Mechanisms Of Organophosphorus Ester Induced Delayed Neurotoxicity In Hens

Organophosphorus compounds (Ops) are widely used as pesticides, oil additives, lubricants, solvents, fire-retardant, nerve agents. These compounds are able to induce a delayed neurodegenerative condition known as organophosphorus ester induced delayed neurotoxicity or neuropathy (OPIDN) in human being and sensitive animals. OPIDN is characterized by 1-3 weeks before the onset of clinical signs and a concurrence of central-periphera] axonopathy. The symptoms of OPIDN include muscular incoordination, ataxia, spasticity, and flaccid paralysis. Paralysis begins distally in the lower limbs and eventually spreads to the upper limbs. Neuropathologic observation showed lesions as internodal swelling and axonal degeneration with subsequent Wallerian-like degeneration of the myelin in the most distal portion of large diameter, long axons in the peripheral nerve and motor and sensory tracts in the spinal cord. The peripheral nerve damage precedes to the spinal cord. Ultrastructural studies show axonal swelling containing aggregations of neurofilaments, microtubules, multivesicular vesicles, and proliferation of smooth endoplasmic retivulum soon after the appearance of clinical signs of OPIDN. This is followed by partial matting and disappearance of neurofilaments from swollen axons. Although a large number of toxicology studies have been done attempting to reveal the actual mechanism of OPIDN, it remains unclear. Focusing on its molecular mechanism, we investigated the dynamics changes of axon by morphological observations, axonal cytoskeletal components on protein and mRNA levels by molecular biological techniques in different nerve tissues after dosing of methamidophos and TOCP following the Abou-Donia's hypothesis that abnormalmodification of cytoskeletal proteins may involve in the occurrence of OPIDN. OPIDN model had been developed in 10-month-old hens by both subcutaneous administration and gavage at diiferent dose rate of methamidophos, and also induced by gavage with single dosage of 750mg/kg tri-ortho-cresyl phosphate (TOCP) respectively. The ultrastructural changes of peripheral nerves (including sural and sciatic nerves) by morphological observations under electron microscopy at different time points i.e. 2, 10, 23d after manifesting neurotoxicity respectively, and at day 21 post-treatment of TOCP. The protein levels of cytoskeletal components including high molecular weight neurofilament (NF-H), middle molecular weight neurofilament (NF-M), low molecular weight neurofilament (NF-L), a -tubulin, P -tubulin and P -actin in sciatic nerves, cerebrum and spinal cord, and mRNA levels of those with glyceraldehydes-3-phoaphate dehydrogenase (GAPDH) as inner inference in cerebrum and spinal cord of hens at same time points were analyzed by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) and immunoblotting , and reverse transcription- polymerase chain reaction (RT-PCR) respectively. The nerve functional repair by continuously injecting 900u/kg nerve growth factor (NGF) intramuscularly once a day for 30 days after post-exposure methamidophos and TOCP was examined and estimated by neuroelectrophysiologic parameters. The alterations of protein expression profiles in nervous systems of hens were also observed by two-dimensional gel electrophoresis. The sciatic nerves, cerebrum and spinal cord of hens at lOd after manifesting neurotoxicity in methamidophos group and at day 10 post-treatment of TOCP, as well hens in control groups were homogenized with lysis solution, centrifugated, the supernatant were subjected to isoelectric focusing (IEF) electrophoresis and dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) respectively. The protein spots were stained with silver, and analyzed by Imagemaster 2D Platinum software. The results were as follows:1. OPIDN model induced by methamidophos and TOCPAll hens were died of seriously acute cholinergic crisis after exposure to 100,125 and 150mg/kg methomidophos by gavage, and others treated with 25,50,75 mg/kg methamidophos also died within 96 hours due to acute poisoning and absorption impairment. There were not alive in hens by 200, 250 mg/kg methamidophos subcutaneous administration. A few hens could exhibit slight neurotoxic clinical signs of OPIDN after administering subcutaneously to 100-150mg/kg, 50mg/kg and 50mg/kg methamidophos at 1, 3, 4 days respectively. 31.4% of the hens could induce OPIDN by daily subcutaneous administration of 30mg/kg methamidophos for more than 15 days, and there was no correlation between the accumulated dosages of methamidophos and severity of OPIDN.Hens began to appear slightly abnormal gait on day 6 with daily progression, and reach total paralysis at day 11 after 750mg/kg TOCP exposure by gavage.2. Morphological observations of peripheral nervesElecton micrographs of peripheral in methamidophos and TOCP groups showed axonal degeneration, different dosage of methomidophos and TOCP differed in the severity of axonal damage. Elevation of electronic density with dense bodies, myelin destructions with onion-bulb and bubble form protrusion on th myelin sheath and axolemma border of myelinated axons at day 2, the myelin sheath changes above, decrease of electronic density, dense-sparse zones due to crosslink and dissolution of neurofilaments and microtubules at day 10, the scatter and disappearance of cytoskeleton, myelin ovoids, intra-axonal vacuoles at day 23 were seen respectively after dose of methomidophos. Degenerated axons filled with myelin debris at day 21 after TOCP exposure was also noted. More heavy degeneration of axon and serious damage of myelin sheath were observed in sural nerve than in sciatic nerves.3. Dynamics changes of cytoskeletal proteins levels in peripheral nervous system and central nervous systemSciatic nerves: All neurofilaments showed earlier elevation in its levels and followed by fall at day 10, 23 after appearance of OPIDN induced by methamidophos, and decrease of three neurofilaments were noted in supernatant of sciatic nerves at different time points following OPIDN appearance. The levels of a-tubulin, P -tubulin were decrease both in pellet and supernatant of sciatic nerves. P -actin level in pellet of sciatic nerve increased at earlier stage, and came back to control levels, that in supernatant significant decreased at day 23 compared to control.All the cytoskeleton proteins were lessened both in pellet and supernatant after exposure to 750mg/kg TOCP.Spinal cords: NF-H was lowered in pellet and increased in supernatant of spinal cord following OPIDN appearance induced by mehamidophos, but the NF-M was different. The levels of NF-L reduced at day 2 and followed by falling to control levels in pellet, that was elevated significantly at day 2,10 and followed by going down in supernatant. Little change of a-tubulin was noted, 3-tubulin level was much more higher than that of control at day 2, 10, and returned to the control levels at day 23 in pellet. Both of the a -tubulin and P -tubulin showed increase at day 2, 10, then decrease in supernatant. P -actin levels increased significantly as compared to control hens in pellet and supernatant.The different changes of cytoskeletal proteins was observed following by TOCP exposure. Apart from NF-L obvious increase in pellet, the levels of three neurofilaments in supernatant and NF-H, NF-M in pellet were decreased compared to the control levels. There was no significant difference in a -tubulin levels of pellet between TOCP and control group. All of a -tubulin, P -tubulin in supernatant and P -tubulin in pellet was showed decrease. Little change of P -actin was observed both in pellet and supernatant following TOCP exposure.Cerebrum: The significant decreased in pellet and little change in supernatant of NF-H were observed at every time points after manifesting neurotoxicity. There was only remarkable increase at day 23 in pellet among of NF-M in cerebrum. NF-L levels both in pellet and supernatant were lower than those of control. Decrease of a -tubulin in supernatant at day 2,10 and unobvious changes in pellet were observed. Decrease of P -tubulin in pellet at day 10,23 and increase in supernatant at day 2,10 were seen, and P -tubulin in pellet was showed decrease. Little change of P -actinwas observed both in pellet and supernatant of cerebrum following OPIDN appearance after methamidophos.In cerebrum of hens with OPIDN induced by TOCP, same change tendency ofneurofilamets with methamidophos exposure was observed, but the increase of NF-Hin pellet and decrease of NF-L were much more obvious. TOCP made the levels ofa -tubulin elevation in supernatant, {3 -tubulin in pellet decrease, and P -actinremarkable increase in supernatant.4. Dynamics changes of mRNA levels of cytoskeletal proteins in central nervous system.Spinal cord: The mRNA levels of cytoskeletal proteins remained unchanged after methamidophos exposure. NF-M. 3 -tubulin mRNA levels were lessened remarkably following TOCP exposue, Other cytoskeletal proteins were also affected.Cerebrum: A little decrease of a -tubulin and significant descent of NF-L, P -tubulin were observed after methamidophos administration. Three neurofilaments and P -tubulin were significantly lowered after treating with TOCP when compare to control, and the most evident reduction was P -tubulin among those.5.Treatment of NGFThe clinical symptoms of hens both in treatment group and control exposure to methamidophos was lessened, the recovery of hens treating with NGF was faster than those in control, but there was no significant difference between them. Hen exposure to 750mg/kg TOCP remained paralysis after NGF administration.Hens following methamidophos and TOCP exposure showed much more susceptible and slow to pain respectively, all of them was significant repaired after NGF treatment for 30 days to some extent.Slow nerve conduction velocity (NCV), long latency, low amplitude, and long duration of the compound action potential (CAP) were observed after exposure to methamidophos and TOCP. Fast NCV, short latency and were seen by giving NGF treatment. Other parameters remained unchangeable.6. Alterations of protein expression profiles in nervous systems of hens bytwo-dimensional gel electrophoresisThe protein spots of sciatic nerves were lessened by 20% and 30% respectively in methamidophos and TOCP groups, when comparing to control. There were 46% and 35% protein spots in methamidophous and TOCP groups respectively to match with that of control. The little changes of protein spots of hens' cerebrum both in methamidophos and TOCP groups were observed, the ratio of matching to control were 61% and 65% respectively. In spinal cord of hens, the number of protein spots in methamidophos was slightly increased, while obviously decreased (25%) in TOCP groups, with 64% and 48% protein spots could match to control respectively.Conclusions:1. Parts of hens could induce OPIDN by daily subcutaneous exposure to 30mg/kg methamidophos for more than 15 days, and there was no correlation between the accumulated dosages of methamidophos and severity of OPIDN. 750mg/kg TOCP exposure could result in hen's paralysis.2. Axon and myelin sheath of peripheral nerve of hens were seriously damaged following methamidophos and TOCP administration, especial in distal nerve.3. Methamidophos and TOCP could damage the neurofilaments, a -tubulin, & -tubulin and 3 -actin in sciatic nerves, cerebrum and spinal cord at different levels, of which neurofilaments was the most.4. The damage degree of cytoskeletal proteins by methamidophos and TOCP differed according to various nerve tissues, and the sciatic nerve was easier to be affected.5. The effect on mRNA level also varied according to different nerve tissues and cytoskeletal proteins. The cerebrum was easier to affected by OPs than spinal cord, and the most decrease in P -tubulin was seen.6. TOCP could exert much more damage on protein and mRNA levels of cytoskeleton than methamidophos.7. NGF administration for 30 days attributed to recovery of OPIDN to some extent.8. Protein expression profiles in nervous systems of hens could be affected by methomidophos and TOCP. Sciatic nerve were the most sensitive to OPs among three never tissues, and the effect induced by TOCP was much seriously than that of methamidophos.9. Basing on our results, we speculated that the OPs might have much more influences on degradation levels than transcription of cytoskeletal protein.