摘要
Background AFB_(1)-8,9-exo-epoxide(AFBO)is the highly toxic product of Aflatoxin B_(1)(AFB_(1)).Glutathione S-transferases(GSTs)play pivotal roles in detoxifying AFB_(1) by catalyzing the conjugation of AFBO with glutathione(GSH).Although there are over 20 GST isozymes that have been identified in chicken,GST isozymes involved in the detoxification process of AFB_(1) have not been identified yet.The objective of this study was to determine which GST isozymes played key role in detoxification of AFB_(1).Results A total of 17 pcDNA3.1(+)-GST isozyme plasmids were constructed and the GST isozyme genes were overexpressed by 80–2,500,000 folds in the chicken Leghorn male hepatoma(LMH)cells.Compared to the AFB_(1) treatment,overexpression of GSTA2X,GSTA3,GSTT1L,GSTZ1-1,and GSTZ1-2 increased the cell viability by 6.5%–17.0%in LMH cells.Moreover,overexpression of five GST isozymes reduced the release of lactate dehydrogenase and reactive oxygen species by 8.8%–64.4%,and 57.2%–77.6%,respectively,as well as enhanced the production AFBO-GSH by 15.8%–19.6%,thus mitigating DNA damage induced by AFB_(1).After comprehensive evaluation of various indicators,GSTA2X displayed the best detoxification effects against AFB_(1).GSTA2X was expressed in Pichia pastoris X-33 and its enzymatic properties for catalyzing the conjugation of AFBO with GSH showed that the optimum temperature and pH were 20–25℃ and 7.6–8.6 as well as the enzymatic kinetic parameter V_(max) was 0.23 nmol/min/mg and the Michaelis constant was 86.05μmol/L with the AFB_(1) as substrate.Conclusions In conclusion,GSTA2X,GSTA3,GSTT1L,GSTZ1-1,and GSTZ1-2 played key roles in AFB_(1) detoxification,which will provide new remediation strategies to prevent aflatoxicosis in chickens.
基金
supported by the Chinese Natural Science Foundation Projects 32072775,32272915 and 32472949
the National Key Research and Development Programs of China(2023YFD1301003 and 2023YFD1301005)
the Fundamental Research Funds for the Central Universities(2662023DKPY002)。
