Characterization of Nucleoside and DNA Adducts Formed by S-(1-Acetoxymethyl)glutathione and Implications for Dihalomethane−Glutathione Conjugates

Abstract

S-(1-Acetoxymethyl)glutathione (GSCH(2)OAc) was synthesized and used as a model for the reaction of glutathione (GSH)-dihaloalkane conjugates with nucleosides 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 deoxyguanosine. GSCH(2)OAc was incubated with the three remaining deoxyribonucleosides to identify other possible adducts. Adducts to all three nucleosides were found using electrospray ionization mass spectrometry (ESI MS). The adduct of GSCH(2)OAc and deoxyadenosine was formed in yield of up to 0.05% and was identified as S-[1-(N(7)-deoxyadenosinyl)methyl]GSH. The pyrimidine deoxyribonucleoside adducts were formed more efficiently, resulting in yields of 1 and 2% for the GSCH(2)OAc adducts derived from thymidine and deoxycytidine, respectively, but their lability prevented their structural identification by (1)H NMR. On the basis of the available UV spectra, we propose the structures S-[1-(N(3)-thymidinyl)methyl]GSH and S-[1-(N(4)-deoxycytidinyl)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 digests were completed in less than 2 h with a two-step method, which consisted of a 15 min incubation of DNA with high concentrations of deoxyribonuclease II and phosphodiesterase II at pH 4.5, followed by incubation of resulting nucleotides with acid phosphatase. Analysis of the hydrolysis products by HPLC-ESI-MS indicated the presence of the thymidine adduct.

Detection of DNA Adducts Derived from the Reactive Metabolite of Furan, cis-2-Butene-1,4-dial

“…Following 18 h incubation at 37 °C, the samples were precipitated with ethanol at ambient temperature and washed twice with 70% ethanol. This DNA was then subjected to enzymatic hydrolysis, employing the method reported by Dennehy and Loeppky (28,29). -8) were added to each sample.…”

Hypermutation and unique mutational signatures of occupational cholangiocarcinoma in printing workers exposed to haloalkanes

“…Mutations with transcriptional strand bias are known to occur in cancer genomes as a result of exposure to abundant mutagens and the formation of bulky DNA adducts, such as in smoking-related lung cancer and ultraviolet-associated melanoma ( 25 , 26 ). We thus suspected that some agents make abundant adducts on G residues and that the transcription-coupled DNA repair machinery preferentially repairs transcribed G residues, that is, sense C residues ( 27 ), inducing strand-biased mutations. These results are consistent with the epidemiological observation that these workers had been exposed to high concentrations of chemical compounds.…”

Analysis of the Kinetic Mechanism of Haloalkane Conjugation by Mammalian θ-Class Glutathione Transferases

“…In conclusion, we still do not have all of the desired mechanistic information relevant to GSH-dihalomethane reactions, due to the difficulties in working with the DNA adducts (44,45). 2 Clearly, CH 2 Cl 2 can be activated to a mutagen, without an obligate role for HCHO.…”