Mortality is one final effect of toxins. The experiment was planned in way to cover a full spectrum of mortality. Rats used in experiments were sacrificed within thirty minutes after intoxication. This short period of paraoxon incidence should ensure that any observations are due to the acute toxicity and cholinergic crisis phases, rather than other mechanisms such as apoptosis arising later [22].

No mortality was observed up to a dose of 170 nmol/kg of body weight (b.wt.). Above 170 nmol, the mortality rose abruptly. A dose of 250 nmol/kg b.wt. of paraoxon was found to be the LD₅₀ for the given time interval. Doubling the LD₅₀ dose up to 500 nmol/kg b.wt. of paraoxon led to overall mortality within ten minutes. Symptomatic manifestations of intoxication were observed starting at the 170 nmol/kg b.wt. dose. Some animals exposed to this dose manifested tonic-clonic seizures, but the symptomatic manifestations caused by the 170 nmol/kg b.wt. dose was slight in comparison with a 250 nmol/kg b.wt. dose. The latter caused strong abrupt tonic-clonic seizures within 5 minutes and overall deterioration of shape. The deaths occurred after quite a long period: 20 minutes. The highest tested dose was 500 nmol/kg b.wt. of paraoxon per animal. Tonic-clonic seizures were of similar level as observed at a dose of 250 nmol/kg b.wt, however animals in this cohort manifested tonic-clonic seizures within 5 minutes and died within 10 minutes after paraoxon intoxication. No other specific symptoms were clearly visible. Every exitus was confirmed by proven cardiac arrest and persisting mydriasis. The symptomatic manifestation observed during experiment correlates with the expected from human cases [23].

Cholinesterase activity was assayed in the way described previously [17]. Faraday's laws of electrolysis were used to calculate cholinesterase activity. Current measured in the reaction mixture grew linearly for approximately 5 minutes. The total electric charge flowed through the system was estimated from the area under the curve. The total flowed charge was simply recalculated to give enzyme activity. The resulting data are summarized in Table 2.

The level of cholinesterases in blood is an important marker of intoxication and strongly correlates with AChE level in CNS as well as PNS [24, 25]. Here, a deterioration of overall shape was observed when at least 50% of cholinesterase activity is inhibited. This corresponded to a dose of less than 170 nmol/kg b.wt. Cholinergic crises happened when more than 80% of cholinesterases were inhibited. This responded at 170 nmol/kg b.wt. of paraoxon. Deaths were observed when inhibition surpassed 90%. This corresponded to doses of 250 and 500 nmol/kg b.wt. Death was caused by a dose of 500 nmol/kg b.wt. per animal. The corresponding inhibition level was nearly 95%. The differences were statistically relevance (ANOVA with Scheffe test, Origin 8 software, OriginLab). Only cholinesterasese activity levels caused by doses renging from 65 – 125 nmol/kg b.wt. and doses 250 – 500 nmol/kg b.wt. were indistinguishable from each other at a probability level P = 0.05. All other groups were found different with each other at a probability level P = 0.05.

The examination of total levels of low molecular weight antioxidants was used as a marker of resulting oxidative stress. The anodic wave showed no significant change for any assayed plasma sample. The achieved current was equal to 2,940 ± 154 nA at a voltage of 665 ± 48 mV. The data suggest no oxidative stress in the laboratory animals during the experiments. It seems that the paraoxon toxicology pathway was based only on short term neurotoxicity.

This study suggests a quite extensive tolerance of organisms to small decreases in cholinesterase levels. On the other hand, the findings of acute poisoning by organophosphate are not useful for assessing shock states and consequences over a long term [26]. Slight inhibition of cholinesterases could be even positive in oxidative stress conditions, for example, by reducing e.g. interleukin-1 beta in Alzheimer's disease [27] and bronchoconstriction during anaphylactic shock [28]. Many drugs such as tacrine and its derivates are also based on inhibition of AChE [14, 29]. Here, we propose that body possesses the capability to resist overdosing by anticholinergic compounds at quite high levels. It is an important fact, when we consider the limited efficacy of currently used antidotes [30] and their limited penetration inside the brain [31].

Male Wistar rats were purchased from Velaz Ltd. (Prague, Czech Republic). Animals were of the same age, weighing 250 – 300 g. The animals were maintained under SATP conditions and 50±10% humidity. Illumination lasted from 7 a.m. to 7 p.m. Animals were allowed to ingest chow and water without any restriction. Animals were housed in the Animal House of the Faculty of Military Health Sciences in Hradec Kralove. Maintenance of animals and all experiments were supervised by the Ethical Committee of the Faculty of Military Health Sciences and a veterinary surgeon.

The laboratory rats were intoxicated with paraxon via intramuscular injection into the caudal thigh muscles. The paraoxon has been diluted with saline to a volume of 200 μL. This volume was not exceeded in order to prevent backward discharge. The weight of individual animals was taken into account when the paraoxon solutions were prepared. Each animal was been unmistakable marked and attentively observed. Every clinical symptom was registered.

Amperometric principles were used for evaluation of cholinesterase activities. Freshly collected blood was mixed with 0.02 M Tris buffer pH 7.6 in a 1:20 ratio. The mixture was left to incubate for 5 minutes in laboratory temperature. No clouds were observed after cell disruption by Tris buffer. The mixtures were not centrifuged in order to retain activity without any losses. Blood lysate (0.5 mL) was mixed with 1 mM acetylthiocholine chloride (ATChCl, 1.5 mL) in phosphate buffered saline (PBS). The mixture was placed into a reaction cell and mixed with a magnetic stirrer coated with Teflon. An electrochemical sensor strip with platinum working and platinum auxiliary electrodes and silver covered by silver chloride as reference electrode (BVT Brno, Czech Republic) was used throughout the experiments for measuring enzyme activity. The sensor was connected to an EmStat device (Houten, The Netherlands) and the applied voltage between working and reference electrode was adjusted up to +500 mV. Since the measuring principle was chronoamperometry, voltage was constant for the all experiments. The current was measured as described in the literature [17]. Percent of inhibition (I) is the percent decrease of cholinesterase activity in comparison with intact blood. It was calculated according to reference [12].

Cyclic voltammetry was performed in order to estimate the total level of low molecular weight antioxidants (LMWA). The cited studies were slightly adapted to perform the assay [24, 32-34]. The electrochemical sensor (the same type as above) was fixed horizontally and the electrodes were overlaid with 20 μL of undiluted plasma. Cyclic voltammetry was measured in a range -0.5 to 1.1 V with a scan rate of 50 mV/s.