Excessive oxidation is widely accepted as a precursor to deleterious cellular function. On the other hand, an awareness of the role of reductive stress as a similar pathological insult is emerging. Here we report early dynamic changes in compartmentalized glutathione (GSH) redox potentials in living cells in response to exogenously supplied thiol-based antioxidants. Noninvasive monitoring of intracellular thiol-disulfide exchange via a genetically encoded biosensor targeted to cytosol and mitochondria revealed unexpectedly rapid oxidation of the mitochondrial matrix in response to GSH ethyl ester or N-acetyl-l-cysteine. Oxidation of the probe occurred within seconds in a concentration-dependent manner and was attenuated with the membrane-permeable ROS scavenger tiron. In contrast, the cytosolic sensor did not respond to similar treatments. Surprisingly, the immediate mitochondrial oxidation was not abrogated by depolarization of mitochondrial membrane potential or inhibition of mitochondrial GSH uptake. After detection of elevated levels of mitochondrial ROS, we systematically inhibited multisubunit protein complexes of the mitochondrial respiratory chain and determined that respiratory complex III is a downstream target of thiol-based compounds. Disabling complex III with myxothiazol completely blocked matrix oxidation induced with GSH ethyl ester or N-acetyl-l-cysteine. Our findings provide new evidence of a functional link between exogenous thiol-containing antioxidants and mitochondrial respiration.

Prebiotic galacto‐oligosaccharides (GOS) are non‐digestible food ingredients that are thought to benefit the host indirectly by stimulating the growth and/or activity of beneficial bacteria in the colon. Herein, human adenocarcinoma derived LS174T cells, which exhibit a goblet cell like phenotype, were used to examine possible direct effects of prebiotic GOS on goblet cell function. LS174T cells were treated with GOS and the expression of goblet cell secretory product genes mucin 2 (MUC2), trefoil factor 3 (TFF3), resistin like molecule beta (RETNLB) and Golgi sulfotransferase genes, carbohydrate (N‐acetylglucosamine 6‐O) sulfotransferase 5 (CHST5) and galactose 3‐O‐sulfotransferase 2 (GAL3ST2), was determined by qRT‐PCR and Western blot analysis. QRT‐PCR analysis demonstrated that MUC2 and CHST5 expression was upregulated 2–4 fold while RETNLB and TFF3 expression were upregulated 6–8 fold at 72h. Immunofluorescence microscopy and Western blot data revealed that GOS treatment increased the level of RETNLB, TFF3 and CHST5 at 96h treatment. In addition, IL‐13 with GOS resulted in synergistic induction of RETNLB and CHST5. GOS treatment did not alter IL‐8 secretion. Collectively, the data indicate that GOS may directly enhance mucosal barrier function by modulating the expression of goblet cell products independent of an inflammatory response.