Perfluoroalkyl compounds (PFCs) are a group of chemical surfactants most notably used in non-stick and stain-resistance applications. Due to their wide-spread use and inherent resistance to degradation, several PFCs have become persistent environmental contaminants. Despite the high concentrations of PFCs reported in wild birds and their eggs, very little is known about the toxicological effects they have on avian species. This thesis investigates the developmental toxicity of PFCs in an avian model species: the domestic chicken (Gallus gallus domesticus). Egg injection experiments were performed to assess the in ovo toxicity of perfluorooctane sulfonate (technical grade, T-PFOS), perfluorooctanoic acid (PFOA), perfluorodecane sulfonate (PFDS) and perfluoroundecanoic acid (PFUdA). Real-time RT-PCR was then used to measure the transcription of candidate biomarker genes in the liver tissue of day 20 embryos. Candidate genes were selected based on their responsiveness to PFC exposure in previously conducted in vitro screening assays. In ovo exposure to PFOS resulted in a dose-dependent decrease in embryo pipping success (a measure of hatching success) with an LD50 of 93 μg/g (3.54 μg/g-672,910 μg/g, 95% confidence interval), however the expression of peroxisome proliferator-activated receptor alpha (PPARα)-regulated genes was not affected in liver tissue as hypothesized. PFOA, PFDS and PFUdA had no effect on the pipping success of chicken embryos. The expression of cytochrome P450 1A4 (CYP1A4) and liver fatty acid binding protein (L-FABP) mRNA increased in embryo liver tissue following in ovo exposure to PFUdA but was only statistically significant at 10 μg/g, which is several orders of magnitude higher than concentrations reported in wild bird eggs. The isomer-specific accumulation of PFOS in chicken embryo livers was also investigated using an in-port derivatization gas-chromatography/mass spectrometry (GC-MS) method. Prior to incubation, chicken eggs were injected with T-PFOS, composed of 63% linear isomer (L-PFOS) and 37.3% branched isomers. The isomer profiles in day-20 embryo liver tissue showed up to 20% enrichment in the proportion of L-PFOS, compared to T-PFOS, with a corresponding decrease in the proportion of branched isomers. This enrichment was inversely proportional to dose. Finally, the transcriptional profiles of cultured chicken embryonic hepatocytes (CEH) exposed to either T-PFOS or L-PFOS were compared using Agilent 4x44k Chicken (V2) Gene Expression microarrays. At equal concentrations (10 μM), T-PFOS altered the expression of significantly more genes (340 genes, >1.5 fold change, false discovery rate adjusted p<0.05) compared to L-PFOS (130 genes). Functional analysis showed that L-PFOS and T-PFOS affected genes involved in lipid metabolism, cellular growth and proliferation, and cell-cell signaling. Pathway and interactome analysis suggested that gene expression may be affected through RXR, oxidative stress response, TP53 signaling, MYC signaling, Wnt/β-catenin signaling and PPARγ and SREBP receptors. In all functional categories and pathways examined, T-PFOS had a more pronounced disruptive effect on transctional regulation than L-PFOS. In summary, egg injection experiments showed that T-PFOS (but not linear PFOA, PFDS or PFUdA) may affect the hatching success of the chicken at environmentally relevant concentrations. It was also demonstrated that the accumulation of PFOS in embryonic liver is isomer specific, and leads to an enrichment of L-PFOS. The increased transcriptional disruption caused by T-PFOS in cultured hepatocytes over L-PFOS suggests that the branched isomers may be largely responsible for the toxicological effects of PFOS. Combined, the results from this thesis demonstrate the importance of considering PFOS isomer burdens during risk assessment. In addition, gene expression analysis identified several candidate mechanisms for PFOS toxicity.

Perfluoroalkyl compounds (PFCs) are a group of chemical surfactants most notably used in non-stick and stain-resistance applications. Due to their wide-spread use and inherent resistance to degradation, several PFCs have become persistent environmental contaminants. Despite the high concentrations of PFCs reported in wild birds and their eggs, very little is known about the toxicological effects they have on avian species. This thesis investigates the developmental toxicity of PFCs in an avian model species: the domestic chicken (Gallus gallus domesticus). Egg injection experiments were performed to assess the in ovo toxicity of perfluorooctane sulfonate (technical grade, T-PFOS), perfluorooctanoic acid (PFOA), perfluorodecane sulfonate (PFDS) and perfluoroundecanoic acid (PFUdA). Real-time RT-PCR was then used to measure the transcription of candidate biomarker genes in the liver tissue of day 20 embryos. Candidate genes were selected based on their responsiveness to PFC exposure in previously conducted in vitro screening assays. In ovo exposure to PFOS resulted in a dose-dependent decrease in embryo pipping success (a measure of hatching success) with an LD50 of 93 μg/g (3.54 μg/g-672,910 μg/g, 95% confidence interval), however the expression of peroxisome proliferator-activated receptor alpha (PPARα)-regulated genes was not affected in liver tissue as hypothesized. PFOA, PFDS and PFUdA had no effect on the pipping success of chicken embryos. The expression of cytochrome P450 1A4 (CYP1A4) and liver fatty acid binding protein (L-FABP) mRNA increased in embryo liver tissue following in ovo exposure to PFUdA but was only statistically significant at 10 μg/g, which is several orders of magnitude higher than concentrations reported in wild bird eggs. The isomer-specific accumulation of PFOS in chicken embryo livers was also investigated using an in-port derivatization gas-chromatography/mass spectrometry (GC-MS) method. Prior to incubation, chicken eggs were injected with T-PFOS, composed of 63% linear isomer (L-PFOS) and 37.3% branched isomers. The isomer profiles in day-20 embryo liver tissue showed up to 20% enrichment in the proportion of L-PFOS, compared to T-PFOS, with a corresponding decrease in the proportion of branched isomers. This enrichment was inversely proportional to dose. Finally, the transcriptional profiles of cultured chicken embryonic hepatocytes (CEH) exposed to either T-PFOS or L-PFOS were compared using Agilent 4x44k Chicken (V2) Gene Expression microarrays. At equal concentrations (10 μM), T-PFOS altered the expression of significantly more genes (340 genes, >1.5 fold change, false discovery rate adjusted p<0.05) compared to L-PFOS (130 genes). Functional analysis showed that L-PFOS and T-PFOS affected genes involved in lipid metabolism, cellular growth and proliferation, and cell-cell signaling. Pathway and interactome analysis suggested that gene expression may be affected through RXR, oxidative stress response, TP53 signaling, MYC signaling, Wnt/β-catenin signaling and PPARγ and SREBP receptors. In all functional categories and pathways examined, T-PFOS had a more pronounced disruptive effect on transctional regulation than L-PFOS. In summary, egg injection experiments showed that T-PFOS (but not linear PFOA, PFDS or PFUdA) may affect the hatching success of the chicken at environmentally relevant concentrations. It was also demonstrated that the accumulation of PFOS in embryonic liver is isomer specific, and leads to an enrichment of L-PFOS. The increased transcriptional disruption caused by T-PFOS in cultured hepatocytes over L-PFOS suggests that the branched isomers may be largely responsible for the toxicological effects of PFOS. Combined, the results from this thesis demonstrate the importance of considering PFOS isomer burdens during risk assessment. In addition, gene expression analysis identified several candidate mechanisms for PFOS toxicity.

Perfluoroalkyl compounds (PFCs) are a group of chemical surfactants most notably used in non-stick and stain-resistance applications. Due to their wide-spread use and inherent resistance to degradation, several PFCs have become persistent environmental contaminants. Despite the high concentrations of PFCs reported in wild birds and their eggs, very little is known about the toxicological effects they have on avian species. This thesis investigates the developmental toxicity of PFCs in an avian model species: the domestic chicken (Gallus gallus domesticus). Egg injection experiments were performed to assess the in ovo toxicity of perfluorooctane sulfonate (technical grade, T-PFOS), perfluorooctanoic acid (PFOA), perfluorodecane sulfonate (PFDS) and perfluoroundecanoic acid (PFUdA). Real-time RT-PCR was then used to measure the transcription of candidate biomarker genes in the liver tissue of day 20 embryos. Candidate genes were selected based on their responsiveness to PFC exposure in previously conducted in vitro screening assays. In ovo exposure to PFOS resulted in a dose-dependent decrease in embryo pipping success (a measure of hatching success) with an LD50 of 93 μg/g (3.54 μg/g-672,910 μg/g, 95% confidence interval), however the expression of peroxisome proliferator-activated receptor alpha (PPARα)-regulated genes was not affected in liver tissue as hypothesized. PFOA, PFDS and PFUdA had no effect on the pipping success of chicken embryos. The expression of cytochrome P450 1A4 (CYP1A4) and liver fatty acid binding protein (L-FABP) mRNA increased in embryo liver tissue following in ovo exposure to PFUdA but was only statistically significant at 10 μg/g, which is several orders of magnitude higher than concentrations reported in wild bird eggs. The isomer-specific accumulation of PFOS in chicken embryo livers was also investigated using an in-port derivatization gas-chromatography/mass spectrometry (GC-MS) method. Prior to incubation, chicken eggs were injected with T-PFOS, composed of 63% linear isomer (L-PFOS) and 37.3% branched isomers. The isomer profiles in day-20 embryo liver tissue showed up to 20% enrichment in the proportion of L-PFOS, compared to T-PFOS, with a corresponding decrease in the proportion of branched isomers. This enrichment was inversely proportional to dose. Finally, the transcriptional profiles of cultured chicken embryonic hepatocytes (CEH) exposed to either T-PFOS or L-PFOS were compared using Agilent 4x44k Chicken (V2) Gene Expression microarrays. At equal concentrations (10 μM), T-PFOS altered the expression of significantly more genes (340 genes, >1.5 fold change, false discovery rate adjusted p<0.05) compared to L-PFOS (130 genes). Functional analysis showed that L-PFOS and T-PFOS affected genes involved in lipid metabolism, cellular growth and proliferation, and cell-cell signaling. Pathway and interactome analysis suggested that gene expression may be affected through RXR, oxidative stress response, TP53 signaling, MYC signaling, Wnt/β-catenin signaling and PPARγ and SREBP receptors. In all functional categories and pathways examined, T-PFOS had a more pronounced disruptive effect on transctional regulation than L-PFOS. In summary, egg injection experiments showed that T-PFOS (but not linear PFOA, PFDS or PFUdA) may affect the hatching success of the chicken at environmentally relevant concentrations. It was also demonstrated that the accumulation of PFOS in embryonic liver is isomer specific, and leads to an enrichment of L-PFOS. The increased transcriptional disruption caused by T-PFOS in cultured hepatocytes over L-PFOS suggests that the branched isomers may be largely responsible for the toxicological effects of PFOS. Combined, the results from this thesis demonstrate the importance of considering PFOS isomer burdens during risk assessment. In addition, gene expression analysis identified several candidate mechanisms for PFOS toxicity.

Background: Exposure to perfluorooctane sulfonic acid (PFOS) or to perfluorooctanoic acid (PFOA) increases mouse and human peroxisome proliferator–activated receptor alpha (PPARα) subtype activity, which influences lipid metabolism. Because cholesterol is the substrate from which testosterone is synthesized, exposure to these substances has the potential to alter testosterone concentrations. Objectives: We explored associations of total testosterone and sex hormone–binding globulin (SHBG) concentrations at age 15 years with prenatal exposures to PFOS, PFOA, perfluorohexane sulfonic acid (PFHxS), and perfluoronanoic acid (PFNA) in females. Methods: Prenatal concentrations of the perfluoroalkyl acids (PFAAs) were measured in serum collected from pregnant mothers at enrollment (1991–1992) in the Avon Longitudinal Study of Parents and Children (ALSPAC). The median gestational age when the maternal blood sample was obtained was 16 weeks (interquartile range, 11–28 weeks). Total testosterone and SHBG concentrations were measured in serum obtained from their daughters at 15 years of age. Associations between prenatal PFAAs concentrations and reproductive outcomes were estimated using linear regression models (n = 72). Results: Adjusted total testosterone concentrations were on average 0.18-nmol/L (95% CI: 0.01, 0.35) higher in daughters with prenatal PFOS in the upper concentration tertile compared with daughters with prenatal PFOS in the lower tertile. Adjusted total testosterone concentrations were also higher in daughters with prenatal concentrations of PFOA (β = 0.24; 95% CI: 0.05, 0.43) and PFHxS (β = 0.18; 95% CI: 0.00, 0.35) in the upper tertile compared with daughters with concentrations in the lower tertile. We did not find evidence of associations between PFNA and total testosterone or between any of the PFAAs and SHBG. Conclusions: Our findings were based on a small study sample and should be interpreted with caution. However, they suggest that prenatal exposure to some PFAAs may alter testosterone concentrations in females.