Combined Ascorbic Acid And Sodium Nitrite Treatment Induces Oxidative DNA Damage-associated Gastric Carcinoma In Vitro

BackgroundSodium nitrite (NaNO2) and ascorbic acid (AsA) are used widely as a food additive to preserve and tinge color-cured meat and fish. It is well known that nitrite reacts with secondary amines in food under acid conditions to produce mutagenic and carcinogenic N-nitroso compounds. Ascorbic acid is a known antioxidant, which has a potential to reduce chemicals. The formation of N-nitroso compounds can be inhibited by AsA, which means that AsA has antimutagenic or anticarcinogenic activity. However, when antioxidants such as ascorbic acid were simultaneously given with sodium nitrite to rats, forestomach and esophagus carcinogenesis were strongly enhanced. These results suggest that NaNO2is a weak promoter and AsA is strong copromoter of rat forestomach carcinogenesis, and that combined treatment has carcinogenic potential.ObjectiveThe aim of this study is to investigate the carcinogenesis in gastric epithelial cells of the combination of NaNO2and AsA, explore whether DNA oxidate damage contribute to this process, and analyze its relationship with mutant P53and P21(WAF1/CIP1) proteins.Methods1. Treatment with NaNO2and AsA in gastric epithelial cells (GES-1) respectively with different concentration. Analyze the effect of NaNO2and AsA on GES-1 proliferation using3-(4,5-Dimethylthiazol-2-yl)-2.5-diphenyltetrazolium bromide (MTT).2. Treatment combination with NaNO2(2000mg/L) and AsA (100mg/L) in GES-1. The cellular morphology was observed with inverted microscope at24hours.3. Treatment combination with NaNO2(2000mg/L) and AsA (100mg/L) in GES-1. The level of8-OHdG was assessed with immunofluorescence at24hours.4. Treatment combination with NaNO2(2000mg/L) and AsA (100mg/L) in GES-1. The expression level of mutant P53and P21(WAF1/CIP1) proteins were assessed with Western blot.Results1. Low concentration of AsA (25mg/L) will promote the proliferation of GES-1while high concentration of AsA (100mg/L and200mg/L) will inhibit it. Also, low concentration of NaNO2(125mg/L) will promote the proliferation of GES-1while high concentration of NaNO2(1000mg/L) will inhibit it. The effect was dependent on time and concentration.2. Compared to the control group, AsA group and NaNO2group, the adherent cells decreased and the suspension cells increased, the cells surrounding debris, nuclei and cytoplasm boundaries blur, and the status of nuclear and cytoplasmic fusion in the combination group.3. The expression level of8-OHdG was higher in combination group when compared to the control group, AsA group and NaNO2group (P<0.05).4. The expression level of P21(WAF1/CIP1) protein was lower in combination group when compared to the control group, AsA group and NaNO2group (P<0.05). The expression level of mutant P53protein was higher in combination group when compared to the control group, AsA group and NaNO2group (P<0.05).ConclusionThe study indicated that the combination of NaNO2with AsA will promote gastric carcinoma due to oxidative DNA damage and the abnormal expression of mutant P53and P21(WAF1/CIP1) proteins.