Molecular Target of Endocrine Disruption in Human Luteinizing Granulosa Cells by 2 3 7 8-Tetrachlorodibenzo-p-Dioxin: Inhibition of Estradiol Secretio
Department of Population Health and Reproduction, School of Veterinary Medicine (F.M.M., A.J.C.), California Regional Primate Research Center (C.A.V.V.), and Center for Health and the Environment (J.W.O., B.L.L.), University of California, Davis,*6')1d?, 百拇医药
Abstract*6')1d?, 百拇医药
Estradiol (E2) production by human luteinized granulosa cells (hLGC) is inhibited by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). The molecular target of TCDD toxicity has not been identified. The decrease in E2 is ameliorated by androgen substrate addition and is not associated with changes in aromatase cytochrome P450 (P450arom) activity or protein expression. An antihuman 17-hydroxylase/17,20-lyase cytochrome P450 (P450c17) antisera and a direct radiometric assay of 17,20-lyase activity were used to test the hypothesis that TCDD targets P450c17, thereby decreasing substrate availability for E2 synthesis by hLGC. P450c17 expression and 17,20-lyase activity were detected in hLGC with high levels of E2 secretion. Western immunoblot analysis demonstrated that TCDD treatment of hLGC decreased the expression of P450c17 by as much 50% (P < 0.05). TCDD exposure induced a 65% decrease in 17,20-lyase activity (P < 0.05), but no changes were seen in P450arom or in nicotinamide adenine dinucleotide phosphate (reduced)-cytochrome P450 oxidoreductase (reductase). Furthermore, the decreases in P450c17 and 17,20-lyase were proportional to the inhibition of E2 secretion. We conclude that the molecular target for endocrine disruption of hLGC by TCDD is P450c17, specifically decreasing the supply of androgens for E2 synthesis, and that it does not involve either P450arom or the redox partner protein reductase.
Introduction?ky, 百拇医药
2,3,7,8-TETRACHLORODIBENZO-p-DIOXIN (TCDD) is a potent toxicant with the ability to disrupt endocrine and reproductive systems. It is a common environmental contaminant and is recognized generally to be one of the most active congeners of the polychlorinated aromatic hydrocarbon group (1, 2). A single dose of TCDD induces abortion and is accompanied by a decrease in serum estradiol (E2) in the laboratory macaque (3, 4, 5). This acute response is followed by chronic arrest of follicular growth, characterized by anovulation and decreased ovarian steroidogenesis (6). The dangers associated with human TCDD exposure are exacerbated by its extremely long half-life in the body (7, 8). Despite numerous studies to characterize the general toxicity of TCDD in reproductive and other tissues, little is known or understood concerning the mechanisms by which TCDD exerts its effect at the cellular or molecular level.?ky, 百拇医药
Studies investigating the molecular events that drive TCDD-induced cellular toxicity have been hampered by two major factors. Firstly, like many toxicants, the effects of TCDD differ among species, and the choice of animal models is crucial. Second, the ability to decipher mechanisms at the molecular level requires a relevant cellular or in vitro model system and the development of the proper tools. The use of human luteinizing granulosa cells (hLGC) in vitro has provided us with a useful model with which to study endocrine disruption in human tissues exposed to TCDD (9). Several studies in this laboratory as well others demonstrated that TCDD decreased E2 production by hLGC (10, 11, 12, 13, 14, 15). Logically, the effects of TCDD and other endocrine disruptors are more likely to be acutely evident if they alter a rate-limiting reaction rather than a reaction that has unlimited capability of metabolizing available substrate.
The results of our prior experiments suggested that the target for TCDD toxicity in hLGC is 17,20-lyase activity (14, 15), but we lacked the means to specifically test this hypothesis at that time. We recently raised antisera against recombinant human cytochrome P450 17{alpha} -hydroxylase/17,20-lyase (16) and adopted the use of a radiometric assay previously developed (17) to directly estimate 17,20-lyase activity (18). Therefore, with these critical tools in hand, the present experiments were designed to test the hypothesis that TCDD inhibits estrogen synthesis by hLGC through effects on P450c17 by examining both protein expression and 17,20-lyase activity. In addition, as both P450c17 and P450arom activities are totally dependent on redox support by the flavoprotein nicotinamide adenine dinucleotide phosphate (reduced) (NADPH)-cytochrome P450 oxidoreductase (reductase), the level of this essential redox partner protein was measured in control and treated cells.x#7.:(, http://www.100md.com
Materials and Methods
Chemicals?mso, 百拇医药
DMEM, antibiotic-antimycotic (10,000 U/ml penicillin G sodium, 10 mg/ml streptomycin sulfate, and 25 µg/ml amphotericin B as a fungizone), and fetal bovine and calf sera were purchased from Life Technologies, Inc. (Grand Island, NY). Pregnyl, a commercial human chorionic gonadotropin (hCG) was purchased from Organon, Inc. (West Orange, NJ). TCDD was a gift from Dr. Steven Safe (Texas A&M University, College Station, TX). 5-Pregnen-3ß, 17{alpha} -diol-20-one [17{alpha} - hydroxypregnenolone (17P5)] was purchased from Steraloids (Wilton, NH). [1,214C]Acetic acid (54 mCi/mmol) was purchased from NEN Life Science Products (Boston, MA). [21-3H]17P5 (25.9 µCi/µmol) was donated by Dr. Angela Brodie (University of Maryland Medical School, Baltimore, MD). All other chemicals were purchased from Sigma-Aldrich (St. Louis, MO).?mso, 百拇医药
hLGC culture?mso, 百拇医药
hLGC were obtained from patients undergoing assisted reproduction treatments at the Northern California Fertility Medical Center (Roseville, CA), and all experiments using them were reviewed and approved by the human subjects advisory committee, University of California-Davis. The cells were processed as previously described (9, 15). hLGC were plated at 5 x 105 cells in 3 ml/60-mm plate or at 106 cells/100-mm plate with 5 ml conditioned medium (DMEM supplemented with antibiotic-antimycotic, 10% noncharcoal-stripped calf serum, and 2 USP/ml hCG).
TCDD treatmentnpz-^fx, 百拇医药
Previous studies determined that a concentration of 10 nM TCDD effectively disrupts E2 production by hLGC (10, 14). Therefore, TCDD (10-5 M) dissolved in dimethylsulfoxide (DMSO) was added directly to each plate at a dilution of 1 µl/ml conditioned medium (10 nM final concentration). TCDD treatment started on the day after recovery. Medium was changed every other day thereafter, and TCDD treatment was added immediately after medium changes. Cells were cultured in TCDD for a total of 10 d; we have previously established that the number of cells at plating does not change with either time or TCDD treatment (10, 13, 14). On the last day of culture, medium was collected and stored frozen at -20 C for hormone analysis. Cells were then harvested by scraping in PBS, pelleted by centrifugation, and stored at -80 C for further processing as described below. Controls included cells treated with DMSO (equal volume) or not.npz-^fx, 百拇医药
Placental tissue
Human term placentas were obtained from patients after normal deliveries in the Obstetrics and Gynecologic Clinic at Sutter Hospital (Davis, CA) and were used as negative control tissues recognized not to express P450c17 (19) in 17,20-lyase activity assays, also after review and approval by the human subjects advisory committee, University of California-Davis. Tissues were collected within 3 h of delivery, transported on ice to the laboratory, and stored at -80 C until used for microsome preparation.xl}78, http://www.100md.com
Microsome-enriched fraction preparationxl}78, http://www.100md.com
Cell pellets from 100-mm dishes were resuspended in 1 ml homogenization buffer [0.1 M KHPO4 (pH 7.4), 20% glycerol, and 5 mM ß-mercaptoethanol] and 0.5 mM of the protease inhibitor phenylmethylsulfonylfluoride; after 10 strokes with a glass-glass homogenator, the cells were briefly sonicated (twice, 3 sec each time). For placenta microsome preparation an additional step of tissue grinding in the same homogenization buffer was performed (twice, 30 sec each time; until homogeneous tissue suspension was observed). Microsomal fractions were prepared by subcellular fractionation (20). The 100,000 x g pellet containing the microsomes was resuspended in 0.1 M KHPO4, 20% glycerol, 5 mM ß-mercaptoethanol, and 1 mM 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate and stored at -80 C. Protein determinations were made of the microsome-enriched preparations using the bicinchoninic acid protein assay reagent (Pierce Chemical Co., Rockford, IL) with BSA as the standard.
RIA for E2+'u)(37, http://www.100md.com
Concentrations of E2 were determined on conditioned medium from control and TCDD-treated cells using available commercial RIA kits (Diagnostic Products, Los Angeles, CA) as previously reported (9, 10, 14, 15). The interassay coefficient of variation was less than 5%.+'u)(37, http://www.100md.com
Western immunoblot+'u)(37, http://www.100md.com
Twenty micrograms of microsomal proteins from control and TCDD-treated cells were separated on an 8% SDS-PAGE and transferred by electroblot onto a polyvinylidene difluoride membrane. Membranes were blotted for P450c17 with a polyclonal antisera (1:2000 dilution) raised in chickens, recently validated for primate tissue in our laboratory (16). Further verification of the specificity of the antisera for P450c17 protein was obtained in a separate experiment that included 15 µg hLGC microsomal protein, 10 µg pig testes microsome, and 0.1 µg human recombinant P450c17 (produced in our laboratory). Immunodetectable proteins were visualized with a horseradish peroxidase-linked antichicken secondary antibody (1:5000 dilution) and developed by chemiluminescence (ECL, Amersham Pharmacia Biotech, Arlington Heights, IL). Autoradiographic films were scanned, and the relative amount of protein for each blot was determined by densitometry.
Cytochrome P450 enzyme activity+$l], http://www.100md.com
Two microsomal cytochrome P450 enzyme activities were analyzed: 17,20-lyase and P450arom. Microsomal protein from control and TCDD-treated cells were assayed for 17,20-lyase activity using a procedure adapted in our laboratory and described recently (18), with minor modifications. Briefly, the assay is based on the release of the radiolabeled acetic acid from [21-3H]17P5 after cleavage of the C17–20 bond that therefore provides a direct estimate of 17,20-lyase activity in the sample (Fig. 1) by measurement of radioactivity in the aqueous phase. One hundred micrograms of microsomal proteins from control and TCDD-treated hLGC were incubated at 37 C in assay buffer (50 mM KHPO4, 1 mM EDTA, and 1 mM 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate), at a final volume of 1 ml in the presence of 10.5 µM 17P5 (final concentration), consisting of 7 µM radiolabeled [21-3H]17P5 and 3.5 µM unlabeled 17P5 substrate. Reactions were continued for 6 h. Samples were analyzed in two assays, the internal standards (18.5 nmol/mg·h) for which were within 0.5 nmol/mg·h of each other. The limit of detection for the 17,20-lyase activity was 20 pmol/tube·h. The P450arom assay was performed on 50 µg microsomal protein from control and TCDD-treated hLGC and 100 µg placenta microsomal protein following a protocol described previously (14).
fig.ommitteed4kw{c}^, http://www.100md.com
Figure 1. Schematic representation of the 17,20-lyase reaction. Metabolism of [21-3H]17P5 by P450c17 releases [3H]acetate that is recovered in the aqueous phase after enzymatic cleavage of the side-chain of the substrate.4kw{c}^, http://www.100md.com
NADPH-cytochrome P450 oxidoreductase (reductase) activity4kw{c}^, http://www.100md.com
Reductase activity was determined on microsomal protein from hLGC (n = 6) and from term human placentas (n = 3). This was achieved by monitoring the spectral reduction of cytochrome c at 550 nm as previously reported (20, 21). The resulting data were subsequently corrected to the protein concentration in each sample and were expressed as nanomoles of cytochrome c reduced per minute per milligram of protein.4kw{c}^, http://www.100md.com
Data analysis4kw{c}^, http://www.100md.com
All data are expressed as the mean ± SEM of the treatment groups. Hormonal concentration data were log-transformed before statistical analysis and are expressed as nanograms of E2 per number of cells initially plated per unit of time. Results for the immunoblot analysis of P450c17 are presented as relative units of protein expression for each treatment after the densitometric analysis as explained above. The results for the 17,20-lyase and P450arom activity assays are expressed as picomoles of substrate converted per milligram of protein per hour. Comparisons for lyase activity and the Western blot data were made by one-way ANOVA, followed by a pairwise multiple comparison procedure using the Student-Newman-Keuls method. E2 production in culture was analyzed by two-way ANOVA, using the patient as the experimental block. Differences with P < 0.05 were considered statistically significant.
Results9|8;{, 百拇医药
TCDD effects on hormone production9|8;{, 百拇医药
The E2 concentration (n = 5) in the conditioned medium of untreated cells was not different from the DMSO (vehicle) control (7.1 ± 2.5 vs. 6.0 ± 2.2 ng/ml, respectively; P > 0.7; Fig. 2); thus, all future comparisons reported in reference to TCDD treatment effects were made using the DMSO vehicle-treated cell cultures as the control group. The E2 concentration was significantly reduced by TCDD treatment from 6.0 ± 2.2 to 2.2 ± 0.8 ng/ml (n = 12; P < 0.001). As we previously reported (14), there was a wide range in the level of E2 production among patients (0.1–23.2 ng/ml) necessitating that the data be log-transformed for statistical analysis. However, it is important to note that E2 production by cells from individual patients was inhibited regardless of the level of basal secretion for that patient, and there was no correlation between the level of E2 and the degree of TCDD-induced inhibition.
fig.ommitteed2o, http://www.100md.com
Figure 2. E2 (nanograms per 5 x 105 cells per 48 h) production by hLGC cultured in the presence of TCDD (10 nM), vehicle (DMSO), or control (DMEM, medium only). *, TCDD exposure (10 d) induced a significant decrease in E2 concentration (n = 12; P < 0.05).2o, http://www.100md.com
TCDD effects on P450c17 and reductase protein expression2o, http://www.100md.com
The antihuman P450c17 antiserum was unable to detect the enzyme protein in hLGC microsomal preparations of cells from patients that produced low levels of E2; in general, those producing less than 1 ng/ml. Thus, a strong correlation was observed between the expression of P450c17 and the level of E2 production by cells among different patients. In cells that produced higher levels of E2, P450c17 was apparent as a major immunodetectable band at 50 kDa, corresponding precisely in molecular size to the human recombinant P450c17-positive control (Figs. 3 and 4A). Pig testis microsomal protein, used as a secondary internal reference, also demonstrated a single immunodetectable band, if at a slightly smaller apparent molecular size. Microsomal proteins from cells of patients producing sufficient E2 (>1 ng/ml) were analyzed further for immunodetectable P450c17 after TCDD treatment. Microsomal proteins (20 µg) from control and TCDD-treated cells were run in adjacent lanes, and comparisons were made among the DMSO control and TCDD treatment for hLGC from each patient. TCDD decreased P450c17 protein expression in cells from all patients (Fig. 4A). Based on densitometric analysis of immunodetectable protein levels, TCDD decreased the expression (P < 0.05) of P450c17 in hLGC by over 50% (Fig. 4B). There was no change observed in immunodetectable levels of reductase (data not shown).
fig.ommitteed%v, 百拇医药
Figure 3. Immunodetection of P450c17 using antihuman P450c17 antiserum (1:2000) raised in chickens against recombinant protein. Microsomal protein (15 µg) from hLGC was separated by 8% SDS-PAGE along with pig testicular microsomal protein and human recombinant P450c17. Molecular size markers (MW) are shown at the left.%v, 百拇医药
fig.ommitteed%v, 百拇医药
Figure 4. Expression and activity of P450c17 by hLGC exposed, or not, to TCDD (10 nM). A, Immunoblot analysis of P450c17 expression in hLGC. Microsomal protein (20 µg/lane) from untreated (DMEM, medium only), vehicle control (DMSO), and treated (TCDD) cells is shown along with pig testicular microsomes (10 µg) and recombinant human P450c17 (0.1 µg) positive controls. B, Summary of densitometric analysis of immunoblots for P450c17 expression by hLGC treated with TCDD. Cells from nine different patients were used in the analysis. *, TCDD significantly (P < 0.05) decreased P450c17 expression. C, 17,20-Lyase activity (picomoles per milligram per hour) in hLGC cultured in the presence of TCDD (10 nM), vehicle (DMSO), or control (DMEM, medium only). Enzyme activity was determined radiometrically using [21-3H]17P5 as substrate. Assays were conducted using 100 µg microsomal protein from control and TCDD-treated hLGC (n = 5), which were incubated with 17P5 at 37 C for 6 h. Radioactivity measured in the aqueous phase after chloroform extraction was used as a direct estimate of 17,20-lyase activity. *, TCDD significantly decreased the 17,20-lyase activity of hLGC (P < 0.05).
TCDD effects on microsomal enzyme activity: 17,20-lyase, P450arom, and reductaseh&s#5]y, 百拇医药
The results of immunoblot analysis for P450c17 were verified by direct assessment of 17,20-lyase activity, and these data were compared with estimates of both P450arom and reductase activities in hLGC and human placenta. In contrast to the immunoblot analysis, 17,20-lyase activity was measurable only in microsomal protein from cells producing more than 8 ng/ml E2, which comprised cells from five different patients. Even in cells from these patients, 17,20-lyase activity was 10-fold lower than P450arom activity (188 ± 48 vs. 2296 ± 17 pmol/mg·h, respectively), suggesting strongly that the androgen supply was much more limiting in E2 synthesis than was the rate of aromatization. More importantly, TCDD inhibited 17,20-lyase activity in hLGC by 65% (Fig. 4C; P < 0.05), and this decrease corresponded very closely to the inhibition of E2 production (Fig. 2). In contrast to 17,20-lyase, there was no effect of TCDD on P450arom or reductase activities of hLGC. P450arom and reductase activities in hLGC microsomes were comparable to those obtained with human placenta microsomes (Table 1), which exhibited no detectable 17,20-lyase activity.
fig.ommitteed*;%hr, 百拇医药
Table 1. Microsomal enzyme activities in hLGC*;%hr, 百拇医药
Discussion*;%hr, 百拇医药
TCDD is known to adversely affect reproductive function in several animal models. However, the reproductive consequences of TCDD exposure appear to be highly dependent on species and gender as well as on dose, duration, and time after exposure (6, 12, 22, 23, 24, 25). Therefore, the selection of the experimental model and design plays an important role in the ability to extrapolate results in this area of research. Dasmahapatra et al. (26) demonstrated that the response of rat granulosa cells to TCDD varied with the sexual maturity of the animal at the time of cell collection, the duration of toxicant exposure, and the presence of FSH. TCDD significantly reduced FSH induction of P450arom and cholesterol side-chain cleavage P450, but was without effect on 3ß-hydroxysteroid dehydrogenase. TCDD has also been shown to alter steroidogenesis in the testes of the rat by modifying cholesterol transport to the inner mitochondrial membrane, inhibiting cytochrome P450 side-chain cleavage enzyme and/or P450c17 (27, 28, 29). TCDD also blocks ovulation in the rat, but although some investigators reported that steroidogenesis was altered (30, 31), others found no effect of TCDD on steroid production (32). TCDD apparently decreases estrogen production from porcine granulosa cells in vitro and progesterone from porcine luteal cells (33). In the nonhuman primate, TCDD induces abortion and decreases estrogen secretion by targeting both the trophoblast and the ovary (3, 4, 5, 6). In contrast, comparatively little is known about specific molecular targets of TCDD or its effects on human reproduction.
The in vitro culture of hLGC permits the investigation of the specific effects of TCDD on estrogen production in a relevant human cell type. Studies in this laboratory (10, 11, 13, 14, 15) and others (12) have shown consistently that TCDD inhibits E2 production by hLGC, but the molecular target(s) of toxicity has not been identified. We reasoned that at the very least, TCDD was likely to inhibit the activity of the rate-limiting step in estrogen synthesis by these cells, and the results of the current experiments indicate that this is the 17,20-lyase activity of P450c17. This is consistent with several previously reported observations from studies conducted in our laboratory. First, TCDD treatment resulted in relatively little change in the levels of either progesterone or 17{alpha} -hydroxyprogesterone, suggesting that the enzymatic step in the steroidogenic cascade most affected by TCDD must occur after cytochrome P450 side-chain cleavage and the action of 3ß-hydroxysteroid dehydrogenase on 5-C21 steroids (14). Similarly, neither P450arom protein expression nor activity was affected by TCDD (14), and neither was correlated with E2 secretion. In the same study TCDD did not change the ability of hLGC to metabolize E2, also consistent with the effects of toxicity inhibiting synthesis rather than increasing clearance (14). Furthermore, we and others have shown that the addition of androgens can reverse the negative effect of TCDD on E2 production in vitro, indicating that the supply of substrate for aromatase is limited in TCDD-treated cells (12, 14, 26). The lack of measurable androgens in medium under basal culture conditions (15) also suggests that aromatization occurs more rapidly in hLGC than does the synthesis of androgens. Therefore, we (14, 15), as have others (12), proposed that the 17,20-lyase activity of P450c17 limits the rate of E2 production by hLGC.
In contrast to the lack of effect of TCDD on P450arom and consistent with the above-stated hypothesis, the present results show clearly that both P450c17 protein and 17,20-lyase activity were reduced by toxicant exposure. Just as importantly, the degree of enzyme inhibition correlated closely with the decrease in E2 production. Collectively, these data indicate that the enzymatic step inhibited by TCDD that results in reduced estrogen synthesis involves 17,20-lyase, and that it is brought about at least in part by a decrease in the expression of P450c17. No effect was seen on the microsomal redox partner protein, reductase. Moreover, as another microsomal P450 also involved in the steroidogenic pathway leading to estrogen synthesis, namely P450arom, is not affected by TCDD exposure, this effect appears to be specific for P450c17 expression. Although aromatization is commonly held to be the rate-limiting step in estrogen biosynthesis, this is clearly not consistent with prior (12, 14, 15) or our current results. The data reported herein indicate that there are substantial differences in the capacity of hLGC to synthesize androgens and estrogens; 17,20-lyase was 15-fold lower than P450arom activity in cells exhibiting the highest levels of 17,20-lyase activity. As noted above, hLGC provides a good model of human luteal function (9); thus, we believe that this has great relevance in understanding the control of estrogen production by the human corpus luteum.
In the context of physiological and toxicological relevance to human health, it is important to note that the levels of 17,20-lyase and aromatase activities reported here for hLGC are within the range of values obtained ex vivo. Sano et al. (34) investigated steroid metabolism by human ovarian tissues collected at various stages of the menstrual cycle. Aromatase activity of cells in our study (2300 pmol/mg·h) was higher than that found in late luteal tissue (around 600 pmol/mg·h). However, our estimates were also made on enriched microsomal protein, whereas those by Sano et al. (34) were made on tissue lysates and would therefore be expected to be lower. Similarly, the estimates of 17,20-lyase activity in late luteal tissue were also lower than those found in hLGC. Regardless, aromatase was also higher than 17,20-lyase in human luteal lysates (34). Changes in P450c17 and P450arom transcripts during the menstrual cycle generally support this view (35, 36), and previous studies have also found relatively low levels of P450c17 mRNA in hLGC. However, circulating androgens of luteal origin increase in parallel with E2 in women during the midluteal phase of the menstrual cycle and increase further with E2 immediately after implantation in conceptive menstrual cycles (37). This early pregnancy-associated rise in androgens and E2 is most likely due to an increase in ovarian P450c17 expression in response to hCG that drives steroid synthesis before placental steroidogenic competency is established. The observation that these midluteal and postimplantation rises in androgens are similar within women, but highly variable between women, can be interpreted to indicate that the ability to induce P450c17 varies between individuals. Individual differences in P450c17 also provide a plausible explanation of the observed ethnic differences in androgen levels during the menopausal transition (38). Variability in the level of expression of a primary target of toxicity may also explain individual differences in susceptibility to TCDD toxicity. Therefore, P450c17 may become an especially sensitive target for TCDD-induced endocrine disruption in women during the late luteal phase, early pregnancy, and even later in life when it is perhaps more limiting than P450arom in estrogen synthesis.
It is also significant that the effect of TCDD is specific for P450c17 and does not affect P450arom expression, if only because TCDD induces the transcriptional expression of other microsomal P450s (39, 40). Studies are in progress to determine whether there is a decrease in transcript or perhaps an increased rate of P450c17 degradation, although this seems less likely based on the specificity of the change in levels for this microsomal P450. However, even if P450c17 transcript levels do decline, this does not necessarily imply that the effect of TCDD is directly on CYP17. Putative dioxin-response elements in the human CYP17 gene are fully 3 kb upstream from the transcriptional start site (Dr. Mike Denison, University of California-Davis, unpublished observations). Others have reported a transient inhibition of E2 secretion before stimulation in the first 48 h of culture, but only when micromolar concentrations of TCDD were used (12). In our studies E2 secretion by hLGC is highly variable in the first 2 d of culture, as is the response to TCDD, and a consistent depression begins by d 4 of culture (14). However, in contrast to others (12), in our studies hLGC are cultured in the continued presence of hCG and nanomolar concentrations of TCDD. Studies in vivo and in vitro demonstrated that TCDD decreased gonadotropic support or response (41, 42, 43), and this may still play a role in the response of reproductive tissues to toxic exposure, for which numerous disease consequences have been suspected (44, 45). Although this mechanism could contribute to the reduction of P450c17 noted here, the lack of effect on P450arom argues strongly that is not a major factor, as both P450c17 and P450arom are believed to be responsive to gonadotropins in human ovarian cells (46, 47). It is not known whether P450c17 expression is a target for TCDD in other tissues, such as the adrenal cortex or testes. The means of transcriptional regulation of CYP17 expression is not necessarily the same in the adrenal gland and gonad, even though the same promoter region is used (48). Further studies are needed to elucidate the specific mode of action of TCDD in depressing P450c17 expression in hLGC.
In summary, to our knowledge this is the first report of P450c17 protein expression and 17,20-lyase activity in hLGC, and the demonstration of its susceptibility to TCDD toxicity. TCDD treatment decreased both the protein expression and the 17,20-lyase activity of P450c17 in microsomal fractions from hLGC by more than 50% and 60%, respectively. To our knowledge, this is the first report of a clear, specific molecular target of endocrine disruption resulting from TCDD toxicity in any human cell.r'g)gt, 百拇医药
Acknowledgmentsr'g)gt, 百拇医药
The authors thank Ms. Karen Woodward for assistance with cell preparation and hormone assays, and Dr. Justin Vidal and staff at Sutter Hospital (Davis, CA) for help with the collection and processing of term placental tissue. The authors also acknowledge the generosity of Drs. Angela Brodie and Victor Njar for providing the tracer for the 17,20-lyase activity assays.r'g)gt, 百拇医药
Received August 5, 2002.r'g)gt, 百拇医药
Accepted for publication October 16, 2002.
References\v*, 百拇医药
Poland A, Knutson JC 1982 2,3,7,8-Tetrachlorodibenzo-p-dioxin and related halogenated aromatic hydrocarbons: examination of the mechanism of toxicity. Annu Rev Pharmacol Toxicol 22:517–554\v*, 百拇医药
Safe SH 1995 Modulation of gene expression and endocrine response pathways by 2,3,7,8-tetrachlorodibenzo-p-dioxin and related compounds. Pharmacol Ther 67:247–281\v*, 百拇医药
Hendrickx AG, Peterson PE, Otianga-Owiti GE, Tarantal AF, Dieter J, Lasley B, Overstreet JW 1999 Endocrine and morphological biomarkers of early pregnancy loss in macaques. In: Weinbauer G, Korte R, eds. Reproduction in nonhuman primates: a model system for human reproductive physiology and toxicology. New York, Munich, Berlin: Waxman, Munster; 111–136\v*, 百拇医药
McNulty WP 1984 Fetotoxicity of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) for rhesus macaques (Macaca mulatta). Am J Primatol 6:41–47\v*, 百拇医药
Guo Y, Hendrickx AG, Overstreet JW, Dieter J, Stewart D, Tarantal AF, Laughlin L, Lasley BL 1999 Endocrine biomarkers of early fetal loss in cynomolgus macaques (Macaca fascicularis) following exposure to dioxin. Biol Reprod 60:707–713
Moran FM, Tarara R, Chen J, Santos S, Cheney A, Overstreet JW, Lasley BL 2001 Effect of dioxin on ovarian function in the cynomolgus macaque (M. fascicularis). Reprod Toxicol 15:377–383j!)-}pj, 百拇医药
Pirkle JL, Wolfe WH, Patterson DG, Needham LL, Michalek JE, Miner JC, Peterson MR, Phillips DL 1989 Estimates of the half-life of 2,3,7,8-tetrachlorodibenzo-p-dioxin in Vietnam veterans of Operation Ranch Hand. J Toxicol Environ Health 27:165–171j!)-}pj, 百拇医药
Michalek JE, Pirkle JL, Caudill SP, Tripathi RC, Patterson Jr DG, Needham LL 1996 Pharmacokinetics of TCDD in veterans of operation ranch hand: 10-year follow-up. J Toxicol Environ Health 47:209–220j!)-}pj, 百拇医药
Stewart D, VandeVoort C 1997 Simulation of human luteal endocrine function with granulosa lutein cell culture. J Clin Endocrinol Metab 82:3078–3083j!)-}pj, 百拇医药
Enan E, Morán FM, VandeVoort CA, Stewart DR, Overstreet JW, Lasley BL 1996 Mechanism of toxic action of 2,3,7,8-tetrachlorodibenzo-p-dioxin TCDD in cultured human luteinized granulosa cells. Reprod Toxicol 10:497–508
Moran FM, Enan E, VandeVoort CA, Stewart DR, Conley AJ, Overstreet JW, Lasley BL 1997 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) effects on steroidogenesis of human luteinized granulosa cell in vitro. Biol Reprod 56:99 (Abstract)$1c], 百拇医药
Heimler I, Rawlins RG, Owen H, Hutz RJ 1998 Dioxin perturbs, in a dose- and time-dependent fashion, steroid secretion, and induces apoptosis of human luteinized granulosa cells. Endocrinology 139:4373–4379$1c], 百拇医药
Enan E, Lasley B, Stewart D, Overstreet J, VandeVoort CA 1996 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) modulates function of human luteinizing granulosa cells via cAMP signaling and early reduction of glucose transporting activity. Reprod Toxicol 10:191–198$1c], 百拇医药
Moran FM, Conley AJ, Corbin CJ, Enan E, VandeVoort C, Overstreet JW, Lasley BL 2000 2,3,7,8-Tetrachlorodibenzo-p-dioxin decreases estradiol production without altering the enzyme activity of cytochrome p450 aromatase of human luteinized granulosa cells in vitro. Biol Reprod 62:1102–1108$1c], 百拇医药
Moran FM, Lohstroh P, VandeVoort CA, Chen J, Overstreet JW, Conley AJ, Lasley BL Exogenous steroid substrate modifies the effect of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on estradiol production of human luteinized granulosa cells in vitro. Biol Reprod, in press
Mapes S, Tarantal AF, Parker CR, Moran FM, Bahr JM, Pyter L, Conley AJ 2002 Adrenocortical cytochrome b5 expression during fetal development of the rhesus macaque. Endocrinology 143:1451–1458tyhp, 百拇医药
Grigoryev DN, Kato K, Njar VC, Long BJ, Ling YZ, Wang X, Mohler J, Brodie AM 1999 Cytochrome P450c17-expressing Escherichia coli as a first-step screening system for 17{alpha} -hydroxylase-C17,20-lyase inhibitors. Anal Biochem 267:319–330tyhp, 百拇医药
Moran FM, Ford JJ, Corbin CJ, Mapes SM, Njar VC, Brodie AM, Conley AJ 2002 Regulation of microsomal P450, redox partner proteins and steroidogenesis in the developing testes of the neonatal pig. Endocrinology 143:3361–3369tyhp, 百拇医药
Simpson E, MacDonald P 1981 Endocrine physiology of the placenta. Annu Rev Physiol 43:163–188tyhp, 百拇医药
Corbin CJ, Trant JM, Conley AJ 2001 Porcine gonadal and placental isozymes of aromatase cytochrome P450: sub-cellular distribution and support by NADPH-cytochrome P450 reductase. Mol Cell Endocrinol 172:115–124tyhp, 百拇医药
Guengerich FP2001 Analysis and characterization of enzymes and nucleic acids. In: Hayes AW, ed. Principles and methods of toxicology. 4th ed. Philadelphia: Taylor, Francis; 1634–1635
Peterson RE, Theobald HM, Kimmel GL 1993 Developmental and reproductive toxicity of dioxins and related compounds: cross-species comparisons. Crit Rev Toxicol 23:283–335n^t, 百拇医药
Dickson LC, Buzik SC 1993 Health risks of "dioxins:" a review of environmental and toxicological considerations. Vet Hum Toxicol 35:68–77n^t, 百拇医药
Chaffin C, Peterson R, Hutz R 1996 In utero and lactational exposure of female Holtzman rats to 2,3,7,8-tetrachlorodibenzo-p-dioxin: modulation of the estrogen signal. Biol Reprod 55:62–67n^t, 百拇医药
Rier SE, Martin DC, Bowman RE, Dmowski WP, Becker JL 1993 Endometriosis in rhesus monkeys (Macaca mulatta) following chronic exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin. Fund Appl Toxicol 21:433–441n^t, 百拇医药
Dasmahapatra AK, Wimpee BAB, Trewin AL, Wimpee CF, Ghorai JK, Hutz RJ 2000 Demonstration of 2,3,7,8-tetrachlorodibenzo-p-dioxin attenuation of P450 steroidogenic enzyme mRNAs in rat granulosa cell in vitro by competitive reverse transcriptase-polymerase chain reaction assay. Mol Cell Endocrinol 164:5–18
Mebus CA, Reddy VR, Piper WN 1987 Depression of rat testicular 17-hydroxylase and 17,20-lyase after administration of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Biochem Pharmacol 36:727–731p0[@o|@, 百拇医药
Kleeman JM, Moore RW, Peterson RE 1990 Inhibition of testicular steroidogenesis in 2,3,7,8-tetrachlorodibenzo-p-dioxin-treated rats: evidence that the key lesion occurs prior to or during pregnenolone formation. Toxicol Appl Pharmacol 106:112–125p0[@o|@, 百拇医药
Moore RW, Jefcoate CR, Peterson RE 1991 2,3,7,8-Tetrachlorodibenzo-p-dioxin inhibits steroidogenesis in the rat testis by inhibiting the mobilization of cholesterol to cytochrome P450scc. Toxicol Appl Pharmacol 109:85–97p0[@o|@, 百拇医药
Li X, Johnson DC, Rozman KK 1995 Reproductive effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in female rats: ovulation, hormonal regulation, and possible mechanism(s). Toxicol Appl Pharmacol 133:321–327p0[@o|@, 百拇医药
Ushinohama K, Son D-S, Roby KF, Rozman KK, Terranova PF 2001 Impaired ovulation by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in immature rats treated with equine chorionic gonadotropin. Reprod Toxicol 15:275–280
Son D-S, Ushinohama K, Gao X, Taylor CC, Roby KF, Rozman KK, Terranova PF 1999 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) blocks ovulation by a direct action on the ovary without alteration of ovarian steroidogenesis: lack of a direct effect on ovarian granulosa and thecal-interstitial cell steroidogenesis in vitro. Reprod Toxicol 13:521–530yyjj\0f, 百拇医药
Gregoraszczuk E 2002 Dioxin exposure and porcine reproductive hormonal activity. Cad Saude Publica 18:453–462yyjj\0f, 百拇医药
Sano Y, Suzuki K, Arai K, Okinaga S, Tamaoki B 1981 Changes in enzyme activities related to steroidogenesis in human ovaries during the menstrual cycle. J Clin Endocrinol Metab 52:994–1001yyjj\0f, 百拇医药
Doody K, Lorence M, Mason J, Simpson E 1990 Expression of messenger ribonucleic acid species encoding steroidogenic enzymes in human follicles and corpora lutea throughout the menstrual cycle. J Clin Endocrinol Metab 70:1041–1045yyjj\0f, 百拇医药
Voutilainen R, Tapanainen J, Chung B, Matteson K, Miller W 1986 Hormonal regulation of P450scc (20,22-desmolase) and P450c17 (17{alpha} -hydroxylase/17,20-lyase) in cultured human granulosa cells. J Clin Endocrinol Metab 63:202–207
Castracane VD, Stewart DR, Gimpel T, Overstreet JW, Lasley BL 1998 Maternal serum androgens in human pregnancy: early increases within the cycle of conception. Hum Reprod 13:460–464ez|zwp, 百拇医药
Lasley BL, Santoro N, Randolph J, Gold EB, Crawford S, Weiss G, McConnell DS, Sowers MF 2002 The relationship of circulating dehydroepiandrosterone, testosterone, and estradiol to stages of the menopausal transition and ethnicity. J Clin Endocrinol Metab 87:3760–3767ez|zwp, 百拇医药
Waxman DJ 1999 P450 Gene induction by structurally diverse xenochemicals: central role of nuclear receptors CAR, PXR, and PPAR. Arch Biochem Biophys 369:11–23ez|zwp, 百拇医药
Whitlock Jr JP 1999 Induction of cytochrome P4501A1. Annu Rev Pharmacol Toxicol 39:103–125.ez|zwp, 百拇医药
Gao X, Son D-S, Terranova PF, Rozman KK 1999 Toxic equivalency factors of polychlorinated dibenzo-p-dioxins in an ovulation model: validation of the toxic equivalency concept for one aspect of endocrine disruption. Toxicol Appl Pharmacol 157:107–116ez|zwp, 百拇医药
Hirakawa T, Minegishi T, Abe K, Kishi H, Inoue K, Ibuki Y,Miyamoto K 2000 Effect of 2,3,7,8-tetrachlorodibenzo-p-dioxin on the expression of follicle-stimulating hormone receptors during cell differentiation in cultured granulosa cells. Endocrinology 141:1470–1476
Li X, Johnson DC, Rozman KK 1997 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) increases release of luteinizing hormone and follicle-stimulating hormone from the pituitary of immature female rats in vivo and in vitro. Toxicol Appl Pharmacol 142:264–269\2, http://www.100md.com
Bertazzi PA, Consonni D, Bachetti S, Rubagotti M, Baccarelli A, Zocchetti C, Pesatori AC 2001 Health effects of dioxin exposure: a 20-year mortality study. Am J Epidemiol 153:1031–1044\2, http://www.100md.com
Kogevinas M 2001 Human health effects of dioxins: cancer, reproductive and endocrine system effects. Hum Reprod Update 7:331–339\2, http://www.100md.com
McAllister J, Kerin J, Trant J, Estabrook R, Mason J, Waterman M, Simpson E 1989 Regulation of cholesterol side-chain cleavage and 17{alpha} -hydroxylase/lyase activities in proliferating human theca interna cells in long term monolayer culture. Endocrinology 125:1959–1966\2, http://www.100md.com
Steinkampf M, Mendelson C, Simpson E 1987 Regulation by follicle-stimulating hormone of the synthesis of aromatase cytochrome P-450 in human granulosa cells. Mol Endocrinol 1:465–471\2, http://www.100md.com
Arlotto MD, Michael MD, Kilgore MW, Simpson ER 1996 17{alpha} -hydroxylase gene expression in the bovine ovary: mechanisms regulating expression differ from those in adrenal cells. J Steroid Biochem Mol Biol 59:21–29(F. M. Morán C. A. VandeVoort J. W. Overstreet B. L. Lasley and A. J. Conley)
Abstract*6')1d?, 百拇医药
Estradiol (E2) production by human luteinized granulosa cells (hLGC) is inhibited by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). The molecular target of TCDD toxicity has not been identified. The decrease in E2 is ameliorated by androgen substrate addition and is not associated with changes in aromatase cytochrome P450 (P450arom) activity or protein expression. An antihuman 17-hydroxylase/17,20-lyase cytochrome P450 (P450c17) antisera and a direct radiometric assay of 17,20-lyase activity were used to test the hypothesis that TCDD targets P450c17, thereby decreasing substrate availability for E2 synthesis by hLGC. P450c17 expression and 17,20-lyase activity were detected in hLGC with high levels of E2 secretion. Western immunoblot analysis demonstrated that TCDD treatment of hLGC decreased the expression of P450c17 by as much 50% (P < 0.05). TCDD exposure induced a 65% decrease in 17,20-lyase activity (P < 0.05), but no changes were seen in P450arom or in nicotinamide adenine dinucleotide phosphate (reduced)-cytochrome P450 oxidoreductase (reductase). Furthermore, the decreases in P450c17 and 17,20-lyase were proportional to the inhibition of E2 secretion. We conclude that the molecular target for endocrine disruption of hLGC by TCDD is P450c17, specifically decreasing the supply of androgens for E2 synthesis, and that it does not involve either P450arom or the redox partner protein reductase.
Introduction?ky, 百拇医药
2,3,7,8-TETRACHLORODIBENZO-p-DIOXIN (TCDD) is a potent toxicant with the ability to disrupt endocrine and reproductive systems. It is a common environmental contaminant and is recognized generally to be one of the most active congeners of the polychlorinated aromatic hydrocarbon group (1, 2). A single dose of TCDD induces abortion and is accompanied by a decrease in serum estradiol (E2) in the laboratory macaque (3, 4, 5). This acute response is followed by chronic arrest of follicular growth, characterized by anovulation and decreased ovarian steroidogenesis (6). The dangers associated with human TCDD exposure are exacerbated by its extremely long half-life in the body (7, 8). Despite numerous studies to characterize the general toxicity of TCDD in reproductive and other tissues, little is known or understood concerning the mechanisms by which TCDD exerts its effect at the cellular or molecular level.?ky, 百拇医药
Studies investigating the molecular events that drive TCDD-induced cellular toxicity have been hampered by two major factors. Firstly, like many toxicants, the effects of TCDD differ among species, and the choice of animal models is crucial. Second, the ability to decipher mechanisms at the molecular level requires a relevant cellular or in vitro model system and the development of the proper tools. The use of human luteinizing granulosa cells (hLGC) in vitro has provided us with a useful model with which to study endocrine disruption in human tissues exposed to TCDD (9). Several studies in this laboratory as well others demonstrated that TCDD decreased E2 production by hLGC (10, 11, 12, 13, 14, 15). Logically, the effects of TCDD and other endocrine disruptors are more likely to be acutely evident if they alter a rate-limiting reaction rather than a reaction that has unlimited capability of metabolizing available substrate.
The results of our prior experiments suggested that the target for TCDD toxicity in hLGC is 17,20-lyase activity (14, 15), but we lacked the means to specifically test this hypothesis at that time. We recently raised antisera against recombinant human cytochrome P450 17{alpha} -hydroxylase/17,20-lyase (16) and adopted the use of a radiometric assay previously developed (17) to directly estimate 17,20-lyase activity (18). Therefore, with these critical tools in hand, the present experiments were designed to test the hypothesis that TCDD inhibits estrogen synthesis by hLGC through effects on P450c17 by examining both protein expression and 17,20-lyase activity. In addition, as both P450c17 and P450arom activities are totally dependent on redox support by the flavoprotein nicotinamide adenine dinucleotide phosphate (reduced) (NADPH)-cytochrome P450 oxidoreductase (reductase), the level of this essential redox partner protein was measured in control and treated cells.x#7.:(, http://www.100md.com
Materials and Methods
Chemicals?mso, 百拇医药
DMEM, antibiotic-antimycotic (10,000 U/ml penicillin G sodium, 10 mg/ml streptomycin sulfate, and 25 µg/ml amphotericin B as a fungizone), and fetal bovine and calf sera were purchased from Life Technologies, Inc. (Grand Island, NY). Pregnyl, a commercial human chorionic gonadotropin (hCG) was purchased from Organon, Inc. (West Orange, NJ). TCDD was a gift from Dr. Steven Safe (Texas A&M University, College Station, TX). 5-Pregnen-3ß, 17{alpha} -diol-20-one [17{alpha} - hydroxypregnenolone (17P5)] was purchased from Steraloids (Wilton, NH). [1,214C]Acetic acid (54 mCi/mmol) was purchased from NEN Life Science Products (Boston, MA). [21-3H]17P5 (25.9 µCi/µmol) was donated by Dr. Angela Brodie (University of Maryland Medical School, Baltimore, MD). All other chemicals were purchased from Sigma-Aldrich (St. Louis, MO).?mso, 百拇医药
hLGC culture?mso, 百拇医药
hLGC were obtained from patients undergoing assisted reproduction treatments at the Northern California Fertility Medical Center (Roseville, CA), and all experiments using them were reviewed and approved by the human subjects advisory committee, University of California-Davis. The cells were processed as previously described (9, 15). hLGC were plated at 5 x 105 cells in 3 ml/60-mm plate or at 106 cells/100-mm plate with 5 ml conditioned medium (DMEM supplemented with antibiotic-antimycotic, 10% noncharcoal-stripped calf serum, and 2 USP/ml hCG).
TCDD treatmentnpz-^fx, 百拇医药
Previous studies determined that a concentration of 10 nM TCDD effectively disrupts E2 production by hLGC (10, 14). Therefore, TCDD (10-5 M) dissolved in dimethylsulfoxide (DMSO) was added directly to each plate at a dilution of 1 µl/ml conditioned medium (10 nM final concentration). TCDD treatment started on the day after recovery. Medium was changed every other day thereafter, and TCDD treatment was added immediately after medium changes. Cells were cultured in TCDD for a total of 10 d; we have previously established that the number of cells at plating does not change with either time or TCDD treatment (10, 13, 14). On the last day of culture, medium was collected and stored frozen at -20 C for hormone analysis. Cells were then harvested by scraping in PBS, pelleted by centrifugation, and stored at -80 C for further processing as described below. Controls included cells treated with DMSO (equal volume) or not.npz-^fx, 百拇医药
Placental tissue
Human term placentas were obtained from patients after normal deliveries in the Obstetrics and Gynecologic Clinic at Sutter Hospital (Davis, CA) and were used as negative control tissues recognized not to express P450c17 (19) in 17,20-lyase activity assays, also after review and approval by the human subjects advisory committee, University of California-Davis. Tissues were collected within 3 h of delivery, transported on ice to the laboratory, and stored at -80 C until used for microsome preparation.xl}78, http://www.100md.com
Microsome-enriched fraction preparationxl}78, http://www.100md.com
Cell pellets from 100-mm dishes were resuspended in 1 ml homogenization buffer [0.1 M KHPO4 (pH 7.4), 20% glycerol, and 5 mM ß-mercaptoethanol] and 0.5 mM of the protease inhibitor phenylmethylsulfonylfluoride; after 10 strokes with a glass-glass homogenator, the cells were briefly sonicated (twice, 3 sec each time). For placenta microsome preparation an additional step of tissue grinding in the same homogenization buffer was performed (twice, 30 sec each time; until homogeneous tissue suspension was observed). Microsomal fractions were prepared by subcellular fractionation (20). The 100,000 x g pellet containing the microsomes was resuspended in 0.1 M KHPO4, 20% glycerol, 5 mM ß-mercaptoethanol, and 1 mM 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate and stored at -80 C. Protein determinations were made of the microsome-enriched preparations using the bicinchoninic acid protein assay reagent (Pierce Chemical Co., Rockford, IL) with BSA as the standard.
RIA for E2+'u)(37, http://www.100md.com
Concentrations of E2 were determined on conditioned medium from control and TCDD-treated cells using available commercial RIA kits (Diagnostic Products, Los Angeles, CA) as previously reported (9, 10, 14, 15). The interassay coefficient of variation was less than 5%.+'u)(37, http://www.100md.com
Western immunoblot+'u)(37, http://www.100md.com
Twenty micrograms of microsomal proteins from control and TCDD-treated cells were separated on an 8% SDS-PAGE and transferred by electroblot onto a polyvinylidene difluoride membrane. Membranes were blotted for P450c17 with a polyclonal antisera (1:2000 dilution) raised in chickens, recently validated for primate tissue in our laboratory (16). Further verification of the specificity of the antisera for P450c17 protein was obtained in a separate experiment that included 15 µg hLGC microsomal protein, 10 µg pig testes microsome, and 0.1 µg human recombinant P450c17 (produced in our laboratory). Immunodetectable proteins were visualized with a horseradish peroxidase-linked antichicken secondary antibody (1:5000 dilution) and developed by chemiluminescence (ECL, Amersham Pharmacia Biotech, Arlington Heights, IL). Autoradiographic films were scanned, and the relative amount of protein for each blot was determined by densitometry.
Cytochrome P450 enzyme activity+$l], http://www.100md.com
Two microsomal cytochrome P450 enzyme activities were analyzed: 17,20-lyase and P450arom. Microsomal protein from control and TCDD-treated cells were assayed for 17,20-lyase activity using a procedure adapted in our laboratory and described recently (18), with minor modifications. Briefly, the assay is based on the release of the radiolabeled acetic acid from [21-3H]17P5 after cleavage of the C17–20 bond that therefore provides a direct estimate of 17,20-lyase activity in the sample (Fig. 1) by measurement of radioactivity in the aqueous phase. One hundred micrograms of microsomal proteins from control and TCDD-treated hLGC were incubated at 37 C in assay buffer (50 mM KHPO4, 1 mM EDTA, and 1 mM 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate), at a final volume of 1 ml in the presence of 10.5 µM 17P5 (final concentration), consisting of 7 µM radiolabeled [21-3H]17P5 and 3.5 µM unlabeled 17P5 substrate. Reactions were continued for 6 h. Samples were analyzed in two assays, the internal standards (18.5 nmol/mg·h) for which were within 0.5 nmol/mg·h of each other. The limit of detection for the 17,20-lyase activity was 20 pmol/tube·h. The P450arom assay was performed on 50 µg microsomal protein from control and TCDD-treated hLGC and 100 µg placenta microsomal protein following a protocol described previously (14).
fig.ommitteed4kw{c}^, http://www.100md.com
Figure 1. Schematic representation of the 17,20-lyase reaction. Metabolism of [21-3H]17P5 by P450c17 releases [3H]acetate that is recovered in the aqueous phase after enzymatic cleavage of the side-chain of the substrate.4kw{c}^, http://www.100md.com
NADPH-cytochrome P450 oxidoreductase (reductase) activity4kw{c}^, http://www.100md.com
Reductase activity was determined on microsomal protein from hLGC (n = 6) and from term human placentas (n = 3). This was achieved by monitoring the spectral reduction of cytochrome c at 550 nm as previously reported (20, 21). The resulting data were subsequently corrected to the protein concentration in each sample and were expressed as nanomoles of cytochrome c reduced per minute per milligram of protein.4kw{c}^, http://www.100md.com
Data analysis4kw{c}^, http://www.100md.com
All data are expressed as the mean ± SEM of the treatment groups. Hormonal concentration data were log-transformed before statistical analysis and are expressed as nanograms of E2 per number of cells initially plated per unit of time. Results for the immunoblot analysis of P450c17 are presented as relative units of protein expression for each treatment after the densitometric analysis as explained above. The results for the 17,20-lyase and P450arom activity assays are expressed as picomoles of substrate converted per milligram of protein per hour. Comparisons for lyase activity and the Western blot data were made by one-way ANOVA, followed by a pairwise multiple comparison procedure using the Student-Newman-Keuls method. E2 production in culture was analyzed by two-way ANOVA, using the patient as the experimental block. Differences with P < 0.05 were considered statistically significant.
Results9|8;{, 百拇医药
TCDD effects on hormone production9|8;{, 百拇医药
The E2 concentration (n = 5) in the conditioned medium of untreated cells was not different from the DMSO (vehicle) control (7.1 ± 2.5 vs. 6.0 ± 2.2 ng/ml, respectively; P > 0.7; Fig. 2); thus, all future comparisons reported in reference to TCDD treatment effects were made using the DMSO vehicle-treated cell cultures as the control group. The E2 concentration was significantly reduced by TCDD treatment from 6.0 ± 2.2 to 2.2 ± 0.8 ng/ml (n = 12; P < 0.001). As we previously reported (14), there was a wide range in the level of E2 production among patients (0.1–23.2 ng/ml) necessitating that the data be log-transformed for statistical analysis. However, it is important to note that E2 production by cells from individual patients was inhibited regardless of the level of basal secretion for that patient, and there was no correlation between the level of E2 and the degree of TCDD-induced inhibition.
fig.ommitteed2o, http://www.100md.com
Figure 2. E2 (nanograms per 5 x 105 cells per 48 h) production by hLGC cultured in the presence of TCDD (10 nM), vehicle (DMSO), or control (DMEM, medium only). *, TCDD exposure (10 d) induced a significant decrease in E2 concentration (n = 12; P < 0.05).2o, http://www.100md.com
TCDD effects on P450c17 and reductase protein expression2o, http://www.100md.com
The antihuman P450c17 antiserum was unable to detect the enzyme protein in hLGC microsomal preparations of cells from patients that produced low levels of E2; in general, those producing less than 1 ng/ml. Thus, a strong correlation was observed between the expression of P450c17 and the level of E2 production by cells among different patients. In cells that produced higher levels of E2, P450c17 was apparent as a major immunodetectable band at 50 kDa, corresponding precisely in molecular size to the human recombinant P450c17-positive control (Figs. 3 and 4A). Pig testis microsomal protein, used as a secondary internal reference, also demonstrated a single immunodetectable band, if at a slightly smaller apparent molecular size. Microsomal proteins from cells of patients producing sufficient E2 (>1 ng/ml) were analyzed further for immunodetectable P450c17 after TCDD treatment. Microsomal proteins (20 µg) from control and TCDD-treated cells were run in adjacent lanes, and comparisons were made among the DMSO control and TCDD treatment for hLGC from each patient. TCDD decreased P450c17 protein expression in cells from all patients (Fig. 4A). Based on densitometric analysis of immunodetectable protein levels, TCDD decreased the expression (P < 0.05) of P450c17 in hLGC by over 50% (Fig. 4B). There was no change observed in immunodetectable levels of reductase (data not shown).
fig.ommitteed%v, 百拇医药
Figure 3. Immunodetection of P450c17 using antihuman P450c17 antiserum (1:2000) raised in chickens against recombinant protein. Microsomal protein (15 µg) from hLGC was separated by 8% SDS-PAGE along with pig testicular microsomal protein and human recombinant P450c17. Molecular size markers (MW) are shown at the left.%v, 百拇医药
fig.ommitteed%v, 百拇医药
Figure 4. Expression and activity of P450c17 by hLGC exposed, or not, to TCDD (10 nM). A, Immunoblot analysis of P450c17 expression in hLGC. Microsomal protein (20 µg/lane) from untreated (DMEM, medium only), vehicle control (DMSO), and treated (TCDD) cells is shown along with pig testicular microsomes (10 µg) and recombinant human P450c17 (0.1 µg) positive controls. B, Summary of densitometric analysis of immunoblots for P450c17 expression by hLGC treated with TCDD. Cells from nine different patients were used in the analysis. *, TCDD significantly (P < 0.05) decreased P450c17 expression. C, 17,20-Lyase activity (picomoles per milligram per hour) in hLGC cultured in the presence of TCDD (10 nM), vehicle (DMSO), or control (DMEM, medium only). Enzyme activity was determined radiometrically using [21-3H]17P5 as substrate. Assays were conducted using 100 µg microsomal protein from control and TCDD-treated hLGC (n = 5), which were incubated with 17P5 at 37 C for 6 h. Radioactivity measured in the aqueous phase after chloroform extraction was used as a direct estimate of 17,20-lyase activity. *, TCDD significantly decreased the 17,20-lyase activity of hLGC (P < 0.05).
TCDD effects on microsomal enzyme activity: 17,20-lyase, P450arom, and reductaseh&s#5]y, 百拇医药
The results of immunoblot analysis for P450c17 were verified by direct assessment of 17,20-lyase activity, and these data were compared with estimates of both P450arom and reductase activities in hLGC and human placenta. In contrast to the immunoblot analysis, 17,20-lyase activity was measurable only in microsomal protein from cells producing more than 8 ng/ml E2, which comprised cells from five different patients. Even in cells from these patients, 17,20-lyase activity was 10-fold lower than P450arom activity (188 ± 48 vs. 2296 ± 17 pmol/mg·h, respectively), suggesting strongly that the androgen supply was much more limiting in E2 synthesis than was the rate of aromatization. More importantly, TCDD inhibited 17,20-lyase activity in hLGC by 65% (Fig. 4C; P < 0.05), and this decrease corresponded very closely to the inhibition of E2 production (Fig. 2). In contrast to 17,20-lyase, there was no effect of TCDD on P450arom or reductase activities of hLGC. P450arom and reductase activities in hLGC microsomes were comparable to those obtained with human placenta microsomes (Table 1), which exhibited no detectable 17,20-lyase activity.
fig.ommitteed*;%hr, 百拇医药
Table 1. Microsomal enzyme activities in hLGC*;%hr, 百拇医药
Discussion*;%hr, 百拇医药
TCDD is known to adversely affect reproductive function in several animal models. However, the reproductive consequences of TCDD exposure appear to be highly dependent on species and gender as well as on dose, duration, and time after exposure (6, 12, 22, 23, 24, 25). Therefore, the selection of the experimental model and design plays an important role in the ability to extrapolate results in this area of research. Dasmahapatra et al. (26) demonstrated that the response of rat granulosa cells to TCDD varied with the sexual maturity of the animal at the time of cell collection, the duration of toxicant exposure, and the presence of FSH. TCDD significantly reduced FSH induction of P450arom and cholesterol side-chain cleavage P450, but was without effect on 3ß-hydroxysteroid dehydrogenase. TCDD has also been shown to alter steroidogenesis in the testes of the rat by modifying cholesterol transport to the inner mitochondrial membrane, inhibiting cytochrome P450 side-chain cleavage enzyme and/or P450c17 (27, 28, 29). TCDD also blocks ovulation in the rat, but although some investigators reported that steroidogenesis was altered (30, 31), others found no effect of TCDD on steroid production (32). TCDD apparently decreases estrogen production from porcine granulosa cells in vitro and progesterone from porcine luteal cells (33). In the nonhuman primate, TCDD induces abortion and decreases estrogen secretion by targeting both the trophoblast and the ovary (3, 4, 5, 6). In contrast, comparatively little is known about specific molecular targets of TCDD or its effects on human reproduction.
The in vitro culture of hLGC permits the investigation of the specific effects of TCDD on estrogen production in a relevant human cell type. Studies in this laboratory (10, 11, 13, 14, 15) and others (12) have shown consistently that TCDD inhibits E2 production by hLGC, but the molecular target(s) of toxicity has not been identified. We reasoned that at the very least, TCDD was likely to inhibit the activity of the rate-limiting step in estrogen synthesis by these cells, and the results of the current experiments indicate that this is the 17,20-lyase activity of P450c17. This is consistent with several previously reported observations from studies conducted in our laboratory. First, TCDD treatment resulted in relatively little change in the levels of either progesterone or 17{alpha} -hydroxyprogesterone, suggesting that the enzymatic step in the steroidogenic cascade most affected by TCDD must occur after cytochrome P450 side-chain cleavage and the action of 3ß-hydroxysteroid dehydrogenase on 5-C21 steroids (14). Similarly, neither P450arom protein expression nor activity was affected by TCDD (14), and neither was correlated with E2 secretion. In the same study TCDD did not change the ability of hLGC to metabolize E2, also consistent with the effects of toxicity inhibiting synthesis rather than increasing clearance (14). Furthermore, we and others have shown that the addition of androgens can reverse the negative effect of TCDD on E2 production in vitro, indicating that the supply of substrate for aromatase is limited in TCDD-treated cells (12, 14, 26). The lack of measurable androgens in medium under basal culture conditions (15) also suggests that aromatization occurs more rapidly in hLGC than does the synthesis of androgens. Therefore, we (14, 15), as have others (12), proposed that the 17,20-lyase activity of P450c17 limits the rate of E2 production by hLGC.
In contrast to the lack of effect of TCDD on P450arom and consistent with the above-stated hypothesis, the present results show clearly that both P450c17 protein and 17,20-lyase activity were reduced by toxicant exposure. Just as importantly, the degree of enzyme inhibition correlated closely with the decrease in E2 production. Collectively, these data indicate that the enzymatic step inhibited by TCDD that results in reduced estrogen synthesis involves 17,20-lyase, and that it is brought about at least in part by a decrease in the expression of P450c17. No effect was seen on the microsomal redox partner protein, reductase. Moreover, as another microsomal P450 also involved in the steroidogenic pathway leading to estrogen synthesis, namely P450arom, is not affected by TCDD exposure, this effect appears to be specific for P450c17 expression. Although aromatization is commonly held to be the rate-limiting step in estrogen biosynthesis, this is clearly not consistent with prior (12, 14, 15) or our current results. The data reported herein indicate that there are substantial differences in the capacity of hLGC to synthesize androgens and estrogens; 17,20-lyase was 15-fold lower than P450arom activity in cells exhibiting the highest levels of 17,20-lyase activity. As noted above, hLGC provides a good model of human luteal function (9); thus, we believe that this has great relevance in understanding the control of estrogen production by the human corpus luteum.
In the context of physiological and toxicological relevance to human health, it is important to note that the levels of 17,20-lyase and aromatase activities reported here for hLGC are within the range of values obtained ex vivo. Sano et al. (34) investigated steroid metabolism by human ovarian tissues collected at various stages of the menstrual cycle. Aromatase activity of cells in our study (2300 pmol/mg·h) was higher than that found in late luteal tissue (around 600 pmol/mg·h). However, our estimates were also made on enriched microsomal protein, whereas those by Sano et al. (34) were made on tissue lysates and would therefore be expected to be lower. Similarly, the estimates of 17,20-lyase activity in late luteal tissue were also lower than those found in hLGC. Regardless, aromatase was also higher than 17,20-lyase in human luteal lysates (34). Changes in P450c17 and P450arom transcripts during the menstrual cycle generally support this view (35, 36), and previous studies have also found relatively low levels of P450c17 mRNA in hLGC. However, circulating androgens of luteal origin increase in parallel with E2 in women during the midluteal phase of the menstrual cycle and increase further with E2 immediately after implantation in conceptive menstrual cycles (37). This early pregnancy-associated rise in androgens and E2 is most likely due to an increase in ovarian P450c17 expression in response to hCG that drives steroid synthesis before placental steroidogenic competency is established. The observation that these midluteal and postimplantation rises in androgens are similar within women, but highly variable between women, can be interpreted to indicate that the ability to induce P450c17 varies between individuals. Individual differences in P450c17 also provide a plausible explanation of the observed ethnic differences in androgen levels during the menopausal transition (38). Variability in the level of expression of a primary target of toxicity may also explain individual differences in susceptibility to TCDD toxicity. Therefore, P450c17 may become an especially sensitive target for TCDD-induced endocrine disruption in women during the late luteal phase, early pregnancy, and even later in life when it is perhaps more limiting than P450arom in estrogen synthesis.
It is also significant that the effect of TCDD is specific for P450c17 and does not affect P450arom expression, if only because TCDD induces the transcriptional expression of other microsomal P450s (39, 40). Studies are in progress to determine whether there is a decrease in transcript or perhaps an increased rate of P450c17 degradation, although this seems less likely based on the specificity of the change in levels for this microsomal P450. However, even if P450c17 transcript levels do decline, this does not necessarily imply that the effect of TCDD is directly on CYP17. Putative dioxin-response elements in the human CYP17 gene are fully 3 kb upstream from the transcriptional start site (Dr. Mike Denison, University of California-Davis, unpublished observations). Others have reported a transient inhibition of E2 secretion before stimulation in the first 48 h of culture, but only when micromolar concentrations of TCDD were used (12). In our studies E2 secretion by hLGC is highly variable in the first 2 d of culture, as is the response to TCDD, and a consistent depression begins by d 4 of culture (14). However, in contrast to others (12), in our studies hLGC are cultured in the continued presence of hCG and nanomolar concentrations of TCDD. Studies in vivo and in vitro demonstrated that TCDD decreased gonadotropic support or response (41, 42, 43), and this may still play a role in the response of reproductive tissues to toxic exposure, for which numerous disease consequences have been suspected (44, 45). Although this mechanism could contribute to the reduction of P450c17 noted here, the lack of effect on P450arom argues strongly that is not a major factor, as both P450c17 and P450arom are believed to be responsive to gonadotropins in human ovarian cells (46, 47). It is not known whether P450c17 expression is a target for TCDD in other tissues, such as the adrenal cortex or testes. The means of transcriptional regulation of CYP17 expression is not necessarily the same in the adrenal gland and gonad, even though the same promoter region is used (48). Further studies are needed to elucidate the specific mode of action of TCDD in depressing P450c17 expression in hLGC.
In summary, to our knowledge this is the first report of P450c17 protein expression and 17,20-lyase activity in hLGC, and the demonstration of its susceptibility to TCDD toxicity. TCDD treatment decreased both the protein expression and the 17,20-lyase activity of P450c17 in microsomal fractions from hLGC by more than 50% and 60%, respectively. To our knowledge, this is the first report of a clear, specific molecular target of endocrine disruption resulting from TCDD toxicity in any human cell.r'g)gt, 百拇医药
Acknowledgmentsr'g)gt, 百拇医药
The authors thank Ms. Karen Woodward for assistance with cell preparation and hormone assays, and Dr. Justin Vidal and staff at Sutter Hospital (Davis, CA) for help with the collection and processing of term placental tissue. The authors also acknowledge the generosity of Drs. Angela Brodie and Victor Njar for providing the tracer for the 17,20-lyase activity assays.r'g)gt, 百拇医药
Received August 5, 2002.r'g)gt, 百拇医药
Accepted for publication October 16, 2002.
References\v*, 百拇医药
Poland A, Knutson JC 1982 2,3,7,8-Tetrachlorodibenzo-p-dioxin and related halogenated aromatic hydrocarbons: examination of the mechanism of toxicity. Annu Rev Pharmacol Toxicol 22:517–554\v*, 百拇医药
Safe SH 1995 Modulation of gene expression and endocrine response pathways by 2,3,7,8-tetrachlorodibenzo-p-dioxin and related compounds. Pharmacol Ther 67:247–281\v*, 百拇医药
Hendrickx AG, Peterson PE, Otianga-Owiti GE, Tarantal AF, Dieter J, Lasley B, Overstreet JW 1999 Endocrine and morphological biomarkers of early pregnancy loss in macaques. In: Weinbauer G, Korte R, eds. Reproduction in nonhuman primates: a model system for human reproductive physiology and toxicology. New York, Munich, Berlin: Waxman, Munster; 111–136\v*, 百拇医药
McNulty WP 1984 Fetotoxicity of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) for rhesus macaques (Macaca mulatta). Am J Primatol 6:41–47\v*, 百拇医药
Guo Y, Hendrickx AG, Overstreet JW, Dieter J, Stewart D, Tarantal AF, Laughlin L, Lasley BL 1999 Endocrine biomarkers of early fetal loss in cynomolgus macaques (Macaca fascicularis) following exposure to dioxin. Biol Reprod 60:707–713
Moran FM, Tarara R, Chen J, Santos S, Cheney A, Overstreet JW, Lasley BL 2001 Effect of dioxin on ovarian function in the cynomolgus macaque (M. fascicularis). Reprod Toxicol 15:377–383j!)-}pj, 百拇医药
Pirkle JL, Wolfe WH, Patterson DG, Needham LL, Michalek JE, Miner JC, Peterson MR, Phillips DL 1989 Estimates of the half-life of 2,3,7,8-tetrachlorodibenzo-p-dioxin in Vietnam veterans of Operation Ranch Hand. J Toxicol Environ Health 27:165–171j!)-}pj, 百拇医药
Michalek JE, Pirkle JL, Caudill SP, Tripathi RC, Patterson Jr DG, Needham LL 1996 Pharmacokinetics of TCDD in veterans of operation ranch hand: 10-year follow-up. J Toxicol Environ Health 47:209–220j!)-}pj, 百拇医药
Stewart D, VandeVoort C 1997 Simulation of human luteal endocrine function with granulosa lutein cell culture. J Clin Endocrinol Metab 82:3078–3083j!)-}pj, 百拇医药
Enan E, Morán FM, VandeVoort CA, Stewart DR, Overstreet JW, Lasley BL 1996 Mechanism of toxic action of 2,3,7,8-tetrachlorodibenzo-p-dioxin TCDD in cultured human luteinized granulosa cells. Reprod Toxicol 10:497–508
Moran FM, Enan E, VandeVoort CA, Stewart DR, Conley AJ, Overstreet JW, Lasley BL 1997 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) effects on steroidogenesis of human luteinized granulosa cell in vitro. Biol Reprod 56:99 (Abstract)$1c], 百拇医药
Heimler I, Rawlins RG, Owen H, Hutz RJ 1998 Dioxin perturbs, in a dose- and time-dependent fashion, steroid secretion, and induces apoptosis of human luteinized granulosa cells. Endocrinology 139:4373–4379$1c], 百拇医药
Enan E, Lasley B, Stewart D, Overstreet J, VandeVoort CA 1996 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) modulates function of human luteinizing granulosa cells via cAMP signaling and early reduction of glucose transporting activity. Reprod Toxicol 10:191–198$1c], 百拇医药
Moran FM, Conley AJ, Corbin CJ, Enan E, VandeVoort C, Overstreet JW, Lasley BL 2000 2,3,7,8-Tetrachlorodibenzo-p-dioxin decreases estradiol production without altering the enzyme activity of cytochrome p450 aromatase of human luteinized granulosa cells in vitro. Biol Reprod 62:1102–1108$1c], 百拇医药
Moran FM, Lohstroh P, VandeVoort CA, Chen J, Overstreet JW, Conley AJ, Lasley BL Exogenous steroid substrate modifies the effect of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on estradiol production of human luteinized granulosa cells in vitro. Biol Reprod, in press
Mapes S, Tarantal AF, Parker CR, Moran FM, Bahr JM, Pyter L, Conley AJ 2002 Adrenocortical cytochrome b5 expression during fetal development of the rhesus macaque. Endocrinology 143:1451–1458tyhp, 百拇医药
Grigoryev DN, Kato K, Njar VC, Long BJ, Ling YZ, Wang X, Mohler J, Brodie AM 1999 Cytochrome P450c17-expressing Escherichia coli as a first-step screening system for 17{alpha} -hydroxylase-C17,20-lyase inhibitors. Anal Biochem 267:319–330tyhp, 百拇医药
Moran FM, Ford JJ, Corbin CJ, Mapes SM, Njar VC, Brodie AM, Conley AJ 2002 Regulation of microsomal P450, redox partner proteins and steroidogenesis in the developing testes of the neonatal pig. Endocrinology 143:3361–3369tyhp, 百拇医药
Simpson E, MacDonald P 1981 Endocrine physiology of the placenta. Annu Rev Physiol 43:163–188tyhp, 百拇医药
Corbin CJ, Trant JM, Conley AJ 2001 Porcine gonadal and placental isozymes of aromatase cytochrome P450: sub-cellular distribution and support by NADPH-cytochrome P450 reductase. Mol Cell Endocrinol 172:115–124tyhp, 百拇医药
Guengerich FP2001 Analysis and characterization of enzymes and nucleic acids. In: Hayes AW, ed. Principles and methods of toxicology. 4th ed. Philadelphia: Taylor, Francis; 1634–1635
Peterson RE, Theobald HM, Kimmel GL 1993 Developmental and reproductive toxicity of dioxins and related compounds: cross-species comparisons. Crit Rev Toxicol 23:283–335n^t, 百拇医药
Dickson LC, Buzik SC 1993 Health risks of "dioxins:" a review of environmental and toxicological considerations. Vet Hum Toxicol 35:68–77n^t, 百拇医药
Chaffin C, Peterson R, Hutz R 1996 In utero and lactational exposure of female Holtzman rats to 2,3,7,8-tetrachlorodibenzo-p-dioxin: modulation of the estrogen signal. Biol Reprod 55:62–67n^t, 百拇医药
Rier SE, Martin DC, Bowman RE, Dmowski WP, Becker JL 1993 Endometriosis in rhesus monkeys (Macaca mulatta) following chronic exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin. Fund Appl Toxicol 21:433–441n^t, 百拇医药
Dasmahapatra AK, Wimpee BAB, Trewin AL, Wimpee CF, Ghorai JK, Hutz RJ 2000 Demonstration of 2,3,7,8-tetrachlorodibenzo-p-dioxin attenuation of P450 steroidogenic enzyme mRNAs in rat granulosa cell in vitro by competitive reverse transcriptase-polymerase chain reaction assay. Mol Cell Endocrinol 164:5–18
Mebus CA, Reddy VR, Piper WN 1987 Depression of rat testicular 17-hydroxylase and 17,20-lyase after administration of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Biochem Pharmacol 36:727–731p0[@o|@, 百拇医药
Kleeman JM, Moore RW, Peterson RE 1990 Inhibition of testicular steroidogenesis in 2,3,7,8-tetrachlorodibenzo-p-dioxin-treated rats: evidence that the key lesion occurs prior to or during pregnenolone formation. Toxicol Appl Pharmacol 106:112–125p0[@o|@, 百拇医药
Moore RW, Jefcoate CR, Peterson RE 1991 2,3,7,8-Tetrachlorodibenzo-p-dioxin inhibits steroidogenesis in the rat testis by inhibiting the mobilization of cholesterol to cytochrome P450scc. Toxicol Appl Pharmacol 109:85–97p0[@o|@, 百拇医药
Li X, Johnson DC, Rozman KK 1995 Reproductive effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in female rats: ovulation, hormonal regulation, and possible mechanism(s). Toxicol Appl Pharmacol 133:321–327p0[@o|@, 百拇医药
Ushinohama K, Son D-S, Roby KF, Rozman KK, Terranova PF 2001 Impaired ovulation by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in immature rats treated with equine chorionic gonadotropin. Reprod Toxicol 15:275–280
Son D-S, Ushinohama K, Gao X, Taylor CC, Roby KF, Rozman KK, Terranova PF 1999 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) blocks ovulation by a direct action on the ovary without alteration of ovarian steroidogenesis: lack of a direct effect on ovarian granulosa and thecal-interstitial cell steroidogenesis in vitro. Reprod Toxicol 13:521–530yyjj\0f, 百拇医药
Gregoraszczuk E 2002 Dioxin exposure and porcine reproductive hormonal activity. Cad Saude Publica 18:453–462yyjj\0f, 百拇医药
Sano Y, Suzuki K, Arai K, Okinaga S, Tamaoki B 1981 Changes in enzyme activities related to steroidogenesis in human ovaries during the menstrual cycle. J Clin Endocrinol Metab 52:994–1001yyjj\0f, 百拇医药
Doody K, Lorence M, Mason J, Simpson E 1990 Expression of messenger ribonucleic acid species encoding steroidogenic enzymes in human follicles and corpora lutea throughout the menstrual cycle. J Clin Endocrinol Metab 70:1041–1045yyjj\0f, 百拇医药
Voutilainen R, Tapanainen J, Chung B, Matteson K, Miller W 1986 Hormonal regulation of P450scc (20,22-desmolase) and P450c17 (17{alpha} -hydroxylase/17,20-lyase) in cultured human granulosa cells. J Clin Endocrinol Metab 63:202–207
Castracane VD, Stewart DR, Gimpel T, Overstreet JW, Lasley BL 1998 Maternal serum androgens in human pregnancy: early increases within the cycle of conception. Hum Reprod 13:460–464ez|zwp, 百拇医药
Lasley BL, Santoro N, Randolph J, Gold EB, Crawford S, Weiss G, McConnell DS, Sowers MF 2002 The relationship of circulating dehydroepiandrosterone, testosterone, and estradiol to stages of the menopausal transition and ethnicity. J Clin Endocrinol Metab 87:3760–3767ez|zwp, 百拇医药
Waxman DJ 1999 P450 Gene induction by structurally diverse xenochemicals: central role of nuclear receptors CAR, PXR, and PPAR. Arch Biochem Biophys 369:11–23ez|zwp, 百拇医药
Whitlock Jr JP 1999 Induction of cytochrome P4501A1. Annu Rev Pharmacol Toxicol 39:103–125.ez|zwp, 百拇医药
Gao X, Son D-S, Terranova PF, Rozman KK 1999 Toxic equivalency factors of polychlorinated dibenzo-p-dioxins in an ovulation model: validation of the toxic equivalency concept for one aspect of endocrine disruption. Toxicol Appl Pharmacol 157:107–116ez|zwp, 百拇医药
Hirakawa T, Minegishi T, Abe K, Kishi H, Inoue K, Ibuki Y,Miyamoto K 2000 Effect of 2,3,7,8-tetrachlorodibenzo-p-dioxin on the expression of follicle-stimulating hormone receptors during cell differentiation in cultured granulosa cells. Endocrinology 141:1470–1476
Li X, Johnson DC, Rozman KK 1997 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) increases release of luteinizing hormone and follicle-stimulating hormone from the pituitary of immature female rats in vivo and in vitro. Toxicol Appl Pharmacol 142:264–269\2, http://www.100md.com
Bertazzi PA, Consonni D, Bachetti S, Rubagotti M, Baccarelli A, Zocchetti C, Pesatori AC 2001 Health effects of dioxin exposure: a 20-year mortality study. Am J Epidemiol 153:1031–1044\2, http://www.100md.com
Kogevinas M 2001 Human health effects of dioxins: cancer, reproductive and endocrine system effects. Hum Reprod Update 7:331–339\2, http://www.100md.com
McAllister J, Kerin J, Trant J, Estabrook R, Mason J, Waterman M, Simpson E 1989 Regulation of cholesterol side-chain cleavage and 17{alpha} -hydroxylase/lyase activities in proliferating human theca interna cells in long term monolayer culture. Endocrinology 125:1959–1966\2, http://www.100md.com
Steinkampf M, Mendelson C, Simpson E 1987 Regulation by follicle-stimulating hormone of the synthesis of aromatase cytochrome P-450 in human granulosa cells. Mol Endocrinol 1:465–471\2, http://www.100md.com
Arlotto MD, Michael MD, Kilgore MW, Simpson ER 1996 17{alpha} -hydroxylase gene expression in the bovine ovary: mechanisms regulating expression differ from those in adrenal cells. J Steroid Biochem Mol Biol 59:21–29(F. M. Morán C. A. VandeVoort J. W. Overstreet B. L. Lasley and A. J. Conley)