Comparison of Polymorphisms in Genes Involved in Polycyclic Aromatic Hydrocarbon Metabolism with Urinary Phenanthrene Metabolite Ratios in Smokers

University of Minnesota Duluth, Duluth, Minnesota, United States
Cancer Epidemiology Biomarkers & Prevention (Impact Factor: 4.13). 10/2006; 15(10):1805-11. DOI: 10.1158/1055-9965.EPI-06-0173
Source: PubMed


The hypothesis that interindividual differences among smokers in the metabolism of polycyclic aromatic hydrocarbons (PAH) are related to lung cancer risk has been extensively investigated in the literature. These studies have compared lung cancer risk in groups of smokers with or without polymorphisms in genes involved in PAH metabolism. We believe that carcinogen metabolite phenotyping, involving the actual measurement of PAH metabolites, would be a better way to investigate differences in lung cancer risk. With this goal in mind, we have developed methods for quantifying phenanthrene metabolites in urine. Phenanthrene is the simplest PAH with a bay region, a feature closely associated with carcinogenicity. The urinary metabolite r-1,t-2,3,c-4-tetrahydroxy-1,2,3,4-tetrahydrophenanthrene (PheT) is a measure of metabolic activation, whereas phenanthrols (HOPhe) are a measure of detoxification. In this study, we quantified urinary PheT/HOPhe ratios in 346 smokers who were also genotyped for 11 polymorphisms in genes involved in PAH metabolism: CYP1A1MspI, CYP1A1I462V, CYP1B1R48G, CYP1B1A119S, CYP1B1L432V, CYP1B1N453S, EPHX1Y113H, EPHX1H139R, GSTP1I105V, GSTP1A114V, and GSTM1 null. The geometric mean molar PheT/3-HOPhe ratio was 4.08 (95% confidence interval, 3.79-4.39). Ten percent of the smokers had PheT/3-HOPhe ratios of > or =9.90. We found a significant association between the presence of the CYP1A1I462V polymorphism and high PheT/3-HOPhe ratios (P = 0.02). This effect was particularly strong in females and in combination with the GSTM1 null polymorphism. In contrast, the CYP1B1R48G and CYP1B1A119S polymorphisms were associated with significantly lower PheT/3-HOPhe ratios, particularly in Blacks. There were no consistent significant effects of any of the other polymorphisms on PheT/3-HOPhe ratios. The highest 10% of PheT/3-HOPhe ratios could not be predicted by the presence of any of the 11 polymorphisms individually or by certain combinations. The effects of the CYP1A1I462 polymorphism observed here, particularly in combination with GSTM1 null, are quite consistent with reports in the literature. However, the results of this study indicate that genotyping is not an effective way to predict PAH metabolism at least as represented by PheT/HOPhe ratios.

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    • "Genotyping was performed by using the iPLEX Gold method (Sequenom, San Diego, CA). Similar methods have been reported previously (Hecht et al., 2006). In brief, the method is based on the primer-extension reaction that generates allele-specific products with distinct masses detected by matrixassisted laser desorption/ionization time-of-flight mass spectrometry . "
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    ABSTRACT: Polycyclic aromatic hydrocarbons (PAHs) in cigarette smoke are among the most likely causes of lung cancer. PAHs require metabolic activation to initiate the carcinogenic process. Phenanthrene (Phe), a noncarcinogenic PAH, was used as a surrogate of benzo[α]pyrene and related PAHs to study the metabolic activation of PAHs in smokers. A dose of 10 μg of deuterated Phe ([D₁₀]Phe) was administered to 25 healthy smokers in a crossover design, either as an oral solution or by smoking cigarettes containing [D₁₀]Phe. Phe was deuterated to avoid interference from environmental Phe. Intensive blood and urine sampling was performed to quantitate the formation of deuterated r-1,t-2,3,c-4-tetrahydroxy-1,2,3,4-tetrahydrophenanthrene ([D₁₀]PheT), a biomarker of the diol epoxide metabolic activation pathway. In both the oral and smoking arms approximately 6% of the dose was metabolically converted to diol epoxides, with a large intersubject variability in the formation of [D₁₀]PheT observed. Two diagnostic plots were developed to identify subjects with large systemic exposure and significant lung contribution to metabolic activation. The combination of the two plots led to the identification of subjects with substantial local exposure. These subjects produced, in one single pass of [D₁₀]Phe through the lung, a [D₁₀]PheT exposure equivalent to the systemic exposure of a typical subject and may be an indicator of lung cancer susceptibility. Polymorphisms in PAH-metabolizing genes of the 25 subjects were also investigated. The integration of phenotyping and genotyping results indicated that GSTM1-null subjects produced approximately 2-fold more [D₁₀]PheT than did GSTM1-positive subjects.
    Full-text · Article · Jun 2012 · Journal of Pharmacology and Experimental Therapeutics
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    ABSTRACT: There are major interindividual differences in carcinogenic polycyclic aromatic hydrocarbon (PAH) metabolism in humans, and it has been hypothesized that these differences may be related to cancer risk in smokers and other exposed people. One important pathway of PAH metabolism involves the detoxification of the epoxide and diol epoxide metabolites by reaction with glutathione, catalyzed by glutathione-S-transferases (GSTs). Interindividual differences in these pathways have been examined by genotyping methods, investigating polymorphisms in GSTM1 and GSTP1. We are developing a phenotyping approach to assessing individual differences in PAH metabolism by quantifying human urinary metabolites of the ubiquitous PAH phenanthrene (1). In this study, we developed a method for quantitation of a mercapturic acid, N-acetyl-S-(9,10-dihydro-9-hydroxy-10-phenanthryl)-l-cysteine (PheO-NAC, 12), the end product of the reaction of phenanthrene-9,10-epoxide (11) with glutathione. [D(10)]PheO-NAC was added to the urine as internal standard, and the PheO-NAC fraction was enriched by solid-phase extraction. PheO-NAC was quantified by liquid chromatography electrospray ionization tandem mass spectrometry with selected reaction monitoring. The detection limit was approximately 4 fmol/mL of urine. PheO-NAC was detected in the urine of 46 of 104 smokers, mean (S.D.) 57.9 +/- 144 fmol/mL. PheO-NAC was detected significantly more frequently (P < 0.0001) in subjects who were GSTM1 positive than in those who were GSTM1 null, and the levels of PheO-NAC were significantly higher in the GSTM1 positive subjects, consistent with a role for GSTM1 in the detoxification of phenanthrene-9,10-epoxide. There were no significant relationships between PheO-NAC levels and the occurrence of two GSTP1 polymorphisms. The results of this study provide the first evidence for a PAH-derived mercapturic acid in human urine and should be useful in the development of a phenotyping approach to assess individual differences in PAH metabolism.
    Preview · Article · Oct 2006 · Chemical Research in Toxicology
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    ABSTRACT: Previous studies of the metabolic activation of chemical carcinogens including benzo(a)pyrene have demonstrated that selected aromatic hydrocarbons can undergo a series of biological substitution reactions in tissue cytosol preparations when in the presence of the methyl donor S-adenosyl-L-methionine. Although identification of the methylated metabolites of carcinogens had been tentatively established using liquid chromatographic techniques, confirmation of the identity of these methylated derivatives has remained elusive. In the present study, the carcinogen benzo(a)pyrene was found to undergo methylation reactions in the presence of rat liver cytosol preparations to yield products that were identified as methylated, formyl and hydroxyalkyl derivatives of the parent carcinogen. Identification and confirmation of the identity of the 6-methylbenzo(a)pyrene metabolite was confirmed by both UV detection, gas chromatographic and mass spectral identification, and NMR analysis of metabolic fractions obtained from incubations of benzo(a)pyrene. The results conclusively demonstrate that the carcinogen benzo(a)pyrene undergoes biological substitution reactions to yield 6-methylbenzo(a)pyrene in the presence of cytosolic enzymes. The presence of 6-methylbenzo(a)pyrene as a metabolite of benzo(a)pyrene illustrates and confirms the presence of centers of reactivity in unsubstituted aromatic hydrocarbons. These centers of reactivity serve as sites for biochemical substitution reactions, yielding methyl substituted hydrocarbons. Furthermore, these methylated metabolites of polycyclic aromatic hydrocarbons are further metabolically activated to hydroxyalkyl and formyl derivatives which can be subsequently activated to electrophilic esters leading to DNA and protein reactive species.
    No preview · Article · Aug 2007 · Polycyclic Aromatic Compounds
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