Reproductive toxicity of DDT in the Japanese medaka fish model: Revisiting the impacts of DDT+ on female reproductive health.

The organochlorine pesticide dichlorodiphenyltrichloroethane (DDT) is an endocrine-disrupting compound (EDC) that has been banned by most countries for decades. However, it continues to be detected in nearly all humans and wildlife due to its biological and environmental persistence. The ovarian dysgenesis syndrome hypothesis speculates that exposure to EDCs during sensitive developmental windows such as early gonadal differentiation lead to reproductive disorders later in life. Yet, mechanisms by which DDT affects developing gonads remain unclear due to the inherent challenge of getting developmental exposure data from adults presenting with reproductive disease. The Japanese medaka (Oryzias latipes) is a valuable fish model for sex-specific toxicological studies due to its chromosomal sex determination, external embryonic development, short generation time, and extensively mapped genome. It is well documented that medaka exposed to DDT and its metabolites and byproducts (herein referred to as DDT+) at different developmental time points experience permanent alterations in gonadal morphology, reproductive success, and molecular and hormonal signaling. However, the overwhelming majority of studies focus primarily on functional and morphological outcomes in males and females and have rarely investigated long-term transcriptional or molecular effects. This review summarizes previous experimental findings and the state of our knowledge concerning toxic effects DDT + on reproductive development, fertility, and health in the valuable medaka model. It also identifies gaps in knowledge, emphasizing a need for more focus on molecular mechanisms of ovarian endocrine disruption using enhanced molecular tools that have become increasingly available over the past few decades. Furthermore, DDT forms a myriad of over 45 metabolites and transformation products in biota and the environment, very few of which have been evaluated for environmental abundance or health effects. This reinforces the demand for high throughput and economical in vivo models for predictive toxicology screening, and the Japanese medaka is uniquely positioned to meet this need.

Novel chemical contaminants associated with thirdhand smoke in settled house dust.

Thirdhand smoke (THS) is the persistent and toxic residue from tobacco smoke in indoor environments. A comprehensive understanding of the chemical constituents of THS is necessary to assess the risks of long-term exposure and to establish reliable THS tracers. The objective of this study was to investigate compounds associated with THS through nontargeted analysis (NTA) of settled house dust samples from smokers' and non-smokers' homes, using comprehensive two-dimensional gas chromatography coupled to time-of-flight mass spectrometry (GC×GC/TOF-MS). Compounds that were either only present in dust from smokers' homes or that had significantly larger abundance than in non-smokers' homes were termed qualified compounds. We identified 140 qualified compounds, and of these, 42 compounds were tentatively identified by searching matching mass spectra in NIST electron impact (EI) mass spectral library including 20 compounds confirmed with their authentic standards. Among the 42 compounds, 26 compounds were statistically more abundant (p < 0.10) in dust from homes of smokers; seven were tobacco-specific compounds, two of which (nornicotyrine, 3-ethenylpyridine) have not been reported before in house dust. Two compounds, tris (2-chloroethyl) phosphate (a toxic compound used as a flame retardant and reported in tobacco) and propanoic acid, 2-methyl-, 1-(1,1-dimethylethyl)-2-methyl-1,3-propanediyl ester (highly abundant and reported in exhaled air of smokers), were found in dust from all smokers' homes and in zero non-smokers' homes, making these potential THS tracers, possibly associated with recent smoking. Benzyl methyl ketone was significantly higher in dust in smokers' homes, and was previously reported not as a product of tobacco but rather as a form of methamphetamine. This compound was recently reported in mainstream tobacco smoke condensate through NTA as well. These identified potential tracers and chemical components of THS in this study can be further investigated for use in developing THS contamination and exposure assessments.

Metagenomic analysis of microbial communities and antibiotic resistant genes in the Tijuana river, and potential sources.

The Tijuana River is a transborder river that flows northwest across the border from Baja California in Mexico into Southern California before discharging into the Pacific Ocean. The river is frequently contaminated with raw sewage due to inadequate sanitary infrastructure in Tijuana. To assess the type and degree of microbial contamination, water samples were collected monthly from a near-border and an estuarine site from August 2020 until May 2021. A portion of each sample was used for epifluorescent microscopy and DNA was extracted directly from the rest for shotgun metagenomic sequencing. After sequence quality checking and processing, we used the rapid taxonomic identifier tool Kaiju to characterize the microbial diversity of the metagenomes and matched the sequences against the Comprehensive Antibiotic Resistance Database (CARD) to examine antimicrobial resistance genes (ARGs). Bacterial and viral-like particle (VLP) abundance was consistently higher in the near-border samples than in the estuarine samples, while alpha diversity (within sample biodiversity) was higher in estuarine samples. Beta-diversity analysis found clear compositional separation between samples from the two sites, and the near-border samples were more dissimilar to one another than were the estuarine sites. Near-border samples were dominated by fecal-associated bacteria and bacteria associated with sewage sludge, while estuarine sites were dominated by marine bacteria. ARGs were more abundant at the near-border site, but were also readily detectable in the estuarine samples, and the most abundant ARGs had multi-resistance to beta-lactam antibiotics. SourceTracker analysis identified human feces and sewage sludge to be the largest contributors to the near-border samples, while marine waters dominated estuarine samples except for two sewage overflow dates with high fecal contamination. Overall, our research determined human sewage microbes to be common in the Tijuana River, and the prevalence of ARGs confirms the importance of planned infrastructure treatment upgrades for environmental health.

Development of a Dynamic Network Model to Identify Temporal Patterns of Structural Malformations in Zebrafish Embryos Exposed to a Model Toxicant, Tris(4-chlorophenyl)methanol.

Embryogenesis is a well-coordinated process relying on precise cues and environmental signals that direct spatiotemporal embryonic patterning. Quite often, when one error in this process occurs, others tend to co-occur. We posit that investigating the co-occurrence of these abnormalities over time would yield additional information about the mode of toxicity for chemicals. Here, we use the environmental contaminant tris(4-chlorophenyl)methanol (TCPMOH) as a model toxicant to assess the relationship between exposures and co-occurrence of developmental abnormalities in zebrafish embryos. We propose a dynamic network modeling approach to study the co-occurrence of abnormalities, including pericardial edema, yolk sac edema, cranial malformation, spinal deformity, delayed/failed swim bladder inflation, and mortality induced by TCPMOH exposure. TCPMOH-exposed samples revealed increased abnormality co-occurrence when compared to controls. The abnormalities were represented as nodes in the dynamic network model. Abnormalities with high co-occurrence over time were identified using network centrality scores. We found that the temporal patterns of abnormality co-occurrence varied between exposure groups. In particular, the high TCPMOH exposure group experienced abnormality co-occurrence earlier than the low exposure group. The network model also revealed that pericardial and yolk sac edema are the most common critical nodes among all TCPMOH exposure levels, preceding further abnormalities. Overall, this study introduces a dynamic network model as a tool for assessing developmental toxicology, integrating structural and temporal features with a concentration response.

Tris(4-chlorophenyl)methane and tris(4-chlorophenyl)methanol disrupt pancreatic organogenesis and gene expression in zebrafish embryos.

OBJECTIVES: Tris(4-chlorophenyl) methane (TCPM) and tris(4-chlorophenyl)methanol (TCPMOH) are anthropogenic environmental contaminants believed to be manufacturing byproducts of the organochlorine pesticide dichlorodiphenyltrichloroethane (DDT) due to environmental co-occurrence. TCPM and TCPMOH are persistent, bioaccumulate in the environment, and are detected in human breast milk and adipose tissues. DDT exposures have been previously shown to disrupt insulin signaling and glucoregulation, increasing risk for diabetes. We have previously shown that embryonic exposures organochlorines such as polychlorinated biphenyls disrupted pancreatic development and early embryonic glucoregulatory networks. Here, we determined the impacts of the similar compounds TCPM and TCPMOH on zebrafish pancreatic growth and gene expression following developmental exposures. METHODS: Zebrafish embryos were exposed to 50 nM TCPM or TCPMOH beginning at 24 hr postfertilization (hpf) and exposures were refreshed daily. At 96 hpf, pancreatic growth and islet area were directly visualized in Tg(ptf1a::GFP) and Tg(insulin::GFP) embryos, respectively, using microscopy. Gene expression was assessed at 100 hpf with RNA sequencing. RESULTS: Islet and total pancreas area were reduced by 20.8% and 13% in embryos exposed to 50 nM TCPMOH compared to controls. TCPM did not induce significant morphological changes to the developing pancreas, indicating TCPMOH, but not TCPM, impairs pancreatic development despite similarity in molecular responses. Transcriptomic responses to TCPM and TCPMOH were correlated (R2  = .903), and pathway analysis found downregulation of processes including retinol metabolism, circadian rhythm, and steroid biosynthesis. CONCLUSION: Overall, our data suggest that TCPM and TCPMOH may be hazardous to embryonic growth and development.

Detection, Quantification, and Simplified Wastewater Surveillance Model of SARS-CoV-2 RNA in the Tijuana River.

The COVID-19 pandemic and the detection of SARS-CoV-2 RNA in sewage has expanded global interest in wastewater surveillance. However, many underserved communities throughout the world lack improved sanitation and use informal combined sanitary and storm sewer systems. Sewage is transported via open channels, ditches, and rivers, where it mixes with surface water and/or stormwater. There is a need to develop better methods for the surveillance of pathogens such as SARS-CoV-2 RNA in this context. We developed a simplified surveillance system and monitored flow rates and concentrations of SARS-CoV-2 RNA in the Tijuana River at two locations downstream of the United States-Mexico border in California, United States. SARS-CoV-2 RNA was detected in the upstream location on six out of eight occasions, two of which were at concentrations as high as those reported in untreated wastewater from California sanitary sewer systems. The virus was not detected in any of the eight samples collected at the downstream (estuarine) sampling location, despite the consistent detection of PMMoV RNA. Synchrony was observed between the number of cases reported in Tijuana and the SARS-CoV-2 RNA concentrations measured with the CDC N1 assay when the latter were normalized by the reported flow rates in the river.

Metagenomic Analysis of Microbial Contamination in the U.S. Portion of the Tijuana River Watershed.

The Tijuana River watershed is binational, flowing from Tijuana, Mexico into San Diego and Imperial Beach, USA. Aging sewage and stormwater infrastructure in Tijuana has not kept pace with population growth, causing overflows into this watershed during major rainfall or equipment failures. The public health consequences of this impaired watershed on the surrounding communities remain unknown. Here, we performed untargeted metagenomic sequencing to better characterize the sewage contamination in the Tijuana River, identifying potential pathogens and molecular indicators of antibiotic resistance in surface waters. In 2019-2020, water samples were collected within 48 h of major rainfall events at five transborder flow sites and at the mouth of the river in the US portion of the Tijuana River and estuary. After filtration, DNA was extracted and sequenced, and sequences were run through the Kaiju taxonomic classification program. A pathogen profile of the most abundant disease-causing microbes and viruses present in each of the samples was constructed, and specific markers of fecal contamination were identified and linked to each site. Results from diversity analysis between the sites showed clear distinction as well as similarities between sites and dates, and antibiotic-resistant genes were found at each site. This serves as a baseline characterization of microbial exposures to these local communities.

Association between particulate matter air pollution and heart attacks in San Diego County.

Air pollution is one of the major risk factors contributing to adverse public health outcomes worldwide. Fine particulate matter (PM2.5) has been repeatedly associated with increased risk of cardiovascular events, including heart attacks. Because PM2.5 is unequally distributed with elevated concentrations near high-traffic and industrial zones, PM2.5 is an environmental justice issue of major public health concern. In this study, we reviewed the relationship between PM2.5, emergency visits due to heart attacks, and environmental justice in San Diego County using data from CalEnviroScreen 3.0. Our results indicate that PM2.5, diesel PM emissions, and emergency visits due to heart attacks are weakly, but positively correlated (r = 0.3, R2 < 0.1). Areas classified as environmental justice communities, communities comprised more dominantly of nonwhite populations, and communities closer to the San Diego-Tijuana border are exposed disproportionately to air pollution in San Diego County. Overall, this work demonstrates that there is an association between elevated local PM concentrations in San Diego County communities with emergency hospital visits due to heart attacks, and that these associations are an environmental justice issue disproportionally affecting disadvantaged communities.Implications: Particulate matter is an adverse contributor to overall health throughout the lifespan, contributing to diseases such as asthma, hypertension, stroke, and increased risk of cardiovascular events. Here, we assess the relationship between particulate matter and heart attacks in San Diego County using CalEnviroScreen3.0. Using these tools, we also examine correlations between this relationship and different sociodemographic indicators such as age, race, income, and proximity to the high-traffic U.S.-Mexico border. Overall, we show that specific communities around San Diego are more highly exposed to particulate matter, and that these relationships may be disproportionately contributing to heart attacks in disadvantaged communities.

Associations between Exposures to Perfluoroalkyl Substances and Diabetes, Hyperglycemia, or Insulin Resistance: A Scoping Review.

Per- and polyfluoroalkyl substances (PFASs) are persistent environmental pollutants that are commonly found in the human body due to exposures via drinking water, surfactants used in consumer materials, and aqueous film-forming foams (AFFFs). PFAS exposure has been linked to adverse health effects such as low infant birth weights, cancer, and endocrine disruption, though increasingly studies have demonstrated that they may perturb metabolic processes and contribute to dysfunction. This scoping review summarizes the chemistry of PFAS exposure and the epidemiologic evidence for associations between exposure to per- and polyfluoroalkyl substances and the development of diabetes, hyperglycemia, and/or insulin resistance. We identified 11 studies on gestational diabetes mellitus, 3 studies on type 1 diabetes, 7 studies on type 2 diabetes, 6 studies on prediabetes or unspecified diabetes, and 15 studies on insulin resistance or glucose tolerance using the SCOPUS and PubMed databases. Approximately 24 reported positive associations, 9 negative associations, 2 non-linear associations, and 2 inverse associations, and 8 reported no associations found between PFAS and all diabetes search terms. Cumulatively, these data indicate the need for further studies to better assess these associations between PFAS exposure and diabetes.

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.

Mathematical modeling of the interaction between yolk utilization and fish growth in zebrafish, Danio rerio.

Optimal embryonic development plays a major role in the health of an individual beyond the developmental stage. Nutritional perturbation during development is associated with cardiovascular and metabolic disease later in life. With both nutritional uptake and overall growth being risk factors for eventual health, it is necessary to understand not only the behavior of the processes during development but also their interactions. In this study, we used differential equations, image analyses, curve fittings, parameter estimation and laboratory experiments to quantify the rate of yolk absorption and its effect on early development of a vertebrate model (Danio rerio). Findings from this study establish a nonlinear functional relationship between nutrient absorption and early fish growth. We found that the rate of change in fish length and yolk utilization is logistic, that is the yolk decays rapidly for a period of time before leveling out. An interesting finding from this study is that yolk utilization reaches its maximum at 84 h post-fertilization. We validated our mathematical models against experimental observations, making them powerful tools for replication and future simulations.

The ecotoxicological contaminant tris(4-chlorophenyl)methanol (TCPMOH) impacts embryonic development in zebrafish (Danio rerio).

Tris(4-chlorophenyl)methanol (TCPMOH) is a water contaminant with unknown etiology, but is believed to be a byproduct of DDT manufacturing. It is highly persistent in the environment, and bioaccumulates in marine species. TCPMOH has also been measured in human breast milk, which poses a risk for developing infants. However, almost no toxicity data is currently available. In this study, we investigate the hazard posed by developmental TCPMOH exposures using the zebrafish model (Danio rerio). Zebrafish (Danio rerio) embryos were exposed to 0, 0.1, 0.5, 1, or 5 µM TCPMOH beginning at 24 h post fertilization (hpf). Embryonic mortality and incidence of morphological deformities increased in a concentration-dependent manner with TCPMOH exposure. RNA sequencing assessed changes in gene expression associated with acute (4 hour) exposures to 50 nM TCPMOH. Developmental exposure to TCPMOH decreased expression of ahr2, as well as metabolic enzymes cyp1a1, cyp1b1, cyp1c1, cyp1c2, and cyp2y3 (p<0.05). These findings were concordant with decreased Cyp1a1 induction measured by the ethoxyresorufin-O-deethylase (EROD) assay (p<0.05). Pathways associated with xenobiotic metabolism, lipid metabolism, and transcriptional and translational regulation were decreased. Pathways involved in DNA replication and repair, carbohydrate metabolism, and endocrine function were upregulated. Overall, this study demonstrates that TCPMOH is acutely toxic to zebrafish embryos at elevated concentrations.

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.

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.

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.

The emerging contaminant 3,3'-dichlorobiphenyl (PCB-11) impedes Ahr activation and Cyp1a activity to modify embryotoxicity of Ahr ligands in the zebrafish embryo model (Danio rerio).

3,3'-dichlorobiphenyl (PCB-11) is an emerging PCB congener widely detected in environmental samples and human serum, but its toxicity potential is poorly understood. We assessed the effects of three concentrations of PCB-11 on embryotoxicity and Aryl hydrocarbon receptor (Ahr) pathway interactions in zebrafish embryos (Danio rerio). Wildtype AB or transgenic Tg(gut:GFP) strain zebrafish embryos were exposed to static concentrations of PCB-11 (0, 0.2, 2, or 20 µM) from 24 to 96 h post fertilization (hpf), and gross morphology, Cytochrome P4501a (Cyp1a) activity, and liver development were assessed via microscopy. Ahr interactions were probed via co-exposures with PCB-126 or beta-naphthoflavone (BNF). Embryos exposed to 20 µM PCB-11 were also collected for PCB-11 body burden, qRT-PCR, RNAseq, and histology. Zebrafish exposed to 20 µM PCB-11 absorbed 0.18% PCB-11 per embryo at 28 hpf and 0.61% by 96 hpf, and their media retained 1.36% PCB-11 at 28 hpf and 0.84% at 96 hpf. This concentration did not affect gross morphology, but altered the transcription of xenobiotic metabolism and liver development genes, impeded liver development, and increased hepatocyte vacuole formation. In co-exposures, 20 µM PCB-11 prevented deformities caused by PCB-126 but exacerbated deformities in co-exposures with BNF. This study suggests that PCB-11 can affect liver development, act as a partial agonist/antagonist of the Ahr pathway, and act as an antagonist of Cyp1a activity to modify the toxicity of compounds that interact with the Ahr pathway.

Perfluorobutanesulfonic Acid Disrupts Pancreatic Organogenesis and Regulation of Lipid Metabolism in the Zebrafish, Danio rerio.

Following the phase-out of highly persistent perfluorosulfonates in the United States from non-stick and stain-resistant products in the early 2000s, perfluorobutanesulfonic acid (PFBS) has replaced these compounds as a primary surfactant. Measurements of PFBS in environmental and human samples have been rising in recent years, raising concerns about potential negative health effects. We previously found that embryonic exposures to a related compound, perfluorooctanesulfonic acid (PFOS), decreased pancreas length and insulin-producing islet area in zebrafish embryos (Danio rerio). The objective of this study was to compare the effects of PFBS exposures on pancreatic organogenesis with our previous PFOS findings. Dechorionated zebrafish embryos from two different transgenic fish lines (Tg[insulin:GFP], Tg[ptf1a:GFP]) were exposed to 0 (0.01% DMSO), 16, or 32 µM PFBS daily beginning at 1 day post fertilization (dpf) until 4 and 7 dpf when they were examined using fluorescent microscopy for islet area and morphology, and exocrine pancreas length. PFBS-exposed embryos had significantly increased caudal fin deformities, delayed swim bladder inflation, and impaired yolk utilization. Incidence of fish with significantly stunted growth and truncated exocrine pancreas length was significantly increased, although these two effects occurred independently. Islet morphology revealed an increased incidence of severely hypomorphic islets (areas lower than the 1st percentile of controls) and an elevated occurrence of fragmented islets. RNA-Seq data (4 dpf) also identify disruptions in regulation of lipid homeostasis. Overall, this work demonstrates that PFBS exposure can perturb embryonic development, energy homeostasis, and pancreatic organogenesis.

Pancreatic beta cells are a sensitive target of embryonic exposure to butylparaben in zebrafish (Danio rerio).

BACKGROUND: Butylparaben (butyl p-hydroxybenzoic acid) is a common cosmetic and pharmaceutical preservative reported to induce oxidative stress and endocrine disruption. Embryonic development is sensitive to oxidative stress, with redox potentials playing critical roles in progenitor cell fate decisions. Because pancreatic beta cells have been reported to have low antioxidant gene expression, they may be sensitive targets of oxidative stress. We tested the hypotheses that butylparaben causes oxidative stress in the developing embryo, and that pancreatic beta cells are a sensitive target of butylparaben embryotoxicity. METHODS: Transgenic insulin:GFP zebrafish embryos (Danio rerio) were treated daily with 0, 250, 500, 1,000, and 3,000 nM butylparaben. Pancreatic islet and whole embryo development were examined though 7 days postfertilization, and gene expression was measured by quantitative real-time PCR. Glutathione (GSH) and cysteine redox content were measured at 28 hr postfertilization using HPLC. RESULTS: Butylparaben exposure caused intestinal effusion, pericardial edema, and accelerated yolk utilization. At 250 nM, beta cell area increased by as much as 55%, and increased incidence of two aberrant morphologies were observed-fragmentation of the islet cluster and ectopic beta cells. Butylparaben concentrations of 500 and 1,000 nM increased GSH by 10 and 40%, respectively. Butylparaben exposure downregulated transcription factor pdx1, as well as genes involved in GSH synthesis, while upregulating GSH-disulfide reductase (gsr). CONCLUSIONS: The endocrine pancreas is a sensitive target of embryonic exposure to butylparaben, which also causes developmental deformities and perturbs redox conditions in the embryo.

Nrf2a modulates the embryonic antioxidant response to perfluorooctanesulfonic acid (PFOS) in the zebrafish, Danio rerio.

The glutathione redox system undergoes precise and dynamic changes during embryonic development, protecting against and mitigating oxidative insults. The antioxidant response is coordinately largely by the transcription factor Nuclear factor erythroid-2 (Nrf2), an endogenous sensor for cellular oxidative stress. We have previously demonstrated that impaired Nrf family signaling disrupts the glutathione redox system in the zebrafish embryo, and that impaired Nrf2 function increases embryonic sensitivity to environmental toxicants. Here, we investigated the persistent environmental toxicant and reported pro-oxidant perfluorooctanesulfonic acid (PFOS), and its impact on the embryonic glutathione-mediated redox environment. We further examined whether impaired Nrf2a function exacerbates PFOS-induced oxidative stress and embryotoxicity in the zebrafish, and the potential for Nrf2-PPAR crosstalk in the embryonic adaptive response. Wild-type and nrf2afh318-/- mutant embryos were exposed daily to 0 (0.01% v/v DMSO), 16, 32, or 64 µM PFOS beginning at 3 h post fertilization (hpf). Embryonic glutathione and cysteine redox environments were examined at 72 hpf. Gross embryonic toxicity, antioxidant gene expression, and apoptosis were examined at 96 hpf. Mortality, pericardial edema, and yolk sac utilization were increased in wild-type embryos exposed to PFOS. Embryonic glutathione and cysteine redox couples and gene expression of Nrf2 pathway targets were modulated by both exposure and genotype. Apoptosis was increased in PFOS-exposed wild-type embryos, though not in nrf2a mutants. In silico examination of putative transcription factor binding site suggested potential crosstalk between Nrf2 and PPAR signaling, since expression of PPARs and gene targets was modulated by both PFOS exposure and Nrf2a genotype. Overall, this work demonstrates that nrf2a modulates the embryonic response to PFOS, and that PPAR signaling may play a role in the embryonic adaptive response to PFOS.