Tobacco-specific nitrosamines, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and gene in the BER and NER

Tobacco-specific nitrosamines, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and gene in the BER and NER deficient cell lines. adducts accumulate in pulmonary Type II cells and correlate with lung tumor formation in NNK-treated rats (14). Finally, pyridyloxobutyl DNA adducts also build up in normal lung tissues of lung cancer patients (15), indicating that the formation and persistence of these adducts may be important in human lung cancer associated with tobacco use. The role of specific pyridyloxobutyl DNA adducts in the overall mutagenic and carcinogenic properties of pyridyloxobutylating nitrosamines is usually unknown. We have exhibited that oncogene of tumors induced by NNKOAc in A/J mouse lungs (13). The mutagenic properties of the other pyridyloxobutyl DNA adducts is largely unknown. A preliminary report describing polymerase reactions with vector (21). CHOAGT cells are stably transfected with the pCMV-hAGT expressing human AGT (21). These cell lines have been used to determine the importance of DNA repair pathways in protecting cells against the cytotoxic and genotoxic effects of a variety of DNA damaging brokers (21,24C30). We measured the influence of the repair proteins around the mutational spectrum of NNKOAc as well as the formation and repair of the known pyridyloxobutyl DNA adducts, 7-pobG, vector without an inserted cDNA sequence (CHOpcDNA3) or the same plasmid expressing human AGT (CHOAGT) (21). Western blot analysis confirmed the expression of AGT in CHOAGT cells (21). The AGT activity in the cells was approximately 450 fmol/mg protein (21). The cell lines were maintained in Minimum Essential Medium Alpha Modification (MEM) supplemented with 10% fetal bovine serum (Invitrogen, Carlsbad, CA) and 1% penicillin/streptomycin at 37C in a humidified 5% CO2 incubator. Pre-existing mutants were eliminated by growing the cells in complete medium supplemented with 100 M hypoxanthine, 0.4 M aminopterin and 16 M thymidine as previously described (25). Survival and mutant frequency Cytotoxicity and mutation frequency at the locus was decided according to published methods (33,34). Six-well plates were seeded with 2 105 63238-67-5 IC50 exponentially growing cells 18C24 h prior to treatment. The cells were exposed to 0C48 M NNKOAc for 1 h. Each treatment was performed in duplicate. The cells were washed with PBS and grown in fresh medium for 16C24 h. The cells were then harvested, counted, and replated for measurement of colony forming ability and mutant frequency measurements. Each experiment was repeated a minimum of three times. Colony forming ability was determined by plating 100 cells from each treatment into 6-well plates 63238-67-5 IC50 (33). Two wells were plated per treatment. The plating efficiencies were similar for all those five cell lines. After 6C7 days growth in normal medium, colonies were stained with 0.05% crystal violet and were counted with an aCOLyte colony counter (Synbiosis, Frederick, MD). Cell survival was normalized to the number of colonies from untreated cells. For the mutagenesis assay, each treated culture was plated into 60 mm dishes and grown in normal medium, with subculturing, to allow phenotypic expression of induced mutants. After 7 days, the cells were harvested, counted and plated for determination of cloning efficiency and mutant frequency. The cloning efficiency was determined by seeding 200 cells into 60 mm dishes in normal medium. Colonies were stained with crystal violet after 6C7 days growth and counted. The mutant frequency was determined by plating 100,000 cells into 100 mm dishes in medium made up of 5 g/ml 6-thioguanine to select for mutant colonies. Ten dishes per treatment were plated. Colonies were stained after 8 days of growth in the selection medium and counted with an aCOLyte colony counter. The mutant frequency was expressed as 63238-67-5 IC50 the 6-thioguanine resistant colonies per 100,000 viable cells. The effect Cd55 of cell type on cytotoxicity was decided using analysis of variance at each concentration of NNKOAc. Since the F-test for the simple effect was significant, pair wise comparisons of UV5 and EM9 with AA8 were conducted. At each step of the analysis, the level of significance was 0.05; the calculated mRNA was reversed transcribed.