Dose-Dependency of Toxic Signs and Outcomes of Paraoxon Poisoning in Rats

8 original article Dose-Dependency of Toxic Signs and Outcomes of Paraoxon Poisoning in Rats Žana M. Maksimović1,*, Ranko Škrbić1,2, Miloš P. Stojiljković1,2 ABSTRACT Organophosphorus compounds induce irreversible inhibition of acetylcholinesterase, which then produces clinically manifested muscarinic, nicotinic and central effects. The aim of the study was to analyse the clinical signs of acute paraoxon poisoning in rats and to determine the relationship between the intensity of signs of poisoning and the dose of paraoxon and/or the outcome of poisoning in rats. Animals were treated with either saline or atropine (10 mg/kg intramuscularly). The median subcutaneous lethal dose (LD50) of paraoxon was 0.33 mg/kg and protective ratio of atropine was 2.73. The presence and intensity of signs of poisoning in rats (dyspnoea, lacrimation, exophthalmos, fasciculations, tremor, ataxia, seizures, piloerection, stereotypic movements) were observed and recorded for 4 h after the injection of paraoxon. Intensity of these toxic phenomena was evaluated as: 0 – absent, 1 – mild/moderate, 2 – severe. Fasciculations, seizures and tremor were more intense at higher doses of paraoxon and in non-survivors. In unprotected rats piloerection occurred more often and was more intense at higher doses of paraoxon as well as in non-survivors. In atropine-protected rats, piloerection did not correlate with paraoxon dose or outcome of poisoning. The intensity of fasciculations and seizures were very strong prognostic parameters of the poisoning severity. KEYWORDS organophosphate; insecticide; paraoxon; poisoning; acetylcholinesterase inhibitor; atropine A U T H O R A F F I L I AT I O N S Centre for Biomedical Research, Faculty of Medicine, University of Banja Luka, Banja Luka, the Republic of Srpska, Bosnia and Herzegovina 2 Department of Pharmacology, Toxicology and Clinical Pharmacology, Faculty of Medicine, University of Banja Luka, Banja Luka, the Republic of Srpska, Bosnia and Herzegovina * Corresponding author: Centre for Biomedical Research, Faculty of Medicine, University of Banja Luka, Banja Luka, the Republic of Srpska, Bosnia and Herzegovina; e-mail: zana.maksimovic@med.unibl.org 1 Received: 9 July 2021 Accepted: 23 February 2022 Published online: 29 June 2022 Acta Medica (Hradec Králové) 2022; 65(1): 8–17 https://doi.org/10.14712/18059694.2022.10 © 2022 The Authors. This is an open-access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Paraoxon Toxicity Signs Related to Dose and Outcome INTRODUCTION Acetylcholinesterase (AChE, EC 3.1.1.7) is a very potent enzyme whose role is to break down acetylcholine (ACh) in the synaptic cleft (1, 2). Inhibition of AChE results in the accumulation of ACh and excessive stimulation of cholinergic receptors. AChE inhibitors (AChEI) can be reversible (carbamate compounds) and irreversible (organophosphorus compounds – OPCs) (3–5). OPCs form a stable covalent bond with AChE, which is not spontaneously hydrolysed (6). They are divided into two major groups, nerve agents (tabun, sarin, soman, VX) (7) and organophosphorus insecticides (OPI). Paraoxon (diethyl (4-nitrophenyl) phosphate) is an active metabolite of the highly toxic OPI parathion (8). Among the OPIs, paraoxon is very similar to nerve agents, in terms of its median lethal dose (LD50), profile of inhibition of cholinesterases and general toxicity (9). Acute OPC poisoning manifests itself with muscarinic effects (bronchoconstriction, bronchorroea, bradycardia, hypotension, nausea, vomiting, increased motility of bowels and bladder, miosis, hypersalivation, lacrimation), nicotinic effects (tachycardia, hypertension, fibrillation, fasciculations, skeletal muscle necrosis, mydriasis) and CNS effects (tremor, convulsions, coma, respiratory depression) (10). Intermediate syndrome can occur after 1–4 days and, 1–2 weeks later, organophosphate-induced delayed neuropathy (OPIDN) can be seen. Treatment of OPC poisoning is based on a triple regimen: symptomatic anticholinergic therapy (atropine), AChE reactivators (oximes) and anticonvulsants (mainly diazepam) (11). Atropine as an antimuscarinic drug, alleviates the muscarinic effects of OPC poisoning, but has no impact on the nicotinic ones. Oximes bind to OPC already bound to AChE, which leads to the reactivation of AChE, with variable affinity for different OPCs between oximes. Diazepam inhibits the excitability of the neurons in the CNS; by increasing the effect of GABA, it increases cAMP, decreases the level of cGMP, leading to the cessation of convulsions (11). The aim of the study was to analyse the clinical signs of acute paraoxon poisoning in rats and to determine whether there is a relationship between the intensity of toxicity signs and the dose of paraoxon and/or outcome of poisoning. MATERIAL AND METHODS ANIMALS The study was conducted in adult Wistar rats weighing 200–240 g, purchased from the Faculty of Natural Sciences and Mathematics, University of Banja Luka, the Republic of Srpska. The animals were given water and food ad libitum, kept at a temperature of 20–22 °C, with a 12 h cycle of light and darkness. The study was approved by the Ethics Committee for the Protection and Welfare of Experimental Animals in Biomedical Research, Faculty of Medicine, University of Banja Luka (Decision No 18/1/20). The animals were treated in accordance with the Guide for the Care and Use of Laboratory Animals (12). The study was 9 conducted at the Centre for Biomedical Research, Faculty of Medicine, University of Banja Luka. CHEMICALS Paraoxon was purchased from Sigma Aldrich, St Louis, MO, USA. Paraoxon was dissolved in isopropyl alcohol up to a concentration of 100 mg/mL and final dilution to the desired concentration was made with saline (0.9% NaCl). Atropine sulphate was dissolved in saline to a concentration of 10 mg/mL. The volumes of administered paraoxon and atropine were 1 mL/kg. Paraoxon and atropine were administered subcutaneously (sc) and intramuscularly (im), respectively. Final dilutions were made immediately before application. STUDY DESIGN The LD50 of paraoxon was determined by the “up and down” method according to Litchfield and Wilcoxon (1949) (13). In the first part of the experiment, then following doses of paraoxon were administered: 0.2, 0.3, 0.35, 0.4 mg/kg sc. One minute after paraoxon the saline (1 mL/kg, im) was administered. In the second part of the experiment, the following doses of paraoxon were administered: 0.6, 0.9 and 1.2 mg/kg sc. Atropine 10 mg/kg im was injected 1 minute after paraoxon application. The presence and intensity of signs of paraoxon poisoning in animals were observed and recorded for 4 h. The following signs have been observed: dyspnoea, lacrimation, exophthalmos, fasciculations, tremor, ataxia, seizures, piloerection, stereotypic movements. Their presence and intensity were noted at the minutes: 5, 10, 15, 30, 60, 90, 120, 150, 180, 210 and 240 after paraoxon application. Intensity was evaluated as: 0 – absent, 1 – mild/ moderate, 2 – severe. Signs of poisoning were observed in relation to the dose of paraoxon, as well as the outcome of the poisoning (survival or death). Tab. 1 Time of death from paraoxon administration depending on paraoxon dose. POX dose (mg/kg sc) With saline With atropine Mean ± SD 95% CI 0.2 – – 0.3 16.67 ± 7.51 −1.98–35.31 0.35 22.00 ± 3.61 18.67–25.33 0.4 18.09 ± 6.99 13.39–22.78 All 19.19 ± 6.19 16.37–22.01 0.6 – – 0.9 14.00 ± 3.83 7.91–20.09 1.2 14.00 ± 4.24 7.25–20.75 All Total Time of death (minute) 14.00 ± 3.74 10.87–17.13 17.76 ± 6.04 15.46–20.06 SD: standard deviation; CI: confidence interval; POX: paraoxon; Administered volumes of paraoxon, atropine and saline were 1 mL/kg; Atropine at a dose of 10 mg/kg im was given 1 minute after paraoxon application. 10 Žana M. Maksimović et al. Acta Medica (Hradec Králové) STATISTICS The LD50 and the PR were analysed by the method of Litchfield and Wilcoxon (1949) on Pharm/PCS statistical software. Other analyses were performed on IBM SPPS for Windows, Version 18.0. After the Kolmogorov-Smirnov test showed an unequal distribution of data, appropriate nonparametric tests were applied: Chi-square test (or Fisher exact test) and Kruskal-Wallis test. Statistical significance level was set at p < 0.05. RESULTS The LD50 of paraoxon was 0.33 mg/kg sc (95% CI: 0.31– 0.36). The LD50 of paraoxon when atropine was administered was 0.91 mg/kg sc (95% CI: 0.67–1.25). Therefore, the PR of atropine was 2.73. All deaths occurred during the first hour of poisoning (Table 1). CLINICAL SIGNS OF POISONING 1. Fasciculations Frequency of fasciculations was not correlated with the dose of paraoxon (Table 2). In atropine-protected rats, fasciculations occurred more often in non-survivors (p = 0.023) (Table 3). Fasciculations occurred earlier and were more intense at higher doses of paraoxon (Figure 1). Although the intensity of fasciculations were related to the dose of paraoxon throughout the observed period, the difference was significant in the minutes 10, 15, 30, 210 and 240 (Kruskal-Wallis test, p = 0.035, p = 0.045, p = 0.038, p = 0.014 and p = 0.034, respectively). Due to atropine protection, higher doses of paraoxon could be administered. The intensity of fasciculations depending on paraoxon dose when atropine was administered is shown in Figure 2. Atropine did not influence the Tab. 2 Frequency of signs of poisoning related to paraoxon dose. Paraoxon + Saline Sign Paraoxon (mg/kg, sc) Paraoxon + Atropine Total p* 83.33 85.71 83.33 100.00 100.00 100.00 0.00 33.33 Exophthalmos 83.33 Lacrimation 83.33 0.2 0.3 0.35 0.4 100.00 66.67 100.00 Seizures 66.67 75.00 Tremor 83.33 Fasciculations Piloerection Ataxia Paraoxon (mg/kg, sc) Total p* 50.00 77.78 0.250 100.00 100.00 94.44 1.000 100.00 100.00 100.00 100.00 1.000 0.009 50.00 50.00 0.00 33.33 0.149 95.23 0.498 83.33 100.00 100.00 94.44 1.000 54.76 0.501 16.67 33.33 16.67 22.22 1.000 0.6 0.9 1.2 0.080 100.00 83.33 83.33 0.225 83.33 100.00 97.61 0.143 66.67 75.00 50.00 91.67 100.00 100.00 50.00 50.00 50.00 83.33 58.33 75.00 58.33 66.67 0.691 66.67 33.33 33.33 44.44 0.589 Stereotypy 100.00 66.67 83.33 41.67 69.05 0.044 66.67 0.00 50.00 38.89 0.095 Dyspnoea 33.33 33.33 25.00 16.67 26.19 0.862 50.00 50.00 33.33 44.44 1.000 * Chi-squared test (Fisher exact), bold: statistical significance. Values in the table are in percentages; sc: subcutaneously; im: intramuscularly; Administered volumes of paraoxon, atropine and saline were 1 mL/kg; Atropine at a dose of 10 mg/kg im was given 1 minute after paraoxon application. Tab. 3 Frequency of signs of poisoning related to poisoning outcome. Paraoxon + Saline Sign Rat survived Paraoxon + Atropine Total p* Rat survived Total p* 100.00 77.78 0.023 90.00 100.00 94.44 1.000 1.000 100.00 100.00 100.00 1.000 0.002 40.00 25.00 33.33 0.638 1.000 90.00 100.00 94.44 1.000 54.76 0.062 20.00 25.00 22.22 1.000 61.90 66.67 0.744 70.00 12.50 44.44 0.025 85.71 52.23 69.05 0.043 60.00 12.50 38.89 0.066 28.57 23.81 26.19 1.000 60.00 25.00 44.44 0.239 Yes No Yes No Fasciculations 85.71 85.71 85.71 1.000 50.00 Seizures 66.67 100.00 83.33 0.009 Tremor 95.23 100.00 Piloerection 23.81 76.19 97.61 50.00 Exophthalmos 95.23 95.23 95.23 Lacrimation 71.43 38.10 Ataxia 71.43 Stereotypy Dyspnoea * Chi-squared test (Fisher exact), bold: statistical significance; Values in the table are in percentages; im: intramuscularly; Administered volumes of paraoxon, atropine and saline were 1 mL/kg; Atropine at a dose of 10 mg/kg im was given 1 minute after paraoxon application. Paraoxon Toxicity Signs Related to Dose and Outcome Fig. 1 Intensity of fasciculations in rats depending on paraoxon dose. * Kruskal-Wallis test, red colour: statistical significance; POX: paraoxon; sc: subcutaneously; Minute: minutes 10, 15, 30, 120, 210 and 240 after paraoxon application. Fig. 2 Intensity of fasciculations depending on paraoxon dose in rats protected with atropine. * Kruskal-Wallis test, red colour: statistical significance; POX: paraoxon; sc: subcutaneously; Minute: minutes 5, 10, 90, 120, 180 and 240 after paraoxon application; Atropine at a dose of 10 mg/kg im was given 1 minute after paraoxon application. Fig. 3 Intensity of fasciculations in relation to whether rat treated with paraoxon survived or not. * Fisher exact test, red colour: statistical significance; Minute: minutes 5, 10, 15 and 30 after paraoxon application. 11 12 intensity of fasciculations. Fasciculations occurred earlier and were more intense at higher doses of paraoxon. Although the intensity of fasciculations was related to the dose of paraoxon throughout the observed period, the difference was significant in the minute 60 (data not shown), 90, 120 and 240 (Kruskal-Wallis test, p = 0.033, p = 0.048, p = 0.048 and p = 0.044, respectively). In unprotected rats intensity of fasciculations was in correlation with the outcome of poisoning (higher intensity was in non-survivors) (Figure 3). In atropine-protected rats, fasciculation intensity did not correlate with the outcome of poisoning. 2. Seizures Frequency of seizures was not correlated with the dose of paraoxon (Table 2), but seizures were more often in non-survivors compared to survivors (Table 3). Seizures Žana M. Maksimović et al. Acta Medica (Hradec Králové) occurred earlier and were more intense at higher doses of paraoxon (Figure 4). Although the intensities of seizures were related to the dose of paraoxon throughout the observed period, the difference was significant only at minutes 15, 180 and 210 (Kruskal-Wallis test, p = 0.002, p = 0.024 and p = 0.015, respectively). A clear relation between paraoxon dose and seizure intensity can be seen in atropine-protected rats (Figure 5). Although the intensity of seizures was related to the dose of paraoxon throughout the observed period, the difference was significant only at minutes 10, 15, 210 and 240 (Kruskal-Wallis test, p = 0.031, p = 0.014, p = 0.044 and p = 0.011, respectively). In unprotected rats the intensity of seizures was in correlation with the outcome of poisoning (higher intensity was in non-survivors) (Figure 6). In atropine-protected rats, the intensity of seizures did not correlate with the outcome of poisoning. Fig. 4 Intensity of seizures in rats depending on paraoxon dose. * Kruskal-Wallis test, red colour: statistical significance; POX: paraoxon; sc: subcutaneously; Minute: minutes 10, 15, 90, 180, 210 and 240 after paraoxon application. Fig. 5 Intensity of seizures depending on paraoxon dose in rats protected by atropine. * Kruskal-Wallis test, red colour: statistical significance; POX: paraoxon; sc: subcutaneously; Minute: minutes 10, 15, 90, 180, 210 and 240 after paraoxon application; Atropine at a dose of 10 mg/kg im was given 1 minute after paraoxon application. Paraoxon Toxicity Signs Related to Dose and Outcome 13 Fig. 6 Intensity of seizures in relation to whether rat treated with paraoxon survived or not. * Fisher exact test, red colour: statistical significance; Minute: minutes 5, 10, 15 and 30 after paraoxon application. 3. Tremor Frequency of tremor was not correlated with the paraoxon dose (Table 2) or the outcome of poisoning (Table 3). Tremor occurred earlier and was more intense at higher doses of paraoxon (Figure 7). Although the intensity of tremor was related to the dose of paraoxon throughout the observed period, the difference was significant at minutes 10, 15 and 240 (Kruskal-Wallis test, p = 0.001, p = 0.002 and p = 0.044, respectively). In the atropine-protected rats, although a higher intensity of tremor was observed at higher doses of paraoxon, the difference was not significant, except at minute 10 (χ2 = 9.88, p = 0.007) and 30 (χ2 = 6.00, p = 0.050). In unprotected rats intensity of tremor was in correlation with the outcome of poisoning (higher intensity was in non-survivors) (Figure 8). In atropine-protected rats, the intensity of tremor did not correlate with the outcome of poisoning. 4. Piloerection Piloerection as a clinical sign of poisoning occurred early (within the first half hour of poisoning) and lasted for a short time (Figure 9). Piloerection occurred more often (Table 2) and was more intense at higher doses of paraoxon administered to unprotected rats. The difference was significant at minutes 10 and 15 (Kruskal-Wallis test, p = 0.052 and p = 0.012, respectively). In atropine-protected rats, piloerection did not correlate with paraoxon dose. In unprotected rats intensity of piloerection was in correlation with the outcome of poisoning. Piloerection was Fig. 7 Intensity of tremor in rats depending on paraoxon dose. * Kruskal-Wallis test, red colour: statistical significance; POX: paraoxon; sc: subcutaneously; Minute: minutes 10, 15, 30, 60 and 240 after paraoxon application. 14 Žana M. Maksimović et al. Acta Medica (Hradec Králové) Fig. 8 Intensity of tremor in relation to whether rat treated with paraoxon survived or not. * Fisher exact test, red colour: statistical significance; Minute: minutes 5, 10, 15 and 30 after paraoxon application. Fig. 9 Intensity of piloerection in rats depending on paraoxon dose. * Kruskal-Wallis test, red colour: statistical significance; POX: paraoxon; sc: subcutaneously; Minute: minutes 5, 10, 15 and 30 after paraoxon application. Fig. 10 Intensity of piloerection in relation to whether rat treated with paraoxon survived or not. * Fisher exact test, red colour: statistical significance; Minute: minutes 5, 10, 15 and 30 after paraoxon application. Paraoxon Toxicity Signs Related to Dose and Outcome more often (Table 3) and of higher intensity in non-survivors (Figure 10). In atropine-protected rats, piloerection did not correlate with the outcome of poisoning. The intensity of stereotypical movements, exophthalmos, lacrimation, ataxia and dyspnoea was not related to the dose of paraoxon or to the outcome of poisoning. The above results are not shown. Of the listed signs, stereotypical movements were less often with higher doses of paraoxon (p = 0.044) (Table 2). Related to poisoning outcome, ataxia (p = 0.025) was observed more often in survivors from the group of atropine-protected rats, while stereotypical movements (p = 0.043) were observed more often in survivors from the group of unprotected rats (Table 3). DISCUSSION Due to its extreme toxicity, the World Health Organization (WHO) has classified parathion, parent compound of paraoxon, as a class Ia (extremely hazardous) pesticide (14). Due to its toxicity, it is banned in most developed countries. Tabun, sarin, soman and VX represent OP compounds similar to parathion and paraoxon, with the same mechanism of action (inhibition of acetylcholinesterase) and their extreme toxicity classifies them as nerve agents. Their production, stockpiling, weaponosing and use is strictly prohibited by the 1993 Chemical Weapons Convention (CWC) (7). In undeveloped and developing countries, OPI poisonings, both accidental and intentional, are common (15, 16). In Sri Lanka and China, pesticide poisoning is the most common method of fatal self-harm (17). The LD50 of paraoxon obtained in this study was 0.33 mg/kg sc, which corresponds with the results of other researchers (18). The PR of atropine was 2.73, which is in accordance with the known publications (19). Atropine is effective in blocking the effects of muscarinic but is ineffective against the nicotinic signs of OPC poisoning (20). This antimuscarinic drug is liposoluble and passes the blood-brain barrier (21). Therefore, it to some extent, antagonises the toxic effects of excessive cholinergic stimulation in the brain (22). It seems that a more lipophilic antimuscarinic drug would be more effective than atropine (23). Krutak-Krol and Domino (24) found that the atropine dose of 10 mg/kg im is optimal in experimental studies. The minimum absolute lethal dose of OPCs is 1.3 LD50 (25). The administration of atropine made it possible for rats to survive the absolute lethal dose of paraoxon. That enabled monitoring of signs of poisoning at high doses of paraoxon. As expected, atropine blocked to some extent the muscarinic effects, but not the nicotinic ones. Since different doses of paraoxon were administered in rats treated with saline or atropine, the results are not comparable. However, this makes it possible to compare the signs of poisoning in future studies with other antidotes. In clinical settings, mainly muscarinic signs of OPC poisoning are expected. Bronchoconstriction and bronchorrhea are life-threatening muscarinic effects. Most studies have cited respiratory failure as the leading cause of death (26–28). Dyspnoea was observed as a sign of poisoning in the present experiment. No clear relationship was 15 found between the intensity of dyspnoea and the dose of paraoxon. However, in the recent study, a clear relationship was found between the onset rate of dyspnoea, as well as the overall intensity of dyspnoea and lethal outcome of poisoning (29). Respiratory failure is a consequence of both peripheral and central cholinergic effects (30). Therefore, it is very important to administer an antidote that can cross the blood-brain barrier and prevent central respiratory depression (31). Another muscarinic sign of poisoning that was observed is lacrimation. It is a sign that is easily noticeable. It is not a sign that directly implies whether the animal is endangered or not, but it is a good indicator of excessive muscarinic stimulation. In the treatment of OPC poisoning, the lack of lacrimation is one of the signs of achieving the so-called patient atropinisation (5). The results of this study also support this assumption. Although significantly higher doses of paraoxon were administered, the lacrimation occurred significantly less frequently in rats treated with atropine (22% vs 55%). ACh is also found in the preganglionic nerve endings of the sympathetic nervous system (32). Stimulation of alpha-1-adrenergic receptors also leads to piloerection (33). Therefore, piloerection can serve as an indirect indicator of sympathetic stimulation. The results of this study showed a clear relationship between both the frequency and intensity of piloerection and the dose of paraoxon. Besides, piloerection occurred more often and was of stronger intensity in non-survivors. Tachycardia and hypertension are rarely expected in patients with OPI intoxications and they often mislead physicians in practice. Saadeh et al found tachycardia in as many as 35–60% of patients poisoned by OPCs (34). It means that tachycardia occurs more often than bradycardia, which indicates that it is a prejudice not to expect nicotinic effects in OPC poisonings. Nicotinic signs of poisoning occur as a consequence of excessive stimulation of ganglionic nicotinic receptors (hypertension, tachycardia, diaphoresis) as well as receptors at the neuromuscular junction (fibrillation and fasciculation) (35). In AChEI poisoning, hypertension and tachycardia can also occur as a consequence of excessive stimulation of the locus coeruleus. Stimulation of this cholinergically innervated sympathetic nuclei leads to the centrally-originated hypertension (36, 37). As already mentioned, ACh is a neurotransmitter of the peripheral nervous system, as well. Excessive stimulation of nicotinic receptors at the neuromuscular junction leads to fasciculations, a toxic phenomenon observed in this study. Fasciculations were the most consistent sign of the severity of rat poisoning. They were more intense at higher doses of paraoxon and in non-survivors throughout the observed period. This is in favour of the fact that nicotinic signs of poisoning appear in severe poisonings (38). When sarin was used in a terrorist attack in the crowded subway in Tokyo, over 5,000 people were injured and 12 people died (7, 39). Published reports cited nicotinic signs of poisoning in severely poisoned patients (40, 41). In rats treated with high doses of paraoxon and atropine, fasciculations were more common in survivors. This can be explained by the rapid lethal outcome of poisoning, which 16 left non-survivors without this toxic sign. Fasciculations often did not occur in the first 10 minutes of poisoning, but were present even after 4 hours in all survivors. In other words, it means that the non-survivors died too quickly to develop fasciculations. Experimental studies with antinicotinic drugs showed their significant antidotal efficacy against carbamates and OPCs (21). However, nicotinic receptor blockers are rarely used in clinical practice in the treatment of OPC poisonings, due to serious side effects at therapeutic doses of these drugs, primarily the respiratory muscle depression (42). Tremor is mediated by a variety of neurotransmitters – dopamine, glutamate, serotonin, adenosine and acetylcholine (43). The M2 muscarinic receptors are highly expressed in the nucleus basalis and occipital cortex, then in hippocampus and other cortical regions. Overstimulation of M2 receptors leads to tremor (44). There is a conflicting evidence regarding the role of M3 and M4 receptors in tremor aetiology (45). In this study, a clear relationship was found between the intensity of poisoning, on the one hand and the dose of paraoxon and the outcome of the poisoning, on the other hand. In atropine-protected rats, tremor occurred in all animals. Tremor is often found as a part of the extrapyramidal syndrome that occurs as a consequence of permanent CNS damage in OPC poisoning survivors (21, 46, 47). Stereotypical movements were registered more often in survivors and at lower dose of paraoxon. The heavily poisoned animals had significantly decreased spontaneous motor activity. Thus, the appearance of stereotypical movements could be a good prognostic sign of a positive outcome of poisoning. At the highest doses of paraoxon (0.9 and 1.2 mg/kg), ataxia was more common in survivors. Atropine prevented the death of rats, but not the skeletal muscle fatigue. As a consequence, only surviving rats could attempt to move in the cage and these movements were ataxic. Seizures intensity was directly related to the dose of paraoxon and the lethal outcome of the poisoning. A total of 66.67% of survivors vs 100% of non-survivors had seizures. Seizures occur at the beginning of OPC poisoning due to the excessive cholinergic stimulation of the CNS. There are three treatment periods after the onset of OPC-induced convulsions: muscarinic, gamma-aminobutyric acid A (GABAA)/benzodiazepine and glutamatergic ones (48). During the first one, antimuscarinic drugs (atropine and, preferably, more lipophilic drugs, such as scopolamine) can be efficiently used to stop the seizures, provided the right dose is chosen (49). However, beyond this period antimuscarinic drugs become ineffective in counteracting the seizures, irrespective of the dose applied. In the second phase this could be compensated by the administration of the GABAA/benzodiazepine receptor antagonists, such as barbiturates (e.g., pentobarbital, thiopental sodium) and benzodiazepines (e.g., diazepam or midazolam) (50, 51). In the third phase, these seizures can be stopped by the administration of N-methyl-D-aspartate (NMDA) receptor antagonists, such as memantine, dizocilpine (MK-801) or ketamine (52–55). The reason for this is the fact that in the meanwhile the seizures became glutamatergic in its origin (56). Along with fasciculations, Žana M. Maksimović et al. Acta Medica (Hradec Králové) seizures were the most constant sign of the severity of the poisoning. CONCLUSION Among all the studied signs of paraoxon toxicity, the intensity of fasciculations and seizures were strong prognostic parameters of the severity of poisoning. They are easily observed and are directly related to both the dose of paraoxon and the lethal outcome of the poisoning. Based on the relationship between the frequency and intensity of muscarinic or nicotinic signs and the doses of paraoxon or outcomes of the poisoning, there are two strong prognostic parameters of the severity of poisoning (fasciculations and seizures) and a good prognostic sign of a positive outcome of poisoning (stereotypical movements). These signs of poisoning may be useful to researchers in monitoring the expected treatment outcome. Also, the appearance of nicotinic and central signs of poisoning in patients indicates the severity of poisoning and provides guidance to clinicians on which potential therapy to use. FUNDING This study is partially funded by the Ministry of Scientific and Technological Development, Higher Education and Informational Society of the Government of the Republic of Srpska (Grant No 125 7030). ABBREVIATIONS ACh: acetylcholine; AChE: acetylcholinesterase; AChEI: acetylcholinesterase inhibitor; OPC: organophosphorus compounds; OPI: organophosphate insecticide REFERENCES 1. Brown DA. Acetylcholine and cholinergic receptors. Brain Neurosci Adv 2019 Mar 21; 3: 2398212818820506. 2. Pope CN, Brimijoin S. Cholinesterases and the fine line between poison and remedy. Biochem Pharmacol 2018 Jul; 153: 205–16. 3. Xiao C, Zhou CY, Jiang JH, Yin C. Neural circuits and nicotinic acetylcholine receptors mediate the cholinergic regulation of midbrain dopaminergic neurons and nicotine dependence. Acta Pharmacol Sin 2020 Jan; 41(1): 1–9. 4. Vale A, Lotti M. Organophosphorus and carbamate insecticide poisoning. Handb Clin Neurol 2015; 131: 149–68. 5. Eddleston M. Novel clinical toxicology and pharmacology of organophosphorus insecticide self-poisoning. Annu Rev Pharmacol Toxicol 2019 Jan 6; 59: 341–60. 6. Henretig FM, Kirk MA, McKay CA Jr. Hazardous chemical emergencies and poisonings. N Engl J Med 2019 Apr 25; 380(17): 1638–55. 7. Stojiljković MP. Nerve agents – a clear and present danger to mankind. Scr Med 2019; 50(3): 109–11. 8. Lorke DE, Nurulain SM, Hasan MY, Kuča K, Petroianu GA. Combined pre- and posttreatment of paraoxon exposure. Molecules 2020 Mar 27; 25(7): 1521. 9. Wadia RS, Sadagopan C, Amin RB, Sardesai HV. Neurological manifestations of organophosphate insecticide poisoning. J Neurol Neurosurg Psychiatry 1974 Jul; 37(7): 841–7. 10. Reddy BS, Skaria TG, Polepalli S, et al. Factors associated with outcomes in organophosphate and carbamate poisoning: a retrospective study. Toxicol Res 2020 Feb 7; 36(3): 257–66. Paraoxon Toxicity Signs Related to Dose and Outcome 11. Amend N, Niessen KV, Seeger T, Wille T, Worek F, Thiermann H. Diagnostics and treatment of nerve agent poisoning-current status and future developments. Ann N Y Acad Sci 2020 Nov; 1479(1): 13–28. 12. National Research Council (US) Committee for the Update of the Guide for the Care and Use of Laboratory Animals. Guide for the Care and Use of Laboratory Animals. 8th edition. Washington (DC): National Academies Press (US); 2011. Available from: https://www .ncbi.nlm.nih.gov/books/NBK54050/. 13. Litchfield JT Jr, Wilcoxon F. A simplified method of evaluating dose-effect experiments. J Pharmacol Exp Ther 1949 Jun; 96(2): 99–113. 14. WHO. The WHO recommended classification of pesticides by hazard 2019. Geneva, 2019. (Accessed 2021-May-02 at https://apps.who .int/iris/bitstream/handle/10665/332193/9789240005662-eng .pdf?ua=1.) 15. Amir A, Raza A, Qureshi T, et al. Organophosphate poisoning: demographics, severity scores and outcomes from National Poisoning Control Centre, Karachi. Cureus 2020 May 31; 12(5): e8371. 16. Kaushal J, Khatri M, Arya SK. A treatise on organophosphate pesticide pollution: Current strategies and advancements in their environmental degradation and elimination. Ecotoxicol Environ Saf 2020 Oct 22; 207: 111483. 17. WHO. Health topics: mental health. Geneva, 2004. (Accessed 2021May-02 at https://www.who.int/mental_health/prevention/suicide /en/PesticidesHealth2.pdf.) 18. Misik J, Pavlikova R, Cabal J, Kuca K. Acute toxicity of some nerve agents and pesticides in rats. Drug Chem Toxicol 2015 Jan; 38(1): 32–6. 19. Holmstedt B. Pharmacology of organophosphorus cholinesterase inhibitors. Pharmacol Rev 1959 Sep; 11: 567–688. 20. Parkes MW, Sacra P. Protection against the toxicity of cholinesterase inhibitors by acetylcholine antagonists. Br J Pharmacol Chemother 1954 Sep; 9(3): 299–305. 21. Stojiljković MP, Škrbić R, Jokanović M, Kilibarda V, Bokonjić D, Vulović M. Efficacy of antidotes and their combinations in the treatment of acute carbamate poisoning in rats. Toxicology 2018 Sep 1; 408: 113–24. 22. Kords H, Lüllmann H, Ohnesorge FK, Wassermann O. Action of atropine and some hexane-1.6-bis-ammonium derivatives upon the toxicity of DFP im mice. Eur J Pharmacol 1968 Jul; 3(4): 341–6. 23. Albuquerque EX, Pereira EF, Aracava Y, et al. Effective countermeasure against poisoning by organophosphorus insecticides and nerve agents. Proc Natl Acad Sci U S A 2006 Aug 29; 103(35): 13220–5. 24. Krutak-Krol H, Domino EF. Comparative effects of diazepam and midazolam on paraoxon toxicity in rats. Toxicol Appl Pharmacol 1985 Dec; 81(3 Pt 1): 545–50. 25. Antonijević B, Stojiljković MP, Bokonjić D, Vucinić S. [Antidotal effect of combinations obidoxime/HI-6 and memantine in mice poisoned with soman, dichlorvos or heptenophos]. Vojnosanit Pregl 2011 Dec; 68(12): 1033–40. Serbian. 26. Eddleston M, Eyer P, Worek F, et al. Differences bet organophosphorus insecticides in human self-poisoning: a prospective cohort study. Lancet 2005 Oct 22–28; 366(9495): 1452–9. 27. Namba T, Nolte CT, Jackrel J, Grob D. Poisoning due to organophosphate insecticides. Acute and chronic manifestations. Am J Med 1971; 50(4): 475–92. 28. Ballantyne B, Marrs TC. Overview of the biological and clinical aspects of organophosphates and carbamates. In: Ballantyne B, Marrs TC, eds. Clinical and experimental toxicology of organophosphates and carbamates. Oxford: Butterworth-Heinemann; 1992, p. 3–14. 29. Maksimović ŽM, Duka D, Bednarčuk N, Škrbić R, Stojiljković MP. Onset rate and intensity of signs of organophosphate poisoning related to paraoxon dose and survival in rats. Scr Med 2021 Mar; 52(1): 49–58. 30. Villa AF, Houze P, Monier C, et al. Toxic doses of paraoxon alter the respiratory pattern without causing respiratory failure in rats. Toxicology 2007 Mar 22; 232(1–2): 37–49. 31. Houze P, Pronzola L, Kayouka M, Villa A, Debray M, Baud FJ. Ventilatory effects of low-dose paraoxon result from central muscarinic effects. Toxicol Appl Pharmacol 2008 Dec 1; 233(2): 186–92. 32. Dhanarisi J, Shihana F, Harju K, et al. A pilot clinical study of the neuromuscular blocker rocuronium to reduce the duration of ventilation after organophosphorus insecticide poisoning. Clin Toxicol (Phila) 2020 Apr; 58(4): 254–6. 17 33. Kikuchi-Utsumi K, Ishizaka M, Matsumura N, Nakaki T. Alpha(1A)-adrenergic control of piloerection and palpebral fissure width in rats. Auton Neurosci 2013 Dec; 179(1–2): 148–50. 34. Saadeh AM, Farsakh NA, al-Ali MK. Cardiac manifestations of acute carbamate and organophosphate poisoning. Heart 1997; 77(5): 461–4. 35. Turner SR, Chad JE, Price M, et al. Protection against nerve agent poisoning by a noncompetitive nicotinic antagonist. Toxicol Lett 2011 Sep 25; 206(1): 105–11. 36. Dirnhuber P, Cullumbine H. The effect of anti-cholinesterase angents on the rat’s blood pressure. Br J Pharmacol Chemother 1955 Mar; 10(1): 12–5. 37. Varagić V. The action of eserine on the blood pressure of the rat. Br J Pharmacol Chemother 1955 Sep; 10(3): 349–53. 38. Persson HE, Sjöberg GK, Haines JA, Pronczuk de Garbino J. Poisoning severity score. Grading of acute poisoning. J Toxicol Clin Toxicol 1998; 36(3): 205–13. 39. Yokoyama K, Yamada A, Mimura N. Clinical profiles of patients with sarin poi¬soning after the Tokvo subway attack. Am J Med 1996 May: 100(5): 586. 40. Nozaki H, Aikawa N, Shinozawa Y, Hori S, Fujishima S, Takuma K, et al. Sarin poisoning in Tokyo subway. Lancet 1995 Apr 15: 345(8955): 980–1. 41. Suzuki T, Morita H, Ono K, Maekawa K, Nagai R, Yazaki Y. Sarin poisoning in To¬kyo subway. Lancet 1995 Apr 15; 345(8955): 980. 42. Sheridan RD, Smith AP, Turner SR, Tattersall JE. Nicotinic antagonists in the treatment of nerve agent intoxication. J R Soc Med 2005 Mar; 98(3): 114–5. 43. Collins LE, Galtieri DJ, Brennum LT, et al. Oral tremor induced by the muscarinic agonist pilocarpine is suppressed by the adenosine A2A antagonists MSX-3 and SCH58261, but not the adenosine A1 antagonist DPCPX. Pharmacol Biochem Behav 2010 Feb; 94(4): 561–9. 44. Gomeza J, Shannon H, Kostenis E, et al. Pronounced pharmacologic deficits in M2 muscarinic acetylcholine receptor knockout mice. Proc Natl Acad Sci U S A 1999 Feb 16; 96(4): 1692–7. 45. Scarr E. Muscarinic receptors: their roles in disorders of the central nervous system and potential as therapeutic targets. CNS Neurosci Ther 2012 May; 18(5): 369–79. 46. Jokanović M. Neurotoxic effects of organophosphorus pesticides and possible association with neurodegenerative diseases in man: A review. Toxicology 2018 Dec 1; 410: 125–31. 47. Reji KK, Mathew V, Zachariah A, et al. Extrapyramidal effects of acute organophosphate poisoning. Clin Toxicol (Phila) 2016 Mar; 54(3): 259–65. 48. Stojiljković MP, Jokanović M, Lončar-Stojiljković D, Škrbić R. Prophylactic and therapeutic measures in nerve agents poisonings. In: Gupta RC. Handbook of toxicology of chemical warfare agents. 3rd ed. Cambridge, MA, USA: Academic Press; 2020, p. 1145–1159. 49. Myhrer T, Nguyen NH, Andersen JM, Aas P. Protection against soman-induced seizures in rats: relationship among doses of prophylactics, soman, and adjuncts. Toxicol Appl Pharmacol 2004 May 1; 196(3): 327–36. 50. Shih T, McDonough JH Jr, Koplovitz I. Anticonvulsants for soman-induced seizure activity. J Biomed Sci 1999 Mar–Apr; 6(2): 86–96. 51. Bokonjić D, Rosić N. Anticonvulsive and protective effects of diazepam and midazolam in rats poisoned by highly toxic organophosphorus compounds. Arh Hig Rada Toksikol 1991 Dec; 42(4): 359–65. 52. Spampanato J, Bealer SL, Smolik M, Dudek FE. Delayed adjunctive treatment of organophosphate-induced status epilepticus in rats with phenobarbital, memantine, or dexmedetomidine. J Pharmacol Exp Ther 2020 Oct; 375(1): 59–68. 53. Stojiljković MP, Škrbić R, Jokanović M, Bokonjić D, Kilibarda V, Vulović M. Prophylactic potential of memantine against soman poisoning in rats. Toxicology 2019 Mar 15; 416: 62–74. 54. Weissman BA, Raveh L. Therapy against organophosphate poisoning: the importance of anticholinergic drugs with antiglutamatergic properties. Toxicol Appl Pharmacol 2008 Oct 15; 232(2): 351–8. 55. Stojiljković MP, Škrbić R, Jokanović M, Kilibarda V, Bokonjić DR, Maksimović M. Effects of memantine and its metabolite Mrz 2/373 on soman-induced inhibition of acetylcholinesterase in vitro. Chem Biol Interact 2021 Jun 1; 342: 109463. 56. Rusyniak DE, Nañagas KA. Organophosphate poisoning. Semin Neurol 2004 Jun; 24(2): 197–204.