镉亚急性暴露对小鼠肝肾功能及甲基化水平的影响

Effect on Functions of Liver and Kidney and Expression of Methylation in Mice with Subacute Cadmium Exposure

[目的]初步探讨氯化镉(cadmium chloride,Cd Cl2)亚急性暴露(4周染毒)对小鼠肝肾功能、全基因组甲基化水平及去甲基化酶Tet1表达的影响。[方法]将48只SPF级KM小鼠(昆明小鼠,雌雄各半)随机分为4组:对照组(dd H2O)、Cd Cl2 17.5 mg/kg组、25 mg/kg组、35 mg/kg组。经口灌胃染毒(每天1次、每周5 d),共染毒4周,CO2麻醉法处死动物。测定肝脏和肾脏的脏器系数、血液常规及血液生化检查、肝肾组织病理学检查、全基因组DNA甲基化水平及Tet1酶表达水平。[结果]35 mg/kg染毒组小鼠活动度及精神状态较差。小鼠体重在各染毒周期和染毒剂量组间差异均有统计学意义,且存在交互作用(P<0.05)。与对照组相比,雄性小鼠肝脏质量和肝脏脏器系数在25 mg/kg和35 mg/kg剂量组差异具有统计学意义(P<0.05);血液常规检查结果显示,雌、雄性小鼠35 mg/kg染毒组血液白细胞(WBC)、血小板(PLT)、淋巴细胞(LYM)计数,均高于对照组(P<0.05);雄性小鼠在各个染毒剂量组间,WBC、PLT、LYM、RBC计数差异有统计学意义(P<0.05)。此外,血生化实验结果显示,各剂量组雄性和雌性小鼠中肝功能的主要指标谷丙转氨酶(ALT)、碱性磷酸酶(ALP)差异有统计学意义(P<0.05),肾功能的主要指标尿酸(UA)、血尿素氮(BUN)、肌酐(CR)差异均有统计学意义(P<0.05)。组织病理学分析发现25 mg/kg和35 mg/kg组雄性小鼠肝脏及肾脏呈现明显的病理性改变。Tet1 m RNA及其蛋白表达水平仅在雄性小鼠的肝脏、肾脏中表达的差异有统计学意义(P<0.05)。焦磷酸测序结果显示,雄性小鼠肝脏、肾脏甲基化水平表达差异有统计学意义(P<0.05),且其与Tet1 m RNA表达呈负相关(r=-0.473,P<0.05;r=-0.134,P<0.05)。[结论]Cd Cl2亚急性染毒可以诱导小鼠肝肾功能损伤,且对全基因组甲基化水平和去甲基化酶Tet1表达呈现出性别差异影响,雄性小鼠的肝肾组织Tet1表达参与调控全基因组DNA甲基化水平改变。

[Objective] To examine the effects on liver and kidney functions, DNA methylation, and the expression of demethylation enzyme 1（Tet1） in mice after sub acute exposure（4 weeks） to cadmium chloride. [Methods] Forty-eight SPF grade KM mice were randomly grouped into four groups： control（dd H2O）, Cd Cl2 17.5 mg/kg, 25 mg/kg, and 35 mg/kg groups. The mice were treated with cadmium chloride by gavage, once a day, five days a week, for four weeks. Then they were neutralized after CO2 anesthesia. Items to be measured included liver and kidney organ coefficients, blood routine and biochemical indicators, pathological examinations of liver and kidney tissue samples, whole genome DNA methylation levels, as well as Tet1 m RNA and protein expressions. [Results] The activity and mental state of mice were lower in the 35 mg/kg group. Significant differences in mice weights among all exposure durations and Cd Cl2 exposure doses were observed, and the interaction between exposure duration and dosage was confirmed（P〈0.05）. Compared with the control group, the liver weights and coefficients of the male mice in the 25 mg/kg and 35 mg/kg groups had significant differences（P〈0.05）. In female and male mice, the blood routine indicators such as white blood cell（WBC）, blood platelet（PLT）, and lymphocyte（LYM） had significant differences among all cadmium chloride exposed groups（P〈0.05）; in the male mice, WBC, PLT, LYM, and RBC had differences among all cadmium chloride exposed groups（P〈0.05）. Besides, as for the blood biochemical indicators, alanine aminotransferase（ALT） and alkaline phosphatase（ALP） of liver functions as well as uric acid（UA）, blood urea nitrogen（BUN）, and creatinine（CR） of kidney functions showed significant differences between two mouse genders（P〈0.05）. The results of histopathologic analysis showed that liver and kidney pathological changes of male mice were found in the 25 mg/kg and the 35 mg/kg groups. The Tet1 protein and m RNA expression levels only in the male liver and kidney samples were obviously different among various dose groups（P〈0.05）. The results of pyrosequencing showed that the male mice had significant differences in methylation in kidney and liver among various dose groups（P〈0.05）, which were negatively correlated with Tel1 m RNA expression（r=-0.473, P〈0.05; r=-0.134, P〈0.05）. [Conclusion] Subacute cadmium exposure could induce kidney and renal injury in mice, and show gender differences in expressions of whole genome methylation and demethylation enzyme Tet1. Tet1 could regulate the global methylation in liver and kidney of male mice.