7,12-Dimethylbenz[a]anthracene exposure induces the DNA repair response in neonatal rat ovaries
7,12-Dimethylbenz[a]anthracene exposure induces the DNA repair response in neonatal rat ovaries
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DOI：
10.1016/j.taap.2013.08.013
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发表时间：
2013-11-01
影响因子：
3.8
通讯作者：
Keating, Aileen F.
中科院分区：
文献类型：
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作者：
Ganesan, Shanthi;Bhattacharya, Poulomi;Keating, Aileen F.
7，12-二甲基苯并[a]蒽（DMBA）在所有发育阶段都会破坏卵泡。本研究探讨DMBA诱导的DNA双链断裂（DSB）的形成，随后激活的卵巢DNA修复反应的模型，窦前或排卵前卵泡损失。将出生后第4天（PND）的Fisher 344（F344）大鼠卵巢培养4天，然后单次暴露于溶媒对照（1%DMSO）或DMBA（12.5 nM或75 nM），并在培养物中维持4或8天。或者，在培养开始时将PND 4 F344大鼠卵巢暴露于1 μ M DMBA 2天。分离总RNA或蛋白质，然后进行qPCR或Western印迹以分别定量mRNA或蛋白质水平。使用免疫荧光染色定位和定量γ H2 AX和磷酸化ATM。DMBA暴露增加了caspase 3和gamma H2 AX蛋白。此外，DMBA（12.5 nM和1 μ M）增加了编码Atm、Xrcc 6、Brca 1和Rad 51的mRNA水平。DMBA处理第4天，PARP 1 mRNA表达下降，第8天，PARP 1蛋白表达上升。总ATM以浓度依赖性时间模式增加（75 nM d4; 12.5 nM d8），而pATM定位于大的初级和次级卵泡中，与对照和12.5 nM DMBA相比，75 nM DMBA暴露8天后增加。这些发现支持，尽管有一些浓度效应，DMBA诱导卵巢DNA损伤和DNA修复机制诱导作为一种潜在的机制，以防止卵泡损失。(C)2013年由Elsevier Inc.出版
7,12-Dimethylbenz[a]anthracene (DMBA) destroys ovarian follicles at all stages of development. This study investigated DMBA-induced DNA double strand break (DSB) formation with subsequent activation of the ovarian DNA repair response in models of pre-antral or pre-ovulatory follicle loss. Postnatal day (PND) 4 Fisher 344 (F344) rat ovaries were cultured for 4 days followed by single exposures of vehicle control (1% DMSO) or DMBA (12.5 nM or 75 nM) and maintained in culture for 4 or 8 days. Alternately, PND4 F344 rat ovaries were exposed to 1 mu M DMBA at the start of culture for 2 days. Total RNA or protein was isolated, followed by qPCR or Western blotting to quantify mRNA or protein level, respectively. gamma H2AX and phosphorylated ATM were localized and quantified using immunofluorescence staining. DMBA exposure increased caspase 3 and gamma H2AX protein. Additionally, DMBA (12.5 nM and 1 mu M) increased levels of mRNA encoding Atm, Xrcc6, Brca1 and Rad51. In contrast, Parp1 mRNA was decreased on d4 and increased on d8 of DMBA exposure, while PARP1 protein increased after 8 days of DMBA exposure. Total ATM increased in a concentration-dependent temporal pattern (75 nM d4; 12.5 nM d8), while pATM was localized in large primary and secondary follicles and increased after 8 days of 75 nM DMBA exposure compared to both control and 12.5 nM DMBA. These findings support that, despite some concentration effects, DMBA induces ovarian DNA damage and that DNA repair mechanisms are induced as a potential mechanism to prevent follicle loss. (C) 2013 Published by Elsevier Inc.