Evaluation of the DNA Damaging Potential of Cannabis Cigarette Smoke by the Determination of Acetaldehyde Derived N2-Ethyl-2′-deoxyguanosine Adducts

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• 作者：Rajinder Singh ; Jatinderpal Sandhu ; Balvinder Kaur ; Tina Juren ; William P. Steward ; Dan Segerbck ; Peter B. Farmer
• 刊名：Chemical Research in Toxicology
• 出版年：2009
• 出版时间：June 15, 2009
• 年：2009
• 卷：22
• 期：6
• 页码：1181-1188
• 全文大小：166K
• 年卷期：v.22,no.6(June 15, 2009)
• ISSN：1520-5010

文摘

Acetaldehyde is an ubiquitous genotoxic compound that has been classified as a possible carcinogen to humans. It can react with DNA to form primarily a Schiff base N²-ethylidene-2′-deoxyguanosine (N²-ethylidene-dG) adduct. An online column-switching valve liquid chromatography tandem mass spectrometry (LC-MS/MS) selected reaction monitoring (SRM) method was developed for the determination of N²-ethylidene-dG adducts in DNA following reduction with sodium cyanoborohydride (NaBH₃CN) to the chemically stable N²-ethyl-2′-deoxyguanosine (N²-ethyl-dG) adduct. Accurate quantitation of the adduct was obtained by the addition of the [¹⁵N₅]N²-ethyl-dG stable isotope-labeled internal standard prior to enzymatic hydrolysis of the DNA samples to 2′-deoxynucleosides with the incorporation of NaBH₃CN in the DNA hydrolysis buffer. The method required 50 μg of hydrolyzed DNA on column for the analysis, and the limit of detection for N²-ethyl-dG was 2.0 fmol. The analysis of calf thymus DNA treated in vitro with acetaldehyde (ranging from 0.5 to 100 mM) or with the smoke generated from 1, 5, and 10 cannabis cigarettes showed linear dose-dependent increases in the level of N²-ethyl-dG adducts (r = 0.954 and r = 0.999, respectively). Similar levels (332.8 ± 21.9 vs 348.4 ± 19.1 adducts per 10⁸ 2′-deoxynucleosides) of N²-ethyl-dG adducts were detected following the exposure of calf thymus DNA to 10 tobacco or 10 cannabis cigarettes. No significant difference was found in the levels of N²-ethyl-dG adducts in human lung DNA obtained from nonsmokers (n = 4) and smokers (n = 4) with the average level observed as 13.3 ± 0.7 adducts per 10⁸ 2′-deoxynucleosides. No N²-ethyl-dG adducts were detected in any of the DNA samples following analysis with the omission of NaBH₃CN from the DNA hydrolysis buffer. In conclusion, these results provide evidence for the DNA damaging potential of cannabis smoke, implying that the consumption of cannabis cigarettes may be detrimental to human health with the possibility to initiate cancer development.