Neuropathology of organophosphate-induced delayed neuropathy (OPIDN) in young chicks

To examine the phenomenon of apparent age resistance of young chicks to organophosphate-induced delayed neuropathy (OPIDN), groups of either 2- or 10-week-old chicks were exposed subcutaneously daily for 4 days to the neuropathic organophosphate (OP), di-isopropylfluorophosphate (DFP, 1 mg/kg), the non-neuropathic OP, paraoxon (PO, 0.25 mg/kg) or atropine (20 mg/kg). Subsequently, all birds were examined at post-exposure intervals (calculated from the last day of exposure) for up to 56 days for neurological deficits and morphological lesions in the central and peripheral nervous systems (CNS, PNS). Clinically, none of the birds in the 2-week-old groups, or in the 10-week-old PO or atropine exposed groups had neurological deficits. However, all birds in the 10-week-old DFP exposed group developed ataxia by 7 days post-exposure (DPE) and then progressive paralysis. Therefore, all birds in the 10-week-old groups were killed at 14 DPE. Pathologically, the 2-week-old DFP exposed chicks had increasingly severe lesions of Wallerian-like degeneration predominantly in the spinal cord from 7 DPE and subsequently. In the 10-week-old DFP exposed chicks, the degenerative lesions of OPIDN were first detected in the CNS at 3 DPE and then with equally increasing severity in the CNS and PNS up to 14 DPE. A higher incidence of neuronal necrosis and chromatolysis in ventral motor horn neurons of spinal cord grey matter and in dorsal root ganglia occurred in both the DFP exposed age groups compared with those lesions in other groups. These results demonstrate that after neuropathic DFP exposure, 2-week-old chicks develop pathological lesions in the spinal cord without neurological deficits. In both age groups, onset of degenerative lesions in the spinal cord preceeded those in the PNS. The claim of apparent age resistance of chicks to OPIDN needs to be re-evaluated.     

Phosphorylation of acetylcholinesterase in bovine blood by Paraoxon and Soman

In order to adjust reaction conditions to physiological conditions, phosphorylation of acetylcholinesterase has been investigated, not in highly diluted erythrocyte suspensions or enzyme solutions, but in undiluted whole blood. The inhibition of esteratic activity by paraoxon within 10 to 20 min, by soman within a few minutes, reaches the final level and shows no alteration during the next hours. This deviation from a pseudo first order kinetics is caused by the enzymatic hydrolysis of paraoxon, a competitive reaction by which paraoxon is destroyed rapidly. Soman did not show this effect, evidently because of the high velocity of the phosphorylation reaction.

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Regional blood flow during paraoxon infusion in rabbits

In anesthetized and artificially ventilated rabbits an intravenous infusion of paraoxon (0.8 mg/kg) was given over 30 min. The effects on cardiac output, blood flow to various vascular beds, and on the mass discharge of the postganglionic sympathetic efferents to the spleen and kidney were monitored. Immediately following paraoxon infusion atropine (0.5 mg/kg) was injected intravenously.Within 20 min of commencing the infusion signs of increased cholinergic stimulation were observed. Between the 20th and 25th min mean arterial blood pressure, heart rate, and cardiac output fell markedly. Even before arterial blood pressure fell total peripheral resistance and regional resistance to flow through the subclavian and coeliac arteries increased significantly, whereas resistance to flow through the superior mesenteric and renal arteries decreased transiently. Towards the end of the infusion period total peripheral resistance was below control, increased regional resistance being found only in the vascular beds of the subclavian and splenic arteries. The activity in the splenic sympathetic efferents increased, while the activity in the renal efferents was sharply reduced.While an effective antidote, atropine elicited transient intestinal vasodilation and a further transient decrease in total peripheral resistance. These and other results suggest that muscarinic mechanisms are mainly responsible for the paraoxon-induced changes in regional blood flow and regional sympathetic activity. The vasodilatory effect of atropine in the intestine was probably due to a local autoregulatory mechanism.     

Pharmacokinetics and efficacies of obidoxime and atropine in paraoxon poisoning

The efficacies (ED₅₀) of obidoxime and atropine against paraoxon poisoning in mice were determined by administering the antidotes 5, 20, 40 and 60 min before administration of the organophosphate. With increasing time intervals t between the administration of the antidote and paraoxon, the dose of antidote (ED₅₀), that reduced the lethality of 2 LD₅₀ of paraoxon to 50% increased. The standardized log ED₅₀/t plot was linear and yielded the efficacy half-life. In addition, the blood concentrations c of the antidotes were measured, resulting in a linear log c/t plot. The efficacy half-life was approximately twice the half-life of the antidote in blood. The possible reasons for this phenomenon are discussed.     

Reactivators of organophosphate-inhibited cholinesterase

Two isomeric cyclopentylcarbonyl and two cycloheptylcarbonyl derivatives of 2-hydroxyiminomethyl-1-[3-(1-pyridinio-2-oxapropyl] pyridinium diiodide and 4-hydroxyiminomethyl-1-[3-(1-pyridinio-2-oxapropyl] pyridinium diiodide were prepared and characterized by spectroscopic methods. The inhibitory power (I₅₀) of the investigated oximes was determined using purified bovine erythrocyte AChE and human erythrocyte AChE. Percentage of reactivation after 30 min was estimated after inhibition of human erythrocyte AChE by sarin, VX, tabun, soman, and paraoxon. The in vitro protective indices (p.i. and P₅₀) against inhibition by soman have been calculated using bovine erythrocyte AChE for p.i. and human erythrocyte AChE for P₅₀. Their I₅₀ for human erythrocyte AChE varied from 1.4–9.8 (10^–4 mol · dm^–3) and for bovine erythrocyte AChE in the range of 1.1–17 (10^–5 mol · dm^–3). With 2 × 10^–5 mol · dm^–3 oximes the percent of reactivation was: 0–17% for paraoxon-inhibited AChE, 9–49% for sarin-inhibited AChE, 16–65% for VX-inhibited AChE, 0–8% for tabun-inhibited AChE, and 0–4% for soman-inhibited AChE. The 2-hydroxyimino derivatives protect human erythrocyte AChE and purified bovine erythrocyte AChE from inhibition by soman.     

The manipulation of circadian rhythms

In a study on male Wistar (TNO) rats kept under phase-shifted light-dark (1212) conditions, it was demonstrated that the circadian rhythms of the following functions were effectively synchronized to the new lighting regimen: (a) body temperature, gross motor activity (after 1 week of acclimatization); (b) stomach weight, liver weight, liver glycogen, liver protein and acid phosphatase activity; serum corticosterone, glucose, total protein and inorganic phosphate (after 4–6 weeks; as concluded from the data pooled of a set of identical experiments); (c) the in vivo toxicity of LD₅₀ amounts (i.p.) for antimycin A and of E 600. In the individual experiments the various normal functions appeared to be different with respect to sensitivity to light. The overall pooled results indicated that, except for glycogen, the 24-h means of the normal values in the stomach, liver and serum were slightly decreased when compared to the respective means obtained from standard LD (= light: 6:00–18:00) -conditioned control groups (P < 0.001 for liver weight, liver protein, serum protein and inorganic phosphate). The experimental animals revealed an intermediate or persisting decrease of the growth rate. From the serial biological and toxicological studies performed in the course of 1 year, it is concluded that the synchronizing effect of an altered lighting regimen may be influenced by seasonal factors.Under certain conditions the use of light-manipulated animals will prove to be a useful tool in chronobiology and chronotoxicology.Supported by grants from Deutsche Forschungsgemeinschaft, Sonderforschungsbereich 146 (Versuchstierkunde) and VW-Stiftung, Hannover AZ 11.2000     

Neurotoxicity induced in differentiated SK-N-SH-SY5Y human neuroblastoma cells by organophosphorus compounds

Organophosphorus (OP) compounds used as insecticides and chemical warfare agents are known to cause potent neurotoxic effects in humans and animals. Organophosphorus-induced delayed neuropathy (OPIDN) is currently thought to result from inhibition of neurotoxic esterase (NTE), but the actual molecular and cellular events leading to the development of OPIDN have not been characterized. This investigation examined the effects of OP compounds on the SY5Y human neuroblastoma cells at the cellular level to further characterize cellular targets of OP neurotoxicity. Mipafox and paraoxon were used as OP models that respectively do and do not induce OPIDN. Mipafox (0.05 mM) significantly decreased neurite length in SY5Y cells differentiated with nerve growth factor (NGF) while paraoxon at the same concentration had no effect when evaluated after each of three 4-day developmental windows during which cells were treated daily with OP or vehicle. In contrast, paraoxon but not mipafox altered intracellular calcium ion levels ([Ca(2+)](i)), as seen in three types of experiments. First, immediately following the addition of a single high concentration of OP to the culture, paraoxon caused a transient increase in [Ca(2+)](i), while mipafox up to 2 mM had no effect. Paraoxon hydrolysis products could also increase intracellular Ca(2+) levels, although the pattern of rise was different than it appeared immediately after paraoxon administration. Second, repeated low-level paraoxon treatment (0.05 mM/day for 4 days) decreased basal [Ca(2+)](i) in NGF-differentiated cells, though mipafox had no effect. Third, carbachol, a muscarinic acetylcholine receptor agonist, transiently increased [Ca(2+)](i) in differentiated cells, an affect attenuated by 4-day pretreatment with paraoxon (0.05 mM/day), but not by pretreatment with mipafox. These results indicate that the decrease in neurite extension that resulted from mipafox treatment was not caused by a disruption of Ca(2+) homeostasis. The effects of OPs that cause or do not cause OPIDN were clearly distinguishable, not only by their effects on neurite length, but also by their effects on Ca(2+) homeostasis in differentiated SY5Y cells.     

Paraoxon inhibits GABA uptake in brain synaptosomes

To investigate possible effect of paraoxon (10⁻⁹–10⁻³ M) on GABA uptake, we used rat cerebral cortex synaptosomes. K_m and V_max of GABA uptake were determined in presence of paraoxon (10⁻³ M). Acetylcholine and its antagonists (atropine and mecamylamine) were used for evaluating cholinergic-dependency of uptake. Type of transporter involved was determined by using glial (beta-alanine) and neuronal (DABA) GABA uptake inhibitors. The results of the study showed that paraoxon at low doses (10⁻⁹–10⁻⁶ M) increased and at high doses (10⁻⁵–10⁻³ M) decreased GABA uptake. One millimolar paraoxon significantly decreased V_max (175.2 ± 4.23 vs. 80.4 ± 2.03, P < 0.001) of GABA uptake while had no effect on its K_m. DABA significantly decreased GABA uptake (P < 0.001) while beta-alanine had no effect. In conclusion, present data suggests that paraoxon probably acts as non-competitive antagonist of GABA uptake.     

Protective effect of metoclopramide against organophosphate‐induced apoptosis in the murine skin fibroblast L929

This study was performed to evaluate the protective efficacy of metoclopramide (MCP) against the organophosphates paraoxon (POX)‐ and malathion (MLT)‐induced apoptosis in the murine L929 skin fibroblasts. L929 cells were exposed to either POX (10 nm ) or 1.0 μm MLT in the absence and presence of increased concentrations of MCP. The protective effect of MCP on these organophosphate‐stimulated apoptotic events was evaluated by flow cytometry analysis after staining with annexin‐V/propidium iodide, processing and activation of the executioner caspase‐3, cleavage of the poly‐ADP ribose polymerase, fragmentation of the nucleosomal DNA and disruption of the mitochondrial membrane potential (Δψ). Our results showed that increased doses of MCP alone (≥10 μm ) did not induce apoptosis or activation of caspase‐3. Pretreatment of the cells with MCP attenuated all the apoptotic events triggered by the organophosphate compounds in a dose‐dependent manner reaching ~70–80% protection when they were preincubated at 1 and 5 μm of the drug before the addition of POX and MLT, respectively. Interestingly, MCP did not offer a significant protective effect against the cytotoxicity of tumor necrosis factor‐α, cisplatinum, etoposide or paclitaxel, which stimulate apoptosis by various mechanisms, suggesting that the anti‐apoptotic effect of the drug is specific to organophosphates. The strong and specific anti‐apoptotic activity of subclinical doses of MCP against the cytotoxicity of organophosphate compounds suggests its potential clinical application in treating their poisoning.