蚯蚓细胞色素P450亚酶及代谢组学对土壤亚致死剂量草甘膦除草剂的响应

Responses of cytochrome P450 subenzymes and metabolomics in earthworms exposed to sublethal of glyphosate-based herbicide in soil

以赤子爱胜蚓(Eisenia fetida)为受试生物,通过外源添加污染物的方法,研究了暴露于亚致死剂量草甘膦(添加剂量分别为5、10、20、40 mg·kg^(-1))的土壤中7 d后,蚯蚓生长抑制率、细胞色素P450亚酶(CYP1A2、2C9与3A4)活力及代谢组学对土壤草甘膦的响应,旨在探讨亚致死剂量草甘膦能否对非靶标生物蚯蚓产生不良影响及其毒性作用机制,初步推断其毒性作用阈值,并筛选敏感生物标记物.结果表明:从蚯蚓生长抑制率来看,各处理组与对照组无显著差异;从蚯蚓CYP亚酶活力来看,在高剂量草甘膦(20、40 mg·kg^(-1))处理下,CYP亚酶活力均受到显著抑制;基于代谢组学鉴定并筛选出14个小分子代谢物标记物,其中9个小分子代谢物(分别是1,6-二磷酸果糖、β-D-果糖6-磷酸、丙酮酸、组氨酸、赖氨酸、鸟氨酸、延胡索酸、肌酸、甜菜碱)在5~40 mg·kg^(-1)草甘膦暴露下显著低于对照水平,另外2个小分子代谢物(尿酸与二十-羟-二十烷四烯酸)则在草甘膦处理下显著升高.代谢组学的响应表明,草甘膦暴露(5~40 mg·kg^(-1))致使蚯蚓糖酵解与三羧酸循环减弱,氨基酸代谢受损,嘌呤代谢紊乱,细胞渗透功能受损,对蚯蚓产生了明显毒副作用,其毒副作用还与细胞色素P450酶代谢相关.蚯蚓CYP亚酶活力与小分子代谢物标记物作为敏感生物标记物,可联合诊断土壤亚致死剂量草甘膦污染.

Earthworms(Eisenia fetida) as test organism were exposed to sublethal doses of glyphosate(5, 10, 20, 40 mg·kg^(-1)) for 7 days by spiking the pollutant into soil. The responses of the growth inhibition rate, cytochrome P450(CYP) subenzymes(CYP1 A2, 2 C9 and 3 A4) activities and the metabolomics of earthworms were studied aiming to explore the adverse effects of sublethal doses of glyphosate on non-target organism earthworms and the corresponding toxic mechanism, which can further help to preliminarily infer the toxicity threshold and screen sensitive biomarkers. The results were as follows: for the growth inhibition rate of earthworm, no significant difference between the treatment groups and the control group was observed;all CYP subenzymes activities were significantly inhibited under higher concentration of glyphosate(20, 40 mg·kg^(-1));based on the metabolomics, 14 small molecular metabolite biomarkers were identified and screened;9 small molecule metabolites(1,6-diphosphate fructose, β-d-fructose-6-phosphate, pyruvic acid, histidine, lysine, ornithine, fumarate, creatine and betaine) under 5~40 mg·kg^(-1) glyphosate exposure were significantly lower than those in the control;2 small molecular metabolites(uric acid and eicosa-hydroxy-eicosantetraenoic acid) were significantly increased under glyphosate exposure. The responses of the metabolomics showed that glyphosate exposure(5~40 mg·kg^(-1)) weakened the glycolysis and tricarboxylic acid cycle and impaired the amino acid metabolism, purine metabolism and cell osmotic function in earthworms, thus evoking obvious adverse toxic effects on earthworms. The toxic effect was related to the metabolic pathway of cytochrome P450 enzymes as well. It is suggested that activities of CYP subenzymes of earthworm and small molecular metabolite biomarkers may be used as a set of biomarkers in order to accurately monitor soil glyphosate pollution.