In glutathione-monochlorobimane assay with human microsomes, increased glutathione concentration and pre-incubation with NADPH and ABT overcomes signal reduction
Glutathione (GSH) is a powerful antioxidant that is vital to cell health to prevent oxidative stress and toxicity. One mechanism of detoxification is through conjugation by glutathione S-transferase (GST) to harmful reactive intermediaries. Drugs, food, environmental pollutants, and other substances can enter the body and produce reactive intermediaries when metabolized, and subsequently alter the levels of GSH in cells, possibly causing cell death, making the effect substances have on GSH concentration of vital importance. At CeeTox, a toxicology testing lab in Kalamazoo, MI, the monochlorobimane-GSH assay is used to assess candidate drug compounds toxicity by measuring GSH concentration after metabolism through conjugation to fluorescent dye monochlorobimane (mBCI). When the mBCI-GSH assay was performed with human microsomes, GSH levels were depleted, evidenced by reduced fluorescence signal, giving confounding information on compound toxicity. Through testing many compounds and running the assay using a range of GSH concentrations, a new procedure was developed. By increasing the GSH concentration to 200 J.lM in samples with human microsomes, and adding a pre-incubation step with reduced nicotinamide adenine dinucleotide phosphate (NADPH) and l-aminobenzotriazole (ABT), the signal was higher while still allowing for differences among samples to be seen, providing researchers with a more accurate assessment of the toxicity of new compounds. This study has refined the important mBCI-GSH assay that has been used prolifically to determine an important characteristic of candidate pharmaceutical compounds.
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