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Aim: To investigate the ability of acid and nitric oxide (NO) to induce DNA damage in esophageal cells in vitro.

Background: Esophageal adenocarcinoma (EAC) develops via a metaplasia-dysplasia sequence. The luminal microenvironment is thought to be important in driving progression. For example, acid and bile can alter proliferation and differentiation in ex vivo and in vitro models and can induce DNA damage in vitro. High levels of NO have been demonstrated in the esophagus when salivary nitrite is reduced by acid and ascorbic acid (AA) during reflux. By replicating the luminal environment in vitro we aimed to investigate the ability of acid and NO to induce DNA damage in esophageal cells.

Methods: Luminal nitrite chemistry was replicated in vitro by adding physiological concentrations of nitrite and AA to acidified medium. NO was measured with a NO probe (WPI). Cells were incubated with acidified medium containing AA +/- nitrite, with acidified medium alone or with an NO donor (MAHMA NONOate, Axxora) at pH7.4. Primary esophageal cells from BE patients were treated ex vivo with NO donor in a similar manner. DNA damage was assessed by studying phosphorylation of histone H2AX (γH2AX), a marker of double strand breaks (DSBs) and by comet assay. Intracellular detection of reactive oxygen species (ROS) and NO was with CM-H2DCFDA and DAF-FM diacetate dyes (Axxora), respectively. Long-term survival was assessed by clonogenic assay. Phosphorylation of ATM, ATR and DNA-PKcs was detected by western blotting. p53 mutation analysis was performed by restriction site mutagenesis.

Results: Replication of nitrite chemistry in vitro produced concentrations of NO in excess of 200μM. The level of NO produced from nitrite was dependent on AA concentration and pH. Nitrite chemistry, NO donor or acid alone each induced DSBs in esophageal cells in vitro. Acid-induced DSBs occurred in all cells (p<0.05 for incubations >15min at pH3.5) while NO-induced DSBs occurred in a dose response manner (p<0.05 for doses >25μM). NO-induced DSBs were increased in EAC compared to BE cells (p<0.05 for all doses). NO also induced DSBs in primary esophageal cells ex vivo for 2/10 patients. Co-staining with DAPI showed that NO-induced γH2AX occurs preferentially in S-phase cells, suggesting that the DSBs are due to stalled replication forks. Interestingly, NO-induced DSBs occurred in the absence of ATM, ATR and DNA-PKcs phosphorylation. Incubation with acid resulted in intracellular production of ROS, suggesting that acid induced DSBs are mediated by oxidative stress. Clonogenic assays showed that cells were able to survive these insults. However, no mutations were found in the p53 gene of cells treated with either acid or NO.Conclusions: Both acid and NO have the potential to generate DSBs in the esophagus. If not repaired correctly these lesions could lead to gross genetic and chromosomal abnormalities that may aid in the progression of BE to EAC.

98th AACR Annual Meeting-- Apr 14-18, 2007; Los Angeles, CA