應用質譜儀為基礎的脂質體學探討全氟辛烷磺酸暴露對人類肝癌細胞脂質影響

Abstract

全氟與多氟烷基物質（per- and polyfluoroalkyl substances; PFAS）是一類廣泛應用於產品中的化學物質。其中，全氟辛烷磺酸（perfluorooctanesulfonic acid; PFOS）是最常在人體血清與環境樣本中檢測到的化合物之一。由於其對蛋白質具有高度結合親和力，PFOS 容易在肝臟中累積。PFOS 會引發多種有害健康效應，包括干擾脂質代謝、肝臟脂肪累積，以及肝臟發炎。本研究旨在透過脂質體學方法，分析PFOS暴露對人類肝癌細胞株 HepG2 中含磷酸膽鹼的脂質（phosphorylcholine-containing lipids; PC-CL）的變化，以探討其可能毒性機制。 本研究分別以對照組、1/10 IC₁₀、IC₁₀與IC₂₀ PFOS濃度處理HepG2細胞48 小時。細胞收集後，脂質經由改良的Folch法萃取，並以超高效液相層析串聯三重四極桿質譜儀分析HepG2細胞的PC-CL組成。經圖譜處理後，透過主成分分析與偏最小平方區別分析等多變量統計方法，辨識各暴露劑量組別間脂質體的差異；同時使用Kruskal–Wallis檢定以篩選在不同處理組之間具顯著差異的脂質種類。 在高劑量PFOS暴露組中，單元與雙元不飽和二酰基PC（DPC）下降，而多元不飽和DPC（polyunsaturated-DPC; PUFA-DPC）上升。這些結果顯示PFOS可能干擾肝臟中磷脂醯膽鹼的生合成路徑，包括CDP-膽鹼(CDP-choline)路徑與磷脂醯乙醇胺N-甲基轉移酶(phosphatidylethanolamine N-methyltransferase; PEMT)路徑。此外，PUFA-DPC與P-PC的上升可能反映細胞為了對抗PFOS引發的發炎與氧化壓力而產生的肝保護性反應。 總結而言，本研究運用了質譜為基礎的脂質體學方法，探討PFOS暴露對 HepG2 細胞 PC-CL 脂質組成的影響。研究結果指出，在不引起細胞毒性的PFOS暴露濃度下，細胞可能透過肝保護機制來應對PFOS所造成的發炎與氧化壓力反應。本研究的結果展現出體外實驗在脂質體學研究中作為動物實驗替代方案的潛力，特別是在相對低劑量 PFOS 暴露的情況下。

Per- and polyfluoroalkyl substances (PFASs) are a group of synthetic chemicals widely used in consumer products. Among them, perfluorooctanesulfonic acid (PFOS) is one of the most commonly detected compounds in human serum and environmental samples. Due to its high binding affinity to proteins, PFOS accumulates in the liver. PFOS induces several adverse health effects, including the disruption of lipid metabolism, liver steatosis, and liver inflammation. The aims of this study are to understand the possible mechanisms of PFOS-induced cytotoxicity by analyzing changes of phosphorylcholine-containing lipids (PC-CL) in human hepatoma HepG2 cells exposed to a series dose of PFOS using lipidomic approach. HepG2 cells were treated with vehicle control, 1/10 IC10, IC10 and IC20 of PFOS for 48 hours. PC-CL were extracted with modified Folch method and analyzed by ultra-performance liquid chromatography–triple quadrupole mass spectrometry. After spectral processing, multivariate analysis, including principal component analysis and partial least squares discriminant analysis, were conducted to identify different patterns of lipid across the various treatment groups. The Kruskal–Wallis test was applied to identify lipids that significantly differed among treatment groups. The decreased levels of mono- and di-unsaturated diacyl-PC (DPC) and increased levels of polyunsaturated-DPC (PUFA-DPC) were observed in the high dose group. These findings suggest that PFOS may disrupt the hepatic PC biosynthesis pathway by binding to bioavailable choline, thereby reducing PC production via the CDP-choline pathway and upregulating PEMT activity. This compensatory mechanism may enhance PC biosynthesis through the PEMT pathway. In conclusion, this study applied MS-based lipidomic approach to identify the changes of PC-CL profile in PFOS-treated HepG2 cells. The results suggested that at sublethal doses of PFOS, the hepatoprotective effects may occur due to the inflammation and oxidative stress caused by PFOS. The results of this study demonstrate the potential of in vitro models as alternatives to animal testing in lipidomic research, particularly under conditions of sublethal doses of PFOS exposure.