INTRAVITAL STUDY OF CHLORPYRIFOS EFFECTS ON HIPPOCAMPAL NEURONS OF RATS IN CONDITIONS OF CELL CULTURE
Y. T. Salyha, V. V. Vlizlo
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Institute of Animal Biology NAAS, Lviv 79034, st. Stus 38, Ukraine
Chlorpyrifos belongs to the class of organophosphate compounds which are widely used as pesticides. One of the key mechanisms of chlorpyrifos effect is its ability to inhibit cholinesterases. Research works of scientists from different countries, conducted in recent years have shown the existence of other —cholinesterase-independent mechanisms of influence of this substance on a living organism. Unfortunately, there is the growing amount of scientific data showing that chlorpyrifos is much more dangerous for the animal and human organism, especially for children than previously thought. Nervous system is the most vulnerable target of this toxicant. It is known that chlorpyrifos violates normal activity at different stages of nervous cells development. In connection with this, the search for informative ways to explore the neurotoxicity of this compound and determine its features is very actual.
In this paper, we set a goal to test the method of intravital microscopy of primary hippocampal cell cultures to study their growth, development and functioning under the condition of adding to the incubation medium of chlorpyrifos, as well as to establish the existence of quantitative changes of these parameters depending on the dose and duration of the studied toxicant. To visualize nerve cells we used their genetic modification with the green fluorescent protein (GFP) by the method of magnetofection. We studied the neurotoxic effects of chlorpyrifos on hippocampal cells in vitro, adding the chlorpyrifos into the culture medium in concentrations of 5, 15 and 30 μM. Qualitative and quantitative analysis of neurons incubated during 24, 48 and 72 hours of the experiment was made.
Optimal parameters of the method of intravital study of the chlorpyrifos effect on growth, development and the livelihoods of hippocampal neurons in primary cell culture were chosen.
It was found that chlorpyrifos adding into the culture medium in concentrations of 5, 15 and 30 μM lead to statistically significant (p<0.05) dose-dependent damage and as a consequence — the cell death of hippocampal neurons in vitro. Simultaneously, statistically significant (P<0.05) differences in parameters of neurons viability depending on the duration of studied organophosphorus compound action were observed.
Key words: hippocampus, green fluorescent protein, cell culture, cell death, neurons, Neurotoxicity, transfection, chlorpyrifos
