含氯多氟烷基物質 (Cl-PFASs) 對秀麗隱桿線蟲之生殖毒性及其相關機制:以含氯多氟羧酸 (Cl-PFCAs) 作探討

Abstract

全氟和多氟烷基物質 (Per- and polyfluoroalkyl substances, PFASs) 是一類結構多樣的化學物質，廣泛應用於工業和商業領域。由於其穩定性與持久性，全球環境、食品以及人類和動物的血液中都檢測到PFASs的污染。近年來，含氯多氟烷基物質 (Chlorinated polyfluoroalkyl substances, Cl-PFASs) 因其在受PFASs相關產業影響的環境中持續存在而備受關注。先前研究顯示，PFASs對生長發育、運動和生殖具有毒性作用，然而，關於Cl-PFASs毒性的數據仍然非常有限。基於此背景，本研究以秀麗隱桿線蟲 (Caenorhabditis elegans, C. elegans) 作為模式生物，評估Cl-PFASs的毒性效應，並且以Cl-PFASs中屬含氯多氟羧酸 (Chlorinated polyfluorocarboxylic acids, Cl-PFCAs) 之氯二氟乙酸 (Chlorodifluoroacetic acid, CDFA) 與九氯全氟壬酸 (9-Chloroperfluorononanoic acid, 9Cl-PFNA) 為主研究其毒性效應，並進一步探討CDFA誘導生殖毒性之分子調控機制。本研究結果發現，在比較1和5 μM CDFA以及9Cl-PFNA暴露下之C. elegans生長發育、運動行為和繁殖毒性，發現9Cl-PFNA並不會影響C. elegans的體長，而CDFA於濃度5 μM時會減少體長；9Cl-PFNA和CDFA均會影響運動行為和生殖能力，且CDFA對於身體彎曲次數的影響較9Cl-PFNA顯著，顯示CDFA在運動行為上具有較高的毒性。針對CDFA的研究結果發現，長期暴露於10、50、75和150 μM CDFA 顯著影響C. elegans的生長，而暴露於1、5、10、50、75和150 μM CDFA則顯著影響其運動行為和繁殖。另外，長期暴露於1和150 μM CDFA 也顯著增加C. elegans生殖細胞DNA損傷以及其體內ROS含量。而進一步以qRT-PCR分析發現，1和150 μM CDFA 顯著改變了C. elegans生殖相關基因的表達，結果顯示，CDFA 顯著增加了與DNA損傷反應相關之hus-1、調控DNA損傷誘導的細胞週期停滯之cep-1及其下游與生殖細胞週期停滯相關之phg-1的mRNA表達量，並顯著降低了與生殖細胞的維持和增殖相關之glp-1的mRNA表達量。此外，CDFA也顯著上調了與卵黃蛋白生成相關的卵黃蛋白編碼基因vit-2和vit-6的mRNA表達量，同時下調了卵黃內吞受體蛋白編碼基因rme-2的mRNA表達量。另外，CDFA並不會在經rme-2 RNAi的C. elegans中誘導生殖毒性，顯示rme-2在介導CDFA誘導的生殖毒性效應中扮演關鍵的角色。 結合上述，本研究結果表明，CDFA可對C. elegans的生長發育、運動和生殖產生毒性作用，其中生殖毒性可能透過影響C. elegans DNA損傷所誘導的細胞週期停滯並且干擾卵黃蛋白的內吞作用來介導。本研究結果提供Cl-PFASs相關之毒理學數據，並提供其潛在分子機制解析，可為未來研究因Cl-PFASs環境或食品污染造成之毒性危害提供參考依據。

Per- and polyfluoroalkyl substances (PFASs) are structurally diverse chemicals that are used in a wide range of industrial and commercial applications. Consequently, PFASs contamination has been detected in the environment, food, and the blood of humans and animals worldwide. Recently, chlorinated polyfluoroalkyl substances (Cl-PFASs) have drawn more attention because of their continued presence in the environment affected by PFASs-related industries. Previous studies have demonstrated that PFASs exert toxic effects on development, locomotion, and reproduction. However, studies on the toxicity of Cl-PFASs are very limited. Based on these backgrounds, this study aimed to use Caenorhabditis elegans (C. elegans) as a model organism to evaluate the toxic effects of Cl-PFASs. Specifically, it focused on chlorodifluoroacetic acid (CDFA) and 9-chloroperfluorononanoic acid (9Cl-PFNA), two chlorinated polyfluorocarboxylic acids (Cl-PFCAs) within the Cl-PFASs, to assess their toxicity and further investigate the molecular mechanisms underlying CDFA-induced reproductive toxicity. The results showed that when comparing the toxic effects of 1 and 5 μM CDFA and 9Cl-PFNA exposure on C.elegans growth, locomotion, and reproduction, 9Cl-PFNA did not affect the body length of C. elegans, whereas 5 μM CDFA significantly reduced body length. Both 9Cl-PFNA and CDFA impaired locomotion and reproduction. Compared to 9Cl-PFNA, CDFA had a more pronounced effect on body bend, indicating CDFA has higher locomotor toxicity. Further investigations on CDFA revealed that long-term exposure to 10, 50, 75, and 150 μM CDFA significantly affected the growth, while exposure to 1, 10, 50, 75, and 150 μM CDFA significantly affected the locomotion and reproduction of C. elegans. In addition, long-term exposure to 1 and 150 μM CDFA demonstrated significant increase in C. elegans germline DNA damage and ROS levels. Furthermore, 1 and 150 μM CDFA significantly altered the expression the expression of reproduction-related genes in C. elegans, as determined by qRT-PCR. The results showed that CDFA significantly increased the mRNA levels of hus-1, associated with the DNA damage response; cep-1, which regulates DNA damage-induced cell cycle arrest; and its downstream gene phg-1, related to germ cell cycle arrest. Additionally, CDFA significantly decreased the mRNA level of glp-1, a gene related to the maintenance and proliferation of germ cells. Moreover, CDFA significantly increased the mRNA levels of the vitellogenin-encoding genes vit-2 and vit-6, which are involved in yolk lipoprotein production, while reducing the mRNA level of rme-2, the gene encoding the yolk endocytic receptor. Apart from this, rme-2 RNA interference in C. elegans eliminated the toxic effects on reproduction induced by CDFA, suggesting that rme-2 plays a key role in mediating CDFA-induced reproductive toxicity. In conclusion, our findings suggest that CDFA induce toxic effects on development, locomotion, and reproduction in C. elegans, among which reproductive toxicity potentially mediated through affecting the cell cycle arrest induced by DNA damage and interfering with the endocytosis of yolk protein in C. elegans. This study contributes to a better understanding of the toxicological profile of Cl-PFASs and provides insights into their underlying mechanisms, which can provide a reference for future research on the toxic hazards caused by Cl-PFASs contamination in the environment or food.