CAFFEINE METABOLISM BY HUMAN HEPATIC CYTOCHROMES P450 - CONTRIBUTIONSOF 1A2, 2E1 AND 3A ISOFORMS

Caffeine (CA) N1-, N3- and N7-demethylase, CA 8-hydroxylase and phenac etin O-deethylase activities were measured in microsomes from 18 separ ate human livers which had been characterized previously for a range o f cytochrome P450 (CYP) isoform-specific activities and immunoreactive CYP protein contents. Correlations between the high affinity componen ts of the three separate CA N-demethylations were highly significant ( r = 0.77-0.91, P < 0.001) and each of the three high affinity CA N-dem ethylations correlated significantly (r = 0.64-0.93, P < 0.05-0.001) w ith the high affinity phenacetin O-deethylase, 2-acetylaminofluorene N -hydroxylation and 2-amino-1-methyl-6-phenylimidazo[4 ,5-b]pyridine (P hIP) and 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) mutagenicity (al l predominantly CYP1A2-mediated reactions). Consistent with these obse rvations, cDNA-expressed human CYP1A2 catalyzed the N1-, N3- and N7-de methylation of CA and apparent K-m values were similar (0.24-0.28 mM) for ah three reactions and comparable to those observed previously wit h human liver microsomes. The low affinity components of CA N1- and N7 -demethylation correlated significantly (r = 0.55-0.85, P < 0.05-0.001 ) with immunoreactive CYP2E1 content and the CYP2E1-specific activitie s 4-nitrophenol and chlorzoxazone hydroxylation. Diethyldithiocarbamat e, a selective inhibitor of CYP2E1, inhibited the low affinity CA N1- and N7-demethylation, with IC50 values of 23 mu M and 11 mu M, respect ively. The apparent K-m, values for CA N1- and N7-demethylation by cDN A-expressed CYP2E1 (namely 28 and 43 mM, respectively) were of a simil ar order to those calculated for the low affinity microsomal activitie s. Significant correlations (r = 0.87-0.97, P < 0.001) were observed b etween CA 8-hydroxylation and immunoreactive CYP3A content and the CYP 3A-mediated reactions benzo(a)pyrene hydroxylation, omeprazole sulfoxi dation and aflatoxin B1 mutagenesis. Effects of alpha-naphthoflavone, erythromycin, troleandomycin and nifedipine on microsomal CA 8-hydroxy lation were generally consistent with CYP3A involvement. Taken togethe r with previous data, the results indicate a major involvement of CYP1 A2 in the high affinity component of all three human hepatic CA N-deme thylations. In contrast, CYP2E1 appears to be the main enzyme involved in the low affinity components of CA N1- and N7-demethyIation while C A 8-hydroxylation is catalysed predominantly by a CYP3A isoform(s).