N-亚硝基二甲胺和N-亚硝基二乙胺的小鼠重复给药毒性及遗传毒性研究

Toxicity and genotoxicity of N-nitrosodimethylamine and N-nitrosodiethylamine in mice after repeated administration

目的 连续7 d重复给予C57BL/6J小鼠N-亚硝基二甲胺(NDMA)和N-亚硝基二乙胺(NDEA),观察至首次给药后28 d,评价两者主要毒性靶器官及遗传毒性风险。方法 分设溶媒对照组(0.5%羧甲基纤维素钠),受试物组(分别给予0.75、1.50、3.00 mg·kg^(-1)NDMA或3.25、7.50、15.00 mg·kg^(-1)NDEA)和阳性对照组(200 mg·kg^(-1)甲磺酸乙酯、40 mg·kg^(-1)N-乙基-N-亚硝基脲)。溶媒对照组和受试物组连续7 d每天ig给药1次,阳性对照组连续3 d每天ig给药1次。末次给药后开展小鼠肝、肾和外周血彗星试验,计算每只动物的肝、肾和血细胞尾DNA百分含量(%Tail DNA)和尾距(tail moment)平均值。观察结束后(D28)开展小鼠磷脂酰肌醇聚糖A类(Pig-a)基因突变试验,计算网织红细胞(RETCD24-)和总红细胞(RBCCD24-)的突变率。并在末次给药和恢复期结束后,试剂盒法检测小鼠肝、肾和肺的8-羟基脱氧鸟苷(8-OHdG)水平、实时荧光定量PCR(qRT-PCR)法检测DNA损伤基因(ATM、gH2AX、Nbn、Prkdc、Trp)和肝药酶基因(CYP2E1、CYP2A6)。结果 NDMA剂量为3 mg·kg^(-1)时,动物全部死亡,自连续给药4 d后,NDEA 15 mg·kg^(-1)组小鼠体质量与溶媒对照组相比显著性降低(P<0.001);且NDMA和NDEA组与溶媒对照组在脏器系数、血液生化指标、组织病理学等方面存在显著差异(P<0.05、0.01、0.001),提示NDMA和NDEA对肝脏和肾脏存在一定毒性;NDMA和NDEA组小鼠肝、肾彗星试验结果均为阳性,Pig-a基因突变试验中NDEA高剂量组RETCD24-结果为阳性;与溶媒对照组比较,NDMA和NDEA组小鼠肝、肾和肺中8-OHdG含量显著升高(P<0.05、0.001),DNA损伤和肝药酶基因表达也存在显著差异(P<0.05、0.001)。结论 NDMA分别在1.5 mg·kg^(-1)剂量条件下产生肝脏毒性,0.75、1.50 mg·kg^(-1)剂量条件下使小鼠肝、肾细胞产生致突变性和肝、肾DNA损伤;NDEA可在15 mg·kg^(-1)剂量条件下产生肝脏毒性,并导致肝、肾细胞产生致突变性和DNA损伤;8-OHdG和DNA损伤基因等指标检测结果与NDMA和NDEA遗传毒性结果具有一定关联性。

Objectives Repeated administration of N-nitrosodimethylamine(NDMA)and N-nitrosodiethylamine(NDEA)in C57BL/6J mice for seven consecutive days was observed until 28 days after the first administration,and the main toxic target organs and genotoxic risk of both were evaluated.Methods A solvent control group(0.5%sodium carboxymethyl cellulose),a test group(0.75,1.50,3.00 mg·kg^(−1) NDMA or 3.25,7.50,15.00 mg·kg^(−1) NDEA,respectively)and a positive control group(200 mg·kg^(−1) ethyl mesylate;40 mg·kg^(−1) N-ethyl-n-nitrosourea).The solvent control group and the test group were given ig once a day for seven consecutive days,and the positive control group was given ig once a day for three consecutive days.Comet tests in mouse liver,kidney,and peripheral blood were performed after the final dose,and the average percentage of Tail DNA and Tail Moment in liver,kidney,and blood cells of each animal were calculated.After observation(D28),mouse Pig-a gene mutation test was carried out to calculate the mutation rate of reticulocyte(RETCD24-)and total erythrocyte(RBCCD24-).The expression of 8-hydroxydeoxyguanosine(8-OHdG),DNA damage genes(ATM,gH2AX,Nbn,Prkdc,Trp)and liver drug enzyme genes(CYP2E1,CYP2A6)in liver,kidney and lung were studied after the final administration and recovery period.Results When the dose of NDMA was 3 mg·kg^(−1) all the animals died.After continuous administration for four days,the body weight of mice in the NDEA 15 mg·kg^(−1) group significantly decreased compared to the solvent control group(P<0.001).There were differences in organ coefficient,blood biochemical index and histopathology between NDMA and NDEA and the solvent control group(P<0.05,0.01,and 0.001),suggesting that NDMA and NDEA had certain toxicity to liver and kidney.The results of comet test in liver and kidney of NDMA and NDEA mice were positive,and RETCD24-in Pig-a gene mutation test of high-dose NDEA group was positive.The content of 8-OHdG in liver,kidney and lung of NDMA and NDEA groups was significantly increased(P<0.05,0.001),and there were significant differences in DNA damage and liver drug enzyme gene expression(P<0.05,0.001).Conclusion NDMA produced hepatotoxicity at a dose of 1.5 mg·kg^(−1),and mutagenic and DNA damage in liver and kidney cells at a dose of 0.75 mg·kg^(−1),respectively.NDEA can produce hepatotoxicity at a dose of 15 mg·kg^(−1),and lead to mutagen and DNA damage in liver and kidney cells.It has been proved that the results of 8-OHdG and DNA damage genes are related to the genotoxicity results of NDMA and NDEA.