Hybrid natural orifice transluminal endoscopy surgery (NOTES) to perform bilateral ovariectomy in mares.

OBJECTIVES: To combine laparoscopic techniques with natural orifice transluminal endoscopic surgery (hybrid NOTES) to minimize number and enlargement of paralumbar laparoscopic portals for bilateral ovariectomy in standing, sedated mares. STUDY DESIGN: Case series. ANIMALS: Six horses. MATERIALS AND METHODS: Six mares with palpably normal ovaries were restrained in stocks, sedated, and had caudal epidural anesthesia performed. A 7.5 MHz ultrasound probe was used transvaginally to select placement of vaginotomy. An attempt was made to remove both ovaries with 70 cm esophageal forceps through the vaginotomy, while visualized and transected via ipsilateral paralumbar fossa laparoscopic portals. Surgical time, intraoperative, and postoperative complications were recorded. Vaginoscopy was performed at days 0, 3, 7, and 14 postoperatively. After 14 days, mares were euthanized and necropsied. RESULTS: Hybrid NOTES allowed successful bilateral ovariectomy in all six mares, with a mean surgical time of 70 ± 25 min. In two mares, one ovary was dislodged from the forceps during vaginotomy extraction. Abdominal retrieval of the ovary was successful with reapplication of forceps or a surgeon's hand via vaginotomy. At necropsy, five mares had no adhesions within the abdominal cavity or at the vaginotomy site. One mare in which hand retrieval of the ovary was necessary, developed an adhesion between the bladder and the vaginotomy. CONCLUSIONS: This technique appears to offer a safe alternative for bilateral ovariectomy with minimal postoperative complications. CLINICAL SIGNIFICANCE: Decreased number and size of paralumbar laparoscopic incisions could decrease complications associated with exteriorization of ovaries through the flank.

Embryonic atrazine exposure and later in life behavioral and brain transcriptomic, epigenetic, and pathological alterations in adult male zebrafish.

Atrazine (ATZ), a commonly used pesticide linked to endocrine disruption, cancer, and altered neurochemistry, frequently contaminates water sources at levels above the US Environmental Protection Agency's 3 parts per billion (ppb; µg/L) maximum contaminant level. Adult male zebrafish behavior, brain transcriptome, brain methylation status, and neuropathology were examined to test the hypothesis that embryonic ATZ exposure causes delayed neurotoxicity, according to the developmental origins of health and disease paradigm. Zebrafish (Danio rerio) embryos were exposed to 0 ppb, 0.3 ppb, 3 ppb, or 30 ppb ATZ during embryogenesis (1-72 h post fertilization (hpf)), then rinsed and raised to maturity. At 9 months post fertilization (mpf), males had decreased locomotor parameters during a battery of behavioral tests. Transcriptomic analysis identified altered gene expression in organismal development, cancer, and nervous and reproductive system development and function pathways and networks. The brain was evaluated histopathologically for morphometric differences, and decreased numbers of cells were identified in raphe populations. Global methylation levels were evaluated at 12 mpf, and the body length, body weight, and brain weight were measured at 14 mpf to evaluate effects of ATZ on mature brain size. No significant difference in genome methylation or brain size was observed. The results demonstrate that developmental exposure to ATZ does affect neurodevelopment and neural function in adult male zebrafish and raises concern for possible health effects in humans due to ATZ's environmental presence and persistence. Graphical abstract.

Tubular ectasia of the rete testis in an Angus bull.

An 18-month-old Angus bull presented to Auburn University College of Veterinary Medicine for a routine breeding soundness evaluation and lameness evaluation. He was classified as deferred potential breeder due to a lameness and was donated to the university. Following treatment, the bull's lameness resolved. He passed the breeding soundness examination in accordance with the Society for Theriogenology standards. However, avascular dilated areas at the level of the mediastinum testis of the right testicle were detected via Doppler ultrasonography. A high level of vascularity is routinely seen with neoplasia, such as teratomas. Due to the lack of vascularity, a presumptive diagnosis of tubular ectasia of the rete testis was made. The bull was castrated. The right testicle was submitted for histopathology revealing a definitive diagnosis of tubular ectasia of the rete testis.

Embryonic Zebrafish as a Model for Investigating the Interaction between Environmental Pollutants and Neurodegenerative Disorders.

Environmental pollutants have been linked to neurotoxicity and are proposed to contribute to neurodegenerative disorders. The zebrafish model provides a high-throughput platform for large-scale chemical screening and toxicity assessment and is widely accepted as an important animal model for the investigation of neurodegenerative disorders. Although recent studies explore the roles of environmental pollutants in neurodegenerative disorders in zebrafish models, current knowledge of the mechanisms of environmentally induced neurodegenerative disorders is relatively complex and overlapping. This review primarily discusses utilizing embryonic zebrafish as the model to investigate environmental pollutants-related neurodegenerative disease. We also review current applicable approaches and important biomarkers to unravel the underlying mechanism of environmentally related neurodegenerative disorders. We found embryonic zebrafish to be a powerful tool that provides a platform for evaluating neurotoxicity triggered by environmentally relevant concentrations of neurotoxic compounds. Additionally, using variable approaches to assess neurotoxicity in the embryonic zebrafish allows researchers to have insights into the complex interaction between environmental pollutants and neurodegenerative disorders and, ultimately, an understanding of the underlying mechanisms related to environmental toxicants.

Sinonasal chondrosarcoma in a llama.

A 14-y-old intact female llama (Lama glama) was presented for evaluation of a right maxillary swelling of 3-mo duration. Clinically, the animal had mild nasal discharge, abnormal retropulsion of the right eye, and moderate gingival disease. An incisional biopsy of the maxillary mass revealed pleomorphic and mitotically active neoplastic spindle-to-stellate cells organized in haphazard lacunae embedded in abundant chondroid matrix. Given the poor prognosis, euthanasia was elected. Postmortem examination and sectioning of the head exposed a large solid, white, firm mass that vastly expanded the right infraorbital region, extending to the maxilla, effacing the right nasal conchae and ipsilateral zygomatic bone. Collectively, postmortem dissection, cytology, and histopathology of the primary mass supported a diagnosis of sinonasal chondrosarcoma. To our knowledge, this entity had not been reported previously in this species and should be considered a differential for facial deformities in New World camelids.

Blood transcriptome responses to PFOA and GenX treatment in the marsupial biomedical model Monodelphis domestica.

Introduction: Perfluoroalkyl and poly-fluoroalkyl substances (PFASs) are widely used in industrial and consumer products. Due to their environmental persistence and bioaccumulation, PFASs can be found in the blood of humans and wild animals all over the world. Various fluorinated alternatives such as GenX have been developed to replace the long-chain PFASs, but there is limited information about their potential toxicity. Methods:The current study developed blood culture protocols to assess the response to toxic compounds in the marsupial, Monodelphis domestica. After whole-blood culture conditions were tested and optimized, changes in gene expression in response to PFOA and GenX treatment were assessed. Results: More than 10,000 genes were expressed in the blood transcriptomes with and without treatment. Both PFOA and GenX treatment led to significant changes in the whole blood culture transcriptomes. A total of 578 and 148 differentially expressed genes (DEGs) were detected in the PFOA and GenX treatment groups, 32 of which overlapped. Pathway enrichment analysis revealed that DEGs involved in developmental processes were upregulated after PFOA exposure, while those enriched for metabolic and immune system processes were downregulated. GenX exposure upregulated genes associated with fatty acid transport pathways and inflammatory processes, which is consistent with previous studies using rodent models. Discussion: To our knowledge, this study is the first to investigate the effect of PFASs in a marsupial model. The findings provide supportive evidence for significant transcriptomic alterations, suggesting that this mammalian model may provide a mechanism for exploring the potential toxicity of PFOA and GenX.

Adverse developmental impacts in progeny of zebrafish exposed to the agricultural herbicide atrazine during embryogenesis.

Atrazine (ATZ) is an herbicide commonly used on crops in the Midwestern US and other select global regions. The US Environmental Protection Agency ATZ regulatory limit is 3 parts per billion (ppb; µg/L), but this limit is often exceeded. ATZ has a long half-life, is a common contaminant of drinking water sources, and is indicated as an endocrine disrupting chemical in multiple species. The zebrafish was used to test the hypothesis that an embryonic parental ATZ exposure alters protein levels leading to modifications in morphology and behavior in developing progeny. Zebrafish embryos (F1) were collected from adults (F0) exposed to 0, 0.3, 3, or 30 ppb ATZ during embryogenesis. Differential proteomics, morphology, and behavior assays were completed with offspring aged 120 or 144 h with no additional chemical treatment. Proteomic analysis identified differential expression of proteins associated with neurological development and disease; and organ and organismal morphology, development, and injury, specifically the skeletomuscular system. Head length and ratio of head length to total length was significantly increased in the F1 of 0.3 and 30 ppb ATZ groups (p < 0.05). Based on molecular pathway alterations, further craniofacial morphology assessment found decreased distance for cartilaginous structures, decreased surface area and distance between saccular otoliths, and a more posteriorly positioned notochord (p < 0.05), indicating delayed ossification and skeletal growth. The visual motor response assay showed hyperactivity in progeny of the 30 ppb treatment group for distance moved and of the 0.3 and 30 ppb treatment groups for time spent moving (p < 0.05). Due to the changes in saccular otoliths, an acoustic startle assay was completed and showed decreased response in the 0.3 and 30 ppb treatments (p < 0.05). These findings suggest that a single embryonic parental exposure alters cellular pathways in their progeny that lead to perturbations in craniofacial development and behavior.

Anxiety-related behavior and associated brain transcriptome and epigenome alterations in adult female zebrafish exposed to atrazine during embryogenesis.

Atrazine often contaminates drinking water sources, exceeding the maximum contaminant level established by the US Environmental Protection Agency at 3 parts per billion (ppb; µg/L). Atrazine is linked to endocrine disruption, neurotoxicity, and cancer, with delayed health effects observed after developmental exposure in line with the developmental origins of health and disease (DOHaD) hypothesis. To test the hypothesis that embryonic atrazine exposure induces delayed neurotoxicity in adult female zebrafish (Danio rerio), embryos were exposed to 0, 0.3, 3, or 30 ppb atrazine during embryogenesis (1-72 h post fertilization (hpf)) and raised to adults with no additional atrazine exposure. Behavioral outcomes were tested through a novel tank test, light-dark box, and open field test and indicated female zebrafish had more anxious phenotypes at 9 months post fertilization (mpf). Female brain transcriptomic analysis at 9 mpf found altered gene expression pathways related to organismal injury and cancer with beta-estradiol and estrogen receptor as top upstream regulators. These results were compared to 9 mpf male and 6 mpf female groups with the same atrazine embryonic exposures and showed differences in specific genes that were altered, but similarities in top molecular pathways. Molecular pathways associated with behavior were observed only in the 6 mpf transcriptomic profiles, suggesting prediction of observed behavioral outcomes at 9 mpf. The expression of genes associated with serotonin neurotransmission was also evaluated at 14 mpf to determine persistence; however, no significant changes were observed. Brain global methylation in 12 mpf zebrafish observed an increased percent 5 mC in females with embryonic 0.3 ppb atrazine exposure. Finally, the body length, body weight, and brain weight were determined at 14 mpf and were altered in all treatment groups. These results indicate that embryonic atrazine exposure does cause delayed neurotoxicity within the DOHaD framework, which is significant given atrazine's presence and persistence in the environment.

A Novel Neuraminidase Virus-Like Particle Vaccine Offers Protection Against Heterologous H3N2 Influenza Virus Infection in the Porcine Model.

Influenza A viruses (IAVs) pose a global health threat, contributing to hundreds of thousands of deaths and millions of hospitalizations annually. The two major surface glycoproteins of IAVs, hemagglutinin (HA) and neuraminidase (NA), are important antigens in eliciting neutralizing antibodies and protection against disease. However, NA is generally ignored in the formulation and development of influenza vaccines. In this study, we evaluate the immunogenicity and efficacy against challenge of a novel NA virus-like particles (VLPs) vaccine in the porcine model. We developed an NA2 VLP vaccine containing the NA protein from A/Perth/16/2009 (H3N2) and the matrix 1 (M1) protein from A/MI/73/2015, formulated with a water-in-oil-in-water adjuvant. Responses to NA2 VLPs were compared to a commercial adjuvanted quadrivalent whole inactivated virus (QWIV) swine IAV vaccine. Animals were prime boost vaccinated 21 days apart and challenged four weeks later with an H3N2 swine IAV field isolate, A/swine/NC/KH1552516/2016. Pigs vaccinated with the commercial QWIV vaccine demonstrated high hemagglutination inhibition (HAI) titers but very weak anti-NA antibody titers and subsequently undetectable NA inhibition (NAI) titers. Conversely, NA2 VLP vaccinated pigs demonstrated undetectable HAI titers but high anti-NA antibody titers and NAI titers. Post-challenge, NA2 VLPs and the commercial QWIV vaccine showed similar reductions in virus replication, pulmonary neutrophilic infiltration, and lung inflammation compared to unvaccinated controls. These data suggest that anti-NA immunity following NA2 VLP vaccination offers comparable protection to QWIV swine IAV vaccines inducing primarily anti-HA responses.

Atrazine exposure in zebrafish induces aberrant genome-wide methylation.

Atrazine (ATZ) is the second most common agricultural herbicide used in the United States and is an endocrine disrupting chemical (EDC). Developmental exposure to ATZ can lead to significant behavioral and morphological alterations in exposed animals and their progeny suggesting the involvement of an epigenetic mechanism. Specific epigenetic mechanisms responsible for these alterations, however, are yet to be elucidated. In this study, we exposed zebrafish embryos to 0, 0.3, 3, or 30 ppb (µg/L) of ATZ from 1 to 72 h post fertilization (hpf). Chemical exposure was ceased and zebrafish maintained until 9 months post fertilization (mpf), when whole-genome bisulfite sequencing (WGBS) was performed to assess the effects of embryonic ATZ exposure on DNA methylation in female fish brains. The number of differentially methylated genes (DMGs) increased with increasing treatment concentration. DMGs were enriched in neurological pathways with extensive methylation changes consistently observed in neuroendocrine pathways. Specifically, DMGs with methylation changes in promoter regions showed hypomethylation in estrogen receptor signaling and hypermethylation in androgen signaling. DMGs with methylation changes in genebody were primarily enriched for mitochondrion-related pathways associated with healthy aging. Integrated analysis with transcriptomic data at 9 mpf exhibited a similar trend identifying CABLES1 and NDUFA4 as shared targets at all concentrations. We then compared the predicted upstream regulators of transcriptomic changes with DMGs and identified CALML3 as a common upstream regulator at both 0.3 and 30 ppb that exhibit significant methylation changes. Collectively, our study identified long-lasting DNA methylation changes in genome after embryonic ATZ exposure and elucidated potential gene targets whose aberrant methylation features may drive alterations in gene transcription in long-term.

Developmental atrazine exposure in zebrafish produces the same major metabolites as mammals along with altered behavioral outcomes.

Atrazine (ATZ) is the second most commonly applied agricultural herbicide in the United States. Due to contamination concerns, the U.S. EPA has set the maximum contaminant level in potable water sources at 3 parts per billion (ppb; µg/l). Depending on the time of year and sampling location, water sources often exceed this limit. ATZ is an endocrine disrupting chemical in multiple species observed to target the neuroendocrine system. In this study the zebrafish vertebrate model was used to test the hypothesis that a developmental ATZ exposure generates metabolites similar to those found in mammals and alters morphology and behavior in developing larvae. Adult AB zebrafish were bred, embryos were collected, and exposed to 0, 0.3, 3, or 30 ppb ATZ from 1 to 120 h post fertilization (hpf). Targeted metabolomic analysis found that zebrafish produce the same major ATZ metabolites as mammals: desethyl atrazine (DEA), deisopropyl atrazine (DIA), and diaminochloroatrazine (DACT). The visual motor response test at 120 hpf detected hyperactivity in larvae in the 0.3 ppb treatment group and hypoactivity in the 30 ppb treatment group (p < 0.05). Further analysis into behavior during the dark and light phases showed zebrafish larvae exposed to 0.3 ppb ATZ had an increase in total distance moved in the first light phase and time spent moving in the first dark and light phases (p < 0.05). Alternatively, a decrease in total distance moved was observed in the second and third dark phases in zebrafish exposed to 30 ppb ATZ (p < 0.05). No significant differences were observed for any of the morphological measurements following ATZ exposure from 1 to 120 hpf (p > 0.05). These findings suggest that a ATZ exposure during early development generates metabolite profiles similar to mammals and leads to behavioral alterations supporting ATZ as a neurodevelopmental toxicant.

Developmental toxicity of trichloroethylene in zebrafish (Danio rerio).

Trichloroethylene (TCE), an industrial solvent and degreaser, is an environmental toxicant that contaminates over half of Superfund sites, is a known carcinogen, and is linked to congenital defects and neurodegenerative disease. The developmental toxicity of TCE near ecologically relevant levels needs further characterization in order to better assess health risks of exposure. In this study, the toxicodynamics of TCE in the zebrafish (Danio rerio) model was investigated through the establishment of a LC50 concentration and by monitoring the acute developmental toxicity of ecologically relevant concentrations (0, 5, 50, and 500 parts per billion; ppb) of TCE during two different exposure lengths (1-72 hours post fertilization (hpf) and 1-120 hpf). Acute developmental toxicity was assessed by monitoring survival and hatching, larval morphology, larval heart rate, and behavioral responses during an embryonic photomotor response test and a larval visual motor response test. Embryonic exposure to TCE was associated with decreased percent hatch at 48 hpf, altered larval morphology, increased heart rate, and altered behavioral responses during the photomotor response test and visual motor response test. Larval morphology and behavioral alterations were more pronounced in the 1-120 hpf exposure length trials. The observed alterations suggest developmental TCE toxicity is still a concern at regulatory concentrations and that timing of exposure influences developmental toxicity.

Making Waves: New Developments in Toxicology With the Zebrafish.

The laboratory zebrafish (Danio rerio) is now an accepted model in toxicologic research. The zebrafish model fills a niche between in vitro models and mammalian biomedical models. The developmental characteristics of the small fish are strategically being used by scientists to study topics ranging from high-throughput toxicity screens to toxicity in multi- and transgenerational studies. High-throughput technology has increased the utility of zebrafish embryonic toxicity assays in screening of chemicals and drugs for toxicity or effect. Additionally, advances in behavioral characterization and experimental methodology allow for observation of recognizable phenotypic changes after xenobiotic exposure. Future directions in zebrafish research are predicted to take advantage of CRISPR-Cas9 genome editing methods in creating models of disease and interrogating mechanisms of action with fluorescent reporters or tagged proteins. Zebrafish can also model developmental origins of health and disease and multi- and transgenerational toxicity. The zebrafish has many advantages as a toxicologic model and new methodologies and areas of study continue to expand the usefulness and application of the zebrafish.

An embryonic 100µg/L lead exposure results in sex-specific expression changes in genes associated with the neurological system in female or cancer in male adult zebrafish brains.

Developmental lead (Pb) exposure is linked to neurological health issues. Results from non-human primate and rodent studies suggest detrimental effects of an early life Pb exposure, showing transcriptional disturbances and pathological evidence of Alzheimer's disease in the adult animal brain. To elucidate the impacts of an embryonic Pb exposure on the adult brain, transcriptomic analysis was completed on the brain of zebrafish aged 12months exposed to a control treatment or to an embryonic 100µg/L Pb exposure by sex. In the adult female zebrafish brain, significant changes in expression profiles occurred in a number of genes involved in neurological disease and nervous system development and function. On the other hand, in adult males, a number of genes with significant expression alterations were found to be associated with cancer and tumors. p38 mitogen-activated protein kinase (p38 MAPK) was also indicated as an upstream regulator of observed gene expression changes. Western blot analysis confirmed activation of p38 MAPK in the form of phosphorylated p38 MAPK in the male zebrafish brain. In addition, we compared transcriptomic changes observed in this study to a previous study with an embryonic exposure of 10µg/L Pb by sex, showing unique sets of genes dependent on Pb concentration. Overall, these results show sex-specific and concentration-dependent disturbances of global gene expression patterns in the brain of adult zebrafish exposed to Pb during embryogenesis.

Microarray, IPA and GSEA Analysis in Mice Models.

This protocol details a method to analyze two tissue samples at the transcriptomic level using microarray analysis, ingenuity pathway analysis (IPA) and gene set enrichment analysis (GSEA). Methods such as these provide insight into the mechanisms underlying biological differences across two samples and thus can be applied to interrogate a variety of processes across different tissue samples, conditions, and the like. The full method detailed below can be applied to determine the effects of muscle-specific Notch1 activation in the mdx mouse model and to analyze previously published microarray data of human liposarcoma cell lines.

Embryonic atrazine exposure elicits proteomic, behavioral, and brain abnormalities with developmental time specific gene expression signatures.

Atrazine (ATZ), the second most commonly used herbicide in the United States, is an endocrine disrupting chemical linked to cancer and a common drinking water contaminant. This study further investigates ATZ-related developmental toxicity by testing the following hypotheses in zebrafish: the effects of embryonic ATZ exposure are dependent on timing of exposure; embryonic ATZ exposure alters brain development and function; and embryonic ATZ exposure changes protein abundance in carcinogenesis-related pathways. After exposing embryos to 0, 0.3, 3, or 30 parts per billion (ppb) ATZ, we monitored the expression of cytochrome P450 family 17 subfamily A member 1 (cyp17a1), glyoxalase I (glo1), ring finger protein 14 (rnf14), salt inducible kinase 2 (sik2), tetratricopeptide domain 3 (ttc3), and tumor protein D52 like 1 (tpd52l1) at multiple embryonic time points to determine normal expression and if ATZ exposure altered expression. Only cyp17a1 had normal dynamic expression, but ttc3 and tpd52l1 had ATZ-related expression changes before 72 h. Larvae exposed to 0.3 ppb ATZ had increased brain length, while larvae exposed to 30 ppb ATZ were hypoactive. Proteomic analysis identified altered protein abundance in pathways related to cellular function, neurodevelopment, and genital-tract cancer. The results indicate embryonic ATZ toxicity involves interactions of multiple pathways. SIGNIFICANCE: This is the first report of proteomic alterations following embryonic exposure to atrazine, an environmentally persistent pesticide and common water contaminant. Although the transcriptomic alterations in larval zebrafish with embryonic atrazine exposure have been reported, neither the time at which gene expression changes occur nor the resulting proteomic changes have been investigated. This study seeks to address these knowledge gaps by evaluating atrazine's effect on gene expression through multiple time points during embryogenesis, and correlating changes in gene expression to pathological alterations in brain length and functional changes in behavior. Finally, pathway analysis of the proteomic alterations identifies connections between the molecular changes and functional outcomes associated with embryonic atrazine exposure.

Comparative analytical and toxicological assessment of methylcyclohexanemethanol (MCHM) mixtures associated with the Elk River chemical spill.

On January 9, 2014, a chemical mixture containing crude methylcyclohexanemethanol (MCHM) contaminated the water supply of Charleston, West Virginia. Although the mixture was later identified as a mix of crude MCHM and stripped propylene glycol phenyl ethers, initial risk assessment focused on 4-MCHM, the predominant component of crude MCHM. The mixture's exact composition and the toxicity differences between 4-MCHM, crude MCHM, and the tank mixture were unknown. We analyzed the chemical composition of crude MCHM and the tank mixture via GC/MS and, based on identified spectra, found that crude MCHM and the tank mixture differed in chemical composition. To evaluate acute developmental toxicity, zebrafish embryos were exposed to 0, 1, 6.25, 12.5, 25, 50, or 100 parts per million (ppm; mg/L) of 4-MCHM, crude MCHM, or the tank mixture. The percent mortality and percent hatch, larval morphology alterations, and larval visual motor response test were used to establish toxicity profiles for each of the chemicals or mixtures. The acute toxicity differed between 4-MCHM, crude MCHM and the tank mixture with significant differences in survival, hatching, morphology, and locomotion at levels as low as the short-term screening level of 1 ppm, suggesting a need for further research into human health risks. This study is the first to evaluate the developmental toxicity of the tank mixture and highlights that studies evaluating risk should not assume the effects of 4-MCHM or crude MCHM are representative of the Tank 396 mixture.

Thyroid disrupting effects of halogenated and next generation chemicals on the swim bladder development of zebrafish.

Endocrine disrupting chemicals (EDCs) can alter thyroid function and adversely affect growth and development. Halogenated compounds, such as perfluorinated chemicals commonly used in food packaging, and brominated flame retardants used in a broad range of products from clothing to electronics, can act as thyroid disruptors. Due to the adverse effects of these compounds, there is a need for the development of safer next generation chemicals. The objective of this study was to test the thyroid disruption potential of old use and next generation halogenated chemicals. Zebrafish embryos were exposed to three old use compounds, perfluorooctanoic acid (PFOA), tetrabromobisphenol A (TBBPA) and tris (1,3-dichloro-2-propyl) phosphate (TDCPP) and two next generation chemicals, 9,10-dihydro-9-oxa-10-phosphaphenanthrene-10-oxdie (DOPO) and perfluorobutyric acid (PFBA). Sub-chronic (0-6days post fertilization (dpf)) and chronic (0-28dpf) exposures were conducted at 1% of the concentration known to kill 50% (LC50) of the population. Changes in the surface area of the swim bladder as well as in expression levels of genes involved in the thyroid control of swim bladder inflation were measured. At 6dpf, zebrafish exposed to all halogenated chemicals, both old use and next generation, had smaller posterior swim bladder and increased expression in the gene encoding thyroid peroxidase, tpo and the genes encoding two swim bladder surfactant proteins, sp-a and sp-c. These results mirrored the effects of thyroid hormone-exposed positive controls. Fish exposed to a TPO inhibitor (methimazole, MMI) had a decrease in tpo expression levels at 28dpf. Effects on the anterior swim bladder at 28dpf, after exposure to MMI as well as both old and new halogenated chemicals, were the same, i.e., absence of SB in ∼50% of fish, which were also of smaller body size. Overall, our results suggest thyroid disruption by the halogenated compounds tested via the swim bladder surfactant system. However, with the exception of TBBPA and TDCPP, the concentrations tested (∼5-137ppm) are not likely to be found in the environment.

Atrazine exposure decreases the activity of DNMTs, global DNA methylation levels, and dnmt expression.

Atrazine, a herbicide used on agricultural crops is widely applied in the Midwestern United States as well as other areas of the globe. Atrazine frequently contaminates potable water supplies and is a suspected endocrine disrupting chemical. Previous studies have reported morphological, hormonal, and molecular alterations due to developmental and adulthood atrazine exposure; however, studies examining epigenetic alterations are limited. In this study, the effects of atrazine exposure on DNA methyltransferase (DNMT) activity and kinetics were evaluated. Global DNA methylation levels and dnmt expression in zebrafish larvae exposed to 0, 3, or 30 parts per billion (ppb) atrazine throughout embryogenesis was then assessed. Results indicate that atrazine significantly decreased the activity of maintenance DNMTs and that the inhibition mechanism can be described using non-competitive Michaelis-Menten kinetics. Furthermore, results show that an embryonic atrazine exposure decreases global methylation levels and the expression of dnmt4 and dnmt5. These findings indicate that atrazine exposure can decrease the expression and activity of DNMTs, leading to decreased DNA methylation levels.