Five glutathione S-transferase isozymes played crucial role in the detoxification of aflatoxin B1 in chicken liver

Abstract

AFB1-8,9-exo-epoxide (AFBO) is the highly toxic product of Aflatoxin B1 (AFB1). Glutathione S-transferases (GSTs) play pivotal roles in detoxifying AFB1 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 AFB1 have not been identified yet. The objective of this study was to determine which GST isozymes played key role in detoxification of AFB1. 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 AFB1 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 AFB1. After comprehensive evaluation of various indicators, GSTA2X displayed the best detoxification effects against AFB1. 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 Vmax was 0.23 nmol/min/mg and the Michaelis constant was 86.05 μmol/L with the AFB1 as substrate. In conclusion, GSTA2X, GSTA3, GSTT1L, GSTZ1-1, and GSTZ1-2 played key roles in AFB1 detoxification, which will provide new remediation strategies to prevent aflatoxicosis in chickens.