组蛋白H4第20位赖氨酸甲基化修饰与燃煤砷暴露人群DNA损伤修复的关系

Correlation between histone 4 lysine 20 methylation and DNA damage-repair in coal-burning-borne arsenic-exposed residents

目的观察贵州省燃煤污染型砷中毒病区砷暴露者外周血细胞组蛋白H4第20位赖氨酸（H4K20）甲基化修饰水平及其与血细胞DNA损伤的关系，为揭示砷抑制DNA损伤修复机制研究提供依据。方法以贵州省兴仁县燃煤型砷中毒病区交乐村为调查点，通过健康体检，选择115例砷暴露者为砷暴露组；同时选择相邻的非病区上坝田村53例居民作为对照组。采集观察对象发样、外周血，提取血细胞组蛋白；微波消解-电感耦合等离子质谱（ICP—MS）法测定发样中砷元素的含量；酶联免疫吸附实验（ELISA）检测外周血细胞组蛋白H4K20一、二、三甲基化（H4K20me1、me2、me3）修饰水平；单细胞凝胶电泳（SCGE）法检测DNA损伤。结果发砷含量测定结果：与对照组[中位数（M，四分位数）：0．12（0．08～0．18）μg／g]比较，砷暴露组发砷[M（四分位数）：0．30（0．19-0．46）μg／g]含量明显升高（F=11．968，P〈0．05）。H4K20甲基化修饰水平检测结果：与对照组（0．44±0．14、0．99±0．41、1．06±0．33）比较，砷暴露组H4K20mel修饰水平（0．60±0．29）升高（F=2．513，P〈0．05）、H4K20me2修饰水平（0．75±0．26）降低（F=4．707，P〈0．05）、H4K20me3修饰水平（1．20±0．62）变化不明显（F=0．582，P〉0．05）。DNA损伤检测结果：与对照组[M：2．12、1．16]比较，砷暴露组尾部DNA百分含量（TailDNA％，M：10．75）、Olive尾矩（Olivetailmoment，M：11．69）均明显升高（F=9．307、9．457，P均〈0．05）。相关性分析：观察对象H4K20me1修饰水平与发砷含量呈正相关r=0．214，P〈0．05），H4K20me2修饰水平与其呈负相关关系（r=-0．224，P〈0．05）；H4K20me1修饰水平与DNA损伤水平（TailDNA％、Olivetailmoment）均呈正相关关系（r=0．383、0．380，P均〈0．05），H4K20me2修饰水平与其均呈负相关关系（r=-0．290、-0．298，P均〈0．05）。结论H4K20甲基化修饰响应机体砷暴露，H4K20me1、H4K20me2修饰改变可能参与调控砷暴露对机体造成的DNA损伤修复过程。

Objective To detect the global level of histone 4 lysine 20 （H4K20） methylation and its relation with DNA damage-repair in peripheral blood cell of arsenic-exposed residents in the coal-contaminated arsenism areas in Guizhou, in order to provide a basis to deepen the interpretation of the role of arsenic in inhibiting DNA damage-repair. Methods Jiaole village in coal-burning-borne arsenism areas in Xingren County of Guizhou was selected as the survey point, and 115 cases of arsenic-exposed residents were selected as the arsenic exposed group on the basis of physical examination. Moreover, 53 residents from one village of non-epidemic area neighboring the diseased area were selected as controls. Hair and peripheral blood samples of these subjects were collected, and the histone protein was extracted from the lymphocytes separated from blood samples. The hair samples were digested with microwave digestion instrument, and the hair arsenic content was tested via the inductively coupled plasma-mass spectrometry （ICP-MS） method; the level of H4K20 1, 2, 3 methylation （H4K20me2, me2, me3） in peripheral blood cell was tested by enzyme-linked immunosorbent assay （ELISA）; DNA damage of peripheral blood cell was measured by single cell gel electrophoresis （SCGE）. Results The testing results of hair arsenic contents showed that the arsenic levels of hair in arsenic exposed group [0.30 （0.19 - 0.46） μg/g] were significantly higher than those of control group [0.12 （0.08 - 0.18） μg/g, F = 11.968, P 〈 0.05]. Compared with the eontrol （0.44 ± 0.14, 0.99 ± 0.41, 1.06 ± 0.33）, the level of H4K20mel （0.60 ± 0.29） in arsenic exposed group was higher （F = 2.513, P 〈 0.05）, H4K20me2 （0.75± 0.26） was lower （F = 4.707, P 〈 0.05）, and H4K20me3 （1.20± 0.62） was of no significant difference （F = 0.582, P 〉 0.05）. The detecting results of DNA damage of the lymphocytes separated from peripheral blood showed a statistically significant increase （F = 9.307, 9.457, all P 〈 0.05） in TailDNA% and Olivetailmoment in arsenic exposed group [Median （M）： 10.75, 11.69] compared with those of eontrol group （M： 2.12, 1.16）. The correlation analysis indicated that the arsenic levels of hair of subjects were positively correlated with H4K20mel （r = 0.214, P 〈 0.05） and inversely with H4K20me2 （r = - 0.224, P 〈 0.05）; H4K20mel was positively associated with TailDNA% （r = 0.383, P 〈 0.05） and Olivetailmoment （r = 0.380, P 〈 0.05）; H4K20me2 was inversely associated with TailDNA% （r = - 0.290, P 〈 0.05） and Olivetailmoment （r = - 0.298, P 〈 0.05）. Coneluslon H4K20 methylated modification makes a response to arsenic exposure of human body, the alteration of H4K20mel/me2 may participate in regulating DNA damage-repair induced by arsenic in vivo.