Effects of perfluorooctanesulfonate (PFOS) and perfluorononanoic acid (PFNA) on protein expression and steroidogenesis in the human adrenocortical carcinoma cell line H295R

Abstract

The main aim of this study was to investigate effects of the perfluorinated compounds perfluorooctanesulphonate (PFOS) and perflouorononanoic acid (PFNA) using the human adrenocortical carcinoma cell line – H295R. Perfluorinated compounds (PFCs) are stable chemicals used widely in products such as paint and stain repellants. These compounds are known to be able to act as endocrine disruptors. H295R is a well-known and a much used model because among other reasons of its ability to produce hormones related to the stereoidogenesis. In this study the H295R cells were exposed in 48h to three different concentrations, 150 µM, 175 µM and 200 µM, of PFOS and PFNA in 75cm2 tissue culture flasks. The viability was measured with the alamarBlue ® assay, while four hormones related to the steroidogenesis, oestradiol, testosterone, cortisol and progesterone, were measured by using radioimmunoassay. RNA, DNA and proteins were isolated from the exposed cells, and toxic effects on the cell-model were investigated by using proteomics. 2D gels were performed to separate the proteins, and statistically significant proteins spots were excised and identified by LC-MS/MS followed by on-line database searches, using SwissProt and NCBInr databases. Western blot was used to quantitatively validate the findings. In addition, other proteins were chosen for western blot in the light of knowledge of proteins involved in steroidogenesis and cellular stress. Exposure to PFOS had a cytotoxic effect on the cells at all three concentrations. In addition, exposure to PFOS caused an increased oestradiol at the lowest concentration, but no significant alterations at the two highest concentrations. Testosterone secretion was decreased at the highest concentration, and cortisol and progesterone secretion was decreased at all concentrations. Exposure to PFNA caused a statistically non-significant increased secretion of oestradiol and a statistically significant decreased secretion of testosterone, cortisol and progesterone at all concentrations. Seventeen of the protein spots that showed significant differences in normalized volumes were identified and included proteins involved in transcription/protein synthesis, transport, and stress response. The western blots showed increased vimentin in cells exposed to PFNA, and decreased levels of BAX were found in cells exposed to PFOS. In conclusion, exposure to PFOS and PFNA were found to alter adrenal steroidogenesis and protein expression in the H295R in vitro model. Although the mechanisms of these alterations are not known, the current study might contribute in a better understanding of effects of PFOS and PFNA. The alterations in protein regulation suggested that exposure to these compounds affects several cellular processes, including protein synthesis, stress response and apoptosis. In addition, many of the altered proteins have been reported to be involved in carcinogenesis.