Five glutathione S-transferase isozymes played crucial role in the detoxification of aflatoxin B in chicken liver.

BACKGROUND

AFB-8,9-exo-epoxide (AFBO) is the highly toxic product of Aflatoxin B (AFB). Glutathione S-transferases (GSTs) play pivotal roles in detoxifying AFB 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 have not been identified yet. The objective of this study was to determine which GST isozymes played key role in detoxification of AFB.

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 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. After comprehensive evaluation of various indicators, GSTA2X displayed the best detoxification effects against AFB. 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 °C and 7.6-8.6 as well as the enzymatic kinetic parameter V was 0.23 nmol/min/mg and the Michaelis constant was 86.05 μmol/L with the AFB as substrate.

CONCLUSIONS

In conclusion, GSTA2X, GSTA3, GSTT1L, GSTZ1-1, and GSTZ1-2 played key roles in AFB detoxification, which will provide new remediation strategies to prevent aflatoxicosis in chickens.