Study On The Damage Effect Of Acrylamide On HT22 Mouse Hippocampal Neurons

Acrylamide(ACR) is a kind of water-soluble high alpha, beta unsaturated carbonyl compound. Acrylamide is mainly used for the synthesis of polyacrylamide, which is widely employed in mining, sewage purification and the paper industry. Polyacrylamide is non-toxic, but acrylamide monomer is toxic. Since the scientists found out that acrylamide is generated during foods rich in starch subjected to high temperature frying or baking in 2002, the damage of acrylamide increasingly caused the people’s attention. Animal experiments have confirmed that acrylamide has neurotoxicity, carcinogenicity, genotoxicity, and reproduction toxicity. However, only the neural toxicity was found in the people. The clinical manifestations of Acrylamide neurotoxicity are ataxia, skeletal muscle weakness, and the main pathological characteristics are central and peripheral axons distal swelling and terminal degeneration, which expand proximally.The possible mechanisms about the neural toxicity caused by acrylamide mainly include inhibition of kinesin-based fast axonal transport, alteration of neurotransmitter levels, direct inhibition of neurotransmission,the influence on the cell skeleton system,and the inhibition of energy metabolism on the central nerve system. In addition, the oxidative stress and the promotion of neuron apoptosis may also be potential neurotoxic mechanism of ACR. The previous study in our lab has confirmed that sub-acute exposure to acrylamide can induce functional damage on the young rats’ nerve system and degeneration damage on the cerebral cortex neurons and cerebellar cortex purkinje neurons. Exposure to acrylamide in the embryonic period can lead to the reduced weight of borned rats, the deceleration of growth in the lactation and the influence on the hippocampal neuron. Hippocampus is mainly engaging in a variety of functions such as learning, memory, emotional reaction, its damage could affect learning and memory. Researchs have confirmed that ACR can damage the hippocampus.However, the mechanism still remained unclear.To the end, we carried out the acrylamide toxicity studies in vitro. HT22 cell is a kind of immortalized biochemical hippocampus neuron cell in mice which has typical morphology and characteristics of the neurons. According to the similar phenotypes with neuronal precursor cell, HT22 cell has been widely used on the research of neural toxicity, neuronal damage and protection. At present few studies have been reported about the acrylamide damage model estabolished by HT22 cells in vitro, so we focused on the HT22 cells to study the damage effects of acrylamide on hippocampal neurons, and explore the possible mechanism about neurotoxicity caused by acrylamide.Methods:1. The influence of acrylamide on the viability of HT22 cells and screening of simulation concentration: Different concentrations of acrylamide were added when HT22 cells grow to the logarithmic phase. The proliferation state was detected by MTT assay when the cells were treated with acrylamide for 24 h, 36 h and 48 h. The appropriate concentration was selected for the subsequent experiments. 2. The damage effect of acrylamide on HT22 cells: different concentrations of acrylamide was added to HT22 cells when it grow to the logarithmic phase. After 24 h, the morphological changes were observed under the inverted microscope. Hoechst33258 staining was used to observe the nuclear morphological changes; LDH release was detected by LDH kit. 3. The oxidative stress impact of acrylamide on HT22 cells: the generation of ROS was detected by DCFH- DA fluorescent probe. Superoxide dismutase(SOD) activity was detected by xanthine oxidation test. Malondialdehyde(MDA) content was detected by glucosinolates barbituric acid(TBA) method. The content of reduced glutathione(GSH) was detected by disulfide generation 2 nitro benzoic acid(DTNB) method. 4. The influence of acrylamide on the cell apoptosis: the change of mitochondrial membrane potential was detected by rhodamine 123 staining; the expressions of Bax protein, Bcl-2 protein and Cleaved caspase3 protein were detected by western blot assay.Results:1. When treated with different concentrations of acrylamide(0,1,2, 3, 4, 8, 16mmol/L) for 24 h,48h and 72 h, the cell survival rate reduced with the increase of the concentration and the extension of time. The result illustrated that the impact of acrylamide on HT22 cells survival is time and dose dependent. 2. We chosed 0, 1, 2,3mmol/L as simulation acrylamide concentrations for the follow-up study. Under inverted microscope we observed that the cells became round, the cell process reduced, and refractive index fell. The neclei in the control group were homogeneously and slightly stained, and the cell edge was clear. However, the cells in stimulated groups had hyperchromatic nuclear pyknosis, irregular shape, cracking nuclei, and the number of abnormal nuclei increased compared with the control group. With the increasing of acrylamide concentration, the content of LDH in the culture supernatant was significantly increased(P < 0.05 vs control). 3. When treated with acrylamide for 24 h, the accumulation of intracellular ROS and the content of MDA increased, while the activity of SOD and the content of GSH declined. 4. When treated with acrylamide for 24 h, the mitochondrial membrane potential gradually declined, the expression of Bax protein and Cleaved caspase3 protein increased, while the expression of Bcl-2 protein reduced.Conclusions:1. Acrylamide could inhibit the proliferation of HT22 cells, and its effect was time and dose dependent. 2. Acrylamide could affect the cell morphology, induce the apoptosis of cell nuclei, injure the cell membrane. 3. The possible mechanism about neural toxicity caused by acrylamide may be related to oxidative stress injury by increasing the accumulation of intracellular ROS and the content of MDA, and decreasing the activity of SOD and GSH content. 4. The possible mechanism about neural toxicity caused by acrylamide perhaps still be related to apoptosis by decreasing of the mitochondrial membrane potential, increasing Bax and Cleaved caspase3 protein expression and reducing Bcl-2 protein expression.