title: HBCDD-induced sustained reduction in mitochondrial membrane potential, ATP and steroidogenesis in peripubertal rat Leydig cells.
abstract: 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.
authors.affiliation: Molecular and Reproductive Toxicology Unit, Laboratory for Ecotoxicology, Department of Biology and Ecology, Faculty of Sciences, University of Novi Sad, 21000 Novi Sad, Serbia.
authors.forename: Svetlana
authors.initials: S
authors.lastname: Fa
authors.affiliation: Molecular and Reproductive Toxicology Unit, Laboratory for Ecotoxicology, Department of Biology and Ecology, Faculty of Sciences, University of Novi Sad, 21000 Novi Sad, Serbia.
authors.forename: Kristina
authors.initials: K
authors.lastname: Pogrmic-Majkic
authors.affiliation: Molecular and Reproductive Toxicology Unit, Laboratory for Ecotoxicology, Department of Biology and Ecology, Faculty of Sciences, University of Novi Sad, 21000 Novi Sad, Serbia.
authors.forename: Dragana
authors.initials: D
authors.lastname: Samardzija
authors.affiliation: Molecular and Reproductive Toxicology Unit, Laboratory for Ecotoxicology, Department of Biology and Ecology, Faculty of Sciences, University of Novi Sad, 21000 Novi Sad, Serbia.
authors.forename: Jelena
authors.initials: J
authors.lastname: Hrubik
authors.affiliation: Molecular and Reproductive Toxicology Unit, Laboratory for Ecotoxicology, Department of Biology and Ecology, Faculty of Sciences, University of Novi Sad, 21000 Novi Sad, Serbia.
authors.forename: Branka
authors.initials: B
authors.lastname: Glisic
authors.affiliation: Molecular and Reproductive Toxicology Unit, Laboratory for Ecotoxicology, Department of Biology and Ecology, Faculty of Sciences, University of Novi Sad, 21000 Novi Sad, Serbia.
authors.forename: Radmila
authors.initials: R
authors.lastname: Kovacevic
authors.affiliation: Molecular and Reproductive Toxicology Unit, Laboratory for Ecotoxicology, Department of Biology and Ecology, Faculty of Sciences, University of Novi Sad, 21000 Novi Sad, Serbia. Electronic address: nebojsa.andric@dbe.uns.ac.rs.
authors.forename: Nebojsa
authors.initials: N
authors.lastname: Andric
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism*; D000367:Age Factors; D000818:Animals; D002478:Cells, Cultured; D000242:Cyclic AMP / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D015536:Down-Regulation; D004734:Energy Metabolism / Q000187:drug effects*; D005411:Flame Retardants / Q000633:toxicity*; D015971:Gene Expression Regulation, Enzymologic; D012739:Gonadal Steroid Hormones / Q000096:biosynthesis*; D006842:Hydrocarbons, Brominated / Q000633:toxicity*; D007985:Leydig Cells / Q000187:drug effects* / Q000201:enzymology; D008297:Male; D053078:Membrane Potential, Mitochondrial / Q000187:drug effects*; D008928:Mitochondria / Q000187:drug effects* / Q000378:metabolism; D010750:Phosphoproteins / Q000235:genetics / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D017208:Rats, Wistar; D046468:Sexual Development; D013997:Time Factors; D000098682:Steroidogenic Acute Regulatory Protein
chemical_list: D005411:Flame Retardants; D012739:Gonadal Steroid Hormones; D006842:Hydrocarbons, Brominated; D010750:Phosphoproteins; D012333:RNA, Messenger; D000098682:Steroidogenic Acute Regulatory Protein; C089796:hexabromocyclododecane; D000255:Adenosine Triphosphate; D000242:Cyclic AMP
keywords: ATP; HBCDD; Leydig cell; Mitochondrial membrane potential; Steroidogenesis; cAMP