Genotoxicity evaluation of metronidazole using the piscine micronucleus test by acridine orange fluorescent staining

In this study, genotoxic potential of metronidazole, an antibiotic-antiparasitic agent widely used both clinical and veterinary areas, was evaluated using the piscine micronucleus test. Specimens of Oreochromis niloticus (Pisces: Cichlidae) were exposed to different concentrations (5, 10, 15 mg/L) of metronidazole and 4 mg/L cyclophosphamide (as positive control) for 24, 48 and 72 h. A fluorescent staining technique with acridine orange (AO) was performed to improve the sensitivity of erythrocyte micronucleus assay with fish. AO selectively stains newly formed immature erythrocytes (PCEs) therefore allows to obtain more sensitive results by detection of the micronucleated PCEs differed than mature erythrocytes (NCEs) by their RNA-containing cytoplasm. The ratio of PCE/NCE in peripheral blood was also evaluated to assess cytotoxicity. As a result, it was observed that the frequencies of micronucleated PCEs increased both dose and time dependently while PCE/NCE ratios decreased. Our results revealed that metronidazole has cytotoxic and genotoxic effects on fish. The use of acridine orange staining technique also seems useful in assessment of short-term genotoxic effects of chemicals, when fish are used as experimental animal.