| Summary: | 博士 === 國立臺灣大學 === 獸醫學研究所 === 99 === Acrylamide (ACR) has many applications in industries and for protein analysis. ACR is a neurotoxin to humans and laboratory animals, and has been classified as “2A, probably human carcinogen” by International Agency for Research on Cancer since 1994. The fact that ACR in starchy food after high temperature processing was found in 2002 raises food safety concerns in general population. Epidemiological, in vivo and in vitro studies conclude that ACR influences normal neurological functions. However, toxic effects of ACR on glia cells remain unsolved. This study focused on the determination of the regulatory molecules and functional mechanisms involved in ACR-induced cytotoxicity, including DNA damage responses, cell-cycle arrest and apoptosis in astroglial cells in vitro.
ACR-induced cytotoxic effects on astrocytes (U-1240 MG) and neuronal cells (SH-SY5Y) were examined by measuring the cell viability, lactate dehydrogenase leakage (LDH) assay and oligonucleosomal DNA fragmentation. Both U-1240 MG and SH-SY5Y cells showed cytotoxicity after ACR treatment, however, U-1240 MG cells were less sensitive to ACR than SH-SY5Y cells. ACR-induced cytotoxicity in U-1240 MG cells was further confirmed by detecting the phosphorylated p53 (pp53, DNA damage marker protein) and glial fibrillary acidic protein (GFAP, chemical-induced astrogliotic response marker). Increasing pp53 after 2 mM ACR treatment for 24 h and accumulation of GFAP after 2 mM ACR treatment for 48 h indicated that ACR induced astrogliosis in U-1240 MG cells. U-1240 MG cells proliferation were suppressed at 1 mM ACR for 36 h or 2 mM ACR for 24 h by MTT assay, and also decreased 45.9% of Ki-67 protein expression when treated with 2 mM ACR for 48 h than control. ACR increased DNA damage of U-1240 MG cells in a dose-dependent manner with 48 h exposure, as evaluated by comet assay. Therefore, ACR inhibited cell proliferation and caused DNA damage in U-1240 MG cells.
Analysis of cell-cycle arrest by flow cytometry revealed that the ACR treatment resulted in significant increases in the G0/G1-arrested cells in U-1240 MG cells. Expression of DNA damage-associated/checkpoint-related signaling molecules (pp53, p53, p21, p27, Cdk2, and cyclin D1) were parallelly analyzed by immunoblotting in three human astrocytoma cell lines (U-1240 MG, U-251 MG, and U-87 MG). Under 2 mM ACR treated for 48 h, three cell lines caused marked increases in pp53 and Cdk2, as well as decreases in cyclin D1 and p27. Moreover, increases in p53 and p21 were detected in both U-1240 and U-87 MG cells, whereas no marked change in p53 and a decrease in p21 were observed in U-251 MG cells. Cell-cycle arrest of U-251 MG cells was found in G2/M phase with 2 mM ACR treatment for 48 h which reveals a different response to ACR toxicity than other two cell lines in G0/G1 arrest. To address the involvement of ataxia telangiectasia mutated/ATM-Rad3-related (ATM/ATR) kinase in signaling of ACR-induced G0/G1 arrest, caffeine was used to block the ATM/ATR pathway in U-1240 MG cells. The results showed caffeine significantly attenuated the ACR-induced G0/G1 arrest as well as the expression of DNA damage-associated/checkpoint-related signaling molecules in a dose-dependent manner.
ACR causes apoptosis (with 2 mM treatment for 48 h) by analyses of sub-G1 group arrest in cell cycle, protein expressions of pro-caspase 3 and poly ADP ribose polymerase (PARP), and oligonucleosomal DNA fragmentation. Analysis of apoptosis signaling molecules in U-1240 MG cells, including Bax, Bad, bcl-2, bcl-XL, Bax/bcl-2, Cyt c and Caspase 8, further supported the above observations. Treatment of 2 mM ACR for 72 h caused marked decreases in Bax, Bad and bcl-2 expressions, as well as increases in the Bax/bcl-2 ratio, Cyt c and Caspase 8 expressions. However, no marked change in bcl-XL was observed. The above protein expressions indicated that ACR induced mitochondria-mediated apoptosis in U-1240 MG cells. To address the involvement of ATM/ATR kinase in the signaling of ACR-induced apoptosis, caffeine was applied to block the ATM/ATR pathway in U-1240 MG cells. Caffeine significantly attenuated the ACR-induced expression of apoptosis-related signaling molecules, including pp53, p53, Bax, Bad, bcl-2, Bax/bcl-2, Cyt c, Pro-caspase 3 and Caspase 8, in a dose-dependent manner. These results were concordant with the evaluation of ATM and ATR protein expressions and the observation of the high expression of ATR protein by fluorescence microscopy in ACR-treated U-1240 MG cells. The ultrastructure alterations induced by ACR in U-1240 MG cells were observed by Transmission Electron Microscope. Mitochondria with vesicular matrix compartments and cytoplasmic vacuole formation were noticed while U-1240 MG cells treated with 2 mM ACR for 48 h and cells with chromatin condensation, pyknosis, and swollen mitochondria were observed at 72 h.
These results suggest that ACR exposure leads to astrogliosis, proliferation inhibition, DNA damage, G0/G1 phase arrest and mitochondria-dependent apoptosis in human astrocytoma cells in vitro. These studies clearly demonstrate the critical role of ATR in signaling the ACR-induced cell-cycle arrest and apoptosis in U-1240 MG cells. Whether ACR causes different cytotoxic effects and mechanisams on different astrocytic cell lines and primary cells is suggested for future investigation.
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