7-methylguanine and Lung-Neoplasms

O(6)-Alkylguanine-DNA alkyltransferase (MGMT) repairs DNA adducts that result from alkylation at the O(6) position of guanine. These lesions are mutagenic and toxic and can be produced by a variety of agents including the tobacco-specific nitrosamines, carcinogens present in cigarette smoke. Here, we review some of our work in the context of inter-individual differences in MGMT expression and their potential influence on lung cancer risk. In humans there are marked inter-individual differences in not only levels of DNA damage in the lung (N7-methylguanine) that can arise from exposure to methylating agents but also in MGMT activity in lung tissues. In the presence of such exposure, this variability in MGMT activity may alter cancer susceptibility, particularly as animal models have demonstrated that the complete absence of MGMT activity predisposes to alkylating-agent induced cancer while overexpression is protective. Recent studies have uncovered a series of polymorphisms that affect protein activity or are associated with differences in expression levels. The associations between these (and other) polymorphisms and cancer risk are inconsistent, possibly because of small sample sizes and inter-study differences in lung cancer histology. We have recently analysed a consecutive series of case-control studies and found evidence that lung cancer risk was lower in subjects with the R178 allele.

Topics: Alkylating Agents; Animals; DNA Damage; DNA Modification Methylases; DNA Repair Enzymes; Genetic Variation; Guanine; Humans; Lung; Lung Neoplasms; Polymorphism, Genetic; Risk Factors; Tumor Suppressor Proteins

Tobacco specific nitrosamines such as 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) are genotoxic alkylating agents found within cigarette smoke that induce lung adenocarcinomas in animal models. In humans, adenocarcinomas originate most frequently in the lung periphery. The aim of this study was to determine whether peripheral lung has increased susceptibility to the genotoxic effects of alkylating agents by comparing DNA alkylation damage (N7-methylguanine: N7-meG) and repair (O(6)-alkylguanine DNA alkyltransferase: MGMT) in peripheral relative to central lung tissue.. Macroscopically normal lung tissue, resected from patients undergoing surgery for lung cancer, was sampled at equidistant points from central to peripheral lung along a bronchus. N7-meG levels were determined using an immunoslotblot technique and MGMT activity with a [32P]-labelled oligodeoxynucleotide cleavage assay.. A total of 20 subjects were recruited, 12 males and 8 females with a mean age of 68.7±5.8years. There were 14 former and 6 current smokers with a mean smoking exposure of 34.0±18.3packyears. N7-meG (mean 0.75±0.57/10(6)dG, n=65 samples from 14 patients) and MGMT repair (geometric mean 9.57±1.62fmol/μg DNA, n=79 samples from 16 patients) were detected in all samples assayed. MGMT activity increased towards the lung periphery (r=0.28, p=0.023; n=16) with a highly significant association in current (r=0.53, p=0.008; n=6) but not former smokers (r=0.13; p=0.41; n=10). No correlation was seen with N7-meG levels and lung position (r=-0.18; p=0.21; n=14). N7-meG levels were higher in current compared to former smokers reaching significance in two lung positions including peripheral lung (p=0.047).. The findings in this study do not support the hypothesis that peripheral tissue is more susceptible to the genotoxic effects of alkylating agents than central lung tissue. In addition exposure to cigarette smoke reduced the level of MGMT in central bronchial tissue possibly through increased alkylating agent exposure.

Topics: Aged; DNA Adducts; Female; Guanine; Humans; Immunoblotting; Lung; Lung Neoplasms; Male; O(6)-Methylguanine-DNA Methyltransferase; Smoking

Global analyses of DNA methylation contribute important insights into biology and the wide-ranging role of DNA methylation. We describe the use of online solid-phase extraction and isotope-dilution liquid chromatography/tandem mass spectrometry (LC-MS/MS) for the simultaneous measurement of 5-methyl-2'-deoxycytidine (5-medC) and 2'-deoxycytidine (dC) in DNA. With the incorporation of isotope internal standards and online enrichment techniques, the detection limit of this method was estimated to be as low as 0.065 pg which enables human global DNA methylation detection using only picogram amounts of DNA. This method was applied to assess the optimal amounts of enzymes required for DNA digestion regarding an accurate global DNA methylation determination and completeness of digestion and to determine global methylation in human tumor adjacent lung tissue of 79 lung cancer patients. We further determined methylated (N7-methylguanine (N7-meG), O (6)-methylguanine (O (6)-meG), and N3-methyladenine (N3-meA)) and oxidized DNA lesions (8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG)) in lung cancer patients by LC-MS/MS. Optimization experiments revealed that dC was liberated from DNA much more readily than 5-medC by nuclease P1 and alkaline phosphatase (AP) in DNA, which could lead to an error in the global DNA methylation measurement following digestion with insufficient enzymes. Nuclease P1 showed more differential activity for 5-medC and dC than AP. Global DNA methylation levels in adenocarcinoma and squamous cell carcinoma patients were similar in the range of 3.16-4.01 %. Global DNA methylation levels were not affected by smoking and gender and were not correlated with N7-meG or 8-oxodG in lung cancer patients. Levels of O (6)-meG and N3-meA were however found to be undetectable in all lung tissue samples.

Topics: Adenine; Adenocarcinoma; Aged; Carcinoma, Squamous Cell; Chromatography, Liquid; Deoxycytidine; DNA Methylation; DNA, Neoplasm; Female; Fungal Proteins; Guanine; Humans; Indicator Dilution Techniques; Limit of Detection; Lung Neoplasms; Male; Middle Aged; Reproducibility of Results; Single-Strand Specific DNA and RNA Endonucleases; Solid Phase Extraction; Tandem Mass Spectrometry; Tumor Microenvironment

Nitrosamines are mainly mutagenic through methylation of DNA. 7-Methylguanine (m(7)Gua) is a product of base excision repair and spontaneous depurination of such lesions in DNA and a metabolite from RNA. Associations between urinary excretion of m(7)Gua and risk of lung cancer were examined in a population-based cohort of 25,717 men and 27,972 women aged 50-64 years. During 3-7 years follow-up 260 cases with lung cancer were identified and a subcohort of 263 individuals matched on sex, age and smoking duration was selected for comparison. Urine collected at entry was analyzed for m(7)Gua by HPLC. Effect modification by glutathione-S-transferases GSTM1, GSTM3, GSTT1 and GSTP1 was investigated. We found higher excretion of m(7)Gua among current smokers than among former smokers. The IRR (incidence rate ratio) of lung cancer was 1.20 (95% CI: 1.00-1.43) per doubling of m(7)Gua excretion in unadjusted analysis and 1.12 (95% CI: 0.93-1.35) after adjustment for smoking status, intensity and duration at entry. This association was mainly present among current smokers. Comparing the highest with the lowest tertile of m(7)Gua excretion the IRR of lung cancer was 1.75 (95% CI: 1.04-2.95) irrespective of genotype and 2.75 (95% CI: 1.33-5.81) in subjects with GSTM1 null genotype. If not caused by residual confounding by smoking a possible association between m(7)Gua excretion and lung cancer supports the importance of methylation of guanine. The finding of an association between m(7)Gua excretion and lung cancer risk mainly among current smokers and subjects with GSTM1 null genotype supports causality in this respect.

Topics: Female; Gene Frequency; Genotype; Glutathione S-Transferase pi; Glutathione Transferase; Guanine; Humans; Linear Models; Lung Neoplasms; Male; Middle Aged; Multivariate Analysis; Prospective Studies; Registries; Risk Factors; Smoking

N7-Methylguanine (N7-MeG) DNA adducts are markers of human exposure to methylating agents including tobacco-specific nitrosamines (TSNAs). Repair of this adduct is poor, so levels in lung tissue should reflect variation in both intensity of exposure and in metabolism. N7-MeG adducts in lung DNA from bronchial lavage samples were measured to determine whether levels were higher in smokers than non-smokers, and if levels were modified by genetic variation in carcinogen-metabolising enzymes. Adducts were detected in 38 out of 44 DNA samples by 32P post-labelling of the N7-methyldeoxyguanosine-3'-monophosphate (N7-MedGp) isolated from DNA digests by two-stage HPLC. N7-MeG adduct levels were higher in smokers than in never smokers ((9.99 +/-20.3)x10(-7) versus (0.58+/-0.50)x10(-7) N7-MedGp/deoxyguanosine-3'-monophosphate (dGp); P=0.02) and intermediate in ex-smokers ((5.59+/-15.6)x10(-7) N7-MedGp/dGp). Adduct levels tended to be higher in individuals with GSTM1 null, GSTT1 null or GSTP1 ile/ile genotypes. When genotypes were combined, N7-MedGp levels among GSTM1 null/GSTT1 null individuals (n=6) were higher than among those having at least one wild-type allele of these two genes ((26.1+/-38.0)x10(-7) versus (2.73+/-4.07)x10(-7) N7-MedGp/dGp), although the results were not statistically significant (P=0.13). Adduct levels were highest in individuals with three unfavourable genotypes (GSTM1 null/GSTT1 null and GSTP1 ile/ile) compared with others ((74.5+/-13.1)x10(-7) versus (2.64+/-3.89)x10(-7) N7-MedGp/dGp, P=0.02). N7-MeG adduct levels in DNA isolated from lung tissue thus reflect exposure to cigarette smoke, and genetic variation in carcinogen-metabolising enzymes may modify these levels.

Topics: Adult; Bronchoalveolar Lavage Fluid; Chromatography, High Pressure Liquid; Deoxyguanine Nucleotides; DNA Adducts; Genetic Variation; Genotype; Glutathione Transferase; Guanine; Humans; Lung Neoplasms; Phosphorus Radioisotopes; Smoking

Stereochemical determinants of the tumorigenicity and metabolism of the tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) were investigated using the stereospecifically deuterated isotopomers (4R)-[4-(2)H(1)]NNK and (4S)-[4-(2)H(1)]NNK. Upon ip administration to groups of 20 female A/J mice, NNK and (4S)-[4-(2)H(1)]NNK exhibited similar lung tumorigenicity at three different doses, whereas (4R)-[4-(2)H(1)]NNK was 2-fold less tumorigenic at all three doses. In a parallel experiment, levels of O(6)-methylguanine and 7-methylguanine were 2-fold lower in lung DNA of mice treated with (4R)-[4-(2)H(1)]NNK than in mice treated with NNK or (4S)-[4-(2)H(1)]NNK. To corroborate these in vivo data, the in vitro metabolism of these compounds was investigated using A/J mouse lung microsomes and Spodoptera frugiperda (Sf9)-expressed mouse cytochrome p450s 2A4 and 2A5. Kinetic isotope effects on the apparent V(max) ((D)V) for the product of NNK 4-hydroxylation, OPB, were 2.7 +/- 0.2 and 2.8 +/- 0.4 when (4R)- and (4S)-[4-(2)H(1)]NNK were incubated with mouse lung microsomes, respectively. The (D)V values for OPB formation were 3.2 +/- 0.2 and 2.2 +/- 0.2 when (4R)-[4-(2)H(1)]NNK was the substrate for p2A4 and 2A5, respectively, whereas they were 1.3 +/- 0.1 and 1.1 +/- 0.1 when (4S)-[4-(2)H(1)]NNK was the substrate for these respective enzymes. Analysis of an OPB derivative (10) for deuterium content by LC/MS confirmed the results from the kinetic assays and indicated that p450s 2A4 and 2A5 preferentially abstract the pro-R 4-hydrogen of NNK. The results obtained using Sf9-expressed p450s provide a rationale for the differences observed in the lung tumor and DNA adduct experiments, namely, that the attenuated tumorigenicity of (4R)-[4-(2)H(1)]NNK relative to (4S)-[4-(2)H(1)]NNK is due to prochiral selectivity during p450-catalyzed metabolic activation.

Topics: Animals; Aryl Hydrocarbon Hydroxylases; Carcinogens; Cytochrome P-450 CYP2A6; Deuterium; DNA; DNA Adducts; Dose-Response Relationship, Drug; Female; Guanine; Lung; Lung Neoplasms; Mice; Mice, Inbred A; Microsomes; Mixed Function Oxygenases; Molecular Structure; Nitrosamines; Stereoisomerism; Structure-Activity Relationship

Female A/J mice were maintained on NIH-07 diet or on NIH-07 diet containing butylated hydroxyanisole (BHA, a mixture of 2- and 3-tert-butyl-4-hydroxyanisole), 5 mg/g, for 1 week prior to and during 10 weeks of treatment with N-nitrosodimethylamine (NDMA) or N-nitrosopyrrolidine (NPYR), administered in the drinking water. Twenty weeks after nitrosamine treatment ended, mice were sacrificed and lung adenomas were counted. BHA inhibited NDMA tumorigenesis but enhanced NPYR tumorigenesis. Treatment of A/J mice for three weeks with BHA (5 mg/g) added to semisynthetic diet increased whole lung microsomal alpha-hydroxylation of NDMA and NPYR, as measured by aldehyde production, and increased lung and hepatic glutathione-S-transferase activities. No evidence was found for formation of S-methylglutathione in incubations with NDMA and hepatic supernatants obtained from BHA treated or control mice. Four h after gavage of NDMA, levels of 7-methylguanine in the lung DNA of mice treated with BHA were higher than in the lung DNA of control mice, but levels of O6-methylguanine in the two groups were the same. The results of this study indicate that BHA treatment increases the microsomal metabolic alpha hydroxylation of both NDMA and NPYR, but has differential effects on their tumorigenic activities.

Topics: Animals; Anisoles; Biotransformation; Butylated Hydroxyanisole; Dimethylnitrosamine; Female; Glutathione; Glutathione Transferase; Guanine; Lung; Lung Neoplasms; Mice; Microsomes; Microsomes, Liver; N-Nitrosopyrrolidine; Nitrosamines

The tumorigenic activities and DNA methylating abilities in F344 rats of the tobacco specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and the structurally related nitrosamine N-nitrosodimethylamine (NDMA) were compared. Groups of 30 male rats were given 60 s.c. injections of 0.0055 mmol/kg of either NNK or NDMA over a 20-week period (total dose, 0.33 mmol/kg). The experiment was terminated after 104 weeks. The numbers of rats with tumors were as follows for NNK and NDMA, respectively: liver, 10 and 6; lung 13 and 0; and nasal cavity, 6 and 1. NNK was significantly more tumorigenic than was NDMA toward the lung (P less than 0.01) and nasal cavity (P less than 0.05). Groups of rats were treated with a single s.c. injection of 0.39 mmol/kg or 0.055 mmol/kg of NNK or NDMA and the levels of 7-methylguanine and O6-methylguanine were measured in liver, lung, and nasal mucosa 1-48 h after treatment. In liver and lung, levels of 7-methylguanine and O6-methylguanine in DNA were 3-22 times (P less than 0.001) greater in NDMA treated rats than in NNK treated rats. Levels of methylation induced by NDMA and NNK in the nasal mucosa were similar. The results of this study demonstrate that NNK is a more potent tumorigen than NDMA in the F344 rat and suggest that DNA methylation alone does not account for its strong tumorigenicity in rat lung and nasal mucosa.

Topics: Animals; Dimethylnitrosamine; DNA, Neoplasm; Guanine; Liver Neoplasms; Lung Neoplasms; Male; Methylation; Neoplasms, Experimental; Nicotiana; Nitrosamines; Nose Neoplasms; Plants, Toxic; Rats