SODIUM BENZOATE IN LOCALLY AVAILABLE SOFT DRINKS AND ITS EFFECT ON DNA DAMAGE AND LIVER FUNCTION IN RATS

Sodium benzoate (E211) is used as a preservative in several kinds of food. One of the essential uses of E211 is to preserve non-alcoholic carbonated beverages. The amount of sodium benzoate in 17 local soft drink samples was estimated in the present study. The in vivo effects on biochemical aspects of the liver, kidney function, and DNA damage in lymphocytes were also investigated after oral administration of sodium benzoate in rats over 12 days. The control and experimental groups were fed standard pellet diet and distilled water ad libitum ; and administered distilled water (control) and different concentrations (30, 60, and 120 mg/kg body weight) of sodium benzoate once daily through a stomach tube (0.5 ml), respectively. The alkaline comet assay was performed to investigate the possible DNA damage induced by E211 in lymphocytes. At the end of the experiment, after overnight fasting the rats were sacrificed and blood samples were collected. Animals showed no significant weight loss or gain. The investigation revealed that twelve samples contained a higher level, and the rest had trace or below the recommended maximum permitted concentration (150 mg/l) of E211 as a preservative in the soft drinks. The percentage of tail DNA (p < 0.01) and olive tail movement (p < 0.05) was significantly increased in lymphocytes that were treated with high concentration (120 mg/kg body weight) of sodium benzoate, indicated substantially higher DNA damage (3.5 times) in lymphocytes Evaluation of DNA damage by comet assay: The possible DNA damage induced by sodium benzoate in lymphocytes was measured by using the alkaline comet assay. The comet assay was carried out by a slightly modified method of Tice et al . (38) . Lymphocyte was suspended in 75 µl 0.7% low melting point agarose in PBS at 37 º C and placed on a microscopic slide with a 1% agarose layer. For DNA unwinding, the slides were immersed in lysis solution at 4 º C for 1 hour and then placed in a single row in a horizontal electrophoresis tank containing alkaline buffer at 4 º C for 20 minutes. Then, at the same temperature, the electrophoresis was carried out at 25V, 300 mA for 40 minutes. The slides were washed with a neutralization buffer three times (5 minutes each) after removing the slides from the lysis solution and then visualized after silver staining according to the method of Nadin et al . (39) . All sample preparation steps for electrophoresis were conducted under yellow light to minimize the possibility of damage to cellular DNA. The comet images of both control and sodium benzoate treated animal’s lymphocytes were analyzed microscopically using the software Comet Assay Software Project (CASP, version 1.2.2), established by Konca et al . (40) . The threshold values of CASP parameters were adjusted to achieve the optimum values for our staining procedure; DNA damage was quantified by calculating the comet parameter: percentage of DNA in the comet’s tail (%TD) and olive tail moment (OTM). The Olive tail moment is the percent of DNA in the tail multiplied by the distance between the center of gravity of DNA in the tail and the center of gravity of DNA in the head in the x-direction. The more severe the damage, the higher the percent of DNA fragmentation. Similarly, the longer the comet tails, the higher the DNA fragmentation and the more severe the damage. Thus, OTM, higher Biochemical analysis: Standard laboratory methods