engleski

Testing of lindane cytotoxicity on Chinese Hamster Ovary (CHO-K1) cell line

Organochlorine insecticide lindane (gamma-1, 2, 3, 4, 5, 6-hexachlorocyclohexane) has been extensively used for protection of agricultural products, livestock and trees from variety of pests. Although the use of lindane has been restricted for many years, the chemical stability and earlier widespread use caused its ubiquitous presence in the environment, what enables entering the food chain. Due to its lipophilic characteristics lindane can accumulate in various tissues of living organisms, and can easy reach essential tissues of reproductive system. It is declarated as a highly toxic compound and identified as potential endocrine disruptor. In order to determine its possible toxic effects at ovarian cellular level, in this study Chinese Hamster Ovary (CHO-K1 ; CCL-61) cell line was used. Cells grew in monolayer at 37 º C in the atmosphere of 95% air and 5% CO_2, in the medium Dulbecco's MEM with 5% newborn calf serum. After biomass production in T-bottles, cells were separated in the early logarithmic phase of growth and seeded on multiwell plates in concentration of 2 x 10^4 cells/mL of cultivating medium. Cytotoxic effect of lindane at the concentration range of 20 – 100 micro g/mL was determined by colorimetric methods after 24, 48 and 72 hours. IC_50 value was determined from the slope of % inhibition vs. log dose values. Cell viability and the number of cells were measured by: Trypan Blue exclusion method (IC_50 37, 2 micro g/mL) ; Kenacid Blue R binding method change in total cell protein (IC_50 80, 21 micro g/mL) ; lyzosomal activity by Neutral Red method (IC_50 52, 71 micro g/mL) ; MTT assay, the ability of viable cells to convert a soluble tetrazolium salt into insoluble formazan precipitate (IC_50 68, 64 micro g/mL). Application of in vitro assays in evaluation of the toxicity should be an important contribution in elucidating cellular and molecular mechanisms of lindane.

lindane; CHO-K1 cell line; cytotxicity; Trypan Blue; Kenacid Blue; Neutral Red; MTT assay

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