In Vitro Evaluation of Oxidative Stress Induced by Oxime Reactivators of Acetylcholinesterase in HepG2 Cells

Oxime reactivators of acetylcholinesterase (AChE) are used as causal antidotes for intended and unintended poisoning by organophosphate nerve agents and pesticides. Despite all efforts to develop new AChE reactivators, none of these drug candidates replaced conventional clinically used oximes. In addition to the therapeutic efficacy, determining the safety profile is crucial in preclinical drug evaluation. The exact mechanism of oxime toxicity and the structure–toxicity relationship are subjects of ongoing research, with oxidative stress proposed as a possible mechanism. In the present study, we investigated four promising bispyridinium oxime AChE reactivators, K048, K074, K075, and K203, and their ability to induce oxidative stress in vitro. Cultured human hepatoma cells were exposed to oximes at concentrations corresponding to their IC50 values determined by the MTT assay after 24 h. Their potency to generate reactive oxygen species, interfere with the thiol antioxidant system, and induce lipid peroxidation was evaluated at 1, 4, and 24 h of exposure. Reactivators without a double bond in the four-carbon linker, K048 and K074, showed a greater potential to induce oxidative stress compared with K075 and K203, which contain a double bond. Unlike oximes with a three-carbon-long linker, the number of aldoxime groups attached to the pyridinium moieties does not determine the oxidative stress induction for K048, K074, K075, and K203 oximes. In conclusion, our results emphasize that the structure of oximes plays a critical role in inducing oxidative stress, and this relationship does not correlate with their cytotoxicity expressed as the IC50 value. However, it is important to note that oxidative stress cannot be disregarded as a potential contributor to the side effects associated with oximes.


Validation of LC-MS/MS method for determination of MDA in HepG2 cells
The method was validated according to European Medicines Agency (EMA) Guideline on Biomedical method validation.Intra-day and inter-day accuracy and precision were determined by repeated measurements of HepG2 cell homogenate (section 2.6.)spiked with MDA at following concentrations: 0.1, 0.5, 1.0 and 1.5 μM MDA (n=5).The samples were processed as described in section 2.8.Recovery and matrix effect were evaluated by the post-extraction addition approach (n=2).Three sets of samples were prepared: -Matrix-free standards prepared by derivatization of MDA solutions (in 70% MeOH) mixed with internal standard by 25 mM DNPH (ACN/FA, 98:2 v/v) to reach final MDA concentrations of 0.2 and 1.5 μM.
-"Pre-extracted" samples of blank cell homogenate spiked with MDA at concentrations 0.2 and 1.5 μM (n=2) before the procedure described in section 2.8.
-"Post-extracted" samples of blank cell homogenate processed as described in section 2.8. and subsequently spiked with pre-derivatized mixture of internal standard 0.2 and 1.5 μM MDA solution (n=2) after the SPE step.The accuracy and recovery are expressed as mean ± SD (n=5 or n=2).The precision (n=5) and internal-standard normalized matrix effect (n=2) are expressed as CV.

Flow cytometric detection of cell death
The microcapillary flow cytometry (Muse™ Cell Analyzer, Luminex, Austin, TX) was used to determine cell death.
This method is based on detecting externalization of phosphatidylserine on the surface of cells undergoing apoptosis.
The technique allows quantitative analysis of non-apoptotic, early apoptotic, late apoptotic, and necrotic cells.The reagent mixture also contains the membrane non-permeable dye 7-aminoactinomycin D. This component distinguishes necrotic and late apoptotic cells from the live and early apoptotic cells.The measurement of cell death was carried out according to manufacturer instructions after 1, 4, and 24 h incubation with tested oxime reactivators and with oximefree medium as an untreated control.During the first day of the experiment, cells were seeded into a 24-well plate (TPP) with a density of 15 × 104.After the incubation, cells from each well were collected into the polystyrene tube (TPP) and centrifuged for 5 min at 1000 × g at room temperature.The supernatant was discarded.The cell suspensions  S-5

Antioxidant activity of selected oxime reactivators of acetylcholinesterase
The concentration of oximes used for study of the antioxidant activity is shown in the

Fig. S- 1
Fig. S-1The percentage of intact (white column), early (light grey column), late apoptotic (dark grey column), and necrotic HepG2 cells (black column) determined using microcapillary flow cytometry after 1 h, 4 h and 24 h treatment with oxime acetylcholinesterase reactivators or TBHP (positive control).Control represents the cells incubated with an oxime-free culture medium.One-way analysis of variance (ANOVA) followed by Dunnetʼs multiple comparison test was used for statistical analysis by GraphPad Prism 9 software version 9.3.0.Data are expressed as means ± standard deviation (SD) of three independent measurements (n = 3).Significant differences between oxime-treated and untreated control groups (p ≤ 0.05) and marked by asterisk (*).

3 .
Fig. S-2 Data are expressed in percentage as means ± standard deviation (SD) of three independent measurements (n = 3).

Table S - 1 .
Validation parameters of LC-MS/MS method for the determination of intracellular MDA Table S-2 39 .