Elsevier

Biochemical Pharmacology

Volume 58, Issue 7, 1 October 1999, Pages 1201-1208
Biochemical Pharmacology

Gene Expression and Development
Participation of CYP2C8 in retinoic acid 4-hydroxylation in human hepatic microsomes

https://doi.org/10.1016/S0006-2952(99)00192-6Get rights and content

Abstract

Cytochromes P450 (CYPs) catalyze the 4-hydroxylation of all-trans-retinoic acid (ATRA), an agent used in the treatment of certain malignancies. Literature studies have implicated several CYPs in this reaction, but the relative importance of individual CYPs is unclear. Human microsomal CYPs that contribute to the activity were evaluated by correlation with activities of hepatic drug-metabolizing CYPs, the capacity of cDNA-derived CYPs to catalyze the reaction, and inhibition of the microsomal activity by chemicals. 4-HydroxyATRA formation in microsomes varied 7-fold (8.7 to 61 pmol/mg protein/min) and correlated partially with activities mediated by CYPs 3A, 2C, and 1A (ρ = 0.53 to 0.66). cDNA-derived CYPs 2C8, 2C9, and 3A4, but not 1A1 or 1A2, catalyzed ATRA 4-hydroxylation (2.53, 4.68, and 1.29 pmol/pmol CYP/hr). The Km for the reaction was 9 ± 3 μM in hepatic microsomes (N = 3) and 6 μM in microsomes containing cDNA-derived CYP2C8; by comparison, Km values for the activity mediated by CYPs 2C9 and 3A4 were 100 and 74 μM, respectively. Inhibition of microsomal ATRA 4-hydroxylation was elicited by chemicals that interact with CYP2C8 (paclitaxel and diclofenac), but not those that interact with CYP2C9 (sulfaphenazole, tolbutamide, and torasemide). The CYP3A inhibitor troleandomycin and an anti-CYP3A IgG inhibited the activity slightly. Greater inhibition was produced by the less selective CYP3A inhibitors parathion, quinidine, and ketoconazole; CYP1A inhibitors were ineffective. These findings suggest that CYP2C8 is a major contributor to ATRA 4-hydroxylation in human liver and that 3A subfamily CYPs may be minor participants. Individual variation in CYP2C8 and 3A4 expression may influence ATRA pharmacokinetics and drug interactions during therapy.

Section snippets

Chemicals

[11,13-3H]ATRA (50–60 Ci/mmol) was obtained from Amrad Pharmacia Biotech Australia. [4-14C]Testosterone (56 mCi/mmol) and [1-14C]lauric acid (56 mCi/mmol) were from Amersham. Torasemide was a gift from Boehringer Mannheim, ketoconazole was from Janssen Pharmaceutica, parathion was from Rhone-Poulenc, and cyclophosphamide was from Farmitalia Carlo Erba; diclofenac and sulfaphenazole were from Ciba-Geigy, and tolbutamide, chlorpropamide, and hydroxytolbutamide were provided by Hoechst

Interindividual variation in ATRA metabolism in human liver

Microsomal fractions were derived from excess tissue that was available after the transplantation of cut-down adult livers into pediatric recipients, or the normal margin adjacent to tumor tissue in patients undergoing hepatic resection. ATRA 4-hydroxylation in these livers varied over an approximate 7-fold range (8.7 to 61 pmol 4-hydroxyATRA formed/mg protein/min; Table 1). The recent drug histories of the donors are also shown in Table 1; some were reported in previous studies 23, 24. Five

Discussion

The results of the present investigation implicate a major role for CYP2C8 and a possible minor role for CYP3A4 in the biotransformation of ATRA in human liver. The Michaelis constants for ATRA 4-hydroxylation in three human hepatic microsomal preparations (9.0 ± 3.4 μM) were quite similar to that exhibited by cDNA-expressed CYP2C8 (6 μM) and different from those mediated by cDNA-expressed CYPs 2C9 and 3A4 (>70 μM). Thus, CYP2C8 supported ATRA 4-hydroxylation most efficiently, with CYP2C9 and

Acknowledgements

This work was supported by grants from the New South Wales Cancer Council and the Australian National Health and Medical Research Council. L. N. was the recipient of a Westmead Hospital Initiating Grant and a postgraduate scholarship from the Gastroenterological Society of Australia. The assistance of staff of the Queensland Liver Transplant Service at Princess Alexandra Hospital and the Australian Liver Transplant Center at Royal Prince Alfred Hospital in the supply of human liver tissue for

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