Para-nitrophenol hydroxylation by fish liver microsomes: kinetics and effect of selective cytochrome P450 inhibitors

The study investigated the kinetics of p-nitrophenol hydroxylase (PNPH) in hepatic microsomes obtained from Atlantic salmon ( Salmo salar ). The selective inhibitors for some major mammalian cytochrome P450 (CYP450) were used to investigate the potential inhibitory effect on enzymes involved in p-nitrophenol hydroxylation. The following inhibitors were used: α-naphtoflavone (CYP1A), ellipticine (CYP1A1), furafylline (CYP1A2), 8-methoxypsoralen (8MOP, CYP2A6), 4-methylpyrazole (4MP, CYP2A6/2E1), diallyl sulfide (DAS, CYP2E1), and ketoconazole (CYP3A4). Additionally, the natural steroids 17-beta-oestraiol (E2) and testosterone were investigated as potential inhibitors of PNPH activity. It was found that formation of 4-nitrocatechol from p-nitrophenol followed monophasic kinetics with K m = 0.17 ± 0.03 mM and V max = 21.8 ± 1.05 pmol/min/mg. PNPH activity was competitively inhibited by diallyle sulfide with the K i value of 285.1 ± 94.2 μM μM and uncompetitively by ellipticine with K i value of 65.7 ± 7.8 μM. Moreover, E2 showed an ability to reduce PNPH activity through the mechanism-based inhibition mode. Our results suggest that hepatic microsomes from Atlantic salmon possess CYP2E1-like activity. However, specific isoform-mediated PNPH activity should be identified.