A method for measuring disulfide reduction by cultured mammalian cells: Relative contributions of glutathione-dependent and glutathione-independent mechanisms

A method is described for measuring bioreduction of hydroxyethyl disulfide (HEDS) or alpha-lipoate by human A549 lung, MCF7 mammary, and DU145 prostat e carcinomas as well as rodent tumor cells in vitro. Reduction of HEDS or a lpha-lipoate was measured by removing aliquots of the glucose-containing me dia and measuring the reduced thiol wit DTNB (Ellman's reagent). Addition o f DTNB to cells followed by disulfide addition directly measures the format ion of newly reduced thiol. A549 cells exhibit the highest capacity to redu ce alpha-lipoate, while Q(7) rat hepatoma cells show the highest rate of HE DS 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 reduc ed by the cel cultures. Glucose-6-phosphate deficient CHO cells (E89) do no t reduce alpha-lipoate and reduce HEDS at a much slower rate compared to wi ld-type CHO-K1 cells. Depletion of glutathione prevents the reduction of HE DS. The depletion of glutathione inhibited reduction of alpha-lipoate by 25 % and HEDS by 50% in A549 cells, while GSH depletion did not inhibit alpha- lipoate reduction in Q(7) cells but completely blocked HEDS reduction. Thes e data suggest that the relative participation of the thioltransferase (glu taredoxin) and thioredoxin systems in overall cellular disulfide reduction is cell line specific. The effects of various inhibitors of the thiol-disul fide oxidoreductase enzymes (1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU), a rsenite, and phenylarsine oxide) support this conclusion. (C) 2000 Academic Press.