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Physiol. Genomics 34: 285-303, 2008. First published June 10, 2008; doi:10.1152/physiolgenomics.00225.2007
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Received 28 September 2007; accepted in final form 5 June 2008.
Physiological Genomics 34:285-303 (2008)
1094-8341/08 $8.00 © 2008 American Physiological Society

Comparative hepatic gene expression profiling of rats treated with 1H,1H,2H,2H-heptadecafluorodecan-1-ol or with pentadecafluorooctanoic acid

Anja Julie Nilsen 1, Maria A. Landin 1, Kristin H. Haug 3, Frode Fonnum 2,3, Urs Berger 4 and Harald Osmundsen 1

1 Institute for Oral Biology, University of Oslo, Oslo
2 Department of Protection and Material, Norwegian Defence Research Establishment, Kjeller
3 Department of Medical Biochemistry, University of Oslo, Oslo
4 Norwegian Institute of Air Research, Polar Environment Centre, Tromsø, Norway


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Pentadecafluorooctanoic acid is an established peroxisome proliferator. Little is known about effects of treatment with 1H,1H,2H,2H-heptadecafluorodecan-1-ol, which is metabolized to pentadecafluorooctanoic acid. We compared effects of various dosages (3, 10, or 25 mg/kg body wt) of each of these compounds on hepatic gene expression in rats with microarrays. Microarray data were validated by real-time RT-PCR. Expression data were also correlated with hepatic activities of selected enzymes and with hepatic levels of pentadecafluorooctanoic acid and 1H,1H,2H,2H-heptadecafluorodecan-1-ol. Pentadecafluorooctanoic acid caused the more powerful change in gene expression, in terms of both number of genes affected and extent of change in expression. Across the dosages used pentadecafluorooctanoic acid and 1H,1H,2H,2H-heptadecafluorodecan-1-ol caused significant (P ≤ 0.05) changes in expression for 441 and 105 genes, respectively. With 1H,1H,2H,2H-heptadecafluorodecan-1-ol ~38% of the 105 genes exhibited decreased expression with a dose of 25 mg/kg body wt, these genes also appearing less responsive to treatment at the lower dosages. Bioinformatic analysis suggested that these genes are associated with regulatory functions. With pentadecafluorooctanoic acid, increasing dosage up to 10 mg/kg body wt brought about progressive increase in expression of affected genes. Pathways analysis suggested similar effects of the two compounds on lipid and amino acid metabolism. Marked differences were also found, particularly with respect to effects on genes related to oxidative phosphorylation, oxidative metabolism, free radical scavenging, xenobiotic metabolism, and complement and coagulation cascades.

liver; mitochondria; peroxisome; lipid; fatty acid; signaling; xenobiotic


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
PERFLUOROALKYLATED COMPOUNDS have been extensively used in industry during the past 50 years. Physicochemical properties such as hydrophobicity and oleophobicity, together with their high chemical and thermal stability, have made them attractive for a variety of applications, e.g., cleaning and defatting agents, paints, wax, and anticorrosion agents. Their levels in the environment, and in humans, have increased (10, 19, 28).

Some information on the effects of these compounds on living organisms is available (11, 12). Short-chain perfluorinated fatty acids, among them pentadecafluorooctanoic acid (PFOA), are known to cause hepatomegaly and to stimulate oxidative metabolism, effects that were shown to increase with increasing chain length (22). Limited information on the effects of administration of 1H,1H,2H,2H-heptadecafluorodecan-1-ol (8:2 FTOH) is available. Acute or chronic toxicity of 8:2 FTOH, as for PFOA, is considered low for both rodents and aquatic organisms (14). Exposure of cultured breast cancer cells to 8:2 FTOH suggested that the compound mediates estrogen-like effects (23). An analog, pentadecafluoro-1-octanol, has been shown to be a peroxisome proliferator (18).

8:2 FTOH has been shown to be metabolized to PFOA (24). To establish whether exposure to 8:2 FTOH affects liver differently from PFOA we carried out a study entailing comparative gene expression profiling using DNA microarrays. Rats were given PFOA or 8:2 FTOH at doses of 3, 10, or 25 mg/kg body wt. DNA microarray data were validated with real-time RT-PCR. Follow-up experiments entailing measurements of hepatic activities of selected enzymes and assays of hepatic levels of PFOA and 8:2 FTOH were also carried out.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Reagents
RNALater was purchased from Ambion. PFOA (96% pure), 8:2 FTOH (97% pure), 4-nitrophenylacetate, and 4-nitrophenylbutyrate were purchased from Aldrich Chemical.

Experimental Animals
Male Wistar albino rats were purchased from Møllegaard Laboratories (Roskilde, Denmark). The rats had a body wt of ~150 g when used experimentally. Animals were kept as defined by the Norwegian Gene Technology Act of 1994. Animals were kept under pathogen-free conditions, at constant temperature (22 ± 2°C) and humidity (55 ± 5%), with a 12:12-h alternating light-dark cycle. Fodder (RM3, Special Diet Services, Betchworth, Surrey, UK) and water were supplied ad libitum. The animal protocols used were submitted to, and approved by, the local ethical committee for experimental animals.

Treatments
Rats were given single daily intraperitoneal injections for 10 days of either PFOA or 8:2 FTOH, with a dose of 3, 10, or 25 mg/kg body wt. PFOA or 8:2 FTOH was dissolved in 0.5% (vol/vol) Tween 20; at least six rats were used for each dosage. An identical injection volume was used for all treatments, and control rats were given an equivalent volume of 0.5% (vol/vol) Tween 20 (vehicle) as described by Thibodeaux et al. (31). No evidence of pathological effects was observed after these treatments, in line with earlier findings regarding PFOA (13, 14).

On the ensuing day (day 11), no further injections were given. The animals were killed by decapitation on the subsequent day (day 12 after start of treatment). Tissue from the middle liver lobe from the same animal was generally used for RNA isolation and for enzymatic assays. However, using middle lobes from different animals, given identical treatment, gave consistent results.

Isolation of RNA
For isolation of RNA, samples of livers (~50 mg wet wt) were immediately removed from the middle hepatic lobe (21) and placed in 1 ml of RNALater. Total RNA was extracted with the Qiagen RNAeasy Mini-kit (Qiagen, Hilden, Germany). This yielded RNA fractions exhibiting a ratio of optical density (OD)260 to OD280 of at least 1.7. The quality of isolated RNA was assessed with an Agilent 2100 Bioanalyzer fitted with a RNA 6000 nano chip (Agilent) and by real-time RT-PCR. RNA integrity number (RIN) values of 9.3–9.8 were consistently observed. Such solutions of RNA also gave a signal of appropriate lengths from various primer pairs (e.g., ribosomal protein L27 or β-actin) and were considered suitable for use in microarray analysis.

Enzyme Assays
For assays of enzyme activities, samples of the middle liver lobe were placed in ice-cold 50 mM potassium phosphate buffer, pH 7.2. Subsequent homogenization and centrifugation were carried out as described previously (25).

Assays of activities of catalase (EC 1.11.1.6), palmitoyl-CoA L-carnitine acyltransferase (EC 2.3.1.21), and peroxisomal β-oxidation were carried out with supernatants resulting from 10% (wt/vol) liver homogenates centrifuged at 3,000 gav for 3 min (25). Carboxylesterase (EC 3.1.1.1) activity was measured with 4-nitrophenylacetate or 4-nitrophenylbutyrate as substrate, as described previously (4).

Assay of PFOA and 8:2 FTOH in Liver
Levels of PFOA and 8:2 FTOH in whole livers of rats treated with 10 mg/kg body wt of PFOA or 8:2 FTOH for 10 days as described above were assayed by liquid chromatography-mass spectrometry as described elsewhere (2).

Microarray Analysis of mRNAs in Total Hepatic RNA Isolates
Rat DNA oligo(6K) microarrays were purchased from the NTNU Microarray Core Facility, Trondheim, Norway. These slides had been printed with the Operon v.1.1 rat oligo set (Operon Biotechnologies, Cologne, Germany). Each probe was spotted in triplicate.

cDNA synthesis and Cy3/Cy5 labeling were carried out with the Genisphere 3DNA Array 900 detection kit (Genisphere). Each slide was hybridized with cDNA obtained with 1 µg of total RNA. Samples of total RNA isolated from three different animals (given identical treatment) were pooled and analyzed with three separate microarrays. Although statistically less desirable, this approach was chosen to limit consumption of microarray slides. One sample on each slide was always that of a separate vehicle control. Samples of RNA from treated animals were labeled with Cy5; samples from vehicle controls were Cy3 labeled. Control dye-swap experiments were carried out, as were experiments with untreated control versus untreated control, and found to yield consistent results.

The microarrays were scanned in a Packard Bioscience Scanarray Lite microarray scanner (PerkinElmer Life and Analytical Sciences). The Cy3 and Cy5 fluorescence signals were quantitated with the ScanArray Express v. 2.2 program (PerkinElmer Life and Analytical Sciences).

Availability of Microarray Data
Microarray data files have been deposited in the ArrayExpress database with reference number E-MEXP-1045.

Statistical Analysis
Statistical analysis of microarray data was carried out with Spotfire v. 8 Functional Genomics software (Spotfire) and the Limma Guide Spotfire Application Package (Integromics, Madrid, Spain). The analysis was carried out as single-channel analysis because this is more flexible than two-channel analysis (ratio analysis). With the Spotfire software we have previously found both methods to give identical results (20).

Single-channel analysis replicates are required for false discovery rate (FDR) and ANOVA analysis. To this end, fluorescence intensities (median values, with background subtracted) from each of the two channels were converted to log2 scale, and the log2 values were subjected to z score normalization (5) followed by median subtract normalization.

Expression data were filtered as follows: consistent fluorescence intensities for at least two of the triplicate spots on each array, a minimum signal of 300 for either the vehicle or treated samples, and a change of expression of >50%. FDR of Benjamini and Hochberg (1) was used to correct the selection of genes for false positives. With the one-way ANOVA facility of the software normalized intensities were used to select genes that exhibited statistically significant (P ≤ 0.01 or P ≤ 0.05) changes in levels of expression following treatment with PFOA or 8:2 FTOH. Genes with significantly altered expression were subjected to hierarchical clustering. The results are presented as heat maps (see Figs. 1–Go3).


Figure 1
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  		Fig. 1. Heat map resulting from hierarchical clustering of genes that exhibited significantly altered expression in rats treated with either pentadecafluorooctanoic acid (PFOA) or 1H,1H,2H,2H-heptadecafluorodecan-1-ol (8:2 FTOH). Genes with significantly changed expression in rats treated both with PFOA (P ≤ 0.01) and with 8:2 FTOH (P ≤ 0.05) were subjected to hierarchical clustering with Spotfire v. 8.1. This analysis entailed the 55 genes that are shared between the 272 genes and the 105 genes in the PFOA and 8:2 FTOH populations, respectively. One of the 25-mg triplicates showed evidence of inadequate labeling and was not included in the analysis. The heat map resulting from hierarchical clustering is shown, with gene symbols indicated on right. The 6 lanes labeled "Untreated control" are data from livers of rats given vehicle only. The color codes show low levels of expression as green, intermediate levels as dark shades of green/red, and high levels of expression as red, the lighter shades of red showing genes with the higher levels of expression.

 

Figure 2
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  		Fig. 2. Heat map resulting from hierarchical clustering of genes that exhibited significantly altered expression only in rats treated with PFOA. The 217 genes that exhibited significantly (P ≤ 0.01) changed expression only in livers of rats treated with PFOA were subjected to hierarchical clustering as described in Fig. 1. Of the 272 genes that exhibited significantly changed expression, these 217 genes remained after removal of the 55 genes with significantly changed expression also after treatment with 8:2 FTOH. The heat map resulting from hierarchical clustering is shown. The high number of genes precludes presentation of gene symbols. The 6 lanes labeled "Untreated control" are data from livers of rats given vehicle only. The color codes show low levels of expression as green, intermediate levels as dark shades of green/red, and high levels of expression as red, the lighter shades of red showing genes with the higher levels of expression.

 

Figure 3
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  		Fig. 3. Heat map resulting from hierarchical clustering of genes that exhibited significantly altered expression only in rats treated with 8:2 FTOH. The 50 genes that exhibited significantly (P ≤ 0.05) changed expression only in livers of rats treated with 8:2 FTOH were subjected to hierarchical clustering as described in Fig. 1. Of the 105 genes that exhibited significantly changed expression after treatment with 8:2 FTOH these 50 genes remained after removal of the 55 genes whose expression was significantly changed also after treatment with PFOA. The heat map resulting from hierarchical clustering of these 50 genes is shown, with gene symbols indicated on right. The 6 lanes labeled "Untreated control" are data from livers of rats given vehicle only. The color codes show low levels of expression as green, intermediate levels as dark shades of green/red, and high levels of expression as red, the lighter shades of red showing genes with the higher levels of expression.

 
Statistical evaluation (unpaired t-test with Welch correction) of nonmicroarray data was carried out with GraphPad InStat (GraphPad Software).

Bioinformatic Analysis of Microarray Expression Data
Interpretation of expression data in terms of altered activities of cellular functions and canonical or signal pathways was carried out with Ingenuity Pathways Analysis software (Ingenuity Systems). Functional annotation/classification analysis was also carried out with the DAVID program (6).

Validation of Microarray Results with Real-Time RT-PCR
Levels of selected mRNAs (Acadm, Acox1, Acp1, Apcs, Apoa4, Cntb, Crot1, Cyp4b1, Herpud1, Pct1, Rcn2) were also assayed by real-time quantitative RT-PCR with primers designed with Primer3 (29). cDNA was synthesized by oligo(dT) priming with the First Strand Synthesis Kit (Fermentas, St. Leon-Rot, Germany). Real-time RT-PCR assays were carried out with the Stratagene Mx 3005 Pro RT-PCR system (Stratagene) and Ampliqon master mix (Ampliqon, Rødovre, Denmark). RNA isolated from livers of three separate rats treated with 3 mg/kg of PFOA, 3 mg/kg of 8:2 FTOH, or vehicle was used in these assays. All assays were run in triplicate. Statistical evaluation of the significance of differences between measured threshold cycle (Ct) values was carried out with the REST 2005 program (27).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Effects of Treatment with PFOA or 8:2 FTOH on Hepatic Gene Expression
Treatment with PFOA resulted in significant changes in expression of numerous genes, i.e., 441 genes at P ≤ 0.05 or 272 genes (8 with unknown function) at P ≤ 0.01. Initial analysis had indicated that using the latter, smaller, population of PFOA genes for pathways analysis would not substantially alter resulting conclusions. Also, a smaller number of genes facilitates more succinct presentation of data; these 272 genes were therefore used subsequently.

On treatment with 8:2 FTOH only 105 genes (6 with unknown function) exhibited significantly altered expression at all levels of treatment used at P ≤ 0.05 and 27 genes at P ≤ 0.01. The diminished number of genes was likely caused by the smaller changes in expression resulting from treatment with 8:2 FTOH. For pathways analysis the larger population (105 genes) was preferred.

Subsequent pathways analyses were therefore carried out with the 272- and 105-gene populations from PFOA- and 8:2 FTOH-treated rats, respectively. The PFOA (272 genes) and 8:2 FTOH (105 genes) populations shared 55 genes. Conversely, 217 and 50 genes exhibited significant changes in expression only in rats treated with POFA or 8:2 FTOH, respectively.

The heat map in Fig. 1 shows that most of the shared 55 genes exhibited progressively increased expression as the dose of PFOA increased, the genes positioned at the top middle of the heat map clearly requiring a higher dose for their level of expression to change. A similar trend was also observed with rats given 8:2 FTOH; however, a fraction of those genes least responsive to the treatments exhibited decreased expression in rats given 25 mg 8:2 FTOH/kg body wt (Fig. 1, top right).

Genes with significantly altered expression only after treatment with PFOA.
The 217 genes that were affected only by PFOA were hierarchically clustered, yielding the heat map shown in Fig. 2. The data indicate enhanced expression with dosage increasing up to 10 mg/kg. This dosage appears to be saturating because no further, marked, increase in expression is apparent at a dosage of 25 mg/kg body wt. The phenomenon is also observed with data shown in Fig. 1, but primarily with the genes most sensitive to the treatments (Fig. 1, middle bottom).

Genes with significantly altered expression only after treatment with 8:2 FTOH.
The 50 genes with significantly altered expression only in 8:2 FTOH-treated rats were similarly analyzed. The resulting heat map (Fig. 3) indicates that only 12 of these genes showed increased expression, markedly so only at a dosage of 10 or 25 mg/kg body wt. The levels of expression of the remaining 38 genes were relatively unchanged until the dosage was increased to 10 mg/kg body wt, then decreasing as the dosage was further increased to 25 mg/kg body wt. Also, the genes most responsive to the treatment appear to attain a saturating level of expression at a dosage of 10 mg/kg body wt (Fig. 3, bottom).

Bioinformatics: Interpretation of Microarray Expression Data
Genes with significantly altered expression after treatment with PFOA or 8:2 FTOH.
Pathways analyses with Ingenuity Pathways Analysis software using the 272 (PFOA) and 105 (8:2 FTOH) genes were carried out in the comparative mode to highlight differences between effects of the treatments. Significant associations to cellular functions and canonical pathways are shown in Figs. 4 and 5, respectively. In general, the more significant associations are observed for the PFOA population. The affected genes and associated functions/pathways are shown in Tables 1 and 2, respectively.


Figure 4
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  		Fig. 4. Cellular functions associated with genes with significantly altered expression after treatment with PFOA or 8:2 FTOH. Ingenuity Pathways Analysis was carried out with the 272 genes (P ≤ 0.01) and 105 genes (P ≤ 0.05) with significantly altered expression after treatment with 3, 10, or 25 mg/kg body wt PFOA or 8:2 FTOH, respectively. The levels of significance for the various associations were computed with Ingenuity software. The horizontal line indicates the minimal level of significance required for the association to be judged significant.

 

Figure 5
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  		Fig. 5. Canonical pathways associated with genes with significantly altered expression after treatment with PFOA or 8:2 FTOH. Canonical pathways analysis was carried out with Ingenuity Pathways Analysis software, using the 272 genes (P ≤ 0.01) and 105 genes (P ≤ 0.05) with significantly altered expression after treatment with 3, 10, or 25 mg/kg body wt PFOA or 8:2 FTOH, respectively. The levels of significance for the various associations were computed with Ingenuity software. The horizontal line indicates the minimal level of significance required for the association to be judged significant.

 

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  		Table 1. Cellular functions associated with genes exhibiting altered expression after treatment with PFOA or 8:2 FTOH

 

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  		Table 2. Canonical pathways associated with genes exhibiting altered expression after treatment with PFOA or 8:2 FTOH

 
Results presented in Fig. 4 show significant, and similar, associations with "lipid metabolism" and "small molecule biochemistry." Several other functions are differentially affected; associations with "protein synthesis," "molecular transport," "drug metabolism," and "energy production" exhibited markedly higher significance with genes from PFOA-treated rats. "Gene expression," "cellular development," and "cellular growth and proliferation" showed a markedly higher level of significance associated with the genes from 8:2 FTOH-treated rats. A similar result was found for canonical pathways, e.g., "fatty acid metabolism," amino acid metabolism/degradation, and metabolism of arachidonic and linolenic acid (Fig. 5).

The results presented in Tables 1 and 2 illustrate both that numerous identical genes are affected by both treatments and that different genes also are implicated. As expected, a higher number of genes from PFOA treatment are associated with each category. Nevertheless, for several categories a number of the genes in the 8:2 FTOH populations differ from those found in corresponding PFOA populations, e.g., "lipid metabolism," "amino acid metabolism," and "cell cycle" (Table 1) and "fatty acid metabolism," "pyruvate metabolism," and "glycolysis/gluconeogenesis" (Table 2).

Furthermore, data presented in Fig. 4 and Table 1 suggest that "energy production" and "protein degradation" are only associated with genes from PFOA-treated rats. Similarly, results shown in Fig. 5 and Table 2 indicate that "citrate cycle," "glycine, serine, and threonine metabolism," and "oxidative phosphorylation" are only associated with genes from PFOA-treated rats, while "riboflavin metabolism" is only associated with genes from 8:2 FTOH-treated rats.

Genes with significantly altered expression only in either PFOA- or 8:2 FTOH-treated rats.
Fifty-five genes were shared between the 272 genes from PFOA-treated and the 105 genes from 8:2 FTOH-treated rats, leaving 217 and 50 genes with exclusively altered expression only in either PFOA- or 8:2 FTOH-treated rats, respectively.

Pathways analysis using the latter two populations may therefore highlight any potential functional differences caused by the two treatments. Results from this analysis (Figs. 6 and 7, Tables 3 and 4) confirmed this suggestion. As regards cellular functions, the results presented in Fig. 6 and Table 3 suggest that genes from PFOA-treated rats exhibited more significant associations with "lipid metabolism," "small molecule biochemistry," "molecular transport," "protein synthesis," "amino acid metabolism," "energy production," "nucleic acid metabolism," and "posttranslational modification."


Figure 6
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  		Fig. 6. Cellular functions associated with genes with significantly altered expression only after treatment with either PFOA or 8:2 FTOH. Ingenuity Pathways Analysis was carried out with the 217 genes (P ≤ 0.01) and 50 genes (P ≤ 0.05) with significantly altered expression exclusively after treatment with 3, 10, or 25 mg/kg body wt PFOA or 8:2 FTOH, respectively. The levels of significance for the various associations were computed with Ingenuity software. The horizontal line indicates the minimal level of significance required for the association to be judged significant.

 

Figure 7
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  		Fig. 7. Canonical pathways associated with genes with significantly altered expression only after treatment with either PFOA or 8:2 FTOH. Canonical pathways analysis was carried out with Ingenuity Pathways Analysis software, using the 217 genes (P ≤ 0.01) and 50 genes (P ≤ 0.05) with significantly altered expression exclusively after treatment with 3, 10, or 25 mg/kg body wt PFOA or 8:2 FTOH, respectively. The levels of significance for the various associations were computed with Ingenuity software. The horizontal line indicates the minimal level of significance required for the association to be judged significant.

 

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  		Table 3. Cellular functions associated with genes exhibiting exclusively altered expression on treatment only with either PFOA or 8:2 FTOH

 

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  		Table 4. Canonical pathways associated with genes exhibiting exclusively altered expression on treatment with either PFOA or 8:2 FTOH

 
Association with "free radical scavenging," "carbohydrate metabolism," and "protein degradation" was exclusively found in the PFOA-treated group. The higher significances observed for associations of the PFOA genes are, however, unlikely to be due only to the higher numbers of genes in this group. Additional cellular functions exhibited higher significances with genes from the less numerous 8:2 FTOH-treated group, e.g., "gene expression," "cell-to-cell signaling and interaction," "DNA replication, recombination, and repair," "cell cycle," "cell morphology," "cellular growth and proliferation," "cellular development," "cellular function and maintenance," "cell signaling," and "RNA damage and repair" (Fig. 6). The genes associated with the various cellular functions are shown in Table 3.

Pathways analysis of canonical pathways substantiates this impression. This is shown by results presented in Fig. 7 and Table 4. Gene associations with "fatty acid metabolism," metabolism of valine, leucine, isoleucine, glycine, serine, and threonine, "metabolism of xenobiotics by cytochrome P-450," metabolism of arachidonic and linolenic acid, and glycolysis/gluconeogenesis exhibited much higher significances for genes from PFOA-treated rats. Additionally, for "oxidative phosphorylation," "citrate cycle," and "glutathione metabolism" there were no gene associations among the genes from 8:2 FTOH-treated rats. Conversely, for "lysine degradation" and "riboflavin metabolism" higher significances were observed with genes from 8:2 FTOH-treated rats.

Results from analysis of signaling pathways are presented in Fig. 8 and Table 5. Few significant associations were found. With the full complement of genes from PFOA- or 8:2 FTOH-treated rats a significant association was found only for "protein ubiquitination pathway" with genes from PFOA-treated rats (Fig. 8). With the 217 and 50 genes that only exhibited altered expression exclusively in either PFOA- or 8:2 FTOH-treated rats significant associations were found for "xenobiotic metabolism signaling," "amyotrophic lateral sclerosis signaling," and "protein ubiquitination pathway," but only with the 217 genes from PFOA-treatexd rats (Fig. 8).


Figure 8
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  		Fig. 8. Signaling pathways associated with genes with significantly altered expression after treatment with PFOA or 8:2 FTOH. Signaling pathways analysis was carried out with Ingenuity Pathways Analysis software, using the 272 genes (P ≤ 0.01) and 105 genes (P ≤ 0.05) with significantly altered expression after treatment with 3, 10, or 25 mg/kg body wt PFOA or 8:2 FTOH, respectively (A). Identical analysis was also carried out with the 217 and 50 genes with significantly altered expression only with PFOA or 8:2 FTOH, respectively (B). The level of significance for the various associations was computed with Ingenuity software. The horizontal line indicates the minimal level of significance required for the association to be judged significant.

 

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  		Table 5. Signaling pathways associated with genes exhibiting exclusive altered expression on treatment with either PFOA or 8:2 FTOH

 
Bioinformatic Analysis with DAVID
Additional functional annotation/classification analysis was carried out with DAVID (6). Results for genes with significantly altered expression only in either PFOA (217 genes)- or 8:2 FTOH (55 genes)-treated rats are summarized in Table 6. These results suggest similar profiles in regard to significant metabolic associations. However, a substantial fraction (7–16%) of the 8:2 FTOH genes were significantly associated with regulatory or signaling functions, while PFOA genes showed no significant associations in this respect. Substantial fractions of the 217 PFOA genes, on the other hand, exhibited significant associations with, e.g., "response to stress," "regulation of apoptosis," and "protein biosynthesis," associations that were absent from these 55 of the 8:2 FTOH genes (Table 6).


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  		Table 6. Functional annotations from DAVID software for genes that were significantly differently expressed only on treatment with either 8:2 FTOH or PFOA

 
Real-time RT-PCR Analysis of mRNA Levels: Validation of Microarray Data
The results shown in Table 7 demonstrate that data obtained with real-time RT-PCR for a selection of mRNAs (Acox1, Acadm, Acp1, Apcs, Apoa4, Cltb, Cpt1, Crot1, Cyp4b1, Herpud1, and Rcn2) mirror expression results obtained by microarray analysis for these mRNAs. This result therefore serves to validate our microarray data. Some discrepancies were observed with respect to extent of increase in expression, particularly with genes showing the higher increase. The direction of change was, however, always consistent.


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  		Table 7. Validation of microarray data with real-time RT-PCR

 
Effects of Treatment with PFOA or 8:2 FTOH on Relative Liver Weights
Treatment of rodents with peroxisomal proliferators invariably causes hepatomegaly (9, 22, 30). In the present study significant hepatomegaly was also found. With 10 mg/kg body wt of either PFOA or 8:2 FTOH, the relative liver weight was 5.8 ± 0.3% (n = 6) and 4.5 ± 0.5% (n = 6), respectively (control value 3.6 ± 0.4%). In rats given 25 mg/kg body wt, the corresponding values were 5.5 ± 0.4% (n = 6) and 5.0 ± 0.7% (n = 6). The mean relative liver weights of rats given 10 mg/kg body wt of PFOA or 8:2 FTOH were also significantly different (P = 0.0006), while the means found in rats given 25 mg/kg body wt were not.

PFOA therefore appears to be the more potent in terms of ability to cause hepatomegaly. In parallel to the dose-response effect in regard to gene expression (Figs. 1 and 2), no significant hepatomegaly was observed when the dosage was increased from 10 to 25 mg/kg body wt.

Hepatic Levels of PFOA in Livers of Treated Rats
In livers of rats treated with 10 mg PFOA/kg body wt for 10 days the mean level of PFOA was 62 µg/g liver wet wt (±7, n = 4). Conversely, the hepatic mean level of PFOA in livers of rats given 10 mg 8:2 FTOH/kg body wt was 32 µg PFOA/g liver wet wt (±3, n = 4). In livers of the treated rats no detectable 8:2 FTOH was found 24 h after injection (level of detection was 20 ng/g liver wet wt), suggesting complete metabolism of injected 8:2 FTOH.

Hepatic Enzyme Activities
Hepatic peroxisomal β-oxidation was assayed in rats given 3 or 25 mg/kg body wt of either PFOA or 8:2 FTOH. As expected, appreciable induction of activity was observed, although this phenomenon has not been previously documented with respect to 8:2 FTOH. With 3 mg/kg body wt, the more potent induction was found with POFA. With 25 mg/kg body wt, further increase in activity was observed with both treatments, now without any significant difference between the treatments (Fig. 9). Hepatic catalase activity was not significantly altered in any of the treated rats (results not shown).


Figure 9
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  		Fig. 9. Effects of treatment with PFOA or 8:2 FTOH on hepatic activities of peroxisomal β-oxidation, palmitoyl-CoA L-carnitine acyltransferase, and carboxyl esterases. Hepatic activities of peroxisomal β-oxidation (A), carboxyl esterase with 4-nitrophenylacetate (4-NFA) or 4-nitrophenylbutyrate (4-NFB) as substrate (B), and palmitoyl-CoA L-carnitine acyltransferase (C) in livers of rat treated with the various dosages of POFA or 8:2 FTOH are shown. The effect on carboxyl esterase activity was assayed only in rats given 25 mg/kg body wt POFA or 8:2 FTOH. The plotted values are means derived from assays of 6 different rat livers, with SD indicated. *Significantly different (P ≤ 0.05) from corresponding control mean. Details of treatments and of enzyme assays are given in MATERIALS AND METHODS.

 
Hepatic carboxylesterase activities are also known to be induced after treatment with perfluorinated carboxylic acids (7). The significant increases in carboxylesterase activities in livers of rats treated with 25 mg/kg body wt of PFOA or 8:2 FTOH (Fig. 9) are therefore in line with earlier reports. Treatment with PFOA gave the higher carboxylesterase activity. After treatment with 8:2 FTOH, a significantly increased activity was only found with 4-nitrophenylbutyrate as substrate (Fig. 9).

With 3 mg/kg body wt, only PFOA brought about induction of hepatic palmitoyl-CoA L-carnitine transferase activity, while with 25 mg/kg body wt both 8:2 FTOH and PFOA caused induction of activity. The higher increase in activity was observed in rats treated with PFOA (Fig. 9).

Phenotypic Anchoring of Expression Data
Phenotypic anchoring was attempted for the 441 and 105 genes with significantly altered expression (P ≤ 0.05) after treatment with PFOA or 8:2 FTOH, respectively. To this end, normalized mean values of expression and of measured phenotypic parameters were used in hierarchical clustering analysis with Spotfire software.

The results suggested that a group of 12 of the PFOA population of genes (Aco1, Ephx2, G6pd, Gale, Gpd1, Pfkfb, Psmb2, Rabep, Serp1, Slc25a39, Srebf1, Tspo) clustered closely to peroxisomal β-oxidation, hepatomegaly, and palmitoyl-CoA L-carnitine transferase activity (results not shown). With Ingenuity Pathways Analysis, all but one (Slc25a39) of these genes were found in a network involving Srebf1 (Fig. 10).


Figure 10
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  		Fig. 10. Cluster of genes detected on phenotypic anchoring of genes with significantly changed expression after treatment with PFOA. The 441 genes with significantly altered expression across all dosages of PFOA used were subjected to phenotypic anchoring to measured changes in palmitoyl-CoA L-carnitine transferase activity, activity of peroxisomal β-oxidation, and hepatomegaly with the hierarchical clustering facility of Spotfire Functional Genomics. A group of genes (Aco1, Ephx2, G6pd, Gale, Gpd1, Pfkfb, Psmb2, Rabep, Serp1, Slc25a39, Srebf1, Tspo) were found to cluster close to the phenotypic characteristics used and were clustered as shown with Ingenuity Pathways Analysis. The activity of the genes is shown after a dose of 3 or 10 mg/kg body wt (indicated by red color). The small histograms associated with each of the genes show mean expression ratios (treated/control) after treatment with 3, 10, or 25 mg PFOA/kg body wt.

 
With the 105 genes from 8:2 FTOH-treated animals no such cluster emerged, peroxisomal β-oxidation, hepatomegaly, and palmitoyl-CoA L-carnitine transferase activity now clustering closely to genes coding for peroxisomal proteins, e.g., Acox1, Acaa1, and Dci (results not shown).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Effects of Treatment with Either PFOA or 8:2 FTOH on Hepatic Gene Expression
While treatment with 8:2 FTOH brought about significantly altered expression of a smaller number of genes compared with PFOA, this study also suggested further differences. Some genes, affected by both treatments, showed increased expression with PFOA, while their expression was decreased in rats treated with a higher dose of 8:2 FTOH. These 14 genes (C1s, C4bpb, Cstb, Dgat1, Gale, Mrps36_predicted, Hspbp1, Kif1c, Lypla1, Nucb1, Pgk1, Pim3, Map2k2, and 1 gene with unknown function) are involved in lipid metabolism, innate immunity, and complement pathways. The genes also appeared less responsive to the treatments (Fig. 1, top right).

Diminished expression at the higher dose of 8:2 FTOH (25 mg/kg body wt) was particularly apparent among the genes exhibiting altered expression only on treatment with 8:2 FTOH (Fig. 3). Pathways analysis suggested significant associations with these genes and, e.g., "gene expression," "cell signaling," "cell cycle," "amino acid metabolism," and "lipid metabolism" (results not shown). Most of these genes are among those suggested by DAVID software to be associated with regulatory phenomena (Table 6).

Differences in Response Between PFOA and 8:2 FTOH in Terms of Cellular Functions and Canonical and Signaling Pathways
When comparing associations with cellular functions/canonical pathways using the genes altered by treatment with either PFOA or 8:2 FTOH, the resulting associations between subpopulations of genes and various cellular functions/canonical pathways are similar (Figs. 4 and 5). Some relevant differences are, nevertheless, apparent. Categories in which 8:2 FTOH genes exhibited the higher significances (e.g., "lipid metabolism," "small molecule biochemistry," "gene expression," "cellular development," and "cellular growth and proliferation") comprised substantial fractions of genes exhibiting decreased expression with a high dose of 8:2 FTOH (see Figs. 1 and 3). Hence the effect of 8:2 FTOH on these, often regulatory, functions may be inhibitory.

The corresponding analysis carried out using the genes with selectively altered expression in either PFOA- or 8:2 FTOH-treated rats yielded analogous results despite the gene populations now being totally different (Figs. 6 and 7). Again, differences are apparent, e.g., "carbohydrate metabolism" and "free radical scavenging" emerging without any associations to 8:2 FTOH genes (Fig. 6), suggesting that the corresponding associations found for 8:2 FTOH with the complete gene populations (Fig. 4) were due to genes affected by both treatments. As regards canonical pathways, the genes from PFOA-treated rats invariably showed the higher associations (Fig. 7). Apparently, treatment with PFOA stimulated mitochondrial metabolism more than treatment with 8:2 FTOH, as suggested by the absence of associations for 8:2 FTOH genes and "oxidative phosphorylation," "citrate cycle," and "glycine, serine and threonine metabolism." This also applies to "glutathione metabolism." In summary, the analysis suggests more extensive stimulation of oxidative metabolism, and consequent higher oxidative stress, in PFOA-treated livers (Fig. 7).

With the two complete gene populations only a single significant association was found for signaling pathways, i.e., "protein ubiquitination pathway" with PFOA genes (Fig. 8). This is likely to correlate with the associations found with "protein degradation" shown in Figs. 4 and 6. When the genes selectively affected only by treatment with either PFOA or 8:2 FTOH were analyzed, additional significant associations appeared for PFOA genes, i.e., that of "complement and coagulation cascades" showing the higher level of significance (Table 5 and Fig. 8).

Observed effects of treatment with PFOA on expression of individual genes are on the whole in agreement with those reported by Guruge et al. (13). It is also reassuring to note agreement between conclusions from pathways analysis/functional annotation clustering carried out with Ingenuity and DAVID software (Table 6). These results also show that treatment with 8:2 FTOH, in contrast to PFOA, caused significant changes in expression of genes associated with signaling. A similar phenomenon was suggested by pathways analysis, as indicated by the higher significances of associations with genes from 8:2 FTOH-treated rats for related cellular functions, i.e., "gene expression," "cellular development," and "cellular growth and proliferation" (Figs. 4 and 6) and also "cell signaling" (Fig. 6). It is in this context noteworthy that the estrogen-like effect of 8:2 FTOH was not found with PFOA (23). Also, perfluorinated fatty acids, including PFOA, have been found to inhibit intercellular gap junctional communication (32). No link to this effect is, however, inferred from data presented here.

Changes in Relative Liver Weight, Hepatic Enzyme Activities, and Gene Expression
In terms of changes in gene expression, treatment with PFOA is clearly more potent than treatment with 8:2 FTOH. Similarly, on a dosage basis treatment with PFOA induced more hepatomegaly and gave a more powerful dose-response as regards induction of enzyme activities (Fig. 9). The extent to which this effect of 8:2 FTOH is direct, or mediated via metabolites, e.g., PFOA, is unclear.

While effects of treatment with PFOA on various enzyme activities are well established (18, 26), little information regarding such effects of 8:2 FTOH is available. Although catalase activity was not altered by the treatments (results not shown), expression of the Cat gene was significantly increased in PFOA-treated rats (Table 3). Effects on enzyme activities may therefore deviate from changes in expression of a corresponding gene.

While activity of peroxisomal β-oxidation was significantly increased with both treatments, the higher potency of PFOA is demonstrated by the higher activities in rats given the lower dosage (3 mg/kg body wt) (Fig. 9).

The absence of significant differences between these treatments at the high dosage (25 mg/kg body wt) suggests that a saturating dosage had been administered (Fig. 9). This is similar to the saturating effect on gene expression observed as the dosage of PFOA, in particular, was increased from 10 to 25 mg/kg body wt (Figs. 1 and 2).

Palmitoyl-CoA L-carnitine acyltransferase activity was consistently higher in livers of rats treated with PFOA (Fig. 9). This correlates with increased expression of Cpt1a, Cpt1b, and Cpt2 induced by PFOA and Cpt1a by 8:2 FTOH (Table 2). Similar findings regarding treatment with PFOA have been reported (13).

The higher carboxyl esterase activities in treated rats, particularly in rats given PFOA (Fig. 9), are in agreement with findings of Derbel et al. (7). Which gene(s) codes for these enzyme-proteins is less clear. No significantly increased expression was found for Ces1, Ces2, or Ces3 (results not shown). Expression of Ct1e (not included in any Ingenuity functional category) was, as expected (17), powerfully increased by either treatment (results not shown). Ct1e is therefore a candidate gene.

It is likely that these different effects must result from differential inductive effects of PFOA and 8:2 FTOH on hepatic gene expression. Our measurements of levels of PFOA in livers of treated rats demonstrate that 8:2 FTOH is quickly converted into PFOA and that remaining 8:2 FTOH is completely metabolized within 24 h after injection of the last dose of 8:2 FTOH. Similar results have been reported with mice injected with 8:2 FTOH (15). At this stage it is difficult to delineate direct effects mediated by 8:2 FTOH from effects due to metabolites of 8:2 FTOH, e.g., PFOA.

Phenotypic anchoring of expression data provided some further insight as regards effects caused by administration of PFOA or 8:2 FTOH. These results suggested that treatment with PFOA activated a gene network in which Srebf1 exhibits a key position (Fig. 10), a phenomenon not found with data from mice treated with 8:2 FTOH. This indicates that expression of Srebf1 is involved after treatment with PFOA but not with FTOH. Bioinformatic analysis suggested that genes in this cluster were extensively associated with regulation of cellular growth/apoptosis (results not shown), and they may therefore be associated with PFOA-dependent hepatomegaly.

Through alternative splicing the Srebf1 gene codes for two proteins, SRBP-1a and SRBP-1c, both stimulating transcription of genes coding for enzymes involved in carbohydrate metabolism and/or in lipogenesis (for review see Ref. 8). Transcriptional regulation of the Srebf1 gene is not fully understood, although transcriptional stimulation by insulin, or by sterol depletion or the LXR{alpha} receptor, has been demonstrated (8). The PFOA-dependent increase in transcription of Srebf1 observed in the present study (Fig. 9) may therefore be due to hypercholesterolemia caused by PFOA acting as a peroxisome proliferator-activated receptor-{alpha} (PPAR-{alpha}) agonist (3). The finding that the level of Srebf1 mRNA is only significantly increased if animals are given at least 10 mg PFOA/kg body wt (Fig. 9) supports this suggestion.

Concluding Remarks
The results demonstrate a dose-response effect with respect to hepatomegaly, hepatic enzyme activities, and increased gene expression in the sense that all measured parameters approached saturation with a dosage of 10 mg/kg body wt and higher. Treatment with PFOA appears to cause the more extensive alterations in cellular functions, particularly as regards oxidative metabolism and cellular stress. Treatment with 8:2 FTOH appeared to stimulate regulatory functions in a manner not observed with PFOA treatment. These differences were somewhat unexpected because 8:2 FTOH in the liver presumably is metabolized to PFOA. An acute, exogenous dose of PFOA therefore appears to exert a different, and more potent, effect on gene expression compared with PFOA generated from metabolism of an equivalent amount of injected 8:2 FTOH. This more potent effect of PFOA may, indirectly, be mediated thorough activation of Srebf1 expression.

The human response to PPAR-{alpha} agonists, e.g., PFOA, is less robust compared with that of rodents (for review see Ref. 16). Extrapolations regarding risks to humans from exposure to low levels of PFOA/8:2 FTOH based on studies with rodents will therefore be uncertain. Both PFOA and 8:2 FTOH, however, are persistent environmental pollutants (14) with potentially deleterious effects on susceptible animal species.


   ACKNOWLEDGMENTS
 
The skilful technical assistance of Bente Gehrken and Toril Woldene is highly appreciated.


   FOOTNOTES
 
Address for reprint requests and other correspondence: H. Osmundsen, Inst. for Oral Biology, Box 1052 Blindern, Univ. of Oslo, 0316 Oslo, Norway (e-mail: haraldo{at}odont.uio.no).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


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 DISCUSSION
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