Molecular Mechanism Of Acrylamide-induced Neurotoxicity And Intervention Of Blueberry Anthocyanins Extract Against Acrylamide-induced Toxicity In Vivo

Acrylamide(AA)is a commonly found food contaminant formed during carbohydrates-enriched food heating processing.AA has been categorized as a possible carcinogen Group 2 A by IARC.It is well known that AA has neurotoxic effects both in occupational exposure human and experiment animals.AA also has potential genotoxicity and hepatotoxicity.AA-induced oxidative stress and related pathways may contribute to the multiple toxicity espically neurotoxicity,but the mechanisms remain elusive.The objective of this study is to investigate the neurotoxicity of AA,using a primary astrocytes/microglia/co-culture model.We hypothesize that AA induces oxidative stress leading to neurotoxicity via pathways modification.Moreover,the potential relationship among the pathways were explored using proteomic analysis.Considering to ameliorate AA-induced oxidative stress,we investigated the protection of a blueberry anthocyanin extract(BAE)against AA-induced mitochondrial oxidative damage in mice models as well as the intervention mechanism.The main results and conclusions were made as follows:(1)Neonatal Balb/c mice were sacrificed by cervical dislocation within postnatal 48 hour and the brains were harvested.The meninges were removed and the remaining dissociated tissue was replaced in cultural flask.Oligodendrocytes were removed by complement mediated cytolysis and remaining cells were incubated in culture medium for 9-14 days.After that,dislodged cells were separated by magnetic cell sorting using CD11b microbeads.The positive fraction contained purified microglial cells while the negative fraction was enriched for astrocytes.Purified cells were re-suspended in culture medium for purity check via immunofluorescence.Those whose purity were over 90%could be used in further experiment.(2)AA increased ROS formation,reduced glutathione levels causing successive events associated with oxidative stress including 4-hydroxynonenal-adduct and 8-hydroxy-2-deoxyguanosine adduct formation in both astrocytes and microglia cells.Both Nrf2 and NF-?B pathways were activated but the timing was different.Nrf2 and its downstream antioxidative genes HO1,Nqol,GST acted on earlier stage of both cell types before NF-?B was activated on later stage.After activation of NF-?B,the related cytokines including IL6,TNF-p and IL-1? were released,and the cell viability decreased.Microglia was more sensitive to AA induced toxicity than astrocytes.Higher ROS generation,faster GSH reduction and increased oxidative adduct formation were found in microglia compared with astrocytes.Also Nrf2/NF-?B and its downstream genes were up-regulated much faster and higher in microglia than in astrocytes.(3)AA treatment led to a dose-dependent toxicity in astrocytes/co-cultural cells.Oxidative stress was induced as indicated by an increase of ROS,a decrease of GSH levels,and an increase in the formation of 4-hydroxynonenal-adduct and 8-hydroxy-2-deoxyguanosine-adduct.Both Nrf2 and NF-?B pathway contributed to the initiation of oxidative stress but the timing of two factors was different.Similar to the purity cultural,Nrf2 and its related downstream genes were activated earlier than that in NF-?B pathway.Different from the purity cultural,the timing of Nrf2 and NF-?B activation were faster than that in astrocytes and slower than that in microglia.(4)Based on the analysis of pathways,networks,upstream regulators,function cluster by IPA software,ROS overproduction may be the main reason of the AA-induced neurotoxicity.Nrf2 and NF-?B may contribute to the AA-induced neurotoxicity via balancing the oxidcative status.The activation of Nrf2 and NF-?B may be phosphorylated by MAPK related pathways.These predictions are in line with the previous data in chapter ? and ?.(5)Pretreatment with 50-250 mg/kg b.w./day BAE markedly inhibited ROS formation,and prevented the successive events associated with the mitochondrial damage and dysfunction,including recovered activities of electron transport chain and ATPase,ameliorated depolarization of mitochondrial membrane potential and membrane lipid peroxidation,reduced release of cytochrome c and protection of mitochondria against swelling.In a word,mitochondria are a key target at the organelle level for the protective effect of BAE against AA toxicity.