Fa Svetlana, Pogrmic-Majkic Kristina, Samardzija Dragana, Hrubik Jelena, Glisic Branka, Kovacevic Radmila, Andric Nebojsa
Molecular and Reproductive Toxicology Unit, Laboratory for Ecotoxicology, Department of Biology and Ecology, Faculty of Sciences, University of Novi Sad, 21000 Novi Sad, Serbia.
Molecular and Reproductive Toxicology Unit, Laboratory for Ecotoxicology, Department of Biology and Ecology, Faculty of Sciences, University of Novi Sad, 21000 Novi Sad, Serbia.
Toxicol Appl Pharmacol. 2015 Jan 1;282(1):20-9. doi: 10.1016/j.taap.2014.11.001. Epub 2014 Nov 13.
Hexabromocyclododecane (HBCDD), a brominated flame retardant added to various consumer products, is a ubiquitous environmental contaminant. We have previously shown that 6-hour exposure to HBCDD disturbs basal and human chorionic gonadotropin (hCG)-induced steroidogenesis in rat Leydig cells. Reduction in mitochondrial membrane potential (ΔΨm) and cAMP production was also observed. Here, we further expanded research on the effect of HBCDD on Leydig cells by using a prolonged exposure scenario. Cells were incubated in the presence of HBCDD during 24h and then treated with HBCDD+hCG for additional 2h. Results showed that HBCDD caused a sustained reduction in ATP level after 24h of exposure, which persisted after additional 2-hour treatment with HBCDD+hCG. cAMP and androgen accumulations measured after 2h of HBCDD+hCG treatment were also inhibited. Real-time PCR analysis showed significant inhibition in the expression of genes for steroidogenic enzymes, luteinizing hormone receptor, regulatory and transport proteins, and several transcription factors under both treatment conditions. Western blot analysis revealed a decreased level of 30kDa steroidogenic acute regulatory protein (StAR) after HBCDD+hCG treatment. In addition, HBCDD decreased the conversion of 22-OH cholesterol to pregnenolone and androstenedione to testosterone, indicating loss of the activity of cytochrome P450C11A1 (CYP11A1) and 17β-hydroxysteroid dehydrogenase (HSD17β). Cell survival was not affected, as confirmed by cytotoxicity and trypan blue tests or DNA fragmentation analysis. In summary, our data showed that HBCDD inhibits ATP supply, most likely through a decrease in ΔΨm, and targets multiple sites in the steroidogenic pathway in Leydig cells.
六溴环十二烷（HBCDD）是一种添加到各种消费品中的溴化阻燃剂，是一种普遍存在的环境污染物。我们之前已经表明，6小时暴露于HBCDD会干扰大鼠睾丸间质细胞的基础和人绒毛膜促性腺激素（hCG）诱导的类固醇生成。还观察到线粒体膜电位（ΔΨm）和cAMP产生的降低。在这里，我们通过延长暴露时间进一步扩展了对HBCDD对睾丸间质细胞影响的研究。细胞在HBCDD存在下孵育24小时，然后用HBCDD + hCG再处理2小时。结果表明，暴露24小时后HBCDD导致ATP水平持续降低，在用HBCDD + hCG再处理2小时后仍持续存在。HBCDD + hCG处理2小时后测量的cAMP和雄激素积累也受到抑制。实时PCR分析显示，在两种处理条件下，类固醇生成酶、促黄体生成素受体、调节和转运蛋白以及几种转录因子的基因表达均受到显著抑制。蛋白质印迹分析显示，HBCDD + hCG处理后30kDa类固醇生成急性调节蛋白（StAR）水平降低。此外，HBCDD降低了22-羟基胆固醇向孕烯醇酮的转化以及雄烯二酮向睾酮的转化，表明细胞色素P450C11A1（CYP11A1）和17β-羟基类固醇脱氢酶（HSD17β）的活性丧失。细胞毒性和台盼蓝试验或DNA片段化分析证实，细胞存活未受影响。总之，我们的数据表明，HBCDD抑制ATP供应，最有可能是通过降低ΔΨm实现的，并且靶向睾丸间质细胞类固醇生成途径中的多个位点。
