The Analysis of Acrylamide-induced Toxicity on Rat Primary Astrocyte and Human Glioblastoma Cells
Date Issued
2012
Date
2012
Author(s)
Wang, Yan-Shiu
Abstract
Arylamide (ACR) is common used in industry for clarifying drinking water and protein analysis. ACR contains neurotoxicity, genotoxicity, reproductive toxicity and carcinogenicity. Under high-temperature (120－180oC) cooking, food rich with amino acid, particular asparagine, and reduced glucose can produce ACR through Maillard reaction more than 100 times the limits in drinking water which raises food safety concern.
There are several evdences showed ACR influenced neurons and induced neurotoxicity in human epidemiological studies and animal experiments. However, However, there are limited studies on ACR-induced toxicity of glia cell. Until recently, The role of astrocytes in the brain is no longer simple and passive but plays an active function including influencing neurite growth and transmission of nerve impulses, and controlling microenvironment. Chen et al. [2009, 2010]reported two studies about ACR-induced cytotoxicity on U-1240 MG cell. Further studies showed ACR caused genotoxicity, arrested cell cycle in G0/G1 phase and influenced protein associated with DNA-damage response pathway including ATM/ATR, p53, p21, p27, Cdk2 and cylinD1. Nevertheless, different cell lines (U-1240 MG, U-87 MG and U-251 MG) had different reactions after ACR exposure, for example, U-251 MG cells had no regulation of p53 and p21 protein under ACR treatment but U-1240 MG and U-87 MG cells did. The aim of this study determines which cell line has mostly similar toxic effect with rat primary astrocyte in apoptosis pathway in order to further study the ACR-induced astrocytoxic machnism.
The results in MTT assay showed decreasing cell viability was found after ACR 1 mM under 48 and 72 h and 2 mM under 24 to 72 h exposure among four cells in time-dependent and dose-dependent manners. Interestingly, there were changes in low concentration (0.1 and 0.5 mM) only on U-87 MG and primary astrocyte. Also, ACR didn’t induced LDH leakage on U-1240 MG and U-251 MG but on U-87 MG and primary astrocyte after 48 and 72 h 2 mM ACR exposure in LDH assay. ACR raised the sub-G1 phase analyzed by PI stain and change of mitochrondral potential analyzed by JC-1 stain among four cells. Moreover, the apoptotic mechanism was under explored by measuring the DNA damage response pathway proteins including p53 and pp53 and apoptosis pathway associated-molecular proteins including Bcl-2, Bax, pro-caspase 8, 9 and 3 through western blotting analysis. 2 mM ACR increased p53 and pp53 activity in DNA damage response pathway. The co-treatment of ACR with ATM/ATR inhibitor caffeine can attenuate the increase amount of p53 and pp53. Bcl-2 changed with different cells: up-regulation was found on U-1240 MG and U-251 MG and down-regulation was found on U-87 MG and primary astrocyte after 48 h 2 mM ACR exposure. Besides, different concentration ACR also caused different response: up-regulation of Bcl-2 was found in 0.1 and 0.5 mM concentration and down-regulation was found in 1 and 2 mM concentration on primary astrocyte. The up-regulation of Bax and down-regulation of pro-caspase 3, 8 and 9 were found at 0.1, 0.5, 1 and 2 mM ACR 48 h treatment. In conclusion, U-87 MG had the more similar response with primary astrocyte after ACR exposure. We advise the researchers subsequently studied the machanism of ACR-induced astrocytotocixity that U-87 MG cell line should be the best choice for saving the time and technique of the culture of primary astrocyte.
Subjects
Acrylamide
Neurotoxicity
Apoptosis
Primary astrocyte
SDGs
Type
thesis
File(s)![Thumbnail Image]()
Loading...
Name
ntu-101-R99629025-1.pdf
Size
23.32 KB
Format
Adobe PDF
Checksum
(MD5):b38e50414db250e75e14e17b9c303be6