以體外高內涵影像技術探討全多氟烷基化合物 (PFAS) 對於人體之肝臟脂質堆積與毒性效應

Abstract

全氟烷基化合物 (PFAS)結構具有疏水疏油性，其被廣泛用於廚具、食品包裝、化妝品和家具的保護塗層等日常消費品中。美國環境保護署 (US EPA) CompTox Chemicals Dashboard中存在超過 14,000 種和 PFAS 有關的結構。而這些無所不在、大量的PFAS，長期暴露下可能引起相關人類健康危害。哺乳動物暴露PFAS 後常見的表現型如脂肪肝形成與血清甘油三酯數值降低。然而，大多數 PFAS 的體內毒性和肝臟脂肪堆積潛力在很大程度上仍然未知。故本研究旨在利用 HepG2 細胞研究 PFAS 誘導的肝臟脂質堆積與毒性效應，並利用現有的體內毒性數據描述體外效應。我們從具有代表性的 PFAS 家族中選擇了 20 個 PFAS 物種，包括全氟烷基羧酸鹽(perfluoroalkyl carboxylates, PFCAs)、全氟烷基磺酸鹽(perfluoroalkyl sulfonates, PFSAs)、氟調聚物磺酸鹽(fluorotelomer sulfonates, FTSs)、氟調聚物醇類(fluorotelomer alcohols， FTOH)、短鏈新興 PFAS (GenX)、全氟烷基磺醯胺(perfluoroalkyl sulfonamide , PFOSA)、氟調聚物不飽和羧酸(fluorotelomer unsaturated carboxylic acid, PFUcA)及全氟烷基磺醯胺醇(perfluoroalkyl sulfonamidoethanol)。將 HepG2 細胞接種到 96 孔盤中，於 37 ℃ 和 5% CO2下培養 24 小時後，以各測試化合物濃度為0.01、0.1、1、10 及 100 μM 的處理 48 小時。以螢光染劑對細胞進行染色(細胞核、線粒體和中性脂質)後使用 ImageXpress Micro Confocal 收集高內涵成像 (HCI) 表現型，其中劑量反應相關數據以Hill 模型推導出生物起始劑量(POD)。在所有測試的化合物中，以PFOSA 表現出最強的反應，而 FTOHs、FTS、PFUcA 和短鏈新興 PFAS 化合物表現出較小的反應。總結來說，具有磺酸、胺基相關的官能基團與較長的碳氟鏈的 PFAS 化合物在 HepG2 細胞中較容易產生反應。而本研究中獲得的體外 POD 濃度與大鼠 28 天研究中獲得的血中三酸甘油脂含量的體內 POD 值高度相關(r=0.97；ρ=0.76)(NTP TOX-96 及 TOX- 97)。在反轉錄聚合酶連鎖反應中也可以驗證PFAS存在可能之脂肪堆積的效應。總而言之，我們證明了 HepG2 細胞產生的體外 PODs 可以對應到 PFAS 化合物的體內肝脂肪變性的趨勢，這將有助於未來 PFAS 混合物的健康危害評估。本研究也證明了先前文獻的結果，具有8至12個碳的 PFAS 具有較高相關之生物活性。並相信未來隨著收集更多相關數據，此工具可用於填補 PFAS 和未來混合物 PFAS 研究中的數據空白。

Perfluoroalkyl substances (PFAS) manifest water and lipid-resistant properties, which are widely used in consumer products such as protective coatings for cookware, food packaging, cosmetics, and furniture. More than 14,000 structurally-diverse PFAS structures exist in the U.S. Environmental Protection Agency (US EPA) CompTox Chemicals Dashboard. The ubiquitous exposure to numerous PFAS compounds has raised health concerns in humans. Hepatosteatosis and reduced serum triglyceride levels are the common phenotypes induced by PFAS exposures in mammals. However, the in vivo toxicity and hepatosteatosis potential of most PFAS remain largely unknown. This study aims to investigate the PFAS-induced hepatocellular effects using HepG2 cells and delineate the in vitro effects with existing in vivo toxicity data. We selected 20 PFAS species from the representative PFAS families, including perfluoroalkyl carboxylates (PFCAs), perfluoroalkyl sulfonates (PFSAs), fluorotelomer sulfonates (FTSs), fluorotelomer alcohols (FTOH), short-chain emerging PFAS (GenX), perfluoroalkyl sulfonamide (PFOSA), Fluorotelomer unsaturated carboxylic acid (PFUcA), and perfluoroalkyl sulfonamidoethanol. The HepG2 cells were seeded into a 96-well plate, incubated at 37 ℃ and 5% CO2 for 24 hours, and then treated with test compounds at 0.01, 0.1, 1, 10, or 100 μM for 48 hours. Next, the cells were stained with fluorescent dyes, targeting at cell nucleus, mitochondria, and neutral lipids species.The high-content imaging (HCI) phenotypes were collected using ImageXpress Micro Confocal, where the concentration-response data was fitted with the Hill model to derive the point of departure (POD) concentrations. Among the test substances, PFOSA showed the strongest, while FTOHs, FTS, PFUcA, and short-chain emerging PFAS compounds manifested the least hepatosteatosis potential. Overall, PFAS compounds with functional groups sulfonic acid, amine, or saturated carboxylic acid developed in vitro hepatosteatosis effects in HepG2 cells. The in vitro POD concentrations acquired in this study are highly correlated (r=0.97; ρ= 0.76) with the in vivo POD values of serum triglyceride levels obtained from the 28-day gavage studies in rats (NTP reports TOX-96 and TOX-97). In conclusion, we demonstrated that in vitro PODs generated from HepG2 cells could project in vivo hepatosteatosis potentials of PFAS substances, which would facilitate the mixture health assessment of PFAS substances.It proves the results from previous studies that PFAS with 8-12 carbons have more obvious biological activity. As collecting more data, this tool can be used to fill the data gap in PFAS and in future mixture PFAS study.