Molecular Mechanisms of Perf l uorooctanoate-Induced HepatocyteApoptosis in Mice Using Proteomic TechniquesKan Li, † Jie Sun, † Jingping Yang, ‡ Stephen M. Roberts, § Xuxiang Zhang, † Xinyi Cui,* ,† Si Wei,* ,†and Lena Q. Ma †,∥† State Key Laboratory of Pollution Control and Resource Reuse, School of the Environment, Nanjing University, Nanjing, Jiangsu210046, China‡ School of the Medicine, Nanjing University, Nanjing, Jiangsu 210046, China§ Center for Environmental and Human Toxicology, University of Florida, Gainesville, Florida 32611, United States∥ Soil and Water Science Department, University of Florida, Gainesville, Florida 32611, United States* S Supporting InformationABSTRACT: The stability of perf l uorooctanoate (PFOA)coupled with its wide use cause serious concerns regarding itspotential risk to human health. The molecular mechanisms ofPFOA-induced hepatotoxicity relevant to human health wasinvestigated using both in vivo (mouse model) and in vitro(human hepatocyte cells, HL-7702) techniques. Both male andfemale Balb/c mice were administered PFOA at 0.05, 0.5, or2.5 mg/kg-d for 28-d, with serum PFOA concentrations afterexposure being found at environmentally relevant levels. Liversamples were examined for histology and proteomic changeusing iTRAQ and Western Blotting, showing dose-dependenthepatocyte apoptosis and peroxisome proliferation. At highdoses, genotoxicity resulting from ROS hypergeneration wasdue to suppression of Complex I subunits in the electron transport chain and activation of PPARα in both genders. However, at0.05 mg/kg-d, Complex I suppression occurred only in females, making them more sensitive to PFOA-induced apoptosis. Invitro assays using HL-7702 cells conf i rmed that apoptosis was also induced through a similar mechanism. The dose/gender-dependent toxicity mechanisms help to explain some epidemiological phenomena, i.e., liver cancer is not often associated withPFOA exposure in professional workers. Our results demonstrated that a proteomic approach is a robust tool to exploremolecular mechanisms of toxic chemicals at environmentally relevant levels.■INTRODUCTIONPerf l uoroalkyl substances (PFASs) are synthetic compoundswidely used in industrial and consumer products such as Tef l onand Gore-Tex. 1 Due to their strong carbon−f l uorine bonds,PFASs are stable and accumulate in the environment, so theyare classif i ed as persistent organic pollutants (POPs). 2,3Perf l uorooctanoate (PFOA), is one of the most used PFASsand is abundant in the environment, resulting in humanexposure through food, housedust, and drinking water. 4 PFOAhas been detected in cord blood and breast milk in the generalpopulation 1,4 partially due to its long half-life (∼4 y) in thehuman body. 5,6 Because of its high usage in China, increasingserum levels of PFOA in humans have been observed. Forexample, serum levels increased from 0.08 μg/L in 1987 to 4.3μg/L in 2002 in Shenyang and up to 15.2 μg/L in 2009 wasreported for Nanchang. 7,8 The widespread human exposure toPFOA causes increasing concerns regarding its environmentalhealth risks worldwide.Recent epidemiology studies showed no correlation of PFOAexposure with escalated liver cancer or clinical liver diseaseoccurrence, but a positive correlation with elevated serumalanine transaminase levels, a sign of hepatocyte damage. 9−11 Invitro assays using human hepatocellular carcinoma HepG2 cellsshowed that PFOA caused apoptosis, 12,13 which was proven byproteomic assay using isobaric tags for relative and absolutequantitation (iTRAQ) following PFOA exposure in normalhuman hepatocyte cells (HL-7702). Signs of apoptosis likehepatocyte-nuclear condensation is also observed in PFOA-treated mouse liver. 14 However, the mechanisms of PFOA-induced hepatocyte apoptosis in mammalian hepatocytes arestill unknown. Some attributed this to lipid accumulation in thenucleus of mouse liver, while hyperaccumulation of reactiveoxygen species (ROS), which triggered a mitochondrialsignaling pathway, was the mode of action in HepG2 cells,with ROS origination being unclear. 14 Another critical issue ofReceived: May 25, 2017Revised: September 6, 2017Accepted: September 8, 2017Published: September 8, 2017Articlepubs.acs.org/est© 2017 American Chemical Society 11380 DOI: 10.1021/acs.est.7b02690Environ. Sci. Technol. 2017, 51, 11380−11389Downloaded via SHANGHAI JIAO TONG UNIV on July 5, 2025 at 02:43:23 (UTC).See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles.
