TNT經由ROS介導之內質網壓力及粒線體失衡誘導人類肝臟細胞凋亡;2, 4, 6-Trinitrotoluene induces apoptosis by ROS-mediated endoplasmic reticulum (ER) stress and mitochondrial dysfunction in HepG2 cells

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Title:	TNT經由ROS介導之內質網壓力及粒線體失衡誘導人類肝臟細胞凋亡;2, 4, 6-Trinitrotoluene induces apoptosis by ROS-mediated endoplasmic reticulum (ER) stress and mitochondrial dysfunction in HepG2 cells
Authors:	黃暐婷;Huang,Wei-Ting
Contributors:	生命科學系
Keywords:	2,4,6-三硝基甲苯;細胞凋亡;線粒體功能障礙;內質網壓力;氧化壓力;2,4,6-trinitromethylbenzene;Apoptosis;Mitochondrial dysfunction;ER stress;ROS
Date:	2016-08-29
Issue Date:	2016-10-13 13:04:54 (UTC+8)
Publisher:	國立中央大學
Abstract:	三硝基甲苯 (2, 4, 6,-trinitrotoluene,TNT)，又稱作黃色炸藥。目前被人們廣泛使用於工業開發、軍事等用途。但有許多文獻指出，TNT在其製造、爆炸等過程中，恐會對環境或是生物體造成汙染，引發造血、生殖、神經系統方面的損傷，甚至是肝癌的發生。然而，TNT造成肝毒性的分子機制仍需要被進一步探討。因此，本研究選擇使用人類肝癌細胞株(HepG2)，觀察TNT對其所造成之影響，並更進一步探討其致毒機制。在本研究中，我們發現TNT會導致細胞存活率明顯降低，且有基因損傷(DNA damage)的發生。利用RNA-seq觀察經由不同濃度TNT處理24小時前、後的細胞基因發現TNT會誘導細胞基因發生變異。而參考KEGG資料庫顯示：其恐造成包括線粒體功能障礙、內質網壓力和細胞生長週期停滯等影響。因此，本研究利用JC-1染劑測定粒線體膜電位，證實了TNT確實會破壞粒線體膜電位，導致粒線體功能障礙的發生。此外，TNT也會造成蛋白Bax/Bcl-2的比率上升、procaspase 9/3的表現量降低，促使細胞凋亡。而藉由觀察到內質網壓力信號基因BiP、PDI、PERK、CHOP和oxidase 1 like α (Erol-Lα)信號的提升，也證實了內質網壓力的發生。最後，我們也研究了活性氧化物質(ROS)對於TNT誘導細胞毒性的作用機制，TNT進入細胞後在代謝的過程中，恐產生大量的ROS。除此之外，轉錄因子CHOP能誘導Ero1α的轉錄作用，進一步催化PDI的再氧化，增加胞內的氧化壓力。而藉由抗氧化劑N-acetyl-cysteine (NAC)的預處理能有效降低基因損傷、細胞凋亡和內質網傳遞途境訊號異常的比率。TNT所造成的HepG2細胞DNA損傷，是由於ROS所引起的內質網壓力和粒線體功能失衡所造成。 ;2,4,6-Trinitrotoluene (TNT) has been commonly used as an explosive throughout the world. TNT was reported to cause numerous adverse effects including liver cancer. However, the detailed molecular mechanisms underlying TNT-induced liver toxicity still need to be elucidated. At first, we found that TNT significantly decreased cell viability and induced DNA damage. Thereafter, RNA-seq was utilized to detect the differential genes in comparison with cells before and after TNT (30 g/mL and 80 g/mL) treatment for 24 h. It was observed that TNT induced many differential genes, and the Kyoto Encyclopedia of Genes and Genome (KEGG) analysis showed that the diverse biological functions and metabolic pathways affected included mitochondrial dysfunction, ER signaling stress, and cell cycle arrest. Mitochondrial dysfunction was evidenced by the loss of mitochondrial membrane potential followed by the increase in the ratio of Bax/Bcl-2 and caspase 3/7 activity as well as the decreased expression of procaspase 9/3, indicating that apoptosis had occurred. In addition, the expressions of some ER stress-related signaling genes and proteins including BiP, PDI, PERK, CHOP, and oxidase 1 like α (Erol-Lα) had increased. Next, we investigated the role of ROS in TNT-induced cellular toxicity. The levels of DNA damage, apoptosis, and ER stress-signaling pathways were alleviated when the cells were pretreated with the ROS scavenger N-acetyl-cysteine (NAC). Notably, we also demonstrated that TNT induced ROS overproduction. These results indicated that TNT caused the ROS-dependent apoptosis via ER stress and mitochondrial dysfunction.
Appears in Collections:	[Graduate Institute of Life Science] Electronic Thesis & Dissertation

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