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P 273 Evaluation of the DNA damaging potential of antidote oxime K048 in A549 and HaCaT cells

Pyridinium oximes are used to successful antidotes poisoning by various organophosphate compounds mainly due to their ability to protect unphosphorylated acetylcholinesterase (AChE) and/or reactivate phosphorylated AChE. Previous studies with the K048 oxime (N-(4-(4-hydroxyiminomethylpyridinio)butyl)-4-carbamoyl-pyriminium dibromide), a representative of a new generation of pyridinium oximes, have suggested its acceptable cytotoxicity and low potential for infliction of primary DNA damage in human peripheral blood lymphocytes in vitro and rat blood cells in vivo. In the present study, we evaluated the genotoxic potency of K048 oxime on the human lung adenocarcinoma epithelial cell line A549 and human keratinocyte cell line HaCaT in vitro. Since both cell lines are known for non-neuromuscular AChE expressions, this study also aimed to clarify the mutual relationships between DNA damage and AChE activity. Both cell lines were cultivated in RPMI growth medium supplemented with glutamine, heat-inactivated fetal bovine serum, and antibiotics. Prior to the treatment, cells were seeded in 6-well plates (3x105 cells/ml). The K048 oxime was tested in the concentration range 0.0073 to 2mM, which was well below the IC50 value, established previously. After 30 minutes of treatment, aliquots of cells were used for the preparation of agarose microgels according to standard protocol for the alkaline comet assay. Slides were analysed under fluorescent microscope using the Comet Assay IV analysis system (Perceptive Instruments Ltd., UK). The level of DNA damage was evaluated based on comet tail length, tail intensity and tail moment. Our results show that both cell lines expressed a low level of spontaneous DNA damage. The short exposure time used here was obviously not critical for the induction of severe DNA lesions. The most prominent finding was time concentration- dependent decrease of genotoxicitym which was observed in both cell lines, Primary DNA damage was slightly increased as compared to the negative control only at two lowest concentrations tested. This observation points to a potential antioxidative efficacy of the tested compound, which has to be elucidated in future investigations. Taken together, our results confirm high biocompatibility for the K048 oxime, which has already been recognized

acethylcholinesterase; K048 oxime; comet assay; DNA damage; cell line; HaCaT; A549

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