燃煤型砷中毒人群外周血淋巴细胞组蛋白H3K14乙酰化与遗传损伤关联性研究

The association between histone modification lymphocyte of coal-burning arsenicosis residents Xilan, Zhang A ihua of H3K14ac and genetic damage in peripheral blood

目的探讨燃煤型砷中毒人群外周血淋巴细胞组蛋白H3K14乙酰化（H3K14ac）修饰水平与砷中毒及遗传损伤的关系，为燃煤型砷中毒表观遗传调控机制研究提供新线索。方法以贵州省兴仁县燃煤型砷中毒病区交乐村为调查点，在健康体检基础上选择151例砷中毒患者作为砷中毒组，并按《地方性砷中毒诊断标准》（WS／T211-2001）将其分为轻度（62例）、中度（50例）和重度（39例）3个亚组；同时选择病区78例无砷中毒症状健康者作为对照组；采集全部观察对象外周血、发样及尿样。采用微波消解结合电感耦合等离子质谱检测发样中的砷含量；酸抽提法提取观察对象外周血淋巴细胞组蛋白，并用免疫酶联吸附试验（ELISA）检测组蛋白H3K14ac修饰水平；采用遗传学方法检测外周血淋巴细胞微核（MN）率和染色体畸变（CA）率；采用固相萃取法结合串联四级杆液质联用仪检测尿8-羟基脱氧鸟苷（8-OHdG）水平。结果①观察对象砷暴露水平与燃煤型砷中毒及H3K14ac关系分析结果：砷中毒组发砷[0．27（0．15～0.39）μg／g]高于对照组[0．15（0．08～0．20）μg/g，F=10．736，P〈0．01]，且砷中毒程度与发砷水平呈正相关关系（r=0．363，P〈0．05）；H3K14ac修饰水平与发砷水平呈正相关关系（r=0．385，P〈0．05）。②H3K14ac与燃煤型砷中毒关联性分析结果：砷中毒组H3K14ac修饰水平（4．07±4．03）明显高于对照组（1．62±1．19，F=19．753，P〈0．01），且H3K14ac修饰水平的升高是砷中毒的危险因素[OR（95％CI）=1．779（1．323-2．392），P〈0．01]。③H3K14ac与砷中毒程度关系分析结果：对照组和轻、中、重度砷中毒组组间H3K14ac修饰水平比较差异有统计学意义（F=7．524，P〈0．01）；与对照组比较，轻、中、重度砷中毒组H3K14ac修饰水平（3．70±3．20、4．95±5．47、3．49±2．62）均显著升高（P均〈0．01），但轻、中、重度3个砷中毒组组间其修饰水平比较，差异无统计学意义（P均〉0．05）。④观察对象遗传损伤情况：砷中毒组MN率[（2．03±1．55）‰]、CA率[（12．44±5．01）％]和尿8-OHdG水平[（3．80±3．88）μg/g肌酐]均显著高于对照组[（1．17±0．97）‰、（6．29±2．41）％、（2．33±1.34）μg／g肌酐，Wald或F值：14．121、83．164、6．116，P均〈0．05]。⑤H3K14ac与砷中毒遗传损伤关联性分析结果：H3K14ae修饰水平与人外周血淋巴细胞CA率呈正相关（β=0．84，P〈0．01），与外周血淋巴细胞MN率和尿8-OHdG水平未见显著关联（β=0．10、0．05，P均〉0．05）。结论人外周血淋巴细胞组蛋白H3K14ac修饰水平的增加是燃煤型砷中毒的危险因素，其修饰水平的改变与燃煤型砷中毒人群染色体畸变密切相关。

Objective To explore the association of H3K14 acetylation （ac） with arsenicosis induced by coal-burning and arsenic-induced genetic damage, which might help us to find an biomarker to monitor the arsenicosis and arsenic-induced toxicity. Methods Totally 151 arsenieosis subjects were recruited from Jiaole Village of Xingren County, Guizhou. According to ＂National Principle for Diagnosis of Arsenicosis＂ （WS/T 211-2001 ）, the arsenieosis group was divided into 3 subgroups： mild poisoning （n = 62）, intermediate poisoning （n = 50） and severe poisoning （n = 39）. The control group was comprised of 78 healthy villagers from Jiaole Village who were exhibited no signs of arseniasis. The hair, the urine and the peripheral blood samples were collected from the subjects. The contents of arsenic in the hair samples were analyzed with inductively coupled plasma mass spectrometry. Histones were extracted from human peripheral blood lymphocytes （PBLCs） using the method of acid extraction. The levels of H3K14ac was measured with a sandwich enzyme-linked immunosorbent assay （ELISA）. The rate of micronucleus （MN） and chromosome aberration （CA） of peripheral blood lymphocytes were examined by genetic methods. The levels of urinary 8-hydroxy-2 deoxyguanosine （8-OHdG） in all the subjects were measured with the high performance liquid lhromatography-mass spectrometry （HPLC-MS）. Results ① The association of arsenic-exposure with arsenicosis induced by coal-burning and H3K14ac： The levels of hair arsenic in arsenicosis group [0.27 （0.15 - 0.39） txg/g] was significant higher than that in control group [0.15 （0.08 - 0.20） μg/g, F = 10.736, P 〈 0.01]. The degree of arsenicosis was positive correlation with the hair-arsenic level （r = 0.363, P 〈 0.05）. The levels of H3K14ac was also positive correlation with the hair-arsenic level （r = 0.385, P 〈 0.05）. ②he association of H3K14ac and arsenicosis induced by coal-burning： The levels of H3K14ac in arsenicosis group （4.07 ±4.03） was 2.5-fold higher than that in control group （1.62 ± 1.19, F = 19.753,P 〈 0.01）. H3K14ac was a risk factor of arsenicosis,the risk of arsenicosis increased correspondingly with the levels of H3K14ac [OR （95%CI） = 1.779 （1.323 - 2.392）, P 〈 0.01].③The correlation of H3K14ac and the degree of arsenicosis： Based on the degree of arsenicosis, we found a significant difference in the levels of H3K14ac among the four groups （F = 7.524, P 〈 0.01）. Compared with the non-poisoning group （1.62 ± 1.19）, the levels of H3K14ac in mild poisoning, intermediate poisoning and severe poisoning subgroups （3.70 ±3.20, 4.95 ± 5.47, 3.49 ± 2.62） were increased （all P 〈 0.01）, but there were no significant differences in the levels of H3K14ac between the mild poisoning, intermediate poisoning and severe poisoning subgroups （P 〉 0.05）.④The genetic damage of all subjects： The rate of MN （2.03± 1.55） and CA （12.44 ± 5.01） in arsenicosis group were significantly higher than those in control group （MN： 1.17 ± 0.97, Wald = 14.121; CA： 6.29 ± 2.41, Wald = 83.164, P 〈 0.05）. Urinary 8-OHdG was increased in arsenicosis group than that in control group [（3.80 ± 3.88）, （2.33 ±1.34） μg/g Cr, F = 6.116, P 〈 0.05]. ⑤The association of H3K14ac with genetic damage： The results revealed that H3K14ac modification was positively correlated with the rate of CA （13 = 0.84, P 〈 0.01）. The level of H3K14ac was not significantly associated with the rate of MN and urinary 8-OHdG （MN： 13 = 0.10, P 〉 0.05; 8-OHdG： 13= 0.05, P 〉 0.05）. Conclusions The increase of H3K14ac modification in human peripheral blood lymphocytes is a risk factor of arsenic poisoning. Additionally, the dysregulation of H3K14ac was significant association with arsenic- induced chromosomal aberrations.