Studies comparing the kinetics of cysteine conjugation and protein binding of acetaminophen by hepatic microsomes from male mice

Abstract

A large body of evidence indicates that acetaminophen toxicity is mediated through the formation of the reactive metabolite, N-acetyl- p-benzoquinone imine (NABQI). Two assays have been employed to monitor NABQI formation by hepatic microsomes: the conjugation with a thiol trap, such as cysteine or glutathione, and the binding of NABQI to microsomal proteins. Studies from our laboratory with rat hepatic microsomes have suggested that the two assays may not be equivalent. We now find with mouse hepatic microsomes that there are also marked differences between these two assays. Among these the rate of cysteine conjugation was almost three orders of greater than that of protein binding. Furthermore, ethanol feeding increased protein binding by 97%, but cysteine conjugation by only 33%. Protein binding was linear for 20 min while cysteine conjugation was linear for only 5 min. CO, imidazole and metyrapone inhibited cysteine conjugation much more than protein binding while SKF-525A and KCN had similar effects on both reactions. Both reactions increased linearly with increasing [NADPH] up to 0.32 mM. At higher concentrations, the rate of cysteine conjugation markedly decreased while the rate of protein binding plateaued. The addition of equimolar concentrations of NADH decreased protein binding, but had no effect on cysteine conjugation. NADPH reduced the protein binding of added NABQI while NADH had little effect. The reduction of NABQI back to acetaminophen was equal for NADPH and NADH. These data indicate that the formation of the cysteine conjugate of NABQI has markedly different kinetic characteristics than the microsomal protein binding. These data might suggest that the two reactions are catalyzed in part by different isoforms of cytochrome P-450.