Direct effects of diethylstilbestrol on the gene expression of the cholesterol side-chain cleavage enzyme (P450scc) in testicular Leydig cells Katsuhiko Warita a,b, ⁎, Tomoko Mitsuhashi b , Teruo Sugawara c , Yoshiaki Tabuchi d , Takashi Tanida b,e , Zhi-Yu Wang a , Yoshiki Matsumoto a , Toshifumi Yokoyama b , Hiroshi Kitagawa b , Takanori Miki a , Yoshiki Takeuchi a , Nobuhiko Hoshi b a Department of Anatomy and Neurobiology, Faculty of Medicine, Kagawa University, Kagawa 761-0793, Japan b Department of Animal Science, Graduate School of Agricultural Science, Kobe University, Kobe 657-8501, Japan c Department of Molecular Biochemistry, Graduate School of Medicine, Hokkaido University, Sapporo 060-8638, Japan d Division of Molecular Genetics Research, Life Science Research Center, University of Toyama, Toyama 930-0194, Japan e Department of Anatomy and Neurobiology, Graduate School of Medical Science, Kyoto Prefectural University of Medicine, Kyoto 602-8566, Japan abstract article info Article history: Received 11 August 2009 Accepted 25 June 2010 Keywords: Steroidogenesis Estrogenic endocrine disruptor Leydig cells Cholesterol side-chain cleavage enzyme (P450scc) Histone acetylation Aims: To investigate the precise mechanisms underlying the action of estrogenic endocrine disruptors, we evaluated the direct effects of synthetic estrogen diethylstilbestrol (DES) on steroidogenesis in Leydig cells, with particular emphasis on the expression of the cholesterol side-chain cleavage enzyme P450scc. Furthermore, the mechanism underlying the action of DES was compared with that of endogenous estrogen 17β-estradiol (E2), which has a potency equivalent to that of DES. Main methods: TTE1 Leydig cells were treated with 5×10 -8 μM to 5 μM DES or E2 for 24 h, and P450scc gene expression and the histone modifications underlying their transcriptional activation were examined using reverse transcription-polymerase chain reaction (RT-PCR) and chromatin immunoprecipitation (ChIP), respectively. Key findings: P450scc mRNA expression in the DES-treated and E2-treated cells reduced in inverse proportion to the dose of DES and E2, respectively; however, cAMP stimulation induced a recovery in the expression to a level approximately equal to those in the controls. In the DES-treated cells, ChIP assay revealed histone deacetylation in the P450scc promoter region. Interestingly, E2 did not cause histone deacetylation. Significance: In the early stages of steroidogenesis, DES and E2 directly induced a reduction in P450scc mRNA expression in inverse proportion to their doses, and treatment with cAMP restored the decreased P450scc mRNA expression. Furthermore, DES can induce alterations in the histone modification of the P450scc gene, and natural estrogen and synthetic estrogenic compounds such as DES may induce reproductive disorders through different molecular mechanisms. © 2010 Elsevier Inc. All rights reserved. Introduction Estrogenic endocrine disrupters reduce male reproductive success in laboratory animals (Colborn et al. 1993). Estrogenic agents reduce the weight of reproductive organs, sperm count, and sperm motility in male rodents (Goyal et al. 2001; Toyama et al. 2001); they also induce structural and functional abnormalities in the testes and reproductive tract (Arai et al. 1983; Toppari et al. 1996) by inducing the expression of estrogen receptors (ERs) (Cooke et al. 1991; Fisher et al. 1997; Saunders et al. 1997). Exposure to estrogenic agents during development induces long-term changes in various organs, including persistent molecular alterations (Iguchi et al. 2002; Matsuno et al. 2004). Diethylstilbestrol (DES), a synthetic nonsteroidal estrogen, exhi- bits high estrogenic activity by binding to ERs; therefore, it is used to evaluate the potential toxicity of various chemicals that affect or mimic estrogen activity (Colborn et al. 1993). In male mice, DES exposure causes sustained hypoproduction of follicle-stimulating hormone (FSH) and luteinizing hormone (LH) and long-term dysfunction of the hypothalamo-pituitary-testicular axis (Warita et al. 2006, 2008). The plasma testosterone level was vanishingly low in male mice exposed to high doses of DES. Therefore, we hypothesized that DES directly affects the rate-limiting factor in steroidogenesis of Leydig cells. Steroidogenesis is primarily regulated by the steroidogenic acute regulatory (StAR) protein (Clark et al. 1994; Lin et al. 1995; Sugawara et al. 1995) and cytochrome P450-mediated cholesterol side-chain cleavage enzyme CYP11A1 (P450scc) (Chung et al. 1997; Kramer et al. 1984; Miller 1988). StAR transports cholesterol from intracellular stores to the inner mitochondrial membrane; subsequently, P450scc converts Life Sciences 87 (2010) 281–285 ⁎ Corresponding author. Faculty of Medicine, Kagawa University, Department of Anatomy and Neurobiology, 1750-1 Ikenobe, Miki-cho, Kita-gun, Kagawa 761-0793, Japan. Tel.: +81 87 891 2087; fax: +81 87 891 2088. E-mail address: warita@med.kagawa-u.ac.jp (K. Warita). 0024-3205/$ – see front matter © 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.lfs.2010.06.020 Contents lists available at ScienceDirect Life Sciences journal homepage: www.elsevier.com/locate/lifescie