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The Journal of Clinical Endocrinology & Metabolism Vol. 87, No. 6 2830-2837
Copyright © 2002 by The Endocrine Society

Other Original Articles

Trialkyltin Compounds Enhance Human CG Secretion and Aromatase Activity in Human Placental Choriocarcinoma Cells

Tsuyoshi Nakanishi, Junya Kohroki, Shizuko Suzuki, Junichi Ishizaki, Youhei Hiromori, Shinri Takasuga, Norio Itoh, Yoshiteru Watanabe, Naoki Utoguchi and Keiichi Tanaka

Department of Toxicology (T.N., J.K., S.S., I.J., Y.H., S.T., N.I., K.T.), Graduate School of Pharmaceutical Sciences, Osaka University, Osaka 565-0871, Japan; and Department of Pharmaceutics (Y.W., N.U.), Showa Pharmaceutical University, Tokyo 194-8543, Japan

Address all correspondence and requests for reprints to: Tsuyoshi Nakanishi, Ph.D., Department of Toxicology, Graduate School of Pharmaceutical Sciences, Osaka University, 1-6, Yamadaoka Suita, Osaka 565-0871, Japan. E-mail: . nakanishi{at}phs.osaka-u.ac.jp

Abstract

Human choriocarcinoma cell lines have been used as placental models for the study of endocrine function, including aromatase (CYP19) activity and the secretion of human CG (hCG). In the present study, we investigated the effects of trialkyltin compounds, which are suspected endocrine disrupters, on aromatase activity and hCG secretion in human choriocarcinoma JAR, JEG-3, and BeWo cells. Protein synthesis as measured by 35S-methionine incorporation in all cell lines was markedly decreased by treatment with both tributyltin (TBT) and triphenyltin (TPT) at concentrations above 3 x 10-7 M, due to cytotoxicity. In JAR cells, 35S-methionine uptake was decreased by 50% at 3 x 10-7 M of TBT. At a TPT concentration of 1 x 10-7 M, protein synthesis in JAR cells was not affected, whereas JEG-3 and BeWo cells demonstrated slightly decreases. In all cell lines, both TBT and TPT increased levels of hCG secretion and aromatase activity in a dose- and time-dependent fashion following exposure to nontoxic concentration ranges. In addition, these trialkyltin compounds enhanced 8-bromo-cAMP-induced hCG secretion and aromatase activity in JAR cells. TBT caused dose-related increases in steady-state mRNA levels of both hCGß and CYP19 in JAR cells following 24- or 48-h exposure to nontoxic concentrations of TBT. However, these mRNA changes in JAR cells were not comparable to the changes in both hCG secretion and aromatase activity. These results indicate that the observed trialkyltin-induced alterations in human choriocarcinoma cells are due to other mechanism in addition to a regulation of hCG and CYP19 mRNA levels. Our studies suggest that trialkyltin compounds are potent stimulators of human placental hCG production and aromatase activity in vitro; and the placenta represents a potential target organ for trialkyltin compounds, whose endocrine-disrupting effects might be the result of local changes in hCG and estrogen concentrations in pregnant women.

THE PLACENTA PLAYS a vital role in maintaining pregnancy by delivering oxygen and nutrients from the maternal circulation to the fetus and by returning fetal metabolites to the mother. In addition, the placenta performs many crucial endocrine functions. For example, one of the endocrine functions of the human placenta is production of estrogenic steroids (1). The requires the catalytic activity of an aromatase enzyme complex, converting androgenic to estrogenic steroids. This enzyme complex consists of the microsomal CYP19 enzyme and flavoprotein nicotinamide adenine dinucleotide phosphate reduced form-reductase (2, 3). The latter is responsible for transferring reducing equivalents to CYP19 within the membrane of the endoplasmatic reticulum. The human placenta exhibits a high level of aromatase activity and therefore regulates the balance of estrogens in utero. The alterations of aromatase function in utero has been shown to permanently affect human embryos (4) and increase the risk of both breast and endometrial cancers (5). The human placenta also produces protein hormones, such as human CG (hCG), in addition to steroid hormones. The production and release of hCG is precisely regulated by a number of regulating agents, such as GnRH (6), gonadal steroids (7), epidermal growth factor (8), and cytokines such as IL-1 (9) and IL-6 (10). Maternal serum hCG levels peak between 12 and 13 wk of gestation and then decline to relatively low levels for the remainder of pregnancy. Stimulation from hCG not only governs progesterone production in the corpus luteum during the first trimester (11), but also T production within the fetal testes (12). Given the pivotal functional roles of CYP19 and hCG in sexual development and reproduction, the developmental and reproductive toxicity of environmental contaminants known to have endocrine-disrupting effects might plausibly involve these either or both CYP19 and hCG.

Organotin compounds, such as tributyltin (TBT) and triphenyltin (TPT) have been widely used as biocides, agriculture fungicides, wood preservatives, disinfecting agents in circulating industrial cooling waters, and as antifouling paints for marine vessels (13, 14). Human exposure to these organotin compounds may result from the consumption of organotin-contaminated meat and fish products, occupational exposure during the manufacture and formulation of organotin compounds, or the application and removal of organotin-containing paints (15, 16). Potential exposure of humans to organotins has therefore aroused great concern about potential toxicities. Most of the toxic effects of organotin compounds on sexual development and reproductive function have been documented in mollusks. For example, female neogastropod snails have been observed to suffer irreversible sex organ alterations, a phenomenon known as "imposex" (17). These abnormalities are the result of a masculinization process, in which male sex organs develop, notably a penis and a vas deferens. In certain species, growth of a vas deferens disrupts the structure and function of the oviducts, preventing normal breeding activity and causing population decline. Imposex has now been established as a form of endocrine disruption caused by elevated T titers leading to masculinization in organotin-exposed females (18, 19, 20). The precise mechanism by which T levels are increased has not been fully elucidated, but the weight of evidence suggests that organotin compounds act as competitive inhibitors of aromatase activity (19). Some recent data suggests that organotin compounds may also inhibit the formation of sulfur conjugates of T and active T metabolites (20). However, in humans, no studies have examined disturbances in sexual development and reproductive function, including placental function, as a result of exposure to organotin compounds.

To be able to apply current knowledge regarding the toxicity of trialkyltin compounds on sexual development and reproduction to humans, the present study assessed the possible effect of trialkyltin compounds on placental aromatase activity and hCG secretion using three different human choriocarcinoma cell lines, JAR, JEG-3, and BeWo. The potential toxicity of trialkyltin compounds as endocrine disruptors in humans is discussed.

Materials and Methods

Chemicals and cell culture

Tri-n-butyltin chloride (TBT) and tripheniyltin hydrooxide (TPT) were obtained from Tokyo Kasei Kougyo (Tokyo, Japan). 8-bromo cAMP (8-Br-cAMP) was obtained from Nacalai Tesque (Kyoto, Japan). Human choriocarcinoma cell lines JAR, JEG-3, and BeWo, were obtained from ATCC (Manassas, VA). JAR cells (ATCC no. HTB-144) were cultured in Roswell Park Memorial Institute 1640 medium with 10% FCS, 2 mM L-glutamine, 1 mM pyruvate, and 4.5 g/liter glucose. JEG-3 cells (ATCC no. HTB-36) were cultured in MEM with 10% FCS, 2 mM L-glutamine, and a 0.1 mM MEM nonessential amino acid solution. BeWo cells (ATCC no. CCL-98) were cultured in DMEM with 10% FCS, 2 mM L-glutamine, 1 mM pyruvate, and a 0.1 mM MEM nonessential amino acid solution (Invitrogen, Carlsbad, CA). All lines were maintained in a humidified atmosphere containing 5% CO2 at 37 C. Cells were grown to confluence and medium was changed every 2–3 d. Confluent cells were subcultured after trypsinization. Cells were treated with either a trialkyltin compound in 0.1% dimethylsulfoxide (DMSO) or vehicle alone (0.1% DMSO). In control experiments, this concentration of DMSO was not found to alter 35S-labeled methinine incorporation, hCG secretion, or aromatase activity, in any of the cell lines examined.

Protein synthesis assay

To determine cytotoxicity of trialkyltin compounds, 1 x 103 cells/well were cultured in microtiter plates. After 24 h of culture, cells were treated with various concentrations of trialkyltin compounds. Each culture was pulsed with 50 kilobecquerels of 35S-labeled methionine (Amersham Pharmacia Biotech, Little Chalfort, Buckinghamshire, UK) for 4 h before cells were harvested. 35S-count incorporated into TCA-precipitable materials was determined.

Determination of hCG in culture media

Forty-eight well plates were seeded with 4 x 104 cells/well. After 24 h of culture, cells were treated with various concentrations of trialkyltin compounds in the absence or presence of 8-Br-cAMP for an additional various hours. At the end point of each treatment, cells were washed three times and cultured in fresh medium for another 24 h. Culture supernatant was collected, and hCG was determined using ELISA. Immobilized rabbit polyclonal antibody against intact hCG and primary mouse monoclonal antibody against the ß-subunit of hCG conjugated with rat horseradish peroxidase-labeled polyclonal antibody against mouse IgG1 (Zymed Laboratories, Inc., San Francisco, CA) were used for this technique.

Aromatase assay

Twelve-well plates were used to culture 1.5 x 105 cells/well. After 24 h of culture, cells were treated with various concentrations of trialkyltin compounds in the absence or presence of 8-Br-cAMP for an additional number of hours. At the end point of each treatment, cells were washed three times. Aromatase activity was then determined using the tritium release assay described by Lephart and Simpson (21) with some modifications. This method measures production of 3H2O, which forms as a result of aromatization of substrate [1ß-3H] androst-4-ene-3,17-dione (NEN Life Science Products, Boston MA, ). Serum-free medium containing [1ß-3H] androst-4-ene-3,17-dione solution (54 nM) was prepared and 0.5 ml of this solution was added to each well. Wells containing media alone were also tested to provide control values for aromatase activity. After incubation for 1 h at 37 C in an atmosphere of 5% CO2, each plate was placed on ice and 200 µl of culture medium was withdrawn from each well. The medium was extracted with 500 µl of chloroform, vortexed, then centrifuged for 1 min at 9,000 x g. A 100-µl aliquot of the aqueous phase was mixed with 100 µl of a 5% wt/vol charcoal 0.5% wt/vol dextran T-70 suspension, vortexed, then incubated for 10 min. After centrifugation for 5 min at 9,000 x g, a 150-µl aliquot was removed to measure the level of radioactivity.

Determination of E2

Twelve-well plates were used to culture 1.5 x 105 cells/well. After 24 h of culture, cells were treated with various concentrations of trialkyltin compounds for a further 48 h. At the end point of each treatment, cells were washed three times, and 1 ml of culture medium supplemented with 2 µg androstenedione/ml was added. After incubation for 4 h at 37 C in an atmosphere of 5% CO2, cells were rapidly frozen and thawed, and the total E2 content of the cells and their media was determined by assay with a Correlate-EIA E2 Enzyme Immunoassay kit (Assay Designs, Ann Arbor, MI).

Determination of intracellular cAMP

Ninety-six well plates were used to culture 5 x 104 cells/well. After 24 h of culture, cells were treated with various concentrations of trialkyltin compounds for additional various hours. At the end point of each treatment, cells were washed and the level of intracellular cAMP was determined by assay with a cAMP Enzyme Immunoassay kit (Amersham Pharmacia Biotech, Little Chalfort, Buckinghamshire, UK).

Quantitative RT-PCR

Five micrograms of total RNAs extracted from JAR cells were reverse transcribed using SuperScript II reagent (Invitrogen) and oligo-(deoxythymidine) as primer. The reaction was performed by incubation for 1 h at 50 C in 20 µl of volumes. After termination of cDNA synthesis, reaction mixtures were diluted to 1/10 with TE buffer. Aliquots (2 µl) of diluted reverse transcribed products were amplified in a reaction mixture containing 1x LightCycler-FastStart DNA Master SYBER Green I (Roche Diagnostics, Mannheim, Germany), 4 mM MgCl2, and 0.5 µM of each primer using LightCycler (Roche Diagnostics). After preincubation at 95 C for 10 min, PCR was performed with 35–40 cycles of denaturation at 95 C for 1 sec, annealing at 65 C for 5 sec, and elongation at 72 C for 5 sec. Primers used were as follows: human aromatase, 5'-ATGAATCGGGCCTATGTGGACGTG-3' and 5'-CGATAGCACTTTCGTCCAAAGGG-3', hCGß, 5'-CCGTGTGCATCACCGTCAACA-3' and 5'-GTTGCACACCACCTGAGGCGA-3', and human ß-actin, 5'-CTACGAGCTGCCTGACGG-3' and 5'-GCCACAGGACTCCATGCCC-3'.

Statistics

Data were analyzed using the two-tailed unpaired t test. Control and treatment group data were always obtained from an equal number of replicate experiments. Values of P < 0.05 were considered statistically significant.

Results

Effects of TBT and TPT on proliferation of human choriocarcinoma cells

Protein synthesis assays were conducted to determine whether treatment with TBT and TPT was associated with cytotoxic effects on cell division. Cells were treated with various concentrations of trialkyltin compounds for 48 h, and protein synthesis was evaluated by 35S-labeled methionine incorporation. Exposure to 10-8 to 10-7 M of TBT or 10-8 to 3 x 10-8 M of TPT had no significant effect on the 35S-methionine incorporation of JEG-3 cells and BeWo cells. In JAR cells, protein synthesis was slightly increased at these concentration range of these trialkyltin compounds. At a TBT concentration of 3 x 10-7 M, 35S-methionine incorporation in JAR cells was decreased by 50%, whereas that in JEG-3 cells and BeWo cells was decreased by more than 15%. At a TPT concentration of 10-7 M, protein synthesis in JAR cells was unaffected, compared with decreases by 70% in JEG-3 cells and 80% in BeWo cells. Moreover, exposure to greater than 10-6 M TBT or 3 x 10-7 M TPT strikingly decreased 35S-methionine incorporation in all cell lines by more than 5%. Similar results were obtained in parallel experiments, in which cells were treated with trialkyltin compounds for 24 h or 72 h (data not shown). Using this result as a guide, it was considered that exposure of JAR cells to concentrations less than 3 x 10-7 M TBT or 10-7 M TPT, and exposure of JEG-3 cells and BeWo cells to concentrations less than 10-7 M TBT or TPT would be suitable for investigating the possible effects of these trialkyltin compounds on hCG production and aromatase activity of each cell line.

TBT and TPT stimulate hCG production in human placental choriocarcinoma cells

To investigate the possible effects of trialkyltin compounds TBT and TPT on hCG production, JAR, JEG-3, and BeWo cells were incubated with or without TBT or TPT for 48 h. Both TBT and TPT induced dose-related increases in hCG secretion by all choriocarcinoma cells (Fig. 2Go). Amounts of hCG released increased significantly as TBT concentration increased from 10-8 to 3 x 10-7 M or TPT concentrations increased from 10-8 to 10-7 M (Fig. 2Go). Above these concentration ranges, hCG release declined because of cytotoxicity. This is represented by the degree of 35S-methinine incorporation (Fig. 1Go). The observed induction of hCG secretion demonstrated a strong tendency following treatment with TPT rather than TBT (not significant). The hCG secretion induced by both compounds was significantly greater than positive controls induced by RA, which is known to stimulate the secretion of hCG in placental choriocarcinoma cells (22).



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  		Figure 2. Effect of trialkyltin compounds on hCG secretion in human choriocarcinoma cell lines. Cells treated with varying concentrations of TBT (•), TPT ({circ}), or RA ({diamond}), for 48 h. Results expressed as mean ± SD of triplicate cultures.

 


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  		Figure 1. Cytotoxicity of trialkyltin compounds against human choriocarcinoma cell lines. Cells treated with varying concentrations of either TBT (•) or TPT ({circ}) for 48 h. Results expressed as mean ± SD of triplicate cultures.

 
TBT and TPT enhance aromatase activity in human placental choriocarcinoma cells

We then investigated the possible effect of TBT and TPT on aromatase activity in JAR, JEG-3, and BeWo cells. Some investigators have demonstrated that various agents can effect aromatase expression and activity in choriocarcinoma cells when maintained in serum-free medium (21, 23, 24). Serum-free medium was used because serum has been shown to inhibit the inductive effects of several agents on aromatase expression and activity in several cell types (21, 24). However, studies on phorbol ester stimulation of aromatase expression and activity in choriocarcinoma cells, as well as studies examining cAMP stimulation in human term trophoblast cells, have been performed in the presence of serum (25). Regardless, to acquire more precise information on the toxic effects of xenobiotics through in vitro study, we suggest that future experiments should be performed in the presence of serum to reflect in vivo conditions. Thus, in the present study, choriocarcinoma cells were incubated with or without TBT or TPT for 48 h, in the presence of serum. Both TBT and TPT caused a dose-dependent increase in the aromatase activity of all choriocarcinoma cells at the concentration up to 10-7 M (Fig. 3Go). Exposure to greater than 10-8 M TBT or 3 x 10-8 M TPT caused statistically significant increases in aromatase activity in all choriocarcinoma cell lines examined. Notably, JAR cells exhibited the greatest sensitivity to TBT in aromatase activity, with statistically significant increases (P < 0.005) in aromatase activity in JAR cells observed after treatment with only 3 x 10-9 M TBT (Fig. 3Go). Although a statistically significant increase in aromatase activity was observed in all choriocarcinoma cell lines after exposure to 10-8 M TBT, this was not observed in JAR cells and BeWo cells after treatment with 10-8 M TPT (Fig. 3Go).



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  		Figure 3. Effect of trialkyltin compounds on aromatase activity in human choriocarcinoma cell lines. Cells treated with varying concentrations of TBT (•) and TPT ({circ}) for 48 h. Results expressed as mean ± SD of triplicate cultures.

 
In addition, we measured E2 accumulation within JAR cells and within culture medium after treatment with TBT and TPT. Parallel to the observed increase in aromatase activity, E2 accumulation in JAR cells significantly increased upon cultivation with TBT and TPT (Fig. 4Go). Concentrations under 10-6 M of either trialkyltin compound did not significantly affect aromatase activity in microsomes isolated from JAR cells (data not shown). These results suggest that the trialkyltin compounds tested (TBT and TPT) did not exert effects on the enzyme complex at concentrations below 10-6 M.



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  		Figure 4. Effect of trialkyltin compounds on conversion of androstenedione to E2 by JAR cells. Cells treated with varying concentrations of TBT (•) and TPT ({circ}) for 48 h. E2 content of cell media (A), and cells (B), determined by ELISA. Results expressed as mean ± SD of triplicate cultures.

 
Time course of the effects of trialkyltin compounds on hCG secretion and aromatase activity in JAR cells

Both the level of hCG production and aromatase activity in JAR cells were increased in a time-dependent manner of treatment with 10-7 M of TBT or TPT (Fig. 5Go). The level of hCG production increased significantly after 3 h and 6 h treatment with TPT and TBT, respectively (3 h TPT; 197 ± 36%, 6 h TBT; 205 ± 18%, of control secretion). Conversely, levels of aromatase activity increased slightly but significantly (135 ± 12% of control activity) after 12 h treatment with TBT. Furthermore, 48 h exposure was necessary to achieve significant changes in aromatase activity induced by TPT at least (Fig. 5Go).



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  		Figure 5. Time-course study of effects of trialkyltin compounds on hCG secretion (A) and aromatase activity (B) in JAR cells. JAR cells treated with concentrations of 10-7 M TBT (•) and TPT ({circ}) for indicated periods. Results expressed as mean ± SD of triplicate cultures.

 
Effects of trialkyltin compounds on intracellular cAMP accumulation, and 8-Br-cAMP induced-hCG secretion and aromatase activity in JAR cells

Both hCG production and aromatase activity in the placenta are controlled by cAMP-dependent intracellular signal pathways. To determine whether hCG release and aromatase activity mediated by trialkyltin compounds was related to increased intracellular cAMP, we measured cAMP concentrations in JAR cells after 24 h or 48 h treatment with TBT and TPT. Neither trialkyltin compounds demonstrated any effect on cAMP production in JAR cells at all concentrations tested (Fig. 6Go), suggesting that hCG production and aromatase activity is induced by the trialkyltin compounds through a cAMP-independent pathway.



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  		Figure 6. Effect of trialkyltin compounds on cAMP production in JAR cells. JAR cells cultured with varying concentrations of TBT or TPT for indicated periods. Results expressed as mean ± SD of triplicate cultures. Intracellular cAMP levels for control incubates were 58.2 ± 2.5 fmol/well at 24 h, 84.6 ± 2.9 fmol/well at 48 h.

 
We then examined the effects of these trialkyltin compounds on 8-Br-cAMP (a cAMP homolog) stimulated-hCG production and aromatase activity in JAR cells. As shown in Fig. 7AGo, TBT and TPT combined exhibited synergistic effects on hCG release with 8-Br-cAMP over the examined concentration ranges. TBT exhibited additive effects on aromatase activity with 8-Br-cAMP over the examined concentration range (Fig. 7BGo). However, TPT exhibited additive effects on aromatase activity with 8-Br-cAMP at 100 µM but not at concentrations below 50 µM (Fig. 7BGo). These results clearly indicate that both TBT and TPT enhance hCG secretion and aromatase activity stimulated by intracellular signals via cAMP-dependent pathway.



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  		Figure 7. Effect of trialkyltin compounds on 8-Br-cAMP induced hCG production and aromatase activity in JAR cells. JAR cells stimulated with varying concentrations of 8-Br-cAMP in absence or presence of varying concentrations of TBT or TPT for 48 h. Results expressed as mean ± SD of triplicate cultures.

 
Effects of TBT on mRNA expression of hCGß and CYP19 in JAR cells

We then investigated TBT-induced hCG and aromatase (CYP19) mRNA expression in JAR cells by quantitative real-time RT-PCR (Fig. 8Go). The mRNA level of hCGß increased slightly but not significantly as a result of 24 h treatment of JAR cells with TBT. Exposure to 3 x 10-8 to 3 x 10-7 M of TBT induced a significant increase after 48 h treatment at last. In JAR cell, however, hCG production was markedly increased by 48 h treatment with TBT (3 x 10-8 M; 243 ± 6%, 10-7 M; 518 ± 109%, 3 x 10-7 M; 1250 ± 53% of control expression; Fig. 2Go), whereas the mRNA expression of hCGß was barely increased by 48 h treatment with TBT (3 x 10-8 M; 148 ± 7%, 10-7 M; 212 ± 42%, 3 x 10-7 M; 163 ± 19% of control expression; Fig. 8AGo).



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  		Figure 8. Effect of TBT on expression of hCG ß and CYP19 mRNA in JAR cells. Total RNA isolated from JAR cells treated with varying concentrations of TBT for indicated time periods. Relative hCG ß and CYP19 mRNA levels for each condition determined by quantitative RT-PCR three times for each of three independent cultures as described in Materials and Methods. Results expressed as mean ± SD of three independent cultures. *, P < 0.05 represent values significantly different controls.

 
The mRNA level of CYP19 also increased slightly but significantly as a result of 24 h treatment of JAR cells with TBT at concentrations of 10-7 M (150 ± 1% of control expression) and 3 x 10-7 M (240 ± 35% of control expression). After 48 h treatment with TBT, values increased to 187 ± 26%, 216 ± 18% and 429 ± 111% of control expression at concentrations of 3 x 10-8, 10-7 and 3 x 10-7 M, respectively (Fig. 8BGo). Although aromatase activity was significantly increased after 48 h treatment with 3 x 10-9 M TBT (Fig. 3Go), mRNA expression of CYP19 was not increased (Fig. 8BGo). Neither hCGß nor CYP19 demonstrated increased mRNA expression following treatment with TBT for less than 24 h (data not shown). These data suggest that hCG production and aromatase activity induced by treatment with TBT are due to other mechanisms in addition to increases in mRNA expression.

Discussion

The results of the experiments described in this study show that trialkyltin compounds enhance hCG secretion and aromatase activity in human choriocarcinoma cell lines when they are exposed to nontoxic concentrations. The observed induction of hCG secretion demonstrated a strong tendency toward treatment with TPT rather than TBT, whereas induction of aromatase activity exhibited the reverse trend. In addition, we demonstrated that trialkyltin compounds cause dose- and time-dependent increases in hCG and CYP19 mRNA levels in human choriocarcinoma cells. However, no effects were exerted on intracellular cAMP production, which is involved in an intracellular signals pathway to promote hCG and CYP19 mRNA expression. Furthermore, mRNA changes in JAR cells treated with TBT were not comparable to changes in both hCG secretion and aromatase activity. Consequently, we concluded that the observed trialkyltin-induced alterations in human choriocarcinoma cells are due to other mechanisms in addition to regulation of hCG and CYP19 mRNA levels.

Previously, hCG has been shown to stimulate placental aromatization during in situ perfusion of the placenta (26). In addition, Ackermann et al. (27) have demonstrated that hCG secretion increases when JEG-3 cells are cultivated under serum-free conditions and that the amount of hCG secreted parallels increases in microsomal aromatase activity. Thus, they have suggested that placental aromatase is induced by hCG in an autocrine fashion. As a result of these reports, we expected that increased levels of hCG might enhance aromatase activity in human choriocarcinoma cells induced by trialkyltin compounds. However, when hCG was added to the media of three human choriocarcinoma cell lines, aromatase activity was not affected (data not shown). Indeed, Ackermann et al. (27) did not establish whether hCG could stimulate aromatase activity in JEG-3 cells. Moreover, hCG binds to surface receptors on cells and transmits its message intracellularly via cAMP (28). Nevertheless, intracellular cAMP accumulation is not found in JAR cells following exposure to trialkyltin compounds (Fig. 6Go). Although it remains unclear whether human placental aromatase is induced by hCG, it appears that hCG is not involved in the induction of placental aromatase by trialkyltin compounds.

Recently, organotin compounds have become recognized as endocrine-disrupting chemicals because numerous marine organisms have been shown to exhibit sexual abnormalities following exposure to TBT and TPT. In gastropod mollusks, one of the most sensitive species to organotin compounds, TBT has been demonstrated to induce the superimposition of male sex organs, such as a penis and/or a vas deferens, over female sex organs, a phenomenon known as imposex (17). Although it has been theorized that TBT increases androgen levels through inhibition of aromatase activity and/or a suppression of androgen excretion (18, 19, 20), no clear evidence has been provided to verify this hypothesis. On the contrary, a recent report has demonstrated that aromatase activity is not decreased in gastropods exposed to TBT, even in those exhibiting clear evidence of imposex (29). Although Heidrich et al. (30) demonstrated that TBT inhibited human aromatase activity at concentrations above 10-6 M using human placental microsomes and NADPH experimental system, Cooke (31) reported that TBT did not inhibit aromatase activity at concentrations under 10-6 M. Furthermore, trialkyltin compounds demonstrated no direct effects on the aromatase enzyme complex of JAR cells at concentrations below 10-6 M in the present study. Exactly why the actions of trialkyltin compounds with regard to aromatase activity differ in humans and gastropods is uncertain. It may be due to large variations in the cytochrome P450 enzymes of the two species. However, our findings suggest that the endocrine disrupting action of trialkyltin compounds observed in the present study might involve a mechanism unique to humans.

Many of the endocrine alterations induced by environmental contaminants are thought to occur as a result of altered gene expression, mediated by the binding of environmental contaminants to steroid hormone receptors. If TBT and TPT are capable of activating general steroid hormone receptors, they might induce ER- or AR-mediated gene expression. However, several studies have reported that neither TBT nor TPT exhibit estrogenic activity in a yeast two-hybrid screening system (32) or a MCF-7 human breast cancer cell line (33). In the present study, hCG secretion and aromatase activity in human choriocarcinoma cell lines were not affected by treatment with E2 (data not shown). On the other hand, Himeno et al. (33) have reported induction of AR-mediated transcription by trialkyltin compounds in the LNCaP cell line, an AR human prostate cancer cell line. However, in the present study, hCG secretion and aromatase activity in human choriocarcinoma cells were not affected by treatment with T (data not shown). This suggests that steroid hormone receptors do not mediate trialkyltin-induced hCG secretion and aromatase activity in human choriocarcinoma cells.

Several reports have established the in vivo reproductive toxicity of trialkyltin compounds in rodents (34, 35, 36, 37, 38, 39). However, the teratogenicity of trialkyltin compounds remains unclear because it is difficult to evaluate whether they exert direct action on the fetus or whether embryotoxicity results from maternal toxicity. The present study has demonstrated that trialkyltin compounds alter endocrine function in placental cells in vitro, but whether the endocrine disrupting effects of trialkyltin compounds could (partly) be caused by local changes in estrogen and hCG concentrations of the placenta in vivo remains unclear. The in vivo endocrine effects of environmental contaminants on the human placenta from animal studies are difficult to estimate, particularly those involving rodents, because the endocrine functions of the placenta vary considerably among different species.

Aromatization and the secretion of glycoprotein hormones are very important placental endocrine functions during pregnancy in humans. In primates and various ungulate species such as cows, pigs, and horses, aromatase is expressed in the placenta, in addition to other gonadal tissues (40, 41, 42, 43). By contrast, the placentae of fetal rats and mice do not express aromatase (44, 45). However, glycoprotein hormones, including hCG, are structurally similar between the two species, each consisting of a common {alpha}-subunit and a unique, noncovalently associated ß-subunit. A gene encoding the {alpha}-subunit of glycoprotein hormones is also expressed in the placentae of primates and various ungulate species, but not in rodents (46, 47). It has been suggested that rodents are therefore unsuitable for evaluating the endocrine- disrupting effects of environmental contaminants in the human placenta.

In addition, the tissue-specific expression of aromatase is strictly regulated in humans. Human CYP19 is a single-copy gene composed of 10 exons; exons II-X encode for the aromatase protein, as well as the 3' untranslated region of mRNA common to all estrogen-producing tissues (48). A number of variations of exon I exist. These encode for the 5' untranslated regions of various CYP19 mRNAs, which are selectively expressed in some tissues by alternative splicing (48). The tissue-specific expression of CYP19 in humans appears to be mediated by tissue-specific promoters lying upstream of the respective exon I sequences, and by transcription factors binding to specific regions of each promoter. In placenta, CYP19 is driven by the placental major promoter (I.1) and the transcript contains exon I.1, located on approximately 89-kb upstream from exon II (49). On the other hand, ovarian transcripts contain a sequence at 5'-ends immediately upstream of the translation start site, because expression of the gene in the ovary uses a proximal promoter (II) (49). Recently, Saitoh et al. (50) reported that TBT inhibits aromatase activity and decreases mRNA level in ovarian granulosa cells. In these cells, expression of CYP19 is strongly regulated by steroidogenic tissue-specific transcriptional factor, Ad4Bp/SF-1, via promoter II. In contrast, Ad4Bp/SF-1 is expressed at very low levels in human placenta and may not play an important role in activation of placental major promoter I.1 (51, 52). This suggests that the effects of TBT in these cells were partly caused by association with Ad4Bp/SF-1. It is therefore likely that the action of TBT in JAR cells is induced by a pathway clearly differ from that in ovarian granulosa cells, and giving rise to the promotion of aromatase activity and mRNA expression. From these opposite effects of TBT for aromatase activity and expression in different tissue-derived cells, the regulation of tissue-specific aromatase expression would appear to be strictly controlled.

Results of studies using rapid amplification of cDNA ends cDNA libraries constructed from cattle, horse, and pig placental tissues have indicated that tissue-specific promoters lying upstream of placenta-specific exons also regulate placental expression of CYP19 genes in these species (40). In species in which the 5' flanking regions of CYP19 placenta-specific promoters have been cloned, low sequence identity is seen among species (40, 41). Furthermore, in humans, the placenta-specific promoter of the CYP19 gene lies approximately 90 kb upstream from the site of initiation of translation (49), whereas in bovines, the placenta-specific promoter of the CYP19 gene lies approximately 19 kb upstream of the site of initiation of translation (42). In the pig, even greater complexity is observed because as many as three CYP19 genes encode for tissue-specific isoforms and multiple promoters can initiate translation of some of these isoforms (41, 43). Hence, any evaluation of the endocrine-disrupting effects of environmental contaminants on placental aromatase should account for this complexity.

To our knowledge, this is the first study investigating an interaction between trialkyltin compounds and human placental endocrine functions. Ongoing studies in our laboratory are currently investigating other cell lines expressing both aromatase and hCG. Furthermore, we plan to identify the regulatory region involved in trialkyltin-induced hCG and CYP19 expression in the placenta. These studies will provide useful information for assessing human risk in exposure to trialkyltin compounds in utero.

Acknowledgments

Footnotes

This research was supported in part by a Grant in Aid for Scientific Research (No. 11877400, No. 13470499) from the Ministry of Education, Science, Sports and Culture and Industrial Technology Research Grant Program in ’01 from New Energy and Industrial Technology Development Organization (NEDO) of Japan. It was also supported by grants from the Senri Life Science Foundation and the Showa Shell Sekiyu Foundation for Promotion of Environmental Research.

Abbreviations: 8-Br-cAMP, 8-Bromo-cAMP; CYP19, aromatase; DMSO, dimethylsulfoxide; hCG, human CG; TBT, tributyltin: TPT, triphenyltin.

Received July 20, 2001.

Accepted February 25, 2002.

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