Involvement of CYP3A1, 2B1, and 2E1 in C-8 hydroxylation and CYP 1A2 and flavin-containing monooxygenase in N-demethylation of caffeine; identified by using inducer treated rat liver microsomes that are characterized with testosterone metabolic patterns.
Caffeine (CA) is oxidized by rat liver microsomal enzymes to theobromine (TB), paraxanthine (PX), and theophylline (TP) by N-demethylation and to trimethylurate (TMU) by C-8 hydroxylation, In order to identify the specific enzymes responsible for productions of these primary CA metabolites, liver microsomes enriched with various isoforms of cytochrome P450 (CYP) and flavin-containing monooxygenase (FMO) are prepared by pretreatment of rats with several inducers. The specific increases in various CYP or FMO activities are identified with the diagnostic testosterone metabolic patterns or the thiobenzamide S-oxidation assay. They are then employed to metabolize the CA. Liver microsomes isolated from rats pretreated with phenobarbital (PB-microsomes) did not have increased FMO activity but had increased activities for hydroxylating the testosterone at 6 beta-(CYP3A1), 16 beta-(CYP2B1), and 2 beta-(CYP3A1) positions. This PB-microsomes had increased activity for TMU production from CA (result of C-8 hydroxylation). Liver microsomes isolated from rats pretreated with acetone (AC-microsomes) had a normal level of FMO activity but had enhanced rates of 6 beta-(CYP3A1) and 2 beta-(CYP3A1) hydroxylations of testosterone. The AC-microsomes again had increased activity for production of TMU. Similarly, the liver microsomes isolated from rats pretreated with dexamethasone (DEX-microsomes) had a normal level of FMO activity but had enhanced rates of forming 6 beta-and 2 beta-hydroxytestosterone (Cyp3A1) as well as androstenedione (CYP3A1). The DEX-microsomes again had increased activity for production of TMU only. Liver microsomes isolated from rats pretreated with 3-methylcholanthrene (MC-microsomes), however, had increased FMO activity and also enhanced rates of forming the 7 alpha-(CYP1A1/2, and 2A1), 6 beta-(CYP3A1), and 2 beta-(CYP3A1) hydroxytestosterone. The MC-microsomes had increased activity for producing all of the four primary metabolites of CA, i.e. the N-demethylation metabolites like TB, PX. and TP, as well as the C-8 hydroxylation metabolite TMU. By the process of association of the obtained results, liver microsomes with increased contents of CYP2B1, 3A1, and 2E1 could catalyze the C-8 hydroxylation at an increased rate producing increased amount of TMU. Increased productions of CA N-demethylation metabolites (TB, PX, and TP) are, however, catalyzed by the increased activities of CYP1A2 and FMO which are associated uniquely with the MC-microsomes.
Chung
W G
WG
Department of Pharmacology and Toxicology, College of Medicine, Inha University, Nam-gu, Inchon, South Korea.
Roh
H K
HK
Kim
H M
HM
Cha
Y N
YN
D000818:Animals; D001189:Aryl Hydrocarbon Hydroxylases; D002110:Caffeine / Q000378:metabolism*; D019388:Cytochrome P-450 CYP1A2 / Q000378:metabolism; D019362:Cytochrome P-450 CYP2B1 / Q000378:metabolism; D019392:Cytochrome P-450 CYP2E1 / Q000378:metabolism; D051544:Cytochrome P-450 CYP3A; D003577:Cytochrome P-450 Enzyme System / Q000096:biosynthesis / Q000378:metabolism*; D003907:Dexamethasone / Q000494:pharmacology; D004790:Enzyme Induction / Q000187:drug effects; D006900:Hydroxylation; D066298:In Vitro Techniques; D008297:Male; D008745:Methylation; D008748:Methylcholanthrene / Q000494:pharmacology; D008862:Microsomes, Liver / Q000187:drug effects / Q000378:metabolism*; D006899:Mixed Function Oxygenases / Q000378:metabolism; D010105:Oxygenases / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D013739:Testosterone / Q000378:metabolism; D013854:Thioamides / Q000378:metabolism
D013854:Thioamides; D002110:Caffeine; D013739:Testosterone; D008748:Methylcholanthrene; D003907:Dexamethasone; C015539:thiobenzamide; D003577:Cytochrome P-450 Enzyme System; D006899:Mixed Function Oxygenases; D010105:Oxygenases; D019392:Cytochrome P-450 CYP2E1; C020066:dimethylaniline monooxygenase (N-oxide forming); D001189:Aryl Hydrocarbon Hydroxylases; C104464:CYP3A protein, human; C465138:Cyp3a23-3a1 protein, rat; D019388:Cytochrome P-450 CYP1A2; D019362:Cytochrome P-450 CYP2B1; D051544:Cytochrome P-450 CYP3A