Quantitation of multiple pathways for the metabolism of nephrotoxic cysteine conjugates using selective inhibitors of L-α-hydroxy acid oxidase (L-amino acid oxidase) and cysteine conjugate β-lyase

Abstract

In this study, we have established the selectivity of inhibitors for rat kidney cysteine conjugate β-lyase and L-α-hydroxy acid oxidase (L-amino acid oxidase) and have used these inhibitors to explore the relative roles of these two enzymes in the metabolism of nephrotoxic cysteine conjugates by rat kidney homogenate. In addition, we have investigated the relationship between structure and the metabolism of toxic cysteine conjugates by purified rat kidney L-α-hydroxy acid oxidase. With purified enzyme, S-(1,2,3,4,4-pentachlorobutadienyl)-L-cysteine (PCBDC) was about four times more active than S(1,2-dichlorovinyl)-L-cysteine (DCVC). Three alkyl conjugates were less active than DCVC. Purified L-α-hydroxy acid oxidase was not inhibited by the β-lyase inhibitor aminooxyacetic acid but was inactivated by 2-hydroxy-3-butynoate. PCBDC metabolism in rat kidney homogenate was inhibited 74% by aminooxyacetic acid and 42% by 2-hydroxy-3-butynoate, whereas DCVC metabolism was inhibited 77% by aminooxyacetic acid and 28% by 2-hydroxy-3-butynoate. However, only aminooxyacetic acid inhibited the binding of 35S label from [35S]DCVC. Based on these results we have reached three conclusions. First, L-α-hydroxy acid oxidase plays a significant role in the metabolism of some cysteine conjugates. Second, metabolism of DCVC by L-α-hydroxy acid oxidase does not contribute directly to covalent binding. Third, as much as 65% of DCVC may be metabolized to its corresponding α-keto acid. The results are discussed with regard to the nephrotoxicity of cysteine conjugates.