Characterization of nucleoside and DNA adducts formed by S-(1-acetoxymethyl)glutathione and implications for dihalomethane-glutathione conjugates

S-(1-Acetoxymethyl)glutathione (GSCH(2)OAc) was synthesized and used as a m odel for the reaction of glutathione (GSH)-dihaloalkane conjugates with nuc leosides and DNA. Previously, S-[1-(N-2-deoxyguanosinyl)methyl]GSH had been identified as the major adduct formed in the reaction of GSCH(2)OAc with d eoxyguanosine. GSCH(2)OAc was incubated with the three remaining deoxyribon ucleosides to identify other possible adducts. Adducts to all three nucleos ides were found using electrospray ionization mass spectrometry (ESI MS). T he adduct of GSCH(2)OAc and deoxyadenosine was formed in yield of up to 0.0 5% and was identified as S-[1-(N-7-deoxyadenosinyl)methyl]GSH. The pyrimidi ne deoxyribonucleoside adducts were formed more efficiently, resulting in y ields of 1 and 2% for the GSCH(2)OAc adducts derived from thymidine and deo xycytidine, respectively, but their lability prevented their structural ide ntification by H-1 NMR. On the basis of the available UV spectra, we propos e the structures S-[1-(N-3-thymidinyl)methyl]GSH and S-[1-(N-4-deoxycytidin yl)methyl]GSH. Because adduct degradation occurred most rapidly at alkaline and neutral pH values, an enzymatic DNA digestion procedure was developed for the rapid hydrolysis of DNA to deoxyribonucleosides at acidic pH. DNA d igests were completed in less than 2 h with a two-step method, which consis ted of a 15 min incubation of DNA with high concentrations of deoxyribonucl ease II and phosphodiesterase II at pH 4.5, followed by incubation of resul ting nucleotides with acid phosphatase. Analysis of the hydrolysis products by HPLC-ESI-MS indicated the presence of the thymidine adduct.