engleski

Cytotoxicity of herbicide atrazine on Chinese Hamster Ovary cell in monolayer

Atrazine (2-chloro-4-ethylamino-6-isopropylamino-s-triazine) is a selective triazine herbicide, one of the most widely used herbicides in corn and other crops. Therefore the extensive use leads to its possible presence in ground water, as well as in raw and industrial food products. Even though atrazine is declared to be slightly toxic as toxicity class III, its possible ingestion via the food chain may present a risk for the reproductive process. The safety assessment of pesticides and the introducing these data into a human risk assessment evaluation requires a large number of expensive, regulated tests in different animal species. Currently a wide range of animal replacement alternative methods is available. By introducing in vitro methodology in toxicity testing, it is possible to evaluate the toxic influence of a chemical without using the great amount of experimental animals. In the present study, in order to determine cytotoxic effect of atrazine within the reproductive hormone-dependent tissues, using CHO K1 (Chinese Hamster Ovary) cell line in monolayer (CCL-61) performed the research. Cell viability and number of cells were measured by Trypane Blue exclusion method in Fuchs-Rosenthal heamocytometer, and by colorimetric assays as Kenacid Blue R binding method measured the change in total cell protein, lyzosomal activity measured by Neutral Red method and MTT assay measured the mitochondrial succinate dehydrogenase activity. The cells were maintained at 370C in an atmosphere of 95% air and 5% CO_2 in Dulbecco medium supplemented with 10% fetal calf serum. The production of biomass was in T-bottle, and the cells were separated in the early logarithmic phase of growth. The initial concentrations of CHO cells were 2.5x10^4 cells/mL/well on 6-multiwell plates and after 72 h it succeeded 21x10^5 cells/mL in the control well. Cells were seeded 16 h before the treatment with atrazine in the concentrations of 2.5-80 microg/mL/well. The number of cells in the presence of atrazine is compared with that observed in control cultures and the percent inhibition of growth calculated. The cytotoxicity was determined after 24, 48 and 72 h. The cell growth inhibition ranged after 72 h from 5 to 75% and was dose-responded. The IC_20, IC_50 and IC_80 were measured from the slope of % inhibition vs. log dose values. The values of IC50 for atrazine after 72 h was ~55-60 microg/mL with all applied methods, acceptable for the quantitative determination of cytotoxic effects of atrazine on CHO K1 cell line. As the first-step in toxicity evaluation, by excluding the experiments with animals, application of cell lines from reproductive tissues like the Chinese hamster ovary enables determination of toxicity in the reproductive processes.

atrazine; Chinese Hamster Ovary cell line; cytotoxicity

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