新生SD大鼠联合暴露苯并芘和多氯联苯对血清睾酮水平的影响及表观遗传机制对睾酮合成酶的调控作用

发布时间：2018-05-25 01:10

  本文选题：苯并芘 + 多氯联苯　； 参考：《复旦大学》2012年硕士论文

【摘要】：苯并芘(Benzo(a)pyrene;3,4-Benzypyrene; BaP)是一种多环芳香族碳氢化合物,它主要存在于煤焦油,汽车尾气,各类有机物产生的烟雾,烧烤的食物以及工业污水中。BaP具有雄性生殖毒性,它可以引起睾丸萎缩、精子数量及活动能力下降,降低曲精小管的长度和重量以及减少血浆和睾丸内的睾酮水平。多氯联苯(Polychlorinated biphenyls, PCBs)是一类人工合成的工业有机化合物,被列为12种最为危险的持续性有机污染物(Persistent organic pollutants, POPs)之一。PCBs的雄性生殖毒性和内分泌干扰效应已被大量研究证实。BaP和PCBs经常共存于环境中,且在体外实验中PCB126可通过增强BaP的氧化损伤毒性而表现出与BaP作用的协同效应。与PCB126同为二恶英类多氯联苯的PCB169毒性略低于PCB126,我们的研究假设PCB169与BaP联合暴露具有协同效应。 在BaP的毒作用机制中,BaP进入细胞微粒体中代谢形成环氧化苯并芘可与DNA共价结合形成DNA加合物可造成DNA损伤,而形成DNA加合物的靶点往往位于DNA的甲基化区域；另外,也有不少研究证实,BaP暴露可引起DNA甲基化和组蛋白乙酰化的改变。所以,表观遗传修饰的改变很可能是BaP生殖毒性机制的重要组成部分。 新生期是甲基化形成和维持的关键时期,也是机体受化学物干扰的敏感期。因此,本研究以新生SD大鼠联合暴露BaP和PCB169为模型,评价两种化学物的近期和远期联合生殖毒效应,同时探索表观遗传机制在BaP致生殖功能损伤中的作用。 出生当天(Postnatal Day0, PNDO),将所有雄性SD大鼠混合后,重新分成10只/窝(组内、组间体重变异小于10%)。在PND1,按窝别随机分成对照和处理组,每组30只。从PND1开始连续7天,对照和处理组分别经口给予等量的溶剂对照；或BaP5、10和25mg/kg BW;或PCB1690.25mg/kg BW,或PCB1690.25mg/kg联合BaP5、10和25mg/kg BW.各组大鼠分别于染毒结束后24h(PND8)、青春期(PND35)和成年期(PND90),经麻醉后处死,收集并保存血液、肝脏、睾丸及附睾。 结果一新生期大鼠BaP和PCB169联合暴露影响成年精子数量及血清睾酮水平 BaP暴露后,到PND90,每日精子生成量在BaP10mg/kg,25mg/kg下降；PCB1690.25mg/kg暴露亦导致PND90的每日精子生成量降低；在BaP和PCB169联合暴露的各个剂量组,每日精子生成量显著降低。 BaP暴露后,在PND8和PND35, BaP10,25mg/kg剂量组引起血清中T水平降低,在PND90,仅BaP25mg/kg剂量抑制血清T水平;PCB1690.25mg/kg暴露在PND8, PND35和PND90均未引起血清T水平的变化；BaP和PCB169联合暴露后,在PND8和PND35,各个剂量组的血清T水平均显著降低,而在PND90, PCB169联合BaP10mg/kg和PCB169联合BaP25mg/kg剂量组血清T水平受到抑制。 BaP和PCB169对成年期精子数量和发育各阶段血清睾酮水平的影响无协同作用。 结果二新生期大鼠BaP和PCB169暴露干扰Leydig cells中类固醇合成酶的表达 新生期BaP暴露后,在PND8和PND35, StARmRNA表达在BaP10mg/kg、25mg/kg剂量组下降,在PND90仅在BaP25mg/kg表达降低；17β-HSD mRNA的表达水平仅在PND8的BaP25mg/kg剂量组降低,在PND35和PND90未发生变化；BaP10mg/kg、25mg/kg剂量在PND8显著降低P450c17mRNA水平,各剂量在PND35和PND90未引起P450cl7mRNA变化；P450scc,17β-HSD和AR的mRNA表达在PND8, PND35和PND90均无变化。 PCB1690.25mg/kg暴露后,StAR mRNA表达在PND8和PND35被抑制,在PND90无变化；P450scc、3p-HSD、P450c17、AR的mRNA表达水平在PND8下降,在PND35和PND90均未发生改变；17β-HSD的mRNA表达在PND8,PND35和PND90均无变化。 BaP和PCB169联合暴露后,在PND8, StAR mRNA表达在所有剂量组均降低,在PND35和PND90仅在PCB169联合BaP10mg/kg及PCB169联合BaP25mg/kg剂量组降低；P450scc和P450c17的mRNA在PND8的各剂量组表达下降,在PND35仅PCB169联合BaP25mg/kg剂量组被抑制,在PND90无变化；3p-HSD、17β-HSD及AR的mRNA表达在PND8所有剂量组下降,在PND35和PND90均未受影响。 BaP和PCB169仅在PND8对StAR和P450c17mRNA表达的影响具有协同作用,对其他类固醇合成酶表达的抑制在各个发育阶段均无协同效应。 结果三新生期大鼠BaP和PCB169暴露对StAR, P450c17及AR基因表观遗传修饰的影响 BaP暴露后,StAR启动子组蛋白H3K14的乙酰化水平在PND8, PND35和PND90的各剂量组均发生下降；StAR启动子组蛋白H3的乙酰化水平仅在PND8的BaP25mg/kg剂量下降,在PND35和PND90无变化；P450c17和AR启动子甲基化状态PND8, PND35和PND90未发生改变。 PCB1690.25mg/kg暴露后,StAR组蛋白H3K14和H3乙酰化水平及AR启动子甲基化水平在PND8, PND35和PND90均无影响；P450c17启动子Aval位点的甲基化水平在PND8升高,在PND35和PND90未发生变化。 BaP和PCB169联合暴露后,StAR启动子组蛋白H3K14的乙酰化水平在PND8的各个剂量被显著抑制,在PND35的PCB169联合BaP1Omg/kg和PCB169联合BaP25mg/kg剂量组降低,在PND90的PCB169联合BaP25mg/kg剂量组下降；P450c17启动子的甲基化水平在PND8降低,在PND35和PND90无变化；StAR组蛋白H3乙酰化水平和AR启动子的甲基化水平在发育各时段各剂量组均未发生改变。 BaP和PCB169的协同作用仅体现在PND8对StAR启动子组蛋白H3K14乙酰化的抑制方面。 结论 新生期大鼠BaP暴露可抑制睾丸间质细胞中睾酮合成限速酶系的mRNA表达,在发育过程中可通过诱导StAR mRNA表达的持续下降,来持续抑制血清中的T水平,进而在成年期导致精子生成能力降低。BaP对StAR基因启动子组蛋白H3K14乙酰化水平的持续抑制是StAR mRNA表达持续下降的原因之一。BaP与PCB169联合暴露可在比BaP单独暴露更低的剂量下产生明显的毒效应,但两种化学物的协同效应不明显。
[Abstract]:Benzopyrene (Benzo (a) pyrene; 3,4-Benzypyrene; BaP) is a polycyclic aromatic hydrocarbon, which mainly exists in coal tar, automobile exhaust, smoke produced by various kinds of organic substances, the roasted food and the male reproductive toxicity of.BaP in industrial sewage. It can cause the testicular atrophy, the decrease of sperm quantity and activity, and reduce the essence of semen. The length and weight of the tubules and the reduction of testosterone levels in plasma and testicles. Polychlorinated biphenyls (PCBs) is a synthetic industrial organic compound, which is listed as the male reproductive toxicity and endocrine stem of the 12 most dangerous persistent organic pollutants (Persistent organic pollutants, POPs).PCBs. The disturbing effect has been confirmed by a large number of studies that.BaP and PCBs often coexist in the environment, and in vitro PCB126 can exhibit synergistic effects with BaP by enhancing the oxidative damage toxicity of BaP. The PCB169 toxicity of PCB126 and dioxin polychlorinated biphenyls is slightly lower than PCB126. Our study assumes that PCB169 and BaP are associated with CO exposure. The same effect.
In the toxic mechanism of BaP, BaP enters the cell microsomes to form the epoxidation of benzopyrene and can covalent and combine with DNA to form DNA adducts to cause DNA damage. The target of DNA adducts is often located in the methylation area of DNA. In addition, there are many studies confirmed that BaP storm dew can cause the modification of DNA methylation and histone acetylation. Therefore, epigenetic modification is likely to be an important part of the reproductive toxicity mechanism of BaP.
The new stage is a critical period for the formation and maintenance of methylation. It is also a sensitive period for the organism to be disturbed by chemical substances. Therefore, in this study, the combined exposure of BaP and PCB169 in newborn SD rats was used to evaluate the short-term and long-term combined effects of the two chemicals, and the effect of epigenetic mechanism on the damage of reproductive function caused by BaP was also explored.
On the day of birth (Postnatal Day0, PNDO), all male SD rats were mixed and divided into 10 / nests (within the group, the body weight variation was less than 10%). In PND1, each group was randomly divided into control and treatment groups according to the nests, 30 in each group. For 7 days from PND1, the control and treatment groups were given the same amount of solvent control; or BaP5,10 and 25mg/kg BW; or PC. B1690.25mg/kg BW, or PCB1690.25mg/kg combined with BaP5,10 and 25mg/kg BW. rats were treated with 24h (PND8), puberty (PND35) and adulthood (PND90) after the end of the poisoning. After anesthesia, the rats were sacrificed to collect and preserve the blood, liver, testis and epididymis.
Results the combined exposure of BaP and PCB169 affects the number of adult sperm and serum testosterone.
After BaP exposure, to PND90, the daily sperm production was reduced in BaP10mg/kg, 25mg/kg, and PCB1690.25mg/kg exposure also led to a decrease in the daily sperm production of PND90, and the daily sperm production decreased significantly in each dose group of BaP and PCB169 exposure.
After BaP exposure, the level of serum T decreased in the dose group of PND8 and PND35, and in PND90, the serum T level was inhibited by BaP25mg/kg dose only. PCB1690.25mg/kg exposure in PND8, PND35 and PND90 did not cause changes in serum levels. However, serum T levels were inhibited in PND90, PCB169 combined with BaP10mg/kg and PCB169 combined with BaP25mg/kg dose group.
BaP and PCB169 had no synergistic effect on the number of adult sperm and the serum testosterone levels at various stages of development.
Results two BaP and PCB169 exposure in neonatal rats interfered with the expression of steroid synthase in Leydig cells.
After BaP exposure, in PND8 and PND35, the expression of StARmRNA in BaP10mg/kg, 25mg/kg dose group decreased and the expression of PND90 only decreased in BaP25mg/kg, and the expression level of 17 beta -HSD mRNA decreased only in PND8 BaP25mg/kg dose group. The amount of PND35 and PND90 did not cause P450cl7mRNA change; mRNA expression of P450scc, 17 beta -HSD and AR did not change in PND8, PND35 and PND90.
After PCB1690.25mg/kg exposure, the expression of StAR mRNA was suppressed in PND8 and PND35, and there was no change in PND90; the mRNA expression level of P450scc, 3p-HSD, P450c17, AR was decreased in PND8 and not changed.
After the combined exposure of BaP and PCB169, the expression of StAR mRNA decreased in all dose groups and decreased in PND35 and PND90 only in PCB169 combined BaP10mg/kg and PCB169 combined BaP25mg/kg dose group, and the expression of P450scc and StAR decreased in each dose group. 3 P-HSD, mRNA expression of 17 beta -HSD and AR decreased in all dose groups of PND8, but not in PND35 and PND90.
BaP and PCB169 have synergistic effects only on the effects of PND8 on the expression of StAR and P450c17mRNA, and there is no synergistic effect on the inhibition of the expression of other steroid synthetases at various developmental stages.
Results the effects of BaP and PCB169 exposure on epigenetic modification of StAR, P450c17 and AR genes in three new born rats
After BaP exposure, the acetylation level of the StAR promoter H3K14 was decreased in all the doses of PND8, PND35 and PND90, and the acetylation level of the StAR promoter protein H3 was only decreased in the PND8 BaP25mg/kg dose, and there was no change in PND35 and PND90.
After PCB1690.25mg/kg exposure, the level of StAR histone H3K14 and H3 acetylation and the methylation level of AR promoter had no effect on PND8, PND35 and PND90, and the level of methylation at the Aval loci of P450c17 promoter increased in PND8, and did not change at the PND35 and the Aval.
After the combined exposure of BaP and PCB169, the level of the acetylation of the StAR promoter protein H3K14 was significantly inhibited at every dose of PND8, and decreased in the PND35 PCB169 combined BaP1Omg/kg and PCB169 joint BaP25mg/kg dose group, and decreased in the PND90 PCB169 joint dose group. 0 there was no change; StAR histone H3 acetylation level and AR promoter methylation level did not change in all dose groups at all stages of development.
The synergistic effect of BaP and PCB169 is only reflected in the inhibition of PND8 on StAR promoter histone H3K14 acetylation.
conclusion
BaP exposure in neonatal rats can inhibit the mRNA expression of testosterone synthesis rate limiting enzyme system in Leydig cells. In the process of development, it can continue to inhibit the T level in serum by inducing the continuous decline of StAR mRNA expression, and then in adulthood, spermatogenesis can be reduced by.BaP to the level of H3K14 acetylation of the StAR gene promoter. Persistent inhibition is one of the reasons for the continuous decline in the expression of StAR mRNA. The combined exposure of.BaP and PCB169 can produce significant toxic effects at a lower dose than BaP, but the synergistic effect of the two chemicals is not obvious.
【学位授予单位】：复旦大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R114
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本文编号：1931393