Arch Environ Contam Toxicol (2015) 68:148–158 DOI 10.1007/s00244-014-0058-y
Assessment of Urinary Metabolite Excretion After Rat Acute Exposure to Perfluorooctanoic Acid and Other Peroxisomal Proliferators Marc Rigden • Guillaume Pelletier • Raymond Poon • Jiping Zhu • Christiane Auray-Blais • Rene´ Gagnon • Cariton Kubwabo • Ivana Kosarac • Kaela Lalonde • Sabit Cakmak • Bin Xiao • Karen Leingartner • Ka Lei Ku • Ranjan Bose • Jianli Jiao Received: 11 March 2014 / Accepted: 26 May 2014 / Published online: 12 July 2014 Ó Her Majesty the Queen in Right of Canada 2014
Abstract Perfluorooctanoic acid (PFOA) is a persistent environmental contaminant. Activation of the peroxisome proliferator activated receptor alpha (PPARa) resulting from exposure to PFOA has been extensively studied in rodents. However, marked differences in response to peroxisome proliferators prevent extrapolation of rodent PPARa activation to human health risks and additional molecular mechanisms may also be involved in the biological response to PFOA exposure. To further explore the potential involvement of such additional pathways, the effects of PFOA exposure on urinary metabolites were directly compared with those of other well-known PPARa agonists. Male rats were administered PFOA (10, 33, or 100 mg/kg/d), fenofibrate (100 mg/kg/d), or di(2-ethylhexyl)phthalate (100 mg/kg/d) by gavage for 3 consecutive days and allowed to recover for 4 days, and overnight urine was collected. Greater urinary output was observed exclusively in PFOA-treated rats as the total fraction of M. Rigden G. Pelletier R. Poon J. Zhu C. Kubwabo I. Kosarac K. Lalonde S. Cakmak B. Xiao K. Leingartner K. L. Ku Environmental Health Science and Research Bureau, Health Canada, Ottawa, ON K1A 0L2, Canada G. Pelletier (&) Environmental Health Centre, 50 Colombine Driveway, P.L. 0803B Tunney’s Pasture, Ottawa, ON K1A 0L2, Canada e-mail: [email protected] C. Auray-Blais R. Gagnon Department of Pediatrics, Faculty of Medicine and Health Sciences, Universite´ de Sherbrooke, Sherbrooke, QC J1H 5N4, Canada R. Bose J. Jiao New Substances Assessment and Control Bureau, Health Canada, Ottawa, ON K1A 0K9, Canada
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PFOA excreted in urine increased with the dose administered. Assessment of urinary metabolites (ascorbic acid, quinolinic acid, 8-hydroxy-2’-deoxyguanosine, and malondialdehyde) provided additional information on PFOA’s effects on hepatic glucuronic acid and tryptophan-nicotinamide adenine dinucleotide (NAD) pathways and on oxidative stress, whereas increased liver weight and palmitoylCoA oxidase activity indicative of PPARa activation and peroxisomal proliferation persisted up to day five after the last exposure.
Because of their chemical stabilities and physical properties, perfluorinated compounds are used as surfactants in a wide range of industrial and consumer products. Perfluorooctanoic acid (PFOA) is a persistent contaminant ubiquitously present in the environment and wildlife (Butt et al. 2010; Houde et al. 2006). Although its production is gradually being phased out, PFOA also appears to be a degradation product of other perfluorinated compounds such as 1H, 1H, 2H, 2H perfluorodecanol (8:2 FTOH) (Keranen et al. 2013; Post et al. 2012). Population-biomonitoring studies from America (Seals et al. 2011), Europe (Wilhelm & Holzer 2012), and Asia (Harada et al. 2010) uncovered national and regional differences in serum PFOA levels suggesting that factors associated with the production, sources, and history of exposure play important roles in determining PFOA serum levels (Lindstrom et al. 2011). PFOA is an agonist of the peroxisome proliferator activated receptor alpha (PPARa) (Kennedy et al. 2004) the activation of which in rodents can lead to liver peroxisomal proliferation, hepatomegaly, and hepatocarcinogenesis (Gonzalez & Shah 2008). In cell-based in vitro transactivation assays, PFOA activates PPARa at the 10–100 lM dose range (Maloney & Waxman 1999; Takacs & Abbott
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Fig. 1 Schematic representation of the animal exposure phase
2007; Vanden Heuvel et al. 2006), a potency similar to the pharmacological PPARa agonist fenofibrate (Klaunig et al. 2003). However, PFOA can also interact with additional receptors (Bjork et al. 2011; Peters and Gonzalez 2011), and the exact role and contribution of PPARa activation are not as clearly defined for the increased incidence of Leydig cell and pancreatic acinar cell tumors and the reproductive, developmental, and immunological toxicities observed in rodents (DeWitt et al. 2012; Klaunig et al. 2012; Lau 2012; Post et al. 2012; Stahl et al. 2011; Xie et al. 2002). There are also marked differences between the responses of human and rodent PPARa to PFOA (Albrecht et al. 2013; Bjork & Wallace 2009; Nakamura et al. 2009), which prevents the direct extrapolation of PPARa activation in rodents to human health risks (Guyton et al. 2009; Klaunig et al. 2012; Rosen et al. 2009). To gain further insights into the molecular and metabolic pathways affected by PFOA, we directly compared the excretion of urinary metabolites resulting from exposures to PFOA and other well-known activators of PPARa. Adult male rats were exposed for 3 consecutive days to PFOA, the pharmacological PPARa agonist fenofibrate, or di(2-ethylhexyl)phthalate (DEHP), a plasticizer which is both a PPARa activator and an endocrine disruptor (Gentry et al. 2011). Rats were then allowed to recover for 4 days, and overnight urine was collected. Urinary metabolites providing clues on oxidative stress status and hepatic functions allowed us to monitor and compare the effects of PFOA and other PPARa agonists over time. At necropsy on day 5 after the last exposure, gross examination of major organs was performed and their weight recorded. Liver and serum end points generally associated with PPARa activation and peroxisomal proliferation were measured.
Materials and Methods Animal Treatment Male Sprague-Dawley rats were purchased from Charles River Laboratories (St. Constant, Quebec, Canada) and housed individually in Health Guard cages (Research Equipment, Bryant, Texas, USA) at 22 ± 1 °C and 50 ± 10 % humidity on a 12 hour light cycle (7:00 AM to
7:00 PM) and fed Teklad 2014 rodent diet (Harlan Laboratories, Indianapolis, Indiana, USA) and water ad libitum. Rats were allowed to acclimatize to the facility for 1 week. The animal study protocol was approved by the Health Canada Animal Care Committee and performed according to the guidelines of the Canadian Council on Animal Care. Thirty rats weighing 284 ± 11 g were randomly divided into groups of five, corresponding to the six treatment groups (control, fenofibrate, DEHP, low-dose PFOA [10 mg/kg], medium-dose PFOA [33 mg/kg], and highdose PFOA [100 mg/kg]). Treatment was divided into three phases (see Fig. 1) as follows: (1) baseline phase: All animals were dosed by gavage with 1.0 ml/100 g body weight (bw) of corn oil (Mazola; ACH foods, Memphis, Tennessee, USA) on the mornings of days 1, 2, and 3; (2) dosing phase: on day 4, 5, and 6 animals were administered corn oil containing 100 mg/kg bw fenofibrate, 100 mg/kg bw DEHP, or PFOA at 10, 33, and 100 mg/kg bw (all purchased from Sigma-Aldrich, St. Louis, Missouri, USA); control animals received corn oil only; and (3) recovery phase: animals were then kept for 4 more days with no further dosing. BWs were measured daily, and overnight (4:00 PM to 8:00 AM) urine was collected in ice chilled containers and stored at -80 °C. On the morning of day 11, animals were anesthetized with isoflurane. Serum specimens were prepared and stored at -80 °C. Major organs were weighed. Liver homogenates were prepared in 0.05 M tris/0.15 % KCl (pH 7.4) buffer at 1 g/1.5 ml. Part of the homogenates were centrifuged at 90009g for 10 min to obtain supernatants. Liver homogenates and supernatants were stored at -80 °C until use. Liver Enzymatic Activities and Serum Chemistry Liver 90009g supernatants were used to assess ethoxyresorufin-O-deethylase (EROD) activity as described by Lubet et al. (1985), pentoxyresorufin-O-dealkylase (PROD) activity according to Burke et al. (1985) and glutathione-S-transferases (GST) according to the protocol of Habig et al. (1974). Liver homogenates were used to measure uridine 5’-diphospho-glucuronosyltransferase (UDP-GT) activity as described by Burchell and Weatherill (1981) and palmitoyl-CoA oxidase activity according to Small et al. (1985). Quantitative analyses of the following
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serum analytes were performed with an ABX Pentra 400 clinical chemistry analyzer using Horiba ABX CE marked liquid chemistry reagents (Horiba ABX, Irvine, California, USA) according to the manufacturer’s instructions: calcium (Cat# A11A01633), magnesium (Cat# A11A01646), inorganic phosphate (Cat# A11A01665), aspartate aminotransferase (Cat# A11A01629), alkaline phosphatase (Cat# A11A01626), total protein (Cat# A11A01867), urea nitrogen (Cat# A11A01641), creatinine (Cat# A11A01868), glucose (Cat# A11A01667), cholesterol (Cat# A11A01634), and triglycerides (Cat# A11A01640). Serum uric acid was determined using a colorimetric assay from Pointe Scientific (Canton, Michigan, USA) according to manufacturer’s instructions.
Determination of Ascorbic and Quinolinic Acids in Urine Urine total ascorbic acid (ascorbic and dehydroascorbic acids) was measured with an automated enzymatic procedure (Lee et al. 1997). Urine quinolinic acid (QA) was analyzed by liquid chromatography (LC)/mass spectrometry (MS) (Liao et al. 2010). Urine creatinine was measured using a Roche creatinine PAP assay kit (Roche Diagnostics, Laval, Quebec, Canada). All urinary analyte concentrations were normalized against urinary creatinine.
Determination of MDA and 8-OHdG in Urine For determination of urinary MDA, all containers and glassware were first washed in soap-free detergent and then rinsed with distilled water and ethanol to decrease background interference. Urine sample (2 ll) was added to a water solution (1 mL) containing 1 % (v/v) ethanol and 0.02 % (w/v) 2,6,di-tert-4-methyl-phenol in a screw-cap tube and well mixed. A 1:1 (water:acetic acid) solution containing 0.33 % (v/v) thiobarbituric acid (250 ll) was then added. The tube was capped, the solution well mixed, and derivatization allowed to proceed for 1 h at 95 °C. After cooling to room temperature, 1.25 ml n-butanol was added and mixed thoroughly before centrifugation at 30009g. The MDA reactive fraction extracted in the n-butanol phase and the standard curve were measured by a fluorescent high-performance liquid chromatography (HPLC) method using a Brava C18-ODS column, 250 9 4.6 cm, 5 lm (Mandel Scientific, Guelph, Ontario, Canada) according to a procedure described by Agarwal and Chase (2002). For the determination of 8-hydroxy-2’deoxyguanosine (8-OHdG), urine was first filtered through a 30,000 Da Ultrafree-MC filter (EMD Millipore, Billerica, Massachusetts, USA). The filtrate was then used for the
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assay of 8-OHdG using an enzyme-linked immunosorbent assay (Cosmo Bio, Carlsbad, California, USA). PFOA Quantification in Urine PFOA and 13C4-PFOA standards were purchased from Wellington Laboratories (Guelph, Ontario, Canada). Ammonium acetate (ACS grade) was purchased from BDH (Toronto, Ontario, Canada). OmniSolv acetonitrile, methanol, and HPLC-grade water were obtained from EMD Chemicals (Gibbstown, New Jersey, USA). Methyl tertbutyl ether (MTBE), sodium bicarbonate, and tetrabutylammonium bisulfate were purchased from Sigma-Aldrich. Ten ll of a 1000 ng/ml mixture of internal standards containing 13C-PFOA was added to 1 ml of rat urine and gently mixed. One milliliter of a 0.5 M tetrabutylammonium bisulfate solution (adjusted to pH 10) was added with gentle vortexing to the spiked urine followed by 2 ml of carbonate/bicarbonate buffer, and the solution was again gently mixed. Next, 5 ml of MTBE was added to the spiked urine and the mixture vortexed. The organic and aqueous phases were separated by centrifugation at 3500 rpm for 10 min. The top organic layer was transferred into a clean 15 ml polypropylene tube. Another 5 ml of MTBE was added; the tube was then capped, inverted, and centrifuged for 5 min; and the top organic layer was collected. This procedure was repeated twice. Using a gentle stream of nitrogen, samples were evaporated to dryness at 30 °C and then reconstituted in 1 ml of HPLCgrade water. The extracts were then loaded onto a preconditioned (9 ml methanol and 5 ml of HPLC-grade water) 200 mg Waters Oasis HLB glass cartridge (Milford, Massachusetts, USA). Solid-phase extraction cartridges were allowed to dry for 15 min before solvent elution. The analyte was eluted using 10 mL of methanol. The extracts were concentrated to dryness under a gentle stream of nitrogen and reconstituted in 200 ll of acetonitrile followed by 800 ll of HPLC grade water. The extracts were then transferred by way of precleaned pasteur pipettes before LC/MS/MS analysis. The chromatographic separation of extracts was performed using a Finnigan Surveyor Plus HPLC System (Thermo Electron, San Jose, California, USA). Separation was achieved using a Discovery HS C18 (7.5 cm 9 2.1 mm 9 3 lm) precolumn followed by the analytical column consisting of a Discovery HS C18 column (7.5 cm 9 2.1 mm 9 3 lm). An opti-guard C18 column (10 mm 9 1 mm 9 3 lm) (Sigma-Aldrich) was installed between the pump and the analytical column to trap and delay perfluorinated compounds originating from the solvents and the pumping system. The mobile phase consisted of 20 % acetonitrile in 2 mM ammonium acetate (A) and 90 % acetonitrile in 2 mM ammonium acetate (B). Solvents were not degassed online as the online degasser
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contains teflon, which constitutes for a large source of perfluorinated compounds contamination. The mobile phase was therefore degassed by sparging a gentle flow of ultra-high purity helium through it. Once degassed, the mobile phases were passed directly to the LC pump. Polypropylene tubing supplied the helium gas flow from the helium tank to the solvents mobile-phase reservoirs, and the flow was controlled by a gas regulator. The column temperature was maintained at 40 °C. The gradient elution started with 100 % A for 1 min followed by an 8 min linear gradient to 100 % B, then a 3 min hold at 100 % B, and returned back to 100 % A in 4 min at a flow rate of 200 ml/min. The system was equilibrated for 4 min at the initial conditions before the next injection. Sample injection volume was 25 ll. MS experiments were performed using a Thermo Finnigan TSQ Quantum Ultra EMR triplequadrupole mass spectrometer (Thermo Electron). The samples were analyzed in negative ion electrospray ionization using selective reaction monitoring mode. Xcalibur version 2 (Thermo Electron) was used for data acquisition and processing. The following transitions were monitored: m/z 412.9 ? 368.9 and m/z 416.9 ? 372.0 for PFOA and 13 C4-PFOA (IS), respectively. The method detection limit (MDL) was determined according to United States Environmental Protection Agency Regulation 40 CFR part 136 (Appendix B) method. The limit of quantitation (LOQ) was calculated as ten times the SD associated with seven replicates. The MDL and LOQ were 0.085 and 0.283 ng/ml, respectively, and the average percent recovery was 110.77 %. Statistical Analysis Data set normality was assessed by Shapiro–Wilk test and homogeneity of variance by Levene’s test using SigmaPlot 11.2 (Systat, Chicago, Illinois, USA). End points assessed at the terminal necropsy that satisfied normality and homoscedasticity assumptions were analysed by one-way analysis of variance (ANOVA) followed by Dunnett post hoc test. Data sets that did not satisfy normality and homoscedasticity assumptions were log-transformed, and if they still failed to meet these assumptions, they were then analysed by nonparametric Kruskal–Wallis ANOVA followed by Mann–Whitney U-test. Repeated daily measures did not satisfy normality and homoscedasticity assumptions and were analyzed by Friedman’s nonparametric randomized block ANOVA, which is a nonparametric version of a one-way ANOVA with repeated measures followed by Friedman post hoc test. Test statistics for Friedman’s test is chi-square with the number of repeated measure as degrees of freedom. Differences between control and treatment groups were considered statistically significant at p \ 0.05.
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Fig. 2 Growth curves of the control, fenofibrate, DEHP, and PFOA treatment groups. Error bars on the control and selected treatment groups represent SDs. Treatment groups labeled by # in legend were significantly different from the control group according to nonparametric repeated measure ANOVA, while * signals a significant difference from control group values on a given day, using Friedman’s nonparametric randomized block ANOVA and a p \ 0.05 cut-off for statistical significance
Results Rat body weights decreased after exposure to the medium (33 mg/kg) and high (100 mg/kg) PFOA doses (Fig. 2), but only the latter growth curve was statistically different from the control group (as assessed by Friedman’s nonparametric randomized block ANOVA). No additional symptom of toxicity was observed in any of the PFOA-treated animals, and growth quickly resumed after the last day of exposure. Although there was a slight decrease in food consumption (data not shown), dehydration is the most likely explanation for weight loss because overnight urine volumes were markedly increased, i.e., peaking at 2.5 and 4 times the control urine volume on day 6 for the medium and high PFOA doses, respectively (Fig. 3a). Water consumption peaked at almost two times control value one day later in the high PFOA treatment group (Fig. 3b). Increased water intake was also noted in the medium PFOA treatment group over the next few days, but it did not reach statistical significance. PFOA excretion in urine peaked on day 7 reaching 42, 148, and 517 lg PFOA/ml urine for the low, medium, and high PFOA treatment groups, respectively (Fig. 4a). Because PFOA exposure significantly increased urine output (Fig. 3a), PFOA excretion was also expressed as a ratio on urinary creatinine, which peaked 1 day sooner at 49, 673, and 3240 lg PFOA/mg creatinine in the same treatment groups (Fig. 4b). Interestingly, whereas raw PFOA concentrations in the urine of the low, medium, and high PFOA treatment groups were approximately proportional to the doses administered, creatinine–normalized
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Fig. 3 (a) Overnight urine volume and (b) daily water consumption in controls and animals treated with fenofibrate, DEHP, and PFOA. Error bars on the control and selected treatment groups represent SDs. Treatment groups labeled by # in legend were significantly different from the control group according to nonparametric repeated measure ANOVA, while * signals a significant difference from control group values on a given day, using Friedman’s nonparametric randomized block ANOVA and a p \ 0.05 cut-off for statistical significance
PFOA levels suggested increasing rates of excretion at greater PFOA exposure levels. This hypothesis is further supported by calculation of the total PFOA excretion over the last 6 overnight urine collections assessed, which corresponded to 17 ± 6 %, 36 ± 10 %, and 56 ± 14 % of the total dose administered in the low, medium, and high
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PFOA treatment groups, respectively. Despite large variations in urine volume, the total amount of creatinine excreted in overnight urine collections remained relatively constant; consequently, creatinine was used to normalize the concentration of all other urinary metabolites. Daily urinary ascorbic acid (Fig. 5a), 8-OHdG (Fig. 5c), and MDA (Fig. 5d) measurements after PFOA exposure presented similar patterns where increased metabolite levels already noticeable on days 4 or 5 returned to control values by day 8. High-dose PFOA exposure elicited the most consistent and strongest responses followed by medium-dose PFOA exposure. DEHP and low dose PFOA had no effect, whereas fenofibrate significantly affected 8-OHdG level on a single day. The most pronounced effects were observed for urinary ascorbic acid, whose levels noticeably increased right after the first exposure on day 4 and peaked at 5 and 12 times the control values for the medium and high PFOA doses, respectively. MDA levels peaked on day 5 at approximately three and five times the control values in the same treatment groups. Changes were less pronounced for 8-OHdG with a 2.5-fold increase compared with the control values reached on day 6 after exposure to the high PFOA dose. The two-fold increases over the same days after exposure to medium PFOA dose failed to reach statistical significance, whereas exposure to fenofibrate resulted in a statistically significant two-fold increase on day 5. Urinary QA (Fig. 5b) plateaued at four to five times control levels after exposure to the medium and high PFOA doses, whereas the levels in the low PFOA dose gradually increased to 3 times control levels, reaching statistical significance on day 7, 2 days after the initial response to the medium and high PFOA doses. Interestingly, increased QA levels in all PFOA treatment groups persisted up to day 10, whereas the threefold increase observed after exposure to fenofibrate returned to control values. At termination, animals treated with fenofibrate and PFOA presented significantly increased liver weights with
Fig. 4 PFOA concentration in overnight urine expressed (a) on urine volume and (b) on urinary creatinine concentration in control and PFOAtreated rats. Error bars represent SDs
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Fig. 5 Levels of (a) ascorbic acid, (b) QA, (c) 8-OHdG, and (d) MDA in overnight urine samples from rats treated with fenofibrate, DEHP, or PFOA. Error bars on the control and selected treatment groups represent SDs. Treatment groups labeled by # in legend were significantly different from the control group according
to nonparametric repeated measure ANOVA, while * signals a significant difference from control group values on a given day, using Friedman’s nonparametric randomized block ANOVA and a p \0.05 cut-off for statistical significance
Table 1 Final body weights and relative organ weights (% bw) after treatment with fenofibrate, DEHP, and PFOAa Treatment
Dose (mg/kg)
Body weight (g)
Liver
Heart
Brain
Kidneys
Testis
Thymus
Spleen
Control
0
363 ± 17
4.28 ± 0.20
0.33 ± 0.02
0.54 ± 0.03
0.81 ± 0.02
0.85 ± 0.04
0.19 ± 0.04
0.21 ± 0.03
Fenofibrate
100
357 ± 20
5.73 ± 0.24*
0.34 ± 0.02
0.55 ± 0.03
0.85 ± 0.06
0.86 ± 0.08
0.19 ± 0.03
0.23 ± 0.04
DEHP
100
349 ± 27
4.14 ± 0.34
0.31 ± 0.01
0.59 ± 0.06
0.80 ± 0.05
0.88 ± 0.05
0.21 ± 0.04
0.22 ± 0.04
PFOA
10
356 ± 32
5.73 ± 0.29*
0.31 ± 0.01
0.59 ± 0.04
0.85 ± 0.05
0.87 ± 0.07
0.21 ± 0.06
0.21 ± 0.01
a
33
349 ± 11
6.40 ± 0.34*
0.29 ± 0.01
0.57 ± 0.03
0.79 ± 0.13
0.88 ± 0.09
0.19 ± 0.04
0.22 ± 0.01
100
330 ± 26
6.62 ± 0.47*
0.30 ± 0.02
0.61 ± 0.06
0.87 ± 0.07
0.90 ± 0.04
0.14 ± 0.02
0.22 ± 0.01
Mean ± SD of five animals per group
* One-way ANOVA followed by Dunnett post hoc test; significantly different from control at p \ 0.05
the dose-related increases in the PFOA treatment groups being the most pronounced (Table 1). A dose-related increase in palmitoyl-CoA oxidase activity was also observed in PFOA-treated animals, whereas the modest increases noted in fenofibrate and DEHP-treated rats were not statistically significant (Table 2). Increased hepatic EROD and PROD activities were observed in all PFOAtreated rats, but this reached statistical significance only at
the highest exposure. UDP-glucuronosyltransferase activities were significantly decreased in the fenofibrate and PFOA treatment groups, whereas decreased GST activities were also observed in fenofibrate-treated rats and at medium and high PFOA doses (Table 2). Most of the clinical chemistry parameters assessed in serum at study termination were not significantly affected (Table 3). However, uric acid levels were significantly decreased in the DEHP
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Table 2 Effects of fenofibrate, DEHP, and PFOA on liver enzymesa Treatment
Dose (mg/kg)
Palmitoyl-CoA oxidase (abs/min/g prot)
Phase I drug-metabolising enzymes
Phase II drug-metabolising enzymes
EROD (nmole/min/ mg prot)
UDP-GT (nmole/ min/mg/prot)
PROD (nmole/min/ mg prot)
GST (umole/min/ mg prot)
Control
0
1.02 ± 0.37
0.066 ± 0.020
0.045 ± 0.012
1.69 ± 0.12
1.21 ± 0.11
Fenofibrate
100
1.71 ± 0.58
0.060 ± 0.012
0.046 ± 0.020
1.09 ± 0.14*
0.94 ± 0.13*
DEHP PFOA
100 10
1.40 ± 1.09 3.17 ± 0.65*
0.060 ± 0.016 0.096 ± 0.024
0.039 ± 0.031 0.080 ± 0.024
1.45 ± 0.35 0.88 ± 0.09*
1.27 ± 0.11 1.11 ± 0.13
33
4.89 ± 0.79*
0.080 ± 0.015
0.078 ± 0.031
1.00 ± 0.14*
0.88 ± 0.12*
100
6.11 ± 1.51*
0.113 ± 0.025*
0.107 ± 0.029*
1.12 ± 0.20*
0.94 ± 0.19*
a
Mean ± SD of five animals per group
* One-way ANOVA followed by Dunnett post hoc test; significantly different from controls at p \ 0.05
treatment group and at all PFOA dose levels, again in a dose-dependent manner. Although statistically significant, the increased cholesterol level in fenofibrate-treated rats and alanine aminotransferase activity at medium PFOA dose were most likely spurious observations of dubious biological relevance.
Discussion Although the expected effects of PPARa agonists on serum cholesterol and triglyceride levels were not seen, other hallmark features—such as increased liver weight and hepatic palmitoyl-CoA oxidase activity—were observed at study termination (Tables 1 and 2). This uneven response from parameters usually associated with PPARa activation may have been caused by the different potencies and pharmacokinetics of the compounds tested (Eason et al. 1990; Lau et al. 2007; Pollack et al. 1985), by the short exposure window, or by the 4 days allowed for urine collection before the final necropsy. Despite this, the overall response to PFOA (and, to a lesser extent, fenofibrate and DEHP) was generally compatible with PPARa activation. The impaired bw gains at high PFOA exposure (Fig. 2) were most likely caused by dehydration because severe polyuria was observed in the same treatment group (Fig. 3a). Polyuria quickly subsided after the last day of exposure, and a delayed compensatory increase in water intake was observed (Fig. 3b). Although not statistically significant, the same trends were observed for the medium PFOA treatment group. From day 8 onward, growth resumed at a pace similar to the control group; by the end of the recovery period, urinary volumes and creatinine levels returned to control values suggesting that PFOA’s effects were transient. Although increased urinary output after PFOA administration has been reported before (Goecke et al. 1992), this
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noninvasive end point is infrequently monitored, and this aspect of PFOA toxicity remains relatively unexplored. The severe polyuria observed in the high-dose PFOA treatment group (Fig. 3a) occurred in the absence of effects on kidney weight (Table 1) and serum creatinine and blood urea nitrogen (BUN) levels, which may suggest meaningful impairment of renal functions (Table 3). However, kidney hypertrophy and histological alterations have been reported in rats after exposure to 20 mg PFOA/kg bw for 28 days (Cui et al. 2009). PFOA may also affect kidney functions through the activation of other PPAR subtypes (Cohen 2005), but further investigations will be required to determine if the molecular mechanisms underlying polyuria after rat acute PFOA exposure are also involved in kidney histological alterations observed at a subchronic exposure. PFOA measured in overnight urine collections increased from 17 to 36 to 56 % of the total dose administered in the low, medium, and high PFOA treatment group, respectively. Although the single exposure and smaller doses used in most investigations on PFOA pharmacokinetics may not have allowed this observation (Hanhijarvi et al. 1982; Vanden Heuvel et al. 1991), chronic exposure studies using greater doses also reported increased excretion rates at higher dose levels (Cui et al. 2010; Hanhijarvi et al. 1987). Physiologically based pharmacokinetic models that take into consideration saturable PFOA renal reabsorption (Loccisano et al. 2012) may benefit from a better characterization of the large variations of urinary output after PFOA exposure. Urate transporter 1 (URAT1) is a member of the organic anion transporter family involved in the renal reabsorption of PFOA (Weaver et al. 2010; Yang et al. 2010). URAT1 also mediates the reabsorption of uric acid in the kidney (Enomoto et al. 2002). At high concentrations, PFOA can interfere with uric acid reabsorption (Weaver et al. 2010; Yang et al. 2010) potentially explaining the lower serum uric acid levels observed in PFOA-treated rats (Table 3).
Mean ± SD of five animals per group
* One-way ANOVA followed by Dunnett post hoc test; significantly different from control at p \ 0.05
a
0.90 ± 0.57*
0.73 ± 0.31* 92 ± 25
65 ± 26 45 ± 7
42 ± 4 175 ± 4
185 ± 5 69 ± 8*
53 ± 5 156 ± 18
140 ± 23 393 ± 81
155
343 ± 19 0.37 ± 0.04
0.38 ± 0.04 17.2 ± 1.2
15.0 ± 1.9 5.6 ± 0.3
5.5 ± 0.3 10.2 ± 0.9
11.1 ± 0.9
2.1 ± 0.2
2.2 ± 0.1
10.7 ± 0.4
10.7 ± 0.6
33
100
1.27 ± 0.63*
1.02 ± 0.25* 81 ± 46
97 ± 41 51 ± 9
36 ± 18 166 ± 32
169 ± 16 60 ± 12
63 ± 3 140 ± 17
172 ± 38 311 ± 92
362 ± 99 0.32 ± 0.05
0.37 ± 0.07 16.1 ± 2.7
14.6 ± 2.6 5.2 ± 0.9
5.5 ± 0.2 9.6 ± 0.8
9.5 ± 1.8 1.9 ± 0.3
2.1 ± 0.2
10 PFOA
9.7 ± 1.8
100 DEHP
9.9 ± 1.1
2.33 ± 1.10
1.66 ± 0.37 118 ± 21
124 ± 29 47 ± 15
72 ± 13* 164 ± 17
168 ± 15 51 ± 6
53 ± 16 147 ± 34
129 ± 24 358 ± 118
280 ± 61 0.33 ± 0.04
0.30 ± 0.03 14.2 ± 1.0
16.0 ± 2.3 5.7 ± 0.7
5.2 ± 0.5 9.0 ± 0.8
9.7 ± 1.5 2.0 ± 0.3
2.0 ± 0.2 9.5 ± 1.2
10.0 ± 1.3
0
100
Cont
Feno
Blood urea nitrogen (mg/dl) Total protein (g/ dl) Inorganic phosphate (mg/ dl) Magnesium (mg/dl) Calcium (mg/dl) Dose (mg/ kg) Treatment
Table 3 Serum chemistry after treatment with fenofibrate, DEHP, and PFOAa
Creatinine (mg/dl)
Alkaline phosphatase (U/L)
Aspartate aminotransferase (U/L)
Alanine aminotransferase (U/L)
Glucose (mg/dl)
Cholesterol (mg/dl)
Triglyceride (mg/dl)
Uric acid (mg/dl)
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Fenofibrate can also interfere with URAT1-mediated uric acid reabsorption and lead to lower serum uric acid levels (Uetake et al. 2010). However, the 29 % decrease in serum uric acid level observed in the fenofibrate treatment group was not statistically significant (Table 3). DEHP-treated rats presented significantly lower serum uric acid levels (Table 3), but the interaction of DEHP with URAT1 remains to be characterized. Alternatively, conversion of uric acid to allantoin by hepatic uricase may also contribute to a decrease of serum uric acid levels (Wu et al. 1994). However, reports on DEHP effects on hepatic uricase activity are conflicting because both induction (Jayaraman et al. 1988) and inhibition (Mitchell et al. 1985) were reported, whereas PFOA and fenofibrate do not appear to affect the activity of this hepatic enzyme (Abdellatif et al. 1990; Henninger et al. 1987). Although increased urine excretion in PFOA-treated rats (Fig. 3a) may have played a role in the perturbation of uric acid reabsorption, it was most likely a minor one because the low-dose PFOA, which did not affect urine volumes, already decreased serum uric acid level by 56 % compared with 61 % for the medium and 69 % for the high PFOA treatment groups (Table 3). Increased urinary output may also have affected the excretion of other urinary metabolites. However, all of the metabolites affected after exposure to high-dose PFOA were also significantly affected in the fenofibrate or medium dose PFOA treatment groups on day 5 (Fig. 5) in the absence of significant effects on urinary output (Fig. 3a). Peroxisome proliferator activated receptor alpha agonists can induce oxidative stress in rodent liver, which ultimately contributes to hepatocarcinogenesis (Klaunig et al. 2003). Increased hepatic levels of the biomarker of DNA oxidative damage 8-OHdG have been reported after rat exposure to fenofibrate at 6000 ppm in the diet for 9 weeks (Nishimura et al. 2007) and at 3000 ppm for 13 weeks (Nishimura et al. 2008). Data on PFOA exposure are more equivocal given that increased 8-OHdG level has been reported after exposure to 200 ppm PFOA in food for 2 weeks (Takagi et al. 1991) whereas another study using the same dietary exposure for 9 weeks did not observe such changes (Abdellatif et al. 2003). Assuming a food consumption of 5 g/100 g bw (Pass & Freeth 1993), exposure to 200 ppm PFOA in food corresponds to 10 mg/kg bw approximating our low-dose PFOA treatment group, whereas exposures to 3000 and 6000 ppm fenofibrate represent 150 and 300 mg/kg bw, respectively. In this study, increased urinary 8-OHdG levels were observed only at the highest PFOA dose used (100 mg/kg bw), whereas 8-OHdG levels were significantly increased for a single day in fenofibrate-treated rats (Fig. 5c). Information on the biomarker of lipid peroxidation MDA is more limited and, to our knowledge, this is the first report of
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increased urinary MDA in PFOA-treated rats. The marked increase in MDA excretion (Fig. 5d) clearly suggests lipid peroxidation. Further studies are still needed to correlate urinary MDA and 8-OHdG with oxidative stress occurring in target tissues. Although a dose–response relationships for hepatic phase I and II drug-metabolizing enzymes were not as clear as for palmitoyl-CoA oxidase (Table 2), exposure to highdose PFOA significantly increased EROD and PROD activities suggesting induction of CYP1A1/2 and CYP2B. PFOA exposure also exerted inhibitory effects on UDP-GT and GST activities. These hepatic effects were generally compatible with previous reports (Kawashima et al. 1994, 1995) and may be explained by interaction of PFOA with additional receptors such as the constitutive androstane receptor or the pregnane X receptor (Peters and Gonzalez 2011). It has long been known that exposure to xenobiotics can increase ascorbic acid synthesis and urinary excretion in rats (Longenecker et al. 1940). Although the exact mechanisms involved are not yet fully elucidated, altered UDP-GT and cytochrome P450 activities (most notably CYP2B) have been associated with increased synthesis of ascorbic acid from glucuronic acid (Aranibar et al. 2009; Linster & Van 2007). The increased urinary ascorbic acid levels observed at medium and high PFOA doses (Fig. 5a) may therefore be related to the PFOA-induced perturbation of hepatic UDP-GT and PROD (CYP2B) activities (Table 2). The tryptophan-NAD pathway links tryptophan catabolism via the kynurenine pathway to NADH/NADPH biosynthesis in the liver, thus exerting a significant influence on energy metabolism. PPARa agonists can affect this pathway, resulting in increased urinary levels of metabolites such as n-methylnicotinamide and QA (Delaney et al. 2005; Fukuwatari et al. 2004; Ringeissen et al. 2003; Zhen et al. 2007). The similar four- to fivefold increases compared with control values observed at medium and high PFOA doses may indicate that the response of this parameter had already plateaued (Fig. 5b). QA was also the most sensitive urinary end point identified in this study because it was the only parameter to be significantly affected by the low-dose PFOA and fenofibrate treatment groups according to nonparametric repeated measures ANOVA. In human epidemiological studies, hyperuricemia, decreased glomerular filtration rate, and chronic kidney disease are associated with greater serum PFOA levels (Shankar et al. 2011; Steenland et al. 2010), but it is not yet clear if these increased levels are a cause or a consequence of impaired renal functions (Watkins et al. 2013). These epidemiological observations appear somewhat at odds with the hypouricemia and polyuria reported in this study. However, in addition to legitimate issues about
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extrapolation of PPARa activation in rodent models to human health risks (Guyton et al. 2009; Klaunig et al. 2012; Rosen et al. 2009), there are marked differences in PFOA pharmacokinetics across species (Lau 2012), and human also lacks functional uricase (Uetake et al. 2010) and L-gulonolactone oxidase (Linster & Van 2007) involved in the metabolism of uric acid and ascorbic acid. It is also worth noting that the PFOA doses used in this rat exposure study were several orders of magnitudes greater than actual human exposure (Lau 2012) and that severe polyuria was observed only at the highest PFOA exposure. This high PFOA dose (ten times greater than the low PFOA dose where increases in liver weight and palmitoyl-CoA oxidase activity were unequivocally observed) was also the only dose to robustly affect the excretion of all urinary metabolites assessed in this study. Hence, although the observed polyuria and perturbation of urinary metabolite excretion may provide clues on molecular pathways affected by PFOA, they are unlikely to have meaningful consequences for human health risk assessment of PFOA at environmental levels. In summary, exposure to PFOA led to dose-related increases in liver weight and palmitoyl-CoA oxidase activity, which are characteristic effects of PPARa-induced peroxisomal proliferation. PFOA also affected rat weight gain, a likely result of dehydration caused by a transient increase in urine excretion, which may also impact PFOA pharmacokinetics. The assessment of urinary metabolites identified significant but transitory changes in 8-OHdG, MDA, ascorbic acid and QA levels after exposure to PFOA. These metabolites provided further insights on the effects of acute exposure to PFOA on oxidative stress status and hepatic glucuronic acid and tryptophan-NAD pathways, but further work is needed to clearly delineate the contribution of PPARa and alternative molecular pathways. Acknowledgments This project was funded through Health Canada A-Base. The authors thank Brita Nadeau for excellent technical assistance and Mike Wade and Premkumari Kumarathasan for helpful comments on the manuscript.
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Assessment of Urinary Metabolite Excretion After Rat Acute Exposure to Perfluorooctanoic Acid and Other Peroxisomal Proliferators Marc Rigden • Guillaume Pelletier • Raymond Poon • Jiping Zhu • Christiane Auray-Blais • Rene´ Gagnon • Cariton Kubwabo • Ivana Kosarac • Kaela Lalonde • Sabit Cakmak • Bin Xiao • Karen Leingartner • Ka Lei Ku • Ranjan Bose • Jianli Jiao Received: 11 March 2014 / Accepted: 26 May 2014 / Published online: 12 July 2014 Ó Her Majesty the Queen in Right of Canada 2014
Abstract Perfluorooctanoic acid (PFOA) is a persistent environmental contaminant. Activation of the peroxisome proliferator activated receptor alpha (PPARa) resulting from exposure to PFOA has been extensively studied in rodents. However, marked differences in response to peroxisome proliferators prevent extrapolation of rodent PPARa activation to human health risks and additional molecular mechanisms may also be involved in the biological response to PFOA exposure. To further explore the potential involvement of such additional pathways, the effects of PFOA exposure on urinary metabolites were directly compared with those of other well-known PPARa agonists. Male rats were administered PFOA (10, 33, or 100 mg/kg/d), fenofibrate (100 mg/kg/d), or di(2-ethylhexyl)phthalate (100 mg/kg/d) by gavage for 3 consecutive days and allowed to recover for 4 days, and overnight urine was collected. Greater urinary output was observed exclusively in PFOA-treated rats as the total fraction of M. Rigden G. Pelletier R. Poon J. Zhu C. Kubwabo I. Kosarac K. Lalonde S. Cakmak B. Xiao K. Leingartner K. L. Ku Environmental Health Science and Research Bureau, Health Canada, Ottawa, ON K1A 0L2, Canada G. Pelletier (&) Environmental Health Centre, 50 Colombine Driveway, P.L. 0803B Tunney’s Pasture, Ottawa, ON K1A 0L2, Canada e-mail: [email protected] C. Auray-Blais R. Gagnon Department of Pediatrics, Faculty of Medicine and Health Sciences, Universite´ de Sherbrooke, Sherbrooke, QC J1H 5N4, Canada R. Bose J. Jiao New Substances Assessment and Control Bureau, Health Canada, Ottawa, ON K1A 0K9, Canada
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PFOA excreted in urine increased with the dose administered. Assessment of urinary metabolites (ascorbic acid, quinolinic acid, 8-hydroxy-2’-deoxyguanosine, and malondialdehyde) provided additional information on PFOA’s effects on hepatic glucuronic acid and tryptophan-nicotinamide adenine dinucleotide (NAD) pathways and on oxidative stress, whereas increased liver weight and palmitoylCoA oxidase activity indicative of PPARa activation and peroxisomal proliferation persisted up to day five after the last exposure.
Because of their chemical stabilities and physical properties, perfluorinated compounds are used as surfactants in a wide range of industrial and consumer products. Perfluorooctanoic acid (PFOA) is a persistent contaminant ubiquitously present in the environment and wildlife (Butt et al. 2010; Houde et al. 2006). Although its production is gradually being phased out, PFOA also appears to be a degradation product of other perfluorinated compounds such as 1H, 1H, 2H, 2H perfluorodecanol (8:2 FTOH) (Keranen et al. 2013; Post et al. 2012). Population-biomonitoring studies from America (Seals et al. 2011), Europe (Wilhelm & Holzer 2012), and Asia (Harada et al. 2010) uncovered national and regional differences in serum PFOA levels suggesting that factors associated with the production, sources, and history of exposure play important roles in determining PFOA serum levels (Lindstrom et al. 2011). PFOA is an agonist of the peroxisome proliferator activated receptor alpha (PPARa) (Kennedy et al. 2004) the activation of which in rodents can lead to liver peroxisomal proliferation, hepatomegaly, and hepatocarcinogenesis (Gonzalez & Shah 2008). In cell-based in vitro transactivation assays, PFOA activates PPARa at the 10–100 lM dose range (Maloney & Waxman 1999; Takacs & Abbott
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Fig. 1 Schematic representation of the animal exposure phase
2007; Vanden Heuvel et al. 2006), a potency similar to the pharmacological PPARa agonist fenofibrate (Klaunig et al. 2003). However, PFOA can also interact with additional receptors (Bjork et al. 2011; Peters and Gonzalez 2011), and the exact role and contribution of PPARa activation are not as clearly defined for the increased incidence of Leydig cell and pancreatic acinar cell tumors and the reproductive, developmental, and immunological toxicities observed in rodents (DeWitt et al. 2012; Klaunig et al. 2012; Lau 2012; Post et al. 2012; Stahl et al. 2011; Xie et al. 2002). There are also marked differences between the responses of human and rodent PPARa to PFOA (Albrecht et al. 2013; Bjork & Wallace 2009; Nakamura et al. 2009), which prevents the direct extrapolation of PPARa activation in rodents to human health risks (Guyton et al. 2009; Klaunig et al. 2012; Rosen et al. 2009). To gain further insights into the molecular and metabolic pathways affected by PFOA, we directly compared the excretion of urinary metabolites resulting from exposures to PFOA and other well-known activators of PPARa. Adult male rats were exposed for 3 consecutive days to PFOA, the pharmacological PPARa agonist fenofibrate, or di(2-ethylhexyl)phthalate (DEHP), a plasticizer which is both a PPARa activator and an endocrine disruptor (Gentry et al. 2011). Rats were then allowed to recover for 4 days, and overnight urine was collected. Urinary metabolites providing clues on oxidative stress status and hepatic functions allowed us to monitor and compare the effects of PFOA and other PPARa agonists over time. At necropsy on day 5 after the last exposure, gross examination of major organs was performed and their weight recorded. Liver and serum end points generally associated with PPARa activation and peroxisomal proliferation were measured.
Materials and Methods Animal Treatment Male Sprague-Dawley rats were purchased from Charles River Laboratories (St. Constant, Quebec, Canada) and housed individually in Health Guard cages (Research Equipment, Bryant, Texas, USA) at 22 ± 1 °C and 50 ± 10 % humidity on a 12 hour light cycle (7:00 AM to
7:00 PM) and fed Teklad 2014 rodent diet (Harlan Laboratories, Indianapolis, Indiana, USA) and water ad libitum. Rats were allowed to acclimatize to the facility for 1 week. The animal study protocol was approved by the Health Canada Animal Care Committee and performed according to the guidelines of the Canadian Council on Animal Care. Thirty rats weighing 284 ± 11 g were randomly divided into groups of five, corresponding to the six treatment groups (control, fenofibrate, DEHP, low-dose PFOA [10 mg/kg], medium-dose PFOA [33 mg/kg], and highdose PFOA [100 mg/kg]). Treatment was divided into three phases (see Fig. 1) as follows: (1) baseline phase: All animals were dosed by gavage with 1.0 ml/100 g body weight (bw) of corn oil (Mazola; ACH foods, Memphis, Tennessee, USA) on the mornings of days 1, 2, and 3; (2) dosing phase: on day 4, 5, and 6 animals were administered corn oil containing 100 mg/kg bw fenofibrate, 100 mg/kg bw DEHP, or PFOA at 10, 33, and 100 mg/kg bw (all purchased from Sigma-Aldrich, St. Louis, Missouri, USA); control animals received corn oil only; and (3) recovery phase: animals were then kept for 4 more days with no further dosing. BWs were measured daily, and overnight (4:00 PM to 8:00 AM) urine was collected in ice chilled containers and stored at -80 °C. On the morning of day 11, animals were anesthetized with isoflurane. Serum specimens were prepared and stored at -80 °C. Major organs were weighed. Liver homogenates were prepared in 0.05 M tris/0.15 % KCl (pH 7.4) buffer at 1 g/1.5 ml. Part of the homogenates were centrifuged at 90009g for 10 min to obtain supernatants. Liver homogenates and supernatants were stored at -80 °C until use. Liver Enzymatic Activities and Serum Chemistry Liver 90009g supernatants were used to assess ethoxyresorufin-O-deethylase (EROD) activity as described by Lubet et al. (1985), pentoxyresorufin-O-dealkylase (PROD) activity according to Burke et al. (1985) and glutathione-S-transferases (GST) according to the protocol of Habig et al. (1974). Liver homogenates were used to measure uridine 5’-diphospho-glucuronosyltransferase (UDP-GT) activity as described by Burchell and Weatherill (1981) and palmitoyl-CoA oxidase activity according to Small et al. (1985). Quantitative analyses of the following
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serum analytes were performed with an ABX Pentra 400 clinical chemistry analyzer using Horiba ABX CE marked liquid chemistry reagents (Horiba ABX, Irvine, California, USA) according to the manufacturer’s instructions: calcium (Cat# A11A01633), magnesium (Cat# A11A01646), inorganic phosphate (Cat# A11A01665), aspartate aminotransferase (Cat# A11A01629), alkaline phosphatase (Cat# A11A01626), total protein (Cat# A11A01867), urea nitrogen (Cat# A11A01641), creatinine (Cat# A11A01868), glucose (Cat# A11A01667), cholesterol (Cat# A11A01634), and triglycerides (Cat# A11A01640). Serum uric acid was determined using a colorimetric assay from Pointe Scientific (Canton, Michigan, USA) according to manufacturer’s instructions.
Determination of Ascorbic and Quinolinic Acids in Urine Urine total ascorbic acid (ascorbic and dehydroascorbic acids) was measured with an automated enzymatic procedure (Lee et al. 1997). Urine quinolinic acid (QA) was analyzed by liquid chromatography (LC)/mass spectrometry (MS) (Liao et al. 2010). Urine creatinine was measured using a Roche creatinine PAP assay kit (Roche Diagnostics, Laval, Quebec, Canada). All urinary analyte concentrations were normalized against urinary creatinine.
Determination of MDA and 8-OHdG in Urine For determination of urinary MDA, all containers and glassware were first washed in soap-free detergent and then rinsed with distilled water and ethanol to decrease background interference. Urine sample (2 ll) was added to a water solution (1 mL) containing 1 % (v/v) ethanol and 0.02 % (w/v) 2,6,di-tert-4-methyl-phenol in a screw-cap tube and well mixed. A 1:1 (water:acetic acid) solution containing 0.33 % (v/v) thiobarbituric acid (250 ll) was then added. The tube was capped, the solution well mixed, and derivatization allowed to proceed for 1 h at 95 °C. After cooling to room temperature, 1.25 ml n-butanol was added and mixed thoroughly before centrifugation at 30009g. The MDA reactive fraction extracted in the n-butanol phase and the standard curve were measured by a fluorescent high-performance liquid chromatography (HPLC) method using a Brava C18-ODS column, 250 9 4.6 cm, 5 lm (Mandel Scientific, Guelph, Ontario, Canada) according to a procedure described by Agarwal and Chase (2002). For the determination of 8-hydroxy-2’deoxyguanosine (8-OHdG), urine was first filtered through a 30,000 Da Ultrafree-MC filter (EMD Millipore, Billerica, Massachusetts, USA). The filtrate was then used for the
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assay of 8-OHdG using an enzyme-linked immunosorbent assay (Cosmo Bio, Carlsbad, California, USA). PFOA Quantification in Urine PFOA and 13C4-PFOA standards were purchased from Wellington Laboratories (Guelph, Ontario, Canada). Ammonium acetate (ACS grade) was purchased from BDH (Toronto, Ontario, Canada). OmniSolv acetonitrile, methanol, and HPLC-grade water were obtained from EMD Chemicals (Gibbstown, New Jersey, USA). Methyl tertbutyl ether (MTBE), sodium bicarbonate, and tetrabutylammonium bisulfate were purchased from Sigma-Aldrich. Ten ll of a 1000 ng/ml mixture of internal standards containing 13C-PFOA was added to 1 ml of rat urine and gently mixed. One milliliter of a 0.5 M tetrabutylammonium bisulfate solution (adjusted to pH 10) was added with gentle vortexing to the spiked urine followed by 2 ml of carbonate/bicarbonate buffer, and the solution was again gently mixed. Next, 5 ml of MTBE was added to the spiked urine and the mixture vortexed. The organic and aqueous phases were separated by centrifugation at 3500 rpm for 10 min. The top organic layer was transferred into a clean 15 ml polypropylene tube. Another 5 ml of MTBE was added; the tube was then capped, inverted, and centrifuged for 5 min; and the top organic layer was collected. This procedure was repeated twice. Using a gentle stream of nitrogen, samples were evaporated to dryness at 30 °C and then reconstituted in 1 ml of HPLCgrade water. The extracts were then loaded onto a preconditioned (9 ml methanol and 5 ml of HPLC-grade water) 200 mg Waters Oasis HLB glass cartridge (Milford, Massachusetts, USA). Solid-phase extraction cartridges were allowed to dry for 15 min before solvent elution. The analyte was eluted using 10 mL of methanol. The extracts were concentrated to dryness under a gentle stream of nitrogen and reconstituted in 200 ll of acetonitrile followed by 800 ll of HPLC grade water. The extracts were then transferred by way of precleaned pasteur pipettes before LC/MS/MS analysis. The chromatographic separation of extracts was performed using a Finnigan Surveyor Plus HPLC System (Thermo Electron, San Jose, California, USA). Separation was achieved using a Discovery HS C18 (7.5 cm 9 2.1 mm 9 3 lm) precolumn followed by the analytical column consisting of a Discovery HS C18 column (7.5 cm 9 2.1 mm 9 3 lm). An opti-guard C18 column (10 mm 9 1 mm 9 3 lm) (Sigma-Aldrich) was installed between the pump and the analytical column to trap and delay perfluorinated compounds originating from the solvents and the pumping system. The mobile phase consisted of 20 % acetonitrile in 2 mM ammonium acetate (A) and 90 % acetonitrile in 2 mM ammonium acetate (B). Solvents were not degassed online as the online degasser
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contains teflon, which constitutes for a large source of perfluorinated compounds contamination. The mobile phase was therefore degassed by sparging a gentle flow of ultra-high purity helium through it. Once degassed, the mobile phases were passed directly to the LC pump. Polypropylene tubing supplied the helium gas flow from the helium tank to the solvents mobile-phase reservoirs, and the flow was controlled by a gas regulator. The column temperature was maintained at 40 °C. The gradient elution started with 100 % A for 1 min followed by an 8 min linear gradient to 100 % B, then a 3 min hold at 100 % B, and returned back to 100 % A in 4 min at a flow rate of 200 ml/min. The system was equilibrated for 4 min at the initial conditions before the next injection. Sample injection volume was 25 ll. MS experiments were performed using a Thermo Finnigan TSQ Quantum Ultra EMR triplequadrupole mass spectrometer (Thermo Electron). The samples were analyzed in negative ion electrospray ionization using selective reaction monitoring mode. Xcalibur version 2 (Thermo Electron) was used for data acquisition and processing. The following transitions were monitored: m/z 412.9 ? 368.9 and m/z 416.9 ? 372.0 for PFOA and 13 C4-PFOA (IS), respectively. The method detection limit (MDL) was determined according to United States Environmental Protection Agency Regulation 40 CFR part 136 (Appendix B) method. The limit of quantitation (LOQ) was calculated as ten times the SD associated with seven replicates. The MDL and LOQ were 0.085 and 0.283 ng/ml, respectively, and the average percent recovery was 110.77 %. Statistical Analysis Data set normality was assessed by Shapiro–Wilk test and homogeneity of variance by Levene’s test using SigmaPlot 11.2 (Systat, Chicago, Illinois, USA). End points assessed at the terminal necropsy that satisfied normality and homoscedasticity assumptions were analysed by one-way analysis of variance (ANOVA) followed by Dunnett post hoc test. Data sets that did not satisfy normality and homoscedasticity assumptions were log-transformed, and if they still failed to meet these assumptions, they were then analysed by nonparametric Kruskal–Wallis ANOVA followed by Mann–Whitney U-test. Repeated daily measures did not satisfy normality and homoscedasticity assumptions and were analyzed by Friedman’s nonparametric randomized block ANOVA, which is a nonparametric version of a one-way ANOVA with repeated measures followed by Friedman post hoc test. Test statistics for Friedman’s test is chi-square with the number of repeated measure as degrees of freedom. Differences between control and treatment groups were considered statistically significant at p \ 0.05.
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Fig. 2 Growth curves of the control, fenofibrate, DEHP, and PFOA treatment groups. Error bars on the control and selected treatment groups represent SDs. Treatment groups labeled by # in legend were significantly different from the control group according to nonparametric repeated measure ANOVA, while * signals a significant difference from control group values on a given day, using Friedman’s nonparametric randomized block ANOVA and a p \ 0.05 cut-off for statistical significance
Results Rat body weights decreased after exposure to the medium (33 mg/kg) and high (100 mg/kg) PFOA doses (Fig. 2), but only the latter growth curve was statistically different from the control group (as assessed by Friedman’s nonparametric randomized block ANOVA). No additional symptom of toxicity was observed in any of the PFOA-treated animals, and growth quickly resumed after the last day of exposure. Although there was a slight decrease in food consumption (data not shown), dehydration is the most likely explanation for weight loss because overnight urine volumes were markedly increased, i.e., peaking at 2.5 and 4 times the control urine volume on day 6 for the medium and high PFOA doses, respectively (Fig. 3a). Water consumption peaked at almost two times control value one day later in the high PFOA treatment group (Fig. 3b). Increased water intake was also noted in the medium PFOA treatment group over the next few days, but it did not reach statistical significance. PFOA excretion in urine peaked on day 7 reaching 42, 148, and 517 lg PFOA/ml urine for the low, medium, and high PFOA treatment groups, respectively (Fig. 4a). Because PFOA exposure significantly increased urine output (Fig. 3a), PFOA excretion was also expressed as a ratio on urinary creatinine, which peaked 1 day sooner at 49, 673, and 3240 lg PFOA/mg creatinine in the same treatment groups (Fig. 4b). Interestingly, whereas raw PFOA concentrations in the urine of the low, medium, and high PFOA treatment groups were approximately proportional to the doses administered, creatinine–normalized
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Fig. 3 (a) Overnight urine volume and (b) daily water consumption in controls and animals treated with fenofibrate, DEHP, and PFOA. Error bars on the control and selected treatment groups represent SDs. Treatment groups labeled by # in legend were significantly different from the control group according to nonparametric repeated measure ANOVA, while * signals a significant difference from control group values on a given day, using Friedman’s nonparametric randomized block ANOVA and a p \ 0.05 cut-off for statistical significance
PFOA levels suggested increasing rates of excretion at greater PFOA exposure levels. This hypothesis is further supported by calculation of the total PFOA excretion over the last 6 overnight urine collections assessed, which corresponded to 17 ± 6 %, 36 ± 10 %, and 56 ± 14 % of the total dose administered in the low, medium, and high
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PFOA treatment groups, respectively. Despite large variations in urine volume, the total amount of creatinine excreted in overnight urine collections remained relatively constant; consequently, creatinine was used to normalize the concentration of all other urinary metabolites. Daily urinary ascorbic acid (Fig. 5a), 8-OHdG (Fig. 5c), and MDA (Fig. 5d) measurements after PFOA exposure presented similar patterns where increased metabolite levels already noticeable on days 4 or 5 returned to control values by day 8. High-dose PFOA exposure elicited the most consistent and strongest responses followed by medium-dose PFOA exposure. DEHP and low dose PFOA had no effect, whereas fenofibrate significantly affected 8-OHdG level on a single day. The most pronounced effects were observed for urinary ascorbic acid, whose levels noticeably increased right after the first exposure on day 4 and peaked at 5 and 12 times the control values for the medium and high PFOA doses, respectively. MDA levels peaked on day 5 at approximately three and five times the control values in the same treatment groups. Changes were less pronounced for 8-OHdG with a 2.5-fold increase compared with the control values reached on day 6 after exposure to the high PFOA dose. The two-fold increases over the same days after exposure to medium PFOA dose failed to reach statistical significance, whereas exposure to fenofibrate resulted in a statistically significant two-fold increase on day 5. Urinary QA (Fig. 5b) plateaued at four to five times control levels after exposure to the medium and high PFOA doses, whereas the levels in the low PFOA dose gradually increased to 3 times control levels, reaching statistical significance on day 7, 2 days after the initial response to the medium and high PFOA doses. Interestingly, increased QA levels in all PFOA treatment groups persisted up to day 10, whereas the threefold increase observed after exposure to fenofibrate returned to control values. At termination, animals treated with fenofibrate and PFOA presented significantly increased liver weights with
Fig. 4 PFOA concentration in overnight urine expressed (a) on urine volume and (b) on urinary creatinine concentration in control and PFOAtreated rats. Error bars represent SDs
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Fig. 5 Levels of (a) ascorbic acid, (b) QA, (c) 8-OHdG, and (d) MDA in overnight urine samples from rats treated with fenofibrate, DEHP, or PFOA. Error bars on the control and selected treatment groups represent SDs. Treatment groups labeled by # in legend were significantly different from the control group according
to nonparametric repeated measure ANOVA, while * signals a significant difference from control group values on a given day, using Friedman’s nonparametric randomized block ANOVA and a p \0.05 cut-off for statistical significance
Table 1 Final body weights and relative organ weights (% bw) after treatment with fenofibrate, DEHP, and PFOAa Treatment
Dose (mg/kg)
Body weight (g)
Liver
Heart
Brain
Kidneys
Testis
Thymus
Spleen
Control
0
363 ± 17
4.28 ± 0.20
0.33 ± 0.02
0.54 ± 0.03
0.81 ± 0.02
0.85 ± 0.04
0.19 ± 0.04
0.21 ± 0.03
Fenofibrate
100
357 ± 20
5.73 ± 0.24*
0.34 ± 0.02
0.55 ± 0.03
0.85 ± 0.06
0.86 ± 0.08
0.19 ± 0.03
0.23 ± 0.04
DEHP
100
349 ± 27
4.14 ± 0.34
0.31 ± 0.01
0.59 ± 0.06
0.80 ± 0.05
0.88 ± 0.05
0.21 ± 0.04
0.22 ± 0.04
PFOA
10
356 ± 32
5.73 ± 0.29*
0.31 ± 0.01
0.59 ± 0.04
0.85 ± 0.05
0.87 ± 0.07
0.21 ± 0.06
0.21 ± 0.01
a
33
349 ± 11
6.40 ± 0.34*
0.29 ± 0.01
0.57 ± 0.03
0.79 ± 0.13
0.88 ± 0.09
0.19 ± 0.04
0.22 ± 0.01
100
330 ± 26
6.62 ± 0.47*
0.30 ± 0.02
0.61 ± 0.06
0.87 ± 0.07
0.90 ± 0.04
0.14 ± 0.02
0.22 ± 0.01
Mean ± SD of five animals per group
* One-way ANOVA followed by Dunnett post hoc test; significantly different from control at p \ 0.05
the dose-related increases in the PFOA treatment groups being the most pronounced (Table 1). A dose-related increase in palmitoyl-CoA oxidase activity was also observed in PFOA-treated animals, whereas the modest increases noted in fenofibrate and DEHP-treated rats were not statistically significant (Table 2). Increased hepatic EROD and PROD activities were observed in all PFOAtreated rats, but this reached statistical significance only at
the highest exposure. UDP-glucuronosyltransferase activities were significantly decreased in the fenofibrate and PFOA treatment groups, whereas decreased GST activities were also observed in fenofibrate-treated rats and at medium and high PFOA doses (Table 2). Most of the clinical chemistry parameters assessed in serum at study termination were not significantly affected (Table 3). However, uric acid levels were significantly decreased in the DEHP
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Table 2 Effects of fenofibrate, DEHP, and PFOA on liver enzymesa Treatment
Dose (mg/kg)
Palmitoyl-CoA oxidase (abs/min/g prot)
Phase I drug-metabolising enzymes
Phase II drug-metabolising enzymes
EROD (nmole/min/ mg prot)
UDP-GT (nmole/ min/mg/prot)
PROD (nmole/min/ mg prot)
GST (umole/min/ mg prot)
Control
0
1.02 ± 0.37
0.066 ± 0.020
0.045 ± 0.012
1.69 ± 0.12
1.21 ± 0.11
Fenofibrate
100
1.71 ± 0.58
0.060 ± 0.012
0.046 ± 0.020
1.09 ± 0.14*
0.94 ± 0.13*
DEHP PFOA
100 10
1.40 ± 1.09 3.17 ± 0.65*
0.060 ± 0.016 0.096 ± 0.024
0.039 ± 0.031 0.080 ± 0.024
1.45 ± 0.35 0.88 ± 0.09*
1.27 ± 0.11 1.11 ± 0.13
33
4.89 ± 0.79*
0.080 ± 0.015
0.078 ± 0.031
1.00 ± 0.14*
0.88 ± 0.12*
100
6.11 ± 1.51*
0.113 ± 0.025*
0.107 ± 0.029*
1.12 ± 0.20*
0.94 ± 0.19*
a
Mean ± SD of five animals per group
* One-way ANOVA followed by Dunnett post hoc test; significantly different from controls at p \ 0.05
treatment group and at all PFOA dose levels, again in a dose-dependent manner. Although statistically significant, the increased cholesterol level in fenofibrate-treated rats and alanine aminotransferase activity at medium PFOA dose were most likely spurious observations of dubious biological relevance.
Discussion Although the expected effects of PPARa agonists on serum cholesterol and triglyceride levels were not seen, other hallmark features—such as increased liver weight and hepatic palmitoyl-CoA oxidase activity—were observed at study termination (Tables 1 and 2). This uneven response from parameters usually associated with PPARa activation may have been caused by the different potencies and pharmacokinetics of the compounds tested (Eason et al. 1990; Lau et al. 2007; Pollack et al. 1985), by the short exposure window, or by the 4 days allowed for urine collection before the final necropsy. Despite this, the overall response to PFOA (and, to a lesser extent, fenofibrate and DEHP) was generally compatible with PPARa activation. The impaired bw gains at high PFOA exposure (Fig. 2) were most likely caused by dehydration because severe polyuria was observed in the same treatment group (Fig. 3a). Polyuria quickly subsided after the last day of exposure, and a delayed compensatory increase in water intake was observed (Fig. 3b). Although not statistically significant, the same trends were observed for the medium PFOA treatment group. From day 8 onward, growth resumed at a pace similar to the control group; by the end of the recovery period, urinary volumes and creatinine levels returned to control values suggesting that PFOA’s effects were transient. Although increased urinary output after PFOA administration has been reported before (Goecke et al. 1992), this
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noninvasive end point is infrequently monitored, and this aspect of PFOA toxicity remains relatively unexplored. The severe polyuria observed in the high-dose PFOA treatment group (Fig. 3a) occurred in the absence of effects on kidney weight (Table 1) and serum creatinine and blood urea nitrogen (BUN) levels, which may suggest meaningful impairment of renal functions (Table 3). However, kidney hypertrophy and histological alterations have been reported in rats after exposure to 20 mg PFOA/kg bw for 28 days (Cui et al. 2009). PFOA may also affect kidney functions through the activation of other PPAR subtypes (Cohen 2005), but further investigations will be required to determine if the molecular mechanisms underlying polyuria after rat acute PFOA exposure are also involved in kidney histological alterations observed at a subchronic exposure. PFOA measured in overnight urine collections increased from 17 to 36 to 56 % of the total dose administered in the low, medium, and high PFOA treatment group, respectively. Although the single exposure and smaller doses used in most investigations on PFOA pharmacokinetics may not have allowed this observation (Hanhijarvi et al. 1982; Vanden Heuvel et al. 1991), chronic exposure studies using greater doses also reported increased excretion rates at higher dose levels (Cui et al. 2010; Hanhijarvi et al. 1987). Physiologically based pharmacokinetic models that take into consideration saturable PFOA renal reabsorption (Loccisano et al. 2012) may benefit from a better characterization of the large variations of urinary output after PFOA exposure. Urate transporter 1 (URAT1) is a member of the organic anion transporter family involved in the renal reabsorption of PFOA (Weaver et al. 2010; Yang et al. 2010). URAT1 also mediates the reabsorption of uric acid in the kidney (Enomoto et al. 2002). At high concentrations, PFOA can interfere with uric acid reabsorption (Weaver et al. 2010; Yang et al. 2010) potentially explaining the lower serum uric acid levels observed in PFOA-treated rats (Table 3).
Mean ± SD of five animals per group
* One-way ANOVA followed by Dunnett post hoc test; significantly different from control at p \ 0.05
a
0.90 ± 0.57*
0.73 ± 0.31* 92 ± 25
65 ± 26 45 ± 7
42 ± 4 175 ± 4
185 ± 5 69 ± 8*
53 ± 5 156 ± 18
140 ± 23 393 ± 81
155
343 ± 19 0.37 ± 0.04
0.38 ± 0.04 17.2 ± 1.2
15.0 ± 1.9 5.6 ± 0.3
5.5 ± 0.3 10.2 ± 0.9
11.1 ± 0.9
2.1 ± 0.2
2.2 ± 0.1
10.7 ± 0.4
10.7 ± 0.6
33
100
1.27 ± 0.63*
1.02 ± 0.25* 81 ± 46
97 ± 41 51 ± 9
36 ± 18 166 ± 32
169 ± 16 60 ± 12
63 ± 3 140 ± 17
172 ± 38 311 ± 92
362 ± 99 0.32 ± 0.05
0.37 ± 0.07 16.1 ± 2.7
14.6 ± 2.6 5.2 ± 0.9
5.5 ± 0.2 9.6 ± 0.8
9.5 ± 1.8 1.9 ± 0.3
2.1 ± 0.2
10 PFOA
9.7 ± 1.8
100 DEHP
9.9 ± 1.1
2.33 ± 1.10
1.66 ± 0.37 118 ± 21
124 ± 29 47 ± 15
72 ± 13* 164 ± 17
168 ± 15 51 ± 6
53 ± 16 147 ± 34
129 ± 24 358 ± 118
280 ± 61 0.33 ± 0.04
0.30 ± 0.03 14.2 ± 1.0
16.0 ± 2.3 5.7 ± 0.7
5.2 ± 0.5 9.0 ± 0.8
9.7 ± 1.5 2.0 ± 0.3
2.0 ± 0.2 9.5 ± 1.2
10.0 ± 1.3
0
100
Cont
Feno
Blood urea nitrogen (mg/dl) Total protein (g/ dl) Inorganic phosphate (mg/ dl) Magnesium (mg/dl) Calcium (mg/dl) Dose (mg/ kg) Treatment
Table 3 Serum chemistry after treatment with fenofibrate, DEHP, and PFOAa
Creatinine (mg/dl)
Alkaline phosphatase (U/L)
Aspartate aminotransferase (U/L)
Alanine aminotransferase (U/L)
Glucose (mg/dl)
Cholesterol (mg/dl)
Triglyceride (mg/dl)
Uric acid (mg/dl)
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Fenofibrate can also interfere with URAT1-mediated uric acid reabsorption and lead to lower serum uric acid levels (Uetake et al. 2010). However, the 29 % decrease in serum uric acid level observed in the fenofibrate treatment group was not statistically significant (Table 3). DEHP-treated rats presented significantly lower serum uric acid levels (Table 3), but the interaction of DEHP with URAT1 remains to be characterized. Alternatively, conversion of uric acid to allantoin by hepatic uricase may also contribute to a decrease of serum uric acid levels (Wu et al. 1994). However, reports on DEHP effects on hepatic uricase activity are conflicting because both induction (Jayaraman et al. 1988) and inhibition (Mitchell et al. 1985) were reported, whereas PFOA and fenofibrate do not appear to affect the activity of this hepatic enzyme (Abdellatif et al. 1990; Henninger et al. 1987). Although increased urine excretion in PFOA-treated rats (Fig. 3a) may have played a role in the perturbation of uric acid reabsorption, it was most likely a minor one because the low-dose PFOA, which did not affect urine volumes, already decreased serum uric acid level by 56 % compared with 61 % for the medium and 69 % for the high PFOA treatment groups (Table 3). Increased urinary output may also have affected the excretion of other urinary metabolites. However, all of the metabolites affected after exposure to high-dose PFOA were also significantly affected in the fenofibrate or medium dose PFOA treatment groups on day 5 (Fig. 5) in the absence of significant effects on urinary output (Fig. 3a). Peroxisome proliferator activated receptor alpha agonists can induce oxidative stress in rodent liver, which ultimately contributes to hepatocarcinogenesis (Klaunig et al. 2003). Increased hepatic levels of the biomarker of DNA oxidative damage 8-OHdG have been reported after rat exposure to fenofibrate at 6000 ppm in the diet for 9 weeks (Nishimura et al. 2007) and at 3000 ppm for 13 weeks (Nishimura et al. 2008). Data on PFOA exposure are more equivocal given that increased 8-OHdG level has been reported after exposure to 200 ppm PFOA in food for 2 weeks (Takagi et al. 1991) whereas another study using the same dietary exposure for 9 weeks did not observe such changes (Abdellatif et al. 2003). Assuming a food consumption of 5 g/100 g bw (Pass & Freeth 1993), exposure to 200 ppm PFOA in food corresponds to 10 mg/kg bw approximating our low-dose PFOA treatment group, whereas exposures to 3000 and 6000 ppm fenofibrate represent 150 and 300 mg/kg bw, respectively. In this study, increased urinary 8-OHdG levels were observed only at the highest PFOA dose used (100 mg/kg bw), whereas 8-OHdG levels were significantly increased for a single day in fenofibrate-treated rats (Fig. 5c). Information on the biomarker of lipid peroxidation MDA is more limited and, to our knowledge, this is the first report of
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increased urinary MDA in PFOA-treated rats. The marked increase in MDA excretion (Fig. 5d) clearly suggests lipid peroxidation. Further studies are still needed to correlate urinary MDA and 8-OHdG with oxidative stress occurring in target tissues. Although a dose–response relationships for hepatic phase I and II drug-metabolizing enzymes were not as clear as for palmitoyl-CoA oxidase (Table 2), exposure to highdose PFOA significantly increased EROD and PROD activities suggesting induction of CYP1A1/2 and CYP2B. PFOA exposure also exerted inhibitory effects on UDP-GT and GST activities. These hepatic effects were generally compatible with previous reports (Kawashima et al. 1994, 1995) and may be explained by interaction of PFOA with additional receptors such as the constitutive androstane receptor or the pregnane X receptor (Peters and Gonzalez 2011). It has long been known that exposure to xenobiotics can increase ascorbic acid synthesis and urinary excretion in rats (Longenecker et al. 1940). Although the exact mechanisms involved are not yet fully elucidated, altered UDP-GT and cytochrome P450 activities (most notably CYP2B) have been associated with increased synthesis of ascorbic acid from glucuronic acid (Aranibar et al. 2009; Linster & Van 2007). The increased urinary ascorbic acid levels observed at medium and high PFOA doses (Fig. 5a) may therefore be related to the PFOA-induced perturbation of hepatic UDP-GT and PROD (CYP2B) activities (Table 2). The tryptophan-NAD pathway links tryptophan catabolism via the kynurenine pathway to NADH/NADPH biosynthesis in the liver, thus exerting a significant influence on energy metabolism. PPARa agonists can affect this pathway, resulting in increased urinary levels of metabolites such as n-methylnicotinamide and QA (Delaney et al. 2005; Fukuwatari et al. 2004; Ringeissen et al. 2003; Zhen et al. 2007). The similar four- to fivefold increases compared with control values observed at medium and high PFOA doses may indicate that the response of this parameter had already plateaued (Fig. 5b). QA was also the most sensitive urinary end point identified in this study because it was the only parameter to be significantly affected by the low-dose PFOA and fenofibrate treatment groups according to nonparametric repeated measures ANOVA. In human epidemiological studies, hyperuricemia, decreased glomerular filtration rate, and chronic kidney disease are associated with greater serum PFOA levels (Shankar et al. 2011; Steenland et al. 2010), but it is not yet clear if these increased levels are a cause or a consequence of impaired renal functions (Watkins et al. 2013). These epidemiological observations appear somewhat at odds with the hypouricemia and polyuria reported in this study. However, in addition to legitimate issues about
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extrapolation of PPARa activation in rodent models to human health risks (Guyton et al. 2009; Klaunig et al. 2012; Rosen et al. 2009), there are marked differences in PFOA pharmacokinetics across species (Lau 2012), and human also lacks functional uricase (Uetake et al. 2010) and L-gulonolactone oxidase (Linster & Van 2007) involved in the metabolism of uric acid and ascorbic acid. It is also worth noting that the PFOA doses used in this rat exposure study were several orders of magnitudes greater than actual human exposure (Lau 2012) and that severe polyuria was observed only at the highest PFOA exposure. This high PFOA dose (ten times greater than the low PFOA dose where increases in liver weight and palmitoyl-CoA oxidase activity were unequivocally observed) was also the only dose to robustly affect the excretion of all urinary metabolites assessed in this study. Hence, although the observed polyuria and perturbation of urinary metabolite excretion may provide clues on molecular pathways affected by PFOA, they are unlikely to have meaningful consequences for human health risk assessment of PFOA at environmental levels. In summary, exposure to PFOA led to dose-related increases in liver weight and palmitoyl-CoA oxidase activity, which are characteristic effects of PPARa-induced peroxisomal proliferation. PFOA also affected rat weight gain, a likely result of dehydration caused by a transient increase in urine excretion, which may also impact PFOA pharmacokinetics. The assessment of urinary metabolites identified significant but transitory changes in 8-OHdG, MDA, ascorbic acid and QA levels after exposure to PFOA. These metabolites provided further insights on the effects of acute exposure to PFOA on oxidative stress status and hepatic glucuronic acid and tryptophan-NAD pathways, but further work is needed to clearly delineate the contribution of PPARa and alternative molecular pathways. Acknowledgments This project was funded through Health Canada A-Base. The authors thank Brita Nadeau for excellent technical assistance and Mike Wade and Premkumari Kumarathasan for helpful comments on the manuscript.
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