Organotin compounds cause structure-dependent induction of progesterone in human choriocarcinoma Jar cells

Journal of Steroid Biochemistry & Molecular Biology 155 (2016) 190–198

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Organotin compounds cause structure-dependent induction of progesterone in human choriocarcinoma Jar cells Youhei Hiromori a,b , Hiroki Yui a , Jun-ichi Nishikawa c , Hisamitsu Nagase a , Tsuyoshi Nakanishi a, * a

Laboratory of Hygienic Chemistry and Molecular Toxicology, Gifu Pharmaceutical University, 1-25-4 Daigaku-nishi, Gifu, Gifu, 501-1196, Japan Department of Pharmacy, College of Pharmacy, Kinjo Gakuin University, 2-1723 Omori, Moriyamaku, Nagoya, Aichi, 463-8521, Japan Laboratory of Health Sciences, School of Pharmacy and Pharmaceutical Sciences, Mukogawa Women’s University, 11-68 Kyuban-cho, Koshien, Nishinomiya, Hyogo, 663-8179, Japan b c

A R T I C L E I N F O

A B S T R A C T

Article history: Received 31 March 2014 Received in revised form 10 October 2014 Accepted 14 October 2014 Available online 18 October 2014

Organotin compounds, such as tributyltin (TBT) and triphenyltin (TPT), are typical environmental contaminants and suspected endocrine-disrupting chemicals because they cause masculinization in female mollusks. In addition, previous studies have suggested that the endocrine disruption by organotin compounds leads to activation of peroxisome proliferator-activated receptor (PPAR)g and retinoid X receptor (RXR). However, whether organotin compounds cause crucial toxicities in human development and reproduction is unclear. We here investigated the structure-dependent effect of 12 tin compounds on mRNA transcription of 3b-hydroxysteroid dehydrogenase type I (3b-HSD I) and progesterone production in human choriocarcinoma Jar cells. TBT, TPT, dibutyltin, monophenyltin, tripropyltin, and tricyclohexyltin enhanced progesterone production in a dose-dependent fashion. Although tetraalkyltin compounds such as tetrabutyltin increased progesterone production, the concentrations necessary for activation were 30–100 times greater than those for trialkyltins. All tested active organotins increased 3b-HSD I mRNA transcription. We further investigated the correlation between the agonistic activity of organotin compounds on PPARg and their ability to promote progesterone production. Except for DBTCl2, the active organotins significantly induced the transactivation function of PPARg. In addition, PPARg knockdown significantly suppressed the induction of mRNA transcription of 3b-HSD I by all active organotins except DBTCl2. These results suggest that some organotin compounds promote progesterone biosynthesis in vitro by inducing 3b-HSD I mRNA transcription via the PPARg signaling pathway. The placenta represents a potential target organ for these compounds, whose endocrine-disrupting effects might cause local changes in progesterone concentration in pregnant women. ã 2014 Elsevier Ltd. All rights reserved.

Keywords: Tributyltin (TBT) Triphenyltin (TPT) 3b-Hydroxysteroid dehydrogenase type I Placenta PPARg

1. Introduction Organotin compounds, such as tributyltin (TBT) and triphenyltin (TPT), have been used widely as antifouling biocides for ships and fishing nets [1]. There are many reports of the biological effects of organotin compounds, which vary in their toxic effects on eukaryotes. One of the most notable toxicities in sexual development and reproduction is that of TBT- and

TPT-mediated endocrine disruption in some species of gastropods [2]. This phenomenon is known as ‘imposex’—the superimposition of male genitalia on female animals. Therefore, these organotin compounds are suspected to cause endocrine-disrupting effects in mammals, including humans. Human exposure to organotin compounds may result from consumption of organotin-contaminated meat and fish products or occupational exposure during the manufacture and formulation of organotin compounds or the

Abbreviations: 3b-HSD I, 3b-hydroxysteroid dehydrogenase type I; CL, corpus luteum; DBT, dibutyltin; DMSO, dimethyl sulfoxide; FCS, fetal calf serum; hCG, human chorionic gonadotropin; GR, glucocorticoid receptor; LG, LG100268; LUC, luciferase; MEM, minimal essential medium; PPAR, peroxisome proliferator-activated receptor; RXR, retinoid X receptor; RT-PCR, reverse transcription polymerase chain reaction; Rosi, rosiglitazone; siRNA, small interfering RNA; TBT, tributyltin; TPT, triphenyltin. * Corresponding author. Tel.: +81 58 230 8100; fax: +81 58 230 8117. E-mail address: [email protected] (T. Nakanishi). http://dx.doi.org/10.1016/j.jsbmb.2014.10.010 0960-0760/ ã 2014 Elsevier Ltd. All rights reserved.

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Table. 1 Tin compounds tested in current study. Tin compounds

Abbreviation

Purify (%)

CAS No.

MWa

ClogPb

Source

Trimethyltin chloride Triethyltin bromide Tripropyltin chloride Tributyltin chloride Triphenyltin chloride Tricyclohexyltin hydroxide Trioctyltin hydride Butyltin trichloride Dibutyltin dichloride Tetrabutyltin Phenyltin trichloride Diphenyltin dichloride

TMTCl TETBr TPrTCl TBTCl TPTCl TChTOH TOTH MBTCl3 DBTCl2 TeBT MPTCl3 DPTCl2

>98 >97 >98 >95 >95 >99 >95 >95 >97 >93 >98 >96

1066-45-1 2767-54-6 2279-76-7 1416-22-0 639-58-7 13121-70-5 869-59-0 1118-46-3 683-18-1 1461-25-2 1124-19-2 1135-99-5

199.3 285.8 283.4 325.5 385.5 385.2 459.4 282.2 303.8 347.2 302.2 343.8


0.51 1.27 2.66 4.25 3.57 5.39 10.3 0.41 1.56 10.0 0.77 2.06

Aldrich Chemicals Aldrich Chemicals Merck Tokyo Kasei Kogyo Aldrich Chemicals Aldrich Chemicals Tokyo Kasei Kogyo Aldrich Chemicals Tokyo Kasei Kogyo Aldrich Chemicals Aldrich Chemicals Aldrich Chemicals

a b

MW represents the molecular weight. ClogP represents the calculated logPow.

application and removal of organotin-containing paints [3]. The possible exposure of humans to organotins therefore has prompted great concern about potential toxicities. The placenta plays a vital role in maintaining pregnancy. The production of steroid hormones, such as progesterone and estrogens, is a crucial function of the primate placenta. In humans, by 7 wk of gestation, nearly all progesterone and estrogens in circulation are synthesized by the placenta [4]. In human placenta, steroid biosynthesis is regulated by various steroidogenic enzymes. The enzyme 3b-hydroxysteroid dehydrogenase/isomerase (3b-HSD) catalyzes the conversion of 3-hydroxy-5-ene-steroids (dehydroepiandrosterone and pregnenolone) to 3-oxo-4-ene-steroids (androstenedione and progesterone) [5]. The 3b-HSD enzymes exist in multiple isoforms in humans and rodents. Among these isoforms, type I (3b-HSD I) is expressed exclusively in the placenta. It converts pregnenolone to progesterone to help maintain the uterus in a quiescent state throughout human pregnancy [5]. Whereas placental production of progesterone is required to protect the conceptus during midgestation onward [6], the ingestion of progestins (i.e., natural and synthetic progesterone and testosterone derivatives that produce biologic effects similar to those of progesterone) during pregnancy is associated with an

Group I

CH 3 H 3C

Sn

Sn

Sn

CH 3

Cl

Br

TMTCl

TETBr

Sn

Sn

Cl

Cl

TPrTCl

Sn

Sn

HO

Cl

TChTOH

TBTCl

H

TOTH

TPTCl

Cl

Group II Cl

Sn

Cl

Cl

Cl

MBTCl3 Cl

DBTCl2

Sn

Sn

Sn

Cl

TeBT

TBTCl

Cl

Cl Sn

Cl

Sn

Sn

Cl

Cl

Cl

MPTCl3

DPTCl2

TPTCl

Cl

Sn

Cl

Cl

SnCl4

Fig. 1. Structure of tin compounds in the current study. Group I, comparison of different structures of alkyl and aryl chains in trialkylated and triarylated tin compounds. Group II, comparison of different numbers of alkyl or aryl chains in butyltin and phenyltin compounds. The abbreviation for each compound used is indicated in Table 1.

increased risk of hypospadias [7]. Given the pivotal functional roles of 3b-HSD I, the developmental and reproductive toxicities of environmental contaminants known to have endocrinedisrupting effects plausibly might involve placental 3b-HSD I in humans. In a previous study, we demonstrated that exposure to nontoxic concentrations of some organotin compounds dose-dependently increased the mRNA transcription and production of estradiol to increase the catalytic activity of aromatase, which converts androgen to estrogen, and of 17b-HSD I, which converts low-activity estrone to high-activity estradiol [8–10]. We also identified that TBT and TPT act as nanomolar agonists for both the retinoid X receptor (RXR) and peroxisome proliferator-activated receptor (PPAR)g, which are members of the nuclear receptor superfamily [11]. The promotion of estrogen biosynthesis by the organotin compounds just described involves the activation of RXR rather than PPARg [9,10]. PPARg is expressed abundantly in human trophoblast cells and serves as an essential regulator of placental differentiation and endocrine functions [12]. PPARg is activated by a variety of fatty acids and by a class of synthetic antidiabetic agents, the thiazolidinediones [13]. PPARg regulates the transcription of genes by heterodimerizing with RXR and by binding to the PPAR response elements in the target gene promoter [14]. Human chorionic gonadotoropin (hCG) is a crucial target gene of PPARg in human placenta, and its production and mRNA transcription is ligand-dependently controlled by PPARg [12]. hCG is a luteotropic factor, and its stimulation by hCG governs not only progesterone production in the corpus luteum (CL) during the first trimester [15] but also testosterone production within the fetal testes [16]. Given the pivotal functional roles of hCG in development and reproduction, factors that change PPARg-mediated transcription in the placenta may greatly alter fetal development by disrupting these endocrine functions. Indeed, we found that some organotin compounds, including TBT and TPT, promote hCG production [11]. Therefore, PPARg is a crucial molecular target of organotin compounds in the endocrine disruption of human placenta. To facilitate the application of current knowledge regarding the toxicity of organotin compounds to development and reproduction in humans via the PPARg signaling pathway, we assessed the possible effects of 12 tin compounds on the production of progesterone and mRNA transcription of 3b-HSD I in human placental cells by using human choriocarcinoma Jar cells. Furthermore, we investigated the correlation between the potency of these compounds as agonists for PPARg and progesterone production in Jar cells, and we addressed the potential toxicity of organotin compounds as endocrine disruptors in humans.

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Fig. 2. Effects of tin compounds on progesterone production in Jar cells. Cells were treated with tin compounds at various concentrations (0, 1, 10, and 100 nM of TETBr, TPrTCl, TBTCl, TChTOH, and TPTCl; 0, 0.01, 0.1, and 1 mM of DPTCl2 and TMTCl; and 0, 0.1, 1, and 10 mM of TOTH, SnCl4, MBTCl3, and MPTCl3). Data are expressed relative to the levels of vehicle-treated cells; these levels were set to 1. Results are expressed as means  1 S.D. of triplicate cultures. The progesterone production in vehicle-only cells, calculated from all experiments, was 49.9  1.01 ng/well/4 h (n = 21). Groups I and II correspond to the groups described in the legend for Fig. 1. * (P < 0.05) and ** (P < 0.01) indicate values significantly different from vehicle-control values.

2. Materials and methods 2.1. Chemicals and cell culture The tin compounds tested in this study are listed in Table 1. Octanol–water partition coefficients (logPow) for the tin compounds were calculated by Chemdraw software (PerkinElmer, Waltham, MA). LG100268 (LG) was obtained from Toronto Research Chemicals (North York, Ontario, Canada). Rosiglitazone (Rosi) was purchased from Cayman Chemical (Ann Arbor, MI). All chemicals were dissolved in dimethyl sulfoxide (DMSO; Wako Pure Chemicals, Tokyo). The human choriocarcinoma cell lines Jar and JEG-3 were obtained from American Type Culture Collection (ATCC; Rockville, MD). Jar cells (ATCC no. HTB-144) were cultured in RPMI 1640 medium supplemented with 2 mM L-glutamine, 1 mM pyruvate, 4.5 g/L glucose, and 10% fetal calf serum (FCS). JEG-3 cells (ATCC no. HTB-36) were cultured in minimal essential medium (MEM) supplemented with 2 mM L-glutamine, 0.1 mM MEM nonessential amino acid solution (Invitrogen, Carlsbad, CA), and 10% FCS. To determine the effect of tin compounds on the progesterone production, mRNA transcription and luciferase reporter gene expression, cells were seeded, precultured for 24 h, and then treated with either various concentrations of tin compounds in 0.1% DMSO or vehicle alone (0.1% DMSO) for another 48 h. In control experiments, treatment with 0.1% DMSO did not alter progesterone production, mRNA transcription of target genes, or luciferase reporter gene expression. 2.2. Plasmid construction Full-length cDNA of human PPARg was amplified by reverse transcription polymerase chain reaction (RT-PCR) using mRNA from JEG-3 cells. For the chimeric receptor assay, human PPARg cDNA was fused to the C-terminal end of the GAL4 DNA-binding domain (amino acids 1–147) in the pM expression vector (Clontech, Mountain View, CA) to yield pM-hPPARg. The plasmid constructed was confirmed by sequence analysis. The firefly

luciferase (LUC) reporter construct containing four copies of the GAL4 DNA-binding site (UAS) followed by the thymidine kinase promoter (p4UAS-tk-luc) used in the chimeric receptor assay was a kind gift from Dr. Y. Kamei (National Institute of Health and Nutrition, Japan) [17].

Fig. 3. Effects of tin compounds on the mRNA transcription of 3b-HSD I (HSD3B1) in Jar cells. Total RNA was isolated from Jar cells treated with tin compounds. The doses of each compound were: 100 nM of TETBr, TPrTCl, TBTCl, TChTOH, TPTCl, and DBTCl2; 1 mM of DPTCl2; and 10 mM of TOTH, SnCl4, MPTCl3, and TeBT. The relative mRNA levels for each condition were determined by using quantitative RT-PCR assays for each of three independent cultures. Data are expressed relative to the levels of vehicletreated cells; these levels were set to 1. Results are expressed as means  1 S.D. of three independent cultures. Groups I and II correspond to the groups described in the legend for Fig. 1. * (P < 0.05) and ** (P < 0.01) indicate values significantly different from vehicle-control values.

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Fig. 4. Concentration-dependent effects of the active organotins on 3b-HSD I mRNA transcription in Jar cells. Cells were treated with the organotin compounds at the indicated concentration. Data are expressed relative to the levels of vehicle-treated cells; these levels were set to 1. Results are expressed as means  1 S.D. of three independent cultures. *, value significantly (P < 0.05) different from vehicle-control value.

2.3. Determination of progesterone production Jar cells (3  104 cells/well) were plated in 24-well plates. After 24 h of culture, Jar cells were treated with various concentrations of tin compounds for a further 48 h. At the end point of each treatment, cells were rinsed with fresh serum-free culture medium and then received 0.5 mL of fresh serum-free culture medium supplemented with 10 mM pregnenolone (Wako Pure Chemicals). After incubation for 4 h at 37  C in 5% CO2, culture media were collected, and the total progesterone content was determined by using Correlate-EIA P Enzyme Immunoassay kits (Assay Designs, Ann Arbor, MI). 2.4. Quantitative RT-PCR Jar cells were treated with various tin compounds as described in the previous section but in RPMI 1640 supplemented with 5% charcoal-stripped FCS instead of 10% normal FCS, and then total RNA was extracted from the cells by using TRIzol (Invitrogen). The mRNA transcription of 3b-HSD I in Jar cells was determined by quantitative RT-PCR. We reverse-transcribed 5 mg of total RNA extracted from Jar cells in a total volume of 20 mL by using SuperScript III (Invitrogen) and oligo-(dT) as primer and incubating

for 1 h at 42  C. After termination of cDNA synthesis, each reaction mixture was diluted by adding 80 mL of tris-EDTA buffer. Aliquots (2 mL) of diluted reverse-transcription products were amplified by using LightCycler (Roche Diagnostics, Mannheim, Germany) in a reaction mixture containing QuantiTect SYBR Green PCR Reagent (Qiagen, Valencia, CA) and 0.5 mM of each primer. After preincubation of the reaction mixtures at 95  C for 15 min, real-time PCR amplification was performed for 35–40 cycles of denaturation at 95  C for 15 s, annealing at 65  C for 30 s, and elongation at 72  C for 10 s. The relative amount of mRNA of each gene was expressed as a percentage of the amount of mRNA of the housekeeping gene b-actin. Primers used were: human 3b-HSD I (HSD3B1; GenBank accession no. NM_000862), 50 -CATTGATGTCTTCGGTGTCACTCA-30 and 50 -AGAACTGTCCTCGGATGCTTG-30 , and human b-actin (GenBank accession no. NM_001101), 50 -CCTCGCCTTTGCCGATC30 and 50 -AAGCCGGCCTTG CACAT-30 . 2.5. Transient transfection assay Cells were transfected by using Lipofectamine (Invitrogen) in accordance with the manufacturer’s instructions. JEG-3 cells (3  104 cells) were seeded in 24-well plates and precultured at 37  C for 24 h. The cells then were transfected with pM-hPPARg (5 ng) or p4UAS-

Fig. 5. RXR and PPARg ligands enhance progesterone production and 3b-HSD I mRNA transcription in Jar cells. Cells were treated with LG and Rosi at 100 nM and then the cells were provided for testing progesterone production (A) and determination of 3b-HSD I mRNA transcription (B). Data are expressed relative to the levels of vehicle-treated cells; these levels were set to 1. Results are expressed as means  1 S.D. of three independent cultures. **, value significantly (P < 0.01) different from vehicle-control value.

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Fig. 6. Effect of PPARg knockdown on the mRNA transcription of 3b-HSD I in Jar cells. (A) Quantitative RT-PCR analysis of PPARg in negative-control Jar cells (control siRNA) and PPARg-knockdown Jar cells (PPARg siRNA). (B) Quantitative RT-PCR analysis of 3b-HSD I. (C) Effects of nuclear receptor ligands on the mRNA transcription of 3b-HSD I in negative-control and PPARg-knockdown Jar cells. The relative mRNA levels for each condition were determined by use of quantitative RT-PCR assays for each of three independent cultures. In panels A and B, data are expressed relative to the levels of non-treated cells; these levels were set to 1. In panel C, data are expressed relative to the levels of each vehicle-treated cell for the control siRNA or PPARg siRNA transfection group; each level was set to 1. Results are expressed as means  1 S.D. of three independent cultures. **, values significantly (P < 0.01) different from control siRNA-treated Jar cells. NS, not significant (P  0.05).

tk-luc (10 ng). At 24 h after transfection, the various compounds were added to the transfected cells, which then were cultured in RPMI 1640 supplemented with 1% charcoal-stripped FCS instead of 10% normal FCS. The cells were harvested 24 h later, and extracts were prepared and assayed for firefly LUC activity by using the DualLuciferase Reporter Assay System (Promega, Madison, WI) and a Mithras LB940 luminometer (Berthold Technologies, Wildbad, Germany) according to the manufacturer’s instructions. To normalize firefly LUC activity for transfection and harvesting efficiency, the renilla LUC control reporter construct pGL 4.74 (Promega) was cotransfected as an internal standard in all reporter experiments. Results are expressed as the average relative firefly LUC activity of at least quadruplicate samples. 2.6. RNA interference assay Sequence-specific small interfering RNA (siRNA) targeting PPARg (siPPARg) was purchased from Sigma–Aldrich (St. Louis, MO), and the negative control siRNA was purchased from Qiagen; the sense sequence was 50 -UGCUGACUCCAAAGCUCUGdTdT-30 , and the antisense was 50 -CAGAGCUUUGGAGUCAGCAdTdT-30 . Jar cells (3  105 cells/well) were seeded in 6-well plates and precultured at 37  C for 24 h. The cells then were transfected with siRNA duplexes (20 nM/ well) by using Lipofectamine RNAiMAX (Invitrogen) in accordance

with the manufacturer’s instructions. Jar cells were harvested 24 h after transfection and used for isolation of total RNA. 2.7. Statics Data were analyzed by using Dunnett’s multiple comparisons test or Student’s t test (SPSS Software, Chicago, IL). Control and treatment group data always were obtained from equal numbers of replicate experiments, and experiments were performed independently at least twice. A P value of <0.05 was used to indicate statistical significance. 3. Results 3.1. Effect of organotin compounds on progesterone production in Jar cells To help us interpret our results, we classified these experiments into two groups as follows: Group I experiments compared the effects of various structures of alkyl and aryl chains in trialkylated and triarylated tin compounds; those in Group II assessed the effect of the number of alkyl or aryl chains in butyltin and phenyltin compounds (Fig. 1). We exposed human choriocarcinoma Jar cells to concentrations of the tin compounds at which the

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Fig. 7. Ability of tin compounds to activate GAL–PPARg. JEG-3 cells were cotransfected with p4UAS-tk-luc and pM-hPPARg and then treated with Rosi and tin compounds at various concentrations (0, 1, 10, and 100 nM of TETBr, TPrTCl, TBTCl, TChTOH, TPTCl, and Rosi; 0, 0.01, 0.1, and 1 mM of DPTCl2 and TMTCl; and 0, 0.1, 1, and 10 mM of TOTH, SnCl4, MBTCl3, and MPTCl3). pGL 4.74 was cotransfected as the control for normalization of renilla LUC activity. Groups I and II correspond to the groups described in the legend for Fig. 1. Data are expressed relative to the levels of vehicle-treated cells; these levels were set to 1. Results are expressed as means  1 S.D. of three independent cultures. * (P < 0.05) and ** (P < 0.01) indicate values significantly different from vehicle-control values.

uptake of [3H]thymidine was 10% of that previously reported for the vehicle [10] and evaluated the organotins’ effects on progesterone production. Among the triorganotins (Group I), TPrTCl, TBTCl, TChTOH, and TPTCl significantly (P < 0.05) enhanced progesterone production in a concentration-dependent manner (Fig. 2). Among metabolites of both TBTCl and TPTCl (Group II), DBTCl2, MPTCl3, and DPTCl2 altered progesterone production, and the level of stimulation increased in proportion to the alkylation or arylation of these organotin compounds (tri- > di- > mono-). However, the presence of a fourth alkyl group on the tin atom decreased the potency of the organotin compounds in inducing progesterone production, because TeBT failed to stimulate this placental function at doses of <100 nM (Fig. 2, Group II). The potency of tin compounds for induction of progesterone production was completely independent of their molecular weight and calculated logPow (Fig. 2 and Table 1). These results suggest that the magnitude of the organotin-induced effect on progesterone function is related to both the number and structure of the alkyl or aryl groups. 3.2. Effects of organotin compounds and nuclear receptor agonists on the mRNA transcription of 3b-HSD I (HSD3B1) in Jar cells We then investigated the tin-compound-induced mRNA transcription of 3b-HSD I, which is a crucial enzyme for progesterone production in human placenta, at either the concentration that elicited the greatest response in the progesterone production experiments or the maximal nontoxic concentration in Jar cells [10]. The organotin compounds that enhanced progesterone production also significantly (P < 0.05) increased the mRNA transcription of 3b-HSD I. Furthermore, we examined the relationship between the concentration of the active organotins and the induction of 3b-HSD I mRNA transcription. All active organotins increased the mRNA transcription of 3b-HSD I in a concentration-dependent manner (Fig. 4). We previously demonstrated that some organotin compounds also function as agonists for RXR or PPARg to stimulate the mRNA transcription of human

placental hCG and aromatase in human choriocarcinoma cells [9]. To investigate the effects of RXR and PPARg agonists on progesterone production and 3b-HSD I mRNA transcription, we treated Jar cells with LG (RXR agonist) or Rosi (PPARg agonist). Both LG and Rosi enhanced progesterone production and 3b-HSD I mRNA transcription (Fig. 5). These results suggest that the RXR and PPARg signaling pathways may be involved in organotin-induced progesterone production in human placental cells. 3.3. Effect of PPARg knockdown on mRNA transcription of 3b-HSD I in Jar cells To examine the involvement of the PPARg signaling pathway in 3b-HSD I mRNA transcription, we used PPARg siRNA to suppress endogenous PPARg expression in Jar cells. Transfection with PPARg siRNA led to an approximately 75% decrease in PPARg mRNA transcription. In addition, PPARg knockdown caused an approximately 25% decrease in 3b-HSD I mRNA transcription in Jar cells untreated by organotins (Fig. 6A and B). No significant effect on the mRNA transcription of either PPARg or 3b-HSD I occurred in Jar cells transfected with control siRNA (Fig. 6A and B). We then examined the effect of PPARg knockdown on 3b-HSD I mRNA transcription induced by LG and Rosi in Jar cells. Both LG- and Rosiinduced 3b-HSD I mRNA transcription was significantly (P < 0.05) lower in PPARg-knockdown cells than in control siRNA-transfected cells (Fig. 6C). These results suggest that the PPARg signaling pathway in human placenta is involved in the 3b-HSD I mRNA transcription induced by agonists of both RXR and PPARg. 3.4. Potential correlation between organotin-induced 3b-HSD I mRNA transcription and PPARg agonist activity We previously investigated the functional potency of butyltin and phenyltin compounds to transactivate mouse PPARg [11]. However, in our previous study, we used mouse PPARg rather than the human receptor to evaluate the potency of butyltins and phenyltins as PPARg agonists, and we did not include TMTCl, TETBr,

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Fig. 8. Effect of PPARg knockdown on 3b-HSD I mRNA transcription induced by organotin compounds in Jar cells. Total RNA was isolated from negative-control Jar cells (open bars) and PPARg-knockdown Jar cells (solid bar) treated with tin compounds for 48 h. The doses of each compound were: 100 nM of TBTCl, TPTCl, TPrTCl, TCHTOH, and DBTCl2; 1 mM of DPTCl2; and 10 mM of MPTCl3 and TeBT. The relative mRNA levels for each condition were determined by using quantitative RTPCR assays for each of the three independent cultures. Data are expressed relative to the levels of the corresponding vehicle-treated control for each control siRNA or PPARg siRNA transfection group; each level was set to 1. Results are expressed as means  1 S.D. of three independent cultures. * (P < 0.05) and ** (P < 0.01) indicate values significantly different from those of negative-control Jar cells. y (P < 0.05) indicates values significantly different from vehicle-treated PPARg siRNA transfection group. NS, not significant (P  0.05).

TPrTCl, TChTOH, or TOTH among our test compounds. To investigate the correlation between organotin-induced 3b-HSD I mRNA transcription and the compounds’ agonistic activity for human PPARg, we investigated the ability of organotin compounds to activate human PPARg by using a LUC reporter system that incorporated a chimeric receptor comprising human PPARg and the DNA-binding domain of the yeast transcription factor GAL4. Among the Group I compounds, consistent with our previous observations, 100 nM of either TBTCl or TPTCl significantly (P < 0.05) induced the transactivation function of human PPARg. In addition, 100-nM doses of TPrTCl and TChTOH were significantly active. TMTCl, TETBr, and TOTH (which had no effect on progesterone production) induced no significant activation of human PPARg in the tested range (Fig. 7). Among the Group II organotins, consistent with our previous observations, 10 mM of MPTCl3 and 1 mM of DPTCl2 significantly (P < 0.05) induced the transactivation function of PPARg. In contrast, SnCl4, MBTCl3, DBTCl2, and TeBT lacked significant activating function in the tested concentration range (Fig. 7). To further confirm whether the PPARg signaling pathway was a crucial pathway for organotin-induced 3b-HSD I mRNA transcription, we used PPARg siRNA to suppress endogenous PPARg expression in Jar cells. Although PPARg knockdown in Jar cells did not entirely abolish 3b-HSD I mRNA transcription induced by TBTCl, TPTCl and TeBT, the mRNA transcription induced by the active organotins except for DBTCl2 was significantly (P < 0.05) suppressed (Fig. 8). These results suggest that the human placental 3b-HSD I mRNA transcription induced by organotins (except DBTCl2) reflected the PPARg agonistic activity of the compounds and that the primary pathways of organotin-induced 3b-HSD I mRNA transcription are PPARg-dependent. 4. Discussion Our current study demonstrated that organotin compounds alter progesterone biosynthesis in human placental cells in vitro. Although several reports have established the in vivo reproductive toxicity of organotin compounds in rodents [18], no reports

address whether organotin-induced production of placental progesterone is associated with hypospadias risk. Accordingly, the association between hypospadias risk and organotin-compound-induced local changes in progesterone concentrations of the placenta in vivo remains unclear. Furthermore, the in vivo effects of environmental contaminants are difficult to estimate from animal studies, particularly those involving rodents, because the endocrine functions of the placenta vary considerably among different species. In rodents, progesterone production by the CL is required throughout gestation [19]. During the first 8–9 days of gestation in rodents, pituitary prolactin regulates corpus luteal functions; thereafter, placental lactogens 1 and 2, which are produced by trophoblastic giant cells, are the primary regulators [20]. Consequently, in contrast to the process in humans, the pituitary gland is not required for the initiation and maintenance of pregnancy in rodents. Maintenance of the human CL depends on hCG, which is produced by trophoblasts. After 8 weeks of gestation, placental progesterone production by the syncytiotrophoblast is sufficient to maintain pregnancy [21]. It has been suggested that data from rodents are therefore unsuitable for extrapolating the effects of environmental contaminants on progesterone biosynthesis in human placenta. The regulation of progesterone biosynthesis in placenta is very important for human pregnancy, because normal parturition is associated with a functional withdrawal of progesterone via the loss of progesterone receptor expression in both the decidua [22] and placenta [23]. Indeed, impairment of progesterone action prior to the end of pregnancy has been associated with preterm birth and is the leading cause of perinatal mortality and morbidity in women [24]. Consequently, there is an urgent need to establish effective tools to evaluate the endocrine-disrupting effects and teratogenicity of environmental contaminants that induce changes in the local progesterone concentrations of the placenta in vivo. Knocking down PPARg markedly—but not significantly—decreased the steady-state mRNA transcription of 3b-HSD I in Jar cells (P = 0.055; Fig. 6B). This result suggests that PPARg is essential for 3b-HSD I mRNA transcription and progesterone production in human placenta. PPARg, which is a ligand-activated transcription factor, forms obligate heterodimers with RXR and binds to the PPAR response elements in the target gene promoter [14]. The PPARg–RXR heterodimer can be activated by agonists of either PPARg or RXR, or both, in a more-than-additive fashion [14,25]. However, RXR, another ligand-activated transcription factor, can form not only homodimers, but also heterodimers with a number of other nuclear receptors. Therefore, in addition to the PPARg– RXR heterodimer, RXR agonists can activate homodimers or other heterodimers such as PPARa–RXR, PPARd–RXR, liver X receptor– RXR, and farnesoid X-activated receptor–RXR [14,25]. However, in our current study, knockdown of PPARg expression entirely abolished the 3b-HSD I mRNA transcription induced by RXR agonist LG, similar to its effect after treatment with the PPARg agonist Rosi (Fig. 6C). There results suggest that the 3b-HSD I mRNA transcription induced by LG may involve the activation of the PPARg–RXR heterodimer principally and that the heterodimer may be required for the regulation of 3b-HSD I expression in human placenta. In the current study, we examined the functional potency of 12 tin compounds as agonists of human PPARg The potency of tin compounds for induction of progesterone production, 3b-HSD I mRNA transcription and human PPARg transactivation was completely independent of their molecular weight and calculated logPow (Figs. 2, 3, 4, 7 and Table 1). Consistent with our previous experiments using mouse PPARg [11], an approximately 10-fold lower concentration of TPTCl (10 nM) than of TBTCl (100 nM) was needed to elicit similar responses to human PPARg (Fig. 7). In

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addition, the di- and monosubstituted organotins DBT and MBT provided no significant activation, whereas DPT and MPT were moderately active in the micromolar range (Fig. 7), suggesting that phenyltins are more potent for both binding and activating human PPARg than are butyltins. The organotins tested in the current study were almost similar in their functional potency toward human PPARg to that for RXR in our previous study [9]. Among the triorganotins (Group I) other than TBTCl and TPTCl, TPrTCl and TChTOH were active, whereas TMTCl, TETBr and TOTH failed to transactivate human PPARg. However, by contrast with the results of RXR transactivation, TeBT failed to transactivate human PPARg. These results suggest that the potency of the effects induced by trialkyltin compounds is related to the length of the alkyl groups and the presence of a fourth alkyl group on the tin atom decreases the potency of the organotin compounds’ effects on human PPARg, and, as with RXR [9] and mouse PPARg [11], the order of potency is tri- > tetra- > di- > monosubstituted. The progesterone production induced by the organotins tested in the current study almost paralleled the results of 3b-HSD I mRNA transcription, suggesting that the induction of 3b-HSD I mRNA transcription is a critical event for progesterone production induced by organotins in human placenta. However, 10 nM TPrTCl and 100 nM DPTCl2 significantly increased the progesterone production without the induction of 3b-HSD I mRNA transcription. On the other hand, 1 mM TeBT failed to stimulate the progesterone production despite of its induction of 3b-HSD I mRNA transcription (Figs. 2 and 4). Although it remains unclear why the progesterone production induced by these organotins did not parallel the results of 3b-HSD I mRNA transcription, these organotins might directly affect the catalytic activity of 3b-HSD I. We further investigated the potential correlation between organotin-induced 3b-HSD I mRNA transcription and their PPARg agonistic activity. The results of 3b-HSD I mRNA transcription almost paralleled the compounds’ functional potency regarding human PPARg. In addition, PPARg knockdown significantly suppressed the 3b-HSD I mRNA transcription that was stimulated by the organotin compounds that induced progesterone production, suggesting that the principal pathways of organotin-induced progesterone production and 3b-HSD I mRNA transcription are PPARg-dependent. However, the 3b-HSD I mRNA transcription induced by 10 mM TeBT was comparable to that induced by 100 nM Rosi (Figs. 3 and 5B), although TeBT had little ability to transactivate human PPARg (Fig. 7). In addition, 100 nM TBTCl and TPrTCl were more potent than was 100 nM Rosi at inducing 3b-HSD I mRNA transcription (Figs. 3 and 5B), although these organotins were less potent in the transactivation of human PPARg (Fig. 7). Although these observations seem contradictory, both RXR agonists and PPARg agonists induce placental progesterone production and 3b-HSD I mRNA transcription because both events are upregulated by RXR agonist (Fig. 5). Therefore, organotins may activate PPARg–RXR heterodimers by acting as agonists for PPARg or RXR or both, leading to the induction of progesterone production subsequent to 3b-HSD I mRNA transcription. DBTCl2 induced neither RXR nor PPARg transactivation but significantly both progesterone production and mRNA transcription of 3b-HSD I (Figs. 2 and 4). It remains unclear why DBTCl2 enhanced the activity of 3b-HSD I through its mRNA transcription. At least, the induction appears due to a mechanism other than the activation of RXR and PPARg. In addition, Knocking down PPARg did not entirely abolish 3b-HSD I mRNA transcription induced by TBTCl, TPTCl and TeBT (Fig. 8). Some of these organotins act as ligands for glucocorticoid receptor (GR) [26]. Dexamethasone, a potent synthetic GR agonist, inhibits the catalytic activity and mRNA transcription of 3b-HSD I at 100 nM in rat Leydig cells [27].

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Therefore, the effect of these organotins might involve disruption of GR function. In summary, we have provided the first evidence that 3b-HSD I mRNA transcription in human placenta is dependent on PPARg We also show that various organotin compounds potentially promote progesterone production to enhance 3b-HSD I mRNA transcription in human placenta through the PPARg signaling pathway. However, DBT-induced effects on progesterone production and 3b-HSD I mRNA transcription do not appear to involve either the RXR or PPARg signaling pathway. Consequently, we conclude that the observed organotin-induced alterations in Jar cells are due to mechanisms in addition to regulation of 3b-HSD I mRNA levels. The toxic mechanisms of organotin compounds appear very intricate. For example, organotin compounds have been shown to act potentially as inhibitors of steroidogenic enzymes [18], as an inhibitor of proteasome [28], and as an enhancer of histone acetyltransferase [29]. Future studies need to clarify the precise mechanism of action of organotin compounds in human endocrine disruption in vitro and in vivo.

References [1] I.J. Boyer, Toxicity of dibutyltin, tributyltin and other organotin compounds to humans and to experimental animals, Toxicology 55 (1989) 253–298. [2] T. Horiguchi, H. Shiraishi, M. Shimizu, M. Morita, Effects of triphenyltin chloride and five other organotin compounds on the development of imposex in the rock shell, Thais clavigera, Environ. Pollut. 95 (1997) 85–91. [3] K. Kannan, S. Tanabe, R. Tatsukawa, Occurrence of butyltin residues in certain foodstuffs, Bull. Environ. Contam. Toxicol. 55 (1995) 510–516. [4] A.H. Payne, D.B. Hales, Overview of steroidogenic enzymes in the pathway from cholesterol to active steroid hormones, Endocr. Rev. 25 (2004) 947–970. [5] J. Simard, M.L. Ricketts, S. Gingras, P. Soucy, F.A. Feltus, M.H. Melner, Molecular biology of the 3b-hydroxysteroid dehydrogenase/delta5-delta4 isomerase gene family, Endocr. Rev. 26 (2005) 525–582. [6] A. Malassine, J.L. Frendo, D. Evain-Brion, A comparison of placental development and endocrine functions between the human and mouse model, Hum. Reprod. Update 9 (2003) 531–539. [7] S.L. Carmichael, G.M. Shaw, C. Laurent, M.S. Croughan, R.S. Olney, E.J. Lammer, Maternal progestin intake and risk of hypospadias, Arch. Pediatr. Adolesc. Med. 159 (2005) 957–962. [8] T. Nakanishi, J. Kohroki, S. Suzuki, J. Ishizaki, Y. Hiromori, S. Takasuga, N. Itoh, Y. Watanabe, N. Utoguchi, K. Tanaka, Trialkyltin compounds enhance human CG secretion and aromatase activity in human placental choriocarcinoma cells, J. Clin. Endocrinol. Metab. 87 (2002) 2830–2837. [9] T. Nakanishi, J. Nishikawa, Y. Hiromori, H. Yokoyama, M. Koyanagi, S. Takasuga, J. Ishizaki, M. Watanabe, S. Isa, N. Utoguchi, N. Itoh, Y. Kohno, T. Nishihara, K. Tanaka, Trialkyltin compounds bind retinoid X receptor to alter human placental endocrine functions, Mol. Endocrinol. 19 (2005) 2502–2516. [10] T. Nakanishi, Y. Hiromori, H. Yokoyama, M. Koyanagi, N. Itoh, J. Nishikawa, K. Tanaka, Organotin compounds enhance 17b-hydroxysteroid dehydrogenase type I activity in human choriocarcinoma JAr cells: potential promotion of 17b-estradiol biosynthesis in human placenta, Biochem. Pharmacol. 71 (2006) 1349–1357. [11] Y. Hiromori, J. Nishikawa, I. Yoshida, H. Nagase, T. Nakanishi, Structuredependent activation of peroxisome proliferator-activated receptor (PPAR)g by organotin compounds, Chem. Biol. Interact. 180 (2009) 238–244. [12] A. Tarrade, K. Schoonjans, J. Guibourdenche, J.M. Bidart, M. Vidaud, J. Auwerx, C. Rochette-Egly, D. Evain-Brion, PPARg/RXRa heterodimers are involved in human CG beta synthesis and human trophoblast differentiation, Endocrinology 142 (2001) 4504–4514. [13] J.M. Lehmann, L.B. Moore, T.A. Smith-Oliver, W.O. Wilkison, T.M. Willson, S.A. Kliewer, An antidiabetic thiazolidinedione is a high affinity ligand for peroxisome proliferator-activated receptorg (PPARg), J. Biol. Chem. 270 (1995) 12953–12956. [14] S.A. Kliewer, K. Umesono, D.J. Noonan, R.A. Heyman, R.M. Evans, Convergence of 9-cis retinoic acid and peroxisome proliferator signalling pathways through heterodimer formation of their receptors, Nature 358 (1992) 771–774. [15] T. Yoshimi, C.A. Strott, J.R. Marshall, M.B. Lipsett, Corpus luteum function in early pregnancy, J. Clin. Endocrinol. Metab. 29 (1969) 225–230. [16] I.T. Huhtaniemi, C.C. Korenbrot, R.B. Jaffe, HCG binding and stimulation of testosterone biosynthesis in the human fetal testis, J. Clin. Endocrinol. Metab. 44 (1977) 963–967. [17] Y. Kamei, H. Ohizumi, Y. Fujitani, T. Nemoto, T. Tanaka, N. Takahashi, T. Kawada, M. Miyoshi, O. Ezaki, A. Kakizuka, PPARg coactivator 1b/ERR ligand 1 is an ERR protein ligand, whose expression induces a high-energy expenditure and antagonizes obesity, Proc. Natl. Acad. Sci. U. S. A. 100 (2003) 12378–12383.

198

Y. Hiromori et al. / Journal of Steroid Biochemistry & Molecular Biology 155 (2016) 190–198

[18] V.S. Delgado Filho, P.F. Lopes, P.L. Podratz, J.B. Graceli, Triorganotin as a compound with potential reproductive toxicity in mammals, Braz. J. Med. Biol. Res. 44 (2011) 958–965. [19] J.F. Strauss 3rd, F. Martinez, M. Kiriakidou, Placental steroid hormone synthesis: unique features and unanswered questions, Biol. Reprod. 54 (1996) 303–311. [20] S.S. Galosy, F. Talamantes, Luteotropic actions of placental lactogens at midpregnancy in the mouse, Endocrinology 136 (1995) 3993–4003. [21] J.L. Jameson, A.N. Hollenberg, Regulation of chorionic gonadotropin gene expression, Endocr. Rev. 14 (1993) 203–221. [22] A.G. Brown, R.S. Leite, J.F. Strauss 3rd, Mechanisms underlying functional progesterone withdrawal at parturition, Ann. N. Y. Acad. Sci. 1034 (2004) 36–49. [23] Y.G. Shanker, A.J. Rao, Progesterone receptor expression in the human placenta, Mol. Hum. Reprod. 5 (1999) 481–486. [24] M.S. Kramer, K. Demissie, H. Yang, R.W. Platt, R. Sauve, R. Liston, The contribution of mild and moderate preterm birth to infant mortality. Fetal and

[25]

[26]

[27] [28]

[29]

Infant Health Study Group of the Canadian Perinatal Surveillance System, JAMA 284 (2000) 843–849. T. Nakanishi, Endocrine disruption induced by organotin compounds; organotins function as a powerful agonist for nuclear receptors rather than an aromatase inhibitor, J. Toxicol. Sci. 33 (2008) 269–276. C. Gumy, C. Chandsawangbhuwana, A.A. Dzyakanchuk, D.V. Kratschmar, M.E. Baker, A. Odermatt, Dibutyltin disrupts glucocorticoid receptor function and impairs glucocorticoid-induced suppression of cytokine production, PLoS one 3 (2008) e3545. Y.C. Xiao, Y.D. Huang, D.O. Hardy, X.K. Li, R.S. Ge, Glucocorticoid suppresses steroidogenesis in rat progenitor Leydig cells, J. Androl. 31 (2010) 365–371. G. Shi, D. Chen, G. Zhai, M.S. Chen, Q.C. Cui, Q. Zhou, B. He, Q.P. Dou, G. Jiang, The proteasome is a molecular target of environmental toxic organotins, Environ. Health Perspect. 117 (2009) 379–386. S. Osada, J. Nishikawa, T. Nakanishi, K. Tanaka, T. Nishihara, Some organotin compounds enhance histone acetyltransferase activity, Toxicol. Lett. 155 (2005) 329–335.