丙烯酰胺对大鼠脊髓组织蛋白激酶的影响
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摘要
前言
丙烯酰胺(Acrylamide, ACR)是一种水溶性的、乙烯基单体,主要用于生产聚丙烯酰胺(polyacrylamide,PACR),后者可作为良好的凝结、增稠、吸水剂,广泛用于废水处理、采油、造纸、纺织、皮革、采矿、医药等行业。近年来随着石油工业的迅速发展,丙烯酰胺需求量不断增加。
尽管动物实验显示ACR有神经毒性、致肿瘤毒性和生殖毒性,但只有神经毒性是经过职业暴露人群流行病学研究证实的。ACR神经毒性主要表现为中枢-周围性远端轴索病,临床表现为共济失调、骨骼肌无力,其病理特征是远端轴突肿胀、变性、神经丝(neurofilament,NF)聚集,但发病机理尚未完全阐明。我们通过建立ACR亚慢性中毒大鼠模型,测定大鼠脊髓中钙调蛋白(calmodulin, CaM)、Ca2+/钙调素依赖型蛋白激酶Ⅱ(Ca2+/CaM—dependent protein kinascⅡ, CaMKⅡ)、蛋白激酶A(proteinkinaseA, PKA)、环磷酸腺苷(3'-5'-cyclic adenosine monophosphate, cAMP)、蛋白激酶C(proteinkinase C, KC)和周期蛋白依赖性的蛋白激酶5(cyclin dependent kinasc 5, CDK5)以及CDK5相关因子p35含量,为ACR中毒神经病发病机制研究提供资料,为ACR职业中毒的预防和治疗提供可以借鉴的基础资料。
方法：
1雄性成年健康Wistar大鼠,体重180-220g,ACR经腹腔注射染毒,染毒剂量分别为20、40mg/kg,连续8周(每周染毒3次),建立ACR亚慢性中毒神经病动物模型。
2分离脊髓组织,制备匀浆,高速离心。用SDS-PAGE和Western-Blotting方法测定胞浆蛋白中的PKA、PKC、CDK5以及p35的含量。用ELISA检测试剂盒测定cAMP和CaM含量变化。
3取大鼠脊髓组织胞浆蛋白,利用放射性同位素技术研究ACR染毒对大鼠脊髓中CaMKⅡ、PKA和PKC活性的影响。
结果
1 ACR动物模型的建立：动物经20、40 mg/kg ACR腹腔染毒后,体重增长减缓,步态得分增高；染毒终止时,低、高剂量组分别呈轻度和重度受损步态。ACR亚慢中毒神经病动物模型建立。
2脊髓组织中蛋白激酶含量：
1)CaM蛋白含量在ACR亚慢性中毒大鼠脊髓的胞浆蛋白中明显降低,与对照组相比,20mg/kg ACR染毒组降低了21%,40 mg/kg ACR染毒组降低了17%,差异有统计学意义(P<0.01)。
2)PKA和cAMP蛋白含量在ACR亚慢性中毒大鼠脊髓的胞浆蛋白中没有明显变化(P>0.05)。
3)PKC蛋白含量在ACR亚慢性中毒大鼠脊髓组织的胞浆蛋白中明显降低,与对照组相比,40mg/kg ACR染毒组降低了46%,差别有统计学意义(p<0.05)。与20mg/kg组相比,40mg/kg ACR染毒组降低了41%,差别有统计学意义(p<0.05)。
4)CDK5和p35蛋白含量在ACR亚慢性中毒大鼠脊髓组织的胞浆蛋白中明显降低,与对照组相比,40mg/kg ACR染毒组CDK5和p35蛋白含量分别降低了23%和46%,差别均有统计学意义(p<0.05)。
3脊髓组织中蛋白激酶活性
1)CaMKⅡ蛋白活性在ACR亚慢性中毒大鼠脊髓组织的胞浆蛋白中明显升高,与对照组相比,20mg/kg、40mg/kg ACR染毒组分别升高了123%和261%,差别均有统计学意义(p<0.05)。
2)PKA蛋白活性在ACR亚慢性中毒大鼠脊髓组织的胞浆蛋白中明显升高,与对照组和20mg/kg染毒组相比,40mg/mg染毒组分别升高了57%和50%(p<0.01),差异具有统计学意义。
3)PKC活性在ACR亚慢性中毒大鼠脊髓组织的胞浆蛋白中明显升高,与对照组相比,40mg/kg染毒组升高了170%,与20mg/kg染毒组相比,40mg/kg染毒组升高了160%(p<0.01),差异有统计学意义。
结论
1、ACR染毒可以影响大鼠脊髓的CaM、PKC和CDK5及其P35的含量。
2、ACR染毒可明显提高CaMKⅡ、PKA,PKC的活性。
3、ACR染毒可明显降低CDK5和P35的含量。
4. ACR的神经病发病机制可能与脊髓蛋白激酶改变有关。
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.
Methods
1 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.
Results
1 The establishment of the animal model
After 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 cord
1) 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 cord
1) 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).
Conclusions
1 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.
丙烯酰胺(Acrylamide, ACR)是一种水溶性的、乙烯基单体,主要用于生产聚丙烯酰胺(polyacrylamide,PACR),后者可作为良好的凝结、增稠、吸水剂,广泛用于废水处理、采油、造纸、纺织、皮革、采矿、医药等行业。近年来随着石油工业的迅速发展,丙烯酰胺需求量不断增加。
尽管动物实验显示ACR有神经毒性、致肿瘤毒性和生殖毒性,但只有神经毒性是经过职业暴露人群流行病学研究证实的。ACR神经毒性主要表现为中枢-周围性远端轴索病,临床表现为共济失调、骨骼肌无力,其病理特征是远端轴突肿胀、变性、神经丝(neurofilament,NF)聚集,但发病机理尚未完全阐明。我们通过建立ACR亚慢性中毒大鼠模型,测定大鼠脊髓中钙调蛋白(calmodulin, CaM)、Ca2+/钙调素依赖型蛋白激酶Ⅱ(Ca2+/CaM—dependent protein kinascⅡ, CaMKⅡ)、蛋白激酶A(proteinkinaseA, PKA)、环磷酸腺苷(3'-5'-cyclic adenosine monophosphate, cAMP)、蛋白激酶C(proteinkinase C, KC)和周期蛋白依赖性的蛋白激酶5(cyclin dependent kinasc 5, CDK5)以及CDK5相关因子p35含量,为ACR中毒神经病发病机制研究提供资料,为ACR职业中毒的预防和治疗提供可以借鉴的基础资料。
方法：
1雄性成年健康Wistar大鼠,体重180-220g,ACR经腹腔注射染毒,染毒剂量分别为20、40mg/kg,连续8周(每周染毒3次),建立ACR亚慢性中毒神经病动物模型。
2分离脊髓组织,制备匀浆,高速离心。用SDS-PAGE和Western-Blotting方法测定胞浆蛋白中的PKA、PKC、CDK5以及p35的含量。用ELISA检测试剂盒测定cAMP和CaM含量变化。
3取大鼠脊髓组织胞浆蛋白,利用放射性同位素技术研究ACR染毒对大鼠脊髓中CaMKⅡ、PKA和PKC活性的影响。
结果
1 ACR动物模型的建立：动物经20、40 mg/kg ACR腹腔染毒后,体重增长减缓,步态得分增高；染毒终止时,低、高剂量组分别呈轻度和重度受损步态。ACR亚慢中毒神经病动物模型建立。
2脊髓组织中蛋白激酶含量：
1)CaM蛋白含量在ACR亚慢性中毒大鼠脊髓的胞浆蛋白中明显降低,与对照组相比,20mg/kg ACR染毒组降低了21%,40 mg/kg ACR染毒组降低了17%,差异有统计学意义(P<0.01)。
2)PKA和cAMP蛋白含量在ACR亚慢性中毒大鼠脊髓的胞浆蛋白中没有明显变化(P>0.05)。
3)PKC蛋白含量在ACR亚慢性中毒大鼠脊髓组织的胞浆蛋白中明显降低,与对照组相比,40mg/kg ACR染毒组降低了46%,差别有统计学意义(p<0.05)。与20mg/kg组相比,40mg/kg ACR染毒组降低了41%,差别有统计学意义(p<0.05)。
4)CDK5和p35蛋白含量在ACR亚慢性中毒大鼠脊髓组织的胞浆蛋白中明显降低,与对照组相比,40mg/kg ACR染毒组CDK5和p35蛋白含量分别降低了23%和46%,差别均有统计学意义(p<0.05)。
3脊髓组织中蛋白激酶活性
1)CaMKⅡ蛋白活性在ACR亚慢性中毒大鼠脊髓组织的胞浆蛋白中明显升高,与对照组相比,20mg/kg、40mg/kg ACR染毒组分别升高了123%和261%,差别均有统计学意义(p<0.05)。
2)PKA蛋白活性在ACR亚慢性中毒大鼠脊髓组织的胞浆蛋白中明显升高,与对照组和20mg/kg染毒组相比,40mg/mg染毒组分别升高了57%和50%(p<0.01),差异具有统计学意义。
3)PKC活性在ACR亚慢性中毒大鼠脊髓组织的胞浆蛋白中明显升高,与对照组相比,40mg/kg染毒组升高了170%,与20mg/kg染毒组相比,40mg/kg染毒组升高了160%(p<0.01),差异有统计学意义。
结论
1、ACR染毒可以影响大鼠脊髓的CaM、PKC和CDK5及其P35的含量。
2、ACR染毒可明显提高CaMKⅡ、PKA,PKC的活性。
3、ACR染毒可明显降低CDK5和P35的含量。
4. ACR的神经病发病机制可能与脊髓蛋白激酶改变有关。
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.
Methods
1 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.
Results
1 The establishment of the animal model
After 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 cord
1) 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 cord
1) 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).
Conclusions
1 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.
引文
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[32]Xiwen H, Jing L, Tao C, Ke Y. Studies on biochemical mechanism of neurotoxicity induced by acrylamide in rats[J]. Biomed Environ Sci, 1992,5(3):276-281
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[34]任锐,李百祥.染铝对大鼠大脑皮层中CaM和CaMKⅡ蛋白表达影响[J].毒理学杂志,2005,9(19)：338-339.
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[36]Makoto H, Nobuhisa I, Takaomi C. Saido. Understandingmolecu2larmechanisms of p roteolysis in Alzheimer's disease:Progress to2ward therapeutic interventions [J].B iochim ica et B iophysica Acta,2005,1751(1):60-67.
[37]Gupta RP, Bing G, Hong JS, Abou-Donia MB.cDNA cloning and sequencing of Ca2+/calmodulin-dependent protein kinase Ⅱalpha subunit and its mRNA expression in diisopropyl phosphorofluoridate (DFP)-treated hen central nervous system[J]. Mol Cell Biochem,1998,181(1-2):29-39.
[38]Gupta A and Tsai LH. Neuroscience. A kinase to dampen the effects of cocaine? [J].Science,2001,292(5515):236-237.
[39]Bonkale WL, Fastbom J, Wiehager B, et al. Impaired G-protein-stimulated adenylyl cyclase activity in Alzheimer's disease brain is not accompanied by reduced cyclic-AMP-dependent protein kinase A activity [J].Brain Res,1996, 737(1-2):155-161.
[40]Xu A, Hawkins C, Narayanan N. Ontogeny of sarcoplasmic reticulum protein phosphorylation by Ca 2+/calmodulin2dependent protein kinase. J Mol Cell Cardiol,1997,29:405-418.
[41]Cassabona G.Inteacellualr signal modulation;A pivotal rolefor protein kinase C[J].Prog Neuro Psychopharmacol Biol Psychiatry,1997,21 (3):407-409.
[42]Giasson BI, Cromlish JA, Athlan ES, et.al. Activation of cyclicAMP-dependentprotein kinase in okadaic acid-treated neurons potentiatesneurofilament fragmentation and stimulates phosphorylation of Ser2 inthe low-molecular-mass neurofilament subunit[J]. J Neurochem 1996,66(3):1207-1213.
[43]Hisanaga S, Gonda Y, Inagaki M, Ikai A, et.al.Effects of phosphorylation of the neurofilament L protein on filamentous structures[J].Cell Regul, 1990,1(2):237-248.
[44]Tanii H, Hayashi M, Hashimoto K.Neurofilament degradation in the nervous system of rats intoxicated with acrylamide, related compounds or 2,5-hexanedione[J]. Arch Toxicol,1988,62(1)70-75.
[46]Damodaran TV, Abdel-Rahman A, El-Sourady MH,etc. Differential alteration of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA in the central nervous system of hens treated with diisopropylphosphorofluoridate (DFP)[J]. Neurochem Int,2002,40(4):371-379.
[46]Damodaran TV, Abdel-Rahman A, El-Sourady MH,etc. Differential alteration of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA in the central nervous system of hens treated with diisopropylphosphorofluoridate (DFP)[J]. Neurochem Int,2002,40(4):371-379.
[47]Patzke H,Tsai LH. CDK5 sinks into ALS[J]. Trends Neurosci,2002,25(1):8-10.
[48]Nakamura S, Kawamoto Y, Nakano S, et al. p35nck5a and cyclin-dependent kinase 5 colocalize in Lewy bodies of brains withParkinson's disease[J]. Acta Neuropathol,1997,94(2):153-157.
[49]Borghi R, Giliberto L, Assini A, et al. Increase of cdk5 is related to neurofibrillary pathology in progressive supranuclear palsy[J]. Neurology,2002, 58(4):589-592.
[50]Meyerson M,Enders GH,Wu CL,et al.A family of hu-man cdc2-related protein kinases[J].EMBO J,1992,11 (8):2909-2917.
[51]Smith DS, Greer PL, Tsai LH. CDK5 on the brain[J]. CellGrowth Differ, 2001,12(6):277-283.
[52]Tarricone C, Dhavan R, Peng J, et al. A Structure and regulation of the CDK52p25 (nck5a) complex. Mol Cel,2001,8(3):657-669.
[53]Elhanany E, Jaffe H, Link WT, et al. Identification of endogenously phosphorylated KSP sites in the high-molecular-weight rat neurofilament protein[J]. J Neurochem,1994,63(6):2324-2335.
[54]Hisanage S, MastuokaY, Nishiawza K, et.al. Phosphorylation of nantieve and reassembled neuorouflamentcomposed of NF-L,NF-M,NF-M,and NF-H by the catalytic subunit of cAMP-dependent protein kinase[J]. Mol BiloCell,1994,5(2):161-172.
[55]Patrick GN, Zhou P, Kwon YT, et al. p35, the neuronal-specific activator of cyclin-dependent kinase 5 (Cdk5) is degraded by the ubiquitin-proteasome pathway[J]. J Biol Chem,1998,273(37):24057-24064.
[56]Alvarez A, Toro R, Caceres A et al.Inhibition of tau phosphorylatingprotein kinase cdk5 prevents beta2amyloid induced neuronal death [J].FEBS Lett, 1999,459(3):421-426.
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