In GSH-mBCI Assay, Increased GSH Concentration and Pre-Incubation with NADPH and ABT Overcomes Signal Reduction

Abstract

Glutathione (GSH) is a powerful antioxidant that prevents cell toxicity through conjugation to harmful reactive intermediaries and foreign compounds. The reaction is catalyzed by glutathione S-transferase (GST). GSH can be depleted after the metabolism of pharmaceutical drugs. Because maintaining GSH concentration in cells is vitally important, the effect that candidate drugs have on GSH levels is assessed in preliminary stages of drug development. The GSH-monochlorobimane (mBCl) assay has been developed to measure GSH reduction after the metabolism of compounds by cytochrome P450 enzymes (CYP450s) in human and rat microsomes. Controls of 1-aminobenzotriazole (+ABT) are used to assure that the drug was metabolized by CYP450s. NADPH was used to keep GSH in the active reduced form; a negative NAPDH control was used as well (NADPH). Early assays with human microsomes had large GSH reductions, caused by the microsomes themselves. This study aimed to mediate that reduction, producing a viable assay to assess new compounds’ toxicity.