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[35S]-LABELING OF THE SALMONELLA TYPHIMURIUM GLUTATHIONE POOL TO ASSESS GLUTATHIONE-MEDIATED DNA BINDING BY 1,2-DIBROMOETHANE
Citation:
Ross, M. K. AND R A. Pegram. [35S]-LABELING OF THE SALMONELLA TYPHIMURIUM GLUTATHIONE POOL TO ASSESS GLUTATHIONE-MEDIATED DNA BINDING BY 1,2-DIBROMOETHANE. CHEMICO-BIOLOGICAL INTERACTIONS. Elsevier Ireland Limited, Limerick, Ireland, 146(1):39-49, (2003).
Impact/Purpose:
The purpose of this study was to metabolically radiolabel the in vivo glutathione pool of Salmonella typhimurium with a [35S]-label and to examine the glutathione-mediated bioactivation of a model haloalkane, 1,2-dibromoethane, by measuring the binding of [35S]-label to DNA.
Description:
Biotransformation of drugs and environmental chemicals to reactive intermediates is often studied with the use of radiolabeled compounds that are synthesized by expensive and technically difficult procedures. In general, glutathione (GSH) conjugation serves as a detoxification mechanism, and conjugation of reactive intermediates with GSH is often a surrogate marker of reactive species formation. However, several halogenated alkanes can be bioactivated by GSH to yield highly reactive GSH conjugates, some of which are DNA-reactive (e.g., conjugates of 1,2-dibromoethane). The purpose of this study was to metabolically radiolabel the in vivo GSH pool of Salmonella typhimurium with a [35S]-label and to examine the GSH-mediated bioactivation of a model haloalkane, 1,2-dibromoethane, by measuring the binding of [35S]-label to DNA. The strain of Salmonella used in this study had been transformed previously with the gene that codes for rat glutathione transferase theta 1-1 (GSTT1-1), an enzyme that can catalyze formation of genotoxic GSH conjugates. Bacteria were grown to mid-log phase and then incubated with [35S]-L-cysteine in minimal medium (thio-free) until stationary phase of growth was reached. At this stage, the specific activity of Salmonella GSH was estimated to be 7.1 mCi/mmol by derivatization and subsequent HPLC analysis, and GSTT1-1 enzyme activity was still demonstrable in Salmonella cytosol following growth in a minimal medium. The [35S]-labeled bacteria were then exposed to 1,2-dibromoethane (1 mM), and the Salmonella DNA was subsequently purified to quantify [35S]-binding to DNA. Compared to a control Salmonella strain that does not express rat GSTT1-1, DNA from the GSTT1-1-expressing bacteria had significantly greater amounts of covalently bound [35S]-label. Neutral thermal hydrolysis of the DNA yielded a single [35S]-labeled adduct with a similar tR as S-[2-(N7-guanyl)ethyl]GSH, following HPLC analysis of the hydrolysate. Thus, this [35S]-radiolabeling protocol may prove useful for studying the DNA reactivity of GSH conjugates of other halogenated alkanes in a cellular context that maintains GSH at normal physiological levels. This is also, to our knowledge, the first demonstration of de novo incorporation of [35S]-L-cysteine into the bacterial GSH pool.