Kostka Grażyna, Urbanek-Olejnik Katarzyna, Liszewska Monika, Winczura Alicja
Department of Toxicology and Risk Assessment, National Institute of Public Health-National Institute of Hygiene, Chocimska 24, 00-791, Warsaw, Poland.
Department of Molecular Biology, Institute of Biochemistry and Biophysics Polish Academy of Sciences, Pawińskiego 5a, Warsaw, Poland.
Environ Toxicol. 2016 May;31(5):584-92. doi: 10.1002/tox.22071. Epub 2014 Nov 20.
The aim of the study was to investigate the early effect of acute dichlorodiphenyltrichloroethane (DDT) exposure on the methylation status of the promoter region of two tumor suppressor genes: p53 and p16(INK4a) (p16) in rat liver. We analyzed their transcript and protein expression profiles concurrently with the examination of transcriptional and protein expression levels of DNA (cytosine-5)-methyltransferase 1 (Dnmt1). Male Wistar rats were treated with a single dose of DDT (57 mg kg(-1) of body weight) and the methylation status of p53 and p16 genes was examined after 24 h using methylation-sensitive restriction analysis-MSRA. The obtained results indicate that DDT induced alternations in methylation of the promoter region in both p53 and p16 genes. In all the tested samples, the promoter CpG islands of p53 (-261, -179, and -450) were methylated within 100% as compared to control samples (0%). The methylation status of the p16 promoter (-11 and +77) was also altered due to exposure to DDT. Methylated cytosines were detectable in 75% of the tested DNA samples. The Real-time PCR and western blot analyses showed a decrease in mRNA and protein levels of p53, respectively, which was related to the increase in DNA synthesis. These relationships were also observed for mRNA and protein expressions of p16, although to a slighter extent. We also showed that hypermethylation in the promoter region of both tumor suppressor genes was consistent with an increased Dnmt1 mRNA level, and this relationship was further confirmed at the protein level of DNMT1. Concluding, our data suggests that epigenetically mediated changes in gene expression may play an important role in the mechanism of DDT toxicity, including carcinogenic action.
本研究的目的是调查急性接触二氯二苯三氯乙烷（DDT）对大鼠肝脏中两个肿瘤抑制基因p53和p16（INK4a）（p16）启动子区域甲基化状态的早期影响。我们在分析它们的转录本和蛋白质表达谱的同时，检测了DNA（胞嘧啶-5）-甲基转移酶1（Dnmt1）的转录和蛋白质表达水平。给雄性Wistar大鼠单次注射DDT（57 mg kg⁻¹体重），24小时后使用甲基化敏感限制性分析-MSRA检测p53和p16基因的甲基化状态。所得结果表明，DDT诱导了p53和p16基因启动子区域甲基化的改变。在所有测试样本中，与对照样本（0%）相比，p53（-261、-179和-450）的启动子CpG岛100%发生了甲基化。由于接触DDT，p16启动子（-11和+77）的甲基化状态也发生了改变。在75%的测试DNA样本中可检测到甲基化的胞嘧啶。实时PCR和蛋白质印迹分析表明，p53的mRNA和蛋白质水平分别降低，这与DNA合成的增加有关。p16的mRNA和蛋白质表达也观察到了这些关系，尽管程度较轻。我们还表明，两个肿瘤抑制基因启动子区域的高甲基化与Dnmt1 mRNA水平的增加一致，并且这种关系在DNMT1的蛋白质水平上得到了进一步证实。总之，我们的数据表明，基因表达的表观遗传介导变化可能在DDT毒性机制（包括致癌作用）中起重要作用。
