Amstutz V H, Mircheva A, Cengo A, Dubois L J, Sijm D T H M, Vrolijk M F
Department of Pharmacology and Toxicology, Faculty of Medicine, Health & Life Science, Maastricht University, PO Box 616, Maastricht, 6200 MD, 6229 ER, The Netherlands.
Department of Precision Medicine, GROW Research Institute for Oncology and Reproduction, Maastricht University, Maastricht, 6229 ER, The Netherlands.
Sci Rep. 2025 Jul 1;15(1):21914. doi: 10.1038/s41598-025-07503-7.
Per- and polyfluoroalkyl substances (PFAS) hepatotoxicity is well documented, especially for legacy compounds such as PFHxS, PFOA, PFOS, and PFNA. However, the mechanism(s) involved are yet to be fully understood. The present study aims to investigate the origin of PFAS-induced formation of reactive oxygen species (ROS) and their relevance for the decrease of cell viability of HepG2 cells after exposure to PFASs. Moreover, a structure-activity relationship was assessed using PFASs with different headgroups (carboxylic, sulfonic, and alcoholic) and variable carbon-chain lengths (4-10 C). The link between ROS generation and cell viability was assessed using two antioxidants: quercetin, a generic antioxidant, and mito-tempo, a mitochondria-targeted antioxidant. Both antioxidants were demonstrated to be effective in reducing PFAS-induced ROS generation. The mechanism behind PFAS-induced ROS might be headgroup-dependent, as quercetin increased cell viability after both perfluoroalkyl carboxylic acids (PFCA) and perfluorosulfonic acids (PFSA) exposure, while mito-tempo only improved cell viability after PFCAs exposure. The two major sources of ROS generation in HepG2 cells are the peroxisomes and mitochondria. However, exposure to PFASs did not impact peroxisomal or mitochondrial activity after 24 h. Uncommon sources of ROS generation, such as lysosomal leakage or lipid peroxidation, have been demonstrated to result from previously generated ROS and not from PFASs exposure. Indeed, lysosomal leakage caused by PFASs exposure is negated by either quercetin or mito-tempo treatment, while lipid peroxidation only occurs after 24 h of exposure, long after the initial ROS generation by PFASs. This indicates that both events are a result of previously generated ROS. However, exposure to both PFOA and PFOS was demonstrated to reduce catalase activity in HepG2. In conclusion, the present study demonstrates that ROS generation after PFASs exposure might be due to inhibition of HepG2 endogenous antioxidants. Moreover, a headgroup-dependent mechanism of action has been observed, indicating that PFCAs and PFSAs exposure might lead to hepatotoxicity through different pathways.
全氟和多氟烷基物质（PFAS）的肝毒性已有充分记录，尤其是对于诸如全氟己烷磺酸（PFHxS）、全氟辛酸（PFOA）、全氟辛烷磺酸（PFOS）和全氟壬酸（PFNA）等传统化合物。然而，其中涉及的机制尚未完全明了。本研究旨在探究PFAS诱导的活性氧（ROS）形成的起源及其与HepG2细胞暴露于PFAS后细胞活力降低的相关性。此外，使用具有不同头部基团（羧酸、磺酸和醇）以及可变碳链长度（4 - 10个碳）的PFAS评估了构效关系。使用两种抗氧化剂评估了ROS生成与细胞活力之间的联系：槲皮素，一种通用抗氧化剂，以及线粒体靶向抗氧化剂米托坦。两种抗氧化剂均被证明可有效减少PFAS诱导的ROS生成。PFAS诱导ROS生成的机制可能取决于头部基团，因为槲皮素在全氟烷基羧酸（PFCA）和全氟磺酸（PFSA）暴露后均能提高细胞活力，而米托坦仅在PFCA暴露后改善细胞活力。HepG2细胞中ROS生成的两个主要来源是过氧化物酶体和线粒体。然而，暴露于PFAS 24小时后并未影响过氧化物酶体或线粒体的活性。已证明不常见的ROS生成来源，如溶酶体渗漏或脂质过氧化，是由先前生成的ROS导致，而非PFAS暴露所致。实际上，PFAS暴露引起的溶酶体渗漏可通过槲皮素或米托坦处理消除，而脂质过氧化仅在暴露24小时后发生，这是在PFAS最初生成ROS很久之后。这表明这两个事件都是先前生成的ROS的结果。然而，已证明暴露于PFOA和PFOS均会降低HepG2中的过氧化氢酶活性。总之，本研究表明PFAS暴露后ROS生成可能是由于HepG2内源性抗氧化剂受到抑制。此外，已观察到一种取决于头部基团的作用机制，表明PFCA和PFSA暴露可能通过不同途径导致肝毒性。
