engleski

Imidacloprid toxicity on cell culture and organism models

Modern agriculture has a great environmental impact and the ecological consequences of pesticide use are often of major concern. Pesticides are among the agricultural tools most popularly associated with environmental and human health harm. This research is focused on neonicotinoids which belong to the class of recently developed pesticides for both fast-acting veterinary and crop protection. Imidacloprid (IMI) fits to this group of nicotine-related insecticides, which preferentially act as agonists of the insect nicotinic acetylcholine receptor. A preliminary testing of IMI stability and possible metabolite production was performed with UV-Vis spectrophotometer and HPLC before proceeding with further testing on aquatic invertebrates and cell culture. Consequently, we have explored the baseline toxicity of IMI and its commercial formulation (Confidor 200SL – Bayer Crop Science) in both non-target species Gammarus fossarum (Amphipoda) and in mammalian cell culture model. We tested the effect of different doses of IMI and Confidor (0.4- 2 μM) on animal survival, biochemical biomarkers (i.e. enzymatic activities) and lipid peroxidation (LP). Also, we explored the effects of IMI (1 and 4 mM) on F11 neuronal cell cultures. It was analysed cell proliferation, mitochondrial activity, enzymatic activities and LP. Our data demonstrated an overall low toxicity and mortality rate of IMI in G. fossarum after 24 h exposure. The commercial formulation Confidor demonstrated an increase in mortality at higher concentrations. We found that low concentrations of IMI (0.4 μM) resulted in increased LP levels, while no effects were observed on glutathione-S-transferase (GST) and catalase (CAT) activities. Higher concentration of Confidor (1-2μM) induced an increase GST activity and LP. Furthermore, in cells the results showed relevant cytotoxic effect after 24 and 48h incubation with IMI (4 mM). We established that higher concentration of IMI after 48h induced slight increase in CAT activity and significant increase of LP. In addition, was identified a substantial loss of mitochondrial membrane potential (MMP) with the use of JC-1 fluorescent dye. This loss is a key parameter of mitochondrial function and an indicator of cell death. Control cells show a high MMP, so that JC-1 forms aggregates that cause red fluorescence. On the other hand IMI-treated cells suffer from a low potential, so that JC-1 remains monomeric with green fluorescence. The difference in terms of the MMP of control and treated cells was quantified by fluorescence microscopy. The used spectrophotometric and fluorometric measurements demonstrate the complexity of analytical methods necessary to evaluate potential harmful effects of neonicotinoids during in vivo and in vitro testing.

F11 cells; amphipods; neurons; neonicotinoids; imidacloprid

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