褪黑素对双酚A致成年雄性大鼠生殖细胞DNA损伤的拮抗作用及机制研究
发布时间：2018-09-04 08:06
【摘要】：目的：环境内分泌干扰物双酚A（BPA）是工业生产环氧树脂、聚碳酸酯和聚苯乙烯树脂等的前体物质。因加入BPA可以使聚碳酸酯等塑料产品变得无色透明、耐用和防摔，BPA被广泛应用于婴幼儿奶瓶、食品包装材料、塑料餐具和医疗器械等塑料行业。塑料制品的大量使用使得BPA在我们的生活中广泛存在。因BPA的化学结构与明确的人类致癌物己烯雌酚相似，BPA可能的遗传毒性已经引起了国内外学者的广泛关注。然而，关于BPA遗传毒性的研究结果仍存在争议，至今尚未得到统一的结论。目前，有关BPA对雄性大鼠生殖细胞遗传毒性的研究报道相对较少。本研究以BPA为研究对象，通过建立BPA亚急性暴露的动物实验模型探索BPA暴露对成年期SD雄性大鼠生殖细胞的遗传毒性和抗氧化剂褪黑素（MT）对BPA所致毒性效应的拮抗作用，以探讨其可能的机制。 方法：SPF级8周龄健康雄性SD大鼠，随机分为：(1)正常对照组；（2）MT处理组，，MT10mg/kg体重剂量腹腔注射；（3）BPA处理组，BPA200mg/kg体重剂量经口灌胃染毒；（4）MT预处理+BPA组，大鼠经10mg/kg的MT预处理30min后再灌胃200mg/kg剂量的BPA。每组10只动物，连续处理10天。末次给药24h后，硫代巴比妥酸法测定睾丸组织中MDA含量，黄嘌呤氧化酶法测定SOD活力；分离睾丸精母细胞，碱性彗星实验检测精母细胞DNA损伤，染色质扩散免疫染色γH2AX检测粗线期精母细胞常染色体上γH2AX阳性焦点数量；流式细胞检测睾丸细胞群中不同DNA含量亚细胞群的细胞数量；TUNEL荧光染色检测生殖细胞凋亡；睾丸组织HE染色观察睾丸组织病理形态学变化。 结果：①200mg/kg体重剂量的BPA暴露10天对SD大鼠生长无明显抑制作用，大鼠体重增量、生殖器官睾丸和附睾的重量及精子数量均未出现明显变化（P＞0.05）。 ②200mg/kg体重剂量的BPA处理SD大鼠10天引起了氧化损伤，睾丸组织中MDA含量升高， SOD活力降低（P＜0.01）。 ③BPA暴露引起了睾丸精母细胞DNA损伤。精母细胞彗星参数TL, Tail DNA%, TM和OTM与对照组比较均显著增加（P＜0.01）；SD大鼠粗线期精母细胞常染色体上γH2AX阳性焦点数量与对照组比较明显增多（P＜0.01）。 ④BPA处理显著降低了睾丸细胞群中4C-DNA含量亚细胞群的细胞数量（P＜0.05）。尚未引起生殖细胞凋亡增多和睾丸组织病理形态学的变化。 ⑤10mg/kg体重剂量的MT对SD大鼠进行预处理后减轻了氧化损伤，和BPA处理组比较睾丸组织内MDA含量降低，SOD活力升高（P＜0.05）。 ⑥MT预处理拮抗了BPA暴露引起的精母细胞DNA损伤。显著降低了DNA损伤的程度（P＜0.01）和粗线期精母细胞常染色体上γH2AX阳性焦点的数量（P＜0.05）；并增加了睾丸细胞群中4C-DNA含量亚细胞群的细胞数量，与单独的BPA处理组比较，差异有统计学意义（P＜0.05）。 结论：200mg/kg BPA亚急性暴露10天可引起大鼠精母细胞DNA损伤和生殖细胞比例的改变，但尚未引起细胞周期阻滞、生殖细胞凋亡和睾丸组织形态学变化等严重的DNA损伤不可修复的应答事件。BPA诱发的DNA损伤与睾丸组织中MDA含量升高，SOD活力降低有关。MT10mg/kg体重剂量预处理则可拮抗BPA诱发的损伤。结果提示，氧化应激可能是BPA导致生殖细胞DNA损伤的机制之一，在BPA环境和职业暴露所致遗传毒性的防治药物中，MT具有潜在的应用价值。
[Abstract]:OBJECTIVE: Bisphenol A (BPA), an environmental endocrine disruptor, is a precursor in the industrial production of epoxy resin, polycarbonate and polystyrene resins. BPA is widely used in the plastics of baby bottles, food packaging materials, plastic tableware and medical instruments because BPA can make polycarbonate and other plastic products colorless, transparent, durable and fall-proof. Material industry. The extensive use of plastic products makes BPA widespread in our lives. Due to the chemical structure of BPA is similar to the clear human carcinogen diethylstilbestrol, the possible genetic toxicity of BPA has attracted wide attention of scholars at home and abroad. However, the results of the study on the genetic toxicity of BPA are still controversial and have not yet been unified. Conclusion. At present, there are relatively few reports about the genetic toxicity of BPA on male rat germ cells. This study was conducted to explore the genetic toxicity of BPA exposure to adult SD male rats germ cells and the toxic effects of antioxidant melatonin (MT) on BPA by establishing an animal model of subacute exposure to BPA. The antagonistic effect was explored to explore its possible mechanism.
METHODS: SPF grade 8-week-old healthy male SD rats were randomly divided into: (1) normal control group; (2) MT treatment group, MT 10 mg/kg body weight dose intraperitoneal injection; (3) BPA treatment group, BPA 200 mg/kg body weight dose by oral administration; (4) MT pretreatment + BPA group, rats were pretreated with 10 mg/kg MT for 30 minutes and then gavaged with 200 mg/kg BPA. Twenty-four hours after the last administration, MDA content in testicular tissue was measured by thiobarbituric acid, SOD activity was measured by xanthine oxidase; testicular spermatocytes were isolated, DNA damage in spermatocytes was detected by alkaline comet assay, and the number of gamma H2AX positive foci on pachytene spermatocytes was detected by chromatin diffuse immunostaining (CDI). TUNEL fluorescence staining was used to detect the apoptosis of germ cells and HE staining was used to observe the pathological changes of testicular tissue.
RESULTS: 1. The growth of SD rats was not significantly inhibited after exposure to BPA at 200 mg/kg body weight for 10 days. There were no significant changes in body weight gain, testicular and epididymal weight of reproductive organs and sperm number (P > 0.05).
(2) BPA treatment with 200 mg/kg body weight caused oxidative damage in SD rats for 10 days, MDA content in testicular tissue increased and SOD activity decreased (P < 0.01).
(3) BPA exposure induced DNA damage in testicular spermatocytes. The comet parameters TL, Tail DNA, TM and OTM of spermatocytes were significantly increased compared with the control group (P < 0.01), and the number of positive focal gamma H2AX on the pachytene spermatocytes of SD rats was significantly increased compared with the control group (P < 0.01).
(4) BPA treatment significantly decreased the number of 4C-DNA subpopulations in testicular cell population (P
MT at 10 mg/kg body weight reduced oxidative damage in SD rats after pretreatment. Compared with BPA treatment group, MDA content in testicular tissue decreased and SOD activity increased (P < 0.05).
_MT pretreatment antagonized DNA damage in spermatocytes induced by BPA exposure, significantly reduced the degree of DNA damage (P < 0.01) and the number of gamma H2AX positive foci on pachytene spermatocytes (P Academic significance (P < 0.05).
CONCLUSION: Subacute exposure of 200 mg/kg BPA for 10 days can induce DNA damage in rat spermatocytes and changes in the proportions of germ cells, but it has not yet caused irreparable events such as cell cycle arrest, germ cell apoptosis and histomorphological changes of testis. The results suggest that oxidative stress may be one of the mechanisms of DNA damage in germ cells induced by BPA. MT has potential application value in the prevention and treatment of genetic toxicity induced by BPA environmental and occupational exposure.
【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2013
【分类号】：R114
[Abstract]:OBJECTIVE: Bisphenol A (BPA), an environmental endocrine disruptor, is a precursor in the industrial production of epoxy resin, polycarbonate and polystyrene resins. BPA is widely used in the plastics of baby bottles, food packaging materials, plastic tableware and medical instruments because BPA can make polycarbonate and other plastic products colorless, transparent, durable and fall-proof. Material industry. The extensive use of plastic products makes BPA widespread in our lives. Due to the chemical structure of BPA is similar to the clear human carcinogen diethylstilbestrol, the possible genetic toxicity of BPA has attracted wide attention of scholars at home and abroad. However, the results of the study on the genetic toxicity of BPA are still controversial and have not yet been unified. Conclusion. At present, there are relatively few reports about the genetic toxicity of BPA on male rat germ cells. This study was conducted to explore the genetic toxicity of BPA exposure to adult SD male rats germ cells and the toxic effects of antioxidant melatonin (MT) on BPA by establishing an animal model of subacute exposure to BPA. The antagonistic effect was explored to explore its possible mechanism.
METHODS: SPF grade 8-week-old healthy male SD rats were randomly divided into: (1) normal control group; (2) MT treatment group, MT 10 mg/kg body weight dose intraperitoneal injection; (3) BPA treatment group, BPA 200 mg/kg body weight dose by oral administration; (4) MT pretreatment + BPA group, rats were pretreated with 10 mg/kg MT for 30 minutes and then gavaged with 200 mg/kg BPA. Twenty-four hours after the last administration, MDA content in testicular tissue was measured by thiobarbituric acid, SOD activity was measured by xanthine oxidase; testicular spermatocytes were isolated, DNA damage in spermatocytes was detected by alkaline comet assay, and the number of gamma H2AX positive foci on pachytene spermatocytes was detected by chromatin diffuse immunostaining (CDI). TUNEL fluorescence staining was used to detect the apoptosis of germ cells and HE staining was used to observe the pathological changes of testicular tissue.
RESULTS: 1. The growth of SD rats was not significantly inhibited after exposure to BPA at 200 mg/kg body weight for 10 days. There were no significant changes in body weight gain, testicular and epididymal weight of reproductive organs and sperm number (P > 0.05).
(2) BPA treatment with 200 mg/kg body weight caused oxidative damage in SD rats for 10 days, MDA content in testicular tissue increased and SOD activity decreased (P < 0.01).
(3) BPA exposure induced DNA damage in testicular spermatocytes. The comet parameters TL, Tail DNA, TM and OTM of spermatocytes were significantly increased compared with the control group (P < 0.01), and the number of positive focal gamma H2AX on the pachytene spermatocytes of SD rats was significantly increased compared with the control group (P < 0.01).
(4) BPA treatment significantly decreased the number of 4C-DNA subpopulations in testicular cell population (P
MT at 10 mg/kg body weight reduced oxidative damage in SD rats after pretreatment. Compared with BPA treatment group, MDA content in testicular tissue decreased and SOD activity increased (P < 0.05).
_MT pretreatment antagonized DNA damage in spermatocytes induced by BPA exposure, significantly reduced the degree of DNA damage (P < 0.01) and the number of gamma H2AX positive foci on pachytene spermatocytes (P Academic significance (P < 0.05).
CONCLUSION: Subacute exposure of 200 mg/kg BPA for 10 days can induce DNA damage in rat spermatocytes and changes in the proportions of germ cells, but it has not yet caused irreparable events such as cell cycle arrest, germ cell apoptosis and histomorphological changes of testis. The results suggest that oxidative stress may be one of the mechanisms of DNA damage in germ cells induced by BPA. MT has potential application value in the prevention and treatment of genetic toxicity induced by BPA environmental and occupational exposure.
【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2013
【分类号】：R114
【参考文献】
相关期刊论文 前4条
1 武红娟;陈于;赵奇;王艳;刘露;王帅;;褪黑素对丙烯酰胺致大鼠睾丸细胞DNA损伤的拮抗作用[J];上海交通大学学报(医学版);2012年08期
2 逄兵,吴向东,任道风,金泰^
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