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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.

Aim: Oximes play an important role in the elimination of toxic effects caused by chemical warfare agents, especially in the cases resistant to the atropine treatment alone. K048 oxime as one of the promising antidote in humans recently showed an acceptable genotoxic profile. Present study is focused on potential hepatoprotective effects of K048 effects in vivo. Methods: We selected a model of tabun- poisoned rats, which were given K048 oxime to relieve symptoms of tabun-poisoning. The levels of primary DNA damage in liver cells were studied using the alkaline comet assay 1, 6 and 24 h after the treatments. Sixty male adult Wistar rats were divided in five groups according to the treatment: (1) tabun-poisoned (75% LD50, s.c.), (2) K048-treated (25% LD50 i.p.), (3) atropine-treated (10 mg kg-1, i.p.) after tabun poisoning (75% LD50, s.c.), (4) concomitant treatment with K048 (25% LD50, i.p.) and atropine (10 mg kg-1, i.p.) after tabun poisoning (75% LD50, s.c.), and (5) negative controls given saline only (2 mL kg-1, i.p.). Results: Tabun itself caused the highest level of primary DNA damage in hepatocytes 24 h after its administration. K048 oxime had an acceptable genotoxicity profile at each time point. Its hepatoprotective effects were obvious at each time point studied, and in all cases the levels of primary DNA damage measured in oxime-treated groups were lower or similar as compared to negative control. Minor differences in the genotoxicity observed between the time-points studied could be explained by the metabolism of the compound. Concomitant administration of K048 with atropine changed its genotoxicity profile in the hepatocytes, especially 6 h after treatment. Conclusions: K048 effectively counteracted the poisoning of rats by tabun, both alone and in combination with atropine. Hepatoprotective effects speak in favour of K048 as a promising molecule for therapeutic treatment against fatal poisoning with chemical warfare agents.