The findings that genotoxic carcinogens preferentially accumulate in mitochondria and effectively bind to mtDNA, the observations that mitochondria are altered in cancer cells, and the knowledge of the crucial role of mitochondria in apoptosis, suggest that mtDNA mutations may play an important role in the carcinogenic process. The purpose of this study was to investigate the formation of mtDNA adducts in rats chronically treated with the tobacco-specific carcinogen NNK and its carcinogenic metabolite (S)-NNAL, and to compare the levels of mtDNA and nDNA adducts in the same tissue samples. This is the first study to demonstrate effective binding of NNK and (S)-NNAL metabolites to the mitochondrial genome.
We focused on NNK and (S
)-NNAL-treated animals because of a number of indications that (S
)-NNAL, but not (R
)-NNAL, is important in lung tumorigenesis by NNK. These include predominant formation of (S
)-NNAL from NNK in lung and liver microsomes and cytosol (41
), stereoselective retention of (S
)-NNAL in the lung (43
), and a striking similarity between NNK and (S
)-NNAL in the formation of POB-DNA adducts (11
) – the pathway believed to be an important mechanism of NNK carcinogenesis in rodents (45
) and likely in smokers (47
Overall, the levels of mtDNA and nDNA adducts were higher in the lung than in the liver of both NNK and (S
)-NNAL-treated rats, reproducing the results that we obtained previously upon analysis of total POB- and PHB-DNA adducts in the same animals (11
). When compared in the same samples, mtDNA adducts were higher than nDNA adducts in the lung, but not in the liver. Since lung is the primary target for NNK- and (S
)-NNAL-induced carcinogenesis, our findings are consistent with the results of a study in which preferential binding of N-nitrosodimethylamine and N-nitrosodiethylamine to mtDNA was demonstrated only for tumor susceptible tissues, while there was no difference in binding of these carcinogens to mtDNA or nDNA in non-tumor susceptible tissues (49
). The difference between the levels of mtDNA and nDNA adducts in the lung observed in our study is also in agreement with reports on the relative efficiency of nDNA and mtDNA binding for nitroso compounds and benzo[a]pyrene (BaP). Thus, we found that after 20 weeks of treatment with NNK, levels of 7-POB-Gua, O6
-POB-dGuo, and O2
-POB-dThd in lung mtDNA were 2.08, 4.54, and 2.23 times, respectively, higher than the levels of these adducts in nDNA. In other studies, treatment of laboratory animals with N-methyl-N-nitrosourea (50
) and N-nitrosodimethylamine (51
) led to 3–7 times more efficient methylation of liver and kidney mtDNA compared to nDNA. Some in vitro
experiments on preferential accumulation of carcinogens in mitochondria demonstrated that BaP and its carcinogenic dihydrodiol epoxide derivative bind to mtDNA of animal cells about 50 times more than to nDNA (52
). An in vivo
study, however, demonstrated that the levels of mtDNA adducts in the liver, lung, and kidney of rats treated with BaP were only 2 times higher than those of nDNA adducts (37
). In the same study, treatment of rats with cigarette smoke produced results comparable to BaP treatment.
The steady accumulation of mtDNA adducts observed in the rat lung and liver over the 20-week period of NNK- and (S
)-NNAL-treatment is in contrast with the results obtained upon analysis of nDNA adducts. The fact that nDNA adducts reach maximum values after 10–16 weeks of treatment and subsequently decline is indicative of a shift of balance between adduct formation and DNA repair (11
). In the case of mtDNA, continuous accumulation of adducts points to the inefficiency or lack of appropriate mtDNA repair mechanisms. Thus, nuclear O6
-POB-dGuo is efficiently repaired by AGT (54
), while in mitochondria AGT repairs methyl and ethyl adducts, but not bulky ones (56
). Similarly, O2
-POB-dThd is probably a nucleotide excision repair (NER) substrate in nDNA, while mitochondria are deficient in the NER pathway (reviewed in (21
)). Similarly, the mechanism of 7-POB-Gua repair, yet unknown in nDNA, is apparently not effective in mitochondria. Of the measured adducts, O6
-POB-dGuo has been shown to be highly mutagenic (55
). Accumulation of this adduct in lung mtDNA might play an important role in the induction of lung tumors by NNK in laboratory animals, and potentially in smokers.
The biological plausibility of the role of mtDNA mutations in the carcinogenic process is supported by the critical role of mitochondria in cellular energy production, apoptosis, and cellular growth and differentiation. In addition to these theoretical considerations, there is growing experimental evidence of a relationship between mtDNA mutations and cancer. Such evidence includes propagation of mtDNA mutations in cancer cells, changes in the cell surface produced by mtDNA mutations, suppression of the tumorigenic phenotype by the fusion of cancer cells with cytoplasts from non-tumorigenic cells, and appearance of rearranged and normal segments of mtDNA in nuclear genomes of various cell species (reviewed in (20
)). Furthermore, mathematical models demonstrate that both random distribution of mutated mtDNA among daughter cells and relaxed replication independent of the cell cycle, can lead to clonal expansion of a single mtDNA mutation during biological time scales (57
). Each of 2 to 10 mtDNA molecules present in a mitochondrion encodes 2 ribosomal RNA, 22 mitochondrial transfer RNA, and 13 mitochondrial proteins (subunits of respiratory complexes I, III, IV, and V) (59
). Many mutational hotspots occur in the non-coding D-loop of mtDNA, which serves as the main site for mtDNA replication and transcription. On the other hand, mtDNA lacks introns, and all non-D-loop mutations will occur in sequences coding for functionally important enzymes in the respiratory chain complex.
In summary, we demonstrate here that the chronic treatment of rats with NNK and (S)-NNAL at low doses gives higher levels of POB and PHB adducts in mtDNA than in nDNA of the lung, but not the liver. In both the lung and the liver, mtDNA adducts accumulated over the course of treatment, indicating inefficient repair of these adducts in mtDNA. This is the first study to examine the formation of NNK- and (S)-NNAL-derived adducts in rat mtDNA. The results support the hypothesis that preferential binding of tobacco carcinogens to mtDNA of the lung might be functionally important in the development of smoking-induced lung cancer.