Formation and Mass Spectrometric Analysis of DNA and Nucleoside Adducts by S-(1-Acetoxymethyl)glutathione and by Glutathione S-Transferase-Mediated Activation of Dihalomethanes

• Published in: Chemical research in toxicology 2004-01, Vol.17 (1), p.45-54
• Main Authors: Marsch, Glenn A, Botta, Sisir, Martin, Martha V, McCormick, W. Andrew, Guengerich, F. Peter
• Format: Article
• Language: English
• Subjects: Animals; Chromatography, High Pressure Liquid; Cyclic Nucleotide Phosphodiesterases, Type 2; dihalomethanes; DNA Adducts - analysis; DNA Adducts - chemical synthesis; DNA Adducts - toxicity; Endodeoxyribonucleases; Escherichia coli - genetics; Escherichia coli - metabolism; Glutathione - analogs & derivatives; Glutathione - chemistry; Glutathione Transferase - chemistry; Glutathione Transferase - genetics; Glutathione Transferase - metabolism; Humans; Hydrocarbons, Brominated - chemistry; Hydrocarbons, Brominated - toxicity; Isoenzymes - chemistry; Isoenzymes - metabolism; Methylene Chloride - chemistry; Methylene Chloride - toxicity; Molecular Structure; Phosphoric Diester Hydrolases; Rats; S-(1-acetoxymethyl)glutathione; Spectrometry, Mass, Electrospray Ionization; Transfection
• ISSN: 0893-228X; 1520-5010
• DOI: 10.1021/tx034156z

The dihalomethane CH2Cl2 is an industrial solvent of potential concern to humans because of its potential genotoxicity and carcinogenicity. To characterize DNA damage by dihalomethanes, a rapid DNA digestion under acidic conditions was developed to identify alkali labile DNA−dihalomethane nucleoside adducts using HPLC-electrospray mass spectrometry. DNA digestion worked best using pH 5.0 sodium acetate buffer, a 30 min incubation with DNase II and phosphodiesterase II, and a 2 h acid phosphatase digest. DNA was modified with S-(1-acetoxymethyl)glutathione (GSCH2OAc), a reagent modeling activated dihalomethanes. Adducts to G, A, and T were detected at high ratios of GSCH2OAc/DNA following digestion of the DNA with the procedure used here. The relative efficacy of adduct formation was G > T > A ≫ C. The four DNA nucleosides were also reacted with the dihalomethanes CH2Cl2 and CH2Br2 in the presence of glutathione (GSH) and GSH S-transferases from bacteria (DM11), rat (GST 5-5), and human (GST T1-1) under conditions that produce mutations in bacteria. All enzymes formed adducts to all four nucleosides, with dGuo being the most readily modified nucleoside. Thus, the pattern paralleled the results obtained with the model compounds GSCH2OAc and DNA. CH2Cl2 and CH2Br2 yielded similar amounts of adducts under these conditions. The relative efficiency of adduct formation by GSH transferases was rat 5-5 > human T1-1 > bacterial DM11, showing that human GSH transferase T1-1 can form dihalomethane adducts under the conditions used. Although the lability of DNA adducts has precluded more sophisticated experiments and in vivo studies have not yet been possible, the work collectively demonstrates the ability of several GSH transferases to generate DNA adducts from dihalomethanes, with G being the preferred site of adduction in both this and the GSCH2OAc model system.