The influence of repair pathways on the cytotoxicity and mutagenicity induced by the pyridyloxobutylation pathway of tobacco-specific nitrosamines

Abstract

Tobacco-specific nitrosamines, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone and N'-nitrosonornicotine, are considered to be human carcinogens. Both compounds are metabolized to pyridyloxobutylating intermediates that react with DNA to form adducts such as 7-[4-(3-pyridyl)-4-oxobut-1-yl]guanine, O(2)-[4-(3-pyridyl)-4-oxobut-1-yl]cytosine, O(2)-[4-(3-pyridyl)-4-oxobut-1-yl]-2'-deoxythymidine (O(2)-pobdT), O(6)-[4-(3-pyridyl)-4-oxobut-1-yl]-2'-deoxyguanosine (O(6)-pobdG), and 4-hydroxy-1-(3-pyridyl)-1-butanone-releasing adducts. The role of specific DNA adducts in the overall genotoxic activity of the pyridyloxobutylation pathway is not known. One adduct, O(6)-pobdG, is mutagenic. To characterize the mutagenic and cytotoxic properties of pyridyloxobutyl DNA adducts, the impact of DNA repair pathways on the cytotoxic and mutagenic properties of the model pyridyloxobutylating agent, 4-(acetoxymethylnitrosamino)-1-(3-pyridyl)-1-butanone (NNKOAc), was investigated in Chinese hamster ovary cell lines proficient or deficient in O(6)-alkylguanine DNA alkyltransferase (AGT), deficient in both AGT and base excision repair (BER), or deficient in both AGT and nucleotide excision repair (NER). The repair of the four pyridyloxobutyl DNA adducts was determined in the same cell lines via sensitive LC-MS/MS methods. NNKOAc was more cytotoxic in the cell lines lacking AGT, BER, and NER repair pathways. It also induced more mutations in the hprt gene in the BER- and NER-deficient cell lines. However, AGT expression did not influence NNKOAc's mutagenicity despite efficient repair of O(6)-pobdG. Analysis of the hprt mutational spectra indicated that NNKOAc primarily caused point mutations at AT base pairs. GC to AT transition mutations were a minor contributor to the overall mutation spectrum, providing a rationale for the observation that AGT does not protect against the overall mutagenic properties of NNKOAc in this model system. The only adduct affected by the absence of effective NER was O(2)-pobdT. Slower repair of O(2)-pobdT in NER-deficient cells was associated with increased AT to TA transversion mutations, supporting the hypothesis that these mutations are caused by O(2)-pobdT. Together, these data support a hypothesis that the pyridyloxobutylation pathway generates multiple mutagenic and toxic adducts.

Figure 1

A. Proposed activation pathways for NNN, NNK and NNKOAc. B. Structures of characterized pyridyloxobutyl DNA adducts.

Figure 2. Cytotoxicity (A, C) and hprt mutagenic activity (B, D) in CHO cell lines following a 1 h treatment with increasing concentrations of NNKOAc

The values represent the average of four separate experiments. The error bars represent the standard deviation. AA8: normal NER, no AGT; UV5: deficient in NER, no AGT; EM9: deficient in XRCC1 (BER), no AGT; CHO^pcDNA3: empty vector; CHO^AGT: vector containing hAGT. * indicates that the treated cell lines were statistically significantly different from the parent cell lines at that concentration, p < 0.05.

Figure 3. Initial levels of pyridyloxobutyl DNA adducts in CHO cell lines following a 1 h treatment with 32 μM NNKOAc

The values represent averages of 3 replicates. The error bars represent the standard deviation. AA8: normal NER, no AGT; UV5: deficient in NER, no AGT; CHO^pcDNA3: empty vector; CHO^AGT: vector containing hAGT. Adduct levels were determined by LC-MS/MS analysis of enzymatic hydrolysates of DNA isolated from NNKOAc-treated CHO cell lines.

Figure 4. Time course of adduct repair following a 1 h exposure to 8 μM NNKOAc

The values equal the average of 3–5 replicates. The error bars represent the standard deviation. Adduct levels are normalized relative to the dG (O²-pobC, 7-pobG, O²-pobdT and O⁶-pobdG) or G (O⁶-pobG) concentrations in the enzyme or acid hydrolysates, respectively. AA8: normal NER, no AGT; UV5: deficient in NER, no AGT; EM9: deficient in XRCC1 (BER), no AGT; CHO^pcDNA3: empty vector; CHO^AGT: vector containing hAGT. * indicates that the treated cell lines were statistically significantly different from the parent cell lines at that concentration, p < 0.05.

Figure 5. Excision of O⁶-pobdG adduct by human repair factors

Substrate DNA (0.8 nM) was incubated with RPA (140 nM), XPA (65 nM), XPC•hR23B (5 nM), TFIIH (~ 12 nM), XPF•ERCC1 (4 nM), and XPG (3 nM) for 2 h at 30°C. DNA alone (lanes 1, 3, 5) and complete reactions (lanes 2, 4, 6) are shown for undamaged DNA (lanes 1–2), O⁶-pobdG DNA (lanes 3–4), and T[6-4]T photoproduct (lanes 5–6). Brackets indicate excision products visualized by autoradiography after resolution in 10% sequencing gel. Levels of excision in this experiment were 0.8% (UM), 1.1% (O⁶-pobdG), and 32.7% T[6-4]T.