Study On The Role And Molecular Mechanisms Of CYP3A1in BDE47-induced Testicular Toxicity In Rats

Polybrominated diphenyl ethers (PBDEs) are a class of flameretardant chemicals frequently applied to textiles, furniture, and electronic and electrical items. Currently, PBDEs are recognized as environmental pollutants of global concern because their levels in the environment and in humans have increased markedly over the past several decades.2,2’,4,4’-tetrabromodiphenyl ether (BDE47) is the dominant PBDE congener in humans, wildlife, and the environment. In animals, BDE47has been identified as a developmental, reproductive, and neurological toxicant and disrupter of multiple endocrine systems. Semen mobility was negatively related to BDE47concentration in human blood plasma. Biotransformation is an important process in vivo for xenobiotics, especially lipophilic chemicals. BDE47could be metabolized to many metabolites mediated by (Cytochrome P450, CYP450), especially CYP3A1. However, the relationship between BDE47and male reproductive dysfunction and realted molecular mechanisms are still unclear.In this study, dexamethasone (DEX), a known inducer of CYP3A1, was used to evaluate the effect of BDE47on CYP3A1induction. The activtity of CYP3A1and the metabolites of BDE47were investigated. To explore the role of CYP3A1in BDE47-induced testicular toxicity, we analysed the morphological changes and apoptosis of germ cells on testicular seminiferous tubule epithelial, and then the key signaling pathway for apoptosis of germ cells was estimated. In addition, the animal reproductive disruption model was esatablished via administration of BDE47by oral to adult male SD rats on high fat diet (HFD). The effect of HFD on the distribution and metabolism of BDE47in vivo was assessed. To reveal the relationship between dysfunction of glycolipid metabolism and testicular toxicity and related molecular mechanism, the effects of HFD or BDE47on the blood glucose, insulin and testesterone were evaluated, the effects of BDE47or HFD on the expression of CYP3A1. The results will help provide insights into the role of biotransformation or endocrine disruptting in environmental toxicity and provide new clues to the prevention and control of environmental pollutant-induced biological damages. Part Ⅰ:Effects and mechanisms of CYP3A1induction onthe testicular toxicity of BDE47in male rats Obiective:To explore the role of Cytochrome P4503A1(CYP3A1) in the formation of oxidative metabolites of BDE47and its induced testicular toxicity was investigated in SD rats. Methods:The animal reproductive disruption model was esatablished via administration of BDE47by oral to adult male SD rats. Dexamethasone (DEX), an inducer of CYP3A1expression, was used to be a tool to confirm the metabolic activation of CYP3A1to BDE47. The expression and activity of hepatic CYP3A1, and the hydroxylated metabolites of BDE47in plasma, liver and testis were dectected by gas chromatography-mass spectrometry (GC-MS). Flow cytometry assay was used to compare apoptosis induced by BDE47or3-OH-BDE47. In addition, the testicular pathological observation, reactive oxygen species (ROS), apoptosis, spermatogenic processes, as well as the expression of apoptosis-related proteins were evaluated.Results:BDE47significantly increased the expression and activity of CYP3A1in rat liver, and3-OH-BDE47, the major oxidative metabolite of BDE47, dose-dependently increased in rat liver, serum, and testis, which was aggravated by DEX, an inducer of CYP3A1. Additionally, testicular3-OH-BDE47and ROS in seminiferous tubules increased especially when BDE47was administered in combination with DEX, which was confirmed in GC-1and GC-2cells that3-OH-BDE47induced more ROS production an cell apoptosis via the upregulation of FAS/FASL, p-p53and caspase3, As a result, daily sperm production dose-dependently decreased, consistent with histological observations in giant cells and vacuolar spaces and increase in TUNEL-positive apoptotic germ cells, which was aggravated by DEX.Part Ⅱ:Effects and mechanisms of CYP3A1in BDE47-induced testicular toxicity in male rats on a high fat dietObiective:To invstigate the effects of HFD on the induction of CYP3A1mediated by BDE47and its distribution and metabolism, explore the role of CYP3A1in BDE47-induced dysfunction of glycolipid metabolism and testicular toxicity and related molecular mechanism.Methods:The animal reproductive disruption model was esatablished via administration of BDE47by oral to adult male SD rats on a normal diet or high fat diet. The expression of hepatic CYP3A1, and the hydroxylated metabolites of BDE47in plasma, liver and testis were dectected by gas chromatography-mass spectrometry (GC-MS). The effects of BDE47on metabolism of glucose and lipid were evaluted by the levles of glucose, insulin and testestone in rat serum. The level of serum testesterone was detected by radioimmunoassay. The changes of testicular morphology and concentration of sperm in the epididymis were evaluated by light microscopy. The expression of glucose transporters, insulin signaling and steroidogenesis related proteins in the rat testis were evaluted by immunbloting.Results:HFD could promote the accumulation of BDE47in the rats, thus significantly increased its metabolites in the target organs. In addition, BDE47could induce the expression of CYP3A1, which was enhanced by HDF in the examination of immunochemistry. On a normal deit, the exposure of BDE47resulted in elevated serum glucose and insulin, liver cells steatsis. In addtion, the exposure of BDE47resulted in decreased in serum testesterone, giant cells and vacuolar spaces in the seminiferous epithelium and reduced sperm concentration in the epididymis. BDE47could upregulate the expreesion of hepatic TNFa, which might contribute to insulin resistance. BDE47could induce the expression of DAX-1and inhibit the exprssion of GLUT-2, GLUT-8, IRβ, StAR and3β-HSD. All these effects were aggravated after BDE47exposure on a high fat diet. Insulin-mediated induction of DAX-1in Leydig cells of testis may be a key regulatory step of serum sex hormone level in insulin-resistant states.ConclusionTo explore the effects of BDE47on reproductive endocrine and molecular mechanism, the biotransformation and endocrine disrupting of BDE47were evaluated by establishing male SD rat models with BDE47exposure combinated with DEX or a high fat diet. Many new results were observed as follows:1. BDE47could induce the exprssion of CYP3A1, DEX could enhance this induction. CYP3A1was a major enzyme invovled the metabolism of BDE47, and3-OH-BDE47was its major active metabolite.2. Exposure of BDE47resulted in reduces testesterone in rat serum, increased ROS, apoptotic germ cells in the seminiferous epithelium, which was aggravated by DEX. The results suggested that CYP3A1played an important role in BDE47-induced reproductive dysfunction in adult rats.3. HFD could enhance the induction of CYP3A1mediated by BDE47. It could not only promote the production of hydroxylated metabolites of BDE47, but also aggravate the dysfunction of glycolipid metabolism. CYP3A1should mediate the combined effects of BDE47and HDF in the testicular toxicity of male rats.4. BDE47could be metabolized by CYP3A1to product the active metabolite3-OH-BDE47which could damage the blood-testis barrier to let more active metabolite enter into the testis, and subsequently induced germ cell apoptosis via the upregulation of FAS/FASL, p-p53and cleaved-caspase3. In addition, the hyperinsulinemia induced by BDE47could upregulate the expression DAX-1which lead to inhibit the expression of StAR and3β-HSD, resulting in the decreased serum testesterone and sperm concentration. Both direct and indirect effects may contribute to BDE47-induced testicular toxicity.