亚致死剂量乙草胺对蚯蚓的毒性效应及敏感生物标记物

Toxic effects of sublethal acetochlor with sublethal doses on earthworms in soil using multiendpoints system

本研究以赤子爱胜蚓为受试生物,采用外源添加污染物的方法,将受试生物暴露于含亚致死剂量乙草胺(添加浓度分别为1、2、4、8 mg·kg^(-1))的土壤中7 d,研究蚯蚓生长抑制率、细胞色素P450同工酶(CYP1A2、2C9和3A4)活力及代谢组学对乙草胺的响应,从个体、酶、小分子标记物3个层次探讨亚致死剂量乙草胺对蚯蚓的毒性效应,初步推断其毒性作用阈值,筛选敏感生物标记物,探讨其致毒机理。结果表明:乙草胺暴露下,与对照相比,蚯蚓体重抑制率无明显差异,但CYP1A2、2C9和3A4活力受到明显抑制,10组小分子代谢物(1,6-二磷酸果糖、胞苷酸、尿苷酸、腺苷酸、腺苷、黄嘌呤、延胡索酸、二羟基戊二酸、鸟氨酸与16-羟二十烷四烯酸)水平显著降低;另有6组小分子代谢物(腺苷琥珀酸、琥珀酸、精氨酸、色氨酸、天冬酰胺与苯丙氨酸)水平在2~8 mg·kg^(-1)乙草胺暴露下显著升高。乙草胺暴露导致蚯蚓受到氧化损伤,糖酵解功能减弱,三羧酸循环失衡,嘌呤及嘧啶代谢紊乱,氨基酸代谢受损。与个体水平的受试终点相比,CYP同工酶活力与上述16个小分子代谢物对乙草胺暴露的响应更为敏感。建议将CYP同工酶(1A2、2C9及3A4)活力与上述小分子代谢物为一组生物标记物,可以多指标、多层次联合诊断土壤乙草胺污染的生态毒性效应。其诊断结果将更为精准。

In this study,we examined the toxic effect of sublethal doses of acetochlor(1,2,4,8 mg·kg^(-1))on earthworms by exogenous addition.The growth inhibition rate,cytochrome P450 isozymes(CYP1 A2,2 C9 and 3 A4)activities and the metabolomics were analyzed after seven days of exposure,to infer the toxicity threshold of acetochlor,screen the sensitive biomarkers from the levels of the individual,detoxified enzymes and small molecular metabolites,and elucidate the underlying toxicity mechanism.The results showed that CYP1 A2,2 C9 and 3 A4 activities were all significantly inhibited,and that the levels of ten metabolites(fructose-6-diphosphate,cytosine monophosphate,uridine monophosphate,adenosine monophosphate,adenosine,xanthine,fumaric acid,dihydroxyglutaric acid,ornithine and 16-hydroxyeicosatetraenoic acid)were significantly decreased by acetochlor exposure.The levels of six metabolites(adenosine succinic acid,succinic acid,arginine,tryptophan,asparagine and phenylalanine)were significantly increased when earthworms being exposed to 2-8 mg·kg^(-1)acetochlor.Acetochlor exposure caused oxidative damage to earthworms,weakened the glycolysis,disturbed the tricarboxylic acid cycle,disordered the purine and pyrimidine metabolism,and impaired the amino acids metabolism.Compared with the end point at individual level,the above 16 small molecule metabolites and CYP isozymes activities were more sensitive to acetochlor exposure.It was thus recommended that CYP isozymes(1 A2,2 C9,and 3 A4)activities and small molecular metabolites could be used as a set of biomarkers to diagnose the acetochlor pollution,given their high sensitivity and accuracy.