探討 PFOS及其前驅物 N-MeFOSE-M 與 PFOSA 於人類肝癌細胞株之生物轉化關係與對代謝物及脂質干擾

Abstract

全多氟烷基化合物 (Per- and polyfluoroalkyl substances, PFAS) 是一類具高度持久與生物累積性的環境污染物，已被過去研究指出廣泛存在於環境及人體內。全氟辛烷磺酸 (Perfluorooctanesulfonic acid, PFOS) 因具有肝毒性、致癌性及代謝功能障礙相關脂肪肝病 (metabolic dysfunction-associated steatotic liver disease, MASLD) 等多種健康效應而受到國際間高度關注。然而，相較於 PFOS 已有許多動物與人體流行病學毒理學證據，其前驅物如全氟辛烷磺醯胺 (Perfluorooctanesulfonamide, PFOSA)及N-甲基全氟辛烷磺醯胺乙醇(N-methylperfluorooctanesulfonamidoethanol, N-MeFOSE-M) 之健康效應與毒性機制仍缺乏研究。此外，此類前驅物仍應用於日常消費產品中，並可能透過飲用水、食物、室內灰塵等途徑暴露，加上此類前驅物可經動物體內生物轉化生成不易降解之 PFOS ，因此僅依據 PFOS 的毒理資料可能造成實際健康風險低估。故本研究以 HepaRG 肝癌細胞株之體外模型，利用非標定代謝體學與脂質體學分析，探討 N-MeFOSE-M 、PFOSA 與 PFOS 暴露後之生物轉化現象、代謝干擾及其潛在毒性作用機制。研究結果顯示，暴露最高濃度 N-MeFOSE-M 與 PFOSA 後，於細胞內偵測到顯著上升之 PFOS 訊號，證實兩種前驅物可於HepaRG 細胞內發生生物轉化生成 PFOS 。細胞內外代謝體與脂質體分析結果顯示，三種 PFAS 皆對細胞內代謝物及脂質造成相較於細胞外更顯著之擾動。共同差異特徵分析發現脂質類別佔最高比例，其中固醇 20:3;O2 (Sterol 20:3;O2, ST 20:3;O2) 與脂肪酸 12:1 (Short fatty ester 12:1, SFE 12:1) 皆於呈現上升趨勢，並且結合細胞內外共同差異表現特徵之趨勢變化，皆顯示出在細胞內發生脂質累積的可能性。進一步結合富集途徑分析結果， N-MeFOSE-M 、 PFOS 與 PFOSA 皆造成脂質代謝干擾、脂質運輸及脂肪酸氧化相關途徑，以及共同干擾SLC介導之跨膜運輸、葡萄糖穩態及尿素循環等途徑，其中部分共同差異特徵於不同 PFAS 暴露下呈現不同變化趨勢，顯示其共同毒性效應可能涉及不同作用機制。綜上所述，本研究補充了 PFOS 前驅物 N-MeFOSE-M 與 PFOSA 於體外肝細胞模型中生物轉化及代謝干擾效應之研究缺口，並可作為後續 PFOS 前驅物毒性評估及作用機制研究之重要參考。

Per- and polyfluoroalkyl substances (PFAS) are a class of environmental contaminants characterized by high persistence and bioaccumulation potential, and previous studies have demonstrated their widespread presence in both the environment and human populations. Perfluorooctanesulfonic acid (PFOS) has received considerable international attention due to its association with various adverse health effects, including hepatotoxicity, carcinogenicity, and metabolic dysfunction-associated steatotic liver disease (MASLD). However, although substantial toxicological and epidemiological evidence is available for PFOS, the health effects and toxicological mechanisms of its precursors, Perfluorooctanesulfonamide (PFOSA) and N-methylperfluorooctanesulfonamidoethanol (N-MeFOSE-M), remain insufficiently investigated. Furthermore, these precursors are still used in consumer products and may contribute to human exposure through drinking water, food, and indoor dust. Since these precursors can undergo biotransformation into persistent PFOS, health risk assessments based solely on PFOS toxicological data may underestimate actual risks. Therefore, this study used an in vitro model HepaRG cells and applied untargeted metabolomics and lipidomics analyses to investigate the biotransformation, metabolic perturbations, and potential toxicological mechanisms induced by N-MeFOSE-M, PFOSA, and PFOS exposure. The results showed that significantly increased intracellular PFOS signals were detected following exposure to the highest concentrations of N-MeFOSE-M and PFOSA, confirming that both precursors can be biotransformed into PFOS within HepaRG cells. Intracellular and extracellular metabolomic and lipidomic analyses revealed that all three PFAS induced more pronounced perturbations in intracellular metabolites and lipids than in extracellular components. Analysis of shared differential features showed that lipids constituted the largest proportion of altered features, among which the Sterol 20:3;O2 (ST 20:3;O2) and the short fatty ester 12:1 (SFE 12:1) exhibited increasing trends. Together with the opposite patterns observed in shared differential features between intracellular and extracellular, these findings suggest the potential occurrence of intracellular lipid accumulation. Further integration with pathway enrichment analysis demonstrated that N-MeFOSE-M, PFOS, and PFOSA commonly disrupted pathways related to lipid metabolism, lipid transport, and fatty acid oxidation, as well as SLC-mediated transmembrane transport, glucose homeostasis, and urea cycle and associated pathways. Notably, some shared differential features exhibited distinct response patterns among PFAS exposures, suggesting that similar outcomes may arise through different underlying mechanisms. Overall, this study addresses important knowledge gaps regarding the biotransformation and metabolic effects of the PFOS precursors N-MeFOSE-M and PFOSA in an in vitro HepaRG cell model, and provides scientific evidence to support future PFAS health risk assessment.