Nitric Oxide and Acid Induce Double-Strand DNA Breaks in Barrett's Esophagus Carcinogenesis via Distinct Mechanisms

Background & Aims: The luminal microenvironment including acid and nitric oxide (NO) has been implicated in Barrett's esophagus carcinogenesis. We investigated the ability of acid and NO to induce DNA damage in esophageal cells. Methods: Transformed and primary Barrett's esophagus and adenocarcinoma cells were exposed to either acid, (pH 3.5), ± antioxidant or NO from a donor or generated by acidification of nitrite in the presence of ascorbate ± NO scavenger. Phosphorylation of histone H2AX and the neutral comet assay were used to detect DNA double-strand breaks (DSBs). Intracellular levels of reactive oxygen species and NO were detected with fluorescent dyes. Mitochondrial viability was measured with a rhodamine dye. Long-term survival was assessed by clonogenic assay. Results: Exposure to acid (pH 3.5) for ≥15 minutes induced DSBs in all cell lines (P < .05). There was a concomitant increase in intracellular reactive oxygen species in the absence of mitochondrial damage, and pretreatment with antioxidants inhibited DNA damage. Exposure to physiologic concentrations of NO produced from the NO donor or acidification of salivary nitrite induced DSBs in a dose- (>25 μmol/L) and cell-dependent manner (adenocarcinoma >Barrett's esophagus, P < .05). This occurred preferentially in S-phase cells consistent with stalled replication forks and was blocked with a NO scavenger. NO also induced DSBs in primary Barrett's esophagus cells treated ex vivo. Cells were able to survive when exposed to acid and NO. Conclusions: Both acid and NO have the potential to generate DSBs in the esophagus and via distinct mechanisms.