Helicobacter pylori-induced oxidative stress and DNA damage in a primary culture of human gastric mucosal cells

Helicobacter pylori has been identified in the pathogenesis of chronic active gastritis and peptic ulcer disease and is epidemiologically linked to gastric cancer and lymphoma. Our previous studies have demonstrated enhanced production of reactive oxygen species (ROS) in cultured gastric adenocarcinoma cells (ATCC CRL/1739) in association with H. pylori. Recently, we have isolated and cultured normal human gastric mucosal cells (GMC) from H. pylori-negative endoscopic biopsies. The integrity of these mucosal cells was determined by periodic acid–Schiff staining. We assessed the effects of various H. pylori strains including 60190 (a 87-kDa cytotoxin producing strain), ATCC 43504, and 60190-v1 (in which the cytotoxin gene has been disrupted) on the primary culture of human gastric mucosal cells. The induction of ROS and DNA fragmentation in the mucosal cells in association with these H. pylori strains were assessed by cytochrome c reduction (an index of superoxide anion production), hydroxyl radical production, and DNA fragmentation. Following incubation of the mucosal cells with 1:0.5 and 1:1 ratios of H. pylori strain 60190, approximately 6.2- and 9.9-fold increases were observed in cytochrome c reduction, respectively, as compared to mucosal cells in the absence of H. pylori, demonstrating the production of superoxide anion. The detection of hydroxyl radicals based on the formation of 2,3-dihydroxybenzoic acid and 2,5-dihydroxybenzoic acid was determined by using a high-performance liquid chromatograph equipped with an electrochemical detector. Approximately 3.5- and 7.7-fold increases in hydroxyl radical production were observed following incubation of the mucosal cells with 1:0.5 and 1:1 ratios of H. pylori, respectively. Approximately 3.6- and 4.5-fold increases in DNA fragmentation were observed in gastric mucosal cells following incubation with 1:0.5 and 1:1 ratios of H. pylori, respectively. The effects of culture supernatant preparations from H. pylori strains 60190 and 60190-v1 on the enhanced production of ROS and increased DNA fragmentation in mucosal cells were also investigated. Culture supernatant preparations, the prime source of the 87-kDa cytotoxin, from both H. pylori strains 60190 and 60190-v1 were extracted under identical conditions to determine the role of 87-kDa cytotoxin on the enhanced production of ROS and DNA fragmentation. The cytotoxin rich-H. pylori strain 60190 induced greater production of ROS and DNA fragmentation in mucosal cells as compared to the supernatant preparation from H. pylori strain 60190-v1, in which the cytotoxin gene has been disrupted. This study demonstrates that H. pylori induces enhanced production of ROS and DNA damage in association with human gastric mucosal cells andthat the 87-kDa cytotoxin protein plays a prime role in the induction of oxidative stress and DNA damage.