A Method for Measuring Disulfide Reduction by Cultured Mammalian Cells: Relative Contributions of Glutathione-Dependent and Glutathione-Independent Mechanisms

Abstract

A method is described for measuring bioreduction of hydroxyethyl disulfide (HEDS) or α-lipoate by human A549 lung, MCF7 mammary, and DU145 prostate carcinomas as well as rodent tumor cells in vitro. Reduction of HEDS or α-lipoate was measured by removing aliquots of the glucose-containing media and measuring the reduced thiol with DTNB (Ellman's reagent). Addition of DTNB to cells followed by disulfide addition directly measures the formation of newly reduced thiol. A549 cells exhibit the highest capacity to reduce α-lipoate, while Q7 rat hepatoma cells show the highest rate of HEDS reduction. Millimolar quantities of reduced thiol are produced for both substrates. Oxidized dithiothreitol and cystamine were reduced to a lesser degree. DTNB, glutathione disulfide, and cystine were only marginally reduced by the cell cultures. Glucose-6-phosphate deficient CHO cells (E89) do not reduce α-lipoate and reduce HEDS at a much slower rate compared to wild-type CHO-K1 cells. Depletion of glutathione prevents the reduction of HEDS. The depletion of glutathione inhibited reduction of α-lipoate by 25% and HEDS by 50% in A549 cells, while GSH depletion did not inhibit α-lipoate reduction in Q7 cells but completely blocked HEDS reduction. These data suggest that the relative participation of the thioltransferase (glutaredoxin) and thioredoxin systems in overall cellular disulfide reduction is cell line specific. The effects of various inhibitors of the thiol–disulfide oxidoreductase enzymes (1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU), arsenite, and phenylarsine oxide) support this conclusion.