Caffeine metabolism by microsomal and cDNA-expressed cytochrome P450 1A from scup (Stenotomus chrysops)

Consequences of CYP1A induction depend in part on the substrates metabolized. We are employing model compounds to determine the substrate specificity of teleost CYP1As, and to assess functional similarities to both CYP1A1 and CYP1A2, vis-à-vis the structural relationships among these proteins. Caffeine (CA; 1,3,7-trimethylxanthine) is a natural alkaloid that is N-demethylated predominantly by CYP1A2 in mammals, to paraxanthine (PX; N-3-demethylation) and theobromine (TB; N-1-demethylation). Theophylline (TP; N-7-demethylation) and 1,3,7-trimethyluric acid (TMU; 8-hydroxylation) are formed largely by other catalysts. BNF-induced scup liver microsomes oxidized CA primarily to PX (173 pmol/min/mg) and TB (50 pmol/min/mg). TP and TMU formation was not detected. Polyclonal and the 1A1-specific monoclonal (1-12-3) antibodies to scup CYP1A inhibited CA metabolism by 80-90%. Chinese hamster V79 cells stably expressing scup CYP1A metabolized CA to PX (major) and TB (minor). The CA metabolite profile was remarkably similar to that formed by V79 cells expressing human CYP1A2. Liver microsomes from Aroclor-treated rats oxidized CA to PX, TB, TP and TMU, all at rates between 25 and 60 pmol/min/mg. CA N-demethylations by rat liver microsomes were inhibited about 40%, and 8-hydroxylation (TMU) was inhibited 90% by Mab 1-12-3, indicating involvement of rat CYP1A1. The results establish that teleost CYP1As metabolize caffeine, with regiospecificity apparently more similar to CYP1A2s than to CYP1A1s. [Support: EPA R-823890-01 and Sea Grant NA46RG0470, R/P-60.]