Perfluorooctanoic acid affects the activity of the hepatocyte nuclear factor 4 alpha (HNF4α)

Abstract

Perfluorooctanoic acid (PFOA) is an industrial chemical that is a global contaminant of water, soil and foodstuff. Numerous animal studies have revealed that PFOA has embryotoxic and hepatotoxic effects in rodents. On the molecular level, the adverse effects of PFOA have been correlated with the PFOA-mediated activation of peroxisome proliferator-activated receptor alpha (PPARα), however, the toxicological relevance of this mode of action for humans is under debate.

In this study, a proteomic approach was chosen to screen for molecular targets affected by PFOA in human liver cells. Treatment of the human liver cell line HepG2 with 25 μM PFOA resulted in 51 deregulated proteins in a two-dimensional gel experiment, and 36 of these proteins were identified by mass spectrometry. Network analysis revealed that these proteins are primarily involved in lipid metabolism and cancer. The hepatocyte nuclear factor 4α (HNF4α), but not PPARα, was the key regulator of the network. Indeed, subsequent western blot analysis revealed that the amount of HNF4α as well as of its target HNF1α was downregulated in PFOA-treated HepG2 cells. Moreover, PFOA was shown to inhibit HNF4α-dependent gene transcription. Thus, this study provides first experimental evidence that HNF4α is negatively affected by PFOA.

Introduction

Perfluorooctanoic acid (PFOA) is the most important member of the class of perfluoroalkyl acids which are used in the production of fluoropolymers and fluoroelastomers. The substance is stable towards thermal or chemical degradation due to the extraordinary stability of the carbonfluorine bond. Moreover, no biological system is known to have the capacity to degrade PFOA. Therefore, these substances persist in the environment (Suja et al., 2009). Humans are mainly exposed to PFOA via oral uptake of contaminated drinking water and food. PFOA is readily absorbed and accumulates in blood serum. The substance is not metabolised and possesses a very low elimination rate.

PFOA is toxicologically well characterised (Kudo and Kawashima, 2003, Lau et al., 2006). Chronic exposure of rats with PFOA resulted in an increased incidence of tumours in testis, pancreas and liver (Biegel et al., 2001). On the molecular level, the adverse effects induced by PFOA have been ascribed to the PFOA-mediated activation of the peroxisome proliferator-activated receptor alpha (PPARα) which is a ligand-dependent transcription factor. PPARα regulates numerous genes whose products are primarily involved in lipid metabolism and energy homeostasis (Martin et al., 1997, Peters et al., 1998). Mono- and polyunsaturated fatty acids are natural ligands of PPARα. Moreover, chemicals such as PFOA which are structurally related to fatty acids have been shown to activate PPARα (Vanden Heuvel, 1996). However, activation of PPARα is obviously not the only relevant mode of action for PFOA since some PFOA-mediated effects have also been observed in PPARα knockout mice (Wolf et al., 2008). Moreover, recent studies have shown that other nuclear receptors such as constitutive androstan receptor (CAR) or pregnane X receptor (PXR) may be activated by PFOA (Ren et al., 2009, Rosen et al., 2008b). Finally, most of the molecular data have been obtained with rodent models, raising the question to which degree these data are relevant to humans. As an example, PPARα is expressed at high levels in liver of rodents, whereas the expression level of PPARα in human liver is about 10-fold lower compared to mouse liver (Palmer et al., 1998).

This study was conducted in order to elucidate metabolic or signalling pathways that are affected by PFOA in human hepatocellular carcinoma cells as the liver is the primary target organ. For this purpose, we have chosen a hypothesis-free proteomic screening method to examine the effects of PFOA on the molecular level and to gain insights into the mode of action of PFOA in human liver cells.