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Polymorphic drug metabolism: studies with recombinant Chinese hamster cells and analyses in human populations
T Yokoi1, M Sawada, T Kamataki
1Division of Drug Metabolism, Faculty of Pharmaceutical Sciences, Hokkaido University, Japan.
Abstract:
Most promutagens and procarcinogens exert their genotoxicity after undergoing metabolic activation. Metabolism of chemicals is an important factor in limiting the extent of the action of a chemical. In this study, we established cell lines which carried cDNAs coding for human CYP1A2 and N-acetyltransferase (NAT); the latter functions as O-acetyltransferase for N-hydroxyarylamines formed by CYP1A2. A cell line which expresses CYP1A2 together with P450 reductase activated aflatoxin B1, but not heterocyclic amines. A cell line which carries CYP1A2 and polymorphic NAT (NAT2) in addition to P450 reductase efficiently activated IQ and some other heterocyclic amines. However, a cell line which carries CYP1A2 and monomorphic NAT (NAT1) had only low activity toward the same heterocyclic amines. In order to determine the presence of and frequency of genetic polymorphisms of CYP1A2 and NAT2 in humans, we performed in caffeine phenotyping test on 205 Japanese volunteers. Analyses of metabolic ratios of urinary metabolites showed a bimodal distribution, indicating that about 86% and 91% of Japanese were extensive metabolizers (EM) of CYP1A2 and NAT2, respectively. The genotype NAT2 determined by the PCR-RFLP method agreed completely with the phenotype. To determine the mechanism of the differences in CYP1A2 activity, genomic DNA from peripheral lymphocytes of poor metabolizers (PM) and EM was subjected to DNA sequencing. No differences in nucleotide sequence were observed between PMs and EMs in the exons, exon-intron junctions and 5'-flanking region of the CYP1A2 gene.(ABSTRACT TRUNCATED AT 250 WORDS)
Insights
This study developed cell lines to investigate chemical metabolism, finding that specific enzymes like CYP1A2 and NAT2 are crucial for activating heterocyclic amines and that most Japanese individuals are extensive metabolizers of these enzymes.
Area of Science:
- Biochemistry
- Toxicology
- Pharmacogenetics
Background:
- Chemicals often require metabolic activation to exert genotoxicity.
- Cytochrome P450 1A2 (CYP1A2) and N-acetyltransferase (NAT) enzymes play key roles in metabolizing promutagens and procarcinogens.
- Genetic variations in these enzymes can influence individual susceptibility to chemical toxicity.
Purpose of the Study:
- To establish and characterize cell lines expressing human CYP1A2 and NAT enzymes for studying metabolic activation.
- To investigate the role of polymorphic NAT2 versus monomorphic NAT1 in the activation of heterocyclic amines.
- To determine the frequency of genetic polymorphisms in CYP1A2 and NAT2 in a Japanese population.
Main Methods:
- Development of cell lines co-expressing CYP1A2, P450 reductase, and either NAT1 or NAT2.
- Activation assays using aflatoxin B1 and heterocyclic amines (e.g., IQ).
- Caffeine phenotyping tests on 205 Japanese volunteers to assess CYP1A2 and NAT2 metabolic activity.
- Genotyping of NAT2 using PCR-RFLP and sequencing of CYP1A2 in extensive and poor metabolizers.
Main Results:
- Cell lines expressing CYP1A2 and NAT2 efficiently activated heterocyclic amines, while those with NAT1 showed low activity.
- Caffeine phenotyping revealed that approximately 86% and 91% of Japanese volunteers were extensive metabolizers (EM) of CYP1A2 and NAT2, respectively.
- NAT2 genotype analysis confirmed phenotype, and CYP1A2 sequencing found no sequence variations in EMs versus poor metabolizers (PMs) in coding and regulatory regions.
Conclusions:
- The combination of CYP1A2 and NAT2 is critical for the metabolic activation of certain heterocyclic amines.
- The Japanese population studied exhibits a high prevalence of extensive metabolizers for both CYP1A2 and NAT2.
- CYP1A2 polymorphisms influencing metabolic activity in this population do not appear to stem from sequence variations in the analyzed gene regions.