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Tire tread particles are microplastics (< 5 mm) and leach organic chemicals into aquatic environments. It is important to understand the behavior of tire wear compounds in sunlight-exposed waters in terms of their persistence, removal, and transformation. Therefore, we conducted photolysis experiments with leachates from laboratory-generated tire tread particles (TTP) over 72 h in a solar simulator to evaluate the behavior of leached compounds and fluorescent components over time. Compared to initial leachates, simulated sunlight exposure resulted in ∼12 % decrease in dissolved organic carbon, 11 % reduction in the total fluorescence of leachates, and ∼30 % removal of the 213 chromatographic features detected by nontargeted analysis (NTA) using comprehensive two-dimensional gas chromatography coupled to time-of-flight mass spectrometry. A decrease in total chemical abundance determined by NTA was observed, with normalized peak areas decreasing by 36.4% in the 72 h photoirradiated samples and by 13.6% in the dark samples. Fifty-three compounds were tentatively identifiable based on mass spectral matching and among them, 12 compounds were confirmed with authentic standards. Among the 53 compounds, 19 compounds were photo-labile, 27 were photo-resistant, and 7 were photo-transformation products. NTA also identified compounds previously unreported as tire-related compounds. Parallel factor analysis (PARAFAC) modeling of three-dimensional excitation-emission-matrix (EEM) data identified five fluorescent components. PARAFAC component C4 (excitation/emission peak at 285/445 nm) was found to be a fluorescent analog for 6PPD. Rapid double exponential decay kinetics were observed for the 6PPD-like component during photoirradiation. Similarly, the peak fluorescence of commercially available 6PPD exposed to simulated sunlight was reduced by >90 % in the first 0.5 h of photoirradiation. 6PPD photodegradation resulted in the production of a fluorescent transformation product resembling PARAFAC Component C2 (with emission at 360 nm). These results prove that EEM fluorescence analyses can serve as a rapid method for kinetics analysis of 6PPD, and may be combined with NTA compound tentative identification to track the behavior of other TTP-derived compounds in experimental studies., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier Ltd.)

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., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:Jennifer M. Cossaboon reports financial support was provided by National Institute of Environmental Health Sciences. Karilyn E. Sant reports financial support was provided by University of Southern California Sea Grant and the National Institute of Environmental Health Sciences. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Summary: Understanding the pathways and biological processes underlying differential gene expression is fundamental for characterizing gene expression changes in response to an experimental condition. Zebrafish, with a transcriptome closely mirroring that of humans, are frequently utilized as a model for human development and disease. However, a challenge arises due to the incomplete annotations of zebrafish pathways and biological processes, with more comprehensive annotations existing in humans. This incompleteness may result in biased functional enrichment findings and loss of knowledge. danRerLib, a versatile Python package for zebrafish transcriptomics researchers, overcomes this challenge and provides a suite of tools to be executed in Python including gene ID mapping, orthology mapping for the zebrafish and human taxonomy, and functional enrichment analysis utilizing the latest updated Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) databases. danRerLib enables functional enrichment analysis for GO and KEGG pathways, even when they lack direct zebrafish annotations through the orthology of human-annotated functional annotations. This approach enables researchers to extend their analysis to a wider range of pathways, elucidating additional mechanisms of interest and greater insight into experimental results., Availability and Implementation: danRerLib, along with comprehensive documentation and tutorials, is freely available. The source code is available at https://github.com/sdsucomptox/danrerlib/ with associated documentation and tutorials at https://sdsucomptox.github.io/danrerlib/. The package has been developed with Python 3.9 and is available for installation on the package management systems PIP (https://pypi.org/project/danrerlib/) and Conda (https://anaconda.org/sdsu&#95;comptox/danrerlib) with additional installation instructions on the documentation website., Competing Interests: None declared., (© The Author(s) 2024. Published by Oxford University Press.)

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., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

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., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Tire tread particles (TTP) are environmentally prevalent microplastics and generate toxic aqueous leachate. We determined the total carbon and nitrogen leachate concentrations and chemical profiles from micron (∼32 μm) and centimeter (∼1 cm) TTP leachate over 12 days. Dissolved organic carbon (DOC) and total dissolved nitrogen (TDN) were used to measure the concentration of leached compounds. Nontargeted chemical analysis by comprehensive two-dimensional gas chromatography coupled to time-of-flight mass spectrometry (GC×GC/TOF-MS) was used to compare the chemical profiles of leachates. After leaching for 12 days, DOC was 4.0 times higher in the micron TTP leachate than in the centimeter TTP leachate, and TDN was 2.6 times higher. The total GC×GC/TOF-MS chromatographic feature peak area was 2.9 times greater in the micron TTP leachate than the centimeter TTP leachate, and similarly, the total relative abundance of 54 tentatively identified compounds was 3.3 times greater. We identified frequently measured tire-related chemicals, such as 6PPD, N-cyclohexyl-N'-phenylurea (CPU), and hexa(methoxymethyl)melamine (HMMM), but nearly 50% of detected chemicals were not previously reported in tire literature or lacked toxicity information. Overall, the results demonstrate that smaller TTP have a greater potential to leach chemicals into aquatic systems, but a significant portion of these chemicals are not well-studied and require further risk assessment., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier Ltd.. All rights reserved.)

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.

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 (R 2  = .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., (© 2022 The Authors. Birth Defects Research published by Wiley Periodicals LLC.)

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.

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., Competing Interests: The authors declare no competing financial interest., (© 2022 The Authors. Published by American Chemical Society.)

Air pollution is one of the major risk factors contributing to adverse public health outcomes worldwide. Fine particulate matter (PM 2.5 ) has been repeatedly associated with increased risk of cardiovascular events, including heart attacks. Because PM 2.5 is unequally distributed with elevated concentrations near high-traffic and industrial zones, PM 2.5 is an environmental justice issue of major public health concern. In this study, we reviewed the relationship between PM 2.5 , emergency visits due to heart attacks, and environmental justice in San Diego County using data from CalEnviroScreen 3.0. Our results indicate that PM 2.5 , diesel PM emissions, and emergency visits due to heart attacks are weakly, but positively correlated (r = 0.3, R 2  < 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.

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.

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 PPARβ/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 PPARβ/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., (Copyright © 2018. Published by Elsevier Inc.)

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., (Copyright © 2021 The Authors. Published by Elsevier B.V. All rights reserved.)

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., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2021. Published by The Company of Biologists Ltd.)

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., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

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., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

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.

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., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

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.

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., (© 2018 Wiley Periodicals, Inc.)

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 nrf2a fh318-/- 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., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Mono(2-ethylhexyl) phthalate (MEHP) is the bioactive metabolite of di(2-ethylhexyl) phthalate, a plasticizing agent and persistent environmental contaminant associated with obesity, developmental abnormalities, and oxidative stress. Nrf2 (Nfe2l2) is a transcription factor that regulates cytoprotective genes as part of the adaptive antioxidant response. We previously identified the pancreas as a sensitive target of oxidative stress during embryonic development. The goals of this study were to 1) characterize the effects of MEHP exposure on pancreatic development, and 2) determine whether oxidative stress contributes to MEHP embryotoxicity. Zebrafish (Danio rerio) embryos from AB wildtype and Tg(ins:GFP;nrf2a fh318/fh318 ) were exposed to 0 or 200 μg/L MEHP at 3 h post fertilization (hpf) through 168 hpf to assess pancreatic organogenesis. MEHP exposure significantly decreased β-cell area at all timepoints (48, 72, 96, 168 hpf), but Nrf2a did not significantly protect against islet hypomorphism. Tg(gcga:GFP) embryos exposed to MEHP showed a decrease in α-cell area in the islet across the same timepoints. Tg(ptf1a:GFP) embryos were assessed at 80 and 168 hpf for exocrine pancreas length. MEHP exposure decreased growth of the exocrine pancreas. Expression of pancreas genes insa, sst2 and ptf1a was significantly reduced by MEHP exposure compared to controls. Glutathione (GSH) concentrations and redox potentials were quantified at 72 hpf by HPLC, but no significant changes were observed. However, expression of the GSH-related genes gstp1 and gsr were significantly altered by MEHP exposure. These data indicate that the developing pancreas is a sensitive target tissue of embryonic exposure to MEHP., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Purpose of Review: Developmental toxicity assessments often focus on structural outcomes and overlook subtle metabolic differences which occur during the early embryonic period. Deviant embryonic nutrition can result in later-life disease, including diabetes, obesity, and cardiovascular disease. Prior to placenta-mediated nutrient exchange, the human embryo requires maternally supplied nutritional substrates for growth, called yolk. Here, we compare the biology of the human and zebrafish yolk and review examples of toxicant-mediated perturbation of yolk defects, composition, and utilization., Recent Findings: Zebrafish embryos, like human embryos, have a protruding yolk sac that serves as a nutritional cache. Aberrant yolk morphology is a common qualitative finding in fish embryotoxicity studies, but quantitative assessment and characterization provides an opportunity to uncover mechanistic targets of toxicant effects on embryonic nutrition. The zebrafish and the study of its yolk sac is an excellent model for uncovering toxicant disruptions to early embryonic nutrition and has potential to discover mechanistic insights into the developmental origins of health and disease.

Redox signaling is important for embryogenesis, guiding pathways that govern processes crucial for embryo patterning, including cell polarization, proliferation, and apoptosis. Exposure to pro-oxidants during this period can be deleterious, resulting in altered physiology, teratogenesis, later-life diseases, or lethality. We previously reported that the glutathione antioxidant defense system becomes increasingly robust, including a doubling of total glutathione and dynamic shifts in the glutathione redox potential at specific stages during embryonic development in the zebrafish, Danio rerio. However, the mechanisms underlying these changes are unclear, as is the effectiveness of the glutathione system in ameliorating oxidative insults to the embryo at different stages. Here, we examine how the glutathione system responds to the model pro-oxidants tert-butylhydroperoxide and tert-butylhydroquinone at different developmental stages, and the role of Nuclear factor erythroid 2-related factor (Nrf) proteins in regulating developmental glutathione redox status. Embryos became increasingly sensitive to pro-oxidants after 72h post-fertilization (hpf), after which the duration of the recovery period for the glutathione redox potential was increased. To determine whether the doubling of glutathione or the dynamic changes in glutathione redox potential are mediated by zebrafish paralogs of Nrf transcription factors, morpholino oligonucleotides were used to knock down translation of Nrf1 and Nrf2 (nrf1a, nrf1b, nrf2a, nrf2b). Knockdown of Nrf1a or Nrf1b perturbed glutathione redox state until 72 hpf. Knockdown of Nrf2 paralogs also perturbed glutathione redox state but did not significantly affect the response of glutathione to pro-oxidants. Nrf1b morphants had decreased gene expression of glutathione synthesis enzymes, while hsp70 increased in Nrf2b morphants. This work demonstrates that despite having a more robust glutathione system, embryos become more sensitive to oxidative stress later in development, and that neither Nrf1 nor Nrf2 alone appear to be essential for the response and recovery of glutathione to oxidative insults., (Copyright © 2017. Published by Elsevier B.V.)

There is compelling evidence that epigenetic modifications link developmental environmental insults to adult disease susceptibility. Animal studies have associated perinatal bisphenol A (BPA) exposure to altered DNA methylation, but these studies are often limited to candidate gene and global non-loci-specific approaches. By using an epigenome-wide discovery platform, we elucidated epigenetic alterations in liver tissue from adult mice offspring (10 months) following perinatal BPA exposure at human physiologically relevant doses (50-ng, 50-μg, and 50-mg BPA/kg diet). Biological pathway analysis identified an enrichment of significant differentially methylated regions in metabolic pathways among females. Furthermore, through the use of top enriched biological pathways, 4 candidate genes were chosen to assess DNA methylation as a mediating factor linking the association of perinatal BPA exposure to metabolic phenotypes previously observed in female offspring. DNA methylation status at Janus kinase-2 (Jak-2), retinoid X receptor (Rxr), regulatory factor x-associated protein (Rfxap), and transmembrane protein 238 (Tmem238) was used within a mediational regression analysis. DNA methylation in all four of the candidate genes was identified as a mediator in the mechanistic pathway of developmental BPA exposure and female-specific energy expenditure, body weight, and body fat phenotypes. Data generated from this study are crucial for deciphering the mechanistic role of epigenetics in the pathogenesis of chronic disease and the development of epigenetic-based prevention and therapeutic strategies for complex human disease., (Copyright © 2017 by the Endocrine Society.)

Perfluorooctanesulfonic acid (PFOS) is a ubiquitous environmental contaminant, previously utilized as a non-stick application for consumer products and firefighting foam. It can cross the placenta, and has been repeatedly associated with increased risk for diabetes in epidemiological studies. Here, we sought to establish the hazard posed by embryonic PFOS exposures on the developing pancreas in a model vertebrate embryo, and develop criteria for an adverse outcome pathway (AOP) framework to study the developmental origins of metabolic dysfunction. Zebrafish (Danio rerio) embryos were exposed to 16, 32, or 64 μM PFOS beginning at the mid-blastula transition. We assessed embryo health, size, and islet morphology in Tg(insulin-GFP) embryos at 48, 96 and 168 hpf, and pancreas length in Tg(ptf1a-GFP) embryos at 96 and 168 hpf. QPCR was used to measure gene expression of endocrine and exocrine hormones, digestive peptides, and transcription factors to determine whether these could be used as a predictive measure in an AOP. Embryos exposed to PFOS showed anomalous islet morphology and decreased islet size and pancreas length in a U-shaped dose-response curve, which resemble congenital defects associated with increased risk for diabetes in humans. Expression of genes encoding islet hormones and exocrine digestive peptides followed a similar pattern, as did total larval growth. Our results demonstrate that embryonic PFOS exposures can disrupt pancreatic organogenesis in ways that mimic human congenital defects known to predispose individuals to diabetes; however, future study of the association between these defects and metabolic dysfunction are needed to establish an improved AOP framework., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

The pancreatic islets, largely comprised of insulin-producing beta cells, play a critical role in endocrine signaling and glucose homeostasis. Because they have low levels of antioxidant defenses and a high perfusion rate, the endocrine islets may be a highly susceptible target tissue of chemical exposures. However, this endpoint, as well as the integrity of the surrounding exocrine pancreas, is often overlooked in studies of developmental toxicology. Disruption of development by toxicants can alter cell fate and migration, resulting in structural alterations that are difficult to detect in mammalian embryo systems, but that are easily observed in the zebrafish embryo model ( Danio rerio ). Using endogenously expressed fluorescent protein markers for developing zebrafish beta cells and exocrine pancreas tissue, we documented differences in islet area and incidence rates of islet morphological variants in zebrafish embryos between 48 and 96 h post fertilization (hpf), raised under control conditions commonly used in embryotoxicity assays. We identified critical windows for chemical exposures during which increased incidences of endocrine pancreas abnormalities were observed following exposure to cyclopamine (2-12 hpf), Mono-2-ethylhexyl phthalate (MEHP) (3-48 hpf), and Perfluorooctanesulfonic acid (PFOS) (3-48 hpf). Both islet area and length of the exocrine pancreas were sensitive to oxidative stress from exposure to the oxidant tert-butyl hydroperoxide during a highly proliferative critical window (72 hpf). Finally, pancreatic dysmorphogenesis following developmental exposures is discussed with respect to human disease., Competing Interests: Conflicts of Interest: The authors declare no conflict of interest.

Mono-2-ethylhexl phthalate (MEHP) is the primary metabolite of di-2-ethylhexyl phthalate (DEHP), a ubiquitous contaminant in plastics. This study sought to determine how structural defects caused by MEHP in mouse whole embryo culture were related to temporal and spatial patterns of redox state and gene expression. MEHP reduced morphology scores along with increased incidence of neural tube defects. Glutathione (GSH) and cysteine (Cys) concentrations fluctuated spatially and temporally in embryo (EMB) and visceral yolk sac (VYS) across the 24h culture. Redox potentials (Eh) for GSSG/GSH were increased by MEHP in EMB (12h) but not in VYS. CySS/CyS Eh in EMB and VYS were significantly increased at 3h and 24h, respectively. Gene expression at 6h showed that MEHP induced selective alterations in EMB and VYS for oxidative phosphorylation and energy metabolism pathways. Overall, MEHP affects neurulation, alters Eh, and spatially alters the expression of metabolic genes in the early organogenesis-stage mouse conceptus., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Developmental exposure to the endocrine-active compound bisphenol A (BPA) has been linked to epigenotoxic and potential carcinogenic effects in rodent liver, prostate, and mammary glands. A dose-dependent increase in hepatic tumors in 10-month mice perinatally exposed to one of three doses of BPA (50 ng, 50 µg, or 50 mg BPA/kg chow) was previously reported. These tumors represent early-onset disease and lack classical sexual dimorphism in incidence. Here, adult epigenome-wide liver DNA methylation profiles to identify gene promoters associated with perinatal BPA exposure and disease in 10-month mice with and without liver tumors were investigated. Mice with hepatic tumors showed 12,822 (1.8%) probes with differential methylation as compared with non-tumor animals, of which 8,656 (67.5%) were hypomethylated. A significant enrichment of differential methylation in Gene Ontology (GO) terms and biological processes related to morphogenesis and development, and epigenomic alteration were observed. Pathway enrichment revealed a predominance of hypermethylated neuronal signaling pathways linked to energy regulation and metabolic function, supporting metabolic consequences in the liver via BPA-induced disruption of neuronal signaling pathways. Hypothesis-driven pathway analysis revealed mouse and human genes linked to BPA exposure related to intracellular Jak/STAT and MAPK signaling pathways. Taken together, these findings are indicators of the relevance of the hepatic tumor phenotype seen in BPA-exposed mice to human health. This work demonstrated that epigenome-wide discovery experiments in animal models were effective tools for identification and understanding of paralagous epimutations salient to human disease. Environ. Mol. Mutagen. 57:435-446, 2016. © 2016 Wiley Periodicals, Inc., (© 2016 Wiley Periodicals, Inc.)

Histiotrophic nutrition pathways (HNPs) are processes by which the organogenesis-stage conceptus obtains nutrients, amino acids, vitamins and cofactors required for protein biosynthesis and metabolic activities. Nutrients are captured from the maternal milieu as whole proteins and cargoes via receptor-mediated endocytosis in the visceral yolk sac (VYS), degraded by lysosomal proteolysis and delivered to the developing embryo (EMB). Several nutrients obtained by HNPs are required substrates for one-carbon (C1) metabolism and supply methyl groups required for epigenetic processes, including DNA and histone methylation. Increased availability of methyl donors has been associated with reduced risk for neural tube defects (NTDs). Here, we show that mono-2-ethylhexyl phthalate (MEHP) treatment (100 or 250μM) alters HNPs, C1 metabolism and epigenetic programming in the organogenesis-stage conceptus. Specifically, 3-h MEHP treatment of mouse EMBs in whole culture resulted in dose-dependent reduction of HNP activity in the conceptus. To observe nutrient consequences of decreased HNP function, C1 components and substrates and epigenetic outcomes were quantified at 24h. Treatment with 100-μM MEHP resulted in decreased dietary methyl donor concentrations, while treatment with 100- or 250-μM MEHP resulted in dose-dependent elevated C1 products and substrates. In MEHP-treated EMBs with NTDs, H3K4 methylation was significantly increased, while no effects were seen in treated VYS. DNA methylation was reduced in MEHP-treated EMB with and without NTDs. This research suggests that environmental toxicants such as MEHP decrease embryonic nutrition in a time-dependent manner and that epigenetic consequences of HNP disruption may be exacerbated in EMB with NTDs., (Copyright © 2015 Elsevier Inc. All rights reserved.)

The process of embryonic nutrition in rodent conceptuses during organogenesis has been shown to involve a dominant histiotrophic mechanism where essential developmental substrates and micronutrients are supplied as whole maternal proteins or cargoes associated with proteins. The histiotrophic nutrition pathways (HNP) responsible for uptake and initial processing of proteins across maternal-conceptal interfaces involve uptake via receptor mediated endocytosis and protein degradation via lysosomal proteolysis. Chemical inhibition of either process can lead to growth deficits and malformation in the embryo (EMB), but selective inhibition of either HNP component will elicit a different subset of developmental perturbations. In vitro, whole embryo culture exposure of GD10 or GD11 rat conceptuses to the natural protease inhibitor, leupeptin, leads to significant reductions in all measured embryonic growth parameters as well as a myriad of other effects. Leupeptin doses of 10 μM or 20 μM over a 26-h period (GD10-GD11) and 50 μM over a 3 h pulse period produced significant decreases in the clearance of FITC-albumin from culture media. The near complete loss of acid soluble fluorescence and increased total visceral yolk sac (VYS) protein content confirmed the selective inhibition of proteolysis. Inhibition of lysosomal proteolysis thus deprives the developing EMB of essential nutrient amino acids producing conditions akin to amino acid starvation, but may also cause direct effects on pathways critical for normal growth and differentiation. Following leupeptin exposure for 26 or 6 h, total glutathione (GSH) concentrations dropped significantly in the VYS, but only slightly in yolk sac (YSF) and amniotic (AF) fluids. Cys concentrations increased in VYS and EMB, but dropped in YSF and AF fluids. Redox potentials (Eh) for the glutathione disulfide (GSSG)/glutathione (GSH) redox couple trended significantly toward the positive, confirming the net oxidation of conceptual tissues following leupeptin treatment. Analysis of the thiol proteome showed few alterations to specific pathways mapped to the Kyoto Encyclopedia of Genes and Genomes Pathway database, but did reveal significant increases in concentrations of proteins associated with glycolysis/gluconeogenesis in the VYS and decreased concentrations proteins associated with ribosome biogenesis and function in the EMB. A subset of proteins elevated by >2-23-fold in the VYS were identified as serum (blood) proteins and represent the maternal-side proteins captured by the VYS and which are not degraded in the lysosomes as a result of leupeptin's inhibitory action. The observed constellation of proteins decreased in the EMB by leupeptin represent proteins from several adaptive pathways that are commonly altered in responses to amino acid starvation. These studies show clear differential responses to protease inhibition in VYS and EMB during organogenesis and suggest the possibility for additional roles of redox regulation, cellular adaptations and metabolic insufficiency caused by protease inhibition., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Exposures to co-planar PCBs and dioxins have been associated with diabetes in epidemiologic studies. Individuals may be predisposed to diseases such as diabetes as a result of exposure to environmental contaminants during early life, resulting in dysmorphic pancreatic islets or metabolically fragile β-cells. We tested the hypothesis that embryonic exposure to a model Ahr-ligand, PCB-126 would cause structural and/or functional alterations to the developing primary pancreatic islet in the zebrafish (Danio rerio). To assess β-cell development, transgenic zebrafish embryos (Tg(ins:GFP) and Tg(ins:mcherry) were exposed to nominal concentrations of 2 or 5nM PCB-126 or DMSO from 24-48h post fertilization (hpf), and imaged via time-lapse microscopy from 80-102hpf. We identified defects including hypomorphic islets, altered islet migration, islet fragmentation, and formation of ectopic β-cells. As we recently showed the transcription factor Nrf2a is protective in PCB-126 embryotoxicity, we then assessed the transcriptional function of the islets in wildtype and nrf2a(fh318/fh318) mutant embryos. We measured gene expression of preproinsulin a, somatostatin2, pdx1, ghrelin, and glucagon. Expression of preproinsulin a increased with PCB treatment in wildtype embryos, while expression of all measured pancreas genes was altered by the nrf2a mutant genotype, suggesting misregulation of the glucose homeostasis axis in those embryos, independent of PCB treatment. This study shows that embryonic exposure to PCB-126 can result in deviant development of the pancreatic islet and suggests that Nrf2a plays a role in regulating glucose homeostasis during development., (Copyright © 2015 Elsevier Inc. All rights reserved.)

The embryotoxicity of co-planar PCBs is regulated by the aryl hydrocarbon receptor (Ahr), and has been reported to involve oxidative stress. Ahr participates in crosstalk with another transcription factor, Nfe2l2, or Nrf2. Nrf2 binds to antioxidant response elements to regulate the adaptive response to oxidative stress. To explore aspects of the crosstalk between Nrf2 and Ahr and its impact on development, we used zebrafish (Danio rerio) with a mutated DNA binding domain in Nrf2a (nrf2a(fh318/fh318)), rendering these embryos more sensitive to oxidative stress. Embryos were exposed to 2 nM or 5 nM PCB126 at 24 h post fertilization (prim-5 stage of pharyngula) and examined for gene expression and morphology at 4 days post fertilization (dpf; protruding - mouth stage). Nrf2a mutant eleutheroembryos were more sensitive to PCB126 toxicity at 4 dpf, and in the absence of treatment also displayed some subtle developmental differences from wildtype embryos, including delayed inflation of the swim bladder and smaller yolk sacs. We used qPCR to measure changes in expression of the nrf gene family, keap1a, keap1b, the ahr gene family, and known target genes. cyp1a induction by PCB126 was enhanced in the Nrf2a mutants (156-fold in wildtypes vs. 228-fold in mutants exposed to 5 nM). Decreased expression of heme oxygenase (decycling) 1 (hmox1) in the Nrf2a mutants was accompanied by increased nrf2b expression. Target genes of Nrf2a and AhR2, NAD(P)H:quinone oxidoreductase 1 (nqo1) and glutathione S-transferase, alpha-like (gsta1), showed a 2-5-fold increase in expression in the Nrf2a mutants as compared to wildtype. This study elucidates the interaction between two important transcription factor pathways in the developmental toxicity of co-planar PCBs., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Ethanol (EtOH) is a reactive oxygen-generating teratogen involved in the etiology of structural and functional developmental defects. Embryonic nutrition, redox environment, and changes in the thiol proteome following EtOH exposures (1.56.0 mg/ml) were studied in rat whole embryo culture. Glutathione (GSH) and cysteine (Cys) concentrations with their respective intracellular redox potentials (Eh) were determined using high-performance liquid chromatography. EtOH reduced GSH and Cys concentrations in embryo (EMB) and visceral yolk sac (VYS) tissues, and also in yolk sac and amniotic fluids. These changes produced greater oxidation as indicated by increasingly positive Eh values. EtOH reduced histiotrophic nutrition pathway activities as measured by the clearance of fluorescin isothiocyanate (FITC)-albumin from culture media. A significant decrease in total FITC clearance was observed at all concentrations, reaching approximately 50% at the highest dose. EtOH-induced changes to the thiol proteome were measured in EMBs and VYSs using isotope-coded affinity tags. Decreased concentrations for specific proteins from cytoskeletal dynamics and endocytosis pathways (α-actinin, α-tubulin, cubilin, and actin-related protein 2); nuclear translocation (Ran and RanBP1); and maintenance of receptor-mediated endocytosis (cubilin) were observed. Kyoto encyclopedia of genes and genomes (KEGG) pathway analysis also identified a decrease in ribosomal proteins in both EMB and VYS. Results show that EtOH interferes with nutrient uptake to reduce availability of amino acids and micronutrients required by the conceptus. Intracellular antioxidants such as GSH and Cys are depleted following EtOH and Eh values increase. Thiol proteome analysis in the EMB and VYS show selectively altered actin/cytoskeleton, endocytosis, ribosome biogenesis and function, nuclear transport, and stress-related responses., (© The Author 2015. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

Developmental signals that control growth and differentiation are regulated by environmental factors that generate reactive oxygen species (ROS) and alter steady-state redox environments in tissues and fluids. Protein thiols are selectively oxidized and reduced in distinct spatial and temporal patterns in conjunction with changes in glutathione/glutathione disulfide (GSH/GSSG) and cysteine/cystine (Cys/CySS) redox potentials (E(h)) to regulate developmental signaling. The purpose of this study was to measure compartment-specific thiol redox status in cultured organogenesis-stage rat conceptuses and to evaluate the impact of thiol oxidation on the redox proteome. The visceral yolk sac (VYS) has the highest initial (0 h) total intracellular GSH (GSH+2GSSG) concentration (5.5 mM) and the lowest Eh (-223 mV) as determined by HPLC analysis. Total embryo (EMB) GSH concentrations ranged lower (3.2 mM) and were only slightly more oxidized than the VYS. Total GSH concentrations in yolk sac fluid (YSF) and amniotic fluid (AF) are >500-fold lower than in tissues and are highly oxidized (YSF E(h)=-121 mV and AF E(h)=-49 mV). Steady-state total Cys concentrations (Cys+2CySS) were significantly lower than GSH in tissues but were otherwise equal in VYS and EMB near 0.5 mM. On gestational day 11, total GSH and Cys concentrations in EMB and VYS increase significantly over the 6h time course while E(h) remains relatively constant. The Eh (GSH/GSSG) in YSF and AF become more reduced over time while E(h) (Cys/CySS) become more oxidized. Addition of L-buthionine-S,R-sulfoximine (BS0) to selectively inhibit GSH synthesis and mimic the effects of some GSH-depleting environmental chemicals significantly decreased VYS and EMB GSH and Cys concentrations and increased Eh over the 6h exposure period, showing a greater overall oxidation. In the YSF, BSO caused a significant increase in total Cys concentrations to 1.7 mM but did not significantly change the E(h) for Cys/CySS. A significant net oxidation was seen in the BSO-treated AF compartment after 6 h. Biotinylated iodoacetamide (BIAM) labeling of proteins revealed the significant thiol oxidation of many EMB proteins following BSO treatment. Quantitative changes in the thiol proteome, associated with developmentally relevant pathways, were detected using isotope coded affinity tag (ICAT) labeling and mass spectroscopy. Adaptive pathways were selectively enriched with increased concentrations of proteins involved in mRNA processing (splicesome) and mRNA stabilization (glycolysis, GAPDH), as well as protein synthesis (aminoacyl-tRNA) and protein folding (antigen processing, Hsp70, protein disulfide isomerase). These results show the ability of chemical and environmental modulators to selectively alter compartmental intracellular and extracellular GSH and Cys concentrations and change their corresponding E(h) within the intact viable conceptus. The altered E(h) were also of sufficient magnitude to alter the redox proteome and change relative protein concentrations, suggesting that the mechanistic links through which environmental factors inform and regulate developmental signaling pathways may be discovered using systems developmental biology techniques., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Epigenetic modifications, including DNA methylation, contribute to the transcriptional regulation of developmental genes that control growth and differentiation during embryogenesis. The methyl donor, S-adenosylmethionine (SAM), is biosynthesized from methionine and adenosine triphosphate by methionine adenosyltransferase 2a (Mat2a) in the one-carbon (C1) metabolism pathway. SAM biosynthesis requires a steady supply of nutrients, vitamins and cofactors obtained by the developing conceptus through histiotrophic nutrition pathways (HNPs). The visceral yolk sac (VYS) captures proteins and their substrate cargos by receptor-mediated endocytosis and degrades them using lysosomal proteases. We hypothesize that leupeptin, a protease inhibitor, reduces the availability of methionine and C1 substrates, restricting SAM biosynthesis and altering patterns of DNA methylation. Rat conceptuses were exposed to 50 and 100 μM leupeptin in whole embryo culture for periods of 26 h from gestational day (GD) 10 or 6 h on GD11. After 6 h on GD11, the 100-μM leupeptin treatment significantly decreased methionine in embryo (EMB) and VYS, reduced Mat2a protein levels and inhibited Mat2a specific activity, all of which produced a significant 52% reduction of SAM in the VYS. The 50- and 100-μM leupeptin treatments significantly decreased global methylation levels by 6%-9% in EMB and by 11%-15% in VYS following both 6- and 26-h exposure periods. This study demonstrates that HNP disruption alters C1 activity and significantly reduces global DNA methylation during organogenesis. Because epigenetic reprogramming is crucial for normal differentiation and growth, these findings suggest a possible mechanism through which nutrients and environmental factors may alter early developmental regulation., (Copyright © 2013. Published by Elsevier Inc.)

DNA methylation is the most extensively studied mechanism of epigenetic gene regulation. Increasing evidence indicates that DNA methylation is labile in response to nutritional and environmental influences. Alterations in DNA methylation profiles can lead to changes in gene expression, resulting in diverse phenotypes with the potential for increased disease risk. The primary methyl donor for DNA methylation is S-adenosylmethionine (SAM), a species generated in the cyclical cellular process called one-carbon metabolism. One-carbon metabolism is catalyzed by several enzymes in the presence of dietary micronutrients, including folate, choline, betaine and other B vitamins. For this reason, nutrition status, particularly micronutrient intake, has been a focal point when investigating epigenetic mechanisms. Although animal evidence linking nutrition and DNA methylation is fairly extensive, epidemiological evidence is less comprehensive. This review serves to integrate studies of the animal in vivo with human epidemiological data pertaining to nutritional regulation of DNA methylation and to further identify areas in which current knowledge is limited., (Copyright © 2012 Elsevier Inc. All rights reserved.)

DNA methylation is an epigenetic form of gene regulation that is universally important throughout the life course, especially during in utero and postnatal development. DNA methylation aids in cell cycle regulation and cellular differentiation processes. Previous studies have demonstrated that DNA methylation profiles may be altered by diet and the environment, and that these profiles are especially vulnerable during development. Thus, it is important to understand the role of DNA methylation in developmental governance and subsequent disease progression. A variety of molecular methods exist to assay for global, gene-specific, and epigenome-wide methylation. Here we describe these methods and discuss their relative strengths and limitations.