Studies described in this thesis were aimed at characterizing the mechanism(s) of aflatoxin B1 (AFB1) pulmonary carcinogenesis by addressing the formation, prevention, and repair of AFB1-induced oxidative DNA damage. The ability of AFB1 to cause oxidative DNA damage in different lung cell types of the A/J mouse was examined. The formation of 8-hydroxy-2’-deoxyguanosine (8-OHdG) in freshly isolated mouse lung alveolar macrophages, alveolar type II cells, and nonciliated bronchial epithelial (Clara) cells, was assessed by high performance liquid chromatography with electrochemical detection. An increase in 8-OHdG formation occurred in macrophage and Clara cell preparations isolated from A/J mice two hours following in vivo treatment with a single tumourigenic dose of AFB1. Prior treatment with polyethylene glycol-conjugated catalase (PEG-CAT) prevented the AFB1-induced increase in 8-OHdG levels in all mouse lung cell preparations. These results support the possibility that oxidative DNA damage in mouse lung cells contributes to AFB1 carcinogenicity. Mouse lung tumourigenesis was assessed following treatment of A/J mice with PEG-CAT and/or AFB1. Unexpectedly, the mean number of tumours per mouse and tumour size in the PEG-CAT + AFB1 group were greater than those of the group treated with AFB1 alone. There was no difference in K-ras exon 1 mutation spectrum or in the histological diagnosis of tumours between treatment groups. In vitro incubation with mouse liver catalase (CAT) resulted in conversion of [3H]AFB1 into a DNA-binding species, a possible explanation for the results observed in vivo. These results demonstrate that PEG-CAT is not protective against AFB1 carcinogenicity in mouse lung despite preventing DNA oxidation. The effect of in vivo treatment of mice with AFB1 on pulmonary and hepatic base excision repair (BER) activities and levels of 8-oxoguanine DNA glycosylase (OGG1) was investigated. AFB1 treatment increased 8-OHdG levels and BER activity in mouse lung, but did not significantly affect either in liver. Levels of OGG1 immunoreactive protein were increased in both mouse lung and liver. These results indicate that oxidative DNA damage may be an important mechanism in the carcinogenicity of AFB1. However, BER activity is increased by AFB1 treatment, possibly representing a compensatory response to the production of oxidative DNA damage. / Thesis (Ph.D, Pharmacology & Toxicology) -- Queen's University, 2008-12-12 10:00:44.81

The tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is believed to play a role in human lung cancer induced by tobacco smoking. NNK biotransformation may involve the enzymes prostaglandin H synthase (PHS)-1, PHS-2 and cytochrome 450 (CYP) 2F. PHS activity is thought to be important in extrahepatic tissues, where CYP activity is low. The CYP2F subfamily contains a single functional enzyme in humans (CYP2F1) and goats (CYP2F3); these enzymes are preferentially expressed in the lung, with little or no expression in other organs. The role of these enzymes in the pulmonary biotransformation of NNK was investigated. 4.2 µM [5-3H]NNK was incubated with human lung microsomes under NADPH-dependent and arachidonic acid-dependent conditions. Metabolites reflective of NNK α-carbon hydroxylation, N-oxidation and carbonyl reduction were detected in the presence of NADPH, and metabolite levels for all three biotransformation pathways were lower in the presence of arachidonic acid compared with NADPH (p<0.05, N=4). Incubation of microsomes with the PHS-1 selective inhibitor SC-560 and the PHS-2 selective inhibitor NS-398 did not change NNK biotransformation either in the presence of NADPH or in the presence of arachidonic acid (p>0.05, N=4). Incubation of [5-3H]NNK with ovine PHS-1 or PHS-2 did not result in formation of α-carbon hydroxylation or N­-oxidation metabolites; 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) was measurable only in the presence of PHS-2. Incubation of goat recombinant CYP2F3 with [5-3H]NNK resulted in formation of keto acid, keto alcohol and NNK-N-oxide (65.0%, 17.5% and 30.0% (µmol enzyme)-1 minute-1, respectively). Metabolite formation was inhibited by 3-methylindole (3-MI), a mechanism-based inactivator of CYP2F3. Based on an N value of 3, incubation of human lung microsomes with 3-MI inhibited N-oxidation (p<0.05) but did not alter NNK bioactivation or carbonyl reduction (p>0.05). However, when metabolite formation was examined in lung microsomes from different individuals, decreases in NNK biotransformation (ranging from 19.6 to 68.5%) were observed and were more pronounced in some patients than others, suggesting inter-individual variability in CYP2F1 activity. These studies demonstrate the ability of CYP2F to biotransform NNK and suggest inter-individual variability in the importance of CYP2F1 for this activity in human lung. They also strongly argue against the involvement of PHS enzymes. / Thesis (Master, Pharmacology & Toxicology) -- Queen's University, 2007-12-30 16:12:58.228

Studies described in this thesis were at aimed at characterizing the mechanisms involved in the pulmonary carcinogenicity of the tobacco-specific nitrosamine, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), by addressing two critical determinants of carcinogenicity; biotransformation and DNA repair. The contributions of cytochrome P450 (CYP) 2A13 and CYP2A6 to NNK biotransformation in human lung microsomes were investigated. Based on total bioactivation and detoxification of NNK and its keto-reduced metabolite, 4 (methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL), subjects could be classified as either high or low bioactivators and detoxifiers. Data from all of 29 individuals revealed no correlations between levels of CYP2A mRNA, enzyme activity or immunoinhibition and the degree of total NNK bioactivation or detoxification. However, subgroups were identified for whom CYP2A13 mRNA correlated with total NNK and NNAL bioactivation (n=4) and NNAL detoxification (n=5). Although results do not support CYP2A13 or CYP2A6 as predominant contributors to NNK metabolism in lung of all individuals, CYP2A13 appears to be important in some. The involvement of nucleotide excision repair (NER) in the repair of NNK-induced DNA pyridyloxobutylation was assessed. Extracts from NER-deficient cells were less active at repairing pyridyloxobutyl (POB) adducts on plasmid DNA than were extracts from normal cells, and NER-deficient cells were more susceptible to 4-(acetoxymethylnitrosamino)-1-(3-pyridyl)-1-butanone (NNKOAc)-induced cytotoxicity, demonstrating the participation of NER in the repair of POB-DNA adducts. The role of DNA repair in contributing to inter-organ susceptibility to NNK-induced carcinogenesis was investigated. POB adduct repair was greater in extracts from mouse liver than lung, and activities in lungs of NNK-treated mice were lower than those of saline-treated mice, while repair was 3 times higher in livers of NNK-treated mice relative to control. NNK treatment decreased incision of POB adducts by 92 % in lung extracts and increased incision by 169 % in liver extracts. In addition, NNK altered the levels and binding to POB damage of key incision proteins. These results suggest that lower NER incision activity and NNK-mediated alterations in levels and activities of incision proteins contribute to the relative susceptibility of mouse lung to NNK-induced carcinogenesis. / Thesis (Ph.D, Pharmacology & Toxicology) -- Queen's University, 2008-11-13 14:10:01.603