Effects of perfluorooctane sulfonate on genes controlling hepatic fatty acid metabolism in livers of chicken embryos

Effects of perfluorooctane sulfonate on genes controlling hepatic fatty acid metabolism in livers... Per- and polyfluoroalkyl substances (PFAS) are synthetic surfactants with a wide variety of applications; however, due to their stability, they are particularly resistant to degradation and, as such, are classed as persistent organic pollutants. Perfluorooctane sulfonate (PFOS) is one such PFAS that is still detectable in a range of different environmental settings, despite its use now being regulated in numerous countries. Elevated levels of PFOS have been detected in various avian species, and the impact of this on avian health is of interest when determining acceptable levels of PFOS in the environment. Due to its similarities to naturally occurring fatty acids, PFOS has potential to disrupt a range of biological pathways, particularly those associated with lipid metabolism, and this has been shown in various species. In this study, we have investigated how in ovo exposure to environ- mentally relevant levels of PFOS affects expression of genes involved in lipid metabolism of developing chicken embryos. We have found a broad suppression of transcription of genes involved in fatty acid oxidation and PPAR-mediated transcription with more significant effects apparent at lower doses of PFOS. These results highlight the need for more research investigating the biological impacts of low levels of PFAS to properly inform environmental policy governing their regulation. . . . . . Keywords Perfluorooctane sulfonate PFOS In ovo Chicken Beta oxidation qPCR array Introduction compounds. Per- and polyfluoroalkyl substances (PFAS) are anthropogenic compounds used commercially for their stabil- The presence of persistent organic pollutants (POPs) is ever ity and surfactant properties. One of the major PFAS is increasing in the environment due to technological develop- perfluorooctane sulfonate (PFOS), which consists of a ment and growing commercialization of synthetic perfluorinated eight-carbon backbone and a sulphonate group. This makes this PFAS amphiphilic and a good surfactant with many fields of application. PFAS are ideal flame-retardants Responsible editor: Philippe Garrigues because of their carbon-fluorine bond, which is one of the Electronic supplementary material The online version of this article strongest in organic chemistry. This is also what renders them (https://doi.org/10.1007/s11356-018-2358-7) contains supplementary resilient to biological degradation, strong acids and alkalis and material, which is available to authorized users. photolysis. Industrial and commercial uses include stain repel- * Nikolai Scherbak lent, non-stick coatings and flame retardants for example in nikolai.scherbak@oru.se firefighting foam, clothes and upholstery, cooking utensils, food wrappers and electronics (Renner 2001). School of Biomedical Sciences, Charles Sturt University, Wagga The chemical and biological stability explains measurable Wagga, Australia environmental levels of different PFAS (i.e. PFOS) in differ- The Life Science Center, School of Science and Technology, Örebro ent matrices all over the globe. PFOS can be found in humans University, Örebro, Sweden worldwide (Kärrman et al. 2007), and is detected far from Present address: The Walter and Eliza Hall Institute, Department of manufacturing facilities in environments such as in high arctic Medical Biology, The University of Melbourne, Parkville, Australia icecaps, high-altitude lakes, off-shore waters, and deep-sea MTM Research Center, School of Science and Technology, Örebro oceans. PFOS is detectable in various animal species (Houde University, Örebro, Sweden et al. 2011), and is found in high concentrations in top predator Present address: Clinical Neurochemistry Laboratory, Sahlgrenska avian species. A study of eggs and developing embryos of the University Hospital, Mölndal, Sweden Environ Sci Pollut Res (2018) 25:23074–23081 23075 great cormorant and herring gull showed PFOS concentra- dimethyl sulfoxide (DMSO; Sigma-Aldrich, Darmstadt, tions in the μg/g wet weight in whole egg and liver (Nordén Germany) to final concentrations of either 0.1 or 1.0 mg/ml. et al. 2013). Studies of the effects of environmentally compa- On the fourth day of incubation, a single microinjection of 1 µl rable concentrations of PFOS on development of White of this PFOS solution per gram of egg was aseptically added Leghorn chicken showed reduced embryo survival, signifi- into air sacs of eggs resulting in treatment concentrations of 0.1 cant immunological, neurological and morphological changes respectively 1.0 µg of PFOS per 1g of egg. All treatments were in treated embryos compared to controls (Nordén et al. 2016; done in four replicates. Eggs injected with 1 µl/g egg of DMSO Peden-Adams et al. 2009). PFOS-induced immunotoxicity only (5%; n = 4) were used as controls. Holes were sealed with has been noted in various species at environmentally relevant paraffin and eggs put in an incubator at 37.5 °C and 60% hu- doses (DeWitt et al. 2012), and has the potential to impact the midity.Eggswereturnedin a6-hcycleandsacrificed1day fitness of wild species, particularly if faced with environmen- before expected pipping, i.e. 19 days post incubation start. tal challengessuchasinfection. Obtained liver samples were preserved at − 80 °C in RNA Hepatotoxicity is another important feature of PFOS expo- stabilisation solution (RNAlater®, Invitrogen/ThermoFisher, sure, with hepatomegaly, necrosis, and lipid accumulation MA, USA) until use. The experimental protocol was approved found in various animal models (Bijland et al. 2011;Du et by the Swedish Board of Agriculture, Jönköping, Sweden. al. 2009; Peden-Adams et al. 2009; Wan et al. 2012; Wang et al. 2014), although the mechanism behind this is still not clear. RNA purification and qPCR Transcriptomic analysis of the livers of 6-week-old chicken (Gallus gallus) that were subcuticularly exposed to low doses Approximately 15 mg of chicken embryo liver tissue was used of PFOS showed changes in expression of genes mainly in- for RNA purification using RNeasy® mini kit (QIAGEN, volved in electron and oxygen transport, and the metabolism Hilden, Germany) according to manufacturer protocol. RNA of lipids and fatty acids (Yeung et al. 2007). Other studies have was checked for purity and quantified using a spectrophotom- also assessed changes to gene expression using microarrays eter (NanoDrop® 2000; NanoDrop Technologies, (Martin et al. 2007; O'Brien et al. 2011), or investigation of Wilmington, DE). RNA integrity was confirmed with gel elec- small subsets of genes (Cwinn et al. 2008; O'Brien et al. trophoresis using a 1.2% (w/v) agarose gel. Complementary 2009); however, these have given conflicting results regarding DNA (cDNA) was synthesised using RT2 First Strand Kit® how PFOS impacts lipid metabolism. Moreover, direct com- (QIAGEN) according to manufacturer instructions using parison of these studies is challenging, due to large variations 0.5 μg purified RNA from each sample. Using qPCR, samples in the dose of PFOS used, with many studies using levels well were analysed using Chicken Fatty Acid Metabolism RT2 in excess of that found environmentally. Despite the varied profiler PCR array® (Catalogue PAGG-007Z; QIAGEN). results in the literature, hepatic steatosis is one of the more These arrays come in 96-well plate format, which include 84 commonly documented effects of PFOS exposure, which wells containing primers for genes of interest, 5 wells contain- strongly suggests liver lipid metabolism is indeed disrupted. ing housekeeping genes suggested by QIAGEN, and addition- To address how gene expression changes contribute to this, al controls to analyse genomic DNA contamination, reverse approaches with greater sensitivity than conventional micro- transcription efficiency and PCR array reproducibility. For the arrays, and greater depth and resolution than smaller focused list of analysed genes, see Table S1 in Supporting information, gene studies, are required. In the current study, we investigat- and for information on the RT2 profiler system see https:// ed the effects of environmentally relevant concentrations of dataanalysis.sabiosciences.com/pcr/documents/ PFOS on expression of genes controlling liver fatty acid me- RT2ProfilerDataAnalysisHandbook.pdf. tabolism of chicken embryos using the Chicken Fatty Acid One plate was used to analyse each biological sample, and Metabolism RT2 profiler PCR array®. an electronic pipetting system was used to limit any variation caused by pipetting technique. The qPCR program was set to 40 cycles consisting of the following temperatures and time Material and methods intervals: an initial denaturation at 95 °C for 5 minutes, followed by 15 s at 95 and 60 °C for 1 min for 40 cycles using Egg incubation and exposure an Applied Biosystems® 9700 thermocycler (Applied Biosystems, Carlsbad, CA). Each run was completed with Treatments were done as previously described by Nordén et al. melting curve analysis to confirm a single amplified product. (2016). Fertilised, unincubated eggs from White Leghorn chicken (Gallus gallus) were purchased from Ova Production, Data analysis Vittinge, Sweden and kept at 10–12 °C until incubation. Potassium salt of PFOS (Chemica 98%, Lot 77.282, approxi- The analysis software RT2 Profiler PCR array Data Analysis® mately 21% branched isomer) was dissolved in 5% solution of (QIAGEN) version 3.5 was used for interpretation of PCR 23076 Environ Sci Pollut Res (2018) 25:23074–23081 array data. In brief, data was first normalised against the geo- were both more strongly affected by the 1.0 μg/g of egg dose metric mean of a panel of housekeeping genes suggested by of PFOS (Table 1). Additionally, ACAT2 (FR = − 2.68; p = QIAGEN to generate a ΔCt value. As the differences between 0.025; 0.1 μg/g PFOS) was also downregulated by 1.0 μg/g the geometric means of all control and test groups were within PFOS (FR = − 2.22; p =0.08). the limits suggested by QIAGEN, we opted to use all five The genes selected for our analysis were those specifically genes in the panel for normalisation (ACTB, H6PD, HMBS, targeted to metabolic pathways, so we performed enrichment RPL4 and UBC). The software then calculates an average ΔCt analyses to determine which pathways were most influenced value for each of the control and treatments groups, as well as by our selected genes (Table S2). Of these pathways, a subset a standard deviation (SD) to assess variability. The average showed a proportionally large number of genes affected by ΔCt values are used to calculate fold change (FC), which is treatment with either dose of PFOS, particularly butanoate the ratio of relative gene expression between the control group metabolism, PPAR signalling pathway, fatty acid degradation, and the test group, using the formula 2^(ΔΔCt), where valine, leucine and isoleucine degradation and fatty acid me- ΔΔCt = average ΔCt(test group) – average ΔCt(control tabolism (Table 2). Full expression data for all genes tested in group). For the purposes of this study, FC is represented as the PCR array can be found in Table S3. fold regulation (FR), where for FC ≥ 1, FR = FC and for FC < 1, FR = − 1/FC. The p values for each gene were determined using a Student’s t test, which was calculated using the aver- Discussion age ΔCt of each test group versus the control group and their associated SD. To focus on gene expression changes that were PFAS such as PFOS are persistent environmental pollutants more likely to be associated with a biological effect, we used a and well known to cause adverse effects on the health of cut-off for differential expression as a FR ± 2 (p ≤ 0.05). various wild and laboratory animals. Although most KEGG pathway analysis of all tested genes, and those dif- European and Northern American countries now regulate pro- ferentially expressed genes at either dose of PFOS, was per- duction of these compounds, they are still actively used in formed using STRING version 10.5. During the pathway other countries, such as China (Fu et al. 2016), and are found analysis, STRING performs a Fisher’s exact test based on to be present in a range of consumer products (Kotthoff et al. the number of specified genes that fall within a particular 2015). Acceptable environmental levels have been debated, pathway category, the number of total genes annotated to that with a recent push to adopt lower thresholds from a number pathway and the total gene number present in the organism of different agencies. Additionally, recent animal experiments being studied. This is then corrected for multiple testing to indicate PFAS doses corresponding to current environmental give a false discovery rate, which is a measure of the likely levels can impact various biological pathways (Lilienthal et al. proportion of false positive gene matches for the specified 2017). In this study, we have used qPCR arrays to examine the pathway (Szklarczyk et al. 2017). effect of PFOS on expression of genes related to lipid metab- olism in livers of chicken embryos, and have found that low doses suppress transcription of genes relating to lipid catabo- Results lism and fatty acid β-oxidation. By using KEGG pathway analysis, the top identified met- As previous studies indicated dysregulated lipid metabolism abolic process affected in our analysis was butanoate metab- after treatment with perfluoronated compounds, we used a olism, which involves processing of short chain fatty acids focused PCR array to analyse 84 genes associated with lipid (SCFAs) and is known to be important for regulating mito- metabolism (Table S1). Normalisation against the five refer- chondrial energy production, lipogenesis and cellular meta- ence genes (ACTB, H6PD, HMBS, RPL4, UBC)revealed a bolic processes including fatty acid oxidation (Schönfeld general downregulation of expression after treatment of eggs and Wojtczak 2016). Additionally, SCFAs, including butyrate, with PFOS at both 0.1 and 1.0 μg/g of egg (Fig. 1a, b). Of have been shown to act as a switch between fatty acid oxida- these 84 genes, we found 22 genes with significant downreg- tion and lipogenesis in a PPARγ-dependent manner (den ulation (fold regulation (FR) ≤− 2; p ≤ 0.05), with four of Besten et al. 2015). Deregulated lipogenesis in the form of these genes (ACAD8, ACSL6, ELOVL3, FABP7) downregu- hepatic steatosis is commonly seen after exposure to PFOS lated in both the 0.1 and 1.0 μg/g treatment groups (Fig. 1c; (Cheng et al. 2016; Lai et al. 2017), as are perturbations to Table 1). A further seven genes with FR ≤−2(p ≤ 0.05) at a fatty acid oxidation (Wan et al. 2012). However, laboratory PFOS dose 0.1 μg/g of egg (ACAA2, ACAT1, ACSM3, CPT2, results are contradictory in relation to this, with some studies DECR1, FABP3, FABP5) were also downregulated to a lesser indicating increased beta oxidation or gene expression of rel- extent (FR ≤− 1.5; p ≤ 0.05) at the higher dose of PFOS evant enzymes (Hu et al. 2005;Nordén et al. 2012; Tan et al. (Table 1;FR ≤− 1.5 and > − 2.0 shown in italics). A similar 2012), while others indicate beta oxidation is supressed (Adinehzadeh and Reo 1998; Bijland et al. 2011; Cheng et effect was seen with ACOT8 and LOC771098,although they Environ Sci Pollut Res (2018) 25:23074–23081 23077 0.1µg/g PFOS vs control 1.0µg/g PFOS vs control FR = -2.0 FR = -2.0 ab p = 0.05 p = 0.05 Log2 (FC of 0.1µg/g PFOS/control Log2 (FC of 1.0µg/g PFOS/control ACAA2 FABP5 ACOT8 ACSBG1 HMGCL ACAD8 ACSM5 HADHA ACSM3 0.1 µg/g 1.0 µg/g ELOVL3 ACSL6 FABP3 PFOS PFOS ACAT1 LOC771098 PPA1 CPT2 DECR1 FABP7 FABP4 HMGCS2 SLC27A1 ACAT2 Fig. 1 Results of gene expression analysis from the Chicken Fatty Acid changes to expression are positioned above the horizontal line. Panel c Metabolism RT2 profiler PCR array®. Panel a, b shows volcano plots shows comparison genes with altered expression (FR ≥ ±2; p ≤ 0.5) by depicting a general downregulation of studied genes after in ovo treatment with 0.1 μg/g (purple) and 1.0 μg/g (green) of PFOS. Genes treatment with 0.1 μg/g (a) and 1.0 μg/g (b) of PFOS. Genes with whose expression was altered at both doses are represented by the inter- greater than two-fold regulation (FR) in expression are shown in green section (pink) (suppression) and red (induction). Genes with significant (p ≤ 0.5) al. 2016). These differences may be a result of the range of (2012) showed that mice exposed to PFOS had both increased concentrations being used, different responses between ani- peroxisomal beta oxidation and decreased mitochondrial beta mal models and cell culture and differences in how the path- oxidation. Importantly, impaired mitochondrial function is ways themselves are assessed. Interestingly, our results indi- proposed as a key event leading to hepatic steatosis (Angrish cate a suppression of transcription of genes involved in beta et al. 2016), such as is seen in PFOS liver toxicity. Moreover, oxidation that is more apparent at lower doses corresponding Wan et al. (2012) also found an increase in total beta oxida- to environmentally relevant concentrations, suggesting that tion, similar to that found in our previous analysis of day 10 metabolic responses to PFOS could differ based on the level embryonic chicken livers (Nordén et al. 2012). Interestingly, of exposure. the only acyl-CoA thioesterase (ACOT) found to be downreg- We also noted a number of the genes found to be differen- ulated by PFOS (at 1.0 μg/g of egg) in this current study was tially expressed in this study relate to mitochondrial beta ox- ACOT8, which is proposed to be the predominant ACOT in- idation. This includes CPT2 and DECR1, which both have volved in negative regulation of peroxisomal beta oxidation crucial roles in positive regulation of this pathway, as well as (Hunt et al. 2014). Together, these data imply PFOS may other positive regulators of beta oxidation, such as HADHA, induce a transition from mitochondrial beta oxidation to per- ACAA2 and ACAT1 (Houten et al. 2016). These were all found oxisomal beta oxidation, which could help to clarify both the to be downregulated at the lower dose of 0.1 μg/g egg of mechanism of PFOS toxicity and explain some of the contra- PFOS (Table 1). Previously, induction of beta oxidation by dictory results found in the literature, including our own pre- PFOS has previously been linked to peroxisomal beta oxida- vious results. Analysis of the gene expression profile of day 10 tion (Hu et al. 2005; Tan et al. 2012); however, Wan et al. embryonic livers would need to be done to clarify if this was -Log10 (p-value -Log10 (p-value 23078 Environ Sci Pollut Res (2018) 25:23074–23081 Table 1 Genes with expression changes of ≥ 2(p ≤ 0.05) after treatment palmitoyltransferase 1a (CPT1A) transcription seems to be with PFOS. Expression changes of significantly regulated genes in both regulated through PPARα (Honda et al. 2016). Similar stud- treatment conditions. Values in italics indicate significant (p ≤ 0.5) ies of PPAR-related effects of PFOS on the embryonic expression changes, with fold regulation < 2. Values in grey indicate where there were no statistically significant (p > 0.5) changes to chicken liver have given varied responses, with studies in expression both 18 and 21 days chicken embryos indicating no signif- icant changes to expression of PPAR-induced genes Gene symbol PFOS 0.1 μg/g egg PFOS 1.0 μg/g egg (O'Brien et al. 2009; Strömqvist et al. 2012). It should be fold regulation P value Fold regulation P value noted, however, that the study conducted at the same time point as ours used doses of PFOS 20-fold higher than the ACAA2 − 2.0106 0.00168 − 1.9936 0.002946 upper concentration used in the present study. ACAD8 − 2.3604 0.02053 − 2.2698 0.030889 In rodent and chicken studies, and in cell culture models of ACAT1 − 2.3008 0.020093 − 1.9327 0.041243 various species, most studies have found that PFOS causes ACAT2 − 2.6831 0.025219 − 2.2171 0.082354 induction of PPAR-mediated transcription, particularly ACOT8 − 1.7545 0.03791 − 2.084 0.01941 PPARα (Bjork et al. 2011; Elcombe et al. 2012; Strömqvist A A ACSBG1 − 2.37 0.025374 − 1.39 0.340918 et al. 2012). Interestingly, a study by Wang et al. (2014) ACSL6 − 3.954 0.013228 − 2.9527 0.03381 showed that both PPARα and CPT1A gene expression were ACSM3 − 2.1211 0.017689 − 1.7454 0.039628 impaired by PFOS only in mice that were fed a high-fat diet. ACSM5 − 1.4078 0.891541 − 5.3785 0.020546 This observation may have relevance to studies such as ours, CPT2 − 2.0424 0.015545 − 1.8386 0.032922 considering the relatively high in ovo fat content, and may DECR1 − 2.0763 0.006687 − 1.8092 0.024563 also go partway toward explaining differing results seen both ELOVL3 − 3.8073 0.009259 − 2.715 0.034701 between and within different species models. Furthermore, FABP3 − 2.236 0.029092 − 1.9463 0.048893 both avian and rodent studies indicate that PPAR-mediated a A FABP4 − 4.1192 0.017246 − 1.73 0.118269 effects are not solely responsible for toxic and disruptive ef- FABP5 − 3.5536 0.002826 − 1.8447 0.022399 fects of PFOS (Abbott et al. 2009; O'Brien et al. 2009; Rosen FABP7 − 2.904 0.035071 − 2.7541 0.038439 et al. 2010), indicating other transcription factors may be in- HADHA − 2.0065 0.042016 − 1.6934 0.087043 volved. One particularly interesting candidate is hepatocyte HMGCL − 2.7223 0.021643 − 1.6228 0.06323 nuclear factor 4α (HNF-4α), a transcription factor with HMGCS2 − 1.1307 0.441867 − 2.455 0.033322 known effects on lipid metabolism through regulating both LOC771098 − 1.5118 0.002207 − 2.0853 0.016339 PPARα and CPT1A gene transcription (Karagianni and PPA1 − 2.774 0.034539 − 1.5339 0.158511 Talianidis 2015; Martinez Jimenez et al. 2010). Binding of PFOS to HNF-4α is believed to disrupt the normal lipid bind- SLC27A1 − 2.1635 0.033117 − 1.6206 0.085314 ing required for its stabilisation, with PFOS treatment induc- Analysed data had C > 30 so results should be interpreted with caution ing degradation of both the mouse and human proteins (Beggs et al. 2016). The ability of PFOS to induce degradation of the case, or whether the differences between the two studies HNF-4α, or other transcription factors, could help explain were due to different transcriptional responses induced by the downregulation of multiple genes related to lipid metabo- PFOS at those developmental time points. lism seen here. Moreover, knockout of HNF-4α induces liver The broad transcriptional repression seen in this study steatosis in mouse models (Hayhurst et al. 2001), similar to could be explained by PFOS binding to, and interfering that seen after PFOS treatment. Follow-up studies would be with, relevant transcription factors. One possible mechanism required to see whether this effect was also seen in avian suggested by the KEGG pathway analysis in our study is species. PPAR-mediated regulation, and indeed there are a number of Our results indicate broad suppression of transcription studies that implicate PPAR as being responsible for the of genes associated with lipid metabolism after in ovo ex- metabolic disruption seen after PFOS exposure (Cwinn et posure of chicken embryos to PFOS, particularly at lower, al. 2008;Fanget al. 2012; Lai et al. 2017), although, like environmentally relevant doses. There are, however, some beta-oxidation, the directionality of this response is still de- limitations of these results. Firstly, we only analysed two bated. Interestingly, the second most significantly affected environmentally relevant doses of PFOS. This decision pathway in our KEGG analysis was PPAR signalling, with was made based on our previous work, which suggested approximately one third of the genes tested being signifi- that these doses were sufficient to cause changes to lipid cantly repressed by two-fold or more. Although there are metabolism (Nordén et al. 2012). However, in the previous fewer studies investigating the links between PPAR and beta study, we noted the most profound effect at 0.3 μg/g of egg oxidation in chicken, there is indication that there are some dose of PFOS, an intermediate dose to those used here. As similarities to other model organisms. In particular, carnitine most of the statistically significant changes to gene Environ Sci Pollut Res (2018) 25:23074–23081 23079 Table 2 Signalling pathways affected by PFOS treatment. KEGG Fraction of affected genes refers to the number of differentially affected pathway information generated from analysis in STRING using genes genes compared to the total number of genes in that pathway that were whose expression showed ≥ two-fold regulation (p ≤ 0.05) at either of analysed in the array (refer Table S2). The false discovery rate (calculated the two administered doses of PFOS, including the proportion of genes by STRING) is an indication of the likely proportion of false positive analysed within these pathways that met the aforementioned criteria. gene matches for the specified pathway KEGG Pathway description Observed genes Fraction of False ID affected discovery genes rate 650 Butanoate metabolism ACAT1, ACAT2, ACSM3, ACSM5, HMGCS2, HADHA, HMGCL 7 of 13 6.43E-14 3320 PPAR signalling pathway ACSBG1, ACSL6, CPT2, FABP3, FABP4, FABP5, FABP7, SLC27A1 8 of 28 2.01E-13 71 Fatty acid degradation ACAA2, ACAT1, ACAT2, ACSBG1, ACSL6, CPT2, HADHA 7 of 22 3.28E-13 280 Valine, leucine and isoleucine ACAA2, ACAD8, ACAT1, ACAT2, HMGCS2, HADHA, HMGCL 7 of 14 1.73E-12 degradation 1212 Fatty acid metabolism ACAA2, ACAT1, ACAT2, ACSBG1, ACSL6, CPT2, HADHA 7 of 27 2.00E-12 72 Synthesis and degradation ACAT1, ACAT2, HMGCS2, HMGCL 4 of 7 7.63E-09 of ketone bodies 1100 Metabolic pathways ACAA2, ACAD8, ACAT1, ACAT2, ACOT8, ACSBG1, ACSL6, 12 of 40 9.17E-09 ACSM3, ACSM5, HMGCS2, HADHA, HMGCL 900 Terpenoid backbone biosynthesis ACAT1, ACAT2, HMGCS2 3 of 4 2.22E-05 640 Propanoate metabolism ACAT1, ACAT2, HADHA 3 of 8 0.000115 380 Tryptophan metabolism ACAT1, ACAT2, HADHA 3 of 5 0.000188 1120 Microbial metabolism in diverse ACAA2, ACAT1, ACAT2, HADHA 4 of 10 0.000188 environments 310 Lysine degradation ACAT1, ACAT2, HADHA 3 of 5 0.000237 4146 Peroxisome ACOT8, ACSL6, HMGCL 3 of 16 0.00107 1200 Carbon metabolism ACAT1, ACAT2, HADHA 3 of 8 0.0017 62 Fatty acid elongation ACAA2, HADHA 2 of 4 0.00271 630 Glyoxylate and dicarboxylate ACAT1, ACAT2 2 of 4 0.00456 metabolism 620 Pyruvate metabolism ACAT1, ACAT2 2 of 6 0.00917 4920 Adipocytokine signalling ACSBG1, ACSL6 2 of 13 0.0213 pathway expression noted in this study were relatively small, we For these results to have relevance in relation to wild bird may have found more definitive results if that dose had species found to be affected by PFOS, we would also suggest also been used in this study. Secondly, due to the cost of investigating whether similar effects are seen in other avian the arrays, we were only able to analyse four individuals species. As we do not yet have full coverage of the genomes of per treatment group and we were not able to perform du- wild avian species, a metabolomics-based study would pres- plicate plates. However, this is a robust commercially de- ently be the most appropriate method to investigate this. That signed assay that has been used in a wide range of studies, said, the Avian Phylogenomics Consortium is currently work- and is equipped with various controls, including PCR re- ing to sequence all known avian species (Zhang 2015), and producibility. This control showed little variability both this knowledge would enable comparative gene expression within and between plates, giving us confidence in the studies between species. If similar results as seen here are assay and the results it generated. Moreover, the fact that found in wild avian species, changes to expression of key the responses were consistent enough to give statistically metabolic enzymes such as CPT2 and DECR1 could poten- significant data despite the small sample size is encourag- tially act as environmental markers of PFOS exposure. Such ing. Thirdly, as we were unable to determine the sex of the applications would, however, need to take into account wheth- embryos studied, we cannot exclude that sex differences er other common pollutants have overlapping effects. have contributed to these results. Follow up studies will A better understanding of species-specific effects of PFAS need to take this into account. Lastly, it should also be and the doses at which they occur is important when consid- notedthatwe onlyanalysedgeneexpressiondata and,as ering both acceptable levels of these compounds in the envi- such, further studies, such as metabolomics or proteomics, ronment and safe exposure levels for persons with occupation- are required to confirm whether these changes relate to a al contact with PFAS. Although we did not directly measure hepatic liver PFOS concentrations, similarly designed functional impairment of lipid metabolism. 23080 Environ Sci Pollut Res (2018) 25:23074–23081 Angrish MM, Kaiser JP, McQueen CA, Chorley BN (2016) Tipping the previous studies have shown these concentrations are approx- balance: hepatotoxicity and the 4 apical key events of hepatic imately equivalent to the administered dose at the time point steatosis. Toxicol Sci 150:261–268. https://doi.org/10.1093/toxsci/ we measured (Nordén et al. 2016; O'Brien et al. 2009). As kfw018 these concentrations are comparable to those found in envi- Beggs K, McGreal S, McCarthy A, Gunewardena S, Lampe J, Lau C, Apte U (2016) The role of hepatocyte nuclear factor 4-alpha in ronmental analyses of wild birds, it is important that future perfluorooctanoic acid- and perfluorooctanesulfonic acid-induced studies are done to determine whether similar effects are seen hepatocellular dysfunction. Toxicol Appl Pharmacol 304:18–29 in these species, and whether chicken can continue to be used Bijland S, Rensen PCN, Pieterman EJ, Maas ACE, van der Hoorn JW, as a model for environmental exposure. 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Toxicology 288:8–17. https://doi.org/10.1016/j.tox. 2011.06.012 Conclusion Cheng J, Lv S, Nie S, Liu J, Tong S, Kang N, Xiao Y, Dong Q, Huang C, Yang D (2016) Chronic perfluorooctane sulfonate (PFOS) exposure induces hepatic steatosis in zebrafish. Aquat Toxicol 176:45–52. In this study, we investigated the influence of perfluorooctane https://doi.org/10.1016/j.aquatox.2016.04.013 sulfonate (PFOS) on genes associated to fatty acid metabolism Cwinn MA, Jones SP, Kennedy SW (2008) Exposure to perfluorooctane in developing chicken embryos. Liver samples from embryos sulfonate or fenofibrate causes PPAR-α dependent transcriptional responses in chicken embryo hepatocytes. Comparative treated with PFOS showed downregulation of the majority of Biochemistry and Physiology Part C: Toxicology & Pharmacology genes involved in metabolism of fatty acids and this effect was 148:165–171. https://doi.org/10.1016/j.cbpc.2008.05.002 more pronounced at the lower of the two tested doses of DeWitt JC, Peden-Adams MM, Keller JM, Germolec DR (2012) PFOS. Our findings shows that environmentally relevant con- Immunotoxicity of perfluorinated compounds: recent developments. centrations of perfluorooctane sulfonate could impact energy Toxicol Pathol 40:300–311. https://doi.org/10.1177/ metabolism in livers of developing chicken embryos, and sug- Du Y, Shi X, Liu C, Yu K, Zhou B (2009) Chronic effects of water-borne gest further functional studies should be performed to confirm PFOS exposure on growth, survival and hepatotoxicity in zebrafish: the physiological impact of this. a partial life-cycle test. Chemosphere 74:723–729. https://doi.org/ 10.1016/j.chemosphere.2008.09.075 Acknowledgements This work was supported by Örebro University, Elcombe CR, Elcombe BM, Foster JR, Chang S-C, Ehresman DJ, grants from Magnus Bergvalls Stiftelse and by the EnForce project, Butenhoff JL (2012) Hepatocellular hypertrophy and cell prolifera- funded by Knowledge Foundation. We also would like to acknowledge tion in Sprague–Dawley rats from dietary exposure to potassium Steffen Keiter for critical reading of the manuscript and for his valuable perfluorooctanesulfonate results from increased expression of comments. Furthermore, we would like to thank the reviewers for their xenosensor nuclear receptors PPARα and CAR/PXR. 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Effects of perfluorooctane sulfonate on genes controlling hepatic fatty acid metabolism in livers of chicken embryos

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Environment; Environment, general; Environmental Chemistry; Ecotoxicology; Environmental Health; Atmospheric Protection/Air Quality Control/Air Pollution; Waste Water Technology / Water Pollution Control / Water Management / Aquatic Pollution
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Abstract

Per- and polyfluoroalkyl substances (PFAS) are synthetic surfactants with a wide variety of applications; however, due to their stability, they are particularly resistant to degradation and, as such, are classed as persistent organic pollutants. Perfluorooctane sulfonate (PFOS) is one such PFAS that is still detectable in a range of different environmental settings, despite its use now being regulated in numerous countries. Elevated levels of PFOS have been detected in various avian species, and the impact of this on avian health is of interest when determining acceptable levels of PFOS in the environment. Due to its similarities to naturally occurring fatty acids, PFOS has potential to disrupt a range of biological pathways, particularly those associated with lipid metabolism, and this has been shown in various species. In this study, we have investigated how in ovo exposure to environ- mentally relevant levels of PFOS affects expression of genes involved in lipid metabolism of developing chicken embryos. We have found a broad suppression of transcription of genes involved in fatty acid oxidation and PPAR-mediated transcription with more significant effects apparent at lower doses of PFOS. These results highlight the need for more research investigating the biological impacts of low levels of PFAS to properly inform environmental policy governing their regulation. . . . . . Keywords Perfluorooctane sulfonate PFOS In ovo Chicken Beta oxidation qPCR array Introduction compounds. Per- and polyfluoroalkyl substances (PFAS) are anthropogenic compounds used commercially for their stabil- The presence of persistent organic pollutants (POPs) is ever ity and surfactant properties. One of the major PFAS is increasing in the environment due to technological develop- perfluorooctane sulfonate (PFOS), which consists of a ment and growing commercialization of synthetic perfluorinated eight-carbon backbone and a sulphonate group. This makes this PFAS amphiphilic and a good surfactant with many fields of application. PFAS are ideal flame-retardants Responsible editor: Philippe Garrigues because of their carbon-fluorine bond, which is one of the Electronic supplementary material The online version of this article strongest in organic chemistry. This is also what renders them (https://doi.org/10.1007/s11356-018-2358-7) contains supplementary resilient to biological degradation, strong acids and alkalis and material, which is available to authorized users. photolysis. Industrial and commercial uses include stain repel- * Nikolai Scherbak lent, non-stick coatings and flame retardants for example in nikolai.scherbak@oru.se firefighting foam, clothes and upholstery, cooking utensils, food wrappers and electronics (Renner 2001). School of Biomedical Sciences, Charles Sturt University, Wagga The chemical and biological stability explains measurable Wagga, Australia environmental levels of different PFAS (i.e. PFOS) in differ- The Life Science Center, School of Science and Technology, Örebro ent matrices all over the globe. PFOS can be found in humans University, Örebro, Sweden worldwide (Kärrman et al. 2007), and is detected far from Present address: The Walter and Eliza Hall Institute, Department of manufacturing facilities in environments such as in high arctic Medical Biology, The University of Melbourne, Parkville, Australia icecaps, high-altitude lakes, off-shore waters, and deep-sea MTM Research Center, School of Science and Technology, Örebro oceans. PFOS is detectable in various animal species (Houde University, Örebro, Sweden et al. 2011), and is found in high concentrations in top predator Present address: Clinical Neurochemistry Laboratory, Sahlgrenska avian species. A study of eggs and developing embryos of the University Hospital, Mölndal, Sweden Environ Sci Pollut Res (2018) 25:23074–23081 23075 great cormorant and herring gull showed PFOS concentra- dimethyl sulfoxide (DMSO; Sigma-Aldrich, Darmstadt, tions in the μg/g wet weight in whole egg and liver (Nordén Germany) to final concentrations of either 0.1 or 1.0 mg/ml. et al. 2013). Studies of the effects of environmentally compa- On the fourth day of incubation, a single microinjection of 1 µl rable concentrations of PFOS on development of White of this PFOS solution per gram of egg was aseptically added Leghorn chicken showed reduced embryo survival, signifi- into air sacs of eggs resulting in treatment concentrations of 0.1 cant immunological, neurological and morphological changes respectively 1.0 µg of PFOS per 1g of egg. All treatments were in treated embryos compared to controls (Nordén et al. 2016; done in four replicates. Eggs injected with 1 µl/g egg of DMSO Peden-Adams et al. 2009). PFOS-induced immunotoxicity only (5%; n = 4) were used as controls. Holes were sealed with has been noted in various species at environmentally relevant paraffin and eggs put in an incubator at 37.5 °C and 60% hu- doses (DeWitt et al. 2012), and has the potential to impact the midity.Eggswereturnedin a6-hcycleandsacrificed1day fitness of wild species, particularly if faced with environmen- before expected pipping, i.e. 19 days post incubation start. tal challengessuchasinfection. Obtained liver samples were preserved at − 80 °C in RNA Hepatotoxicity is another important feature of PFOS expo- stabilisation solution (RNAlater®, Invitrogen/ThermoFisher, sure, with hepatomegaly, necrosis, and lipid accumulation MA, USA) until use. The experimental protocol was approved found in various animal models (Bijland et al. 2011;Du et by the Swedish Board of Agriculture, Jönköping, Sweden. al. 2009; Peden-Adams et al. 2009; Wan et al. 2012; Wang et al. 2014), although the mechanism behind this is still not clear. RNA purification and qPCR Transcriptomic analysis of the livers of 6-week-old chicken (Gallus gallus) that were subcuticularly exposed to low doses Approximately 15 mg of chicken embryo liver tissue was used of PFOS showed changes in expression of genes mainly in- for RNA purification using RNeasy® mini kit (QIAGEN, volved in electron and oxygen transport, and the metabolism Hilden, Germany) according to manufacturer protocol. RNA of lipids and fatty acids (Yeung et al. 2007). Other studies have was checked for purity and quantified using a spectrophotom- also assessed changes to gene expression using microarrays eter (NanoDrop® 2000; NanoDrop Technologies, (Martin et al. 2007; O'Brien et al. 2011), or investigation of Wilmington, DE). RNA integrity was confirmed with gel elec- small subsets of genes (Cwinn et al. 2008; O'Brien et al. trophoresis using a 1.2% (w/v) agarose gel. Complementary 2009); however, these have given conflicting results regarding DNA (cDNA) was synthesised using RT2 First Strand Kit® how PFOS impacts lipid metabolism. Moreover, direct com- (QIAGEN) according to manufacturer instructions using parison of these studies is challenging, due to large variations 0.5 μg purified RNA from each sample. Using qPCR, samples in the dose of PFOS used, with many studies using levels well were analysed using Chicken Fatty Acid Metabolism RT2 in excess of that found environmentally. Despite the varied profiler PCR array® (Catalogue PAGG-007Z; QIAGEN). results in the literature, hepatic steatosis is one of the more These arrays come in 96-well plate format, which include 84 commonly documented effects of PFOS exposure, which wells containing primers for genes of interest, 5 wells contain- strongly suggests liver lipid metabolism is indeed disrupted. ing housekeeping genes suggested by QIAGEN, and addition- To address how gene expression changes contribute to this, al controls to analyse genomic DNA contamination, reverse approaches with greater sensitivity than conventional micro- transcription efficiency and PCR array reproducibility. For the arrays, and greater depth and resolution than smaller focused list of analysed genes, see Table S1 in Supporting information, gene studies, are required. In the current study, we investigat- and for information on the RT2 profiler system see https:// ed the effects of environmentally relevant concentrations of dataanalysis.sabiosciences.com/pcr/documents/ PFOS on expression of genes controlling liver fatty acid me- RT2ProfilerDataAnalysisHandbook.pdf. tabolism of chicken embryos using the Chicken Fatty Acid One plate was used to analyse each biological sample, and Metabolism RT2 profiler PCR array®. an electronic pipetting system was used to limit any variation caused by pipetting technique. The qPCR program was set to 40 cycles consisting of the following temperatures and time Material and methods intervals: an initial denaturation at 95 °C for 5 minutes, followed by 15 s at 95 and 60 °C for 1 min for 40 cycles using Egg incubation and exposure an Applied Biosystems® 9700 thermocycler (Applied Biosystems, Carlsbad, CA). Each run was completed with Treatments were done as previously described by Nordén et al. melting curve analysis to confirm a single amplified product. (2016). Fertilised, unincubated eggs from White Leghorn chicken (Gallus gallus) were purchased from Ova Production, Data analysis Vittinge, Sweden and kept at 10–12 °C until incubation. Potassium salt of PFOS (Chemica 98%, Lot 77.282, approxi- The analysis software RT2 Profiler PCR array Data Analysis® mately 21% branched isomer) was dissolved in 5% solution of (QIAGEN) version 3.5 was used for interpretation of PCR 23076 Environ Sci Pollut Res (2018) 25:23074–23081 array data. In brief, data was first normalised against the geo- were both more strongly affected by the 1.0 μg/g of egg dose metric mean of a panel of housekeeping genes suggested by of PFOS (Table 1). Additionally, ACAT2 (FR = − 2.68; p = QIAGEN to generate a ΔCt value. As the differences between 0.025; 0.1 μg/g PFOS) was also downregulated by 1.0 μg/g the geometric means of all control and test groups were within PFOS (FR = − 2.22; p =0.08). the limits suggested by QIAGEN, we opted to use all five The genes selected for our analysis were those specifically genes in the panel for normalisation (ACTB, H6PD, HMBS, targeted to metabolic pathways, so we performed enrichment RPL4 and UBC). The software then calculates an average ΔCt analyses to determine which pathways were most influenced value for each of the control and treatments groups, as well as by our selected genes (Table S2). Of these pathways, a subset a standard deviation (SD) to assess variability. The average showed a proportionally large number of genes affected by ΔCt values are used to calculate fold change (FC), which is treatment with either dose of PFOS, particularly butanoate the ratio of relative gene expression between the control group metabolism, PPAR signalling pathway, fatty acid degradation, and the test group, using the formula 2^(ΔΔCt), where valine, leucine and isoleucine degradation and fatty acid me- ΔΔCt = average ΔCt(test group) – average ΔCt(control tabolism (Table 2). Full expression data for all genes tested in group). For the purposes of this study, FC is represented as the PCR array can be found in Table S3. fold regulation (FR), where for FC ≥ 1, FR = FC and for FC < 1, FR = − 1/FC. The p values for each gene were determined using a Student’s t test, which was calculated using the aver- Discussion age ΔCt of each test group versus the control group and their associated SD. To focus on gene expression changes that were PFAS such as PFOS are persistent environmental pollutants more likely to be associated with a biological effect, we used a and well known to cause adverse effects on the health of cut-off for differential expression as a FR ± 2 (p ≤ 0.05). various wild and laboratory animals. Although most KEGG pathway analysis of all tested genes, and those dif- European and Northern American countries now regulate pro- ferentially expressed genes at either dose of PFOS, was per- duction of these compounds, they are still actively used in formed using STRING version 10.5. During the pathway other countries, such as China (Fu et al. 2016), and are found analysis, STRING performs a Fisher’s exact test based on to be present in a range of consumer products (Kotthoff et al. the number of specified genes that fall within a particular 2015). Acceptable environmental levels have been debated, pathway category, the number of total genes annotated to that with a recent push to adopt lower thresholds from a number pathway and the total gene number present in the organism of different agencies. Additionally, recent animal experiments being studied. This is then corrected for multiple testing to indicate PFAS doses corresponding to current environmental give a false discovery rate, which is a measure of the likely levels can impact various biological pathways (Lilienthal et al. proportion of false positive gene matches for the specified 2017). In this study, we have used qPCR arrays to examine the pathway (Szklarczyk et al. 2017). effect of PFOS on expression of genes related to lipid metab- olism in livers of chicken embryos, and have found that low doses suppress transcription of genes relating to lipid catabo- Results lism and fatty acid β-oxidation. By using KEGG pathway analysis, the top identified met- As previous studies indicated dysregulated lipid metabolism abolic process affected in our analysis was butanoate metab- after treatment with perfluoronated compounds, we used a olism, which involves processing of short chain fatty acids focused PCR array to analyse 84 genes associated with lipid (SCFAs) and is known to be important for regulating mito- metabolism (Table S1). Normalisation against the five refer- chondrial energy production, lipogenesis and cellular meta- ence genes (ACTB, H6PD, HMBS, RPL4, UBC)revealed a bolic processes including fatty acid oxidation (Schönfeld general downregulation of expression after treatment of eggs and Wojtczak 2016). Additionally, SCFAs, including butyrate, with PFOS at both 0.1 and 1.0 μg/g of egg (Fig. 1a, b). Of have been shown to act as a switch between fatty acid oxida- these 84 genes, we found 22 genes with significant downreg- tion and lipogenesis in a PPARγ-dependent manner (den ulation (fold regulation (FR) ≤− 2; p ≤ 0.05), with four of Besten et al. 2015). Deregulated lipogenesis in the form of these genes (ACAD8, ACSL6, ELOVL3, FABP7) downregu- hepatic steatosis is commonly seen after exposure to PFOS lated in both the 0.1 and 1.0 μg/g treatment groups (Fig. 1c; (Cheng et al. 2016; Lai et al. 2017), as are perturbations to Table 1). A further seven genes with FR ≤−2(p ≤ 0.05) at a fatty acid oxidation (Wan et al. 2012). However, laboratory PFOS dose 0.1 μg/g of egg (ACAA2, ACAT1, ACSM3, CPT2, results are contradictory in relation to this, with some studies DECR1, FABP3, FABP5) were also downregulated to a lesser indicating increased beta oxidation or gene expression of rel- extent (FR ≤− 1.5; p ≤ 0.05) at the higher dose of PFOS evant enzymes (Hu et al. 2005;Nordén et al. 2012; Tan et al. (Table 1;FR ≤− 1.5 and > − 2.0 shown in italics). A similar 2012), while others indicate beta oxidation is supressed (Adinehzadeh and Reo 1998; Bijland et al. 2011; Cheng et effect was seen with ACOT8 and LOC771098,although they Environ Sci Pollut Res (2018) 25:23074–23081 23077 0.1µg/g PFOS vs control 1.0µg/g PFOS vs control FR = -2.0 FR = -2.0 ab p = 0.05 p = 0.05 Log2 (FC of 0.1µg/g PFOS/control Log2 (FC of 1.0µg/g PFOS/control ACAA2 FABP5 ACOT8 ACSBG1 HMGCL ACAD8 ACSM5 HADHA ACSM3 0.1 µg/g 1.0 µg/g ELOVL3 ACSL6 FABP3 PFOS PFOS ACAT1 LOC771098 PPA1 CPT2 DECR1 FABP7 FABP4 HMGCS2 SLC27A1 ACAT2 Fig. 1 Results of gene expression analysis from the Chicken Fatty Acid changes to expression are positioned above the horizontal line. Panel c Metabolism RT2 profiler PCR array®. Panel a, b shows volcano plots shows comparison genes with altered expression (FR ≥ ±2; p ≤ 0.5) by depicting a general downregulation of studied genes after in ovo treatment with 0.1 μg/g (purple) and 1.0 μg/g (green) of PFOS. Genes treatment with 0.1 μg/g (a) and 1.0 μg/g (b) of PFOS. Genes with whose expression was altered at both doses are represented by the inter- greater than two-fold regulation (FR) in expression are shown in green section (pink) (suppression) and red (induction). Genes with significant (p ≤ 0.5) al. 2016). These differences may be a result of the range of (2012) showed that mice exposed to PFOS had both increased concentrations being used, different responses between ani- peroxisomal beta oxidation and decreased mitochondrial beta mal models and cell culture and differences in how the path- oxidation. Importantly, impaired mitochondrial function is ways themselves are assessed. Interestingly, our results indi- proposed as a key event leading to hepatic steatosis (Angrish cate a suppression of transcription of genes involved in beta et al. 2016), such as is seen in PFOS liver toxicity. Moreover, oxidation that is more apparent at lower doses corresponding Wan et al. (2012) also found an increase in total beta oxida- to environmentally relevant concentrations, suggesting that tion, similar to that found in our previous analysis of day 10 metabolic responses to PFOS could differ based on the level embryonic chicken livers (Nordén et al. 2012). Interestingly, of exposure. the only acyl-CoA thioesterase (ACOT) found to be downreg- We also noted a number of the genes found to be differen- ulated by PFOS (at 1.0 μg/g of egg) in this current study was tially expressed in this study relate to mitochondrial beta ox- ACOT8, which is proposed to be the predominant ACOT in- idation. This includes CPT2 and DECR1, which both have volved in negative regulation of peroxisomal beta oxidation crucial roles in positive regulation of this pathway, as well as (Hunt et al. 2014). Together, these data imply PFOS may other positive regulators of beta oxidation, such as HADHA, induce a transition from mitochondrial beta oxidation to per- ACAA2 and ACAT1 (Houten et al. 2016). These were all found oxisomal beta oxidation, which could help to clarify both the to be downregulated at the lower dose of 0.1 μg/g egg of mechanism of PFOS toxicity and explain some of the contra- PFOS (Table 1). Previously, induction of beta oxidation by dictory results found in the literature, including our own pre- PFOS has previously been linked to peroxisomal beta oxida- vious results. Analysis of the gene expression profile of day 10 tion (Hu et al. 2005; Tan et al. 2012); however, Wan et al. embryonic livers would need to be done to clarify if this was -Log10 (p-value -Log10 (p-value 23078 Environ Sci Pollut Res (2018) 25:23074–23081 Table 1 Genes with expression changes of ≥ 2(p ≤ 0.05) after treatment palmitoyltransferase 1a (CPT1A) transcription seems to be with PFOS. Expression changes of significantly regulated genes in both regulated through PPARα (Honda et al. 2016). Similar stud- treatment conditions. Values in italics indicate significant (p ≤ 0.5) ies of PPAR-related effects of PFOS on the embryonic expression changes, with fold regulation < 2. Values in grey indicate where there were no statistically significant (p > 0.5) changes to chicken liver have given varied responses, with studies in expression both 18 and 21 days chicken embryos indicating no signif- icant changes to expression of PPAR-induced genes Gene symbol PFOS 0.1 μg/g egg PFOS 1.0 μg/g egg (O'Brien et al. 2009; Strömqvist et al. 2012). It should be fold regulation P value Fold regulation P value noted, however, that the study conducted at the same time point as ours used doses of PFOS 20-fold higher than the ACAA2 − 2.0106 0.00168 − 1.9936 0.002946 upper concentration used in the present study. ACAD8 − 2.3604 0.02053 − 2.2698 0.030889 In rodent and chicken studies, and in cell culture models of ACAT1 − 2.3008 0.020093 − 1.9327 0.041243 various species, most studies have found that PFOS causes ACAT2 − 2.6831 0.025219 − 2.2171 0.082354 induction of PPAR-mediated transcription, particularly ACOT8 − 1.7545 0.03791 − 2.084 0.01941 PPARα (Bjork et al. 2011; Elcombe et al. 2012; Strömqvist A A ACSBG1 − 2.37 0.025374 − 1.39 0.340918 et al. 2012). Interestingly, a study by Wang et al. (2014) ACSL6 − 3.954 0.013228 − 2.9527 0.03381 showed that both PPARα and CPT1A gene expression were ACSM3 − 2.1211 0.017689 − 1.7454 0.039628 impaired by PFOS only in mice that were fed a high-fat diet. ACSM5 − 1.4078 0.891541 − 5.3785 0.020546 This observation may have relevance to studies such as ours, CPT2 − 2.0424 0.015545 − 1.8386 0.032922 considering the relatively high in ovo fat content, and may DECR1 − 2.0763 0.006687 − 1.8092 0.024563 also go partway toward explaining differing results seen both ELOVL3 − 3.8073 0.009259 − 2.715 0.034701 between and within different species models. Furthermore, FABP3 − 2.236 0.029092 − 1.9463 0.048893 both avian and rodent studies indicate that PPAR-mediated a A FABP4 − 4.1192 0.017246 − 1.73 0.118269 effects are not solely responsible for toxic and disruptive ef- FABP5 − 3.5536 0.002826 − 1.8447 0.022399 fects of PFOS (Abbott et al. 2009; O'Brien et al. 2009; Rosen FABP7 − 2.904 0.035071 − 2.7541 0.038439 et al. 2010), indicating other transcription factors may be in- HADHA − 2.0065 0.042016 − 1.6934 0.087043 volved. One particularly interesting candidate is hepatocyte HMGCL − 2.7223 0.021643 − 1.6228 0.06323 nuclear factor 4α (HNF-4α), a transcription factor with HMGCS2 − 1.1307 0.441867 − 2.455 0.033322 known effects on lipid metabolism through regulating both LOC771098 − 1.5118 0.002207 − 2.0853 0.016339 PPARα and CPT1A gene transcription (Karagianni and PPA1 − 2.774 0.034539 − 1.5339 0.158511 Talianidis 2015; Martinez Jimenez et al. 2010). Binding of PFOS to HNF-4α is believed to disrupt the normal lipid bind- SLC27A1 − 2.1635 0.033117 − 1.6206 0.085314 ing required for its stabilisation, with PFOS treatment induc- Analysed data had C > 30 so results should be interpreted with caution ing degradation of both the mouse and human proteins (Beggs et al. 2016). The ability of PFOS to induce degradation of the case, or whether the differences between the two studies HNF-4α, or other transcription factors, could help explain were due to different transcriptional responses induced by the downregulation of multiple genes related to lipid metabo- PFOS at those developmental time points. lism seen here. Moreover, knockout of HNF-4α induces liver The broad transcriptional repression seen in this study steatosis in mouse models (Hayhurst et al. 2001), similar to could be explained by PFOS binding to, and interfering that seen after PFOS treatment. Follow-up studies would be with, relevant transcription factors. One possible mechanism required to see whether this effect was also seen in avian suggested by the KEGG pathway analysis in our study is species. PPAR-mediated regulation, and indeed there are a number of Our results indicate broad suppression of transcription studies that implicate PPAR as being responsible for the of genes associated with lipid metabolism after in ovo ex- metabolic disruption seen after PFOS exposure (Cwinn et posure of chicken embryos to PFOS, particularly at lower, al. 2008;Fanget al. 2012; Lai et al. 2017), although, like environmentally relevant doses. There are, however, some beta-oxidation, the directionality of this response is still de- limitations of these results. Firstly, we only analysed two bated. Interestingly, the second most significantly affected environmentally relevant doses of PFOS. This decision pathway in our KEGG analysis was PPAR signalling, with was made based on our previous work, which suggested approximately one third of the genes tested being signifi- that these doses were sufficient to cause changes to lipid cantly repressed by two-fold or more. Although there are metabolism (Nordén et al. 2012). However, in the previous fewer studies investigating the links between PPAR and beta study, we noted the most profound effect at 0.3 μg/g of egg oxidation in chicken, there is indication that there are some dose of PFOS, an intermediate dose to those used here. As similarities to other model organisms. In particular, carnitine most of the statistically significant changes to gene Environ Sci Pollut Res (2018) 25:23074–23081 23079 Table 2 Signalling pathways affected by PFOS treatment. KEGG Fraction of affected genes refers to the number of differentially affected pathway information generated from analysis in STRING using genes genes compared to the total number of genes in that pathway that were whose expression showed ≥ two-fold regulation (p ≤ 0.05) at either of analysed in the array (refer Table S2). The false discovery rate (calculated the two administered doses of PFOS, including the proportion of genes by STRING) is an indication of the likely proportion of false positive analysed within these pathways that met the aforementioned criteria. gene matches for the specified pathway KEGG Pathway description Observed genes Fraction of False ID affected discovery genes rate 650 Butanoate metabolism ACAT1, ACAT2, ACSM3, ACSM5, HMGCS2, HADHA, HMGCL 7 of 13 6.43E-14 3320 PPAR signalling pathway ACSBG1, ACSL6, CPT2, FABP3, FABP4, FABP5, FABP7, SLC27A1 8 of 28 2.01E-13 71 Fatty acid degradation ACAA2, ACAT1, ACAT2, ACSBG1, ACSL6, CPT2, HADHA 7 of 22 3.28E-13 280 Valine, leucine and isoleucine ACAA2, ACAD8, ACAT1, ACAT2, HMGCS2, HADHA, HMGCL 7 of 14 1.73E-12 degradation 1212 Fatty acid metabolism ACAA2, ACAT1, ACAT2, ACSBG1, ACSL6, CPT2, HADHA 7 of 27 2.00E-12 72 Synthesis and degradation ACAT1, ACAT2, HMGCS2, HMGCL 4 of 7 7.63E-09 of ketone bodies 1100 Metabolic pathways ACAA2, ACAD8, ACAT1, ACAT2, ACOT8, ACSBG1, ACSL6, 12 of 40 9.17E-09 ACSM3, ACSM5, HMGCS2, HADHA, HMGCL 900 Terpenoid backbone biosynthesis ACAT1, ACAT2, HMGCS2 3 of 4 2.22E-05 640 Propanoate metabolism ACAT1, ACAT2, HADHA 3 of 8 0.000115 380 Tryptophan metabolism ACAT1, ACAT2, HADHA 3 of 5 0.000188 1120 Microbial metabolism in diverse ACAA2, ACAT1, ACAT2, HADHA 4 of 10 0.000188 environments 310 Lysine degradation ACAT1, ACAT2, HADHA 3 of 5 0.000237 4146 Peroxisome ACOT8, ACSL6, HMGCL 3 of 16 0.00107 1200 Carbon metabolism ACAT1, ACAT2, HADHA 3 of 8 0.0017 62 Fatty acid elongation ACAA2, HADHA 2 of 4 0.00271 630 Glyoxylate and dicarboxylate ACAT1, ACAT2 2 of 4 0.00456 metabolism 620 Pyruvate metabolism ACAT1, ACAT2 2 of 6 0.00917 4920 Adipocytokine signalling ACSBG1, ACSL6 2 of 13 0.0213 pathway expression noted in this study were relatively small, we For these results to have relevance in relation to wild bird may have found more definitive results if that dose had species found to be affected by PFOS, we would also suggest also been used in this study. Secondly, due to the cost of investigating whether similar effects are seen in other avian the arrays, we were only able to analyse four individuals species. As we do not yet have full coverage of the genomes of per treatment group and we were not able to perform du- wild avian species, a metabolomics-based study would pres- plicate plates. However, this is a robust commercially de- ently be the most appropriate method to investigate this. That signed assay that has been used in a wide range of studies, said, the Avian Phylogenomics Consortium is currently work- and is equipped with various controls, including PCR re- ing to sequence all known avian species (Zhang 2015), and producibility. This control showed little variability both this knowledge would enable comparative gene expression within and between plates, giving us confidence in the studies between species. If similar results as seen here are assay and the results it generated. Moreover, the fact that found in wild avian species, changes to expression of key the responses were consistent enough to give statistically metabolic enzymes such as CPT2 and DECR1 could poten- significant data despite the small sample size is encourag- tially act as environmental markers of PFOS exposure. Such ing. Thirdly, as we were unable to determine the sex of the applications would, however, need to take into account wheth- embryos studied, we cannot exclude that sex differences er other common pollutants have overlapping effects. have contributed to these results. Follow up studies will A better understanding of species-specific effects of PFAS need to take this into account. Lastly, it should also be and the doses at which they occur is important when consid- notedthatwe onlyanalysedgeneexpressiondata and,as ering both acceptable levels of these compounds in the envi- such, further studies, such as metabolomics or proteomics, ronment and safe exposure levels for persons with occupation- are required to confirm whether these changes relate to a al contact with PFAS. Although we did not directly measure hepatic liver PFOS concentrations, similarly designed functional impairment of lipid metabolism. 23080 Environ Sci Pollut Res (2018) 25:23074–23081 Angrish MM, Kaiser JP, McQueen CA, Chorley BN (2016) Tipping the previous studies have shown these concentrations are approx- balance: hepatotoxicity and the 4 apical key events of hepatic imately equivalent to the administered dose at the time point steatosis. Toxicol Sci 150:261–268. https://doi.org/10.1093/toxsci/ we measured (Nordén et al. 2016; O'Brien et al. 2009). As kfw018 these concentrations are comparable to those found in envi- Beggs K, McGreal S, McCarthy A, Gunewardena S, Lampe J, Lau C, Apte U (2016) The role of hepatocyte nuclear factor 4-alpha in ronmental analyses of wild birds, it is important that future perfluorooctanoic acid- and perfluorooctanesulfonic acid-induced studies are done to determine whether similar effects are seen hepatocellular dysfunction. Toxicol Appl Pharmacol 304:18–29 in these species, and whether chicken can continue to be used Bijland S, Rensen PCN, Pieterman EJ, Maas ACE, van der Hoorn JW, as a model for environmental exposure. 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Chemosphere 74:723–729. https://doi.org/ 10.1016/j.chemosphere.2008.09.075 Acknowledgements This work was supported by Örebro University, Elcombe CR, Elcombe BM, Foster JR, Chang S-C, Ehresman DJ, grants from Magnus Bergvalls Stiftelse and by the EnForce project, Butenhoff JL (2012) Hepatocellular hypertrophy and cell prolifera- funded by Knowledge Foundation. We also would like to acknowledge tion in Sprague–Dawley rats from dietary exposure to potassium Steffen Keiter for critical reading of the manuscript and for his valuable perfluorooctanesulfonate results from increased expression of comments. Furthermore, we would like to thank the reviewers for their xenosensor nuclear receptors PPARα and CAR/PXR. 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