BLTK1 Murine Leydig Cells: A Novel Steroidogenic Model for Evaluating the Effects of Reproductive and Developmental Toxicants

Toxicological Sciences, Jun 2012

Leydig cells are the primary site of androgen biosynthesis in males. Several environmental toxicants target steroidogenesis resulting in both developmental and reproductive effects including testicular dysgenesis syndrome. The aim of this study was to evaluate the effect of several structurally diverse endocrine disrupting compounds (EDCs) on steroidogenesis in a novel BLTK1 murine Leydig cell model. We demonstrate that BLTK1 cells possess a fully functional steroidogenic pathway that produces low basal levels of testosterone (T) and express all the necessary steroidogenic enzymes including Star, Cyp11a1, Cyp17a1, Hsd3b1, Hsd17b3, and Srd5a1. Recombinant human chorionic gonadotropin (rhCG) and forskolin (FSK) elicited concentration- and time-dependent induction of 3′,5′-cyclic adenosine monophosphate, progesterone (P), and T, as well as the differential expression of Star, Hsd3b6, Hsd17b3, and Srd5a1 messenger RNA levels. The evaluation of several structurally diverse male reproductive toxicants including 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), atrazine, prochloraz, triclosan, monoethylhexyl phthalate (MEHP), glyphosate, and RDX in BLTK1 cells suggests different modes of action perturb steroidogenesis. For example, prochloraz and triclosan antifungals reduced rhCG induction of T, consistent with published in vivo data but did not alter basal T levels. In contrast, atrazine and MEHP elicited modest induction of basal T but antagonized rhCG-mediated induction of T levels, whereas TCDD, glyphosate, and RDX had no effect on basal or rhCG induction of T in BLTK1 cells. These results suggest that BLTK1 cells maintain rhCG-inducible steroidogenesis and are a viable in vitro Leydig cell model to evaluate the effects of EDCs on steroidogenesis. This model can also be used to elucidate the different mechanisms underlying toxicant-mediated disruption of steroidogenesis.

A PDF file should load here. If you do not see its contents the file may be temporarily unavailable at the journal website or you do not have a PDF plug-in installed and enabled in your browser.

Alternatively, you can download the file locally and open with any standalone PDF reader:

https://toxsci.oxfordjournals.org/content/127/2/391.full.pdf

BLTK1 Murine Leydig Cells: A Novel Steroidogenic Model for Evaluating the Effects of Reproductive and Developmental Toxicants

Agnes L. Forgacs 0 2 Qi Ding 2 Rosemary G. Jaremba 2 Ilpo T. Huhtaniemi 1 Nafis A. Rahman 1 Timothy R. Zacharewski 0 2 0 Center for Integrative Toxicology, Michigan State University , East Lansing, Michigan 48824 1 Department of Physiology, University of Turku , Turku, Finland 2 Department of Biochemistry & Molecular Biology, Michigan State University , East Lansing, Michigan 48824 Leydig cells are the primary site of androgen biosynthesis in males. Several environmental toxicants target steroidogenesis resulting in both developmental and reproductive effects including testicular dysgenesis syndrome. The aim of this study was to evaluate the effect of several structurally diverse endocrine disrupting compounds (EDCs) on steroidogenesis in a novel BLTK1 murine Leydig cell model. We demonstrate that BLTK1 cells possess a fully functional steroidogenic pathway that produces low basal levels of testosterone (T) and express all the necessary steroidogenic enzymes including Star, Cyp11a1, Cyp17a1, Hsd3b1, Hsd17b3, and Srd5a1. Recombinant human chorionic gonadotropin (rhCG) and forskolin (FSK) elicited concentration- and time-dependent induction of 3#,5#-cyclic adenosine monophosphate, progesterone (P), and T, as well as the differential expression of Star, Hsd3b6, Hsd17b3, and Srd5a1 messenger RNA levels. The evaluation of several structurally diverse male reproductive toxicants including 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), atrazine, prochloraz, triclosan, monoethylhexyl phthalate (MEHP), glyphosate, and RDX in BLTK1 cells suggests different modes of action perturb steroidogenesis. For example, prochloraz and triclosan antifungals reduced rhCG induction of T, consistent with published in vivo data but did not alter basal T levels. In contrast, atrazine and MEHP elicited modest induction of basal T but antagonized rhCG-mediated induction of T levels, whereas TCDD, glyphosate, and RDX had no effect on basal or rhCG induction of T in BLTK1 cells. These results suggest that BLTK1 cells maintain rhCG-inducible steroidogenesis and are a viable in vitro Leydig cell model to evaluate the effects of EDCs on steroidogenesis. This model can also be used to elucidate the different mechanisms underlying toxicant-mediated disruption of steroidogenesis. - Steroidogenesis is a potential target for drugs, chemicals, natural products, and environmental contaminants that adversely impact reproductive development and fertility (Sanderson, 2006; Whitehead and Rice, 2006). The mechanisms involved in altering steroid levels remain largely unknown but could involve disrupting enzyme expression and/or activity that affects testosterone (T) biosynthesis (Vinggaard et al., 2006). For example, fungicides, pesticides, and phthalates elicit reproductive tract abnormalities, compromise reproductive fitness, and cause cancer as a result of altering steroid levels (Gray et al., 2006; Scott et al., 2009; Vinggaard et al., 2006; Wilson et al., 2008). Developmental exposure to high doses of endocrine disrupting compounds (EDCs) has been associated with testicular dysgenesis syndrome in humans, comprised of adverse effects including cryptorchidism, hypospadias, low sperm count, and testicular cancer, such effects have been characterized in rodents and wildlife species with similar etiology (Edwards et al., 2006; Sharpe, 2006; Skakkebaek et al., 2001; Wohlfahrt-Veje et al., 2009). Current approaches to identify developmental and reproductive toxicants involve examining offspring after dosing pregnant animals to maternally toxic levels (Kimmel et al., 1993). Apical endpoints such as nipple retention, anogenital distance, vaginal opening, and balano-preputial separation are assessed in addition to evaluating reproductive fitness (Cooper, 2009; Marty et al., 2009). To comply with the European Commissions Registration, Evaluation, Authorization, and Restriction of Chemicals initiative, approximately $2.9 billion and 41 million animals are required for reproductive toxicity testing alone using current approaches (Breithaupt, 2006; Brown, 2003; Hartung and Rovida, 2009; Hofer et al., 2004). Such animal-based testing is expensive, time consuming, generally descriptive, poses ethical concerns and does not significantly contribute to the elucidation of mechanisms (Andersen and Krewski, 2009; National Research Council, 2007; Daston, 2007; Daston et al., 2010; Holsapple et al., 2009; Knudsen and Daston, 2010). Furthermore, they are contrary to the replacement, reduction, and refinement of animal testing. Alternative short-term assays, such as the sliced testes assay and the human H295R adrenal corticocarcinoma cellbased assay used in tier 1 of the Endocrine Disruptor Screening Program, still require animals or are based on adrenal steroidogenesis, which is not the primary site of sex hormone production (Borgert et al., 2011; Harding et al., 2006; Hecker et al., 2006; Higley et al., 2010). Therefore, in vitro gonadal-based steroidogenesis assays that support ranking and prioritization for further investigation are needed. The Toxicity Testing in the 21st Century: A Vision and a Strategy report proposed testing should be less reliant on whole animal studies with more effort devoted to systems oriented computational models developed using in vitro and in vivo data that could screen chemicals, metabolites, and mixtures for potential toxicity (National Research Council, 2007). In response, the U.S. Environmental Protection Agency (USEPA), the National Institute of Environmental Health Sciences National Toxicology Program, and the U.S. Food and Drug Administration have initiated ToxCast, Tox21, and EDSP21 programs in collaboration with the National Institute of Health Chemical Genomics Center, as well as other highthroughput service providers, to use in silico and in vitro assays to screen chemicals for potential toxicity (Dix et al., 2007; Shukla et al., 2010; USEPA, 2011). These programs use informatic platforms to integrate data from thousands of chemicals that are robotically screened in diverse assays to develop predictive computational models of toxicity. Similarly, the European Union has established ChemScreen and ReProTect to develop simple rapid in vitro/in silico screening systems for reproductive toxicants (www.chemscreen.eu; www.reprotect.eu; Schenk et al., 2010). Pathways and overrepresented gene functions identified in dose-response genomic and highthroughput screening studies may also be used to determine points of departure in provisional peer reviewed toxicity values (http://hhpprtv.ornl.gov) for chemicals not comprehensively evaluated by the Integrated Risk Information System (http:// www.epa.gov/iris/index.html) or where there is minimal safety data. Despite this broad spectrum of programs, which includes assays for cell viability, apoptosis, mitochondrial function, receptor-mediated activity, and DNA damage in human, mouse, and rat cell lines, as well as cell-free systems, none assesses steroidogenesis in a high-throughput format (USEPA, 2007). In mammals, the induction of steroidogenesis has been thoroughly studied. Luteinizing hormone (LH) secreted from the anterior pituitary induces gonadal steroidogenesis. LH binds G-protein-coupled LH-human chorionic gonadotropin (hCG) receptors (LHCGR) on the surface of Leydig cells. Subsequent dissociation of the Gas subunit stimulates adenylyl cyclase activity producing 3#,5#-cyclic adenosine monophosphate (cAMP) that induces gene expression and enzyme activity increasing steroid biosynthesis. BLTK1 cells, isolated from a testicular tumor that developed in a transgenic mouse expressing the mouse inhibin a promoter/simian virus 40 T-antigen fusion gene (Kananen et al., 1996; Rahman and Huhtaniemi, 2004), retain functional LHCGR-mediated steroidogenesis. They are stable over multiple passages, easily transfected, and exhibit excellent growth characteristics. In this study, recombinant human chorionic gonadotropin (rhCG) and forskolin (FSK) are used as positive controls for the induction of steroidogenesis, as measured by increases in progesterone (P), T, and 17b-estradiol (E2) levels in media. We investigate murine BLTK1 Leydig cells as a novel model for evaluating the effects of chemicals on steroidogenesis. Our results demonstrate that BLTK1 cells can be used to screen substances that alter intracellular cAMP, steroidogenic gene expression, and sex steroid levels. MATERIALS AND METHODS Cell culture and treatment. Mouse Leydig BLTK1 (BLT-1 cells, clone K1) cells were isolated from a testicular tumor that developed in a transgenic mouse expressing the mouse inhibin a promoter/simian virus 40 T-antigen fusion gene (Rahman and Huhtaniemi, 2004). Cells were maintained in phenol redfree Dulbeccos modified Eagle medium nutrient mixture F-12 (DMEM/F-12 media) (Invitrogen, Carlsbad, CA) with 10% fetal bovine serum (Thermo Scientific HyClone, Logan, UT), 100 U/ml penicillin and 100 lg/ml streptomycin (Invitrogen) and incubated under standard conditions (5% CO2, 37 C). For the evaluation of steroidogenic enzyme and receptor expression, cells were grown to 80% confluency and harvested without any treatment. For 3#,5#-cyclic adenosine monophosphate (cAMP), progesterone (P), testosterone (T), and estradiol (E2) determination, cells were grown to 80% confluency, transferred into 24-well tissue culture plates (Sarstedt, Newton, NC), and incubated overnight. Cells were treated with dimethyl sulfoxide (DMSO) (Sigma, St Louis, MO) vehicle, or 0.1, 0.3, 1, 3, 10, 30, or 100 ng/ml rhCG (obtained from A.F. Parlow, National Institute of Diabetes and Digestive and Kidney Diseases National Hormone & Peptide Program, Harbor-UCLA Medical Center, Torrance, CA) or 0.1, 0.3, 1, 3, 10, 30, or 100lM FSK (Sigma), and media were collected at indicated times. Time course studies were conducted with DMSO vehicle, 3 ng/ml rhCG, or 10lM FSK, and media were collected at 1, 2, 4, 8, 12, 24, or 48 h. Gene expression studies used the same study design, concentrations, and time points with cells seeded into T-25 flasks (Sarstedt). MTT assay. BLTK1 cells in 96-well plates (Sarstedt) were treated with 1, 3, 10, 30, 100, 300, or 600 ng/ml rhCG (positive control), 1, 3, 10, 30, 100, 300, or 600lM FSK (positive control), or 1, 3, 10, 30, 100, 300, or 600lM of test compound in triplicate, respectively. Media were aspirated after 24 h and replaced with 50 ll of fresh MTT reagent (5 mg/ml thiazolyl blue tetrazolium bromide [Sigma] in PBS). Following a 3 h incubation, MTT reagent was aspirated and replaced with 150 ll DMSO. Cells were incubated for 2 h followed by absorbance measurements at 595 and 650 nm (A595A650) using an Emax precision microplate reader (Molecular Devices, Sunnyvale, CA). Results are reported as percentage of control calculated from the relative absorbance of treated versus DMSO controls where 100% indicates no cytotoxicity. Western blotting. Lysates were prepared in lysis buffer (50mM Tris-HCl, pH 7.4, 150mM NaCl, 1mM EDTA, 0.5% Triton, and protease inhibitor tablet [Roche Diagnostics, Indianapolis, IN]). Protein concentration was determined spectrophotometrically by Bradford assay (Bio-Rad Laboratories, Hercules, CA). Extracts (50 lg) were resolved on a 10% SDS/polyacrylamide gel and transferred to Immobilon membrane (Millipore, Billerica, MA). Membranes were incubated with 1 lg/ml primary antibody (rabbit anti-StAR [Santa Cruz Biotechnology, Santa Cruz, CA; Catalog No. 25806], rabbit anti-Cyp11a1 [Millipore; Catalog No. AB1244], goat anti-Cyp17a1 [Santa Cruz; Catalog No. SC-46081], goat anti-3bHSD [Santa Cruz; Catalog No. SC-30820], goat antiSrd5a1 [Santa Cruz; Catalog No. SC-20399], and rabbit anti-Cyp19a1 [Abcam, Cambridge, MA; Catalog No. ab51924]) at 4 C overnight. Membranes were washed and incubated with donkey anti-rabbit or donkey anti-goat IgG conjugated with horseradish peroxidase (Santa Cruz; Catalog No. SC-2077 and SC-2056, respectively) for 1 h. Enhanced Chemiluminescence (Thermo Scientific Pierce, Rockford, IL) was used to visualize detected protein. Enzyme immunoassays. Enzyme immunoassay (EIA) kits for T (Cayman Chemical Company, Ann Arbor, MI; limit of detection 6 pg/ml, cross-reactivity 27% for 5a-dihydrotestosterone and 19% for 5b-dihydrotestosterone), E2 (Cayman Chemical Company; limit of detection 20 pg/ml, cross-reactivity 14% for estradiol-3-glucuronide, 12% for estrone, and 10% for estradiol 17-glucuronide), and P (ALPCO Diagnostics, Salem, NH) were used according to the manufacturers instructions. Intracellular cAMP was measured by EIA (Cayman Chemical Company; limit of detection 3 pmol/ml cAMP, crossreactivity 1.5% for cyclic guanosine monophosphate) following hydrochloric acid extraction. All kits consisted of 96-well precoated antibody plates, for which samples compete for binding with conjugated hormone. Following incubation, plates were washed and measured at 420 nm for cAMP, T, and E2 or 405 nm for P, using an Emax precision microplate reader (Molecular Devices). Standards (0.3750 pmol/ml cAMP, 060 ng/ml P, 3.9500 pg/ml T, or 6.6 4000 pg/ml E2) were used to generate a standard curve for quantification. RNA isolation and gene expression. Total RNA was extracted from cell pellets using RNeasy Mini Kits (Qiagen, Valencia, CA) according to the manufacturers protocol with an additional RNase-free DNase (Qiagen) digestion. RNA was quantified at 260 nm (A260) and purity assessed using the A260/A280 ratio, as well as by denaturing gel electrophoresis. First-strand complementary DNA (cDNA) was synthesized from RNA (1 lg) using SuperScript II reverse transcriptase (Invitrogen) and anchored oligo-dT primer (Invitrogen) as described by the manufacturer. For real-time PCR (RT-PCR) evaluation of steroidogenic enzyme and receptor expression, the cDNA was used as template for PCR amplification with gene-specific primers (Supplementary table 1). Quantitative RT-PCR (QRT-PCR) was used to quantify concentration- and time-dependent expression of specific genes. Reactions in 96-well plates consisted of 30 ll, including 1 ll of cDNA template, 0.1lM forward and reverse gene-specific primers, 3mM MgCl2, 1.0mM deoxynucleotide triphosphates, 0.025 IU AmpliTaq Gold, and 1 X SYBR Green PCR Buffer (Applied Biosystems, Foster City, CA) using an Applied Biosystems PRISM 7500 Sequence Detection System. Dissociation curve analysis assured single product amplification. To control for differences in RNA loading, quality, and cDNA synthesis, samples were standardized to the geometric mean of three housekeeping genes: ActB, Gapdh, and Hprt (Vandesompele et al., 2002). Results were quantified using a standard curve generated on the same 96-well plate and amplified by using purified cDNA product as template specific for each gene (serial 103 dilutions from 108 to 101 copies). The slope of the standard curve was used to assess amplification efficiency as described by the manufacturer with all amplification efficiencies > 90%. Fold changes were calculated relative to time-matched vehicle. Relative expression was scaled such that time-matched vehicle control expression equaled one for graphing purposes. Dose-response modeling and statistical analyses. The ToxResponse modeler uses particle swarm optimization to identify the best fit across five model classes: sigmoidal, exponential, linear, quadratic, and Gaussian (Burgoon and Zacharewski, 2008). The best fitting model was then used to calculate half maximal effective concentration (EC50) values. All statistical analyses were carried out using SAS v9.1 (SAS Institute, Cary, NC) by ANOVA, with Dunnetts or Tukeys post hoc tests for concentration-response and time course data, respectively. Differences between treatment groups were considered significant when p < 0.05 relative to time-matched DMSO control. Steroidogenic Enzyme Expression in BLTK1 Cells Steroidogenesis involves the conversion of cholesterol to progestogens (pregnenolone, progesterone [P], 17a-hydroxypregnenolone, and 17a-hydroxyprogesterone), followed by further metabolism to androgens (dehydroepiandrosterone [DHEA], androstenedione, testosterone [T], and 5a-dihydrotestoeterone) and estrogens including estrone and 17b-estradiol (Fig. 1). Steroidogenic enzyme messenger RNA (mRNA) and protein were detected in BLTK1 cells by RT-PCR and/or Western blotting, confirming the expression of all required steroidogenic enzymes (Fig. 2A). In addition, mRNA for several potential regulatory factors including LHCGR (data not shown), estrogen receptor (ER), androgen receptor (AR), and steroidogenic factor 1 (SF-1), peroxisome proliferatoractivated receptors (PPARa and PPARc), the pregnane X receptor (PXR), and the aryl hydrocarbon receptor (AhR) were also detected (Fig. 2B). However, mRNA for progesterone receptor, glucocorticoid receptor, or the liver receptor homolog 1 was not detected in BLTK1 cells despite verification of RT-PCR primer specificity and functionality in mouse Hepa1c1c7 cells (data not shown). Induction of Steroidogenesis by FSK and rhCG Temporal profiles of intracellular cAMP as well as P and T levels in media were evaluated in response to 3 ng/ml rhCG or 10lM FSK by EIA (Fig. 3). Intracellular cAMP was induced by FSK at 30 min (~120 pmol/ml, ~10-fold) and by 1 h in response to rhCG (635 pmol/ml, 60-fold). However, levels quickly diminish such that no intracellular cAMP was detected by 8 h. Maximum P levels (200 ng/ml, eightfold) were observed at 2 h in response to rhCG and FSK, followed by a steady decline due to metabolism to androgens and estrogens. In contrast, T levels gradually increased reaching a maximum of ~200 pg/ml (sevenfold) at 48 h, with significant increases detected as early as 1 h posttreatment. Concentration-dependent induction of intracellular cAMP and secreted P and T was evaluated at 4 h when cAMP can still be detected (Figs. 4AC). 17b-Estradiol (E2) was evaluated at 48 h (Fig. 4D) as it was not consistently detected at 4 h (data not shown). cAMP, P, and T were induced 25-, 10-, and 4-fold, respectively, at 4 h, whereas E2 was induced ~fourfold by 48 h. The EC50 for cAMP induction was > 24 ng/ml for rhCG and > 29lM for FSK. Meanwhile, EC50 values of 1 ng/ml rhCG and 9lM FSK were conserved for both P and T induction, whereas E2 EC50 values were 10 ng/ml for rhCG and 9lM for FSK. Intracellular cAMP levels are not only regulated by synthesis but also degradation, which is regulated by cyclic nucleotide phosphodiesterase enzymes (Chen et al., 2007). The phosphodiesterase inhibitor IBMX maximizes cAMP levels in order to further induce steroidogenesis. However, IBMX cotreatment with rhCG or FSK did not increase T levels further, albeit rhCG and FSK potencies were greater (Fig. 5; T EC50 with and without IBMX by FSK: 0.1lM vs. 9.4lM and rhCG: 0.1 ng/ml vs. 0.9 ng/ml, respectively). rhCG- and FSK-Elicited Steroidogenic Gene Expression The effect of rhCG and FSK on the gene expression of steroidogenic enzymes including steroidogenic acute regulatory protein (Star) was evaluated by QRT-PCR at 24 h. rhCG elicited concentration-dependent induction of Star, Hsd3b6, Hsd17b3, and Srd5a1 mRNA levels (Fig. 6). In contrast, only Star and Cyp17a1 mRNA exhibited concentration-dependent induction by FSK at 24 h. Star, the rate-limiting step in steroidogenesis, was induced ~20-fold by rhCG and ~10-fold by FSK. Temporal gene expression (Fig. 6) also revealed differences in the induction kinetics of Star, Hsd3b6, and Cyp19a1 by rhCG and FSK. Most notably were differences in the induced levels of Hsd17b3 and Srd5a1. These differences are likely a result of differences in pathway activation. FSK directly induces cAMP levels by the activating adenylyl cyclase, whereas rhCG induction via the LHCGR activates adenylyl cyclase and can also activate alternate signal transduction pathways such as the release of arachidonic acid and its metabolites (Ronco et al., 2002; Wang and Stocco, 1999). Furthermore, Cyp17a1 induction was only seen with FSK, while rhCG inhibited expression at 2 and 24 h. There were negligible effects on Cyp11a1 and Hsd3b1 in both concentration FIG. 3. Temporal rhCG- and FSK-induced cAMP, P and T levels. Intracellular cAMP (A) as well as P (B) and T (C) levels in media were evaluated in response to 3 ng/ml rhCG (light bars) or 10lM FSK (dark bars) relative to DMSO vehicle controls (white bars). Both treatments elicited similar temporal profiles consisting of maximal cAMP levels reached early at 0.51 h followed by decrease and was undetectable by 8 h, whereas P reached maximum induction at 2 h followed by a gradual decrease over time. In contrast, T levels steadily increased throughout the time course. Data represent mean SE of three replicates, *p < 0.05 vs. DMSO controls. response and temporal evaluation (data not shown). More detailed studies are warranted to elucidate the mechanisms responsible for the differential effects of rhCG and FSK on steroidogenic gene expression. Effects of Endocrine Disrupting Compounds on Testosterone Levels The effect of selected EDCs, reported to elicit reproductive toxicity (Table 1), was assessed in BLKT1 cells by evaluating T levels. They include the triazine herbicide atrazine (PogrmicMajkic et al., 2010; Trentacoste et al., 2001), the broad spectrum herbicidal active ingredient glyphosate used in commercially available Roundup (Romano et al., 2010; Walsh et al., 2000), the conazole antifungal prochloraz (Kjaerstad et al., 2010; Vinggaard et al., 2005), the antifungal/antibacterial triclosan (Kumar et al., 2009; Zorrilla et al., 2009), and the diethylhexyl phthalate (DEHP) metabolite monoethylhexyl phthalate (MEHP) (Fan et al., 2010; Ge et al., 2007; Gunnarsson et al., 2008). None of these chemicals elicited significant cytotoxicity in the concentration range examined (Supplementary fig. S1). Compounds were evaluated for concentration-dependent effect on basal T levels and at a single cotreatment concentration to investigate potential interactions with rhCG-mediated stimulation of T levels (Fig. 7). T levels were induced twofold (60 pg/ml) and 10-fold (250 pg/ml) by atrazine and MEHP, respectively. No other examined chemical altered basal T levels. However, steroidogenesis in Leydig cells is stimulated by LH via the LHCGR during late gestation first measurable at gestational day (GD) 15 and peaking at GD 18 in the mouse (OShaughnessy et al., 2006), and throughout gestation in humans, with a similar programming window for androgen-dependent masculinization extrapolated between humans and rodents (Welsh et al., 2008). LH stimulation is also crucial during puberty and adulthood to stimulate T biosynthesis to support Sertoli cell spermatogenesis. Consequently, cotreatment studies with 3 ng/ml rhCG were conducted to mimic Leydig cell stimulation. Atrazine (300lM) inhibited rhCG-induced T levels ~30% (rhCG alone ~100 pg/ml T vs. Atrazine rhCG ~70 pg/ml T). Similarly, 100lM MEHP inhibited rhCG-elicited T induction ~ 20% (rhCG alone ~100 pg/ml vs. MEHP cotreatment ~80 pg/ml). In contrast, prochloraz did not significantly alter basal T levels but cotreatment decreased rhCG-induced T to control levels (rhCG ~100 pg/ml vs. prochloraz rhCG ~50 pg/ml vs. DMSO ~40 pg/ml). Similarly, 30lM triclosan had no effect, yet cotreatment decreased rhCG-elicited T induction ~25% (rhCG alone ~100 pg/ml vs. triclosan rhCG cotreatment ~75 pg/ml). Glyphosate, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), and RDX did not induce or alter rhCG induction of T. The in vitro evaluation of steroidogenesis is important for screening potential reproductive and developmental toxicants (Sanderson, 2006; Whitehead and Rice, 2006). Current models include human H295R adrenocarcinoma cells and several rodent Leydig cell lines including MA-10, mLTC, and R2C. Human H295R cells (American Type Culture Collection CLR-2128) were isolated from a pluripotent adenocortical carcinoma in a 48-year-old black female, produce detectable basal aldosterone, cortisol, androgens, and E2 (Gracia et al., 2006), and are part of the Endocrine Disruptor Screening Program to screen for effects on T and E2 biosynthesis (USEPA, 2009). However, H295R cells do not express functional adrenalcorticotropic hormone receptors (a member of the cell surface 7-transmembrane domain superfamily of G-protein-coupled receptors), and therefore, steroidogenesis must be stimulated using FSK. Moreover, besides glucocorticoids and mineralocorticoids, the adrenal cortex normally only produces dehydroepiandrosterone sulfate and androstenedione (precursors of T) and does not express the 17-beta-hydroxysteroid dehydrogenase necessary for T biosynthesis (Hinson and Raven, 1996). As such, it has been suggested that H295R cells have the characteristics of zonally undifferentiated human fetal adrenal cells (Gracia et al., 2006). Only Leydig cells synthesize T and E2 directly from cholesterol (Scott et al., 2009). In ovarian steroidogenesis, theca and FIG. 5. Effect of the phosphodiesterase inhibitor IBMX on rhCG- and FSK-elicited induction of T. Concentration-dependent induction of cAMP (A and B) and T (C and D) by rhCG (0.1100 ng/ml) or FSK (0.1100lM) alone (dark bars) or in the presence of 0.2mM IBMX (light bars) for 4 h. Although IBMX did not affect T induction, cAMP levels were significantly increased, and the potency of rhCG and FSK were enhanced. Data represent mean SE of three independent replicates, *p < 0.05 versus DMSO controls. FIG. 6. Concentration- and time-dependent induction of steroidogenic gene expression. The concentration-dependent (24 h, left column) and temporal (right column) effects of rhCG (ng/ml, light bars) and FSK (lM, dark bars) on Star, Cyp17a1, Hsd3b6, Hsd17b3, Srd5a1, and Cyp19a1 mRNA levels were examined using QRT-PCR. Cyp11a1 mRNA expression was not affected by treatment (data not shown). Data represent mean SE of three replicates, *p < 0.05 versus DMSO controls. granulosa cells produce P, while only theca cells metabolize P to T, and subsequent aromatization to estrogens or 5a reduction to DHT occurs in granulosa cells (Havelock et al., 2004). Steroidogenic theca cell line development has not been successful, and as such, rodent models are of Leydig cell origin such as mouse MA-10 and mLTC-1 models as well as rat R2C cells. MA-10 and mLTC1 cells were established from transplantable M548OP Leydig tumors carried in C57Bl/6 mice that exhibit P induction by rhCG but lacked the enzymes required for subsequent metabolism to T (Ascoli, 1981; Freeman and Ascoli, 1981; Rebois, 1982). However, recent studies report mLTC-1 cells can produce low levels of androgens, with 100 IU/ml hCG eliciting a maximum of 600 pmol/well P and 10 pmol/well T in a similar 24-well plating format as used herein (Panesar et al., 2003). These levels are lower than those observed in resting unstimulated BLTK1 cells. Rat R2C cells lack functional LHCGR expression and are not rhCG inducible but exhibit high basal steroidogenesis activity (Stocco and Chen, 1991). In contrast, BLTK1 cells produce progestogens, androgens, and E2 in response to trophic hormone stimulus via LHCGR, the 7-transmembrane G-protein-coupled cell surface receptor, similar to T biosynthesis in vivo following LH secretion from the anterior pituitary (Chen et al., 2007). Although human steroidogenesis prefers the D5 pathway over the D4 pathway in mice, the enzymatic steps are highly conserved facilitating extrapolation of murine BLTK1 results to humans (Scott et al., 2009). Gestation is the critical period of exposure to EDCs that targets fetal Leydig cells causing reproductive and development toxicity. Fetal Leydig cells are observed as early as GD 12.5 in mice or week 6 in humans (OShaughnessy et al., 2006; Rahman and Huhtaniemi, 2004). Murine fetal Leydig cells do not express measurable LHCGR until GD 15.5, and murine fetal T production is LH independent (Yao and Barsoum, 2007). However, human fetal Leydig cells do express LHCGR and are dependent on LH and hCG stimulation of steroidogenesis for masculinization during development (OShaughnessy et al., 2006). Though BLTK1 cells are not murine fetal Leydig cells, they retain rhCG-inducible steroidogenesis characteristic of murine adult Leydig cells and both fetal and adult human Leydig cells. The ability to evaluate rhCGinducible steroidogenesis provides a unique functional feature that is important for screening EDC interactions that may occur during human fetal and adult exposure. During gestation, LH stimulates Leydig cell proliferation and T production, whereas during puberty and adulthood, LH stimulates T biosynthesis to indirectly support spermatogenesis by contributing to Sertoli cell stimulation. rhCG also promotes cell proliferation or apoptosis, but no such effects were observed on BLTK1 cells (data not shown). rhCG is used clinically as an LH substitute to treat LH deficiency and in research to stimulate steroidogenesis (Hakola et al., 1998), due to its longer half-life (~24 h compared with ~4 h for LH) (Yen et al., 1968). Like LH, rhCG induces steroidogenesis via the LHCGR and initiates the same signaling pathways resulting in Atrazine Molecular weight (g/mol) 215.68 Chemical name Triclosan 2,4,4#-trichloro-2#-hydroxydiphenyl ether Prochloraz N-propyl-N-(2,4,6-trichlorophenoxy) ethyl-imidazole-1-carboxamide 2,3,7,8-tetrachlorodibenzo-p-dioxin Glyphosate N-(phosphonomethyl) glycine 1071-83-6 169.07 2-(2-ethylhexoxycarbonyl) benzoic acid 1,3,5-trinitroperhydro-1,3,5-triazine Structure Reproductive toxicity Decreased serum T, dysregulation of steroidogenesis (Pogrmic-Majkic et al., 2010; Trentacoste et al., 2001) Antiandrogenic, decreased T production by Leydig cells, decreased serum T levels (Kumar et al., 2009; Zorrilla et al., 2009) Decreased serum T levels, AR antagonism, altered androgen-dependent gene expression (Kjaerstad et al., 2010; Vinggaard et al., 2005) Decreased intratesticular T, inhibition of steroidogenic gene expression (Adamsson et al., 2009; Fukuzawa et al., 2004; Lai et al., 2005) Inhibition of steroidogenic enzymes expression, decreased T production (Romano et al., 2010; Walsh et al., 2000) Decreased serum T levels, Leydig cell hyperplasia (Ge et al., 2007; Gunnarsson et al., 2008) Altered reproductive capacity (Mukhi and Patino, 2008; Zhang et al., 2006, 2008) a robust in vivo and in vitro steroidogenic response (Dufau et al., 1984; Hakola et al., 1998; Payne and Youngblood, 1995). Interestingly, LH/rhCG levels required for maximum cAMP synthesis are 15-fold higher than the level needed for maximal T production (Chen et al., 2007), suggesting that minimal changes in cAMP can affect steroidogenesis. Our expression prior to any conclusions regarding the steroidogenic effects of these chemicals. In summary, current protocols and models are inadequate to screen the universe of chemicals, metabolites, and mixtures that may alter steroidogenesis (Sanderson, 2006; Whitehead and Rice, 2006; Wilson et al., 2008). BLTK1 cells are a novel complementary rhCG-inducible Leydig-based model that can be used to assess effects on steroidogenic gene expression, intracellular cAMP, and P, T, and E2 levels in media. Their consistent response characteristics and inducibility over 30 passages also make this cell line attractive for high-throughput screening. Comprehensive characterization of effects on intermediate steroid biosynthesis, including pregnenolone, 17-hydroxyprogesterone, DHEA, androstenedione, estrone, and DHT, as well as the differential expression of steroidogenic enzymes will also facilitate the elucidation of modes of action relevant to adverse outcome pathways in humans and other relevant species. SUPPLEMENTARY DATA Supplementary data are available online at http://toxsci. oxfordjournals.org/. ACKNOWLEDGMENTS The authors would like to thank Michelle DSouza for technical assistance as well as Dr Anna Kopec, Michelle Angrish and Rance Nault for the critical reading of the manuscript. Adamsson, A., Simanainen, U., Viluksela, M., Paranko, J., and Toppari, J. (2009). The effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin on foetal male rat steroidogenesis. Int. J. Androl. 32, 575585. Andersen, M. E., and Krewski, D. (2009). Toxicity testing in the 21st century: Bringing the vision to life. Toxicol. Sci. 107, 324330. Ascoli, M. (1981). Characterization of several clonal lines of cultured Leydig tumor cells: Gonadotropin receptors and steroidogenic responses. Endocrinology 108, 8895. Borgert, C. J., Mihaich, E. M., Quill, T. F., Marty, M. S., Levine, S. L., and Becker, R. A. (2011). Evaluation of EPAs Tier 1 Endocrine Screening Battery and recommendations for improving the interpretation of screening results. Regul. Toxicol. Pharmacol. 59, 397411. Breithaupt, H. (2006). The costs of REACH. REACH is largely welcomed, but the requirement to test existing chemicals for adverse effects is not good news for all. EMBO Rep. 7, 968971. Brown, V. J. (2003). REACHing for chemical safety. Environ. Health Perspect. 111, A766A769. Burgoon, L. D., and Zacharewski, T. R. (2008). Automated quantitative doseresponse modeling and point of departure determination for large toxicogenomic and high-throughput screening data sets. Toxicol. Sci. 104, 412418. Chen, H., Midzak, A., Luo, L., and Zirkin, B. R. (2007). In Aging and the Decline of Androgen Production (A. H. Payne and M. P. Hardy, Eds.). Humana Press Inc., Totowa, NJ. Clewell, R. A., Campbell, J. L., Ross, S. M., Gaido, K. W., Clewell, H. J., III, and Andersen, M. E. (2010). Assessing the relevance of in vitro measures of phthalate inhibition of steroidogenesis for in vivo response. Toxicol. In Vitro 24, 327334. Cooper, R. L. (2009). Current developments in reproductive toxicity testing of pesticides. Reprod. Toxicol. 28, 180187. Daston, G. P. (2007). Genomics and developmental risk assessment. Birth Defects Res. 79, 17.


This is a preview of a remote PDF: https://toxsci.oxfordjournals.org/content/127/2/391.full.pdf

Agnes L. Forgacs, Qi Ding, Rosemary G. Jaremba, Ilpo T. Huhtaniemi, Nafis A. Rahman, Timothy R. Zacharewski. BLTK1 Murine Leydig Cells: A Novel Steroidogenic Model for Evaluating the Effects of Reproductive and Developmental Toxicants, Toxicological Sciences, 2012, 391-402, DOI: 10.1093/toxsci/kfs121