Maternal PFOS exposure affects offspring development in Nrf2-dependent and independent ways in zebrafish (Danio rerio).

Perfluorooctanesulfonic acid (PFOS) is a ubiquitous legacy environmental contaminant detected broadly in human samples and water supplies. PFOS can cross the placenta and has been detected in cord blood and breastmilk samples, underscoring the importance of understanding the impacts of maternal PFOS exposure during early development. This study aimed to investigate the effects of a preconception exposure to PFOS on developmental endpoints in offspring, as well as examine the role of the transcription factor Nuclear factor erythroid-2-related factor (Nrf2a) in mediating these effects. This transcription factor regulates the expression of several genes that protect cells against oxidative stress including during embryonic development. Adult female zebrafish were exposed to 0.02, 0.08 or 0.14 mg/L PFOS for 1 week (duration of one cycle of oocyte maturation) and then paired with unexposed males from Nrf2a mutant or wildtype strains. Embryos were collected for two weeks or until completion of 5 breeding events. PFOS was maternally transferred to offspring independent of genotype throughout all breeding events in a dose-dependent manner, ranging from 2.77 to 23.72 ng/embryo in Nrf2a wildtype and 2.40 to 15.80 ng/embryo in Nrf2a mutants. Although embryo viability at collection was not impacted by maternal PFOS exposure, developmental effects related to nutrient uptake, growth and pancreatic ß-cell morphology were observed and differed based on genotype. Triglyceride levels were increased in Nrf2a wildtype eggs from the highest PFOS group. In Nrf2a wildtype larvae there was a decrease in yolk sac uptake while in Nrf2a mutants there was an increase. Additionally, there was a significant decrease in pancreatic ß-cell (islet) area in wildtype larvae from the 0.14 mg/L PFOS accompanied by an increase in the prevalence of abnormal islet morphologies compared to controls. Abnormal morphology was also observed in the 0.02 and 0.08 mg/L PFOS groups. Interestingly, in Nrf2a mutants there was a significant increase in the pancreatic ß-cell area in the 0.02 and 0.08 mg/L PFOS groups and no changes in the prevalence of abnormal islet morphologies. These results suggest that the regulation of processes like nutrient consumption, growth and pancreatic ß-cell development are at least partially modulated by the presence of a functional Nrf2a transcriptomic response. Overall, preconception exposure to environmental pollutants, such as PFOS, may impact the maturing oocyte and cause subtle changes that can ultimately impact offspring health and development.

Comparing the effects of developmental exposure to alpha lipoic acid (ALA) and perfluorooctanesulfonic acid (PFOS) in zebrafish (Danio rerio).

Alpha lipoic acid (ALA) is a dietary supplement that has been used to treat a wide range of diseases, including obesity and diabetes, and have lipid-lowering effects, making it a potential candidate for mitigating dyslipidemia resulting from exposures to the per- and polyfluoroalkyl substance (PFAS) family member perfluorooctanesulfonic acid (PFOS). ALA can be considered a non-fluorinated structural analog to PFOS due to their similar 8-carbon chain and amphipathic structure, but, unlike PFOS, is rapidly metabolized. PFOS has been shown to reduce pancreatic islet area and induce ß-cell lipotoxicity, indicating that changes in ß-cell lipid microenvironment is a mechanism contributing to hypomorphic islets. Due to structural similarities, we hypothesized that ALA may compete with PFOS for binding to proteins and distribution throughout the body to mitigate the effects of PFOS exposure. However, ALA alone reduced islet area and fish length, with several morphological endpoints indicating additive toxicity in the co-exposures. Individually, ALA and PFOS increased fatty acid uptake from the yolk. ALA alone increased liver lipid accumulation, altered fatty acid profiling and modulated PPARÉ£ pathway signaling. Together, this work demonstrates that ALA and PFOS have similar effects on lipid uptake and metabolism during embryonic development in zebrafish.

Dietary exposure to the food preservative tert-Butylhydroquinone (tBHQ) impairs zebrafish (Danio rerio) survival, growth, organ development, and gene expression in Nrf2a-dependent and independent ways.

Tert-Butylhydroquinone (tBHQ), a preservative used to prevent oxidative deterioration of oil, fat, and meat products, has been linked to both chemoprotective and adverse effects. This study investigates the impact of dietary tBHQ consumption on survival, growth parameters, organ development, and gene expression in zebrafish (Danio rerio). As tBHQ activates the transcription factor nuclear factor erythroid 2-related factor 2 (Nrf2a), a zebrafish line with a mutation in the DNA-binding domain of Nrf2a was used to identify Nrf2a-dependent vs independent effects. Homozygous Nrf2a wildtype (wt) and mutant (m) larvae were fed a diet containing 5% tBHQ or a control diet. Survival and growth parameters were assessed at 15 days and at 5 months, and samples were collected for RNA sequencing at 5 months. Dietary exposure to tBHQ throughout the larval and juvenile periods negatively impacted growth and survival. RNA-seq analysis found differentially expressed genes related to growth and development and upregulation of several immune system-related pathways. The findings herein demonstrate that dietary tBHQ exposure may impair growth and survival in both Nrf2a dependent and independent manners.

Developmental impacts of Nrf2 activation by dimethyl fumarate (DMF) in the developing zebrafish (Danio rerio) embryo.

Dimethyl fumarate (DMF) is pharmaceutical activator of the transcription factor nuclear factor erythroid 2-related factor 2 (Nrf2), which regulates of many cellular antioxidant response pathways, and has been used to treat inflammatory diseases such as multiple sclerosis. However, DMF has been shown to produce adverse effects on offspring in animal studies and as such is not recommended for use during pregnancy. The goal of this work is to better understand how these adverse effects are initiated and the role of DMF-induced Nrf2 activation during three critical windows of development in embryonic zebrafish (Danio rerio): pharyngula, hatching, and protruding-mouth stages. To evaluate Nrf2 activation, wildtype zebrafish, and mutant zebrafish (nrf2afh318/fh318) embryos with a loss of function mutation in Nrf2a, the co-ortholog to human Nrf2, were treated for 6 h with DMF (0-20 µM) beginning at the pharyngula, hatching, or protruding-mouth stage and assessed for survival and morphology. Nrf2a mutant fish had an increase in survival, however, morphology studies demonstrated Nrf2a mutant fish had more severe deformities occurring with exposures during the hatching stage. To verify Nrf2 cellular localization and downstream impacts on protein-S-glutathionylation in situ, a concentration below the LOAEL was chosen (7 µM) for immunohistochemistry and S-glutathionylation. Embryos were imaged via epifluorescence microscopy studies, the Nrf2a protein in the body tissue was decreased with DMF only when exposed at the hatching stage, while total protein S-glutathionylation was modulated by Nrf2a activity and DMF during the pharyngula and protruding-mouth stage. The pancreatic islet and liver were further analyzed via confocal microscopy. Pancreatic islets and liver also had tissue specific differences with Nrf2a protein expression and protein S-glutathionylation. This work demonstrates how critical windows of exposure and Nrf2a activity may influence toxicity of DMF and highlights tissue-specific changes in Nrf2a protein levels and S-glutathionylation in pancreatic islet and liver during embryonic development.

The Zebrafish (Danio rerio) Embryo Model as a Tool to Assess Drinking Water Treatment Efficacy for Freshwater Impacted by Crude Oil Spill.

Traditional approaches toward evaluating oil spill mitigation effectiveness in drinking water supplies using analytical chemistry can overlook residual hydrocarbons and treatment byproducts of unknown toxicity. Zebrafish (Danio rerio) were used to address this limitation by evaluating the reduction in toxicity to fish exposed to laboratory solutions of dissolved crude oil constituents treated with 3 mg/L ozone (O3 ) with or without a peroxone-based advanced oxidation process using 0.5 M H2 O2 /M O3 or 1 M H2 O2 /M O3 . Crude oil water mixtures (OWMs) were generated using three mixing protocols-orbital (OWM-Orb), rapid (OWM-Rap), and impeller (OWM-Imp) and contained dissolved total aromatic concentrations of 106-1019 µg/L. In a first experiment, embryos were exposed at 24 h post fertilization (hpf) to OWM-Orb or OWM-Rap diluted to 25%-50% of full-strength samples and in a second experiment, to untreated or treated OWM-Imp mixtures at 50% dilutions. Toxicity profiles included body length, pericardial area, and swim bladder inflation, and these varied depending on the OWM preparation, with OWM-Rap resulting in the most toxicity, followed by OWM-Imp and then OWM-Orb. Zebrafish exposed to a 50% dilution of OWM-Imp resulted in 6% shorter body length, 83% increased pericardial area, and no swim bladder inflation, but exposure to a 50% dilution of OWM-Imp treated with O3 alone or with 0.5 M H2 O2 /M O3 resulted in normal zebrafish development and average total aromatic destruction of 54%-57%. Additional aromatic removal occurred with O3 + 1 M H2 O2 /M O3 but without further attenuation of toxicity to zebrafish. This study demonstrates using zebrafish as an additional evaluation component for modeling the effectiveness of freshwater oil spill treatment methods. Environ Toxicol Chem 2022;41:2822-2834. © 2022 SETAC.

Nrf2a dependent and independent effects of early life exposure to 3,3'-dichlorobiphenyl (PCB-11) in zebrafish (Danio rerio).

The environmental pollutant 3,3'-dichlorobiphenyl (PCB-11) is a lower-chlorinated polychlorinated biphenyl (PCB) congener present in air and water samples. Both PCB-11 and its metabolite, 4-PCB-11-Sulfate, are detected in humans, including in pregnant women. Previous research in zebrafish (Danio rerio) has shown that 0.2 µM exposures to 4-PCB-11-Sulfate starting at 1 day post fertilization (dpf) increase hepatic neutral lipid accumulation in larvae at 15 dpf. Here, we explored whether nuclear factor erythroid 2-related factor 2 (Nrf2), known as the master-regulator of the adaptive response to oxidative stress, contributes to metabolic impacts of 4-PCB-11-Sulfate. For this work, embryos were collected from homozygous wildtype or Nrf2a mutant adult zebrafish that also express GFP in pancreatic ß-cells, rendering Tg(ins:GFP;nrf2afh318+/+) and Tg(ins:GFP;nrf2afh318-/-) lines. Exposures were conducted from 1-15 dpf to either 0.05% DMSO or DMSO-matched 0.2 µM 4-PCB-11-Sulfate, and at 15 dpf subsets of larvae were imaged for overall morphology, primary pancreatic islet area, and collected for fatty acid profiling and RNAseq. At 15 dpf, independent of genotype, fish exposed to 4-PCB-11-Sulfate survived significantly more at 80-85% compared to 65-73% survival for unexposed fish, and had primary pancreatic islets 8% larger compared to unexposed fish. Fish growth at 15 dpf was dependent on genotype, with Nrf2a mutant fish a significant 3-5% shorter than wildtype fish, and an interaction effect was observed where Nrf2a mutant fish exposed to 4-PCB-11-Sulfate experienced a significant 29% decrease in the omega-3 fatty acid DHA compared to unexposed mutant fish. RNAseq revealed 308 differentially expressed genes, most of which were dependent on genotype. These findings suggest that Nrf2a plays an important role in growth as well as for DHA production in the presence of 4-PCB-11-Sulfate. Further research would be beneficial to understand the importance of Nrf2a throughout the lifecourse, especially in the context of toxicant exposures.

The Nrf2a pathway impacts zebrafish offspring development with maternal preconception exposure to perfluorobutanesulfonic acid.

Since the voluntary phaseout of perfluorooctanesulfonic acid (PFOS), smaller congeners, such as perfluorobutanesulfonic acid (PFBS) have served as industrial replacements and been detected in contaminated aquifers. This study sought to examine the effects of a maternal preconception PFBS exposure on the development of eggs and healthy offspring. Adult female zebrafish received a one-week waterborne exposure of 0.08, 0.14, and 0.25 mg/L PFBS. After which, females were bred with non-exposed males and embryos collected over 5 successful breeding events. PFBS concentrations were detected in levels ranging from 99 to 253 pg/embryo in the first collection but were below the limit of quantitation by fourth and fifth clutches. Therefore, data were subsequently binned into early collection embryos in which PFBS was detected and late collections, in which PFBS was below quantitation. In the early collection, embryo 24 h survival was significantly reduced. In the late collection, embryo development was impacted with unique patterns emerging between Nrf2a wildtype and mutant larvae. Additionally, the impact of nutrient loading into the embryos was assessed through measurement of fatty acid profiles, total cholesterol, and triglyceride content. There were no clear dose-dependent effects, but again unique patterns were observed between the genotypes. Preconception PFBS exposures were found to alter egg and embryo development, which is mediated by direct toxicant loading in the eggs, nutrient loading into eggs, and the function of Nrf2a. These findings provide insight into the reproductive and developmental effects of PFBS and identify maternal preconception as a novel critical window of exposure.

Relationships between type 2 diabetes, cell dysfunction, and redox signaling: A meta-analysis of single-cell gene expression of human pancreatic α- and ß-cells.

BACKGROUND: Type 2 diabetes mellitus (T2DM) is a chronic disease characterized by insulin resistance and failure of ß-cells to meet the metabolic demand for insulin. Recent advances in single-cell RNA sequencing (sc-RNA-Seq) have allowed for in-depth studies to further understand the underlying cellular mechanisms of T2DM. In ß-cells, redox signaling is critical for insulin production. A meta-analysis of human pancreas islet sc-RNA-Seq data was conducted to evaluate how T2DM may modify the transcriptomes of α- and ß-cells. METHODS: Annotated sc-RNA-Seq data from six studies of human pancreatic islets from metabolically healthy and donors with T2DM were collected. α- and ß-cells, subpopulations of proliferating α-cells, immature, and senescent ß-cells were identified based on expression levels of key marker genes. Each dataset was analyzed individually before combining, using weighted comparisons. Pathways of significant genes and individual redox-related gene expression were then evaluated to further understand the role that redox signaling may play in T2DM-induced ß-cell dysfunction. RESULTS: α- and ß-cells from T2DM donors modified genes involved in energy metabolism, immune response, autophagy, and cellular stress. α- and ß-cells also had an increased nuclear factor erythroid 2-related factor 2 (NFE2L2)-mediated antioxidant response in T2DM donors. The proportion of immature and senescent ß-cells increased in T2DM donors, and in immature and senescent ß-cells, genes regulated by NFE2L2 were further upregulated. CONCLUSIONS: These findings suggest that NFE2L2 plays a role in ß-cell maturation and dysfunction. Redox singling maybe a key pathway for ß-cell restoration and T2DM therapeutics.

Modulation of PPAR signaling disrupts pancreas development in the zebrafish, Danio rerio.

Peroxisome Proliferator Activated Receptors (PPARs) are transcription factors that regulate processes such as lipid and glucose metabolism. Synthetic PPAR ligands, designed as therapeutics for metabolic disease, provide a tool to assess the relationship between PPAR activity and pancreas development in vivo, an area that remains poorly characterized. Here, we aim to assess the effects of PPAR agonists and antagonists on gene expression, embryonic morphology and pancreas development in transgenic zebrafish embryos. To evaluate developmental perturbations, we assessed gross body and pancreas morphology at 4 days post fertilization (dpf) in response to developmental exposures with PPARα, PPARγ, and PPARß/δ agonists and antagonists at 0, 0.01, 0.1, 1, and 10 µM concentrations. All ligand exposures, with the exception of the PPARα agonist, resulted in significantly altered fish length and yolk sac area. PPARγ agonist and antagonist had higher incidence of darkened yolk sac and craniofacial deformities, whereas PPARα antagonist had higher incidence of pericardial edema and death. Significantly reduced endocrine pancreas area was observed in both PPARγ ligands and PPARα agonist exposed embryos, some of which also exhibited aberrant endocrine pancreas morphology. Both PPARß/δ ligands caused reduced exocrine pancreas length and novel aberrant phenotype, and disrupted gene expression of pancreatic targets pdx1, gcga, and try. Lipid staining was performed at 8 dpf and revealed altered lipid accumulation consistent with isoform function. These data indicate chronic exposure to synthetic ligands may induce morphological and pancreatic defects in zebrafish embryos.

Dibenzyl trisulfide binds to and competitively inhibits the cytochrome P450 1A1 active site without impacting the expression of the aryl hydrocarbon receptor.

The toxicological manifestation of many pollutants relies upon their binding to the aryl hydrocarbon receptor (AHR), and it follows a cascade of reactions culminating in an elevated expression of cytochrome P450 (CYP) 1 enzymes. CYP1A1 and CYP1B1 are associated with enhanced carcinogenesis when chronically exposed to certain polyaromatic hydrocarbons, and their inhibition may lead to chemoprevention. We evaluated dibenzyl trisulfide (DTS), expressed in the ethnomedical plant, Petiveria alliacea, for such potential chemoprevention. Using recombinant human CYP1A1 and CYP1B1 bactosomes on a fluorogenic assay, we first demonstrated that DTS moderately inhibited both enzymes with half maximal inhibitory concentration (IC50) values of 1.3 ± 0.3 and 1.7 ± 0.3 µM, respectively. Against CYP1A1, DTS was a reversible, competitive inhibitor with an apparent inhibitory constant (Ki) of 4.55 ± 0.37 µM. In silico molecular modeling showed that DTS binds with an affinity of -39.8 kJ·mol-1, situated inside the binding pocket, approximately 4.3 Å away from the heme group, exhibiting interactions with phenylalanine residue 123 (Phe-123), Phe-224, and Phe-258. Lastly, zebrafish (Danio rerio) embryos were exposed to 0.08-0.8 µM DTS from 24 to 96 h post fertilization (hpf) with the in vivo ethoxyresorufin-O-deethylase (EROD) assay, and, at 96 hpf, DTS significantly suppressed EROD CYP1A activity in a dose-dependent manner, with up to 60% suppression in the highest 0.8 µM exposure group. DTS had no impact on gene transcription levels for cyp1a and aryl hydrocarbon receptor 2 (ahr2). In co-exposure experiments, DTS suppressed CYP1A activity induced by both B[a]P and PCB-126, although these reductions were not significant. Taken together, these results demonstrate that DTS is a direct, reversible, competitive inhibitor of the carcinogen-activating CYP1A enzyme, binding in the active site pocket close to the heme site, and shows potential in chemoprevention.

Maternal preconception PFOS exposure of Drosophila melanogaster alters reproductive capacity, development, morphology and nutrient regulation.

Perfluorooctanesulfonic acid (PFOS) is a persistent synthetic surfactant widely detected in the environment. Developmental PFOS exposures are associated with low birth weight and chronic exposures increase risk for obesity and type 2 diabetes. As an obesogen, PFOS poses a major public health exposure risk and much remains to be understood about the critical windows of exposure and mechanisms impacted, especially during preconception. Here, we leverage evolutionarily conserved pathways and processes in the fruit fly Drosophila melanogaster (wild-type Canton-S and megalin-UAS RNAi transgenic fly lines) to investigate the window of maternal preconception exposure to PFOS on reproductive and developmental toxicity, and examine receptor (megalin)-mediated endocytosis of nutrients and PFOS into the oocyte as a potential mechanism. Preconception exposure to 2 ng PFOS/female resulted in an internal concentration of 0.081 ng/fly over two days post exposure, no mortality and reduced megalin transcription. The number of eggs laid 1-3 days post exposure was reduced and contained 0.018 ng PFOS/egg. Following heat shock, PFOS was significantly reduced in eggs from megalin-knockdown transgenic females. Cholesterol and triglycerides were increased in eggs laid immediately following PFOS exposure by non-heat shocked transgenic females whereas decreased cholesterol and increased protein levels were found in eggs laid by heat shocked transgenic females. Preconception exposure likewise increased cholesterol in early emerging wildtype F1 adults and also resulted in progeny with a substantial developmental delay, a reduction in adult weights, and altered transcription of Drosophila insulin-like peptide genes. These findings support an interaction between PFOS and megalin that interferes with normal nutrient transport during oocyte maturation and embryogenesis, which may be associated with later in life developmental delay and reduced weight.

Using Monochlorobimane to Visualize Glutathione Utilization in the Developing Zebrafish (Danio rerio) Embryo.

Glutathione (GSH) plays fundamental roles in cellular redox buffering and is a common detoxification pathway for excretion of xenobiotics. This is especially crucial during vertebrate embryogenesis, when an organism is at one of its most vulnerable life stages. Importantly, GSH content and redox potential can dictate cell fate decisions, which can have profound consequences if altered by early life xenobiotic exposures. Owing to technical limitations, the best available method to detect and quantify changes in GSH has been high-pressure liquid chromatography, a terminal method that prevents suborganism-level resolution of these changes in developing embryos. Here, we describe a protocol that leverages the transparent nature of zebrafish embryos and the compatibility of monochlorobimane with the zebrafish GSH-S-transferase enzymes, to allow for the visualization of changes in GSH via S-glutathionylation in a live, developing embryo. This method can find broad application in developmental biology and toxicology. © 2021 Wiley Periodicals LLC.

Developmental exposures to perfluorooctanesulfonic acid (PFOS) impact embryonic nutrition, pancreatic morphology, and adiposity in the zebrafish, Danio rerio.

Perfluorooctanesulfonic acid (PFOS) is a persistent environmental contaminant previously found in consumer surfactants and industrial fire-fighting foams. PFOS has been widely implicated in metabolic dysfunction across the lifespan, including diabetes and obesity. However, the contributions of the embryonic environment to metabolic disease remain uncharacterized. This study seeks to identify perturbations in embryonic metabolism, pancreas development, and adiposity due to developmental and subchronic PFOS exposures and their persistence into later larval and juvenile periods. Zebrafish embryos were exposed to 16 or 32 µM PFOS developmentally (1-5 days post fertilization; dpf) or subchronically (1-15 dpf). Embryonic fatty acid and macronutrient concentrations and expression of peroxisome proliferator-activated receptor (PPAR) isoforms were quantified in embryos. Pancreatic islet morphometry was assessed at 15 and 30 dpf, and adiposity and fish behavior were assessed at 15 dpf. Concentrations of lauric (C12:0) and myristic (C14:0) saturated fatty acids were increased by PFOS at 4 dpf, and PPAR gene expression was reduced. Incidence of aberrant islet morphologies, principal islet areas, and adiposity were increased in 15 dpf larvae and 30 dpf juvenile fish. Together, these data suggest that the embryonic period is a susceptible window of metabolic programming in response to PFOS exposures, and that these early exposures alone can have persisting effects later in the lifecourse.

Modulating glutathione thiol status alters pancreatic ß-cell morphogenesis in the developing zebrafish (Danio rerio) embryo.

Emerging evidence suggests that redox-active chemicals perturb pancreatic islet development. To better understand potential mechanisms for this, we used zebrafish (Danio rerio) embryos to investigate roles of glutathione (GSH; predominant cellular redox buffer) and the transcription factor Nrf2a (Nfe2l2a; zebrafish Nrf2 co-ortholog) in islet morphogenesis. We delineated critical windows of susceptibility to redox disruption of ß-cell morphogenesis, interrogating embryos at 24, 48 and 72 h post fertilization (hpf) and visualized Nrf2a expression in the pancreas using whole-mount immunohistochemistry at 96 hpf. Chemical GSH modulation at 48 hpf induced significant islet morphology changes at 96 hpf. Pro-oxidant exposures to tert-butylhydroperoxide (77.6 µM; 10-min at 48 hpf) or tert-butylhydroquinone (1 µM; 48-56 hpf) decreased ß-cell cluster area at 96 hpf. Conversely, exposures to antioxidant N-acetylcysteine (bolsters GSH pools; 100 µM; 48-72 hpf) or sulforaphane (activates Nrf2a; 20 µM; 48-72 hpf) significantly increased islet areas. Nrf2a was also stabilized in ß-cells: 10-min exposures to 77.6 µM tert-butylhydroperoxide significantly increased Nrf2a protein compared to control islet cells that largely lack stabilized Nrf2a; 10-min exposures to higher (776 µM) tert-butylhydroperoxide concentration stabilized Nrf2a throughout the pancreas. Using biotinylated-GSH to visualize in situ protein glutathionylation, islet cells displayed high protein glutathionylation, indicating oxidized GSH pools. The 10-min high (776 µM) tert-butylhydroperoxide exposure (induced Nrf2a globally) decreased global protein glutathionylation at 96 hpf. Mutant fish expressing inactive Nrf2a were protected against tert-butylhydroperoxide-induced abnormal islet morphology. Our data indicate that disrupted redox homeostasis and Nrf2a stabilization during pancreatic ß-cell development impact morphogenesis, with implications for disease states at later life stages. Our work identifies a potential molecular target (Nrf2) that mediates abnormal ß-cell morphology in response to redox disruptions. Moreover, our findings imply that developmental exposure to exogenous stressors at distinct windows of susceptibility could diminish the reserve redox capacity of ß-cells, rendering them vulnerable to later-life stresses and disease.

Embryonic exposures to mono-2-ethylhexyl phthalate induce larval steatosis in zebrafish independent of Nrf2a signaling.

Mono-2-ethylhexyl phthalate (MEHP) is the primary metabolite of the ubiquitous plasticizer and toxicant, di-2-ethylhexyl phthalate. MEHP exposure has been linked to abnormal development, increased oxidative stress, and metabolic syndrome in vertebrates. Nuclear factor, Erythroid 2 Like 2 (Nrf2), is a transcription factor that regulates gene expression in response to oxidative stress. We investigated the role of Nrf2a in larval steatosis following embryonic exposure to MEHP. Wild-type and nrf2a mutant (m) zebrafish embryos were exposed to 0 or 200 µg/l MEHP from 6 to either 96 (histology) or 120 hours post fertilization (hpf). At 120 hpf, exposures were ceased and fish were maintained in clean conditions until 15 days post fertilization (dpf). At 15 dpf, fish lengths and lipid content were examined, and the expression of genes involved in the antioxidant response and lipid processing was quantified. At 96 hpf, a subset of animals treated with MEHP had vacuolization in the liver. At 15 dpf, deficient Nrf2a signaling attenuated fish length by 7.7%. MEHP exposure increased hepatic steatosis and increased expression of peroxisome proliferator-activated receptor alpha target fabp1a1. Cumulatively, these data indicate that developmental exposure alone to MEHP may increase risk for hepatic steatosis and that Nrf2a does not play a major role in this phenotype.

Per- and Polyfluoroalkyl Substances and Obesity, Type 2 Diabetes and Non-alcoholic Fatty Liver Disease: A Review of Epidemiologic Findings.

Per- and polyfluoroalkyl substances, a group of fluoro-surfactants widely detected in the environment, wildlife and humans, have been linked to adverse health effects. A growing body of literature has addressed their effects on obesity, diabetes and non-alcoholic fatty liver disease/ non-alcoholic steatohepatitis. This review summarizes the brief historical use and chemistry of per- and polyfluoroalkyl substances, routes of human exposure, as well as the epidemiologic evidence for associations between exposure to per- and polyfluoroalkyl substances and the development of obesity, diabetes and non-alcoholic fatty liver disease/ non-alcoholic steatohepatitis. We identified 22 studies on obesity and 32 studies on diabetes, while only 1 study was found for non-alcoholic fatty liver disease/ non-alcoholic steatohepatitis by searching PubMed for human studies. Approximately 2/3 of studies reported positive associations between per- and polyfluoroalkyl substances exposure and the prevalence of obesity and/or type 2 diabetes. Causal links between per- and polyfluoroalkyl substances and obesity, diabetes and non-alcoholic fatty liver disease/ non-alcoholic steatohepatitis, however, require further large-scale prospective cohort studies combined with mechanistic laboratory studies to better assess these associations.

Chemical Characterization of a Legacy Aqueous Film-Forming Foam Sample and Developmental Toxicity in Zebrafish (Danio rerio).

BACKGROUND: Drinking water contamination related to the use of aqueous film-forming foam (AFFF) has been documented at hundreds of military bases, airports, and firefighter training facilities. AFFF has historically contained high levels of long-chain per- and polyfluoroalkyl substances (PFAS), which pose serious health concerns. However, the composition and toxicity of legacy AFFF mixtures are unknown, presenting great uncertainties in risk assessment and affected communities. OBJECTIVES: This study aimed to determine the fluorinated and nonfluorinated chemical composition of a legacy AFFF sample and its toxicity in zebrafish embryos. METHODS: A sample of legacy AFFF (3% application formulation, manufactured before 2001) was provided by the Massachusetts Department of Environmental Protection. High resolution mass spectrometry (HRMS) was used to identify PFAS and nonfluorinated compounds, and a commercial laboratory measured 24 PFAS by a modified U.S. EPA Method 537.1. AFFF toxicity was assessed in zebrafish embryos in comparison with four major constituents: perfluorooctanesulfonic acid (PFOS); perfluorohexanesulfonic acid (PFHxS); sodium dodecyl sulfate (SDS); and sodium tetradecyl sulfate (TDS). End points included LC50 values, and sublethal effects on growth, yolk utilization, and pancreas and liver development. RESULTS: We identified more than 100 PFAS. Of the PFAS detected, PFOS was measured at the highest concentration (9,410mg/L) followed by PFHxS (1,500mg/L). Fourteen nonfluorinated compounds were identified with dodecyl sulfate and tetradecyl sulfate the most abundant at 547.8 and 496.4mg/L, respectively. An LC50 of 7.41×10-4% AFFF was calculated, representing a dilution of the 3% formulation. TDS was the most toxic of the constituents tested but could not predict the AFFF phenotype in larval zebrafish. PFOS exposure recapitulated the reduction in length but could not predict effects on development of the liver, which was the tissue most sensitive to AFFF. DISCUSSION: To our knowledge, this research is the first characterization of the chemical composition and toxicity of legacy AFFF, which has important implications for regulatory toxicology. https://doi.org/10.1289/EHP6470.

Perfluorooctanesulfonic acid (PFOS) and perfluorobutanesulfonic acid (PFBS) impaired reproduction and altered offspring physiological functions in Caenorhabditis elegans.

Perfluorobutanesulfonic acid (PFBS), a shorter chain Per- and polyfluoroalkyl substances (PFASs) cognate of perfluorooctanesulfonic acid (PFOS), has been used as replacement for the toxic surfactant PFOS. However, emerging evidences suggest safety concerns for PFBS and its effect on reproductive health is still understudied. Therefore, the current work aimed to investigate the effect of PFBS, in comparison to PFOS, on reproductive health using Caenorhabditis elegans as an in vivo animal model. PFOS (≥10 µM) and PFBS (≥1000 µM) significantly impaired the reproduction capacity of C. elegans, represented as reduced brood size (total egg number) and progeny number (hatched offspring number), without affecting the hatchability. Additionally, the preconception exposure of PFOS and PFBS significantly altered the embryonic nutrient loading and composition, which further led to abnormalities in growth rate, body size and locomotive activity in F1 offspring. Though the effective exposure concentration of PFBS was approximately 100 times higher than PFOS, the internal concentration of PFBS was lower than that of PFOS to produce the similar effects of PFOS. In conclusion, PFOS and PFBS significantly impaired the reproductive capacities in C. elegans and the preconception exposure of these two compounds can lead to offspring physiological dysfunctions.

The sulfate metabolite of 3,3'-dichlorobiphenyl (PCB-11) impairs Cyp1a activity and increases hepatic neutral lipids in zebrafish larvae (Danio rerio).

The environmental contaminant 3,3'-dichlorobiphenyl (PCB-11) is widely detected in environmental samples, and this parent compound along with its metabolites 4-OH-PCB-11 and 4-PCB-11-Sulfate are detected in human serum. Our previous research in zebrafish (Danio rerio) embryos shows exposure to 20 µM PCB-11 inhibits Cyp1a enzyme activity and perturbs lipid metabolism pathways. In this study, wildtype AB embryos underwent acute exposures from 1 to 4 days post fertilization (dpf) to 0.002-20 µM 4-OH-PCB-11 or 0.2-20 µM 4-PCB-11-Sulfate, with and without co-exposures to 100 µg/L benzo[a]pyrene (B[a]P) or 5 nM 3,3',4,4',5-pentachlorobiphenyl (PCB-126), and were assessed for in vivo EROD activity and morphometrics. Chronic exposures from 1 to 15 dpf to assess lipid accumulation using Oil-Red-O staining were also conducted with 0.2 µM parent or metabolite compounds, alongside a co-exposure experiment of 0.002-0.2 µM 4-PCB-11-Sulfate and 10 µg/L B[a]P. For acute experiments, 2 and 20 µM 4-OH-PCB-11 was lethal but no Cyp1a or morphological effects were observed at lower concentrations; 20 µM 4-PCB-11-Sulfate significantly lowered the Cyp1a activity of B[a]P and PCB-126 but did not alter morphological development. For chronic experiments, 0.2 µM 4-PCB-11-Sulfate significantly increased lipid accumulation 30% in single exposures and 44% in co-exposures with B[a]P. Further long-term studies would better elucidate the effects of this contaminant, particularly in the context of environmentally-relevant mixtures.