Abstract
Human cytochrome P450 2A13 (CYP2A13), which is highly efficient in the metabolic activation of a major tobacco-specific carcinogen, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), may play important roles in xenobiotic toxicity and tobacco-related tumorigenesis in the respiratory tract. The aim of this study was to identify any genetic polymorphisms of theCYP2A13 gene, which may alter the metabolic capacities of the enzyme. Polymerase chain reaction (PCR) single-strand conformational polymorphism analysis was used to identify single-nucleotide polymorphisms (SNPs) in all of the exons and at the exon-intron boundaries, and PCR-restriction fragment length polymorphism analysis and DNA sequencing were used to determine the frequencies of the newly identified variant alleles in the four major ethnic groups. Blood spot DNA from more than 100 individuals was used for these analyses. Seven variant alleles were found, but only one SNP was detected in the coding region, in exon 5, leading to an Arg257Cys amino acid change. The frequencies of the Arg257Cys allele in white, black, Hispanic, and Asian individuals are 1.9%, 14.4%, 5.8%, and 7.7%, respectively. Functional analysis of the variant protein was performed following its heterologous expression. The Arg257Cys variant was 37 to 56% less active than the wild-type Arg-257 protein toward all substrates tested. With NNK, Cys-257 had higher Km and lowerVmax values than did Arg-257, with a >2-fold decrease in catalytic efficiency. The Arg257Cys mutation could provide some protection against xenobiotic toxicity in the respiratory tract to individuals who are homozygous for the Cys-257 allele.
Footnotes
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This work was supported in part by Research Grant ES07462 from the National Institute of Environmental Health Sciences, National Institutes of Health (Bethesda, MD).
- Abbreviations:
- NNK
- 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone
- P450
- cytochrome P450
- SNP
- single-nucleotide polymorphism
- PCR
- polymerase chain reaction
- SSCP
- single-strand conformational polymorphism
- NP-10
- Nonidet P-10
- RFLP
- restriction fragment length polymorphism
- bp
- base pair(s)
- HMPA
- hexamethylphosphoramide
- PEG
- polyethylene glycol
- HTP
- hydroxyapatite
- Received February 21, 2002.
- Accepted March 25, 2002.
- The American Society for Pharmacology and Experimental Therapeutics
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