| 14727918 |
Marsch GA, Botta S, Martin MV, McCormick WA, Guengerich FP: Formation and mass spectrometric analysis of DNA and nucleoside adducts by S-(1-acetoxymethyl) glutathione and by glutathione S-transferase-mediated activation of dihalomethanes. Chem Res Toxicol. 2004 Jan;17(1):45-54.
The dihalomethane CH (2) Cl (2) 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 (GSCH (2) OAc), a reagent modeling activated dihalomethanes. Adducts to G, A, and T were detected at high ratios of GSCH (2) OAc/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 CH (2) Cl (2) and CH (2) Br (2) 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 GSCH (2) OAc and DNA. CH (2) Cl (2) and CH (2) Br (2) 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 GSCH (2) OAc model system. |
1(0,0,0,1) |