Oxidative inactivation of cytochrome P-450 1A (CYP1A) stimulated by 3,3 ',4,4 '-tetrachlorobiphenyl: Production of reactive oxygen by vertebrate CYP1As

Microsomal cytochrome P-450 1A (CYP1A) in a vertebrate model (the teleost f ish scup) is inactivated by the aryl hydrocarbon receptor agonist 3,3',4,4' -tetrachlorobiphenyl (TCB). Here, the mechanism of CYP1A inactivation and i ts relationship to reactive oxygen species (ROS) formation were examined by using liver microsomes from soup and rat and expressed human CYP1As. In vi tro inactivation of scup CYP1A activity 7-ethoxyresorufin O-deethylation by TCB was time dependent, NADPH dependent, oxygen dependent, and irreversibl e. TCB increased microsomal NADPH oxidation rates, and CYP1A inactivation w as lessened by adding cytochrome c. CYP1A inactivation was accompanied by l oss of spectral P-450, a variable loss of heme and a variable appearance of P-420. Rates of scup liver microsomal metabolism of TCB were < 0.5 pmol/mi n/mg, 25-fold less than the rate of P-450 loss. Non-heme iron chelators, an tioxidant enzymes, and ROS scavengers had no influence on inactivation. Ina ctivation was accelerated by H2O2 and azide but not by hydroxylamine or ami notriazole. TCB also inactivated rat liver microsomal CYP1A, apparently CYP 1A1, Adding TCB to scup or rat liver microsomes containing induced levels o f CYP1A, but not control microsomes, stimulated formation of ROS; formation rates correlated with native CYP1A1 content. TCB stimulated ROS formation by baculovirus-expressed human CYP1A1 but not CYP1A2. The results indicate that TCB uncouples the catalytic cycle of CYP1A, ostensibly CYP1A1, resulti ng in formation of ROS within the active site. These ROS may inactivate CYP 1A or escape from the enzyme. ROS formed by CYP1A1 may contribute to the to xicity of planar halogenated aromatic hydrocarbons.