Identification of Formaldehyde-Induced DNA-RNA Cross-Links in the A/J Mouse Lung Tumorigenesis Model

Abstract

4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is a potent lung carcinogen present in tobacco products, and exposure to it is likely one of the factors contributing to the development of lung cancer in cigarette smokers. To exert its carcinogenic effects, NNK must be metabolically activated into highly reactive species generating a wide spectrum of DNA damage. We have identified a new class of DNA adducts, DNA-RNA cross-links found for the first time in NNK-treated mice lung DNA using our improved high-resolution accurate mass segmented full scan data-dependent neutral loss MS³ screening strategy. The levels of these DNA-RNA cross-links were found to be significantly higher in NNK-treated mice compared to the corresponding controls, which is consistent with higher levels of formaldehyde due to NNK metabolism as compared to endogenous levels. We hypothesize that this DNA-RNA cross-linking occurs through reaction with NNK-generated formaldehyde and speculate that this phenomenon has broad implications for NNK-induced carcinogenesis. The structures of these cross-links were characterized using high-resolution LC-MS² and LC-MS³ accurate mass spectral analysis and comparison to a newly synthesized standard. Taken together, our data demonstrate a previously unknown link between DNA-RNA cross-link adducts and NNK and provide a unique opportunity to further investigate how these novel NNK-derived DNA-RNA cross-links contribute to carcinogenesis in the future.

Fig. 1.

Putative DNA-RNA crosslink detected in NNK-treated mice lung DNA. (A.I) Extracted ion chromatogram, (A.II) MS², and (A.III) MS³ triggered scan events for m/z 547.2012 at 22.4 min (The corresponding events are marked with the asterisk *). (B) MS² spectrum of m/z 547.2012 at 22.4 min. and (C) MS³ spectrum of m/z 431.1536 (loss of deoxyribose, dR) from m/z 547.2012 at 22.4 min.

Figure 2.

Proposed structures of the MS² and MS³ fragment ions of the putative formaldehyde-induced DNA-RNA crosslink, guanosine-CH₂-dA, detected in A/J mice lung DNA.

Fig. 3.

Putative DNA-DNA crosslink detected in NNK-treated mice lung DNA (A) Extracted ion chromatogram, MS², and MS³ triggered scan events for dG-CH₂-dG (The corresponding events are marked with the asterisk *) (B) MS² spectrum of dG-CH₂-dG (C) MS³ spectrum of dG-CH₂-dG.

Fig. 4.

(A) MS² fragmentation of guanosine-CH₂-dA detected in vitro (B) MS² fragmentation of guanosine-CH₂-dA detected in NNK-treated mice lung DNA.

Fig. 5.

(A) Extracted ion chromatograms of guanosine-CH₂-dG (*) in an NNK-treated mouse sample (B) MS² spectrum of guanosine-CH₂-dG (C) MS³ spectrum of guanosine-CH₂-dG.

Fig. 6.

(A) Relative levels of guanosine-CH₂-dA - and (B) guanosine-CH₂-dG in NNK-treated mouse samples (6 h) compared to the corresponding control. Levels were normalized to [D₃] O⁶-methyl-dG as internal standard. Error bars represent standard deviation (SD) of 3 technical replicate measurements.

Fig. 7.

Immunodetection and dot blot of DNA:RNA hybrids in mice lung DNA. (A) DNA:RNA hybrids in NNK-treated mice and control with and without RNase H treatment. (B) Levels of input DNA detected using dsDNA-specific antibody. (C) Relative levels of DNA:RNA hybrids in NNK-treated mice compared to the corresponding control mice. Levels are normalized against dsDNA signals. Error bars represent the standard deviation of the triplicate measurements.