RESUMO
BACKGROUND: After accounting for smoking history, lung cancer incidence is greater in African Americans than Whites. In the multiethnic cohort, total nicotine equivalents (TNE) are higher in African Americans than Whites at similar reported cigarettes per day. Greater toxicant uptake per cigarette may contribute to the greater lung cancer risk of African Americans. METHODS: In a nested case-control lung cancer study within the Southern Community Cohort, smoking-related biomarkers were measured in 259 cases and 503 controls (40% White; 56% African American). TNE, the trans-3-hydroxycotinine/cotinine ratio, 4-(methylnitrosamino)-1-3-(pyridyl)-1-butanol (NNAL), mercapturic acid metabolites of volatile organic compounds, phenanthrene metabolites, cadmium (Cd), and (Z)-7-(1R,2R,3R,5S)-3,5-dihydroxy-2-[(E,3S)-3-hydroxyoct-1-enyl]cyclopenyl]hept-5-enoic acid were quantified in urine. Unconditional logistic regression was used to estimate the ORs and 95% confidence intervals (CI) for each biomarker and lung cancer risk. RESULTS: TNE, NNAL, and Cd were higher in cases than controls (adjusted for age, race, sex, body mass index, and cigarettes per day). Among cases, these levels were higher in African Americans compared with Whites. After accounting for age, sex, body mass index, and pack-years, a one-SD increase in log-TNE (OR = 1.30; 95% CI, 1.10-1.54) and log-NNAL (OR = 1.27; 95% CI, 1.03-1.58 with TNE adjustment) was associated with lung cancer risk. In this study, in which NNAL concentration is relatively high, the association for log-TNE was attenuated after adjustment for log-NNAL. CONCLUSIONS: Smoking-related biomarkers provide additional information for lung cancer risk in smokers beyond smoking pack-years. IMPACT: Urinary NNAL, TNE, and Cd concentrations in current smokers, particularly African American smokers, may be useful for predicting lung cancer risk.
Assuntos
Negro ou Afro-Americano , Neoplasias Pulmonares , População Branca , Humanos , Masculino , Neoplasias Pulmonares/urina , Neoplasias Pulmonares/etiologia , Neoplasias Pulmonares/epidemiologia , Feminino , Negro ou Afro-Americano/estatística & dados numéricos , Pessoa de Meia-Idade , Estudos de Casos e Controles , População Branca/estatística & dados numéricos , Idoso , Biomarcadores Tumorais/urina , Estudos de Coortes , Fatores de Risco , Biomarcadores/urina , Fumar Cigarros/urina , Fumar Cigarros/efeitos adversos , Fumar Cigarros/etnologia , BrancosRESUMO
Smoking intensity varies across smokers and is influenced by individual variability in the metabolism of nicotine, the major addictive agent in tobacco. Therefore, lung cancer risk, which varies by racial ethnic group, is influenced by the primary catalyst of nicotine metabolism, cytochrome P450 2A6 (CYP2A6). In smokers, CYP2A6 catalyzes nicotine 5'-oxidation. In vitro, CYP2A6 also catalyzes, to a much lower extent, 2'-oxidation, which leads to the formation of 4-hydroxy-4-(3-pyridyl) butanoic acid (hydroxy acid). The urinary concentration of hydroxy acid has been quantified in only a few small studies of White smokers. To quantitatively assess the importance of nicotine 2'-oxidation in smokers, an LC-MS/MS-based method was developed for the analysis of nicotine and ten metabolites in urine. The concentrations of nicotine and these metabolites were measured in 303 smokers (99 Whites, 99 Native Hawaiians, and 105 Japanese Americans), and the relative metabolism of nicotine by four pathways was determined. Metabolism by these pathways was also compared across quartiles of CYP2A6 activity (measured as the plasma ratio of 3-hydroxycotinine to cotinine). As reported previously and consistent with their average CYP2A6 activity, nicotine 5'-oxidation was highest in Whites and lowest in Japanese Americans. Nicotine N-glucuronidation and N-oxidation increased with decreasing CYP2A6 activity. However, the relative urinary concentration of hydroxy acid (mean, 2.3%; 95% CI, 2.2-2.4%) did not vary by ethnic group or by CYP2A6 activity. In summary, CYP2A6 is not an important catalyst of nicotine 2'-oxidation in smokers, nor does nicotine 2'-oxidation compensate for decreased CYP2A6 activity.
Assuntos
Asiático , Nicotina , Humanos , Nicotina/metabolismo , Ácido Butírico , Havaiano Nativo ou Outro Ilhéu do Pacífico , Cromatografia Líquida , Brancos , Espectrometria de Massas em Tandem , Cotinina/metabolismo , Citocromo P-450 CYP2A6RESUMO
Carcinogen and toxicant uptake by e-cigarette users have not been fully evaluated. In the study reported here, we recruited 30 e-cigarette users, 63 nonsmokers, and 33 cigarette smokers who gave monthly urine samples over a period of 4-6 months. Their product use status was confirmed by measurements of exhaled CO, urinary total nicotine equivalents, cyanoethyl mercapturic acid (CEMA), and total 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol. Urinary biomarkers of exposure to the carcinogens acrolein (3-hydroxypropyl mercapturic acid, 3-HPMA), benzene (S-phenyl mercapturic acid, SPMA), acrylonitrile (CEMA), and a combination of crotonaldehyde, methyl vinyl ketone, and methacrolein (3-hydroxy-1-methylpropyl mercapturic acid, HMPMA) were quantified at each visit. Data from subject visits with CEMA > 27 pmol/mL were excluded from the statistical analysis of the results because of possible unreported exposures to volatile combustion products such as secondhand cigarette smoke or marijuana smoke exposure; this left 22 e-cigarette users with 4 or more monthly visits and all 63 nonsmokers. Geometric mean levels of 3-HPMA (1249 versus 679.3 pmol/mL urine) were significantly higher (P = 0.003) in e-cigarette users than in nonsmokers, whereas levels of SPMA, CEMA, and HMPMA did not differ between these two groups. All analytes were significantly higher in cigarette smokers than in either e-cigarette users or nonsmokers. The results of this unique multimonth longitudinal study demonstrate consistent significantly higher uptake of the carcinogen acrolein in e-cigarette users versus nonsmokers, presenting a warning signal regarding e-cigarette use.
Assuntos
Acroleína , Sistemas Eletrônicos de Liberação de Nicotina , Humanos , Acroleína/metabolismo , Fumantes , Acetilcisteína/metabolismo , Estudos Longitudinais , Carcinógenos/análise , Biomarcadores/urinaRESUMO
The Multiethnic Cohort Study has demonstrated that the risk for lung cancer in cigarette smokers among three ethnic groups is highest in Native Hawaiians, intermediate in Whites, and lowest in Japanese Americans. We hypothesized that differences in levels of DNA adducts in oral cells of cigarette smokers would be related to these differing risks of lung cancer. Therefore, we used liquid chromatography-nanoelectrospray ionization-high resolution tandem mass spectrometry to quantify the acrolein-DNA adduct (8R/S)-3-(2'-deoxyribos-1'-yl)-5,6,7,8-tetrahydro-8-hydroxypyrimido[1,2-a]purine-10(3H)-one (γ-OH-Acr-dGuo, 1) and the lipid peroxidation-related DNA adduct 1,N6-etheno-dAdo (εdAdo, 2) in DNA obtained by oral rinse from 101 Native Hawaiians, 101 Whites, and 79 Japanese Americans. Levels of urinary biomarkers of nicotine, acrolein, acrylonitrile, and a mixture of crotonaldehyde, methyl vinyl ketone, and methacrolein were also quantified. Whites had significantly higher levels of γ-OH-Acr-dGuo than Japanese Americans and Native Hawaiians after adjusting for age and sex. There was no significant difference in levels of this DNA adduct between Japanese Americans and Native Hawaiians, which is not consistent with the high lung cancer risk of Native Hawaiians. Levels of εdAdo were modestly higher in Whites and Native Hawaiians than in Japanese Americans. The lower level of DNA adducts in the oral cells of Japanese American cigarette smokers than Whites is consistent with their lower risk for lung cancer. The higher levels of εdAdo, but not γ-OH-Acr-dGuo, in Native Hawaiian versus Japanese American cigarette smokers suggest that lipid peroxidation and related processes may be involved in their high risk for lung cancer, but further studies are required.
Assuntos
Acrilonitrila , Neoplasias Pulmonares , Produtos do Tabaco , Acroleína/química , Estudos de Coortes , DNA , Adutos de DNA , Etnicidade , Humanos , Peroxidação de Lipídeos , Neoplasias Pulmonares/urina , Nicotina/urina , Purinas , Fumantes , FumarRESUMO
INTRODUCTION: Studies suggest tobacco and cannabis co-users may experience greater toxicant exposure than exclusive cigarette (ET) smokers. No study has systematically tested differences in toxicant exposure among co-users, exclusive cannabis (ECa) smokers, and ET smokers. AIMS AND METHODS: Adult daily cigarette smokers and/or weekly cannabis smokers completed two laboratory visits. Co-users (n = 19) tested positive for urinary 11-nor-9-carboxy-Δâ9-tetrahydrocannabinol (THCCOOH), self-reported cannabis use ≥1 per week, and smoked ≥5 cigarettes per day (CPD). ET smokers (n = 18) denied past month cannabis use, tested negative for urinary THCCOOH and smoked ≥5 CPD. ECa smokers (n = 16) tested positive for urinary THCCOOH, self-reported cannabis use ≥1 per week, and denied past month tobacco use (NicAlert <3). Self-reported tobacco and cannabis use were collected at both visits. First morning urinary tobacco and combustion-related biomarkers of exposure were compared following a cannabis or tobacco smoking session (visit 2). RESULTS: Co-users and ET smokers had higher levels of exhaled carbon monoxide, total nicotine equivalents, metabolites of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNAL), and all four measured mercapturic acids (measures of volatile organic compounds) than ECa smokers (ps < .005). ET smokers (geometric mean = 7220.2 pmol/mg) had higher levels of 2-hydroxypropylmercapturic acid than co-users (geometric mean = 5348.7 adjusted p = .009). Phenanthrene tetraol did not differ by group (p > .05). CONCLUSIONS: Co-users and ET smokers demonstrated comparable levels of biomarkers of exposure to harmful constituents despite smoking similar amounts of tobacco. ECa smokers demonstrated lower levels of toxicant exposure for most biomarkers. IMPLICATIONS: Although ECa smokers are exposed to significantly lower levels of harmful constituents compared with co-users and exclusive cigarette smokers, this group is still exposed to higher levels of toxicants than observed in studies of nonsmokers. Additionally, these three groups were exposed to similar levels of phenanthrene tetraol. It is important to account for cannabis use in studies examining biomarkers of exposure among cigarette smokers. Additionally, further research is needed examining exposure to harmful chemicals among various types of cannabis and tobacco users.
Assuntos
Cannabis , Sistemas Eletrônicos de Liberação de Nicotina , Produtos do Tabaco , Adulto , Biomarcadores , Humanos , Projetos Piloto , FumantesRESUMO
Nicotine is the most abundant alkaloid in tobacco accounting for 95% of the alkaloid content. There are also several minor tobacco alkaloids; among these are nornicotine, anatabine, and anabasine. We developed and applied a 96 well plate-based capillary LC-tandem mass spectrometry method for the analysis of nornicotine, anatabine, and anabasine in urine. The method was validated with regard to accuracy and precision. Anabasine was quantifiable to low levels with a limit of quantitation (LOQ) of 0.2 ng/mL even when nicotine, which is isobaric, is present at concentrations >2500-fold higher than anabasine. This attribute of the method is important since anatabine and anabasine in urine have been proposed as biomarkers of tobacco use for individuals using nicotine replacement therapies. In the present study, we analyzed the three minor tobacco alkaloids in urine from 827 smokers with a wide range of tobacco exposures. Nornicotine (LOQ 0.6 ng/mL) was detected in all samples, and anatabine (LOQ, 0.15 ng/mL) and anabasine were detected in 97.7% of the samples. The median urinary concentrations of nornicotine, anatabine, and anabasine were 98.9, 4.02, and 5.53 ng/mL. Total nicotine equivalents (TNE) were well correlated with anatabine (r(2) = 0.714) and anabasine (r(2) = 0.760). TNE was most highly correlated with nornicotine, which is also a metabolite of nicotine. Urine samples from a subset of subjects (n = 110) were analyzed for the presence of glucuronide conjugates by quantifying any increase in anatabine and anabasine concentrations after ß-glucuronidase treatment. The median ratio of the glucuronidated to free anatabine was 0.74 (range, 0.1 to 10.9), and the median ratio of glucuronidated to free anabasine was 0.3 (range, 0.1 to 2.9). To our knowledge, this is the largest population of smokers for whom the urinary concentrations of these three tobacco alkaloids has been reported.
Assuntos
Alcaloides/urina , Anabasina/urina , Nicotiana/química , Nicotina/análogos & derivados , Piridinas/urina , Fumar/urina , Adolescente , Cromatografia Líquida de Alta Pressão , Humanos , Estrutura Molecular , Nicotina/urina , Espectrometria de Massas em TandemRESUMO
OBJECTIVES: We studied the impact of clean indoor air law exemptions and preemption policies on the prevalence of a tobacco-specific lung carcinogen-4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK)--among nonsmoking bar and restaurant workers. METHODS: secondhand smoke were compared with results from participants who were exposed to it. RESULTS: Participants exposed to workplace secondhand smoke were more likely to have any detectable level of NNAL (P=.005) and higher mean levels of NNAL (P < .001) compared with nonexposed participants. Increased levels of NNAL were also associated with hours of a single workplace exposure (P=.005). CONCLUSIONS: Nonsmoking employees left unprotected from workplace secondhand smoke exposure had elevated levels of a tobacco-specific carcinogen in their bodies. All workers--including bar and restaurant workers--should be protected from indoor workplace exposure to cancer-causing secondhand smoke.
Assuntos
Carcinógenos/análise , Nitrosaminas/urina , Exposição Ocupacional/estatística & dados numéricos , Política Pública , Restaurantes , Poluição por Fumaça de Tabaco/prevenção & controle , Adolescente , Adulto , Biomarcadores/urina , Estudos Transversais , Feminino , Humanos , Masculino , Análise Multivariada , Exposição Ocupacional/legislação & jurisprudência , Exposição Ocupacional/prevenção & controle , Oregon , Análise de Regressão , Restaurantes/legislação & jurisprudência , Risco , Fatores de Tempo , Poluição por Fumaça de Tabaco/efeitos adversos , Poluição por Fumaça de Tabaco/legislação & jurisprudênciaRESUMO
Liquid chromatography with electrospray tandem mass spectrometry (LC/ESI-MS/MS) was employed to quantify O6-[4-oxo-4-(3-pyridyl)butyl]-2'-deoxyguanosine (O6-pobdG), a mutagenic adduct formed by pyridyloxobutylating nitrosamines. Selected reaction monitoring (SRM) of the neutral loss of the sugar from protonated molecules of the adduct, [M + H - 116]+, was utilized for detection of O6-pobdG in pyridyloxobutylated DNA from both in vitro and in vivo sources. Quantitation was based on isotope dilution with synthetic O6-[1,2,2-2H3-4-oxo-4-(3-pyridyl)butyl]-2'-deoxyguanosine. The detection limits in this study were less than 5 fmol of pure standard and 50 fmol in 1.5 mg of DNA. This method was validated by comparing adduct levels measured with the LC/ESI-MS/MS method to those obtained with radiochemical methods in DNA alkylated with the model pyridyloxobutylating agent, [5-3H]4-(acetoxymethylnitrosamino)-1-(3-pyridyl)-1-butanone ([5-3H]NNKOAc). The pyridyloxobutyl 2'-deoxyguanosine adduct coeluting with the deuterated standard disappeared when NNKOAc-treated DNA had been reacted with the repair protein, O6-alkylguanine-DNA alkyltransferase. This result confirms that the coeluting peak is solely O6-pobdG. Preliminary studies with liver DNA isolated from NNKOAc-treated mice demonstrated that this method can be used to quantify O6-pobG in DNA from in vivo sources. The improved sensitivity and specificity of adduct detection afforded by this LC/ESI-MS/MS method will allow us to explore the role of O6-pobdG in the toxicological properties of pyridyloxobutylating nitrosamines.
Assuntos
Cromatografia Líquida/métodos , Adutos de DNA/análise , Desoxiguanosina/análogos & derivados , Desoxiguanosina/análise , Mutagênicos/análise , Nicotiana/toxicidade , Nitrosaminas/química , Piridinas/análise , Espectrometria de Massas por Ionização por Electrospray/métodos , Animais , Adutos de DNA/química , Desoxiguanosina/química , Feminino , Guanina/análogos & derivados , Camundongos , Mutagênicos/química , Nitrosaminas/toxicidade , Piridinas/químicaRESUMO
The tobacco specific nitrosamine, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), a potent pulmonary carcinogen, both methylates and pyridyloxobutylates DNA. Both reaction pathways generate promutagenic O6-alkylguanine adducts. These adducts, O6-methylguanine (O6-mG) and O6-[4-oxo-4-(3-pyridyl)butyl]guanine (O6-pobG), are repaired by O6-alkylguanine-DNA alkyltransferase (AGT). In this report, we demonstrate that pyridyloxobutyl DNA adducts are repaired by AGT in a reaction that results in pyridyloxobutyl transfer to the active site cysteine. Because minor changes within the binding pocket of AGT can alter the ability of this protein to repair bulky O6-alkylguanine adducts relative to O6-mG, we explored the ability of AGTs from different species as well as several human AGT variants and mutants to discriminate between O6-mG or O6-pobG adducts. We incubated proteins with equal molar amounts of oligodeoxynucleotides containing site specifically incorporated O6-mG or O6-pobG and measured repair. Bacterial AGTs poorly repaired O6-pobG. Mouse and rat AGT repaired both adducts at comparable rates. Wild-type human AGT, variant I143V/K178R, and mutant N157H repaired O6-mG approximately twice as fast as O6-pobG. Human variant G160R and mutants P140K, Y158H, G156A, and E166G did not repair O6-pobG until all of the O6-mG was removed. To understand the role of adduct structure on relative repair rates, the competition experiments were repeated with two other bulky O6-alkylguanine adducts, O6-butylguanine (O6-buG) and O6-benzylguanine (O6-bzG). The proteins displayed similar repair preference of O6-mG relative to O6-buG as observed with O6-pobG. In contrast, all of the mammalian proteins, except the mutant P140K, preferentially repaired O6-bzG. These studies indicate that the rate of repair of O6-pobG is highly dependent on protein structure. Inefficient repair of O6-pobG by bacterial AGT explains the high mutagenic activity of this adduct in bacterial systems. In addition, differences observed in the repair of this adduct by mammalian proteins may translate into differences in sensitivity to the mutagenic and carcinogenic effects of NNK or other pyridyloxobutylating nitrosamines.
Assuntos
Carcinógenos/química , Adutos de DNA/química , Guanina/análogos & derivados , Guanina/química , Nitrosaminas/química , O(6)-Metilguanina-DNA Metiltransferase/química , Animais , Bovinos , Reparo do DNA , Humanos , Cinética , Camundongos , O(6)-Metilguanina-DNA Metiltransferase/classificação , Ratos , Especificidade da EspécieRESUMO
4-Hydroxy-1-(3-pyridyl)-1-butanone (HPB) is a metabolite of the tobacco specific nitrosamines, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and N'-nitrosonornicotine (NNN). HPB is also a breakdown product of covalently bound pyridyloxobutyl adducts resulting from NNK and NNN exposure. HPB released from DNA or hemoglobin has been used as an important dosimeter of tobacco specific nitrosamine exposure in a variety of studies. This compound is not reactive with cellular nucleophiles under biological conditions. We have discovered that HPB reacts with nucleophiles under acidic conditions to form cyclic tetrahydrofuranyl reaction products. Dithiothreitol, 2-mercaptoethanol, and N-acetylcysteine all reacted with HPB under these reaction conditions. In addition, reactions were observed with buffer chemicals such as 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid and tris(hydroxymethyl)aminomethane. The resulting cyclic adducts were unstable at room temperature. Their half-lives were significantly longer under neutral conditions than under acidic conditions. NMR studies established that the cyclic form of HPB, 2-hydroxy-2-(3-pyridyl)-2,3,4,5-THF, is present at significant concentrations in acidic solutions. The observation of this cyclic compound suggests that the reaction with nucleophiles may occur via a cyclic oxonium ion intermediate. This reaction was significant in our biological samples; there was up to 40% conversion of [5-(3)H]HPB to cyclic DTT-derived compounds when acidic DNA repair reactions containing [5-(3)H]pyridyloxobutylated DNA were stored overnight at -20 degrees C. Therefore, long-term storage of acid hydrolysates of pyridyloxobutylated DNA or protein for the analysis of HPB-releasing adducts could result in an underestimation of HPB-releasing adduct in those samples. In addition, these observations provide a mild synthetic method to prepare large quantities of cyclic 2-(3-pyridyl)-2,3,4,5-THF adducts predicted to result from pyridyloxobutylation of important cellular nucleophiles as a result of NNK and/or NNN exposure.
Assuntos
Butanonas/química , Nicotiana/química , Nitrosaminas/química , Piridinas/química , Compostos de Sulfidrila/química , Cromatografia Líquida de Alta Pressão , Concentração de Íons de Hidrogênio , Espectroscopia de Ressonância Magnética , Fatores de TempoRESUMO
The tobacco-specific nitrosamine, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), is a potent pulmonary carcinogen. This unsymmetric nitrosamine can be metabolically activated to lung DNA methylating and pyridyloxobutylating intermediates. The methyl DNA adducts are well characterized. The pyridyloxobutyl adducts are unstable under DNA hydrolysis conditions and decompose to release 4-hydroxy-1-(3-pyridyl)-1-butanone (HPB). One of the HPB-releasing adducts,O6-[4-oxo-4-(3-pyridyl)butyl]guanine (O6-pobG), has been detected in DNA reacted in vitro with the model pyridyloxobutylating agent, 4-(acetoxymethylnitrosamino)-1-(3-pyridyl)-1-butanone (NNKOAc). To determine whether this adduct was formed in vivo, A/J mice were treated with 10 mumol of [5-3H]NNK and sacrificed 24 h postinjection. The mutagenic O6-pobG was detected in liver but not lung DNA from these animals. Since 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) is a major metabolite of NNK, it is also possible that these animals are activating NNAL to a pyridylhydroxybutylating agent. Therefore, we also measured the levels of O6-[4-hydroxy-4-(3-pyridyl)butyl]guanine (O6-phbG) in these DNA samples. While radioactivity did coelute with synthetic standard for this potential NNAL adduct in one lung DNA sample, significant levels of O6-phbG were not detected in any other lung or liver DNA samples. The pyridyloxobutyl adduct, O6-pobG, was also observed in lung and liver DNA from mice treated with 4.2 mumol of [5-3H]NNKOAc in the presence but not absence of 2.5 mumol of O6-benzylguanine, a known depletor of the repair protein O6-alkylguanine-DNA alkyltransferase (AGT). These data indicate that this adduct is formed in vivo but is repaired in part by AGT. Cell-free extracts from A/J mouse lung and liver were used to determine the relative rate of O6-alkylguanine repair. O6-mG and O6-pobG were removed from DNA to the same extent in a competitive assay, suggesting that low levels of O6-pobG in lungs of NNK-treated mice did not result from preferential repair of O6-pobG by AGT. It is more likely that initial levels of O6-pobG are much lower than initial levels of O6-mG in lung DNA from NNK-treated A/J mice. These data are consistent with previous studies, which indicate that DNA methylation is the critical pathway for NNK-induced lung carcinogenesis in A/J mice.
Assuntos
Carcinógenos/toxicidade , Adutos de DNA/análise , DNA/efeitos dos fármacos , Guanina/análogos & derivados , Guanina/análise , Nitrosaminas/toxicidade , Animais , Feminino , Fígado/química , Fígado/efeitos dos fármacos , Pulmão/química , Pulmão/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos A , Piridinas/toxicidadeRESUMO
Mitogen-activated protein kinases (MAPKs) play a central role in transmitting stress-induced signals stimulated by genotoxic agents. The present study is the first to investigate the mechanisms by which genotoxic alkylating agents modulate MAPKs by directly measuring the effects of methylating agents on MAPK activity, DNA methylation, and intracellular glutathione levels. The effects of acetoxymethylmethylnitrosamine (AMMN), N-nitroso-N-methylurethane (NMUR), and N-methyl-N-nitrosourea (MNU) on these parameters were compared in a fetal rat lung cell line model (MP48). These compounds were chosen because they methylate DNA via a methanediazonium intermediate and, therefore, should induce similar cellular methylation patterns, although they produce different side products upon decomposition. All three compounds stimulated the activation of the stress-activated MAPKs, c-Jun N-terminal kinase, and p38. In contrast to what has been reported for other methylating agents, these compounds also stimulated the activation of extracellular signal regulated kinase (ERK), a MAPK typically activated by mitogenic agents. O6-methylguanine (O6-mG) is widely considered to be the critical toxic lesion induced by methylating agents, including AMMN, NMUR, and MNU, which form DNA adducts through SN1 reactions. O6-mG does not appear to be a key regulator of MAPK activity by these compounds, however. There is no direct relationship between the levels of O6-mG and the levels of MAPK activation, and formation of O6-mG does not appear to be sufficient to stimulate MAPK activation. The present studies also indicate that depletion of glutathione is not required or sufficient to stimulate MAPK activation by the methylating agents investigated here. The use of a pharmacological inhibitor indicates that these methylating agents activate ERK through a signaling pathway that requires the ERK kinase MEK. Altogether, these data indicate that genotoxic methylating agents activate MAPKs through mechanisms that are likely to involve the alkylation of cellular targets other than DNA.