ABSTRACT
Cancer is a major cause of death of humans and animals. Cancer develops as a function of age, environment, diet and genetic makeup, whether in man or animal. It remains a major chronic health problem associated with genotoxic substances. Generally, cancer cells develop from normal cells due to unrepaired damage of DNA. Ethidium bromide is an intercalating agent commonly used as a fluorescent tag (nucleic acid stain) in molecular biology laboratories to visualize nucleic acid bands in electrophoresis and in other methods of gel-based nucleic acid separation. It has been found to be a mutagen but whether it is a carcinogen in animals and humans has remained controversial and murky.
In this study, biochemical, histopathological and histochemical evaluation of ethidium bromide for carcinogenic potential in the colon, kidney, liver and lungs of Wistar albino rats were investigated. Ninety six rats were used. They were divided into eight groups, consisting of twelve rats each. Group A served as the control and was administered distilled water orally. The other groups, B, C, D, E, F, G and H were administered ethidium bromide at 5mg, 10mg, 20mg, 40mg, 60mg, 80mg, and 100mg per kilogram body weight respectively once a week for twenty-eight weeks. At the end of the twenty-eight weeks, the rats were sacrificed and blood samples were obtained by cardiac puncture. The kidney, liver, lungs and colon were excised for biochemical analyses, as well as histopathological and histochemical examinations.
Biochemical analyses revealed that relative to the controls, there were dose-dependent reduction in the activities of antioxidant enzymes: catalase, superoxide dismutase and glutathione peroxidase. Biochemical analysis also showed a dose-dependent reduction in the level of total protein. A dose-dependent increase in malondialdehyde level in the organs of the ethidium bromide treated rats was observed. The results showed that there were increase in lactate dehydrogenase activities in the blood and organs in a dose-dependent fashion. Significant dose-dependent decreases (p≤0.05) in weight gain were also observed in ethidium bromide treated rats when compared to the control rats. Dose dependent curve revealed that the antioxidant enzymes (catalase, glutathione peroxidase and superoxide dismustase) had significant negative correlation with ethidium bromide dose (r = -0.8336 to -0.9963, -0.9328 to 0.9824, -0.8295 to -0.9845; p<0.001) while lactate dehydrogenase and malondialdehyde had significant positive correlation with ethidium bromide dose (r = +0.9476 to +0.9954, +0.9794 to +0.9933; p<0.001) in the colon, kidney, liver and lungs. Analysis of the various biochemical parameters revealed negative correlation between the antioxidant and lipid peroxidation in the organs (r = -0.9027 to -0.9854, -0.9502 to -0.9864, -0.7456 to -0.9919; p<0.001). Light microscopy of hematoxylin and eosin stained sections of the colon, liver, kidney, and lungs of the rats exposed to different doses of ethidium bromide revealed evidence of precancerous and/or cancer related changes namely hyperchromatic nuclei, increased nuclei/cytoplasm ratio, pleomorphism and abnormal mitosis. Immunohistochemistry carried out on the sections of the colon, kidney, liver and lung after the light microscopy examination revealed the presence of tumor markers, namely cytokeratin 20 (CK 20) in the colon, alpha-fetoproteins (AFP) in the liver, cluster of differentaiation (CD) 10 in the kidney and CD 56 in the lungs. Immunohistochemistry done on organs from the control group rats were negative to these tumor markers. The results strongly suggest that ethidium bromide is an agent that causes cancer in the colon, liver, kidney and lungs of Wistar albino rats.
