Oxidized extracellular E_h Cys/CySS induces ROS production in monocytes but has no effect on cellular E_h GSH/GSSG.

<p>U937 cells were lysed 2 h and 8 h after exposure to −80 mV and −46 mV Cys redox states. Cellular concentrations of GSH and GSSG were determined by HPLC. GSH levels (A) and E_h GSH/GSSG (B) were not significantly different between 80 mV and −46 mV treatments. In (C), U937 cells were pre-incubated with an ROS-sensitive dye, DCFH-DA (100 µM) for 30 min, before treating with −80 mV and −46 mV redox media for 5 min at 37°C. Oxidation of DCFH-DA to fluorescent DCF was measured on a microplate reader. Cells exposed to oxidized Cys/CySS redox (−46 mV) show a 5-fold increase in ROS production compared to cells exposed to a physiological redox potential of −80 mV (*P<0.001). Pre-treating cells with 0.25 mM 4-acetamide-4′-amleimidylstilbene-2,2′-disulfonic acid (AMS), a non-permeant alkylating agent, attenuated the increase in ROS production (P<0.001). As a positive control, ROS production was measured in monocytes treated with glucose oxidase (2 units); an enzyme system that generates H₂0₂ (*P<0.001). NAC pre-treatment attenuated glucose oxidase-induced ROS production (*P<0.001). Data are mean+SE of 4 replicates of a representative experiment repeated 3 times.