Department of Medical Biochemistry, Trakya University School of Medicine, Edirne, Turkey.
Drug Chem Toxicol. 2022 Jul;45(4):1536-1543. doi: 10.1080/01480545.2020.1846548. Epub 2020 Nov 16.
Acrylamide is a harmful chemical, and its metabolism occurs mainly in the liver. Acrylamide can form adducts on proteins. Protein homeostasis is vital for metabolic and secretory functions of the liver. No study has investigated the effect of acrylamide on the ubiquitin-proteasome system (UPS). Also, the effect of acrylamide on autophagy and its regulation is not fully known. We aimed to investigate the effects of acrylamide on the UPS, autophagy, mammalian target of rapamycin (mTOR), and heat shock protein 70 (HSP70) in HepG2 cells as well as to examine the effects of -acetylcysteine and curcumin on these parameters in acrylamide-treated cells. HepG2 cells were initially treated with variable concentrations of acrylamide (0.01-0.1-1-10 mM) for 24 hours. Then, HepG2 cells were treated with 5 mM -acetylcysteine and 6.79 µM curcumin in the presence of 10 mM acrylamide for 24 hours. Cell viability was evaluated by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-tetrazolium bromide assay. Ubiquitinated protein, mTOR, microtubule-associated proteins 1 A/1B light chain 3B-II (LC3B-II), and HSP70 levels were measured by immunoblotting. Acrylamide at 10 mM concentration, without any significant change at lower concentrations, caused an increase in ubiquitinated protein, LC3B-II, and HSP70 levels and a decrease in mTOR phosphorylation. Furthermore, 5 mM -acetylcysteine caused a decrease in ubiquitinated protein and HSP70 levels; however, 6.79 µM curcumin did not affect 10 mM in acrylamide-treated cells. Our study showed that acrylamide at high concentration inhibits UPS and mTOR, activates autophagy, and increases HSP70 levels in HepG2 cells, and -acetylcysteine reduces UPS inhibition and HSP70 levels in acrylamide-treated cells.
丙烯酰胺是一种有害化学物质，其代谢主要发生在肝脏中。丙烯酰胺可以在蛋白质上形成加合物。蛋白质的动态平衡对于肝脏的代谢和分泌功能至关重要。目前还没有研究调查丙烯酰胺对泛素蛋白酶体系统（UPS）的影响。此外，丙烯酰胺对自噬及其调节的影响也不完全清楚。我们旨在研究丙烯酰胺对 HepG2 细胞 UPS、自噬、哺乳动物雷帕霉素靶蛋白（mTOR）和热休克蛋白 70（HSP70）的影响，并研究 -乙酰半胱氨酸和姜黄素对丙烯酰胺处理细胞中这些参数的影响。首先，将 HepG2 细胞用不同浓度的丙烯酰胺（0.01-0.1-1-10 mM）处理 24 小时。然后，在用 10 mM 丙烯酰胺处理的同时，用 5 mM -乙酰半胱氨酸和 6.79 µM 姜黄素处理 HepG2 细胞 24 小时。通过 3-(4,5-二甲基-2-噻唑基)-2,5-二苯基-2-四唑溴盐测定法评估细胞活力。通过免疫印迹法测量泛素化蛋白、mTOR、微管相关蛋白 1A/1B 轻链 3B-II（LC3B-II）和 HSP70 水平。在没有任何明显变化的情况下，10 mM 浓度的丙烯酰胺导致泛素化蛋白、LC3B-II 和 HSP70 水平增加，mTOR 磷酸化减少。此外，5 mM -乙酰半胱氨酸导致泛素化蛋白和 HSP70 水平降低；然而，6.79 µM 姜黄素对丙烯酰胺处理细胞中的 10 mM 没有影响。我们的研究表明，高浓度的丙烯酰胺抑制 UPS 和 mTOR，激活自噬，并增加 HepG2 细胞中的 HSP70 水平，-乙酰半胱氨酸降低丙烯酰胺处理细胞中的 UPS 抑制和 HSP70 水平。
