| Tri-ortho-cresyl phosphate (TOCP) is widely used in the plastics industry and can induce a delayed neurodegenerative condition known as organophosphorus easter-induced delayed neurotoxicity (OPIDN) in human beings and sensitive animals. Cytoskeletal proteins are very important in maintaining the normal structure and function of neuron. In order to make appropriate toxic models of adult hens treated with TOCP and inquire the toxic mechanisms of OPIDN, cytoskeletal proteins, such as: high molecular weight neurofilament(NF-H), medium molecular weight neurofilament(NF-M) and light molecular weight neurofilament(NF-L), as well as α-tubulin, β-tubulin, microtubule associated protein 2(MAP-2) and β-actin were investigated in cerebrum and spinal cord of hens treated with TOCP.Eighteen adult Roman hens were randomly divided into 3 groups: 375 mg·kg-1 group, 750 mg·kg-1 group, and control group(n= 6 each group). TOCP was dissolved in corn oil and administered by gavage at single dosages of 0.65 ml·kg-1. Six control hens received an equivalent volume of corn oil. Their neurobehavioral functions were examined carefully, and body weights were measured every four days . After the hens were sacrificed on day 21, the cerebrums and spinal cords was dissected, and separated into pellet and supernatant after homogeneization and centrifugation, then the levels of NF-H, NF-M and NF-L, as well as α-tubulin, β-tubulin, MAP-2 and β-actin were analyzed by SDS-PAGE and western blotting.The results were following:1. body weight and neurobehavioral function tests showed:(1) all treated groups had significant decrease of body weight: 750 mg·kg-1 group decreased 11.6%(P <0.05)on day 13, 15.1%(P <0.01)on day 17, and 23.8%(P < 0.01)on day 21, repectively; 375 mg·kg-1 group decreased 12.1%(P <0.05)on day 17, and 2\.9%(P <0.01)on day 21, repectively。(2) delayed neurotoxicitic symptoms: hens in 750 mg·kg-1 group began to take on slightly abnormal gait on day 5 with daily progression, reached total paralysis on day 15; hens of 375 mg·kg-1 TOCP exposure had slightly abnormal gait on day 7 and paralysis on day 17 respectively. All hens exposed to TOCP still kept paralysis till sacrifice. In contrast, no clinical signs of delayed neurotoxicity were observed in control hens.2. SDS-PAGE and Western Blotting:(1) the changes of cytoskeletal proteins levels in cerebrums of hen with TOCP-induced delayed neurotoxicity:In cerebrum pellet: NF-H decreased by 23% and 47% in 375 and 750 mg·kg-1 group, respectively(P <0.01); NF-M failed by 47% in 750 mg·kg-1 group(P <0.01); NF-L enhanced by 17% in 750 mg·kg-1 group(P <0.05).In cerebrum supernatant: NF-H increased by 215% in 750 mg·kg-1 group(P < 0.01); NF-M ascended by 248% and 233% in 375 and 750 mg·kg-1 group, respectively(P <0.01); NF-L enhanced by 220% and 197% in 375 and 750 mg·kg-1 group, respectively(P <0.01).No significant changes were found in the amount of α-tubulin and β-tubulin in cerebrum pellet and supernatant between the treated and control animals(P >0.05).MAP-2 increased both in cerebrum pellet and supernatant. In pellet, that were 280% and 360% in 375 and 750 mg·kg-1 group, respectively(P < 0.01); in supernatant, that were 160% and 204% , respectively(P <0.01).No remarkable changes were found in the amount of β-actin in cerebrum pellet and supernatant between the treated and control animals(P >0.05).(2) the changes of cytoskeletal proteins contents in spinal cords of hen with TOCP-induced delayed neurotoxicity:In spinal cord pellet: NF-H decreased by 36% and 55%(P <0.01); NF-M reduced by 36% and 47% in 375 and 750 mg·kg-1 group, respectively(P <0.01); NF-L enhanced by 49% in 750 mg·kg-1 group(P <0.01).In spinal cord supernatant: NF-H dropped by 22% in 750 mg·kg-1 group(P < 0.05); NF-M increased by 71% in 375 mg·kg-1 group(P <0.01); NF-L enhanced by 249% in 375 mg·kg-1 group(P <0.01).No remarkable changes were found in the amount of α-tubulin and β-tubulin in spinal cord pellet and supernatant between the treated and control animals(P >0.05).MAP-2 reduced both in spinal cord pellet and supernatant. In pellet, that were 50% and 43%(P <0.01); in supernatant, that were 28% and 55% in 375 and 750 mg·kg-1 group, respectively(P <0.01).No significant changes were found in the amount of β-actin in spinal cord pellet, but in spinal cord supernatant, the level of β-actin increased by 37.5% in 375 mg·kg-1 group(P <0.01).The results suggest that:1. It is the first time that the level of neurofilament subunits are found affected in cerebrum and spinal cord of hens with TOCP-induced delayed neurotoxicity, and the changes of neurofilaments are detected more obvious than those of microtubulin and microfilament.2. It is the first time that MAP-2 contents are found influenced in cerebrum and spinal cord of hens with TOCP-induced delayed neurotoxicity, and the alterations of MAP-2 are proved more distinct than those of microtubulin.3. β-actin contents are found impactted in cerebrum and spinal cord of hens with TOCP-induced delayed neurotoxicity, so it might not be regarded as the normalization in gene expression experiments of nervous tissue.4. The changes of cytoskeletal proteins in hen cerebrum and spinal cord may contribute to the occurrence and development of TOCP-induced delayed neurotoxicity.
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