Relative contribution of hepatic CYP3A and CYP2E1 to oxidative stress upon CHIP E3 ligase knockdown

Abstract

Liver microsomal cytochromes P450 (P450s) are responsible for drug‐metabolism. CYP3A4 and CYP2E1 incur ubiquitin (Ub)‐dependent proteasomal degradation, wherein CHIP is an active E3 Ub‐ligase. We found that upon shRNA‐mediated CHIP‐knockdown (CHIP‐KD; ≈ 80%), functional parent and inactive ubiquitinated CYP3A and CYP2E1 species are stabilized. CHIP knockout (CHIP‐KO) is associated with enhanced oxidative stress and premature ageing in mice (Min et al.). To determine whether CYPs 3A and 2E1, both prone to oxidative uncoupling/radical production, could contribute to oxidative stress upon CHIP‐KD we monitored the levels of hepatic 15‐F2t‐isoprostane (15‐IP), a lipid peroxidation indicator. Upon CHIP‐KD of cultured rat hepatocytes, a 2.5‐fold 15‐IP increase was observed. To determine the relative contribution of CYP3A and CYP2E1 to this increase, we employed relatively selective functional probes. CYP3A inhibition with ketoconazole (KTZ, 10 μM) decreased 15‐IP levels by ≈15%, whereas CYP2E1 inhibition with 4‐methylpyrazole (4MP, 2.5 mM) caused a corresponding ≈80% decrease. Similar 15‐IP increases were observed in cultured CHIP+/− relative to CHIP+/+ mouse hepatocytes that were also largely abolished by 4MP but not KTZ. These findings reveal that such P450 stabilization is also pathophysiologically relevant: CYP2E1 rather than CYP3A is the major contributor to hepatocellular oxidative stress.