二氯乙醇致脂肪變性與致死性的機轉探討

Exploring the mechanism of 2-chloroethanol-induced Steatosis and Lethality in Mice

周宗欣(Tzung-Hsin Chou)

指導教授 ： 華國泰

國立臺灣大學/醫學院/毒理學研究所/博士(2020年)

https://doi.org/10.6342/NTU202001710

摘要

二氯乙醇2-choloroethanol (2CE)為氯乙烯及1,2-二氯乙烷之中間代謝物，是一種工業及農業中常見之毒物。暴露氯乙烯及1,2-二氯乙烷會導致肝臟脂肪變性以及脂肪性肝炎，然而此致病機轉目前尚未十分清楚。本研究以二氯乙醇及其可能的代謝物二氯乙醛，研究ICR小鼠暴露後之組織學變化，發現小鼠暴露二氯乙醇及二氯乙醛的表徵明顯不同。二氯乙醇導致嚴重之系統性多器官脂肪變性，而二氯乙醛並無類似變化。其次，二氯乙醇可以導致低血酮低血糖，此表徵類似先天粒線體脂肪酸代謝異常疾病，意味二氯乙醇藉由抑制粒線體脂肪酸代謝功能導致全身性脂肪變性。本研究進一步利用代謝體學研究，先分析小鼠尿中檸檬酸循環代謝物，以及利用脂類組學分析小鼠暴露2CE後肝臟組織之脂肪成份，確認小鼠暴露二氯乙醇後導致肝臟磷脂絲胺酸顯著降低，此結果與粒線體能量代謝異常相符合。因此，再進一步利用大鼠肝細胞株H4-II-E-C3(CRL-1600)研究粒線體與功能，發現二氯乙醇抑制粒線體長鏈脂肪酸的氧化能力呈現劑量時間效應。同時，二氯乙醇可以導致細胞自噬及內質網網壓力，此與粒線體能量生產不足之結果相符合。最後本研究利用二甲基亞碸處理大鼠細胞，嘗試藉由二甲基亞碸調控粒線體電子傳遞以及氧化還原壓力，探討二氯乙醇暴露後之可能救治方式，本研究可以提供工業暴露二氯乙醇、氯乙烯及1,2-二氯乙烷後之急毒性救治基礎。

關鍵字

二氯乙醇 ； 脂肪酸氧化 ； 脂肪變性 ； 毒物

並列摘要

2-Chloroethanol (2CE) is a highly toxic, common metabolite of vinyl chloride and 1,2-dichloroethane that induces toxicant-associated fatty liver disease (TAFLD) and steatohepatitis (TASH). Currently, the mechanism by which 2CE induces TAFLD is not well characterized. In this study, we first evaluated the action of 2CE and its metabolite chloroacetaldehyde (CAA) in ICR mice, and verified by histology and biochemistry that it follows different mechanisms. Second, ICR mice exposed to the median lethal dose (LD50) of 2CE (130 mg/kg) for 24 h presented with hypoketotic hypoglycemia with systemic lipid dysregulation, indicating mitochondrial fatty acid oxidation (FAO) dysfunction. A metabolomics approach with target urinary organic acid analysis through gas chromatography-mass spectrometry and lipid profiling of samples from mouse livers by liquid chromatography-mass spectrometry, further supported the occurrence of 2CE-induced mitochondrial dysfunction. Furthermore, exposing a rat liver cell line (H4-II-E-C3(CRL-1600)) to 2CE for 24 h at a dose equal to the IC50 value for lethality resulted in decreased mitochondrial FAO, confirming defective mitochondrial function. These mitochondrial dysfunctions could be reversed by 0.33% dimethyl sulfoxide (DMSO), suggesting a role of DMSO in reversing 2CE-induced FAO inhibition. Both pre- and post-treatment with DMSO reversed 2CE cytotoxicity, indicating the existence of a salvage window. Pretreatment with DMSO also improved ICR mouse survival after acute 2CE exposure (24 h). 2CE-induced steatosis highlights the link between TAFLD and impaired mitochondrial FAO, which could be reversed by low-dose DMSO.

並列關鍵字

2-chloroethanol ； Fatty acid oxidation ； Steatosis ； Toxicant

參考文獻

Andrews JE, Courtney KD, Donaldson WE (1983) The effects of ethylene chlorohydrin on fatty acid synthesis. Journal of environmental science and health Part B, Pesticides, food contaminants, and agricultural wastes 18(3):351-67 doi:10.1080/03601238309372374

Ardail D, Gasnier F, Lermé F, Simonot C, Louisot P, Gateau-Roesch O (1993) Involvement of mitochondrial contact sites in the subcellular compartmentalization of phospholipid biosynthetic enzymes. J Biol Chem 268(34):25985-92

Cave M, Falkner KC, Ray M, et al. (2010) Toxicant-associated steatohepatitis in vinyl chloride workers. Hepatology 51(2):474-81 doi:10.1002/hep.23321

Chang SC, Heacock PN, Clancey CJ, Dowhan W (1998) The PEL1 gene (renamed PGS1) encodes the phosphatidylglycero-phosphate synthase of Saccharomyces cerevisiae. J Biol Chem 273(16):9829-36 doi:10.1074/jbc.273.16.9829

Chen D, Zhang XY, Shi Y (2006) Identification and functional characterization of hCLS1, a human cardiolipin synthase localized in mitochondria. Biochem J 398(2):169-76 doi:10.1042/bj20060303