The Effect Of Protein Kinase Of Spinal Cord In Acrylamide Intoxicated Rats

Monomeric acrylamide (ACR) is a water-soluble, vinyl monomer that is used primarily to produce polyacrylamides which widely used as binding agent, thickening agent or bibulous in different industry such as wastewater treatment, petroleum extract, paper making, mining, medicine. The requirement of ACR is enhancing with the rapid developmemt of petroleum industry.In addition, ACR is widely found in potato-based and grain-based foods that have been prepared at high temperature (e.g., French fries, potato chips and some breads)Although experimental animal studies have implicated carcinogenicity and reproductive toxicity as possible consequences of ACR exposure, neurotoxicity is the only outcome confirmed by epidemiological studies on occupationally exposed population group. ACR is known to produce central-peripheral distal axonopathy, which is characterized by ataxia and distal skeletal muscle weakness. The major pathological hallmarks are distal swellings and secondary degeneration both in experimental animals and human companioning excessive accumulation of neurofilaments (NFs) in the distal swollen axon. However, the mechanisms of ACR axonopathy remain unknown.Based on the pathological alterations, We builted ACR-intoxicated rats models and investigated the changes of calmodulin (calmodulin, CaM),calmodulin kinaseⅡ(Ca2+/caM-dependent protein kinascⅡ, CaMKⅡ), protein kinase A (protein kinaseA, PKA), cyclic adenosine monophosphate (cAMP), protein kinase C (proteinkinase C, PKC) and cyclin-dependent protein kinase 5 (cyclin dependent kinasc 5, CDK5) and CDK5-related factor.These changes will provide basic data for the neurotoxicity of ACR and other poisons. They also will provide information for prevention and treatment for occupational poisoning. Methods1 Male Wistar rats, weighing 180-220g, were randomly assigned to 3 groups (n =9 rats per group). They were administered ACR by intraperitoneal injection to produce animal model of axonopathy, according to the following daily dosing schedules:20 or 40mg/kg/d for continuous 8 weeks (3 times per week).2 The tissues of spinal cord were homogenized in ice-cold homogenizing buffer containing buffer (20 mM Tris-HCL buffer(PH 7.4), 1mM DTT,5 mM EGTA,2 mM EDTA,10% glycerol,1 mM MgCl2,1 mM PMSF,2μg/ml aprotinin, and 2μg/ml leupeptin), homogenized for 2min, then centrifuging at 700×g 2 times for 4℃for 15 min, collect supernatant, and then centrifuging at 27,000×g for 60 min at 4℃. The relative levels of PKA, PKC and CDK5 and CDK5-related factor in the cytosolic fractions of spinal cord were determined by SDS-PAGE and immunoblotting. The relative levels of CaM, cAMP in the cytosolic fractions of spinal cord were determined by ELISA kits.3 The activities of CaMKII, PKA and PKC were determined by using corresponding radioactivated 32P assay kits in corresponding cytosolic fractions of spinal cord of control and experimental group rats.Results1 The establishment of the animal modelAfter 8 weeks of ACR treatment at 20 or 40mg/kg dose-rate, the rats showed neurological deficits of completely different levels; i.e., slight neurotoxicity (slight ataxia, hopping gait and foot splay) or severe neurotoxicity (dragged their feet as they walked, foot splay or paralysis). In addition, both the low and high dose-rate produced progressive retardation of body weight gain and increasing gait score. After 8 weeks treatment, all the rats in 40mg/kg dose-rate group exhibited severe neurotoxicity and those of 20mg/kg dose-rate group, slight neurotoxicity.2 The contents of protein kinase in spinal cord1) The contents of CaM were significantly decreased(p<0.05) treatment with 20mg/kg and 40mg/kg (by27% and 17%,respectively) in the cytoplasmic fraction of spinal cord.2) The contents of PKA were not changed compared with the control group in cytoplasmic fraction.3) The contents of cAMP was not changed compared with the control group in the cytosolic fraction.4) The contents of PKC were significantly decreased(p<0.05) treatment with 20mg/kg and 40mg/kg(by46% and41% respectively) in the cytoplasmic fraction.5) The contents of CDK5 were significantly decreased(p<0.05) treatment with 40mg/kg (by23%) in the cytoplasmic fraction. The contents of P35 were significantly decreased(p<0.05) treatment with 40mg/kg (by46%) in the cytoplasmic fraction.3 The activities of protein kinase in spinal cord1) The activities of CaMKⅡwere significantly increased (p＜0.01)treatment with 20mg/kg and 40 mg/kg 123% and 261%, respectively) in the cytosolic fraction.2) The activities of PKA were significantly increased (p<0.01) in the cytosolic fraction treatment with 20mg/kg and 40 mg/kg (57% and 50%, respectively)3) The activities of PKC were significantly increased (p<0.01)treatment with 20mg/kg and 40 mg/kg (160% and170%, respectively).Conclusions1 ACR induced can effeced the contents of CaM,PKC and CDK5 and P35.2 ACR induced can significantly increased the activities of CaMKII,PKA,PKC.3 ACR induced can significantly decreased the contents of CDK5 and P35.4 There was a relationship between ACR-induced neuropathy and protein kinase changed.