High Perfluorooctanoic Acid Exposure Induces Autophagy Blockage and Disturbs Intracellular Vesicle Fusion in the Liver

Arch Toxicol (2017) 91:247–258 DOI 10.1007/s00204-016-1675-1 MOLECULAR TOXICOLOGY High perfluorooctanoic acid exposure induces autophagy blockage and disturbs intracellular vesicle fusion in the liver Shengmin Yan1 · Hongxia Zhang1 · Xuejiang Guo2 · Jianshe Wang1 · Jiayin Dai1 Received: 25 October 2015 / Accepted: 28 January 2016 / Published online: 15 February 2016 © Springer-Verlag Berlin Heidelberg 2016 Abstract Perfluorooctanoic acid (PFOA) has been PFOA on cell viability. Although these findings demon- shown to cause hepatotoxicity and other toxicological strate that PFOA blocked autophagy and disturbed intracel- effects. Though PPARα activation by PFOA in the liver lular vesicle fusion in the liver, the changes in autophagy has been well accepted as an important mechanism of were observed only at high cytotoxic concentrations of PFOA-induced hepatotoxicity, several pieces of evidence PFOA, suggesting that autophagy may not be a primary have shown that the hepatotoxic effects of PFOA may not target or mode of toxicity. Furthermore, since altered liver be fully explained by PPARα activation. In this study, we autophagy was not observed at concentrations of PFOA observed autophagosome accumulation in mouse livers as associated with human exposures, the relevance of these well as HepG2 cells after PFOA exposure. Further in vitro findings must be questioned. study revealed that the accumulation of autophagosomes was not caused by autophagic flux stimulation. In addition, Keywords Perfluorooctanoic acid · Autophagy · we observed that PFOA exposure affected the proteolytic Proteome · Vesicle fusion activity of HepG2 cells while significant dysfunction of lysosomes was not detected. Quantitative proteomic analy- sis of crude lysosomal fractions from HepG2 cells treated Introduction with PFOA revealed that 54 differentially expressed proteins were related to autophagy or vesicular trafficking and Perfluoroalkyl acids (PFAAs) are widely used anthropo- fusion. The proteomic results were further validated in the genic compounds, which have been incorporated into many cells in vitro and livers in vivo after PFOA exposure, which products over the past six decades due to their unique physi- implied potential dysfunction at the late stage of autophagy. cal and chemical characteristics (Lau et al. 2007; Lind- However, in HepG2 cells, it seemed that further inhibi- strom et al. 2011). Because many of these compounds fit tion of autophagy did not significantly alter the effects of the defining characteristics of persistent organic pollutants (POPs), concerns regarding their impact on human health and the environment have increased (Lindstrom et al. 2011). Electronic supplementary material The online version of this As one of the most widely known PFAAs, the toxicologi- article (doi:10.1007/s00204-016-1675-1) contains supplementary cal effects of perfluorooctanoic acid (PFOA) have been material, which is available to authorized users. extensively studied, with hepatotoxicity found in rodents * Jiayin Dai even at low-dose exposure (Lau et al. 2007; Yan et al. 2014). [email protected] Medical surveillance studies have also found PFOA exposure to be positively associated with serum alanine ami- 1 Key Laboratory of Animal Ecology and Conservation notransferase (ALT) levels and negatively related to serum Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, People’s Republic of China high-density lipoprotein (HDL) levels in fluorochemical production workers (Gallo et al. 2012; Wang et al. 2012). 2 State Key Laboratory of Reproductive Medicine, Nanjing Medical University, Nanjing 210029, Activation of peroxisome proliferator-activated receptor α People’s Republic of China (PPARα) has been widely accepted as a critical mechanism 1 3 248 Arch Toxicol (2017) 91:247–258 for PFOA-induced hepatomegaly and disturbance of lipid Earlier reviewed studies suggest that autophagy also metabolism (Lau et al. 2007; Takacs and Abbott 2007), plays an important physiological role in the liver, particu- and our previous study observed significant activation of larly in regard to energy and nutrient balance, misfolded PPARα in livers of mice exposed to PFOA for 28 days (Yan proteins removal, and major subcellular organelle turno- et al. 2015). However, liver weight increase as well as per- ver under normal and pathophysiological conditions (Rau- turbation of fatty acid metabolism still could be observed tou et al. 2010; Yin et al. 2008). However, little is known in PFOA-exposed PPARα-null mice (Minata et al. 2010; about the connection between autophagy and the toxico- Rosen et al. 2008; Wolf et al. 2008), and this evidence logical effects of PFOA. In the present study, we assessed implies that there may be potential mechanisms other than the occurrence of autophagy and its potential relevance to PPARα activation in PFOA-induced hepatotoxicity. hepatotoxic effects after PFOA exposure. We demonstrated Autophagy is an evolutionarily conserved catabolic that the dysfunction of lysosome–autophagosome fusion cellular process dependent on lysosomes for its execu- led to the blockage of autophagy after PFOA exposure. tion and includes macroautophagy, microautophagy, and chaperone-mediated autophagy (Kim and Lee 2014; Malhi et al. 2010). Macroautophagy (referred to as autophagy Materials and methods hereafter) is characterized by the formation of a double- membrane-bound structure that sequesters cellular orga- Animal treatment nelles or cytoplasm into a structure called the autophagosome, which then fuses with the lysosome for degradation Male Balb/c mice (aged 6–8 weeks) were purchased from of the sequestered material (Kim and Lee 2014; Malhi et al. Beijing Vital River Experimental Animals Centre (Beijing, 2010). At least four major protein complexes are required China), with all experimental manipulations described in for the formation of autophagosomes: Atg1–Unc-51-like our previous study (Yan et al. 2014). Briefly, mice were kinase (ULK) complex, which initiates formation; class randomly divided into six groups and dosed with either III phosphatidylinositol 3-kinase (PI3K) or Vps34 com- Milli-Q water or PFOA diluted in Milli-Q water at doses plexes, which are required for autophagosome nucleation; of 0.08, 0.31, 1.25, 5, and 20 mg/kg/day via gavage for autophagy-related (Atg) protein 9 (Atg9) and its traffick- 28 days. The PFOA doses were chosen based on earlier ing machinery, which may play an important role in deliv- toxicological studies and our previous experiments (Yan ering vesicles and lipids to expand the autophagosome et al. 2014). All animal treatments were approved by the membranes at all stages; and ubiquitin-like proteins Atg12 Committee on the Ethics of Animal Experiments from the and Atg8/LC3 and their conjugation machinery, which Institute of Zoology, Chinese Academy of Sciences (Permit are required for autophagosome expansion and closure Number: EET-015-08-2012) and in accordance with the (Dall’Armi et al. 2013; Lamb et al. 2013). Guiding Principles in the Use of Animals in Toxicology, Autophagy occurs at low basal levels in most cells and which were adopted by the Society of Toxicology in 1989. contributes to the regular turnover of cytoplasmic compo- Full materials and methods were described in supple- nents (Rautou et al. 2010; Shintani and Klionsky 2004). It mentary information. can be rapidly induced by a change in environmental conditions, such as nutrient depletion, growth factor withdrawal, or high bioenergetic demands (Rautou et al. 2010; Shintani Results and Klionsky 2004). In conditions of nutrient deprivation, several cellular signaling pathways are modulated upon dif- PFOA exposure induced autophagosome accumulation ferent nutrient deficiencies, which lead to the initiation of autophagosome formation (Kim and Lee 2014). Autophagy Consistent with the serum ALT levels in our previous report has also been suggested to interplay with apoptosis, which (Yan et al. 2014), the histopathology results with H&E has been well reviewed elsewhere (Delgado et al. 2014; staining also showed significant hepatocyte swelling and Marino et al. 2014). Evidence from mice with systemic or lipid deposits in mice after 28-day exposure, especially tissue-specific deletion of autophagy-related (Atg) genes at high doses of PFOA (Fig. S1). Microtubule-associated suggests that autophagy dysfunction might contribute to protein light chain 3 (LC3) conversion (LC3 I to LC3 II) certain disease phenotypes, such as cancer, neurodegenera- was detected by Western blotting to investigate the occur- tion, liver disease, and metabolic disease (Jiang and Miz- rence of autophagosome accumulation. Both LC3A II and ushima 2014; Shintani and Klionsky 2004). The possible LC3B II were accumulated in livers of mice after 28-day roles of autophagy in health and disease can serve to pro- exposure to PFOA, especially at the middle dose of PFOA tect cells but may also contribute to cell damage (Shintani (Fig. 1a, b). Several autophagy-related genes, especially and Klionsky 2004). Atg12, were stimulated in mouse livers by PFOA (Fig. S2). 1 3 Arch Toxicol (2017) 91:247–258 249 Fig. 1 PFOA exposure induced autophagosome accumulation in levels were analyzed from whole lysates of HepG2 cells after 72 h of mouse livers and HepG2 cells. a LC3A, LC3B, Atg12–Atg5, and exposure to PFOA. Long indicates the representative images of long p62 protein levels were analyzed from whole lysates of livers after exposures, and short indicates short exposures. Band densities of

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