Mechanism of gastroprotection by bismuth subsalicylate against chemically induced oxidative stress in cultured human gastric mucosal cells

Reactive oxygen species (ROS) are implicated in the pathogenesis of chemica lly induced gastric mucosal injury. We have investigated the effects of eth anol, hydrochloric acid (HCl), and sodium hydroxide (NaOH) on: (1) enhanced production of ROS including superoxide anion and hydroxyl radicals, (2) mo dulation of intracellular oxidized states by laser scanning confocal micros copy, and (3) DNA fragmentation, indices of oxidative tissue, and DNA damag e in a primary culture of normal human gastric mucosal cells (GC), which we re isolated and cultured from Helicobacter-pylori-negative endoscopic biops ies from human subjects. The induction of ROS and DNA damage in these cells following exposure to ethanol (15%), HCl (150 mM) and NaOH (150 mM) were a ssessed by cytochrome c reduction (superoxide anion production), HPLC detec tion for enhanced production of hydroxyl radicals, changes in intracellular oxidized states by laser scanning confocal microscopy, and DNA damage by q uantitating DNA fragmentation. Furthermore, the protective ability of bismu th subsalicylate (BSS) was assessed at concentrations of 25, 50, and 100 mg /liter. Incubation of GC with ethanol, HCl, and NaOH increased superoxide a nion production by approximately 8.0-, 6.1- and 7.1-fold and increased hydr oxyl radical production by 13.3-, 9.6-, and 8.9-fold, respectively, compare d to the untreated gastric cells. Incubation of GC with ethanol, HCl, and N aOH increased DNA fragmentation by approximately 6.7-, 3.3-. and 4.8-fold, respectively. Approximately 20.3-, 17.5-, and 13.1-fold increases in fluore scence intensities were observed following incubation of gastric cells with ethanol, HCl, and NaOH, respectively, demonstrating dramatic changes in th e intracellular oxidized states of GC following exposure to these necrotizi ng agents. Preincubation of GC with 25, 50, and 100 mg/liter of BSS decreas ed ethanol-induced increases in intracellular oxidized states in these cell s by 36%, 56%, and 66%, respectively, demonstrating a concentration-depende nt protective ability by BSS. Similar results were observed with respect to BSS in terms of superoxide anion and hydroxyl radical production, and DNA damage. The present study demonstrates that ethanol, HCl, and NaOH induce o xidative stress and DNA damage in GC and that BSS can significantly attenua te gastric injury by scavenging these ROS.