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.