Elsevier

Biochemical Pharmacology

Volume 55, Issue 9, 1 May 1998, Pages 1405-1411
Biochemical Pharmacology

Original Articles
Strain Differences in CYP3A-Mediated C-8 Hydroxylation (1,3,7-Trimethyluric Acid Formation) of Caffeine in Wistar and Dark Agouti Rats: Rapid Metabolism of Caffeine in Debrisoquine Poor Metabolizer Model Rats

https://doi.org/10.1016/S0006-2952(97)00654-0Get rights and content

Abstract

We observed significant strain differences [Dark Agouti (DA) > Wistar] in 1,3,7-trimethyluric acid formation (C-8 hydroxylation) during caffeine metabolism, though not in N-demethylations, in adult male DA and Wistar rats. In contrast, adult female and immature male rats of both DA and Wistar strains did not show significant differences in activity levels of C-8 hydroxylation. Kinetic studies using liver microsomes revealed that adult male DA rats have a larger Vmax for C-8 hydroxylation than do Wistar rats. Troleandomycin (TAO), known as a cytochrome P450 (CYP) 3A inhibitor, and an anti-rat CYP3A2 polyclonal antibody effectively reduced C-8 hydroxylation by rat liver microsomes in a concentration-dependent manner, suggesting that C-8 hydroxylation in rats is mediated largely by an isoform(s) of the CYP3A subfamily. Troleandomycin and the antibody did not inhibit the N-demethylations of caffeine by rat liver microsomes. Treatment of rats with CYP3A inducers caused a marked increase in C-8 hydroxylase activity. These results indicate that the rat CYP3A subfamily is capable of catalyzing C-8 hydroxylation of caffeine as is the case for human CYP3A4. The results of western blotting analysis using anti CYP3A antiserum showed that the staining intensity of the protein band in DA rat liver microsomes was higher than that in Wistar rat liver microsomes. We concluded that marked sex-dependent strain differences in C-8 hydroxylation of caffeine between Wistar and DA rats are due to the differences in the levels of expression of CYP3A in these strains of rats.

Section snippets

Chemicals

Chemicals were obtained from the following sources: caffeine from Wako; theobromine, theophylline, and 8-chlorotheophylline from Tokyo Kasei; paraxanthine and 1,3,7-trimethyluric acid from Funakoshi; TAO, 3-methylcholanthrene, phenobarbital, dexamethasone, and pregnenolone 16α-carbonitrile from the Sigma Chemical Co.; anti-rat CYP3A2 serum for inhibition studies from the Daiichi Pure Chemical Co.; and glucose-6-phosphate, glucose-6-phosphate dehydrogenase, and NADPH from the Oriental Yeast Co.

Results and Discussion

Caffeine N-demethylase and C-8 hydroxylase activities were assayed in liver microsomes from Wistar and DA rats of both sexes (7 weeks old). We determined the amounts of theobromine (N-1 demethylation), paraxanthine (N-3 demethylation), theophylline (N-7 demethylation), and 1,3,7-trimethyluric acid (C-8 hydroxylation) by HPLC after incubating caffeine with rat liver microsomes.

The main metabolite of caffeine in rats was 1,3,7-trimethyluric acid (C-8 hydroxylation formation), which amounted to

Acknowledgements

We thank Dr. H. Iwata, Y. Yamamoto, T. Tasaki, M. Ishizuka, H. Hoshi, Y. Shimamoto, M. Akamatsu, I. Chiba, and K. Hirose for many helpful discussions, Professor W. Jones for useful advice on the writing of this manuscript, and A. Takahashi and S. Yoshino for their secretarial assistance in the preparation of this manuscript. This study was supported, in part, by a Grant-in-Aid for scientific research from the Ministry of Education, Sciences, Sports and Culture of Japan.

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    Present address: Drug Metabolism and Pharmacokinetics Lab., Chugai Pharmaceutical Co., Ltd., 3-41-8, Takada, Toshima-ku, Tokyo 171, Japan.

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