Catalyzation of cocaine N-demethylation by Cytochromes P4502B, P4503A, and P4502D in fish liver

Cocaine N-demethylation by microsomal cytochrome P450s is the principal pathway in cocaine bioactivation and hepatotoxicity. P450 isozymes involved in N-demethylation of cocaine have not been elucidated yet and they differ from species to species. In humans and mice, P4503A contributes to cocaine N-demethylase activity, whereas in rats, both P4503A and P4502B participate. In the present study, contribution of different P450 isozymes to cocaine N-demethylase activity was studied in vitro with fish liver microsomes. The specific cocaine N-demethylase activity was found to be 0.672 +/- 0.22 nmol formaldehyde formed/min/mg protein (mean +/- SD, n = 6). Cocaine N-demethylase exhibited biphasic kinetics, and from the LineweaverBurk plot, two Km values were calculated as 0.085 and 0.205 mM for the high- and low-affinity enzyme. These results indicate that N-demethylation of cocaine in mullet liver microsomes is catalyzed by at least two cytochrome P450 isozymes. Inhibitory effects of cytochrome P450 isozyme-selective chemical inhibitors, ketoconazole, cimetidine, SKF-525A, and quinidine, on cocaine N-demethylase activity were studied at 50, 100, and 500 muM concentrations of these inhibitors. At 100 muM final concentrations, ketoconazole (P4503A inhibitor), SKF-525A (inhibitor of both P4502B and P4503A), and cimetidine (P4503A inhibitor) inhibited N-demethylation activity by 73, 69, and 63%, respectively. Quinidine, P4502D-specific inhibitor, at 100 muM final concentration, reduced N-demethylation activity down to 64%. Aniline, a model substrate for P4502E1, did not alter N-demethylase activity in the final concentration of 100 muM. IC50 values were calculated to be 20 muM for ketoconazole, 48 muM for cimetidine (both specific P4503A inhibitors), 164 muM for quinidine (P4502D inhibitor), and 59 muM for SKF-525A (inhibitor of both P4503A and P4502B). The contribution of P4502B to cocaine N-demethylase activity in mullet liver microsomes was further explored by the use of purified mullet cytochrome P4502B in the reconstituted system containing purified mullet P450 reductase and lipid. The turnover number was calculated as 4.2 nmol HCOH/(min nmol P450). Overall, these results show that P4503A and P4502B are the major P450s responsible for N-demethylation of cocaine, whereas contribution of P4502D is a minor one, and P4502E1 is not involved in the N-demethylation of cocaine in mullet liver microsomes. (C) 2003 Wiley Periodicals, Inc

Inhibitory effects of divalent metal ions on liver microsomal 7-ethoxyresorufin O-deethylase (EROD) activity of leaping mullet

The purpose of the present study was to elucidate in vitro effects of Hg2+, Zn2+, Ni2+ and Cd2+ on cytochrome P4501Al (CYP1A1) dependent EROD activities in leaping mullet liver microsomes. Fish captured from the most polluted part of Izmir Bay, had highly elevated EROD activities, and induced CYP1A1 protein levels as determined by Western blotting. Although all of the metal ions caused inhibition of the initial velocity of the reaction, Hg2+ and Cd2+ exhibited much higher inhibitory effect at lower concentrations and they were evidently more potent inhibitors than others. The inhibitor concentration giving 50% inhibition (IC50 values) of Zn2+, Ni2+, Cd2+ and Hg2+ of initial EROD activity were 107, 16, 1.3 and 0.15 micromolar, respectively. Glutathione (GSH) at 0.5 mM final concentration, completely reversed Ni2+ and Cd2+ inhibition of EROD activity indicating the protective action of GSH

Cytochrome P4501A and associated mixed-function oxidase induction in fish as a biomarker for toxic carcinogenic pollutants in the aquatic environment

Polycyclic aromatic hydrocarbons (PAHs), dioxins, dibenzofurans, and polychlorinated biphenyls (PCBs) present in polluted environment induce cytochrome P4501A (CYP1A) isozyme in fish, which in turn results in a marked increased production of carcinogenic metabolites from PAHs. The induction of hepatic CYP1A in fish by certain classes of chemicals has been suggested as an early warning system, a "most sensitive biological response" for assessing environmental contamination conditions. This has implications for human fish consumption, as well as for the health status of aquatic organisms. Correlation between elevated CYP1A and altered steroid metabolism and decreased reproductive success has been pointed out. The induction of CYP1A and associated enzyme activities has now been confirmed in a number of field studies. Cases where these biomarkers have been studied in field conditions will be presented. Special emphasis will be given to field studies in which the induction of CYP1A activity, 7-ethoxyresonufin O-deethylase (EROD) activities and immunochemical detection of CYP1A in leaping mullet and common sole are used as a biomarker for PAH- and/or PCB-type pollutants along the Izmir Bay on the Aegean Sea