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