Functional characterization of eight human cytochrome P450 1A2 gene variants by recombinant protein expression

Palma, Bernardo Brito; Sousa, Marta Silva E; Vosmeer, C. Ruben; Lastdrager, Jeroen; Rueff, José; Vermeulen, Nico; Kranendonk, Michel

doi:10.1038/tpj.2010.2

Cited by 26 publications

(22 citation statements)

References 49 publications

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“…This approach requires modifications of the N terminus to maximize expression and has been successful in characterizing several CYP1A2 polymorphisms, including CYP1A I386F (CYP1A2*4), CYP1A2 C406Y (CYP1A2*5), CYP1A2 D348N, and CYP1A2 R456H [37, 38]). Interestingly, the allele CYP1A2 C406Y actually confers higher k cat / K m (about 2-fold) and a higher mutagenic response after exposure to low doses of 2-amino-3,5-dimethylimadazo[4,5-f]quinoline (MeIQ) in an Escherichia coli mutagenesis assay [37].…”

Section: Introductionmentioning

confidence: 99%

Activation of aflatoxin B1 by expression of human CYP1A2 polymorphisms in Saccharomyces cerevisiae

Fasullo

Smith

Egner

et al. 2014

Mutation Research/Genetic Toxicology and Environmental Mutagene

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Human susceptibility to environmental carcinogens is highly variable and depends on multiple genetic factors, including polymorphisms in cytochrome P450 genes. Although epidemiological studies have identified individual polymorphisms in cytochrome P450 genes that may alter cancer risk, there is often conflicting data about whether such polymorphisms alter the genotoxicity of environmental carcinogens. This is particularly true of the CYP1A2 polymorphisms that confer differential activation of multiple human carcinogens. To determine whether a single cytochrome P450 polymorphism confers higher levels of carcinogen-associated genotoxicity, we chose an organism that lack enzymes to metabolically activate aflatoxins and expressed individual human P450 genes in budding yeast. We measured the frequencies of recombination, Rad51 foci formation, 7-methoxyresorufin O-demethylase, and the concentrations of carcinogen-associated DNA adducts in DNA repair proficient yeast expressing P450 polymorphisms after exposure to aflatoxin B1 (AFB1). We measured growth of rad4 rad51 cells expressing CYP1A2 polymorphisms while exposed to AFB1. We observed that there was significantly less AFB1-associated genotoxicity in yeast expressing I386F, while yeast expressing CYP1A2 C406Y exhibited intermediate levels of genotoxicity compared to yeast expressing CYP1A2 D348N or wild type. We conclude that differences in carcinogen genotoxicity can be observed in yeast expressing different CYP1A2 alleles. This is the first report that carcinogen-associated P450 polymorphisms can be studied in yeast.

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Section: Introductionmentioning

confidence: 99%

Activation of aflatoxin B1 by expression of human CYP1A2 polymorphisms in Saccharomyces cerevisiae

Fasullo

Smith

Egner

et al. 2014

Mutation Research/Genetic Toxicology and Environmental Mutagene

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“…In human CYPOR, the loss of FAD and FMN from its binding site caused by mutation is the major cause of diminished P450 activity. For example, the R457H and V492E mutations at the FAD-binding site in human CYPOR result in an impaired function of CYP17A1 (17a-hydroxylase/17, 20-lyase), CYP19A1 (aromatase) and CYP1A2 in vitro [23,28–30]. Recently, a study on heme oxygenase I (HO-I) activity also indicated a decrease in CYPOR activity affected HO-I catalytic rate, and rate of bilirubin formation by HO-I enzyme is CYPOR-activity dependent [31].…”

Section: Resultsmentioning

confidence: 99%

Modeling of Anopheles minimus Mosquito NADPH-Cytochrome P450 Oxidoreductase (CYPOR) and Mutagenesis Analysis

Sarapusit

Lertkiatmongkol

Duangkaew

et al. 2013

IJMS

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Malaria is one of the most dangerous mosquito-borne diseases in many tropical countries, including Thailand. Studies in a deltamethrin resistant strain of Anopheles minimus mosquito, suggest cytochrome P450 enzymes contribute to the detoxification of pyrethroid insecticides. Purified A. minimus CYPOR enzyme (AnCYPOR), which is the redox partner of cytochrome P450s, loses flavin-adenosine di-nucleotide (FAD) and FLAVIN mono-nucleotide (FMN) cofactors that affect its enzyme activity. Replacement of leucine residues at positions 86 and 219 with phenylalanines in FMN binding domain increases FMN binding, enzyme stability, and cytochrome c reduction activity. Membrane-Bound L86F/L219F-AnCYPOR increases A. minimus P450-mediated pyrethroid metabolism in vitro. In this study, we constructed a comparative model structure of AnCYPOR using a rat CYPOR structure as a template. Overall model structure is similar to rat CYPOR, with some prominent differences. Based on primary sequence and structural analysis of rat and A. minimus CYPOR, C427R, W678A, and W678H mutations were generated together with L86F/L219F resulting in three soluble Δ55 triple mutants. The C427R triple AnCYPOR mutant retained a higher amount of FAD binding and increased cytochrome c reduction activity compared to wild-type and L86F/L219F-Δ55AnCYPOR double mutant. However W678A and W678H mutations did not increase FAD and NAD(P)H bindings. The L86F/L219F double and C427R triple membrane-bound AnCYPOR mutants supported benzyloxyresorufin O-deakylation (BROD) mediated by mosquito CYP6AA3 with a two-to three-fold increase in efficiency over wild-type AnCYPOR. The use of rat CYPOR in place of AnCYPOR most efficiently supported CYP6AA3-mediated BROD compared to all AnCYPORs.

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“…Moreover, this would not necessarily imply a decrease in the enzyme activity toward the production of caffeine metabolites in carriers of the 22467 deletion, which we did not observe in this study. In fact, Palma et al [2010] recently showed that although CYP1A2 genetic variation appears to cause relatively minor activity changes, some variants can have profound effects on particular CYP1A2-mediated biotransformation in vitro. Interestingly, the authors demonstrated that 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), a carcinogen present in tobacco smoke, was the most discriminative of the substrates assayed [Palma et al, 2010].…”

Section: Discussionmentioning

confidence: 99%

Haplotypes in the 5′‐untranslated region of the CYP1A2gene are inversely associated with lung cancer risk but do not correlate with caffeine metabolism

Gervasini

Ghotbi

Aklillu

et al. 2012

Environ and Mol Mutagen

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In this study, we analyzed the influence of CYP1A2 genetic variation and enzyme activity on lung cancer risk in a high-incidence area. A total of 95 lung cancer patients and 196 controls were genotyped for the -3860G/A, -3113A/G, -2467T/delT, -739T/G, and -163C/A polymorphisms in the 5'-untranslated region of the gene. In addition, a subset of 70 patients and 115 controls were phenotyped by high-performance liquid chromatography determination of the caffeine metabolic ratio (CMR). The -2467T/delT polymorphism and the CYP1A2*1V haplotype (-163C>A, -2467T>delT) were inversely associated with lung cancer risk (odds ratio [OR] = 0.47 [0.2-0.9]; P = 0.02 and OR = 0.13 [0.02-1.0]; P = 0.04; respectively). In addition, the CYP*1A/*1V and *1F (-163C>A)/*1D (-163C>A, -2467T>delT) diplotypes were absent in the patients group, whereas accounting for 7.1% (P = 0.017) and 5.6% (P = 0.037) of controls, respectively. Mean CMR was significantly higher in patients than in controls (10.50 ± 17.31 vs. 6.52 ± 6.26, P = 0.01) but regression analyses did not yield significant ORs for the association with lung cancer risk. Similarly, no significant correlations were found between any genetic variant and enzyme activity. Several CYP1A2 haplotypes and diplotypes containing the -2467delT variant were associated with lower lung cancer risk; however, they did not correlate with significant changes in CYP1A2 metabolic activity toward caffeine.

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Functional characterization of eight human cytochrome P450 1A2 gene variants by recombinant protein expression

Cited by 26 publications

References 49 publications

Activation of aflatoxin B1 by expression of human CYP1A2 polymorphisms in Saccharomyces cerevisiae

Activation of aflatoxin B1 by expression of human CYP1A2 polymorphisms in Saccharomyces cerevisiae

Modeling of Anopheles minimus Mosquito NADPH-Cytochrome P450 Oxidoreductase (CYPOR) and Mutagenesis Analysis

Haplotypes in the 5′‐untranslated region of the CYP1A2gene are inversely associated with lung cancer risk but do not correlate with caffeine metabolism

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