engleski

Effects of paraoxon and oxime K048 on AChE activity and primary DNA damage in A549 and HaCaT cell lines in vitro

Organophosphorus (OP) compound poisoning is an important worldwide clinical and public health problem. Some nerve warfare agents have relatively recently been used in terrorist attacks, while pesticide poisonings represent the leading cause of poisonings reported globally. OPs act primarily as neurotoxins due to the irreversible inhibition of acetylcholinesterase (AChE), but there is increasing evidence for secondary mechanisms of their toxicity. Oximes act as an essential part of antidotal therapy by ensuring the recovery of inhibited AChE. Our recent in vitro and in vivo studies have drawn attention to oxime 1- (4-hydroxyiminomethylpyridinium)-4-(4- carbamoylpyridinium)-butane dibromide (K048) as a very potent reactivator of the nerve agent tabun- and pesticide-inhibited AChE. This study evaluated OP toxicity at molecular level and its potential alleviation by the use of an oxime. For this purpose, as it is one of the most potent AChE-inhibiting compounds available, we selected paraoxon as a model OP and estimated its ability to induce primary DNA damage in two human cell lines known for non- neuromuscular AChE expression. By applying the K048 oxime, we tested its ability to extenuate the effects of paraoxon. Human lung adenocarcinoma epithelial cell line A549 and human keratinocyte cell line HaCaT were cultivated in RPMI growth medium supplemented with glutamine, heat-inactivated fetal bovine serum, and antibiotics. Prior to treatment, cells were seeded in 6-well plates (3x105 cells/mL). The tested concentrations of paraoxon (1 µM), and K048 oxime (0.02 mM) were determined in a preliminary experiment on human erythrocytes. For the genotoxicity testing, we applied the same study design in both cell lines: (1) control group (untreated cells), (2) cells treated with paraoxon (for 10 min), (3) cells treated with K048 (for 30 min), (4) cells treated 10 min with paraoxon followed by a 30- min treatment with K048 oxime. After the treatments, aliquots of cells were used for the preparation of agarose microgels according to standard protocol for the alkaline comet assay. Slides were analyzed 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. AChE activity measurements were performed by using the standardized spectrophotometric Ellman method. At the concentration tested, the K048 oxime did not induce a significant increase of primary DNA damage as compared to the negative control. In contrast, treatment with paraoxon, in spite of the relatively short exposure time, resulted in measurable genotoxicity both on A549 and HaCaT cells. At the same concentration, paraoxon caused a 93% erythrocyte AChE inhibition in our preliminary experiment. The most important observation was the lowering of primary DNA damage in cells treated with both compounds. In both cell lines, when applied after paraoxon, K048 caused a significant decrease of all comet assay parameters as compared to single paraoxon treatment. Furthermore, K048 was shown to reactivate about 87% of paraoxon-inhibited erythrocyte AChE. Apart from its high reactivation potency at the concentration tested, the K048 oxime also possesses antioxidative efficacy, which is an important property for a compound intended for use as an antidote.

oximes; human cell lines; DNA damage; antioxidative potency

nije evidentirano