Cigarette smoking enhances the metabolic activation of the polycyclic aromatic hydrocarbon phenanthrene in humans.

Although it is well established that human cytochrome P450 1 family enzymes are induced by cigarette smoking through activation of the Ah receptor, it is not known whether this leads to increased metabolic activation or detoxification of carcinogenic polycyclic aromatic hydrocarbons (PAH), which are present in cigarette smoke and the general environment. We gave oral doses of deuterated phenanthrene ([D10]Phe), a non-carcinogenic surrogate of carcinogenic PAH such as benzo[a]pyrene, to smokers (N = 170, 1 or 10 µg doses) and non-smokers (N = 57, 1 µg dose). Bioactivation products (dihydrodiol and tetraol) and detoxification products (phenols) of [D10]Phe were determined in 6-h urine to obtain a comprehensive metabolic profile. Cigarette smoking increased the bioactivation of [D10]Phe and decreased its detoxification resulting in significantly different metabolic patterns between smokers and non-smokers (P < 0.01), consistent with increased cancer risk in smokers. The Phe bioactivation ratios ([D10]PheT/total [D9]OHPhe) were significantly higher (2.3 (P < 0.01) to 4.8 (P < 0.001) fold) in smokers than non-smokers. With solid human in vivo evidence, our results for the first time demonstrate that cigarette smoking enhances the metabolic activation of Phe, structurally representative of carcinogenic PAH, in humans, strongly supporting their causal role in cancers caused by smoking. The results suggest potential new methods for identifying smokers who could be at particularly high risk for cancer.

Quantitation of phenanthrene dihydrodiols in the urine of smokers and non-smokers by gas chromatography-negative ion chemical ionization-tandem mass spectrometry.

Polycyclic aromatic hydrocarbons (PAH) are well-established environmental carcinogens likely to be causative agents for some human cancers. Bay-region diol epoxides are ultimate carcinogenic metabolites of multiple PAH. Dihydrodiols are the important intermediate products of this pathway and can be further oxidized to form diol epoxides. We quantified two dihydrodiol metabolites of phenanthrene (Phe), the simplest PAH with a bay-region, in the 6 h urine of smokers (N = 25) and non-smokers (N = 25) using a newly developed and validated analytical method. After hydrolysis by ß-glucuronidase and sulfatase, and solid phase extraction, the sample was silylated and analyzed by gas chromatography-negative ion chemical ionization-tandem mass spectrometry (GC-NICI-MS/MS). Levels (nmol/6h urine) of Phe-1,2-dihydrodiol (Phe-1,2-D) and Phe-3,4-dihydrodiol (Phe-3,4-D) were 2.04 ± 1.52 and 0.51 ± 0.35 , respectively, in smokers, significantly higher than those in non-smokers (1.35 ± 1.11 of Phe-1,2-D, p < 0.05; 0.27 ± 0.25 of Phe-3,4-D, p < 0.005). Cigarette smoking also influenced the regioselective metabolism of Phe, presenting as a significant difference in the urinary distribution pattern of Phe-1,2-D and Phe-3,4-D between smokers and non-smokers: the ratio Phe-3,4-D: Phe-1,2-D increased from 0.20 in non-smokers to 0.28 in smokers (p < 0.01), which can be explained by the induction of the phenanthrene metabolizing enzymes CYP1A2 and CYP1B1 by cigarette smoke. The method described here is the first example of facile quantitation of an intact human dihydrodiol metabolite of any PAH with three or more aromatic rings and will be applicable in clinical and molecular epidemiology studies of PAH metabolism and cancer susceptibility.

Methyl DNA phosphate adduct formation in lung tumor tissue and adjacent normal tissue of lung cancer patients.

The formation of methyl DNA adducts is a critical step in carcinogenesis initiated by the exposure to methylating carcinogens. Methyl DNA phosphate adducts, formed by methylation of the oxygen atoms of the DNA phosphate backbone, have been detected in animals treated with methylating carcinogens. However, detection of these adducts in human tissues has not been reported. We developed an ultrasensitive liquid chromatography-nanoelectrospray ionization-high resolution tandem mass spectrometry method for detecting methyl DNA phosphate adducts. Using 50 µg of human lung DNA, a limit of quantitation of two adducts/1010 nucleobases was achieved. Twenty-two structurally unique methyl DNA phosphate adducts were detected in human lung DNA. The adduct levels were measured in both tumor and adjacent normal tissues from 30 patients with lung cancer, including 13 current smokers and 17 current non-smokers, as confirmed by measurements of urinary cotinine and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol. Levels of total methyl DNA phosphate adducts in normal lung tissues were higher in smokers than non-smokers, with an average of 13 and 8 adducts/109 nucleobases, respectively. Methyl DNA phosphate adducts were also detected in lung tissues from untreated rats with steady-state levels of 5-7 adducts/109 nucleobases over a period of 70 weeks. This is the first study to report the detection of methyl DNA phosphate adducts in human lung tissues. The results provide new insights toward using these DNA adducts as potential biomarkers to study human exposure to environmental methylating carcinogens.

Ultrasensitive High-Resolution Mass Spectrometric Analysis of a DNA Adduct of the Carcinogen Benzo[a]pyrene in Human Lung.

Benzo[a]pyrene (BaP), an archetypical polycyclic aromatic hydrocarbon, is classified as "carcinogenic to humans" and is ubiquitous in the environment, as evident by the measurable levels of BaP metabolites in virtually all human urine samples examined. BaP carcinogenicity is believed to occur mainly through its covalent modification of DNA, resulting in the formation of BPDE-N2-dG, an adduct formed between deoxyguanosine and a diol epoxide metabolite of BaP, with subsequent mutation of critical growth control genes. In spite of the liquid chromatography-mass spectrometry (LC-MS)-based detection of BPDE-N2-dG in BaP-treated rodents, and indirectly through high-performance liquid chromatography (HPLC)-fluorescence detection of BaP-7,8,9,10-tetraols released from human DNA upon acid hydrolysis, BPDE-N2-dG adducts have rarely if ever been observed directly in human samples using LC-MS techniques, even though sophisticated methodologies have been employed which should have had sufficient sensitivity. With this in mind, we developed a liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) methodology employing high-resolution/accurate mass analysis for detecting ultratrace levels of these adducts. These efforts are directly translatable to the development of sensitive detection of other small molecules using trap-based LC-ESI-MS/MS detection. The developed methodology had a limit of detection (LOD) of 1 amol of BPDE-N2-dG on-column, corresponding to 1 BPDE-N2-dG adduct per 1011 nucleotides (1 adduct per 10 human lung cells) using 40 µg of human lung DNA. To our knowledge, this is the most sensitive DNA adduct quantitation method yet reported, exceeding the sensitivity of the 32P-postlabeling assay (∼1 adduct per 1010 nucleotides). Twenty-nine human lung DNA samples resulted in 20 positive measurements above the LOD, with smoker and nonsmoker DNA containing 3.1 and 1.3 BPDE-N2-dG adducts per 1011 nucleotides, respectively.

Longitudinal study of [D10]phenanthrene metabolism by the diol epoxide pathway in smokers.

The extent of metabolism of [D10]phenanthrene to [D(10)]r-1,t-2,3,c-4-tetrahydroxy-1,2,3,4-tetradeuterophenanthrene ([D10]PheT) could be a biomarker of human metabolic activation of carcinogenic polycyclic aromatic hydrocarbons, leading to identification of smokers particularly susceptible to lung cancer. The longitudinal stability of [D10]PheT was evaluated in 24 cigaret smokers given 7-8 oral doses of [D10]phenanthrene (10 µg) over 5.5 months. [D10]PheT in 6 h urine was quantified after each dose. The overall coefficient of variation for 24 subjects was (mean ± S.D.) 27.4% ± 8.83%. Thus, a single administration of [D10]phenanthrene is likely sufficient to determine a smoker's ability to metabolize it to [D10]PheT.

Phenanthrene metabolism in smokers: use of a two-step diagnostic plot approach to identify subjects with extensive metabolic activation.

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.

Metabolism of [D10]phenanthrene to tetraols in smokers for potential lung cancer susceptibility assessment: comparison of oral and inhalation routes of administration.

Polycyclic aromatic hydrocarbons (PAHs) are believed to be among the causative agents for lung cancer in smokers. PAHs require metabolic activation for carcinogenicity. One pathway produces diol epoxides that react with DNA, causing mutations. Because diol epoxides are converted to tetraols, quantitation of tetraols can potentially be used to identify smokers who may be at higher risk for lung cancer. Our approach uses [D(10)]phenanthrene, a labeled version of phenanthrene, a noncarcinogenic PAH structurally analogous to carcinogenic PAH. Although smokers are exposed to PAH by inhalation, oral dosing would be more practical for phenotyping studies. Therefore, we investigated [D(10)]phenanthrene metabolism in smokers after administration by inhalation in cigarette smoke or orally. Sixteen smokers received 10 µg of [D(10)]phenanthrene in a cigarette or orally. Plasma and urine samples were analyzed for [D(10)]r-1,t-2,3,c-4-tetrahydroxy-1,2,3,4-tetrahydrophenanthrene ([D(10)]PheT), the major end product of the diol epoxide pathway, by gas chromatography-negative ion chemical ionization-tandem mass spectrometry. The ratios of [D(10)]PheT (oral dosing/inhalation) in 15 smokers were 1.03 ± 0.32 and 1.02 ± 0.35, based on plasma area under the concentration-time curve (0-∞) and total 48-h urinary excretion, respectively. Overall, there was no significant difference in the extent of [D(10)]PheT formation after the two different routes of exposure in smokers. A large interindividual variation in [D(10)]PheT formation was observed. These results demonstrate that the level of [D(10)]PheT in urine after oral dosing of [D(10)]phenanthrene can be used to assess individual capacity of PAH metabolism by the diol epoxide pathway.

Quantitation of a minor enantiomer of phenanthrene tetraol in human urine: correlations with levels of overall phenanthrene tetraol, benzo[a]pyrene tetraol, and 1-hydroxypyrene.

Polycyclic aromatic hydrocarbons (PAH) are well established carcinogens that are likely to play a role in causing some human cancers. One accepted pathway of PAH metabolic activation is the formation of bay region diol epoxides. Some individuals may be particularly susceptible to PAH carcinogenesis because they metabolically activate PAH more effectively than others. We have used the measurement of urinary phenanthrene tetraols (Phe-tetraols) as a biomarker of PAH exposure plus metabolic activation since bay region diol epoxides are hydrolyzed to tetraols. Because of stereoselectivity in Phe metabolism, Phe-(1R,2S,3R,4S)-tetraol (4) results mainly from the bay region diol epoxide pathway, and Phe-(1S,2R,3S,4R)-tetraol (7) is formed mainly from the reverse diol epoxide pathway, not generally associated with carcinogenicity. The latter pathway accounts for more than 95% of human urinary Phe-tetraol. In most previous studies, Phe-tetraol was quantified without enantiomeric resolution, using a relatively rapid and practical method, applicable to large studies. It was not clear, however, whether measurement of overall unresolved Phe-tetraol would accurately represent the bay region diol epoxide metabolic activation pathway. Therefore, in this study we specifically quantified Phe-(1R,2S,3R,4S)-tetraol (4) by supplementing our usual analysis with chiral HPLC separations and using [(13)C(6)]Phe-(1R,2S,3R,4S)-tetraol as internal standard. We then investigated the relationship of urinary levels of 4 to those of Phe-tetraols (4 + 7), quantified without enantiomeric resolution. We applied these methods to urine samples from cigarette smokers and highly PAH-exposed creosote workers. The results were also compared to levels of benzo[a]pyrene-7,8,9,10-tetraol and 1-hydroxypyrene in the same samples. Levels of 4 were highly correlated with those of 4 + 7 (r > 0.9, P < 0.0001) in both types of urine samples. Strong correlations of 4 and 4 + 7 with benzo[a]pyrene-7,8,9,10-tetraol and 1-hydroxypyrene were also observed. The results of this study demonstrate therefore that practical and convenient measurement of overall Phe-tetraols (4 + 7) in human urine, without enantiomeric resolution, is an excellent indicator of PAH exposure and metabolism by the bay region diol epoxide metabolic activation pathway.

Immediate consequences of cigarette smoking: rapid formation of polycyclic aromatic hydrocarbon diol epoxides.

Polycyclic aromatic hydrocarbons (PAH) are among the likely major causative agents for lung cancer in smokers. PAH require metabolic activation to exert their carcinogenic effects, and one important pathway proceeds through a three-step sequence resulting in the formation of diol epoxides, which react with DNA to produce adducts that can cause mutations and initiate the carcinogenic process. However, no previous published studies have examined this critical pathway in humans specifically exposed to PAH by inhalation of cigarette smoke. This study used a unique approach employing a stable isotope derivative of phenanthrene, the simplest PAH with a bay region, a feature closely associated with PAH carcinogenicity. Twelve subjects each smoked a cigarette to which [D(10)]phenanthrene had been added. Plasma was analyzed for [D(10)]r-1,t-2,3,c-4-tetrahydroxy-1,2,3,4-tetrahydrophenanthrene ([D(10)]PheT), the major end product of the diol epoxide metabolism pathway of phenanthrene. The analysis was performed by gas chromatography--negative ion chemical ionization--tandem mass spectrometry, using [(13)C(6)]PheT as internal standard. The results demonstrated that the three-step pathway resulting in the formation of diol epoxides, as monitored by [D(10)]PheT, occurred with remarkable rapidity. Levels of [D(10)]PheT in plasma of all subjects were maximal at the earliest time points examined, 15-30 min after smoking the cigarette containing [D(10)]phenanthrene, and decreased thereafter. These results demonstrate that the formation of a PAH diol epoxide occurs rapidly in smokers. Because PAH diol epoxides are mutagenic and carcinogenic, the results clearly demonstrate immediate negative health consequences of smoking, which should serve as a major warning to anyone contemplating initiating tobacco use.

Analysis of r-7,t-8,9,c-10-tetrahydroxy-7,8,9,10-tetrahydrobenzo[a]pyrene in human urine: a biomarker for directly assessing carcinogenic polycyclic aromatic hydrocarbon exposure plus metabolic activation.

Polycyclic aromatic hydrocarbons (PAH) are believed to be causative agents for various types of cancers in humans. Benzo[a]pyrene (BaP) is a prototypic carcinogenic PAH, which requires metabolic activation to elicit its detrimental effects. The major end product of its diol epoxide metabolic activation pathway is r-7,t-8,9,c-10-tetrahydroxy-7,8,9,10-tetrahydrobenzo[a]pyrene (trans, anti-BaPT). Individual differences in exposure to, and metabolic activation of, carcinogenic PAH may influence cancer risk. Measurement of PAH metabolites in human urine could provide a direct way to assess individual differences in susceptibility to PAH-related cancer. In this article, we describe a sensitive and reliable method for the quantitation of trans, anti-BaPT in human urine using gas chromatography-negative ion chemical ionization-tandem mass spectrometry (GC-NICI-MS/MS). [(13)C(6)] trans, anti-BaPT was used as the internal standard. The urine was treated with ß-glucuronidase and sulfatase, and then trans, anti-BaPT was enriched by solid-phase extraction with polymeric reversed phase and phenylboronic acid cartridges. The sample was silylated and analyzed by GC-NICI-MS/MS with selected reaction monitoring (SRM) for the trimethylsilyl (TMS) derivatives of trans, anti-BaPT (m/z 446 → m/z 255) and [(13)C(6)]trans, anti-BaPT (m/z 452 → m/z 261). The mean assay recovery was 44%. The instrumental on-column detection limit was about 20 amol of trans, anti-BaPT (as BaPT-TMS). trans, anti-BaPT was readily detected in all urine samples analyzed including those of 30 smokers (0.71 ± 0.64 fmol/mg creatinine) and 30 nonsmokers (0.34 ± 0.2 fmol/mg creatinine) (P = 0.0036). The results of this study demonstrate a highly sensitive and selective method for the quantitation of trans, anti-BaPT in human urine. This is to our knowledge the first study to show that smokers have significantly higher levels of trans, anti-BaPT in their urine than do nonsmokers. This method may be useful as a direct phenotyping approach to assess individual differences in uptake and metabolic activation of carcinogenic PAH.

Preferential glutathione conjugation of a reverse diol epoxide compared with a bay region diol epoxide of benzo[a]pyrene in human hepatocytes.

Many studies have examined the relationship between polymorphisms in glutathione S-transferase genes and cancer in people exposed to polycyclic aromatic hydrocarbons (PAH) such as benzo[a]pyrene (BaP), but the results to date have been modest. Missing from these studies has been an exploration of the formation of the appropriate glutathione conjugates in humans. We incubated human hepatocytes from 10 donors with racemic anti-BaP-7,8-diol-9,10-epoxide (BPDE), believed to be a major ultimate carcinogen of BaP, or with the noncarcinogenic reverse diol epoxide, racemic anti-BaP-9,10-diol-7,8-epoxide (rev-BPDE). Incubations were carried out for 12 or 24 h. We used high-performance liquid chromatography-electrospray ionization-tandem mass spectrometry-selected reaction monitoring at m/z 464 --> m/z 317 to analyze the incubation mixtures for the mercapturic acid products that would result from glutathione conjugation. The standard mercapturic acids were synthesized by reaction of BPDE or rev-BPDE with N-acetylcysteine. We obtained convincing evidence in human hepatocytes for mercapturic acid formation from rev-BPDE in all 10 samples, in amounts up to 17 pmol/ml. However, we could detect mercapturic acids from BPDE in only 1 of 10 samples (0.05 pmol/ml). Taken together with our similar previous results of analyses of phenanthrene metabolites in human hepatocytes and human urine, the results of this study indicate that conjugation of BPDE with glutathione is a minor pathway in humans, indicating that glutathione S-transferase genotyping is not an effective method for assessing risk of PAH-induced cancer in humans, at least with respect to the diol epoxide pathway of PAH carcinogenesis.

Analysis of phenanthrene and benzo[a]pyrene tetraol enantiomers in human urine: relevance to the bay region diol epoxide hypothesis of benzo[a]pyrene carcinogenesis and to biomarker studies.

One widely accepted metabolic activation pathway of the prototypic carcinogenic polycyclic aromatic hydrocarbon (PAH) benzo[a]pyrene (BaP) proceeds through the "bay region diol epoxide" BaP-(7R,8S)-diol-(9S,10R)-epoxide (2). However, few studies have addressed the analysis of human urinary metabolites of BaP, which result from this pathway. Phenanthrene (Phe) is structurally related to BaP, but human exposure to Phe is far greater, and its metabolites can be readily detected in urine. Thus, Phe metabolites have been proposed as biomarkers of PAH exposure and metabolic activation. Phe-tetraols in particular could be biomarkers of the diol epoxide pathway. While BaP-tetraols and Phe-tetraols have been previously quantified in human urine, no published studies have determined their enantiomeric composition. This is important because different enantiomers would result from the bay region diol epoxide and "reverse" diol epoxide pathways, the latter being associated with weak mutagenicity and carcinogenicity. We addressed this problem using chiral HPLC to separate the enantiomers of BaP-7,8,9,10-tetraol and Phe-1,2,3,4-tetraol. Urine samples from smokers were subjected to solid-phase extraction, chiral HPLC, and GC-NICI-MS/MS analysis for silylated Phe-1,2,3,4-tetraols. The results demonstrated that >96% of Phe-1,2,3,4-tetraol in smokers' urine was Phe-(1S,2R,3S,4R)-tetraol (12), resulting from the "reverse" diol epoxide pathway, whereas less than 4% resulted from the "bay region diol epoxide" pathway of Phe metabolism. Urine from creosote workers was similarly analyzed for BaP-7,8,9,10-tetraol enantiomers. In contrast to the results of the Phe-tetraol analyses, 78% of BaP-7,8,9,10-tetraol in these human urine samples was BaP-(7R,8S,9R,10S)-tetraol (3) resulting from the "bay region diol epoxide" pathway of BaP metabolism. These results provide further support for the bay region diol epoxide pathway of BaP metabolism in humans and demonstrate differences in BaP and Phe metabolism, which may be important when considering Phe-tetraols as biomarkers of PAH metabolic activation.

Preferential glutathione conjugation of a reverse diol epoxide compared to a bay region diol epoxide of phenanthrene in human hepatocytes: relevance to molecular epidemiology studies of glutathione-s-transferase polymorphisms and cancer.

Bay region diol epoxides are recognized ultimate carcinogens of polycyclic aromatic hydrocarbons (PAH), and in vitro studies have demonstrated that they can be detoxified by conjugation with glutathione, leading to the widely investigated hypothesis that individuals with low activity forms of glutathione-S-transferases are at higher risk of PAH induced cancer, a hypothesis that has found at most weak support in molecular epidemiology studies. A weakness in this hypothesis was that the mercapturic acids resulting from the conjugation of PAH bay region diol epoxides had never been identified in human urine. We recently analyzed smokers' urine for mercapturic acids derived from phenanthrene, the simplest PAH with a bay region. The only phenanthrene diol epoxide-derived mercapturic acid in smokers' urine was produced from the reverse diol epoxide, anti-phenanthrene-3,4-diol-1,2-epoxide (11), not the bay region diol epoxide, anti-phenanthrene-1,2-diol-3,4-epoxide (10), which does not support the hypothesis noted above. In this study, we extended these results by examining the conjugation of phenanthrene metabolites with glutathione in human hepatocytes. We identified the mercapturic acid N-acetyl-S-(r-4,t-2,3-trihydroxy-1,2,3,4-tetrahydro-c-1-phenanthryl)-L-cysteine (14a), (0.33-35.9 pmol/mL at 10 microM 8, 24 h incubation, N = 10) in all incubations with phenanthrene-3,4-diol (8) and the corresponding diol epoxide 11, but no mercapturic acids were detected in incubations with phenanthrene-1,2-diol (7), and only trace amounts were observed in incubations with the corresponding bay region diol epoxide 10. Taken together with our previous results, these studies clearly demonstrate that glutathione conjugation of a reverse diol epoxide of phenanthrene is favored over conjugation of a bay region diol epoxide. Since reverse diol epoxides of PAH are generally weakly or nonmutagenic/carcinogenic, these results, if generalizable to other PAH, do not support the widely held assumption that glutathione-S-transferases are important in the detoxification of PAH in humans.

Quantitation of pyridylhydroxybutyl-DNA adducts in liver and lung of F-344 rats treated with 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone and enantiomers of its metabolite 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol.

The tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is a potent pulmonary carcinogen in rats and is believed to be one cause of lung cancer in smokers. NNK is metabolized to 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL), which is also a strong lung carcinogen in rats and has a chiral center at its 1-carbon. Previous studies have demonstrated that cytochrome P450-catalyzed alpha-hydroxylation of NNK in the lung leading to the formation of methyl and pyridyloxobutyl (POB)-DNA adducts is critical for its carcinogenicity. alpha-Hydroxylation of NNAL would similarly produce pyridylhydroxybutyl (PHB)-DNA adducts, but these have not been previously investigated in vivo. POB- and PHB-DNA adduct levels can indicate the amounts of pyridyloxobutylating and pyridylhydroxybutylating agents present in tissues of NNK- or NNAL-treated rats at any given point. Therefore, in this study, we developed a sensitive and quantitative liquid chromatography-electrospray ionization-tandem mass spectrometry-selected reaction monitoring method to determine levels of the PHB-DNA adducts O(6)-[4-(3-pyridyl)-4-hydroxybut-1-yl]-2'-deoxyguanosine (O(6)-PHB-dGuo, 10b), O(2)-[4-(3-pyridyl)-4-hydroxybut-1-yl]thymidine (O(2)-PHB-dThd, 11b), and 7-[4-(3-pyridyl)-4-hydroxybut-1-yl]-2'-deoxyguanosine (7-PHB-dGuo, 12b), the latter as the corresponding base 7-[4-(3-pyridyl)-4-hydroxybut-1-yl]-Gua (7-PHB-Gua, 14b) in DNA isolated from liver and lung of rats treated with 10 ppm NNK, (S)-NNAL, or (R)-NNAL in the drinking water for 20 weeks and sacrificed at 1, 2, 5, 10, 16, and 20 weeks. PHB-DNA adduct levels were higher in lung than in liver at each time point, consistent with previous studies of POB-DNA adducts in rats treated with NNK and NNAL in the drinking water. The results showed that NNK and (S)-NNAL behaved in a similar fashion, while (R)-NNAL was strikingly different. In the rats treated with NNK or (S)-NNAL, levels of each adduct at each time point were remarkably similar in lung, and levels of O(2)-PHB-dThd were generally greater than 7-PHB-Gua > O(6)-PHB-dGuo. The highest PHB-DNA adduct levels were found in lung and liver of rats treated with (R)-NNAL, suggesting that there are cytochrome P450s that efficiently catalyze the alpha-methyl hydroxylation of this compound. The results of this study provide further support for our hypothesis that (S)-NNAL is rapidly formed from NNK, sequestered at an unknown site in the lung, and then released and reoxidized to NNK with consequent DNA adduct formation resulting in lung carcinogenicity.

Analysis of phenanthrene diol epoxide mercapturic acid detoxification products in human urine: relevance to molecular epidemiology studies of glutathione S-transferase polymorphisms.

Many studies have investigated the effects of glutathione S-transferase (GST) polymorphisms on cancer incidence in people exposed to carcinogenic polycyclic aromatic hydrocarbons (PAHs). The basis for this is that the carcinogenic bay region diol epoxide metabolites of several PAH are detoxified by GSTs in in vitro studies. However, there are no reports in the literature on the identification in urine of the mercapturic acid metabolites that would result from this process in humans. We addressed this by developing a method for quantitation in human urine of mercapturic acids which would be formed from angular ring diol epoxides of phenanthrene (Phe), the simplest PAH with a bay region, and a common environmental pollutant. We prepared standard mercapturic acids by reactions of syn- or anti-Phe-1,2-diol-3,4-epoxide and syn- or anti-Phe-3,4-diol-1,2-epoxide with N-acetylcysteine. Analysis of human urine conclusively demonstrated that the only detectable mercapturic acid of this type--N-acetyl-S-(r-4,t-2,3-trihydroxy-1,2,3,4-tetrahydro-c/t-1-phenanthryl)-L-cysteine (anti-PheDE-1-NAC)--was derived from the 'reverse diol epoxide', anti-Phe-3,4-diol-1,2-epoxide, and not from the bay region diol epoxides, syn- or anti-Phe-1,2-diol-3,4-epoxide. Levels of anti-PheDE-1-NAC in the urine of 36 smokers were (mean +/- SD) 728 +/- 859 fmol/ml urine. The results of this study provide the first evidence for a mercapturic acid of a PAH diol epoxide in human urine, but it was not derived from a bay region diol epoxide as molecular epidemiologic studies have presumed, but rather from a reverse diol epoxide, representative of metabolites with little if any carcinogenic activity. These results demonstrate the need for integration of genotyping and phenotyping information in molecular epidemiology studies.

Investigation of the reaction of myosmine with sodium nitrite in vitro and in rats.

Previous studies have shown that the minor tobacco alkaloid myosmine (5) reacts with NaNO2 in the presence of acid to yield 4-hydroxy-1-(3-pyridyl)-1-butanone (HPB, 8) via 4-(3-pyridyl)-4-oxobutanediazohydroxide (7). Intermediate 7 is also formed in the metabolism of the tobacco-specific nitrosamines N'-nitrosonornicotine (NNN, 1) and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK, 2), resulting in pyridyloxobutylation of DNA and Hb. These pyridyloxobutyl adducts can be quantified by analyzing HPB released upon acid treatment of DNA or base treatment of Hb. Quantitation of HPB-releasing DNA and Hb adducts has been used to assess the metabolic activation of NNN and NNK in smokers and smokeless tobacco users. Because myosmine is found in the diet as well as in tobacco products, it has been suggested that nitrosation of myosmine could lead to the formation of HPB-releasing adducts in people not exposed to tobacco products. We investigated the nitrosation of myosmine in vitro and in vivo in rats. The reaction of myosmine with NaNO2 under acidic conditions produced HPB, as previously reported. A new product was identified as 3'-oximinomyosmine (11) based on its spectral properties. NNN was not detected. Groups of rats were treated with NNN, NNK, myosmine, NaNO2, or combinations of myosmine and NaNO2. HPB-releasing Hb and DNA adducts were clearly detected in the rats treated with NNN or NNK, but we found no evidence for production of these adducts from the combination of myosmine plus NaNO2. The results of this study do not support the hypothesis that exposure to dietary myosmine could lead to HPB-releasing DNA or Hb adducts in humans.

Quantitation of N-acetyl-S-(9,10-dihydro-9-hydroxy-10-phenanthryl)-L-cysteine in human urine: comparison with glutathione-S-transferase genotypes in smokers.

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.

Identification of an acetaldehyde adduct in human liver DNA and quantitation as N2-ethyldeoxyguanosine.

Acetaldehyde, an ubiquitous mutagen and carcinogen, could be involved in human cancer etiology. Because DNA adducts are important in carcinogenesis, we have used liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) to explore the presence in human liver DNA of the major acetaldehyde DNA adduct, N2-ethylidenedeoxyguanosine (1). DNA was isolated and enzymatically hydrolyzed in the presence of NaBH3CN, which quantitatively converts adduct 1 to N2-ethyldeoxyguanosine (N2-ethyl-dGuo, 2). [15N5]N2-Ethyl-dGuo was synthesized and used as an internal standard. Adduct 2 was enriched from the hydrolysate by solid phase extraction and analyzed by LC-ESI-MS/MS. Clear peaks were observed for adduct 2 in analyses of human liver DNA, calf thymus DNA, and rat liver DNA. These peaks were not observed, or were much smaller, when the NaBH3CN step was omitted. When the DNA was subjected to neutral thermal hydrolysis prior to NaBH3CN treatment, adduct 2 was not observed. Control experiments using [13C2]acetaldehyde demonstrated that adducts 1 and 2 were not formed as artifacts during DNA isolation and analysis. These results strongly indicate that adduct 1 is present in human liver DNA and demonstrate that it can be quantified as adduct 2. Levels of adduct 2 measured in 12 human liver samples were 534 +/- 245 fmol/micromol dGuo (mean +/- SD). The results of this study establish the presence of an acetaldehyde adduct in human liver DNA and suggest that it is a commonly occurring endogenous DNA adduct.