- Email: [email protected]
A brief overview of PFAA modes of action
8
Abstracts / Reproductive Toxicology 33 (2012) 1–10
cholesterol and and/or triglycerides are frequently observed clinical chemistry findings in toxicological studies. Changes in the lipid content of hepatocytes have also been noted. The roles of PPAR␣ and CAR/PXR in mediating these changes have become clearer in recent years. For example, the hypolipidemic effects of perfluorohexanesulfonate (PFHxS) and PFOS can be ascribed to PPAR␣ and PXR mediated changes in the formation and clearance of lipoproteins. A number of rodent immunotoxicology studies have been published on PFOA and PFOS. In general, these studies have provided evidence of effects on inflammatory responses, production of proteins involved in immune responses, lymphoid organ weights, and antibody synthesis. Reported findings have been somewhat inconsistent, and have varied with dose, strain, and dosing methodology. Although observed responses have been shown to be driven, in part, by PPAR␣, the need to study also the role of PPAR␣-independent processes and other factors that may affect the nature of observed responses has become clear. Several perfluoroalkyls have been studied for their potential to disturb reproduction in rats. Overt effects on reproductive function in male and female rats generally have not been observed. Increased early full-liter resorption is one effect noted in female rodents dosed with PFOA during gestation. This may be more the result of an effect on the maintenance of pregnancy in the rodent than an embryotoxic effect. Because there are significant differences between humans and rodents in the maintenance of pregnancy, it is important to develop a better understanding of this observation. Since the first observation of perinatal mortality in a multigeneration study of PFOS in rats, there have been numerous developmental studies undertaken to increase understanding of the potential developmental toxicity of perfluoroalkyls. The prenatal developmental effects of these compounds largely are unremarkable. Postnatal mortality, developmental delays, and stunting of development of mammary tissue have been a major focus of research. A principal role for PPAR␣ in mediating the developmental effects of PFOA in mice has been discovered. How this finding translates to potential human relevance requires additional insight and discussion. In the case of PFOS, post-natal developmental effects appear to be either not mediated by PPAR␣ or at least largely independent of PPAR␣. Potential interference with the functional properties of pulmonary surfactant at birth has been and continues to be a leading hypothesis for the basis of PFOS postnatal developmental effects. Hormonal investigations have focused primarily on the etiology of PFOS-induced hypothyroxinemia in toxicology studies and investigation of changes in sex hormones for PFOS and PFOA. PFOS-induced hypothyroxinemia in rats appears to be the result of increased displacement from serum carrier proteins and increased uptake and elimination by the liver and kidney. Although PFOA has been shown to increase serum estradiol in male rats, PFOS and PFOA decreased serum estradiol in monkeys at relatively high doses. The increase in estradiol in male rats may be the result of a PPAR␣-mediated induction of aromatase. Most of the responses observed with perfluoroalkyls, either as early responses occurring during the course of repeat dosing or as responses occurring at lower doses, do not suggest the nervous system as a primary site of action. It is perhaps for this reason that fewer neurotoxicological investigations have been undertaken. Even after lethal or sub-lethal doses, there has not been compelling evidence of neurotoxicological damage at doses relevant to risk assessment. In summary, in navigating the vast sea of data on the biological interactions of perfluoroalkyls, it appears we should set our sights on expanding our understanding of the molecular biological, metabolic, and physiological bases of responses observed in laboratory toxicology studies. Even more important, however, is
incorporating an understanding of epidemiological investigations as we try to gain perspective on the toxicological data. Translating understanding from toxicological systems into a human context will improve our collective ability to understand potential humanhealth risk from environmental levels of exposures to these agents. doi:10.1016/j.reprotox.2011.11.024 O21 PFAA ecotoxicology John L. Newsted Entrix, Okemos, MI; Michigan State University, East Lansing, MI, United States Perfluorinated compounds (PFCs) have been manufactured for over 50 years and, due to their unique properties of repelling both water and oil, have been used in numerous products that have resulted in their release into the environment. Since 2001 when Giesy and Kannan reported on the global distribution and bioaccumulation of several PFCs in fish, birds and mammalian wildlife, significant effort has been put measurement of these compounds to better characterize their distribution in biotic and abiotic matrices. In addition, development of improved analytical methods has allowed researchers to identify additional perfluorinated compounds and classes in the environment such as the fluorotelomer alcohols and perfluorophosphate surfactants. While analytical methodology has allowed the detection of these compounds at ever decreasing levels, the ecotoxicological significance of these compounds remains largely unknown. Of the perfluorinated chemicals tested to date, perfluorooctane sulfonate (PFOS) and perfluorooctanoic acid (PFOA) have been the most extensively studied in ecologically relevant species. As a result, this presentation will focus principally on PFOS with several objectives in mind: (1) to provide an up-to-date overview of currently available toxicological data for aquatic and terrestrial species, (2) to put these data into context relative to establishing toxicity reference values and water quality criteria for aquatic species and wildlife, (3) to identify data gaps that will need to be addressed reduce the uncertainties in the hazard assessments of this compounds, and (4) draw comparisons between what is know about ecotoxicological effects of PFOS to other PFCs such as PFOA and fluorotelomer alcohols to provide some insight into potential hazard to ecological systems. doi:10.1016/j.reprotox.2011.11.025 O22 A brief overview of PFAA modes of action Chris Corton Integrated Systems Toxicology Division, National Health and Environmental Effects Research Lab, U.S. EPA, Research Triangle Park, NC, United States Several putative modes of action have been suggested to account for the adverse effects induced by perfluorinated alkyl acids (PFAAs). These will be briefly summarized. In particular, in mouse and rat liver PFAAs including perfluorooctanoic acid (PFOA) and perfluorooctane sulfonate (PFOS) elicit transcriptional and phenotypic effects similar to peroxisome proliferator chemicals (PPC) that work through the nuclear receptor peroxisome proliferator-activated receptor alpha (PPAR␣). Recent studies indicate that along with PPAR␣, other nuclear receptors are required for transcriptional changes in the liver after PFAA exposure
Abstracts / Reproductive Toxicology 33 (2012) 1–10
including the constitutive activated receptor (CAR) and other PPAR family members. This session will focus on examining the role of these and other nuclear receptors in toxicities in the liver or extra-hepatic tissues including developmental toxicity, immunotoxicity, and metabolic changes that may be linked to metabolic syndrome. doi:10.1016/j.reprotox.2011.11.026 O23 PPAR involvement in PFAA developmental toxicity Barbara D. Abbott Developmental Toxicology Branch, Toxicity Assessment Division, National Health and Environmental Effects Research Laboratory, Office of Research and Development, U.S. Environmental Protection Agency, Research Triangle Park, NC, United States Perfluoroalkyl acids (PFAAs) are found in the environment and in serum of wildlife and humans. Perfluorooctanoic acid (PFOA), perfluorononanoic acid (PFNA), and perfluorooctane sulfonate (PFOS) are developmentally toxic in rodents. The effects of in utero exposure include increased neonatal death, developmental delay, and deficits in postnatal growth. Members of the PFAA family of compounds were shown to activate peroxisome proliferatoractivated receptor-alpha (PPAR␣) in a transfected Cos-1 cell model. PPAR␣, PPAR/␦ and PPAR␥, are expressed in human and rodent embryos with tissue and developmental stage-specific expression patterns. PPARs have significant physiological roles, regulating energy homeostasis, adipogenesis, lipid metabolism, inflammatory responses, and hematopoiesis. Studies in PPAR␣ knockout (KO) mice revealed a role for PPAR␣ in the induction of developmental toxicity by PFOA and PFNA, but not PFOS. The induction of postnatal lethality by PFOS may be related to effects on lung function. In fetal lung and liver, PFOA and PFOS altered gene expression and in fetal liver both compounds produced profiles typical of PPAR␣ activation. Activation of some genes in liver persisted to PND63. Fetal and neonatal heart also showed altered gene expression after exposure to PFOA. Effects in heart differed from liver and were found at least to PND28. Although it is not clear exactly how changes in gene expression are related to the effects on neonatal survival and growth, perturbation of PPAR␣regulated lipid and glucose homeostasis potentially impact energy availability and utilization. The absence of developmental toxicity in the PPAR␣ KO mouse and alterations in gene expression typical of PPAR␣ activation in the wild type mouse, support a role for PPAR␣ in mediating the developmental toxicity of PFOA and PFNA. The primary cause of PFOS-induced developmental toxicity is PPAR␣-independent and may be an effect on lung function. However, PFOS altered gene expression in a manner similar to PFOA and if effects on PPAR␣-regulated genes are responsible for developmental toxicity, then it is expected that PFOS would still produce developmental toxicity even in absence of an effect on lung function. This abstract does not necessarily reflect US EPA policy. doi:10.1016/j.reprotox.2011.11.027
9
O24 Nuclear receptor involvement in PPAA-induced metabolic changes Mitch B. Rosen Integrated Systems Biology, NHEERL, ORD, U.S. EPA, Research Triangle Park, NC, United States It has been proposed that certain xenobiotics commonly identified in biomonitoring studies may play a role in the incidence of obesity and metabolic syndrome in the United States and other countries. The list of potential “environmental obesogens” includes endocrine disrupting compounds such as Diethylstilbesterol and Bisphenol A, as well as chemicals that present a variety of toxicities in mammals such as the organotins. Interestingly, perfluoroalkyl acids (PFAAs) have also been mentioned as possible environmental obesogens because of their ability to alter energy homeostasis. While it may not be clear how compounds that function as peroxisome proliferator activated receptor alpha (PPAR␣) ligands could induce obesity, the biological activity of PFAAs is not limited to activation of PPAR␣. Many of these compounds also activate the constitutive androstane receptor (CAR) and it is now recognized that CAR influences not only xenobiotic metabolism but also certain aspects of energy metabolism as well. Chronic exposure to PPAR␣ agonists also has the potential to alter energy metabolism in ways that are only first beginning to be understood. For example, chemical or metabolic challenge during gestation could result in PPAR␣-dependent epigenetic modifications which result in persistent alterations in phenotype. This talk will consider the potential effects of chronic PFAA exposure on nuclear receptor regulated energy metabolism. (This abstract does not necessarily reflect EPA policy.) doi:10.1016/j.reprotox.2011.11.028 O25 PPAR involvement in PFAA immunotoxicity J.C. DeWitt 1,∗ , M.M. Peden-Adams 2 , D.E. Keil 2 , S.E. Anderson 3 1
Department of Pharmacology and Toxicology, East Carolina University, Greenville, NC, United States 2 Clinical Laboratory Sciences, University of Nevada, Las Vegas, NV, United States 3 Health Effects Laboratory Division, National Institute for Occupational Safety and Health, Morgantown, WV, United States When immune endpoints are evaluated in experimental animals exposed to PFAAs, alterations in antibody synthesis, inflammatory responses, cytokine production, lymphocyte cellularity, and lymphoid organ weights are reported. These alterations indicate that exposure to PFAAs results in immunomodulation. The mechanisms by which immunomodulation occur have not been elucidated, although ligation of PPAR-alpha, and to some extent PPAR-gamma, is assumed to explain some of the immune changes associated with PFAA exposure. However, increasing experimental evidence suggests that while PPAR-alpha receptor activation is important for mediation of inflammatory responses and many nonimmune parameters, several immune endpoints are affected by PFAA exposure whether or not a functional PPAR-alpha is present. In addition, host phenotype also may impact the effects of PFAA exposure on immune responses. Two strains of PPAR-alpha knockout mice are generally available; one is on a 129 background and the other is on a B6 background. Initial studies with the 129 strain indicate that attenuation of antibody synthesis and decreases in lymphocyte cellularity and lymphoid organ weights observed in
Abstracts / Reproductive Toxicology 33 (2012) 1–10
cholesterol and and/or triglycerides are frequently observed clinical chemistry findings in toxicological studies. Changes in the lipid content of hepatocytes have also been noted. The roles of PPAR␣ and CAR/PXR in mediating these changes have become clearer in recent years. For example, the hypolipidemic effects of perfluorohexanesulfonate (PFHxS) and PFOS can be ascribed to PPAR␣ and PXR mediated changes in the formation and clearance of lipoproteins. A number of rodent immunotoxicology studies have been published on PFOA and PFOS. In general, these studies have provided evidence of effects on inflammatory responses, production of proteins involved in immune responses, lymphoid organ weights, and antibody synthesis. Reported findings have been somewhat inconsistent, and have varied with dose, strain, and dosing methodology. Although observed responses have been shown to be driven, in part, by PPAR␣, the need to study also the role of PPAR␣-independent processes and other factors that may affect the nature of observed responses has become clear. Several perfluoroalkyls have been studied for their potential to disturb reproduction in rats. Overt effects on reproductive function in male and female rats generally have not been observed. Increased early full-liter resorption is one effect noted in female rodents dosed with PFOA during gestation. This may be more the result of an effect on the maintenance of pregnancy in the rodent than an embryotoxic effect. Because there are significant differences between humans and rodents in the maintenance of pregnancy, it is important to develop a better understanding of this observation. Since the first observation of perinatal mortality in a multigeneration study of PFOS in rats, there have been numerous developmental studies undertaken to increase understanding of the potential developmental toxicity of perfluoroalkyls. The prenatal developmental effects of these compounds largely are unremarkable. Postnatal mortality, developmental delays, and stunting of development of mammary tissue have been a major focus of research. A principal role for PPAR␣ in mediating the developmental effects of PFOA in mice has been discovered. How this finding translates to potential human relevance requires additional insight and discussion. In the case of PFOS, post-natal developmental effects appear to be either not mediated by PPAR␣ or at least largely independent of PPAR␣. Potential interference with the functional properties of pulmonary surfactant at birth has been and continues to be a leading hypothesis for the basis of PFOS postnatal developmental effects. Hormonal investigations have focused primarily on the etiology of PFOS-induced hypothyroxinemia in toxicology studies and investigation of changes in sex hormones for PFOS and PFOA. PFOS-induced hypothyroxinemia in rats appears to be the result of increased displacement from serum carrier proteins and increased uptake and elimination by the liver and kidney. Although PFOA has been shown to increase serum estradiol in male rats, PFOS and PFOA decreased serum estradiol in monkeys at relatively high doses. The increase in estradiol in male rats may be the result of a PPAR␣-mediated induction of aromatase. Most of the responses observed with perfluoroalkyls, either as early responses occurring during the course of repeat dosing or as responses occurring at lower doses, do not suggest the nervous system as a primary site of action. It is perhaps for this reason that fewer neurotoxicological investigations have been undertaken. Even after lethal or sub-lethal doses, there has not been compelling evidence of neurotoxicological damage at doses relevant to risk assessment. In summary, in navigating the vast sea of data on the biological interactions of perfluoroalkyls, it appears we should set our sights on expanding our understanding of the molecular biological, metabolic, and physiological bases of responses observed in laboratory toxicology studies. Even more important, however, is
incorporating an understanding of epidemiological investigations as we try to gain perspective on the toxicological data. Translating understanding from toxicological systems into a human context will improve our collective ability to understand potential humanhealth risk from environmental levels of exposures to these agents. doi:10.1016/j.reprotox.2011.11.024 O21 PFAA ecotoxicology John L. Newsted Entrix, Okemos, MI; Michigan State University, East Lansing, MI, United States Perfluorinated compounds (PFCs) have been manufactured for over 50 years and, due to their unique properties of repelling both water and oil, have been used in numerous products that have resulted in their release into the environment. Since 2001 when Giesy and Kannan reported on the global distribution and bioaccumulation of several PFCs in fish, birds and mammalian wildlife, significant effort has been put measurement of these compounds to better characterize their distribution in biotic and abiotic matrices. In addition, development of improved analytical methods has allowed researchers to identify additional perfluorinated compounds and classes in the environment such as the fluorotelomer alcohols and perfluorophosphate surfactants. While analytical methodology has allowed the detection of these compounds at ever decreasing levels, the ecotoxicological significance of these compounds remains largely unknown. Of the perfluorinated chemicals tested to date, perfluorooctane sulfonate (PFOS) and perfluorooctanoic acid (PFOA) have been the most extensively studied in ecologically relevant species. As a result, this presentation will focus principally on PFOS with several objectives in mind: (1) to provide an up-to-date overview of currently available toxicological data for aquatic and terrestrial species, (2) to put these data into context relative to establishing toxicity reference values and water quality criteria for aquatic species and wildlife, (3) to identify data gaps that will need to be addressed reduce the uncertainties in the hazard assessments of this compounds, and (4) draw comparisons between what is know about ecotoxicological effects of PFOS to other PFCs such as PFOA and fluorotelomer alcohols to provide some insight into potential hazard to ecological systems. doi:10.1016/j.reprotox.2011.11.025 O22 A brief overview of PFAA modes of action Chris Corton Integrated Systems Toxicology Division, National Health and Environmental Effects Research Lab, U.S. EPA, Research Triangle Park, NC, United States Several putative modes of action have been suggested to account for the adverse effects induced by perfluorinated alkyl acids (PFAAs). These will be briefly summarized. In particular, in mouse and rat liver PFAAs including perfluorooctanoic acid (PFOA) and perfluorooctane sulfonate (PFOS) elicit transcriptional and phenotypic effects similar to peroxisome proliferator chemicals (PPC) that work through the nuclear receptor peroxisome proliferator-activated receptor alpha (PPAR␣). Recent studies indicate that along with PPAR␣, other nuclear receptors are required for transcriptional changes in the liver after PFAA exposure
Abstracts / Reproductive Toxicology 33 (2012) 1–10
including the constitutive activated receptor (CAR) and other PPAR family members. This session will focus on examining the role of these and other nuclear receptors in toxicities in the liver or extra-hepatic tissues including developmental toxicity, immunotoxicity, and metabolic changes that may be linked to metabolic syndrome. doi:10.1016/j.reprotox.2011.11.026 O23 PPAR involvement in PFAA developmental toxicity Barbara D. Abbott Developmental Toxicology Branch, Toxicity Assessment Division, National Health and Environmental Effects Research Laboratory, Office of Research and Development, U.S. Environmental Protection Agency, Research Triangle Park, NC, United States Perfluoroalkyl acids (PFAAs) are found in the environment and in serum of wildlife and humans. Perfluorooctanoic acid (PFOA), perfluorononanoic acid (PFNA), and perfluorooctane sulfonate (PFOS) are developmentally toxic in rodents. The effects of in utero exposure include increased neonatal death, developmental delay, and deficits in postnatal growth. Members of the PFAA family of compounds were shown to activate peroxisome proliferatoractivated receptor-alpha (PPAR␣) in a transfected Cos-1 cell model. PPAR␣, PPAR/␦ and PPAR␥, are expressed in human and rodent embryos with tissue and developmental stage-specific expression patterns. PPARs have significant physiological roles, regulating energy homeostasis, adipogenesis, lipid metabolism, inflammatory responses, and hematopoiesis. Studies in PPAR␣ knockout (KO) mice revealed a role for PPAR␣ in the induction of developmental toxicity by PFOA and PFNA, but not PFOS. The induction of postnatal lethality by PFOS may be related to effects on lung function. In fetal lung and liver, PFOA and PFOS altered gene expression and in fetal liver both compounds produced profiles typical of PPAR␣ activation. Activation of some genes in liver persisted to PND63. Fetal and neonatal heart also showed altered gene expression after exposure to PFOA. Effects in heart differed from liver and were found at least to PND28. Although it is not clear exactly how changes in gene expression are related to the effects on neonatal survival and growth, perturbation of PPAR␣regulated lipid and glucose homeostasis potentially impact energy availability and utilization. The absence of developmental toxicity in the PPAR␣ KO mouse and alterations in gene expression typical of PPAR␣ activation in the wild type mouse, support a role for PPAR␣ in mediating the developmental toxicity of PFOA and PFNA. The primary cause of PFOS-induced developmental toxicity is PPAR␣-independent and may be an effect on lung function. However, PFOS altered gene expression in a manner similar to PFOA and if effects on PPAR␣-regulated genes are responsible for developmental toxicity, then it is expected that PFOS would still produce developmental toxicity even in absence of an effect on lung function. This abstract does not necessarily reflect US EPA policy. doi:10.1016/j.reprotox.2011.11.027
9
O24 Nuclear receptor involvement in PPAA-induced metabolic changes Mitch B. Rosen Integrated Systems Biology, NHEERL, ORD, U.S. EPA, Research Triangle Park, NC, United States It has been proposed that certain xenobiotics commonly identified in biomonitoring studies may play a role in the incidence of obesity and metabolic syndrome in the United States and other countries. The list of potential “environmental obesogens” includes endocrine disrupting compounds such as Diethylstilbesterol and Bisphenol A, as well as chemicals that present a variety of toxicities in mammals such as the organotins. Interestingly, perfluoroalkyl acids (PFAAs) have also been mentioned as possible environmental obesogens because of their ability to alter energy homeostasis. While it may not be clear how compounds that function as peroxisome proliferator activated receptor alpha (PPAR␣) ligands could induce obesity, the biological activity of PFAAs is not limited to activation of PPAR␣. Many of these compounds also activate the constitutive androstane receptor (CAR) and it is now recognized that CAR influences not only xenobiotic metabolism but also certain aspects of energy metabolism as well. Chronic exposure to PPAR␣ agonists also has the potential to alter energy metabolism in ways that are only first beginning to be understood. For example, chemical or metabolic challenge during gestation could result in PPAR␣-dependent epigenetic modifications which result in persistent alterations in phenotype. This talk will consider the potential effects of chronic PFAA exposure on nuclear receptor regulated energy metabolism. (This abstract does not necessarily reflect EPA policy.) doi:10.1016/j.reprotox.2011.11.028 O25 PPAR involvement in PFAA immunotoxicity J.C. DeWitt 1,∗ , M.M. Peden-Adams 2 , D.E. Keil 2 , S.E. Anderson 3 1
Department of Pharmacology and Toxicology, East Carolina University, Greenville, NC, United States 2 Clinical Laboratory Sciences, University of Nevada, Las Vegas, NV, United States 3 Health Effects Laboratory Division, National Institute for Occupational Safety and Health, Morgantown, WV, United States When immune endpoints are evaluated in experimental animals exposed to PFAAs, alterations in antibody synthesis, inflammatory responses, cytokine production, lymphocyte cellularity, and lymphoid organ weights are reported. These alterations indicate that exposure to PFAAs results in immunomodulation. The mechanisms by which immunomodulation occur have not been elucidated, although ligation of PPAR-alpha, and to some extent PPAR-gamma, is assumed to explain some of the immune changes associated with PFAA exposure. However, increasing experimental evidence suggests that while PPAR-alpha receptor activation is important for mediation of inflammatory responses and many nonimmune parameters, several immune endpoints are affected by PFAA exposure whether or not a functional PPAR-alpha is present. In addition, host phenotype also may impact the effects of PFAA exposure on immune responses. Two strains of PPAR-alpha knockout mice are generally available; one is on a 129 background and the other is on a B6 background. Initial studies with the 129 strain indicate that attenuation of antibody synthesis and decreases in lymphocyte cellularity and lymphoid organ weights observed in