Zamir-Nasta Toraj, Razi Mazdak, Shapour Hasanzadeh, Malekinejad Hassan
Department of Basic Science, Faculty of Veterinary Medicine, Urmia University, P.O. Box 1177, Urmia, Iran.
Department of Pharmacology and Toxicology, Faculty of Pharmacy, Urmia University of Medical Sciences, Urmia, Iran.
Environ Toxicol. 2018 Apr;33(4):385-395. doi: 10.1002/tox.22524. Epub 2017 Dec 23.
This study was done in order to investigate time-dependent effect of AFB1 on expression of genes involving in cell cycle check point machinery at G, S, and M phases. For this purpose, 24 mature male Swiss albino mice were randomly divided into control and test groups. The animals in test group subdivided into three groups, which received the AFB1 at a daily dose of 20 µg/kg body weight, through intraperitoneal (i.p.) route, for 7, 14, and 21 days. The p21, p53, cyclin D1, CDK4, and ERα expressions at both mRNA and protein level were analyzed by using reverse transcription PCR (RT-PCR) and immunohistochemistry, respectively. Moreover, the tubular differentiation (TDI) and spermiogenesis (SPI) indices were analyzed. Finally, the testicular DNA fragmentation was assessed by using DNA Ladder test. Observations revealed that the AFB1 remarkably (P < .05) reduced cyclin D1, Cdk4, and ERα expression at both mRNA and protein levels. Up-regulated p21 and p53 expression was revealed in AFB1-received animals, which developed time dependently. Histological examinations exhibited a significant reduction in TDI and SPI indices. Finally, the AFB1 resulted in severe DNA fragmentation. Our data showed that the AFB1 by down-regulating the cyclin D1, Cdk4, and ERα expression adversely affects cyclin D1/Cdk4 and cyclin D1/ERα interactions. Moreover, the AFB1-induced overexpression of p21 (as a kinase inhibitor), in turn results in cell cycle arrest via inhibiting the Cdk4 interaction with cyclin D1. Finally, the AFB1-induced DNA damage triggers the p53-dependent apoptosis pathway independent to p21 overexpression.
本研究旨在探讨黄曲霉毒素B1（AFB1）对细胞周期G、S和M期检查点机制相关基因表达的时间依赖性影响。为此，将24只成熟雄性瑞士白化小鼠随机分为对照组和试验组。试验组动物再分为三组，通过腹腔注射途径，每天按20 μg/kg体重的剂量给予AFB1，持续7、14和21天。分别采用逆转录聚合酶链反应（RT-PCR）和免疫组织化学方法分析p21、p53、细胞周期蛋白D1、细胞周期蛋白依赖性激酶4（CDK4）和雌激素受体α（ERα）在mRNA和蛋白水平的表达。此外，分析了生精小管分化（TDI）和精子发生（SPI）指数。最后，采用DNA Ladder试验评估睾丸DNA片段化情况。观察结果显示，AFB1在mRNA和蛋白水平均显著（P＜0.05）降低了细胞周期蛋白D1、CDK4和ERα的表达。在接受AFB1的动物中发现p21和p53表达上调，且呈时间依赖性发展。组织学检查显示TDI和SPI指数显著降低。最后，AFB1导致严重的DNA片段化。我们的数据表明，AFB1通过下调细胞周期蛋白D1、CDK4和ERα的表达，对细胞周期蛋白D1/CDK4和细胞周期蛋白D1/ERα相互作用产生不利影响。此外，AFB1诱导的p21（作为一种激酶抑制剂）过表达，进而通过抑制CDK4与细胞周期蛋白D1的相互作用导致细胞周期停滞。最后，AFB1诱导的DNA损伤触发了独立于p21过表达的p53依赖性凋亡途径。
