Perfluorooctanoic acid (PFOA) is an industrial chemical that is used for the fabrication of numerous products with water and dirt repellent properties. In animal experiments, repeated administration of PFOA in rats and mice led to the formation of liver tumors. This is explained at the molecular level by activation of the peroxisome proliferator-activating receptor alpha (PPARα). This mechanism plays a subordinate role in the human liver, so that the liver-toxic effect of PFOA observed in rodents is not considered relevant for humans. The aim of this project was to investigate whether PFOA in human liver cells affects other signaling and/or metabolic pathways in addition to the activation of PPARα, and whether these effects may be of interest for the ongoing assessment of the toxicity of PFOA.
Our own preliminary work had already shown that PFOA negatively affects the stability and/or the activity of the hepatocyte nuclear factor 4 alpha (HNF4α). HNF4α is an important transcription factor in the liver that is involved in the regulation of liver development and in the regulation of numerous metabolic pathways in the liver and other organs. Using computer models, it was shown that PFOA could bind with high affinity in a binding pocket of human HNF4α, which, according to the published crystal structure, is normally occupied by a fatty acid. Replacement of this fatty acid by PFOA could be the cause of the observed structure-destabilizing effect of PFOA on HNF4α. Furthermore it could be shown that PFOA leads to a strong dephosphorylation of Ser304 and Ser313 in HNF4α. Phosphorylation of these two serine residues is associated with the dimerization of HNF4α and is essential for DNA binding and thus for the transcriptional activity of this protein. However, further investigations could not prove that PFOA has drastic effects on the functionality of HNF4α. For example, HNF4α has an essential function in the functionality of pancreatic β-cells, and PFOA had no effect on insulin secretion by these cells, which is regulated by HNF4α.
The results of this project were able to underpin the data from our own preliminary work, which state that PFOA stimulates cellular proliferation by activating intracellular signal cascades. It could be shown that PFOA in human hepatocytes ultimately leads to an increased expression of the proto-oncogene c-FOS via a phosphorylation cascade via MEK1/2, ERK1/2 and ELK-1. This correlated with an altered expression of various genes, the products of which are involved in cell cycle regulation. Overall, the data show that PFOA stimulates the proliferation of human hepatocytes.
Further molecular investigations could show that PFOA does not stimulate any other human nuclear receptors (PPARγ, PPARδ, CAR, PXR, FXR, LXR, RAR, RXR) besides PPARα and also has no significant effects on the human steroid receptors ERα, ERβ and AR. No effects of PFOA on the formation of the sex hormones estradiol, progesterone and testosterone were observed, so that the endocrine effects of PFOA discussed in the literature could not be substantiated by our in vitro studies. However, a strong effect of PFOA on cholesterol metabolism and in particular on the formation and secretion of various bile acids could be observed. In human hepatocytes, PFOA led to an altered expression of numerous genes, the products of which are involved in synthesis, metabolism and transport of cholesterol and bile acids. In addition, PFOA led to a dilatation of bile ducts. Such morphological changes are associated with the clinical picture of cholestasis.
In summary, the results of this project allow a deep insight into the molecular mechanisms of the toxicity of PFOA in human liver cells, in particular with regard to the stability and activity of HNF4α, the stimulation of cellular proliferation, possible endocrine effects, and cholesterol metabolism. It must be emphasized that all effects were only observed with PFOA concentrations of at least 10 µM. These concentrations are at least 1000 times higher than the blood serum concentrations of PFOA measured in the general population. The results of our in vitro studies do not allow the conclusion that PFOA is of toxicological relevance for the human liver with regard to real , at least with regard to the endpoints investigated in this project.