The Effect On Neurofilament Phosphorylation Related Protein Kinases And Proteins Induced By Acrylamide

ObjectiveAcrylamide (ACR) is a kind of widely-used industrial material. It is regarded as a neurotoxicant and human potential carcinogen through the studies of more than thirty years. 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 exact mechanism of action of its neurotoxicity has not been completely clear until now.Based on the pathological alterations, we built ACR-intoxicated rats models and investigated the relative levels of proteins in the tissue of sciatic nerves (SN) to determine the molecular mechanisms. We detected the changes of three protein kinases and four relative proteins, include calmodulin (CAM), protein kinase C (PKC), protein kinase A (PKA), Cyclic Adenosine monophosphate (cAMP), cyclin dependent kinase 5 (CDK5) and CDK5-related factors (P35 and P25) in SN of ACR-treated rats. Moreover, the contents of PKA and PKC in the serum of rats were also tested, which will offer the cues and evidences for exploring the mechanism of ACR neurotoxicity and searching biomarkers.Methods 1. Male Wistar rats weighing 180-220g were divided into three groups, each 9 animals. The rats in group 1 served as control, and received double distilled water (DDW). The animals in group 2 and 3 were given ACR dissolved in DDW(20,40 mg/kg i.p.3 days/week) for 8 weeks.2. Neurobehavioral index were determined per week. The changes of general status, body weight of rats and neurobehavioral function were observed. In addition, we observed abnormal gait and got the gait score.3. The excised nervous tissues of SN were homogenized in ice-cold homogenizing buffer and then centrifuged at a high speed. The supernatant was saved in-80℃.The relative levels of PKA, PKC, CDK5, P35 and P25 in the supernatant of SN were determined by SDS-PAGE and Western Blotting. The contents of CaM and cAMP in SN were determined by ELISA kits.4. The activities of PKA and PKC were determined by using corresponding radioactivated 32P assay kits in corresponding cytosolic fractions of SN of control and experimental group rats.5. The decapitated bloods were sampled, and the serum was separated routinely. The contents of PKA and PKC in the rat serum were investigated by SDS-PAGE and immunoblotting.Results1. The body weight and gait scoreCompared with the control, the body weight of rats lost in the ACR-induced groups, i.e. the body weight of the 5th to 8th week decreased significantly (P<0.05) of high ACR group. Compared with the control, gait score increased remarkably at 8th week (P<0.01) of low ACR group and increased remarkably at 4th to 8th week (P<0.01) of high ACR group. 2. The changes of proteins(1) The change of CaM contents in sciatic nerves:Compared with control, no significant changes observed in the low dose group and high dose group had increased 50%(P<0.01). Compared with the low dose group, CaM content increased by 50%(P<0.01) in the high dose group.(2) The change of cAMP contents in sciatic nerves:The exposure to ACR resulted in a significant decrease in cAMP contents. Compared with the control, the level of cAMP decreased by 11% in the low dose group and by 95%(P<0.01) in the high dose group. Compared with the low dose group, cAMP content decreased by 94%(P<0.01) in the high dose group.(3) The contents and activities of PKA and PKC in sciatic nerves:In comparison with the control rats, the levels of PKA and PKC increased respectively by 18%and 21% in low ACR group, by73% (P<0.01) and 170%(P<0.01) in high ACR group. Compared with the control group, PKA activity had increased 60% (P＜0.01) in low dose group but decreased 50%(P<0.01) in high dose group. While compared with the low dose group, it decreased 220%(P<0.01) in high dose group. Compared with the control, PKC activity was significantly increased in 20 (by 50%, P< 0.01) and 40 (6 folds, P< 0.01) mg/kg ACR group rats, respectively. Compared with the 20 mg/kg ACR group, PKC activity increased 3 folds (P<0.01).(4) The exposure to ACR resulted in a significant decrease in CDK5, P25 and P35 contents in sciatic nerve. In comparison with the control rats, CDK5 in the low dose group decreased 51%(P<0.01), in the high dose group decreased 22%(P<0.05). Compared with the low dose group, CDK5 in the high dose group increased by 59%(P<0.01). In comparison with the control rats, the levels of P25 and P35 in the high dose group decreased respectively by 77%(P<0.01) and 82%(P<0.01). (5) The levels of PKA and PKC in serum significantly increased by 6% and 6%in 20mg/kg ACR rats,by 45%(P<0.01) and 55%(P<0.01)in 40 mg/kg ACR rats, respectively.Conclusions1. Subchronic intoxation of ACR can induce the injury of nerve system. It was showed that the gait abnormal.2. ACR exposure could increase the content of CaM in sciatic nerve tissue, which indicates ACR significantly increased the intracellular Ca2+ concentration.3. The changes of protein kinases in sciatic nerve may be related to ACR induced peripheral nerve toxicity. ACR exposure could significantly change the contents of cAMP, PKA, PKC, CDK5, P35 and P25, and, the activities of PKA and PKC in sciatic nerve. As a result, the phosphorylation state of NFs changed.4. The contents of PKA and PKC were significantly increased in serum of ACR-treated rats, which suggested they might be served as some of the biomarkers of ACR-induced neuropathy for earlier diagnosis.5. The peripheral nerve toxicity mechanism of ACR may be associated with the changes of the above-mentioned proteins.