium + cytosol) L H and F S H were unaffected after 24 h preincubation with either E2 or A1242. After 48 h preincubation, either E2 or A1242 (10 ppm) in combination with G n R H significantly increased total L H . A1242 at 50 ppm significantly decreased total

Experiment 2: effects ofA1242 with or without E2 on GnRH-induced L H release (Figure 3). In agreement with the results from Experiment 1, 48 h preincubation with either E2 or 10 ppm A1242 alone significantly increased ( P < 0.01) GnRH-induced L H release. Preincubation with 0.1 or 1.0 ppm

Estrogenic/Antiestrogenic PCBs • H. T. JANSEN ET AL.

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A1242 remained ineffective. A1242, when coadministered with E2, did not affect the GnRHinduced L H release due to Ez alone. Basal L H release and total L H were not affected by any treatment (data not shown).

Experiment 3: effects of A1242 and E2 on GnRH Dose-Response. The effects of A1242 (5 and 10 ppm) on L H responses to increasing doses of G n R H are depicted in Figure 4. G n R H alone significantly stimulated L H release ( P < 0.01) at 10 -9 t o 10 -7 M in a dose-dependent fashion. G n R H at 10-" or 10 -m M did not significantly affect L H release. Maximal L H stimulation was observed at 10 -8 to

10 -7 M GnRH. Neither E2 nor A1242 affected basal L H release (no GnRH), consistent with the results from Experiment I. H o w e v e r , A1242 at both doses and E2 potentiated GnRH-induced L H release; that is, the G n R H d o s e - r e s p o n s e curves were shifted to the left. E2 and A1242 potentiated ( P < 0.01) the GnRH-induced release of L H at G n R H doses above 10-" M. In addition, significantly ( P < 0.01) more L H was released into the medium at 10-" to 10 -l° M G n R H when cells were exposed to 10 ppm A1242 compared to E2. The maximal increase in L H release in response to 10 -8 to 10 -7 M G n R H alone was further enhanced (P < 0.01) by both concentrations of A1242 and E2; this increase was significantly less

242

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Fig. 5. Effects of a single dose of A1242 on uterine weights of immature rats. Sprague-Dawley rats (n = 5 or 6 per treatment) were administered ip corn oil (oil) or Aroclor 1242 (0.64, 2.56, or 8.0 mg per rat) in corn oil at 20 days of age. Rats were killed on day 21 and uteri excised and weighed. Different letters above bars are significantly different from one another (P < 0.0l).

(P < 0.05) for A1242 when compared to E2 at 10 -7 M GnRH. The interaction of A1242 with GnRH appears to differ from that of E2 with GnRH because the dose-response curves were not parallel. This is reflected as a reduced effectiveness of A1242 in increasing LH release at GnRH concentrations above 10-9 M and as an increased effectiveness of 10 ppm A1242 at GnRH concentrations below 10-9 M. In contrast, E2 responses remained proportional to the controls at GnRH doses above 10-n M.

In vivo studies Single dose effects ofA1242 on uterine weight. The effects of single doses of A1242 on uterine weights are illustrated in Figure 5. The highest dose of A1242 (8 mg) significantly (P < 0.01) increased uterine weight whereas lower doses had no significant effect.

Effects of repeated doses ofA1242 and individual congeners alone, or together with E2, on uterine weights. The effects of vehicle (corn oil), A1242, PCB77, PCB52, OHPCB, and Ez administered as repeated doses at days 20 and 21 on uterine weights of immature rats are illustrated in Figure 6. PCB77 did not significantly affect uterine weight (P < 0.05

vs. controls). A1242, PCB52, OHPCB, and E2 all increased uterine weights (P < 0.01) compared to controls. The increase in uterine weight induced by 80 and 320/zg doses of A1242 did not differ significantly (P > 0.05) from each other and were similar in magnitude to those after single 8.0 mg doses (see Figure 5). Coadministration of A1242 with Ez did not significantly alter the effects of Ez alone. PCB77 when coadministered with E2 or A1242 significantly (P < 0.01) attenuated the uterine weight increases following treatment with either A1242 (320/zg) or E2 alone (Figure 7). As was seen in the previous experiment, PCB77 alone did not affect uterine weight (P > 0.05).

3H-thymidine autoradiography. Tritiated thymidine autoradiograms of uterine sections from animals given vehicle, E2, PCB52, A1242, or PCB77 + E2, are shown in Figure 8. In control rats, labeling of both epithelial and stromal cells was relatively light (8A). E2 treatment resulted in an increase in labeling of both epithelial and stromal cells (8B), dramatically increased the height of the uterine epithelium compared to the controls, and also resulted in an increase in the number of uterine glands. A1242 and PCB52 also stimulated epithelial and stromal labeling over the level seen in control rats (8C,D), although the labeling following either treat-

244

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Fig. 6. Effects of repeated doses of PCBs on uterine weights of immature rats. Sprague-Dawley rats (n = 11 or 14 per treatment) were administered ip corn oil (oil) or PCBs in oil: Aroclor 1242 (80 or 320/xg), PCB77 (3,4,3',4'-TCB; 160/zg), OHPCB (40H-2,4,6-TCB; 250/zg), PCB52 (2,5,2',5'-TCB; 640/xg), or E2 (17/3-estradiol; 1 /xg) at days 20 and 21. Rats were killed on day 22 and the uteri were excised and weighed. Different letters above bars are significantly different from one another (P < 0.01). 70

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Estrogenic/Antiestrogenic PCBs • H. T. JANSENET AL.

245

Fig. 8. Autoradiograms of 3H-thymidine incorporation into immature rat uteri following ip administration, on days 20 and 21, of A. corn oil (control), B. 17fl-estradiol (E2), C. 2,5,2',5'-TCB (PCB52), D. Aroclor 1242, or E. 3,4,3',4'-TCB (PCB77) + E2. Labeling (arrows) in both epithelium (E) and stroma (S) is minimal in control uteri. E2 stimulated labeling in both E and S and also stimulated uterine gland (G) formation. PCB52 and A1242 also stimulated E and S labeling compared to controls, but this labeling was less than seen with E2. PCB77 treatment reduced, but did not abolish, the stimulatory effects of E2. Magnification = 330 x for A through E.

ment in both tissue compartments was less pronounced than in those animals given E2. PCB77 antagonized, but did not completely suppress, the normal increase in epithelial and stromal labeling seen with E2 treatment (8E). PCB77 alone did not affect 3H-thymidine labeling (data not shown).

DISCUSSION The purpose of the present investigation was to evaluate the estrogenic and antiestrogenic proper-

ties of various PCB compounds on the reproductive axis of the female rat. Results from in vitro experiments demonstrate that a complex PCB mixture (A1242) can increase basal gonadotropin release from the pituitary as well as potentiate pituitary reponsiveness to G n R H ; the latter effects were similar to those observed with E2. In vivo experiments confirmed the estrogenicity of A1242 and several ortho-substituted PCB congeners. A non-ortho substituted PCB congener (PCB77) was not estrogenic.

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Indeed, this compound antagonized both the E2 and A1242 increases in uterine weight. The data presented here are the first demonstration that PCBs can directly affect basal LH and FSH secretion by the pituitary and also alter pituitary responsiveness to GnRH. Certain effects, such as the potentiation of GnRH-induced LH and FSH release by 5 to 10 ppm A1242, the increase in maximal LH release induced by GnRH and the shift in the GnRH dose-response curves, were similar in magnitude to those observed after E2 treatment. However, the A1242 effects occurred at doses nearly 4 to 5 orders of magnitude higher than E2. Other effects, such as the potentiation of LH release at 10 -11 to 10 -1° M GnRH, the attenuation of GnRH-induced LH release, and reduction in total LH and FSH (intracellular + medium) by 50 ppm A1242, were clearly different than those observed with E2. The reduction in total LH and FSH was probably not due to a cytotoxic effect of A1242 since cell viability always remained ->90%. Furthermore, the effects on total LH and FSH indicate that A1242 may affect the synthesis and/or degradation of these pituitary hormones. Overall, there appears to be a narrow doseresponse relationship for the A1242 effect on GnRH-induced LH release. Such narrow doseresponse relationships are characteristic of a variety of physiologically important estrogens, estrogen metabolites, and progestins in vitro (29). Indeed, the effective range of E2 concentrations for potentiating 10 -9 M GnRH-induced LH release in rat pituitary cells in vitro is approximately 10-fold (29). Our observation that GnRH-induced LH release was potentiated in a dose-related fashion (approximately 10-fold) by A1242 is therefore consistent with that of other estrogens. A1242, at the doses tested, does not appear to be antiestrogenic. This is based on the observation that coadministration of A1242 with Ez did not inhibit the GnRH-induced increase in LH due to E2 (Figure 3) or the E2 effects on uterine weight (Figure 6). Of the greater than 70 individual congeners present in A1242, each bearing different degrees and patterns of chlorination (30), some (such as PCB77) possess antiestrogenic properties, both in vitro (23) and in vivo (see below). Thus, while the concentrations of these antiestrogenic congeners apparently were not sufficient to block the E2 effects on GnRH-induced LH release, the potential for other complex interactions cannot be ruled out and warrants further investigation. The precise mechanism(s) by which A1242 exerts its estrogenlike action on GnRH-induced LH

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release remains unknown. However, several possibilities exist. First, A1242 may increase GnRH receptor concentrations in a manner similar to that observed with E2 (31,32) as evidenced by an increase in the maximal response to GnRH. Second, A1242, or a constituent thereof, like E2, may be producing effects on post-GnRH receptor mechanisms such as protein kinase C (PKC) or Ca 2+ (33,34). Third, A1242 may affect pituitary LH release and production via mechanisms unrelated to its estrogenic action. This is evidenced by the nonparallelism of the LH responses to A1242 vs. E2 (Figure 4). It is unlikely that a single mechanism can explain the complex series of effects on pituitary gonadotropin secretion following A1242 exposure and it remains to be determined what constituents of the A1242 mixture are responsible for these effects. Experiments utilizing pure PCB congeners would provide more detailed insight into specific mechanisms of action and might also reveal novel cellular actions of these compounds. In addition, classes of responses based on congener structure could be formulated. Taken together, the results from our in vitro studies suggest that A1242 acts as an estrogen to affect pituitary LH and FSH release. The effects of A1242 on cultured anterior pituitary cells predicted net in vivo estrogenic activity. Uterine weight represents a classical bioassay for estrogenicity, yet historical confirmation of increased uterine weight following A1242 is lacking (6,11). Using a simple modification of the dosing regimen described by Ecobichon and MacKenzie (11), we were able to demonstrate the estrogenicity of A1242. A1242 was more effective (by a factor of 12 to 50) when administered as two doses separated by 24 h, rather than as a single dose (11, present study). There are at least two possibilities to explain the difference in the effectiveness of PCB treatment. First, it may relate to the persistence of these compounds (35). The initially injected PCB may still be present at the time of the second injection and therefore the effects observed following a second injection may have reflected an additive effect. Second, the duration of PCB treatment may be an important factor in determining the physiologic effects of these compounds. These observations may be potentially significant in terms of environmental PCB exposure, which tends to occur chronically rather than acutely. Our demonstration that a diortho-substituted congener (PCB52) increases uterine weight in the immature rat confirmed a previous report (11). However, with our dosing regimen, we were able to demonstrate clear estrogenicity at a dose approxi-

Estrogenic/Antiestrogenic PCBs • H. T. JANSEN ET AL.

mately 1/6th of that used previously (11), indicating that PCB52 is more estrogenic than previously reported. The unambiguous estrogenicity of a diortho-substituted, putative PCB metabolite (OHPCB), is in agreement with an earlier report demonstrating both estrogen receptor binding and uterine weight increases following administration of this compound (10). Since the coplanar PCB77 did not affect uterine weight, our data are consistent with the hypothesis that the estrogenicity of PCB congeners appears to be related to ortho-substitution (10,11). The antagonism by PCB77 of the E2 effects on uterine weight represents the first demonstration of antiestrogenic activity of this compound in vivo, an activity that had been suggested based on in vitro studies (23). In addition, the antagonism by PCB77 of the E2-induced increase in uterine cell proliferation is similar to that observed following treatment of human breast cancer cells with the antiestrogens 6-methyl-l,3,8-trichlorodibenzofuran (MCDF) and TCDD, both of which are thought to act through an arylhydrocarbon (Ah) receptor mechanism (36,37). PCB77 may act through similar Ah-receptor mechanisms (21) although its toxic equivalency (determined by measuring induction of hepatocyte aryl hydrocarbon hydroxylase or ethoxyresorufin-Odeethylase activity (P4501A1) (38)) is approximately 1000-fold lower than for TCDD [Safe, personal communication, (38)]. However, the dose of PCB77 required for antiestrogenic effects on uterine weight in this study was only 40 to 80 times greater than that previously shown for TCDD (22). In addition, MCDF, also a weak inducer of P4501A1, exhibits substantial antiestrogenicity (36). Thus, PCB77 and MCDF may both belong to a unique class of anti-estrogens that possess Ah-receptor binding activity, but weak hepatic enzyme inducing capacity. Alternatively, PCB77 may be directly competing with E2 for receptor occupancy or decrease estrogen receptor levels without involvement of the Ah-receptor. Some of the E2-antagonistic activity of PCB77 is potentially due to induction of catabolic monooxygenases, as has been suggested for TCDD (39). The weak enzyme-inducing capacity of PCB77 and MCDF would argue against increased E2 metabolism. In addition, the attenuation of the A1242 effect by PCB77 cannot be due to increased E2 degradation since prepubertal rats have very low levels of circulating E2 and therefore must involve another mechanism. While increased oxidation of certain PCB congeners in the Aroclor mixture might be expected to enhance estrogenicity, since a putative

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hydroxylated metabolite is estrogenic (10, present study), monooxygenase induction could also alter the biotransformation of specific congeners in A1242 to metabolites that have reduced or no estrogenic activity. The role of hepatic enzyme induction by PCBs and the subsequent effects on the estrogenic or antiestrogenic potential of individual congeners remains to be elucidated. To summarize, these results illustrate a complex series of effects of PCBs on estrogen-sensitive tissues both in vitro and in vivo. Our demonstration of direct pituitary effects of A1242, together with the antiestrogenic activity of PCB77 on uterus are novel, and highlight the potential for multiple actions of PCBs on reproductive function. However, predictions of the net reproductive hazard posed by PCBs are made more difficult by the complexity of PCB mixtures found in the environment (40). Nevertheless, these results provide an opportunity to derive the non-Ah receptor toxic equivalencies required for hazard assessment of PCB residues (40,41). Acknowledgment

--

Supported in part by NIH grant HD09659

(TCL).

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