Multiple mutations are required for cancer development, and genome sequencing has revealed that several cancers, including breast, have somatic mutation spectra dominated by C-to-T transitions1–9. Most of these mutations occur at hydrolytically disfavored10 non-methylated cytosines throughout the genome, and are sometimes clustered8. Here, we show that the DNA cytosine deaminase APOBEC3B (A3B) is a likely source of these mutations. A3B mRNA is up-regulated in the majority of primary breast tumors and breast cancer cell lines. Tumors that express high levels of A3B have twice as many mutations as those that express low levels and are more likely to have mutations in TP53. Endogenous A3B protein is predominantly nuclear and the only detectable source of DNA C-to-U editing activity in breast cancer cell line extracts. Knockdown experiments show that endogenous A3B correlates with elevated levels of genomic uracil, increased mutation frequencies, and C-to-T transitions. Furthermore, induced A3B over-expression causes cell cycle deviations, cell death, DNA fragmentation, γ-H2AX accumulation, and C-to-T mutations. Our data suggest a model in which A3B-catalyzed deamination provides a chronic source of DNA damage in breast cancers that could select TP53 inactivation and explain how some tumors evolve rapidly and manifest heterogeneity.

With respect to the compounds analyzed here, e-cigarettes have a more favorable toxicity profile than tobacco cigarettes.

The tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK, 1) is a potent lung carcinogen in laboratory animals and is believed to play a key role in the development of lung cancer in smokers. Metabolic activation of NNK leads to the formation of pyridyloxobutyl DNA adducts, a critical step in its mechanism of carcinogenesis. In addition to DNA nucleobase adducts, DNA phosphate adducts can be formed by pyridyloxobutylation of the oxygen atoms of the internucleotidic phosphodiester linkages. We report the use of a liquid chromatography–nanoelectrospray ionization–high-resolution tandem mass spectrometry technique to characterize 30 novel pyridyloxobutyl DNA phosphate adducts in calf thymus DNA (CT-DNA) treated with 4-(acetoxymethylnitrosamino)-1-(3-pyridyl)-1-butanone (NNKOAc, 2), a regiochemically activated form of NNK. A 15N3-labeled internal standard was synthesized for one of the most abundant phosphate adducts, dCp[4-oxo-4-(3-pyridyl)butyl]dC (CpopC), and this standard was used to quantify CpopC and to estimate the levels of other adducts in the NNKOAc-treated CT-DNA. Formation of DNA phosphate adducts by NNK in vivo was further investigated in rats treated with NNK acutely (0.1 mmol/kg once daily for 4 days by subcutaneous injection) and chronically (5 ppm in drinking water for 10, 30, 50, and 70 weeks). This study provides the first comprehensive structural identification and quantitation of a panel of DNA phosphate adducts of a structurally complex carcinogen and chemical support for future mechanistic studies of tobacco carcinogenesis in humans.

The compound 3-amino-2-quinoxalinecarbonitrile 1,4-dioxide (4) displays potent hypoxia-selective cytotoxicity in cell culture. This compound is structurally similar to the known hypoxia-selective DNA-damaging agent tirapazamine (1, TPZ), but the ability of 4 to cause DNA damage under low-oxygen conditions has not previously been characterized. The results presented here provide the first evidence that 4 causes reductively activated DNA damage under hypoxic conditions. The findings indicate that one-electron reduction of 4 by NADPH:cytochrome P450 reductase yields an oxygen-sensitive intermediate (5). This activated intermediate is rapidly destroyed by reaction with O2 under aerobic conditions, but goes forward to cause DNA damage under low-oxygen conditions. Analysis of the DNA damage indicates that reductive activation of 4 leads to production of a highly reactive, freely diffusible oxidizing radical that causes sequence-independent cleavage of the deoxyribose backbone and oxidative damage to the heterocyclic bases in duplex DNA. On the basis of the experiments reported here, the chemical nature of the DNA damage caused by redox-activated 4 is analogous to that reported previously for TPZ.