| The recent detection of perfluorinated fatty acids (PFFAs) in wildlife from even remote locations has spurred interest in the environmental occurrence and effects of these chemicals. Among them, perfluorooctane sulfonic acid (PFOS) is the end metabolite of a number of perfluorinated fatty acid analogues extensively used in industrial materials and commercial applications. Few studies have been conducted on this novel compound, and its mechanism of action still remains unclear. The amphiphillic nature of PFOS suggests its cell membrane related effects. In the current study, effects of PFOS on membrane fluidity and mitochondrial membrane potential were examined using flow cytometry and effects on membrane permeability were tested using cell bioassay procedures (H4IIE, MCF-7, PLHC-1). PFOS increased plasma membrane fluidity and decreased mitochondrial membrane potential in fish leukocytes in a dose-dependent fashion. The lowest effective concentrations for both membrane fluidity and mitochondrial membrane potential effects of PFOS were 5 to 15 mg/L. This suggests that membrane properties could be used as sensitive biomarkers for PFOS related adverse effects. Besides membrane related effects, studies were also designed to examine modulations of gene expression by PFOS exposure using states of art molecular toxicology techniques. In the current study, the restriction fragment differential display (RFDD-PCR), a mRNA fingerprinting technique, and Affymetrix rat genome U34A chips, the high throughput genomic technique with 8790 genes, were used to identify alterations in gene expression level due to PFOS exposure in vitro and in vivo. RNA samples were extracted from H4IIE rat hepatoma cells and rat livers exposed to PFOS, and prepared for subsequent analysis. Following the RFDD-PCR procedure, 55 bands on sequencing gel were identified as different across treatment groups. All these candidate genes were sub-cloned and sequenced. Gene chip analysis was conducted by hybridizing U34A chips with experimental samples. Approximately 5% of the genes on the chip were identified as differentially expressed in response to PFOS exposure, and clustered as genes coding for fatty acid metabolizing enzymes, drug and xenobiotic metabolizing enzymes, and proteins involved in signal transduction pathways and hormone regulation. Consistent results were obtained from replicate exposures, however, expression profiles of samples prepared in vitro and in vivo showed only limited similarity. The major pathway affected by PFOS is postulated to be peroxisomal fatty acid beta-oxidation, which could be explained by the structural similarity between PFOS and endogenous fatty acids. Comparisons were made between differential display and gene chip techniques based on their specificity, sensitivity, and scope of applications. The mechanistic interpretation derived from these two methods was in agreement, although the results were not directly comparative. |
