Gene Expression Profiles in Rat Liver Treated With Perfluorooctanoic A
http://www.100md.com
《毒物学科学杂志》
Toxico-Biochemistry Section, National Institute of Animal Health, Kannondai 3-1-5, Tsukuba, Ibaraki 305-0856, Japan
Department of Biology and Chemistry, City University of Hong Kong, Tat Chee Avenue, Kowloon, Hong Kong SAR, PR China
Zoology Dept., National Food Safety and Toxicology Center and Center for Integrative Toxicology, Michigan State University, East Lansing, Michigan 48824
Environmental Measurement Group, National Institute of Advance Industrial Science and Technology, Onogawa 16-1, Tsukuba, Ibaraki 305-8569, Japan
ABSTRACT
Perfluorooctanoic acid (PFOA; Pentadecafluorooctanoic acid) is widely used in various industrial applications. It is persistent in the environment and does not appear to undergo further degradation or transformation. PFOA is found in tissues including blood of wildlife and humans; however, the environmental fate and biological effects of PFOA remain unclear. Microarray techniques of gene expression have become a powerful approach for exploring the biological effects of chemicals. Here, the Affymetrix, Inc. rat genome 230 2.0 GeneChip was used to identify alterations in gene regulation in Sprague-Dawley rats treated with five different concentrations of PFOA. Male rats were exposed by daily gavage to 1, 3, 5, 10, or 15 mg PFOA/kg, body weight (bw)/day for 21 days and at the end of the exposure, liver was isolated and total liver RNA were used for the gene chip analysis. Over 500 genes, whose expression was significantly (p < 0.0025) altered by PFOA at two-fold changes compared to control, were examined. The effects were dose-dependent with exposure to 10 mg PFOA/kg, bw/day, causing alteration in expression of the greatest number of genes (over 800). Approximately 106 genes and 38 genes were consistently up- or down-regulated, respectively, in all treatment groups. The largest categories of induced genes were those involved in transport and metabolism of lipids, particularly fatty acids. Other induced genes were involved in cell communication, adhesion, growth, apoptosis, hormone regulatory pathways, proteolysis and peptidolysis and signal transduction. The genes expression of which was suppressed were related to transport of lipids, inflammation and immunity, and especially cell adhesion. Several other genes involved in apoptosis; regulation of hormones; metabolism; and G-protein coupled receptor protein signaling pathways were significantly suppressed.
Key Words: perfluorinated compounds; PFOA; GeneChip; liver gene expression; fatty acid and lipid metabolism.
INTRODUCTION
Perfluorinated compounds (PFCs) have been manufactured and used for various industrial applications for over 50 years (Giesy and Kannan, 2002; Sohlenius et al., 1994). For example, PFOS has been used as a surfactant and surface protector in carpet, leather, paper, and food containers; as well as performance in chemicals in products such as fire-fighting foams, floor polishes, and personal care products (OECD, 2002). The high-energy C-F covalent bonds in PFCs are strong enough to resist hydrolysis, photolysis, biodegradation, and metabolism and thus result in a high degree of environmental persistence and bioaccumulation of PFCs and their derivatives. A number of commercially used PFCs have been shown to occur in the environment, including perfluorooctane sulfonic acid (PFOS), perfluorooctanoic acid (PFOA), perfluorooctane-sulfonamide (PFOSA), perfluorohexanesulfonate (PFHS), perfluorobutanesulfonate (PFBS), and perfluorononanoic acid (PFNA) (Giesy and Kannan, 2002).
The vapor pressure and solubility of PFOS (K+) are 3.31 x 10–4 Pa and 0.5 g/l and those of PFOA (H+) are 70 Pa and 9.5 g/l, respectively (Hekster et al., 2003). Even though there are great differences in the physical and chemical properties of PFOA and PFOS, they were found to be the dominant PFCs in the environment and were found to be widespread in biota, such as wild birds (Giesy and Kannan, 2001), dolphins (Kannan et al., 2001), polar bears and ringed seals (Martin et al., 2002), shell and finfish (Hoff et al., 2003; Van de Vijver et al., 2003), and in human serum and seminal plasma (Guruge et al., 2005; Olsen et al., 1999). They are usually present as a dissociated anion or ion pair in aqueous media; Kannan et al. (2005) has shown that PFOA is less restrained in the body and less bioaccumulated among trophic levels compared with PFOS.
Previous studies have demonstrated that PFOS could affect the membrane fluidity and mitochondrial membrane potential in vitro and in vivo (Hu et al., 2002, 2003). Also, PFOS inhibited gap junction intercellular communication (GJIC) in a dose-dependent manner (Hu et al., 2002) and peroxisomal fatty acid -oxidation was found to be the major pathway affected by PFOS.
Several studies have shown that PFOA has the potential to induce peroxisomal -oxidation in the liver of male rats but not in female rats, whereas PFOA induced the activity in cultured hepatocytes from both male and female rats (Berthiaume and Wallace, 2002; Kudo et al., 2002). Increases in -oxidation of fatty acid, several cytochrome P-450 (CYP450) mediated reactions and inhibition of the secretion of low-density lipoproteins and cholesterol from liver have also been demonstrated (Kennedy et al., 2004). However, the mechanisms of action and metabolic pathways affected by PFOA are still unclear.
Because, there are a number of PFCs with similar structures to which organisms can be exposed simultaneously, risk assessment of these complex mixtures need to be considered. Toward this end, it is important to know if the various PFCs have the potential to elicit similar responses through similar mechanisms and modes of action and whether their risks need to be assessed based on similar joint action or independently. In the present study, the effects of PFOA on liver gene expression in rats was determined by use of microarray techniques to determine effects on gene expression patterns relative to that of unexposed rats. This information was then used to determine which metabolic and signal transduction pathways were affected. This information can then be used to determine the primary pathways that are affected and to infer mechanisms of action. The results of this study can then be compared to those with other PFCs, such as PFOS (Hu et al., 2005a,b).
MATERIALS AND METHODS
Animals and administration.
Seven-week-old male Sprague-Dawley rats were obtained from CLEA Inc. (Tokyo, Japan) and housed at 20–24°C in humidity-controlled (40–60%) facilities at the National Institute of Animal Health, Japan. After acclimatization to a standard diet (MF, Oriental Yeast Co., Japan) for 1 week, rats (225–250 g) were treated daily between 0900 and 1100 h with a single oral gavage of 1 ml/kg body weight (bw) of either vehicle control (0.5% Tween-20) or different doses of pentadecafluorooctanoic acid (perfluorooctanoate; PFOA, purity 95%, CAS Number 335-67-1, Wako Chemicals, Japan). A similar study on PFOS used a dose of 5 mg/kg, bw (Hu et al., 2005a). In this study, 1, 3, 5, 10, 15 mg/kg were used to allow an investigation of the effect of PFOA over a wider range of doses and, at the same time, allow a comparison with the study on PFOS. Six male rats were randomly assigned to one group. Rats in each group were dosed daily with 1, 3, 5, 10, or 15 mg PFOA/kg, bw, daily for 21 days. This experiment was conducted according to the guidelines for animal experiments of the National Institute of Animal Health, Tsukuba, Japan. At the end of exposure, rats were anesthetized with sodium pentabarbital and exsanguinated via the abdominal aorta. The liver was removed and portions were immediately frozen in liquid nitrogen and stored at –80°C until RNA isolation.
RNA extraction.
Total RNA was isolated from livers with Trizol Reagent (Invitrogen, CA) using manufacturer recommended procedures. The reason for choosing liver as the target organ was that perfluorinated fatty acid (PFFAs) mainly accumulated in blood and liver; the major target organ for PFFAs is presumed to be liver (Giesy and Kannan, 2001). In addition, a similar study on the effects of PFOS on gene expression in the rat had been conducted using total liver RNA (Hu et al., 2005a). The ratio of the optical densities from RNA samples measured at 260 and 280 nm was used to evaluate nucleic acid purity and total RNA concentrations were determined by the absorbance at 260 nm. The quality of total RNA was estimated based on the integrity of 28S and 18S rRNA. rRNA was separated using 1% agarose gel electrophoresis, good RNA quality was indicated by the 28s rRNA banding twice the intensity of the 18s rRNA without significant smearing of the rRNA bands. Samples of total RNA from the three rats exposed to the same dose of PFOA were pooled for subsequent use in the GeneChip analysis. Prior to GeneChip analysis, the pooled total RNA samples were purified using the RNeasy Total RNA Mini Kit (Qiagen, Valencia, CA) using manufacturer recommended procedures.
Microarray analysis.
Single and double stranded cDNA was synthesized from total RNA samples using SuperScript II (Invitrogen, CA). High-quality total RNA (16 μg) was used as the starting material and 2 μl of 50 μmol T7- Oligo(dT)24 Primer (5'-GGCCAGTGAATTGTAATACGACTCACTATAGGGAGGCGG-(dT)24 – 3'; Operon, HPLC purified DNA) was used to prime the reaction. After double stranded cDNA clean up and quality check, an in vitro transcription reaction was conducted with the Enzo RNA Transcript Labeling Kit (Affymetrix, Santa Clara, CA) to produce biotin-labeled cRNA from the cDNA. The cRNA was then purified with the RNeasy Mini Kit (Qiagen, Valencia, CA) and fragmented for hybridization analysis. Fifteen microgram aliquants of the fragmented cRNA were hybridized with the Rat Genome 230 2.0 array (Affymetrix, Santa Clara, CA) in hybridization cocktail (0.5 mg/ml cRNA, 50 pM control oligonucleotide B2, 1.5 pM bioB, 5 pM bioC, 25 pM bioD, 100 pM cre, 0.1 mg/ml herring sperm DNA, 0.5 mg/ml acetylated BSA, 100m M MES, 1 M Na+ 20 mM EDTA, 0.01% Tween 20). Hybridization was allowed to proceed overnight (16 h) at 45°C, followed by washing and staining with streptavidin-phycoerythrin (SAPE, Molecular Probes, Eugene, OR). Hybridization assay procedures including preparation of solutions were carried out as described in the Affymetrix GeneChip Expression Analysis Technical Manual. The distribution of fluorescent material on the array was obtained using G2500A GeneArray Scanner (Affymetrix, Santa Clara, CA). Microarray Suite (MAS) version 5.0 and GeneChip Operating Software (GCOS) supplied by Affymetrix was used to perform gene expression analysis.
Scatter plot, gene tree, and condition tree.
Scatter plots were used to visually examine the expression level of genes between the control and PFOA exposed rats. Hierarchical dendrograms were drawn with the Gene tree algorithm of GeneSpring 7 (Silicongenetics, CA). It was similar to phylogenic dendrogram, which was created by clustering the genes according to their expression to the response towards the toxicants. Genes sharing similar expression profiles tended to be clustered together, and the location of a branch containing the genes can be considered a measure of how similar the gene expression was. Condition trees are similar to gene trees, in that they examine the relationships of the expression levels among treatments. Genes were selected for the construction of gene tree if the expression of the gene was two-fold greater or less in the treatments, relative to that in the control (vehicle-exposed) individuals. The horizontal axis shows the clustering of the genes according to their expression profile in the treatment; while the vertical axis showed the clustering according to their expression across treatments.
Pathway analysis.
GeneSpring was used to map genes and their expression values on graphical representations of metabolic pathways with reference to the Kyoto Encyclopedia of Genes and Genomes (www.genome.ad.jp/kegg) and GenMAPP organization (www.genmapp.org). A list of pathways affected, the list of genes affected and the p-value of genes overlapping with pathway values were created.
Statistical analysis.
GeneChip analysis generates very large data sets, it is therefore necessary to evaluate the validity of the data. For the present GeneChip probe array study, the data for each genes represents data from 11–20 probe pairs each approximately 25 bp in length. For each probe pair, one probe is a perfect match while the other has a single mismatch at nucleotide 13. The mismatch probe works as an internal control to evaluate the cross hybridizations between closely related target sequences. The overall target-specific intensity was obtained by the difference between the intensity of perfect match and the mismatch probes. The One-Sided Wilcoxon's Signed Rank test was employed to generate the Detection p-value. If the overall intensity of perfect match were much larger than that of mismatch, the detection p-value would be small. The probe set would be regarded as present if the p-value was less than 0.04 and if the p-value was larger than 0.06, the probe set would be regarded as absent.
The GeneChip probe array system only allows comparison of one treatment hybridizing with the probe set. In a Comparison Analysis, two samples were hybridized to two GeneChip probe arrays of the same type, they were compared against each other in order to detect and quantify changes in gene expression. One GeneChip was for baseline (control) and the other was for the experiment (treatment). Prior to performing data comparison, the data were scaled with the rat230_2norm.msk to 2000 in order to correct for variations in overall intensity and for heterogeneity among the GeneChip probe arrays. Two sets of algorithms were generated and they were used to generate change significance and change quantity metrics for every probe set using Microarray Suite (MAS) version 5.0 (Affymetrix, CA). The change algorithm generated a Change p-value and an associated fold-change value. The second algorithm gave a quantitative estimate of the change in gene expression in the form of Signal Log Ratio. In the present study, the level of gene expression can be regarded as increased if its Change p-value was less than 0.0025 and the gene expression would be considered to be decreased if its Change p-value was greater than 0.9975. This method has been used by other investigators (Hu et al., 2005a). Fold change could be calculated with the following formula: Fold change = 2 (signal log ratio).
RESULTS
The Rat Genome 230 2.0 array is spotted with 31,042 gene probes made of 25-mer single strand oligonucleotides. In the present study, six chips were used. These included one solvent control and five concentrations of PFOA (1, 3, 5, 10, and 15 mg PFOA/kg, bw/day). Comparison analyses of the expression profiles were performed between the control rats and PFOA-treated rats from the GeneChip data. Over 500 genes whose expression was significantly (p < 0.0025) altered by at least two-fold after exposure to PFOA at 1 mg/kg, and at 10 mg/kg PFOA the expression of the greatest number of genes (over 800) was altered (Table 1). The use of two-fold cut-off for significance is conventionally used in other similar studies (Barrans et al., 2001; Kume et al., 2005).
Scatter plots were constructed by comparing a composite sample from PFOA-treated rats with that of a composite sample from unexposed (control) rats (Fig. 1). The vertical position of each gene represents its expression level in the treatment of PFOA at different concentrations, and the horizontal position represents its control condition. Those genes above the diagonal (1:1 regression line) were considered to be induced by PFOA exposure while genes below the diagonal were considered to be suppressed. The number of genes deviating from the 1:1 regression line increased as a function of PFOA dose.
Gene tree analysis created several clusters among the PFOA doses. However, the expression profile of the genes in each cluster was different among treatments. This might be due to different toxico-kinetic mechanisms of different concentrations of PFOA on gene expression. The gene tree analysis classified the patterns of relative responses for all doses relative to the control (Fig. 2). The greatest distance of the node was between the control and the 10 mg PFOA/kg, bw/day exposure. Among the treatment groups, 3 mg and 15 mg PFOA/kg, bw/day were in the same node and this sub-branch was under the node of 5 mg PFOA/kg, bw/day.
The genes altered by the treatment of PFOA of different concentrations could be divided into several large functional categories according to the annotation information and protein information from Protein Knowledgebase provided by Swiss Institute of Bioinformatics (Swiss-Prot) and their biological processes. The genes for which the annotation was unclear such as expressed sequence tags (ESTs) were excluded from the table and when the genes were not fitted into the selected categories, they were regarded as others (Tables 2 and 3). Among the samples from PFOA-treated rats, the largest categories of genes induced were involved in transport and metabolisms of fatty acids and lipids. Other significantly induced genes were involved in cell communication, adhesion, growth, apoptosis, regulation of hormone, proteolysis and peptidolysis and signal transduction. The largest groups of genes suppressed were related to transport, inflammation and immune response and cell adhesion. Several genes involved in apoptosis, regulation of hormone, metabolisms, and G-protein coupled receptor protein signaling pathway were suppressed significantly.
A total of 113 genes and 56 genes were found to be up-regulated and down-regulated, respectively, in at least three of the PFOA doses. Among the genes that showed consistent alternations of gene expression in all treatments, 106 were up-regulated while 38 genes were down-regulated. The genes for which annotation was unclear (ESTs, etc.) were excluded from the table (Table 4). The largest grouping of genes up-regulated by PFOA exposure were for gene products involved in transport and metabolism of lipids, particularly fatty acids. Besides, several genes involved in apoptosis; cell communication, adhesion, growth and cycle; signal transduction; regulation of hormone; and proteolysis and peptidolysis were also induced significantly. Other genes involved in apoptosis, cell adhesion, metabolisms, transport and signal transduction were suppressed significantly.
Pathway analysis was conducted by overlapping the genes with the KEGG pathways in the Kyoto Encyclopedia of Genes and Genomes (www.genome.ad.jp/keg) and GenMAPP organization (www.genmapp.org). The number of common genes with each pathway and the random overlap p-value of the gene list against pathway were obtained. KEGG pathways of fatty acid synthesis (six genes), fatty acid degradation (nine genes), and mitochondrial fatty acid -oxidation (seven genes) were altered significantly (p < 0.0025) by exposure of PFOA. Cholesterol synthesis (four genes) and Krebs-TCA Cycle (three genes) appeared to be altered but the effect was not statistically significant.
The gene expression profiles for rats exposed to 5mg PFOA/kg, bw/day were compared with those reported for rats exposed to PFOS (Hu et al., 2005a) (Tables 5 and 6). Of the 23 genes that were up-regulated by PFOS, 12 genes were also found up-regulated by PFOA. However, seven of the genes were unchanged and four could not be found in the present study. Of the 19 genes for which expression was suppressed by PFOS, only one gene was found to be suppressed and all others but one were not affected by exposure to PFOA.
DISCUSSION
The results of the present study provide insight into how gene expression in livers of rats responded to treatment with several doses of PFOA. Gene expression data was useful in identifying the genes involved in the different biochemical pathways which were affected, however, it should not be regarded as a dose-response relationship because the degree of the alternation in gene expression was not solely due to the administered concentration of PFOA. To interpret the potential for effects in vivo, other parameters, such as the accumulation of PFOA in the target organ, should be considered. Alteration of the expression of a particular gene does not necessarily mean that a particular protein or biochemical pathway would be affected in vivo. However, alteration of a group of genes involved in a particular biochemical pathway would provide strong evidence that PFOA may affect that particular biochemical pathway. Confirmation or isolation of a group of particular proteins or assessment of metabolite flow through the entire target pathway would be the next step in further study and it was not included in the present study.
The number of the genes induced or suppressed was directly proportional to the dose over the range of 1 to 10 mg PFOA/kg, bw/day. However, it was found that the levels of expression of certain genes were different when compared among the concentrations of PFOA. For instance, Resp18, primarily expressed in neural and endocrine tissues, was induced at 1 mg/kg PFOA, was not induced at 3 mg PFOA/kg, bw/day, but was induced again in 5, 10, and 15 mg PFOA/kg, bw/day. In order to understand why a gene was expressed differently at different concentrations of PFOA, the linkage and the pattern of related genes was investigated. However, the linkages of genes were still unclear and it was beyond the scope of the present study to investigate specific pathways in detail. The results of the GeneChip study can, however, be used to design more targeted in vivo studies.
Since the results showed that different doses of PFOA resulted in different gene expression profiles, genes which were up- or down-regulated in all concentrations were selected and analyzed in order to understand the effects of different PFOA doses. Overall, our results showed that a large number of genes associated with lipid or fatty acid metabolism were altered by PFOA and some of the genes were linked with pathways of fatty acid degradation and mitochondrial fatty acid -oxidation in all concentrations of PFOA treated rats. Indeed, similar effects causing interference of fatty acid metabolism had also been found in other studies (Haughom and Spydevold, 1992; Kudo et al., 1999, 2000).
PFOA affected mitochondrial fatty acid -oxidation, fatty acid synthesis and degradation. The genes coding for the enzymes involved in these mechanisms were generally not all induced significantly at the concentrations of PFOA used, except for the genes responsible for the unsaturated fatty acid metabolism (Tables 7 and 8) and for the transfer of fatty acids for oxidation. Specifically, Cpt1b, Cpt2, and Slc25a20 were induced significantly for all PFOA concentrations. Cpt1b catalyzes the transfer of long chain fatty acids to carnitine for translocation across the mitochondrial inner membrane; Cpt2 coding for the inner mitochondrial membrane protein that converted acylcarnitine to acyl-CoA for further fatty acid metabolism and slc25a20 was a carnitine carrier protein which was a component of the mitochondrial inner membrane and transferred fatty acylcarnitines into the mitochondria. The entrance of fatty acids into the mitochondria requires the activation by acyl-CoA synthetase, however, the genes for acyl-CoA synthetase were not induced significantly, and this suggests that PFOA does not undergo activation by acyl-CoA synthetase. The observation of up-regulated Cpt1b, Cpt2, and Slc25a20 implies that there was an increase in the transfer of activated fatty acids or PFOA across the membrane of the mitochondria, as PFOA was not metabolized (Kudo et al., 2001; Vanden Heuvel et al., 1991), the genes coding for the enzymes for the fatty acid -oxidation were not significantly up-regulated among the concentrations of PFOA. PFOA has a similar structure to endogenous fatty acids except that fluorine atoms replace all the hydrogen atoms linked with carbon atoms in the fatty acids. Therefore, it is possible that PFOA could be misguided by the fatty acid metabolism machinery as a substrate because of its structural similarity to endogenous fatty acids, and this effect is similar to those observed for PFOS (Hu et al., 2005a). Although the genes coding for the enzymes for fatty acid degradation, -oxidation and synthesis were not significantly up-regulated among the concentrations of PFOA, the genes coding for the enzymes involved in the metabolism of unsaturated fatty acids were up-regulated. Therefore, there might have been some interactions between PFOA and the pathways that facilitated the degradation of unsaturated fatty acids.
In animal cells, mitochondria and peroxisomes can oxidize fatty acids via -oxidation. There are two major differences between mitochondrial and peroxisomal -oxidation, in terms of its specificity and mechanism (Reddy and Mannaerts, 1994; Wanders et al., 2001). In mammals, the mitochondria oxidize short, medium, and most long chain fatty acids, while peroxisomes oxidize some long chain and very long chain fatty acids. Kudo et al. (2000) demonstrated that PFOA induced peroxisomal -oxidation in in vivo and in vitro studies. In the present study, it was found that the genes coding for peroxisomal oxidation were induced significantly especially at a dose of 10 mg PFOA/kg, bw/day (Fig. 3). Since PFOA is non-biodegradable, the accumulation of PFOA could have resulted in a significant induction of the genes coding for enzymes responsible the degradation of fatty acids. Because the expression of catalase was not induced by PFOA it is possible that potentially toxic hydrogen peroxide was produced in peroxisomes from acyl-CoA that could have caused oxidative stress or oxidation damage to the proteins and DNA. A similar explanation of the effects of PFOS has been previously hypothesized (Hu et al., 2005a). Furthermore, peroxisomes could also oxidize the side chains of eicosanoids, molecules important in short-range signaling, derived from arachidonic acid. Prostaglandins and leukotrienes are representatives of the family of eicosanoid; hence, they might be oxidized and could not perform their normal functions. The gene coding for one of the enzymes responsible for the metabolism of prostaglandins, hpgd, was significantly up-regulated by 3-fold by the 5 mg PFOA/kg, bw/day dose and 4-fold by the 15 mg PFOA/kg, bw/day dose. This observation suggests increased metabolism of prostaglandins.
Both mitochondrial and peroxisomal isomerases were up-regulated. The genes Dci and Decr1, which are involved the mitochondrial -oxidation; and the genes Ech1 and Decr2, which are involved in the peroxisomal -oxidation, were also up-regulated. These isomerases are enzymes involved in the oxidation of the long-chain fatty acids in peroxisomes (Fig. 3). These enzymes, which help to oxidize unsaturated fatty acids through the isomerization of 3-trans-, 5-cis-dienoyl-CoA to 2-trans-, 4-trans-dienoyl-CoA were up-regulated by the peroxisome proliferator [clofibrate or di (2-ethylhexyl) phthalate] (FitzPatrick et al., 1995). Up-regulation of these genes implies that there was increased unsaturated fatty acids metabolism. Therefore, there might be some interaction between PFOA and unsaturated fatty acids, or PFOA was mistaken as the substrate for the unsaturated fatty acid -oxidation in both the mitochondrial and peroxisomal pathways. However, no conclusion could be drawn here because further confirmation of an increased metabolism of unsaturated fatty acids is required.
In the present study, the expression of Cyp4b1 was significantly up-regulated (2.8- to 4.3-fold) among the PFOA treatments (Table 4). Several studies have shown that the induction of the Cyp4b1 gene could be related to bladder cancer in rabbits, mice, and humans (Imaoka et al., 1995, 2000, 2001). Cyp4b1 has an important role in mutagenic activation of procarcinogens in the bladder. Cyp4b1 expression can be induced by benzidine and 2-naphthylamine, which have been identified as carcinogens that can initiate bladder carcinomas (Imaoka et al., 1997). Caspase-11, a critical activator of several other caspase-genes involved in the cytokine regulation of apoptosis, was also induced in PFOA treated animals compared to the controls. Likewise, it is noteworthy that a number of genes involved in immune function, such as interleukin, macroglobulin, and leukocyte activation were down-regulated by at least three or four of the PFOA doses. Additionally, suppression of G-protein associated signaling pathway genes such as Ghsr, Avpr1a, and Hcrtr2 might affect regulation of food intake and energy homeostasis by affecting mechanisms of neuronal transmission.
PFOS, one of the predominant PFCs detected in the environment and accumulated into biota, is structurally analogous to of PFOA except for the terminal sulfonic acid group as compared to the terminal carboxylate of PFOA and a different number of carbon atoms in PFOA, 7 carbon chain attached to the terminal carboxylic acid compared to the 8 carbon chain of PFOS. To determine the similarity of effects on gene expression of these two PFCs, the results of PFOA on gene expression in the current study to that of PFOS were compared (Tables 5 and 6). The genes that were up- or down-regulated by PFOA were quite different from those for which gene expression was altered by PFOS that might be due to the large differences in their physical and chemical properties however it needs further investigations. Cytochrome groups were of interest as they were related to the metabolism of xenobiotics. Up-regulation of Cyp2b15, which is responsible for catalytic reactions involved in drug metabolism, was similar for PFOA and PFOS (Table 5). However, the response of Cyp17a1, which is responsible for steroid hydroxylation and comprises a main component of the steroidogenic pathway, was up-regulated by PFOS about 4-fold more than by PFOA. Two cytochromes Cyp3a18 and Cyp3a11, which are responsible for the conversion and the hydroxylation of testosterone, were up-regulated by PFOS, but not PFOA. Cyp2j4, which is a putative cytochrome p450 monooxygenase enzyme, was up-regulated PFOA but not PFOS. To conclude, the cytochrome genes induced by PFOA were mainly related to xenobiotic metabolisms while those of PFOS were related to both xenobiotic metabolisms as well as steroid metabolisms.
Both PFOS and PFOA up-regulated genes responsible for enzymes involved in fatty acid and lipid metabolism which might be due to their structural similarity to endogenous fatty acids. These compounds may act as structural analogues of endogenous fatty acids and the differences in the gene expression profiles might be due to the different functional groups of PFOA and PFOS, suggesting that further investigation on the toxicity of PFOA is necessary. In this study we observed that PFOA affected both peroxisomal and mitochondrial fatty acids oxidation while PFOS has been reported to mostly affect peroxisomal oxidation (Hu et al., 2005a). The fold change of the enzymes up-regulated by both the 5 mg PFOS/kg, bw/day dose and all doses of PFOA were similar for most of the genes along the peroxisomal fatty acid oxidation pathway except for Acaa1. The fold change of Acaa1 induced by PFOS was approximately twice that of PFOA (Tables 5 and 8).
While there were similarities in modulation of gene expression by PFOS and PFOA, there were also differences. Some of these differences could have been due to different aspects of the design of the two experiments being compared. For instance, the study of PFOS used female rats while that on PFOA used male rats. The reason for choosing male rats was that the elimination rate of PFOA in female was much faster than that of male (Hanhijarvi et al., 1982; Kudo et al., 2002; Vanden Heuvel et al., 1991), the use of male rats will allow this study on PFOA to have more similar toxicokinetics as compared to the previous study on PFOS. Although there was a difference in sex, both PFOA and PFOS induced the genes in the peroxisomal -oxidation pathway.
PFOA has been shown to interfere with fatty acid metabolism and cholesterol synthesis in the liver (Haughom and Spydevold, 1992). Studies of selected enzymes demonstrated a significant decrease in Hmgcr, acyl coenzyme A (CoA) cholesterol acyltranseferase (ACAT) activities, which were related to a lesser concentration of serum cholesterol after 24 h when rats fed PFOA. In the present study, expression of the gene that codes for Hmgcr activity was significantly down-regulated at doses of 5, 10, and 15 mg PFOA/kg, bw/day. Hmgcr is an enzyme involved in mevalonate synthesis which is a rate limiting enzyme in the cholesterol synthesis pathway. This is consistent with previous studies that have found that alteration in the activity of this gene may contribute to the hypolipidemic effect caused by PFOA. Any effects on the expression of the gene that codes for Hmgcr could affect production of lipoprotein particles because of the reduced synthesis of cholesterol together with the increased oxidation of fatty acids in the liver (Haughom and Spydevold, 1992). Cholesterol is an important component of the cell membrane; it stablizes the membrane's fluidity over a bigger temperature interval. Since down-regulation of this gene with a concomitant decrease in Hmgcr activity would result in less cholesterol synthesis, it might alter the cell membrane fludity, an effect that has also been observed to be caused by PFOS (Hu et al., 2003). It may even be hypothesized that increases in membrane fulidity caused by exposure to PFOS result in decreased synthesis of cholesterol by down-regulation of Hmgcr. Another gene, Abcg1, which is responsible for cholesterol transport, was significantly up-regulated (Table 4). Since cholesterol production has been found to be decreased by exposure to both PFOS and PFOA, efflux of excess cholesterol from macrophages would have helped maintain cholesterol homeostasis. Obviously, gene expression studies can not be used to directly investigate effects on cell membrane fluidity. However, recent studies have shown that PFOS can be incorporated into cell membranes resulting in altered cell membrane fluidity and membrane potential (Hu et al., 2003). PFOS also affected gap junction intercellular communication (Hu et al., 2002). Similar effects of PFOA to those of PFOS on membrane fluidity can be postulated, but further research would be necessary to demonstrate this phenomenon. PFOA both up- and down-regulated gene expression that are related to signal transduction and G-protein coupled receptors. This is consistent with the observation that PFOA can inhibit gap junction intercellular communication (Upham et al., 1998).
It has been suggested that both slc22a7 (Oat 2—older name) and slc22a8 (Oat3—older name) are responsible for the urinary elimination of PFOA (Kudo et al., 2002). In previous studies, neither slc22a7 nor slc22a8 were down-regulated by any of the doses of PFOS, however both, slc21a1 (Oatp1) and slc21a5 (Oatp2) were down-regulated by some PFOA doses. The genes slc22a7, slc22a8, slc21a4, slc21a1, and slc21a5 are involved in transport of organic anions and they might also be involved in the urinary elimination of PFOA. Since all of these genes were suppressed, it would be expected that exposure to PFOA would result in a negative feedback that would inhibit elimination of PFOA in the rat.
Here we have reported, for the first time, information on the gene expression profiles of rats receiving different doses of PFOA. These results can be used to understand the biochemical processes affected by PFOA. PFOA enhanced both peroxisomal and mitochondrial fatty acid -oxidation and the peroxisomal -oxidation might create oxidative stress on DNA and protein. Besides, the reduction of cholesterol synthesis observed in in vitro studies would be consistent with the down-regulation of the gene Hmgcr observed in this study. Furthermore, this study suggests that the suppression of organic anion transport genes may explain the delayed urinary clearance of PFOA. There were a number of genes related to tumor progression and inflammation affected by exposure to PFOA, which suggests that exposure to PFOA might enhance the risk of cancer. The outcome of gene data comparison between PFOA and PFOS suggests that the responses in gene expression are similar, but not identical for the two substances. Furthermore the similarities in the effects on lipid metabolism suggest similar modes of action, but the exact mechanisms of the effects may be different and/or may be affected to different degrees by PFOA and PFOS. The responses of genes in the critical pathways are sufficient to consider the development of relative potency factors for PFOS and PFOA to be used in risk assessments. However, differences in the toxicokinetics of PFOA and PFOS would need to be considered if the effects of the two compounds on a single pathway were considered.
ACKNOWLEDGMENTS
This research work was partially supported from the Japanese Ministry of Environment under the Global Environment Conservation Research Fund (2004–2008) to K.S.G. and by a CERG grant (CityU1401/05M) from the Hong Kong Research Grants Council awarded to P.K.S.L. The authors also thank Dr. H. Hoshiba and animal care members of NIAH for their invaluable assistance during the study. Conflict of interest: none declared.
REFERENCES
Barrans, J. D., Stamatios, D., and Liew, C. (2001). Construction of a human cardiovascular cDNA microarray: Portrait of the failing heart. Biochem. Biophys. Res. Commun. 280, 964–969.
Berthiaume, J., and Wallace, K. B. (2002). Perfluorooctanoate, perflourooctanesulfonate, and N-ethyl perfluorooctanesulfonamido ethanol; peroxisome proliferation and mitochondrial biogenesis. Toxicol Lett. 129, 23–32.
FitzPatrick, D. R., Germain-Lee, E., and Valle, D. (1995). Isolation and characterization of rat and human cDNAs encoding a novel putative peroxisomal enoyl-CoA hydratase. Genomics 27, 457–466.
Giesy, J. P., and Kannan, K. (2001). Global distribution of perfluorooctane sulfonate in wildlife. Environ. Sci. Technol. 35, 1339–1342.
Giesy, J. P., and Kannan, K. (2002). Perfluorochemical surfactants in the environment. Environ. Sci. Technol. 36, 146A–152A.
Guruge, K. S., Taniyasu, S., Yamashita, N., Wijeratna, S., Mohotti, K. M., Seneviratne, H. R., Kannan, K., Yamanaka, N., and Miyazaki, S. (2005). Perfluorinated organic compounds in human blood serum and seminal plasma: A study of urban and rural tea worker populations in Sri Lanka J. Environ. Monit. 7, 371–377.
Hanhijarvi, H., Ophaug, R., and Singer, L. (1982). The sex related different in perfluorooctanceate excretion in the rat. Proc. Soc. Exp. Biol. Med. 171, 50–55.
Haughom, B., and Spydevold, O. (1992). The mechanism underlying the hydropolipemic effect of perfluorooctanoic acid (PFOA), perfluorooctane sulphonic acid (PFOSA) and clofibric acid. Biochim. Biophys. Acta 1128, 65–72.
Hekster, F. M., Laane, R. W. P. M., and de Voogt, P. (2003). Environmental and toxicity effects of perfluoroalkylated substances. Rev. Environ. Contam. Toxicol. 179, 99–121.
Hoff, P. T., Van Dongen, W., Esmans, E. L., Blust, R., and DeCoen W. M. (2003). Evaluation of the toxicological effects of perfluorooctane sulfonic acid in the common carp (Cyprinus carpio). Aquat. Toxicol. 62, 349–359.
Hu, W. Y., Jones, P. D., Upham, B. L., Trosko, J. E., Lau, C., and Giesy, J. P. (2002). Inhibition of gap junctional intercellular communication by perfluorinated compounds in rat liver and dolphin kidney epithelial cell lines in vitro and Sprague-Dawley rats in vivo. Toxicol. Sci. 68, 429–436.
Hu, W. Y., Jones, P. D., DeCoen, W., King, L., Frakwe, P., Newsted, J. L., and Giesy, J. P. (2003). Alternations in cell membrane properties caused by perfluorinated compounds. Comp. Biochem. Physiol. 135, 77–88.
Hu, W. Y, Jones, P. D., Celius, T., and Giesy, J. P. (2005a). Identification of genes responsive to PFOS using gene expression profiling. Environ. Toxicol. Pharmcol. 19, 57–70.
Hu, W. Y., Jones, P. D., DeCoen, W., and Giesy, J. P. (2005b). Comparison of gene expression methods to identify genes responsive to perfluorinated sulfonic Acid. Environ. Toxicol. Pharmacol. 19, 153–160.
Imaoka, S., Hiroi, T., Tamura, Y., Yamazaki, H., Shimada, T., Komori, N., Degawa, M., and Funae, Y. (1995). Mutagenic activation of 3-methoxy-4-aminoazobenzene by mouse renal cytochrome-P450 CYP4B1: Cloning and characterization of mouse CYP4B1. Arch. Biochem. Biophys. 321, 255–262.
Imaoka, S., Yoneda, Y., Matsuda, T., Degawa, M., Fukushima, S., and Funae, Y. (1997). Mutagenic activation of urinary bladder carcinogens by CYP4B1 and the presence of CYP4B1 in bladder mucosa. Biochem. Pharmacol. 54, 677–683.
Imaoka, S., Yoneda, Y., Sugimoto, T., Hiroi, T., Yamamoto, K., Nakatani, T., and Funae, Y. (2000). CYP4B1 is a possible risk factor for bladder cancer in humans. Biochem. Biophys. Res. Commun. 277, 776–780.
Imaoka, S., Yoneda, Y., Sugimoto, T., Ikemoto, S., Hiroi, T., Yamamoto, K., Nakatani, T., and Funae, Y. (2001). Androgen regulation of CYP4B1 responsible for mutagenic activation of bladder carcinogens in the rat bladder: Detection of CYP4B1 mRNA by competitive reverse transcription-polymerase chain reaction. Cancer Lett. 166, 119–123.
Kannan, K., Koistinen, J., Beckmen, K., Evans, T., Gorzelany, J. F., Hansen, K. J., Jones, P. D., Helle, E., Nyman, M., and Giesy, J. P. (2001). Accumulation of perfluorooctane sulfonate in marine mammals. Environ. Sci. Technol. 35, 1593–1598.
Kannan, K., Tao, L., Sinclair, E., Pastva, S. D., Jude, D. J., and Giesy, J. P. (2005). Perfluorinated compounds in aquatic organisms at various trophic levels in a Great Lakes food chain. Arch. Environ. Contam. Toxicol. 48, 559–566.
Kennedy, G. L., Butenhoff, J. L., Olsen, G. W., O'Connor, J. C., Seacat, A. M., Perkins, R. G., Biegel, L. B., Murphy, S. R., and Farrar, D. G. (2004). The toxicology of perfluorooctanoate. Crit. Rev. Toxicol. 34, 351–384.
Kudo, N., Mizuguchi, H., and Kawashima, Y. (1999). Alternations by perfluorooctanoic acid of glycerolipid metabolism in rat liver. Chemico-Biol. Interact. 118, 69–83.
Kudo, N., Bandai, N., Suzuki, E., Katakura, M., and Kawashima, Y. (2000). Induction by perfluorinated fatty acids with different carbon chain length of peroxisomal beta-oxidation in the liver of rats. Chemico-Biol. Interact. 124, 119–132.
Kudo, N., Suzuki, E., Katakura, M., Ohmori, K., Noshiro, R., and Kawashima, Y. (2001). Comparison of the elimination between perfluorinated fatty acids with different carbon chain length in rats. Chemico-Biol. Interact. 134, 203–216.
Kudo, N., Katakura, M., Sato, Y., and Kawashima, Y. (2002). Sex hormone-regulated renal transport of perfluorooctanoic acid. Chemico-Biol. Interact. 139, 301–316.
Kume, E., Argua, C., Ishizuka, Y., Takahashi, K., Miwa, S., Itoh, M., Fujimura, H., Toriumi, W., Kitamura, K., and Doi, K. (2005). Gene expression profiling in streptozotocin treated mouse liver using DNA microarray Exp. Toxicol. Pathol. 56, 235–244.
Martin, J. W., Muir, D. C. G., Moody, C. A., Ellis, D. A., Kwan, W. C., Solomon, K. R., and Mabury, S. A. (2002). Collection of airborne fluorinated organics and analysis by gas chromatography/chemical ionization mass spectrometry. Anal. Chem. 74, 584–590.
OECD (Organization for Economic Co-operation and Department). (2002). Co-operation on existing chemicals: Hazard assessment of perfluorooctane sulfonate (PFOS) and its salts. ENV/JM/RD (2002) 17/FINAL.
Olsen, G. W., Burris, J. M., Mandel, J. H., and Zobel, L. R. (1999). Serum perfluorooctane sulfonate and hepatic and lipid clinical chemistry tests in fluorochemical production employees. J. Occup. Environ. Med. 41, 799–806.
Reddy, J. K., and Mannaerts, G. P. (1994). Peroxisomal lipid metabolism. Annu. Rev. Nutr. 14, 343–370.
Sohlenius, A. K., Andersson, K., Bergstand, A., Spydevold, O., and De Pierre, J. W. (1994). Effects of perfluorooctanoic acid—a potent peroxisome proliferators in rat—on Morris hepatoma 7800C1 cells, a rat cell line. Biochim. Biophys. Acta 1213, 63–74.
Upham, B. L., Deocampo, N. D., Wurl, B., and Trosco, J. E. (1998). Inhibition of gap junctional intercellular communication by perfluorinated fatty acids is dependent on the chain length of the fluorinated tail. Int. J. Cancer 78, 491–495.
Vanden Heuvel, J., Kuslikis, B., and Van Rafelghem, M. (1991). Tissue distribution, metabolism, and elimination of perfluorooctanoic acid in male and female rats. Biochem. Toxicol. 6, 83–92.
Van de Vijver, K. I., Hoff, P. T., Van Dongen, W., Esmans, E. L., Blust, R., and DeCoen, W. M. (2003). Exposure patterns of perfluorooctane sulfonate in aquatic invertebrates from the Western Scheldt estuary and the southern North Sea. Environ. Toxicol. Chem. 22, 2037–2041.
Wanders, R. J. A., Vreken, P., Ferdinandusse, S., Jansen, G. A., Waterham, H. R., Van Roermund, C. W. T., and Van Grunsven, E. G. (2001). Peroxisomal fatty alpha- and beta- oxidation in humans: Enzymeology, peroxisomal metabolite transporters and peroxisomal diseases. Biochem. Soc. Trans. 29, 250–267.(Keerthi S. Guruge, Leo W. Y. Yeung, Nori)
Department of Biology and Chemistry, City University of Hong Kong, Tat Chee Avenue, Kowloon, Hong Kong SAR, PR China
Zoology Dept., National Food Safety and Toxicology Center and Center for Integrative Toxicology, Michigan State University, East Lansing, Michigan 48824
Environmental Measurement Group, National Institute of Advance Industrial Science and Technology, Onogawa 16-1, Tsukuba, Ibaraki 305-8569, Japan
ABSTRACT
Perfluorooctanoic acid (PFOA; Pentadecafluorooctanoic acid) is widely used in various industrial applications. It is persistent in the environment and does not appear to undergo further degradation or transformation. PFOA is found in tissues including blood of wildlife and humans; however, the environmental fate and biological effects of PFOA remain unclear. Microarray techniques of gene expression have become a powerful approach for exploring the biological effects of chemicals. Here, the Affymetrix, Inc. rat genome 230 2.0 GeneChip was used to identify alterations in gene regulation in Sprague-Dawley rats treated with five different concentrations of PFOA. Male rats were exposed by daily gavage to 1, 3, 5, 10, or 15 mg PFOA/kg, body weight (bw)/day for 21 days and at the end of the exposure, liver was isolated and total liver RNA were used for the gene chip analysis. Over 500 genes, whose expression was significantly (p < 0.0025) altered by PFOA at two-fold changes compared to control, were examined. The effects were dose-dependent with exposure to 10 mg PFOA/kg, bw/day, causing alteration in expression of the greatest number of genes (over 800). Approximately 106 genes and 38 genes were consistently up- or down-regulated, respectively, in all treatment groups. The largest categories of induced genes were those involved in transport and metabolism of lipids, particularly fatty acids. Other induced genes were involved in cell communication, adhesion, growth, apoptosis, hormone regulatory pathways, proteolysis and peptidolysis and signal transduction. The genes expression of which was suppressed were related to transport of lipids, inflammation and immunity, and especially cell adhesion. Several other genes involved in apoptosis; regulation of hormones; metabolism; and G-protein coupled receptor protein signaling pathways were significantly suppressed.
Key Words: perfluorinated compounds; PFOA; GeneChip; liver gene expression; fatty acid and lipid metabolism.
INTRODUCTION
Perfluorinated compounds (PFCs) have been manufactured and used for various industrial applications for over 50 years (Giesy and Kannan, 2002; Sohlenius et al., 1994). For example, PFOS has been used as a surfactant and surface protector in carpet, leather, paper, and food containers; as well as performance in chemicals in products such as fire-fighting foams, floor polishes, and personal care products (OECD, 2002). The high-energy C-F covalent bonds in PFCs are strong enough to resist hydrolysis, photolysis, biodegradation, and metabolism and thus result in a high degree of environmental persistence and bioaccumulation of PFCs and their derivatives. A number of commercially used PFCs have been shown to occur in the environment, including perfluorooctane sulfonic acid (PFOS), perfluorooctanoic acid (PFOA), perfluorooctane-sulfonamide (PFOSA), perfluorohexanesulfonate (PFHS), perfluorobutanesulfonate (PFBS), and perfluorononanoic acid (PFNA) (Giesy and Kannan, 2002).
The vapor pressure and solubility of PFOS (K+) are 3.31 x 10–4 Pa and 0.5 g/l and those of PFOA (H+) are 70 Pa and 9.5 g/l, respectively (Hekster et al., 2003). Even though there are great differences in the physical and chemical properties of PFOA and PFOS, they were found to be the dominant PFCs in the environment and were found to be widespread in biota, such as wild birds (Giesy and Kannan, 2001), dolphins (Kannan et al., 2001), polar bears and ringed seals (Martin et al., 2002), shell and finfish (Hoff et al., 2003; Van de Vijver et al., 2003), and in human serum and seminal plasma (Guruge et al., 2005; Olsen et al., 1999). They are usually present as a dissociated anion or ion pair in aqueous media; Kannan et al. (2005) has shown that PFOA is less restrained in the body and less bioaccumulated among trophic levels compared with PFOS.
Previous studies have demonstrated that PFOS could affect the membrane fluidity and mitochondrial membrane potential in vitro and in vivo (Hu et al., 2002, 2003). Also, PFOS inhibited gap junction intercellular communication (GJIC) in a dose-dependent manner (Hu et al., 2002) and peroxisomal fatty acid -oxidation was found to be the major pathway affected by PFOS.
Several studies have shown that PFOA has the potential to induce peroxisomal -oxidation in the liver of male rats but not in female rats, whereas PFOA induced the activity in cultured hepatocytes from both male and female rats (Berthiaume and Wallace, 2002; Kudo et al., 2002). Increases in -oxidation of fatty acid, several cytochrome P-450 (CYP450) mediated reactions and inhibition of the secretion of low-density lipoproteins and cholesterol from liver have also been demonstrated (Kennedy et al., 2004). However, the mechanisms of action and metabolic pathways affected by PFOA are still unclear.
Because, there are a number of PFCs with similar structures to which organisms can be exposed simultaneously, risk assessment of these complex mixtures need to be considered. Toward this end, it is important to know if the various PFCs have the potential to elicit similar responses through similar mechanisms and modes of action and whether their risks need to be assessed based on similar joint action or independently. In the present study, the effects of PFOA on liver gene expression in rats was determined by use of microarray techniques to determine effects on gene expression patterns relative to that of unexposed rats. This information was then used to determine which metabolic and signal transduction pathways were affected. This information can then be used to determine the primary pathways that are affected and to infer mechanisms of action. The results of this study can then be compared to those with other PFCs, such as PFOS (Hu et al., 2005a,b).
MATERIALS AND METHODS
Animals and administration.
Seven-week-old male Sprague-Dawley rats were obtained from CLEA Inc. (Tokyo, Japan) and housed at 20–24°C in humidity-controlled (40–60%) facilities at the National Institute of Animal Health, Japan. After acclimatization to a standard diet (MF, Oriental Yeast Co., Japan) for 1 week, rats (225–250 g) were treated daily between 0900 and 1100 h with a single oral gavage of 1 ml/kg body weight (bw) of either vehicle control (0.5% Tween-20) or different doses of pentadecafluorooctanoic acid (perfluorooctanoate; PFOA, purity 95%, CAS Number 335-67-1, Wako Chemicals, Japan). A similar study on PFOS used a dose of 5 mg/kg, bw (Hu et al., 2005a). In this study, 1, 3, 5, 10, 15 mg/kg were used to allow an investigation of the effect of PFOA over a wider range of doses and, at the same time, allow a comparison with the study on PFOS. Six male rats were randomly assigned to one group. Rats in each group were dosed daily with 1, 3, 5, 10, or 15 mg PFOA/kg, bw, daily for 21 days. This experiment was conducted according to the guidelines for animal experiments of the National Institute of Animal Health, Tsukuba, Japan. At the end of exposure, rats were anesthetized with sodium pentabarbital and exsanguinated via the abdominal aorta. The liver was removed and portions were immediately frozen in liquid nitrogen and stored at –80°C until RNA isolation.
RNA extraction.
Total RNA was isolated from livers with Trizol Reagent (Invitrogen, CA) using manufacturer recommended procedures. The reason for choosing liver as the target organ was that perfluorinated fatty acid (PFFAs) mainly accumulated in blood and liver; the major target organ for PFFAs is presumed to be liver (Giesy and Kannan, 2001). In addition, a similar study on the effects of PFOS on gene expression in the rat had been conducted using total liver RNA (Hu et al., 2005a). The ratio of the optical densities from RNA samples measured at 260 and 280 nm was used to evaluate nucleic acid purity and total RNA concentrations were determined by the absorbance at 260 nm. The quality of total RNA was estimated based on the integrity of 28S and 18S rRNA. rRNA was separated using 1% agarose gel electrophoresis, good RNA quality was indicated by the 28s rRNA banding twice the intensity of the 18s rRNA without significant smearing of the rRNA bands. Samples of total RNA from the three rats exposed to the same dose of PFOA were pooled for subsequent use in the GeneChip analysis. Prior to GeneChip analysis, the pooled total RNA samples were purified using the RNeasy Total RNA Mini Kit (Qiagen, Valencia, CA) using manufacturer recommended procedures.
Microarray analysis.
Single and double stranded cDNA was synthesized from total RNA samples using SuperScript II (Invitrogen, CA). High-quality total RNA (16 μg) was used as the starting material and 2 μl of 50 μmol T7- Oligo(dT)24 Primer (5'-GGCCAGTGAATTGTAATACGACTCACTATAGGGAGGCGG-(dT)24 – 3'; Operon, HPLC purified DNA) was used to prime the reaction. After double stranded cDNA clean up and quality check, an in vitro transcription reaction was conducted with the Enzo RNA Transcript Labeling Kit (Affymetrix, Santa Clara, CA) to produce biotin-labeled cRNA from the cDNA. The cRNA was then purified with the RNeasy Mini Kit (Qiagen, Valencia, CA) and fragmented for hybridization analysis. Fifteen microgram aliquants of the fragmented cRNA were hybridized with the Rat Genome 230 2.0 array (Affymetrix, Santa Clara, CA) in hybridization cocktail (0.5 mg/ml cRNA, 50 pM control oligonucleotide B2, 1.5 pM bioB, 5 pM bioC, 25 pM bioD, 100 pM cre, 0.1 mg/ml herring sperm DNA, 0.5 mg/ml acetylated BSA, 100m M MES, 1 M Na+ 20 mM EDTA, 0.01% Tween 20). Hybridization was allowed to proceed overnight (16 h) at 45°C, followed by washing and staining with streptavidin-phycoerythrin (SAPE, Molecular Probes, Eugene, OR). Hybridization assay procedures including preparation of solutions were carried out as described in the Affymetrix GeneChip Expression Analysis Technical Manual. The distribution of fluorescent material on the array was obtained using G2500A GeneArray Scanner (Affymetrix, Santa Clara, CA). Microarray Suite (MAS) version 5.0 and GeneChip Operating Software (GCOS) supplied by Affymetrix was used to perform gene expression analysis.
Scatter plot, gene tree, and condition tree.
Scatter plots were used to visually examine the expression level of genes between the control and PFOA exposed rats. Hierarchical dendrograms were drawn with the Gene tree algorithm of GeneSpring 7 (Silicongenetics, CA). It was similar to phylogenic dendrogram, which was created by clustering the genes according to their expression to the response towards the toxicants. Genes sharing similar expression profiles tended to be clustered together, and the location of a branch containing the genes can be considered a measure of how similar the gene expression was. Condition trees are similar to gene trees, in that they examine the relationships of the expression levels among treatments. Genes were selected for the construction of gene tree if the expression of the gene was two-fold greater or less in the treatments, relative to that in the control (vehicle-exposed) individuals. The horizontal axis shows the clustering of the genes according to their expression profile in the treatment; while the vertical axis showed the clustering according to their expression across treatments.
Pathway analysis.
GeneSpring was used to map genes and their expression values on graphical representations of metabolic pathways with reference to the Kyoto Encyclopedia of Genes and Genomes (www.genome.ad.jp/kegg) and GenMAPP organization (www.genmapp.org). A list of pathways affected, the list of genes affected and the p-value of genes overlapping with pathway values were created.
Statistical analysis.
GeneChip analysis generates very large data sets, it is therefore necessary to evaluate the validity of the data. For the present GeneChip probe array study, the data for each genes represents data from 11–20 probe pairs each approximately 25 bp in length. For each probe pair, one probe is a perfect match while the other has a single mismatch at nucleotide 13. The mismatch probe works as an internal control to evaluate the cross hybridizations between closely related target sequences. The overall target-specific intensity was obtained by the difference between the intensity of perfect match and the mismatch probes. The One-Sided Wilcoxon's Signed Rank test was employed to generate the Detection p-value. If the overall intensity of perfect match were much larger than that of mismatch, the detection p-value would be small. The probe set would be regarded as present if the p-value was less than 0.04 and if the p-value was larger than 0.06, the probe set would be regarded as absent.
The GeneChip probe array system only allows comparison of one treatment hybridizing with the probe set. In a Comparison Analysis, two samples were hybridized to two GeneChip probe arrays of the same type, they were compared against each other in order to detect and quantify changes in gene expression. One GeneChip was for baseline (control) and the other was for the experiment (treatment). Prior to performing data comparison, the data were scaled with the rat230_2norm.msk to 2000 in order to correct for variations in overall intensity and for heterogeneity among the GeneChip probe arrays. Two sets of algorithms were generated and they were used to generate change significance and change quantity metrics for every probe set using Microarray Suite (MAS) version 5.0 (Affymetrix, CA). The change algorithm generated a Change p-value and an associated fold-change value. The second algorithm gave a quantitative estimate of the change in gene expression in the form of Signal Log Ratio. In the present study, the level of gene expression can be regarded as increased if its Change p-value was less than 0.0025 and the gene expression would be considered to be decreased if its Change p-value was greater than 0.9975. This method has been used by other investigators (Hu et al., 2005a). Fold change could be calculated with the following formula: Fold change = 2 (signal log ratio).
RESULTS
The Rat Genome 230 2.0 array is spotted with 31,042 gene probes made of 25-mer single strand oligonucleotides. In the present study, six chips were used. These included one solvent control and five concentrations of PFOA (1, 3, 5, 10, and 15 mg PFOA/kg, bw/day). Comparison analyses of the expression profiles were performed between the control rats and PFOA-treated rats from the GeneChip data. Over 500 genes whose expression was significantly (p < 0.0025) altered by at least two-fold after exposure to PFOA at 1 mg/kg, and at 10 mg/kg PFOA the expression of the greatest number of genes (over 800) was altered (Table 1). The use of two-fold cut-off for significance is conventionally used in other similar studies (Barrans et al., 2001; Kume et al., 2005).
Scatter plots were constructed by comparing a composite sample from PFOA-treated rats with that of a composite sample from unexposed (control) rats (Fig. 1). The vertical position of each gene represents its expression level in the treatment of PFOA at different concentrations, and the horizontal position represents its control condition. Those genes above the diagonal (1:1 regression line) were considered to be induced by PFOA exposure while genes below the diagonal were considered to be suppressed. The number of genes deviating from the 1:1 regression line increased as a function of PFOA dose.
Gene tree analysis created several clusters among the PFOA doses. However, the expression profile of the genes in each cluster was different among treatments. This might be due to different toxico-kinetic mechanisms of different concentrations of PFOA on gene expression. The gene tree analysis classified the patterns of relative responses for all doses relative to the control (Fig. 2). The greatest distance of the node was between the control and the 10 mg PFOA/kg, bw/day exposure. Among the treatment groups, 3 mg and 15 mg PFOA/kg, bw/day were in the same node and this sub-branch was under the node of 5 mg PFOA/kg, bw/day.
The genes altered by the treatment of PFOA of different concentrations could be divided into several large functional categories according to the annotation information and protein information from Protein Knowledgebase provided by Swiss Institute of Bioinformatics (Swiss-Prot) and their biological processes. The genes for which the annotation was unclear such as expressed sequence tags (ESTs) were excluded from the table and when the genes were not fitted into the selected categories, they were regarded as others (Tables 2 and 3). Among the samples from PFOA-treated rats, the largest categories of genes induced were involved in transport and metabolisms of fatty acids and lipids. Other significantly induced genes were involved in cell communication, adhesion, growth, apoptosis, regulation of hormone, proteolysis and peptidolysis and signal transduction. The largest groups of genes suppressed were related to transport, inflammation and immune response and cell adhesion. Several genes involved in apoptosis, regulation of hormone, metabolisms, and G-protein coupled receptor protein signaling pathway were suppressed significantly.
A total of 113 genes and 56 genes were found to be up-regulated and down-regulated, respectively, in at least three of the PFOA doses. Among the genes that showed consistent alternations of gene expression in all treatments, 106 were up-regulated while 38 genes were down-regulated. The genes for which annotation was unclear (ESTs, etc.) were excluded from the table (Table 4). The largest grouping of genes up-regulated by PFOA exposure were for gene products involved in transport and metabolism of lipids, particularly fatty acids. Besides, several genes involved in apoptosis; cell communication, adhesion, growth and cycle; signal transduction; regulation of hormone; and proteolysis and peptidolysis were also induced significantly. Other genes involved in apoptosis, cell adhesion, metabolisms, transport and signal transduction were suppressed significantly.
Pathway analysis was conducted by overlapping the genes with the KEGG pathways in the Kyoto Encyclopedia of Genes and Genomes (www.genome.ad.jp/keg) and GenMAPP organization (www.genmapp.org). The number of common genes with each pathway and the random overlap p-value of the gene list against pathway were obtained. KEGG pathways of fatty acid synthesis (six genes), fatty acid degradation (nine genes), and mitochondrial fatty acid -oxidation (seven genes) were altered significantly (p < 0.0025) by exposure of PFOA. Cholesterol synthesis (four genes) and Krebs-TCA Cycle (three genes) appeared to be altered but the effect was not statistically significant.
The gene expression profiles for rats exposed to 5mg PFOA/kg, bw/day were compared with those reported for rats exposed to PFOS (Hu et al., 2005a) (Tables 5 and 6). Of the 23 genes that were up-regulated by PFOS, 12 genes were also found up-regulated by PFOA. However, seven of the genes were unchanged and four could not be found in the present study. Of the 19 genes for which expression was suppressed by PFOS, only one gene was found to be suppressed and all others but one were not affected by exposure to PFOA.
DISCUSSION
The results of the present study provide insight into how gene expression in livers of rats responded to treatment with several doses of PFOA. Gene expression data was useful in identifying the genes involved in the different biochemical pathways which were affected, however, it should not be regarded as a dose-response relationship because the degree of the alternation in gene expression was not solely due to the administered concentration of PFOA. To interpret the potential for effects in vivo, other parameters, such as the accumulation of PFOA in the target organ, should be considered. Alteration of the expression of a particular gene does not necessarily mean that a particular protein or biochemical pathway would be affected in vivo. However, alteration of a group of genes involved in a particular biochemical pathway would provide strong evidence that PFOA may affect that particular biochemical pathway. Confirmation or isolation of a group of particular proteins or assessment of metabolite flow through the entire target pathway would be the next step in further study and it was not included in the present study.
The number of the genes induced or suppressed was directly proportional to the dose over the range of 1 to 10 mg PFOA/kg, bw/day. However, it was found that the levels of expression of certain genes were different when compared among the concentrations of PFOA. For instance, Resp18, primarily expressed in neural and endocrine tissues, was induced at 1 mg/kg PFOA, was not induced at 3 mg PFOA/kg, bw/day, but was induced again in 5, 10, and 15 mg PFOA/kg, bw/day. In order to understand why a gene was expressed differently at different concentrations of PFOA, the linkage and the pattern of related genes was investigated. However, the linkages of genes were still unclear and it was beyond the scope of the present study to investigate specific pathways in detail. The results of the GeneChip study can, however, be used to design more targeted in vivo studies.
Since the results showed that different doses of PFOA resulted in different gene expression profiles, genes which were up- or down-regulated in all concentrations were selected and analyzed in order to understand the effects of different PFOA doses. Overall, our results showed that a large number of genes associated with lipid or fatty acid metabolism were altered by PFOA and some of the genes were linked with pathways of fatty acid degradation and mitochondrial fatty acid -oxidation in all concentrations of PFOA treated rats. Indeed, similar effects causing interference of fatty acid metabolism had also been found in other studies (Haughom and Spydevold, 1992; Kudo et al., 1999, 2000).
PFOA affected mitochondrial fatty acid -oxidation, fatty acid synthesis and degradation. The genes coding for the enzymes involved in these mechanisms were generally not all induced significantly at the concentrations of PFOA used, except for the genes responsible for the unsaturated fatty acid metabolism (Tables 7 and 8) and for the transfer of fatty acids for oxidation. Specifically, Cpt1b, Cpt2, and Slc25a20 were induced significantly for all PFOA concentrations. Cpt1b catalyzes the transfer of long chain fatty acids to carnitine for translocation across the mitochondrial inner membrane; Cpt2 coding for the inner mitochondrial membrane protein that converted acylcarnitine to acyl-CoA for further fatty acid metabolism and slc25a20 was a carnitine carrier protein which was a component of the mitochondrial inner membrane and transferred fatty acylcarnitines into the mitochondria. The entrance of fatty acids into the mitochondria requires the activation by acyl-CoA synthetase, however, the genes for acyl-CoA synthetase were not induced significantly, and this suggests that PFOA does not undergo activation by acyl-CoA synthetase. The observation of up-regulated Cpt1b, Cpt2, and Slc25a20 implies that there was an increase in the transfer of activated fatty acids or PFOA across the membrane of the mitochondria, as PFOA was not metabolized (Kudo et al., 2001; Vanden Heuvel et al., 1991), the genes coding for the enzymes for the fatty acid -oxidation were not significantly up-regulated among the concentrations of PFOA. PFOA has a similar structure to endogenous fatty acids except that fluorine atoms replace all the hydrogen atoms linked with carbon atoms in the fatty acids. Therefore, it is possible that PFOA could be misguided by the fatty acid metabolism machinery as a substrate because of its structural similarity to endogenous fatty acids, and this effect is similar to those observed for PFOS (Hu et al., 2005a). Although the genes coding for the enzymes for fatty acid degradation, -oxidation and synthesis were not significantly up-regulated among the concentrations of PFOA, the genes coding for the enzymes involved in the metabolism of unsaturated fatty acids were up-regulated. Therefore, there might have been some interactions between PFOA and the pathways that facilitated the degradation of unsaturated fatty acids.
In animal cells, mitochondria and peroxisomes can oxidize fatty acids via -oxidation. There are two major differences between mitochondrial and peroxisomal -oxidation, in terms of its specificity and mechanism (Reddy and Mannaerts, 1994; Wanders et al., 2001). In mammals, the mitochondria oxidize short, medium, and most long chain fatty acids, while peroxisomes oxidize some long chain and very long chain fatty acids. Kudo et al. (2000) demonstrated that PFOA induced peroxisomal -oxidation in in vivo and in vitro studies. In the present study, it was found that the genes coding for peroxisomal oxidation were induced significantly especially at a dose of 10 mg PFOA/kg, bw/day (Fig. 3). Since PFOA is non-biodegradable, the accumulation of PFOA could have resulted in a significant induction of the genes coding for enzymes responsible the degradation of fatty acids. Because the expression of catalase was not induced by PFOA it is possible that potentially toxic hydrogen peroxide was produced in peroxisomes from acyl-CoA that could have caused oxidative stress or oxidation damage to the proteins and DNA. A similar explanation of the effects of PFOS has been previously hypothesized (Hu et al., 2005a). Furthermore, peroxisomes could also oxidize the side chains of eicosanoids, molecules important in short-range signaling, derived from arachidonic acid. Prostaglandins and leukotrienes are representatives of the family of eicosanoid; hence, they might be oxidized and could not perform their normal functions. The gene coding for one of the enzymes responsible for the metabolism of prostaglandins, hpgd, was significantly up-regulated by 3-fold by the 5 mg PFOA/kg, bw/day dose and 4-fold by the 15 mg PFOA/kg, bw/day dose. This observation suggests increased metabolism of prostaglandins.
Both mitochondrial and peroxisomal isomerases were up-regulated. The genes Dci and Decr1, which are involved the mitochondrial -oxidation; and the genes Ech1 and Decr2, which are involved in the peroxisomal -oxidation, were also up-regulated. These isomerases are enzymes involved in the oxidation of the long-chain fatty acids in peroxisomes (Fig. 3). These enzymes, which help to oxidize unsaturated fatty acids through the isomerization of 3-trans-, 5-cis-dienoyl-CoA to 2-trans-, 4-trans-dienoyl-CoA were up-regulated by the peroxisome proliferator [clofibrate or di (2-ethylhexyl) phthalate] (FitzPatrick et al., 1995). Up-regulation of these genes implies that there was increased unsaturated fatty acids metabolism. Therefore, there might be some interaction between PFOA and unsaturated fatty acids, or PFOA was mistaken as the substrate for the unsaturated fatty acid -oxidation in both the mitochondrial and peroxisomal pathways. However, no conclusion could be drawn here because further confirmation of an increased metabolism of unsaturated fatty acids is required.
In the present study, the expression of Cyp4b1 was significantly up-regulated (2.8- to 4.3-fold) among the PFOA treatments (Table 4). Several studies have shown that the induction of the Cyp4b1 gene could be related to bladder cancer in rabbits, mice, and humans (Imaoka et al., 1995, 2000, 2001). Cyp4b1 has an important role in mutagenic activation of procarcinogens in the bladder. Cyp4b1 expression can be induced by benzidine and 2-naphthylamine, which have been identified as carcinogens that can initiate bladder carcinomas (Imaoka et al., 1997). Caspase-11, a critical activator of several other caspase-genes involved in the cytokine regulation of apoptosis, was also induced in PFOA treated animals compared to the controls. Likewise, it is noteworthy that a number of genes involved in immune function, such as interleukin, macroglobulin, and leukocyte activation were down-regulated by at least three or four of the PFOA doses. Additionally, suppression of G-protein associated signaling pathway genes such as Ghsr, Avpr1a, and Hcrtr2 might affect regulation of food intake and energy homeostasis by affecting mechanisms of neuronal transmission.
PFOS, one of the predominant PFCs detected in the environment and accumulated into biota, is structurally analogous to of PFOA except for the terminal sulfonic acid group as compared to the terminal carboxylate of PFOA and a different number of carbon atoms in PFOA, 7 carbon chain attached to the terminal carboxylic acid compared to the 8 carbon chain of PFOS. To determine the similarity of effects on gene expression of these two PFCs, the results of PFOA on gene expression in the current study to that of PFOS were compared (Tables 5 and 6). The genes that were up- or down-regulated by PFOA were quite different from those for which gene expression was altered by PFOS that might be due to the large differences in their physical and chemical properties however it needs further investigations. Cytochrome groups were of interest as they were related to the metabolism of xenobiotics. Up-regulation of Cyp2b15, which is responsible for catalytic reactions involved in drug metabolism, was similar for PFOA and PFOS (Table 5). However, the response of Cyp17a1, which is responsible for steroid hydroxylation and comprises a main component of the steroidogenic pathway, was up-regulated by PFOS about 4-fold more than by PFOA. Two cytochromes Cyp3a18 and Cyp3a11, which are responsible for the conversion and the hydroxylation of testosterone, were up-regulated by PFOS, but not PFOA. Cyp2j4, which is a putative cytochrome p450 monooxygenase enzyme, was up-regulated PFOA but not PFOS. To conclude, the cytochrome genes induced by PFOA were mainly related to xenobiotic metabolisms while those of PFOS were related to both xenobiotic metabolisms as well as steroid metabolisms.
Both PFOS and PFOA up-regulated genes responsible for enzymes involved in fatty acid and lipid metabolism which might be due to their structural similarity to endogenous fatty acids. These compounds may act as structural analogues of endogenous fatty acids and the differences in the gene expression profiles might be due to the different functional groups of PFOA and PFOS, suggesting that further investigation on the toxicity of PFOA is necessary. In this study we observed that PFOA affected both peroxisomal and mitochondrial fatty acids oxidation while PFOS has been reported to mostly affect peroxisomal oxidation (Hu et al., 2005a). The fold change of the enzymes up-regulated by both the 5 mg PFOS/kg, bw/day dose and all doses of PFOA were similar for most of the genes along the peroxisomal fatty acid oxidation pathway except for Acaa1. The fold change of Acaa1 induced by PFOS was approximately twice that of PFOA (Tables 5 and 8).
While there were similarities in modulation of gene expression by PFOS and PFOA, there were also differences. Some of these differences could have been due to different aspects of the design of the two experiments being compared. For instance, the study of PFOS used female rats while that on PFOA used male rats. The reason for choosing male rats was that the elimination rate of PFOA in female was much faster than that of male (Hanhijarvi et al., 1982; Kudo et al., 2002; Vanden Heuvel et al., 1991), the use of male rats will allow this study on PFOA to have more similar toxicokinetics as compared to the previous study on PFOS. Although there was a difference in sex, both PFOA and PFOS induced the genes in the peroxisomal -oxidation pathway.
PFOA has been shown to interfere with fatty acid metabolism and cholesterol synthesis in the liver (Haughom and Spydevold, 1992). Studies of selected enzymes demonstrated a significant decrease in Hmgcr, acyl coenzyme A (CoA) cholesterol acyltranseferase (ACAT) activities, which were related to a lesser concentration of serum cholesterol after 24 h when rats fed PFOA. In the present study, expression of the gene that codes for Hmgcr activity was significantly down-regulated at doses of 5, 10, and 15 mg PFOA/kg, bw/day. Hmgcr is an enzyme involved in mevalonate synthesis which is a rate limiting enzyme in the cholesterol synthesis pathway. This is consistent with previous studies that have found that alteration in the activity of this gene may contribute to the hypolipidemic effect caused by PFOA. Any effects on the expression of the gene that codes for Hmgcr could affect production of lipoprotein particles because of the reduced synthesis of cholesterol together with the increased oxidation of fatty acids in the liver (Haughom and Spydevold, 1992). Cholesterol is an important component of the cell membrane; it stablizes the membrane's fluidity over a bigger temperature interval. Since down-regulation of this gene with a concomitant decrease in Hmgcr activity would result in less cholesterol synthesis, it might alter the cell membrane fludity, an effect that has also been observed to be caused by PFOS (Hu et al., 2003). It may even be hypothesized that increases in membrane fulidity caused by exposure to PFOS result in decreased synthesis of cholesterol by down-regulation of Hmgcr. Another gene, Abcg1, which is responsible for cholesterol transport, was significantly up-regulated (Table 4). Since cholesterol production has been found to be decreased by exposure to both PFOS and PFOA, efflux of excess cholesterol from macrophages would have helped maintain cholesterol homeostasis. Obviously, gene expression studies can not be used to directly investigate effects on cell membrane fluidity. However, recent studies have shown that PFOS can be incorporated into cell membranes resulting in altered cell membrane fluidity and membrane potential (Hu et al., 2003). PFOS also affected gap junction intercellular communication (Hu et al., 2002). Similar effects of PFOA to those of PFOS on membrane fluidity can be postulated, but further research would be necessary to demonstrate this phenomenon. PFOA both up- and down-regulated gene expression that are related to signal transduction and G-protein coupled receptors. This is consistent with the observation that PFOA can inhibit gap junction intercellular communication (Upham et al., 1998).
It has been suggested that both slc22a7 (Oat 2—older name) and slc22a8 (Oat3—older name) are responsible for the urinary elimination of PFOA (Kudo et al., 2002). In previous studies, neither slc22a7 nor slc22a8 were down-regulated by any of the doses of PFOS, however both, slc21a1 (Oatp1) and slc21a5 (Oatp2) were down-regulated by some PFOA doses. The genes slc22a7, slc22a8, slc21a4, slc21a1, and slc21a5 are involved in transport of organic anions and they might also be involved in the urinary elimination of PFOA. Since all of these genes were suppressed, it would be expected that exposure to PFOA would result in a negative feedback that would inhibit elimination of PFOA in the rat.
Here we have reported, for the first time, information on the gene expression profiles of rats receiving different doses of PFOA. These results can be used to understand the biochemical processes affected by PFOA. PFOA enhanced both peroxisomal and mitochondrial fatty acid -oxidation and the peroxisomal -oxidation might create oxidative stress on DNA and protein. Besides, the reduction of cholesterol synthesis observed in in vitro studies would be consistent with the down-regulation of the gene Hmgcr observed in this study. Furthermore, this study suggests that the suppression of organic anion transport genes may explain the delayed urinary clearance of PFOA. There were a number of genes related to tumor progression and inflammation affected by exposure to PFOA, which suggests that exposure to PFOA might enhance the risk of cancer. The outcome of gene data comparison between PFOA and PFOS suggests that the responses in gene expression are similar, but not identical for the two substances. Furthermore the similarities in the effects on lipid metabolism suggest similar modes of action, but the exact mechanisms of the effects may be different and/or may be affected to different degrees by PFOA and PFOS. The responses of genes in the critical pathways are sufficient to consider the development of relative potency factors for PFOS and PFOA to be used in risk assessments. However, differences in the toxicokinetics of PFOA and PFOS would need to be considered if the effects of the two compounds on a single pathway were considered.
ACKNOWLEDGMENTS
This research work was partially supported from the Japanese Ministry of Environment under the Global Environment Conservation Research Fund (2004–2008) to K.S.G. and by a CERG grant (CityU1401/05M) from the Hong Kong Research Grants Council awarded to P.K.S.L. The authors also thank Dr. H. Hoshiba and animal care members of NIAH for their invaluable assistance during the study. Conflict of interest: none declared.
REFERENCES
Barrans, J. D., Stamatios, D., and Liew, C. (2001). Construction of a human cardiovascular cDNA microarray: Portrait of the failing heart. Biochem. Biophys. Res. Commun. 280, 964–969.
Berthiaume, J., and Wallace, K. B. (2002). Perfluorooctanoate, perflourooctanesulfonate, and N-ethyl perfluorooctanesulfonamido ethanol; peroxisome proliferation and mitochondrial biogenesis. Toxicol Lett. 129, 23–32.
FitzPatrick, D. R., Germain-Lee, E., and Valle, D. (1995). Isolation and characterization of rat and human cDNAs encoding a novel putative peroxisomal enoyl-CoA hydratase. Genomics 27, 457–466.
Giesy, J. P., and Kannan, K. (2001). Global distribution of perfluorooctane sulfonate in wildlife. Environ. Sci. Technol. 35, 1339–1342.
Giesy, J. P., and Kannan, K. (2002). Perfluorochemical surfactants in the environment. Environ. Sci. Technol. 36, 146A–152A.
Guruge, K. S., Taniyasu, S., Yamashita, N., Wijeratna, S., Mohotti, K. M., Seneviratne, H. R., Kannan, K., Yamanaka, N., and Miyazaki, S. (2005). Perfluorinated organic compounds in human blood serum and seminal plasma: A study of urban and rural tea worker populations in Sri Lanka J. Environ. Monit. 7, 371–377.
Hanhijarvi, H., Ophaug, R., and Singer, L. (1982). The sex related different in perfluorooctanceate excretion in the rat. Proc. Soc. Exp. Biol. Med. 171, 50–55.
Haughom, B., and Spydevold, O. (1992). The mechanism underlying the hydropolipemic effect of perfluorooctanoic acid (PFOA), perfluorooctane sulphonic acid (PFOSA) and clofibric acid. Biochim. Biophys. Acta 1128, 65–72.
Hekster, F. M., Laane, R. W. P. M., and de Voogt, P. (2003). Environmental and toxicity effects of perfluoroalkylated substances. Rev. Environ. Contam. Toxicol. 179, 99–121.
Hoff, P. T., Van Dongen, W., Esmans, E. L., Blust, R., and DeCoen W. M. (2003). Evaluation of the toxicological effects of perfluorooctane sulfonic acid in the common carp (Cyprinus carpio). Aquat. Toxicol. 62, 349–359.
Hu, W. Y., Jones, P. D., Upham, B. L., Trosko, J. E., Lau, C., and Giesy, J. P. (2002). Inhibition of gap junctional intercellular communication by perfluorinated compounds in rat liver and dolphin kidney epithelial cell lines in vitro and Sprague-Dawley rats in vivo. Toxicol. Sci. 68, 429–436.
Hu, W. Y., Jones, P. D., DeCoen, W., King, L., Frakwe, P., Newsted, J. L., and Giesy, J. P. (2003). Alternations in cell membrane properties caused by perfluorinated compounds. Comp. Biochem. Physiol. 135, 77–88.
Hu, W. Y, Jones, P. D., Celius, T., and Giesy, J. P. (2005a). Identification of genes responsive to PFOS using gene expression profiling. Environ. Toxicol. Pharmcol. 19, 57–70.
Hu, W. Y., Jones, P. D., DeCoen, W., and Giesy, J. P. (2005b). Comparison of gene expression methods to identify genes responsive to perfluorinated sulfonic Acid. Environ. Toxicol. Pharmacol. 19, 153–160.
Imaoka, S., Hiroi, T., Tamura, Y., Yamazaki, H., Shimada, T., Komori, N., Degawa, M., and Funae, Y. (1995). Mutagenic activation of 3-methoxy-4-aminoazobenzene by mouse renal cytochrome-P450 CYP4B1: Cloning and characterization of mouse CYP4B1. Arch. Biochem. Biophys. 321, 255–262.
Imaoka, S., Yoneda, Y., Matsuda, T., Degawa, M., Fukushima, S., and Funae, Y. (1997). Mutagenic activation of urinary bladder carcinogens by CYP4B1 and the presence of CYP4B1 in bladder mucosa. Biochem. Pharmacol. 54, 677–683.
Imaoka, S., Yoneda, Y., Sugimoto, T., Hiroi, T., Yamamoto, K., Nakatani, T., and Funae, Y. (2000). CYP4B1 is a possible risk factor for bladder cancer in humans. Biochem. Biophys. Res. Commun. 277, 776–780.
Imaoka, S., Yoneda, Y., Sugimoto, T., Ikemoto, S., Hiroi, T., Yamamoto, K., Nakatani, T., and Funae, Y. (2001). Androgen regulation of CYP4B1 responsible for mutagenic activation of bladder carcinogens in the rat bladder: Detection of CYP4B1 mRNA by competitive reverse transcription-polymerase chain reaction. Cancer Lett. 166, 119–123.
Kannan, K., Koistinen, J., Beckmen, K., Evans, T., Gorzelany, J. F., Hansen, K. J., Jones, P. D., Helle, E., Nyman, M., and Giesy, J. P. (2001). Accumulation of perfluorooctane sulfonate in marine mammals. Environ. Sci. Technol. 35, 1593–1598.
Kannan, K., Tao, L., Sinclair, E., Pastva, S. D., Jude, D. J., and Giesy, J. P. (2005). Perfluorinated compounds in aquatic organisms at various trophic levels in a Great Lakes food chain. Arch. Environ. Contam. Toxicol. 48, 559–566.
Kennedy, G. L., Butenhoff, J. L., Olsen, G. W., O'Connor, J. C., Seacat, A. M., Perkins, R. G., Biegel, L. B., Murphy, S. R., and Farrar, D. G. (2004). The toxicology of perfluorooctanoate. Crit. Rev. Toxicol. 34, 351–384.
Kudo, N., Mizuguchi, H., and Kawashima, Y. (1999). Alternations by perfluorooctanoic acid of glycerolipid metabolism in rat liver. Chemico-Biol. Interact. 118, 69–83.
Kudo, N., Bandai, N., Suzuki, E., Katakura, M., and Kawashima, Y. (2000). Induction by perfluorinated fatty acids with different carbon chain length of peroxisomal beta-oxidation in the liver of rats. Chemico-Biol. Interact. 124, 119–132.
Kudo, N., Suzuki, E., Katakura, M., Ohmori, K., Noshiro, R., and Kawashima, Y. (2001). Comparison of the elimination between perfluorinated fatty acids with different carbon chain length in rats. Chemico-Biol. Interact. 134, 203–216.
Kudo, N., Katakura, M., Sato, Y., and Kawashima, Y. (2002). Sex hormone-regulated renal transport of perfluorooctanoic acid. Chemico-Biol. Interact. 139, 301–316.
Kume, E., Argua, C., Ishizuka, Y., Takahashi, K., Miwa, S., Itoh, M., Fujimura, H., Toriumi, W., Kitamura, K., and Doi, K. (2005). Gene expression profiling in streptozotocin treated mouse liver using DNA microarray Exp. Toxicol. Pathol. 56, 235–244.
Martin, J. W., Muir, D. C. G., Moody, C. A., Ellis, D. A., Kwan, W. C., Solomon, K. R., and Mabury, S. A. (2002). Collection of airborne fluorinated organics and analysis by gas chromatography/chemical ionization mass spectrometry. Anal. Chem. 74, 584–590.
OECD (Organization for Economic Co-operation and Department). (2002). Co-operation on existing chemicals: Hazard assessment of perfluorooctane sulfonate (PFOS) and its salts. ENV/JM/RD (2002) 17/FINAL.
Olsen, G. W., Burris, J. M., Mandel, J. H., and Zobel, L. R. (1999). Serum perfluorooctane sulfonate and hepatic and lipid clinical chemistry tests in fluorochemical production employees. J. Occup. Environ. Med. 41, 799–806.
Reddy, J. K., and Mannaerts, G. P. (1994). Peroxisomal lipid metabolism. Annu. Rev. Nutr. 14, 343–370.
Sohlenius, A. K., Andersson, K., Bergstand, A., Spydevold, O., and De Pierre, J. W. (1994). Effects of perfluorooctanoic acid—a potent peroxisome proliferators in rat—on Morris hepatoma 7800C1 cells, a rat cell line. Biochim. Biophys. Acta 1213, 63–74.
Upham, B. L., Deocampo, N. D., Wurl, B., and Trosco, J. E. (1998). Inhibition of gap junctional intercellular communication by perfluorinated fatty acids is dependent on the chain length of the fluorinated tail. Int. J. Cancer 78, 491–495.
Vanden Heuvel, J., Kuslikis, B., and Van Rafelghem, M. (1991). Tissue distribution, metabolism, and elimination of perfluorooctanoic acid in male and female rats. Biochem. Toxicol. 6, 83–92.
Van de Vijver, K. I., Hoff, P. T., Van Dongen, W., Esmans, E. L., Blust, R., and DeCoen, W. M. (2003). Exposure patterns of perfluorooctane sulfonate in aquatic invertebrates from the Western Scheldt estuary and the southern North Sea. Environ. Toxicol. Chem. 22, 2037–2041.
Wanders, R. J. A., Vreken, P., Ferdinandusse, S., Jansen, G. A., Waterham, H. R., Van Roermund, C. W. T., and Van Grunsven, E. G. (2001). Peroxisomal fatty alpha- and beta- oxidation in humans: Enzymeology, peroxisomal metabolite transporters and peroxisomal diseases. Biochem. Soc. Trans. 29, 250–267.(Keerthi S. Guruge, Leo W. Y. Yeung, Nori)