Caffeine metabolism by human hepatic cytochromes P450: contributions of 1A2, 2E1 and 3A isoforms.
Caffeine (CA) N1-, N3- and N7-demethylase, CA 8-hydroxylase and phenacetin O-deethylase activities were measured in microsomes from 18 separate human livers which had been characterized previously for a range of cytochrome P450 (CYP) isoform-specific activities and immunoreactive CYP protein contents. Correlations between the high affinity components 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-demethylations correlated significantly (r = 0.64-0.93, P < 0.05-0.001) with the high affinity phenacetin O-deethylase, 2-acetylaminofluorene N-hydroxylation and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) and 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) mutagenicity (all predominantly CYP1A2-mediated reactions). Consistent with these observations, cDNA-expressed human CYP1A2 catalyzed the N1-, N3- and N7-demethylation of CA and apparent Km values were similar (0.24-0.28 mM) for all three reactions and comparable to those observed previously with 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 activities 4-nitrophenol and chlorzoxazone hydroxylation. Diethyldithiocarbamate, a selective inhibitor of CYP2E1, inhibited the low affinity CA N1- and N7-demethylation, with IC50 values of 23 microM and 11 microM, respectively. The apparent Km values for CA N1- and N7-demethylation by cDNA-expressed CYP2E1 (namely 28 and 43 mM, respectively) were of a similar order to those calculated for the low affinity microsomal activities. Significant correlations (r = 0.87-0.97, P < 0.001) were observed between CA 8-hydroxylation and immunoreactive CYP3A content and the CYP3A-mediated reactions benzo(a)pyrene hydroxylation, omeprazole sulfoxidation and aflatoxin B1 mutagenesis. Effects of alpha-naphthoflavone, erythromycin, troleandomycin and nifedipine on microsomal CA 8-hydroxylation were generally consistent with CYP3A involvement. Taken together with previous data, the results indicate a major involvement of CYP1A2 in the high affinity component of all three human hepatic CA N-demethylations. In contrast, CYP2E1 appears to be the main enzyme involved in the low affinity components of CA N1- and N7-demethylation while CA 8-hydroxylation is catalysed predominantly by a CYP3A isoform(s).
Tassaneeyakul
W
W
Department of Clinical Pharmacology, Flinders Medical Centre, Bedford Park, SA, Australia.
Birkett
D J
DJ
McManus
M E
ME
Tassaneeyakul
W
W
Veronese
M E
ME
Andersson
T
T
Tukey
R H
RH
Miners
J O
JO
D002110:Caffeine / Q000493:pharmacokinetics*; D019388:Cytochrome P-450 CYP1A2; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism*; D006801:Humans; D007527:Isoenzymes / Q000378:metabolism*; D008862:Microsomes, Liver / Q000378:metabolism*; D010088:Oxidoreductases / Q000378:metabolism; D013805:Theobromine / Q000378:metabolism; D013806:Theophylline / Q000378:metabolism; D014527:Uric Acid / Q000031:analogs & derivatives / Q000378:metabolism
D007527:Isoenzymes; D014527:Uric Acid; D002110:Caffeine; C039222:1,3,7-trimethyluric acid; D003577:Cytochrome P-450 Enzyme System; D013806:Theophylline; D010088:Oxidoreductases; D019388:Cytochrome P-450 CYP1A2; D013805:Theobromine; C021183:1,7-dimethylxanthine