Machine Learning-Based Toxicological Modeling for Screening Environmental Obesogens.

The emerging presence of environmental obesogens, chemicals that disrupt energy balance and contribute to adipogenesis and obesity, has become a major public health challenge. Molecular initiating events (MIEs) describe biological outcomes resulting from chemical interactions with biomolecules. Machine learning models based on MIEs can predict complex toxic end points due to chemical exposure and improve the interpretability of models. In this study, a system was constructed that integrated six MIEs associated with adipogenesis and obesity. This system showed high accuracy in external validation, with an area under the receiver operating characteristic curve of 0.78. Molecular hydrophobicity (SlogP_VSA) and direct electrostatic interactions (PEOE_VSA) were identified as the two most critical molecular descriptors representing the obesogenic potential of chemicals. This system was further used to predict the obesogenic effects of chemicals on the candidate list of substances of very high concern (SVHCs). Results from 3T3-L1 adipogenesis assays verified that the system correctly predicted obesogenic or nonobesogenic effects of 10 of the 12 SVHCs tested, and identified four novel potential obesogens, including 2-benzotriazol-2-yl-4,6-ditert-butylphenol (UV-320), 4-(1,1,5-trimethylhexyl)phenol (p262-NP), 2-[4-(1,1,3,3-tetramethylbutyl)phenoxy]ethanol (OP1EO) and endosulfan. These validation data suggest that the screening system has good performance in adipogenic prediction.

Methylation and Demethylation of Emerging Contaminants in Plants.

Many contaminants of emerging concern (CECs) have reactive functional groups and may readily undergo biotransformations, such as methylation and demethylation. These transformations have been reported to occur during human metabolism and wastewater treatment, leading to the propagation of CECs. When treated wastewater and biosolids are used in agriculture, CECs and their transformation products (TPs) are introduced into soil-plant systems. However, little is known about whether transformation cycles, such as methylation and demethylation, take place in higher plants and hence affect the fate of CECs in terrestrial ecosystems. In this study, we explored the interconversion between four common CECs (acetaminophen, diazepam, methylparaben, and naproxen) and their methylated or demethylated TPs in Arabidopsis thaliana cells and whole wheat seedlings. The methylation-demethylation cycle occurred in both plant models with demethylation generally taking place at a greater degree than methylation. The transformation rate of demethylation or methylation was dependent on the bond strength of R-CH3, with demethylation of methylparaben or methylation of acetaminophen being more pronounced. Although not explored in this study, these interconversions may exert influences on the behavior and biological activity of CECs, particularly in terrestrial ecosystems. The study findings demonstrated the prevalence of transformation cycles between CECs and their methylated or demethylated TPs in higher plants, contributing to a more complete understanding of risks of CECs in the human-wastewater-soil-plant continuum.

Stereoselective Bioconcentration and Neurotoxicity of Perfluoroethylcyclohexane Sulfonate in Marine Medaka.

Perfluoroethylcyclohexane sulfonate (PFECHS) is an emerging per- and polyfluoroalkyl substance used to replace perfluorooctane sulfonate (PFOS), mainly in aircraft hydraulic fluids. However, previous research indicates the potential neurotoxicity of this replacement chemical. In this study, marine medaka (Oryzias melastigma) was exposed to environmentally relevant concentrations of PFECHS (concentrations: 0, 0.08, 0.26, and 0.91 µg/L) from the embryonic stage for 90 days. After exposure, the brain and eyes of the medaka were collected to investigate the bioconcentration potential of PFECHS stereoisomers and their effects on the nervous systems. The determined bioconcentration factors (BCFs) of PFECHS ranged from 324 ± 97 to 435 ± 89 L/kg and from 454 ± 60 to 576 ± 86 L/kg in the brain and eyes of medaka, respectively. The BCFs of trans-PFECHS were higher than those of cis-PFECHS. PFECHS exposure significantly altered γ-aminobutyric acid (GABA) levels in the medaka brain and disrupted the GABAergic system, as revealed by proteomics, implying that PFECHS can disturb neural signal transduction like PFOS. PFECHS exposure resulted in significant alterations in multiple proteins associated with eye function in medaka. Abnormal locomotion was observed in PFECHS-exposed medaka larvae, which was rescued by adding exogenous GABA, suggesting the involvement of disrupted GABA signaling pathways in PFECHS neurotoxicity.

Perfluorohexanesulfonic Acid (PFHxS) Impairs Lipid Homeostasis in Zebrafish Larvae through Activation of PPARα.

Perfluorohexanesulfonic acid (PFHxS), an emerging short-chain per- and polyfluoroalkyl substance, has been frequently detected in aquatic environments. Adverse outcome pathway studies have shown that perfluorinated compounds impair lipid homeostasis through peroxisome proliferator activated receptors (PPARs). However, many of these studies were performed at high concentrations and may thus be a result of overt toxicity. To better characterize the molecular and key events of PFHxS to biota, early life-stage zebrafish (Danio rerio) were exposed to concentrations detected in the environment (0.01, 0.1, 1, and 10 µg/L). Lipidomic and transcriptomic evaluations were integrated to predict potential molecular targets. PFHxS significantly impaired lipid homeostasis by the dysregulation of glycerophospholipids, fatty acyls, glycerolipids, sphingolipids, prenol lipids, and sterol lipids. Informatic analyses of the lipidome and transcriptome indicated alterations of the PPAR signaling pathway, with downstream changes to retinol, linoleic acid, and glycerophospholipid metabolism. To assess the role of PPARs, potential binding of PFHxS to PPARs was predicted and animals were coexposed to a PPAR antagonist (GW6471). Molecular simulation indicated PFHxS had a 27.1% better binding affinity than oleic acid, an endogenous agonist of PPARα. Antagonist coexposures rescued impaired glycerophosphocholine concentrations altered by PFHxS. These data indicate PPARα activation may be an important molecular initiating event for PFHxS.

Contribution of Immune Responses to the Cardiotoxicity and Hepatotoxicity of Deltamethrin in Early Life Stage Zebrafish (Danio rerio).

Deltamethrin (DM) is a widely used insecticide that has demonstrated developmental toxicity in the early life stages of fish. To better characterize the underlying mechanisms, embryos from Tg(cmlc2:RFP), Tg(apo14:GFP), and Tg(mpx:GFP) transgenic strains of zebrafish were exposed to nominal DM concentrations of 0.1, 1, 10, 25, and 50 µg/L until 120 h post-fertilization (hpf). Heart size increased 56.7%, and liver size was reduced by 17.1% in zebrafish exposed to 22.7 and 24.2 µg/L DM, respectively. RNA sequencing and bioinformatic analyses predicted that key biological processes affected by DM exposure were related to inflammatory responses. Expression of IL-1 protein was increased by 69.0% in the 24.4 µg/L DM treatment, and aggregation of neutrophils in cardiac and hepatic histologic sections was also observed. Coexposure to resatorvid, an anti-inflammatory agent, mitigated inflammatory responses and cardiac toxicity induced by DM and also restored liver biomass. Our data indicated a complex proinflammatory mechanism underlying DM-induced cardiotoxicity and hepatotoxicity which may be important for key events of adverse outcomes and associated risks of DM to early life stages of fish.

Chlorination of Emerging Contaminants for Application in Potable Wastewater Reuse: Disinfection Byproduct Formation, Estrogen Activity, and Cytotoxicity.

With increasing water scarcity, many utilities are considering the potable reuse of wastewater as a source of drinking water. However, not all chemicals are removed in conventional wastewater treatment, and disinfection byproducts (DBPs) can form from these contaminants when disinfectants are applied during or after reuse treatment, especially if applied upstream of advanced treatment processes to control biofouling. We investigated the chlorination of seven priority emerging contaminants (17ß-estradiol, estrone, 17α-ethinylestradiol, bisphenol A (BPA), diclofenac, p-nonylphenol, and triclosan) in ultrapure water, and we also investigated the impact of chlorination on real samples from different treatment stages of an advanced reuse plant to evaluate the role of chlorination on the associated cytotoxicity and estrogenicity. Many DBPs were tentatively identified via liquid chromatography (LC)- and gas chromatography (GC)-high resolution mass spectrometry, including 28 not previously reported. These encompassed chlorinated, brominated, and oxidized analogs of the parent compounds as well as smaller halogenated molecules. Chlorinated BPA was the least cytotoxic of the DBPs formed but was highly estrogenic, whereas chlorinated hormones were highly cytotoxic. Estrogenicity decreased by ∼4-6 orders of magnitude for 17ß-estradiol and estrone following chlorination but increased 2 orders of magnitude for diclofenac. Estrogenicity of chlorinated BPA and p-nonylphenol were ∼50% of the natural/synthetic hormones. Potential seasonal differences in estrogen activity of unreacted vs reacted advanced wastewater treatment field samples were observed.

The Roles of Diet and Habitat Use in Pesticide Bioaccumulation by Juvenile Chinook Salmon: Insights from Stable Isotopes and Fatty Acid Biomarkers.

Stable isotopes (SI) and fatty acid (FA) biomarkers can provide insights regarding trophic pathways and habitats associated with contaminant bioaccumulation. We assessed relationships between SI and FA biomarkers and published data on concentrations of two pesticides [dichlorodiphenyltrichloroethane and degradation products (DDX) and bifenthrin] in juvenile Chinook Salmon (Oncorhynchus tshawytscha) from the Sacramento River and Yolo Bypass floodplain in Northern California near Sacramento. We also conducted SI and FA analyses of zooplankton and macroinvertebrates to determine whether particular trophic pathways and habitats were associated with elevated pesticide concentrations in fish. Relationships between DDX and both sulfur (δ34S) and carbon (δ13C) SI ratios in salmon indicated that diet is a major exposure route for DDX, particularly for individuals with a benthic detrital energy base. Greater use of a benthic detrital energy base likely accounted for the higher frequency of salmon with DDX concentrations > 60 ng/g dw in the Yolo Bypass compared to the Sacramento River. Chironomid larvae and zooplankton were implicated as prey items likely responsible for trophic transfer of DDX to salmon. Sulfur SI ratios enabled identification of hatchery-origin fish that had likely spent insufficient time in the wild to substantially bioaccumulate DDX. Bifenthrin concentration was unrelated to SI or FA biomarkers in salmon, potentially due to aqueous uptake, biotransformation and elimination of the pesticide, or indistinct biomarker compositions among invertebrates with low and high bifenthrin concentrations. One FA [docosahexaenoic acid (DHA)] and DDX were negatively correlated in salmon, potentially due to a greater uptake of DDX from invertebrates with low DHA or effects of DDX on FA metabolism. Trophic biomarkers may be useful indicators of DDX accumulation and effects in juvenile Chinook Salmon in the Sacramento River Delta.

Methylation and Demethylation of Emerging Contaminants Changed Bioaccumulation and Acute Toxicity in Daphnia magna.

Contaminants of emerging concern (CECs) in the environment undergo various transformations, leading to the formation of transformation products (TPs) with a modified ecological risk potential. Although the environmental significance of TPs is increasingly recognized, there has been relatively little research to understand the influences of such transformations on subsequent ecotoxicological safety. In this study, we used four pairs of CECs and their methylated or demethylated derivatives as examples to characterize changes in bioaccumulation and acute toxicity in Daphnia magna, as a result of methylation or demethylation. The experimental results were further compared to quantitative structure-activity relationship (QSAR) predictions. The methylated counterpart in each pair generally showed greater acute toxicity in D. magna, which was attributed to their increased hydrophobicity. For example, the LC50 values of methylparaben (34.4 ± 4.3 mg L-1) and its demethylated product (225.6 ± 17.3 mg L-1) differed about eightfold in D. magna. The methylated derivative generally exhibited greater bioaccumulation than the demethylated counterpart. For instance, the bioaccumulation of methylated acetaminophen was about 33-fold greater than that of acetaminophen. In silico predictions via QSARs aligned well with the experimental results and suggested an increased persistence of the methylated forms. The study findings underline the consequences of simple changes in chemical structures induced by transformations such as methylation and demethylation and highlight the need to consider TPs to achieve a more holistic understanding of the environmental fate and risks of CECs.

Limited Effectiveness of Carbonaceous Sorbents in Sequestering Aged Organic Contaminants in Sediments.

Carbonaceous materials are often proposed for use in restoring soils or sediments contaminated with hydrophobic organic contaminants (HOCs). However, the contamination of most sites is a result of historical events, where HOCs have resided in the solid compartment for many years or decades. The prolonged contact time, or aging, leads to reduced contaminant availability and likely diminished effectiveness of using sorbents. In this study, three different carbonaceous sorbents, i.e., biochars, powdered activated carbon, and granular activated carbon, were amended to a Superfund site marine sediment contaminated with DDT residues from decades ago. The amended sediments were incubated in seawater for up to 1 year, and the freely dissolved concentration (Cfree) and the biota-sediment accumulation factors (BSAFs) for a native polychaete (Neanthes arenaceodentata) were measured. Even though the bulk sediment concentrations were very high (6.4-154.9 µg/g OC), both Cfree and BSAFs were very small, ranging from nd to 1.34 ng/L and from nd to 0.024, respectively. The addition of carbonaceous sorbents, even at 2% (w/w), did not consistently lead to reduced DDT bioaccumulation. The limited effectiveness of carbonaceous sorbents was attributed to the low DDT availability due to prolonged aging, highlighting the need for considering contaminant aging when using sorbents for remediation.

Relationships between Isomeric Metabolism and Regioselective Toxicity of Hydroxychrysenes in Embryos of Japanese Medaka (Oryzias latipes).

Oxygenated polycyclic aromatic hydrocarbons (oxy-PAHs) are ubiquitous contaminants that can be formed through oxidation of parent PAHs. Our previous studies found 2-hydroxychrysene (2-OHCHR) to be significantly more toxic to Japanese medaka embryos than 6-hydroxychrysene (6-OHCHR), an example of regioselective toxicity. We have also previously identified a sensitive developmental window to 2-OHCHR toxicity that closely coincided with liver development, leading us to hypothesize that differences in metabolism may play a role in the regioselective toxicity. To test this hypothesis, Japanese medaka embryos were treated with each isomer for 24 h during liver development (52-76 hpf). Although 6-OHCHR was absorbed 97.2 ± 0.18% faster than 2-OHCHR, it was eliminated 57.7 ± 0.36% faster as a glucuronide conjugate. Pretreatment with cytochrome P450 inhibitor, ketoconazole, reduced anemia by 96.8 ± 3.19% and mortality by 95.2 ± 4.76% in 2-OHCHR treatments. Formation of chrysene-1,2-diol (1,2-CAT) was also reduced by 64.4 ± 2.14% by ketoconazole pretreatment. While pretreatment with UDP-glucuronosyltransferase inhibitor, nilotinib, reduced glucuronidation of 2-OHCHR by 52.4 ± 2.55% and of 6-OHCHR by 63.7 ± 3.19%, it did not alter toxicity for either compound. These results indicate that CYP-mediated activation, potentially to 1,2-CAT, may explain the isomeric differences in developmental toxicity of 2-OHCHR.

Connecting gut microbiome changes with fish health conditions in juvenile Atlantic cod (Gadus morhua) exposed to dispersed crude oil.

Polycyclic aromatic hydrocarbons found in crude oil can impair fish health following sublethal exposure. However, the dysbiosis of microbial communities within the fish host and influence it has on the toxic response of fish following exposure has been less characterized, particularly in marine species. To better understand the effect of dispersed crude oil (DCO) on juvenile Atlantic cod (Gadus morhua) microbiota composition and potential targets of exposure within the gut, fish were exposed to 0.05 ppm DCO for 1, 3, 7, or 28 days and 16 S metagenomic and metatranscriptomic sequencing on the gut and RNA sequencing on intestinal content were conducted. In addition to assessing species composition, richness, and diversity from microbial gut community analysis and transcriptomic profiling, the functional capacity of the microbiome was determined. Mycoplasma and Aliivibrio were the two most abundant genera after DCO exposure and Photobacterium the most abundant genus in controls, after 28 days. Metagenomic profiles were only significantly different between treatments after a 28-day exposure. The top identified pathways were involved in energy and the biosynthesis of carbohydrates, fatty acids, amino acids, and cellular structure. Biological processes following fish transcriptomic profiling shared common pathways with microbial functional annotations such as energy, translation, amide biosynthetic process, and proteolysis. There were 58 differently expressed genes determined from metatranscriptomic profiling after 7 days of exposure. Predicted pathways that were altered included those involved in translation, signal transduction, and Wnt signaling. EIF2 signaling was consistently dysregulated following exposure to DCO, regardless of exposure duration, with impairments in IL-22 signaling and spermine and spermidine biosynthesis in fish after 28 days. Data were consistent with predictions of a potentially reduced immune response related to gastrointestinal disease. Herein, transcriptomic-level responses helped explain the relevance of differences in gut microbial communities in fish following DCO exposure.

The Use of Non-targeted Lipidomics and Histopathology to Characterize the Neurotoxicity of Bifenthrin to Juvenile Rainbow Trout (Oncorhynchus mykiss).

Due to the detection frequencies and measured concentrations in surface water, the type I pyrethroid insecticide, bifenthrin, has been of particular concern within the Sacramento-San Joaquin Delta in California. Concentrations have been detected above levels previously reported to impair neuroendocrine function and induce neurotoxicity to several species of salmonids. Metabolomic and transcriptomic studies indicated impairment of cellular signaling within the brain of exposed animals and potential alteration of lipid metabolism. To better understand the potential impacts of bifenthrin on brain lipids, juvenile rainbow trout (Oncorhynchus mykiss) were exposed to mean bifenthrin concentrations of 28 or 48 ng/L for 14 days, and non-targeted lipidomic profiling in the brain was conducted. Brain tissue sections were also assessed for histopathological insult following bifenthrin treatment. Bifenthrin-exposed trout had a concentration-dependent decrease in the relative abundance of triglycerides (TGs) with levels of phosphatidylcholines (PCs) and phosphatidylethanolamines (PEs) significantly altered following 48 ng/L bifenthrin exposure. An increased incidence of histopathological lesions, such as focal hemorrhages and congestion of blood vessels, was noted in the brains of bifenthrin-treated animals, suggesting an association between altered lipid metabolism and neuronal cell structure and integrity.

Enrofloxacin Induces Intestinal Microbiota-Mediated Immunosuppression in Zebrafish.

The immunosuppressive effects of antibiotics and the potential associations with the intestinal microbiota of the host have been increasingly recognized in recent years. However, the detailed underlying mechanisms of immune interference of antibiotics in environmental organisms remain unclear, particularly at the early life stage of high sensitivity. To better understand the gut microbiome and immune function interactions, the vertebrate model, zebrafish, was treated with environmentally relevant concentrations of a frequently detected antibiotic, enrofloxacin (ENR), ranging from 0.01 to 100 µg/L. 16S ribosomal RNA sequencing indicated diminished diversity, richness, and evenness of intestinal flora following ENR treatment. Twenty-two taxa of gut bacteria including Rickettsiales, Pseudomonadales, and Flavobacteriales were significantly correlated with immunosuppressive biomarkers, including a significant decrease in the abundance of macrophages and neutrophils. To validate the immunomodulatory effects due to altered intestinal microbial populations, zebrafish reared under sterile and non-sterile husbandry conditions were compared after ENR treatment. A significant inhibitory effect was induced by ENR treatment under non-sterile conditions, while the number of macrophages and neutrophils, as well as biomarkers of immunosuppressive effects, were significantly salved in zebrafish under sterile conditions, confirming for the first time that immunosuppression by ENR was closely mediated through alterations of the intestinal microbiome in fish.

Dietary Exposure to Bifenthrin and Fipronil Impacts Swimming Performance in Juvenile Chinook Salmon (Oncorhynchus tshawytscha).

Two commonly used insecticides, bifenthrin and fipronil, can accumulate in the prey of juvenile Chinook salmon, yet the effects of dietary exposure are not understood. Therefore, to better characterize the effect of a dietary exposure route, juvenile Chinook salmon were fed chironomids dosed with a concentration of 9 or 900 ng/g of bifenthrin, fipronil, or their mixture for 25 days at concentrations previously measured in field-collected samples. Chinook were assessed for maximum swimming performance (Umax) using a short-duration constant acceleration test and biochemical responses related to energetic processes (glucose levels) and liver health (aspartate aminotransferase (AST) activity). Chinook exposed to bifenthrin and bifenthrin and fipronil mixtures had a significantly reduced swimming performance, although not when exposed to fipronil alone. The AST activity was significantly increased in bifenthrin and mixture treatments and glucose levels were increased in Chinook following a mixture treatment, although not when exposed to fipronil alone. These findings suggest that there are different metabolic processes between bifenthrin and fipronil following dietary uptake that may influence toxicity. The significant reductions in swimming performance and increased levels of biochemical processes involved in energetics and fish heath could have implications for foraging activity and predator avoidance in wild fish at sensitive life stages.

Antibiotic Chlortetracycline Causes Transgenerational Immunosuppression via NF-κB.

The extensive and increasing global use of antibiotics results in the ubiquitous presence of antibiotics in the environment, which has made them "pseudo persistent organic contaminants." Despite numerous studies showing wide adverse effects of antibiotics on organisms, the chronic environmental risk of their exposure is unknown, and the molecular and cellular mechanisms of antibiotic toxicity remain unclear. Here, we systematically quantified transgenerational immune disturbances after chronic parental exposure to environmental levels of a common antibiotic, chlortetracycline (CTC), using zebrafish as a model. CTC strongly reduced the antibacterial activities of fish offspring by transgenerational immunosuppression. Both innate and adaptive immunities of the offspring were suppressed, showing significant perturbation of macrophages and neutrophils, expression of immune-related genes, and other immune functions. Moreover, these CTC-induced immune effects were either prevented or alleviated by the supplementation with PDTC, an antagonist of nuclear factor-κB (NF-κB), uncovering a seminal role of NF-κB in CTC immunotoxicity. Our results provide the evidence in fish that CTC at environmentally relevant concentrations can be transmitted over multiple generations and weaken the immune defense of offspring, raising concerns on the population hazards and ecological risk of antibiotics in the natural environment.

Brief Oil Exposure Reduces Fitness in Wild Gulf of Mexico Mahi-Mahi (Coryphaena hippurus).

The Deepwater Horizon (DWH) disaster released 3.19 million barrels of crude oil into the Gulf of Mexico (GOM) in 2010, overlapping the habitat of pelagic fish populations. Using mahi-mahi (Coryphaena hippurus)─a highly migratory marine teleost present in the GOM during the spill─as a model species, laboratory experiments demonstrate injuries to physiology and behavior following oil exposure. However, more than a decade postspill, impacts on wild populations remain unknown. To address this gap, we exposed wild mahi-mahi to crude oil or control conditions onboard a research vessel, collected fin clip samples, and tagged them with electronic tags prior to release into the GOM. We demonstrate profound effects on survival and reproduction in the wild. In addition to significant changes in gene expression profiles and predation mortality, we documented altered acceleration and habitat use in the first 8 days oil-exposed individuals were at liberty as well as a cessation of apparent spawning activity for at least 37 days. These data reveal that even a brief and low-dose exposure to crude oil impairs fitness in wild mahi-mahi. These findings offer new perspectives on the lasting impacts of the DWH blowout and provide insight about the impacts of future deep-sea oil spills.

Effects of an environmentally relevant PFAS mixture on dopamine and steroid hormone levels in exposed mice.

In the present study, we investigated the dopaminergic and steroid hormone systems of A/J mice fed environmentally relevant concentrations of a perfluoroalkyl substance (PFAS) mixture over a period of 10 weeks. The PFAS mixture was chosen based on measured PFAS concentrations in earthworms at a Norwegian skiing area (Trondheim) and consisted of eight different PFAS. Dietary exposure to PFAS led to lower total brain dopamine (DA) concentrations in male mice, as compared to control. On the transcript level, brain tyrosine hydroxylase (th) of PFAS exposed males was reduced, compared to the control group. No significant differences were observed on the transcript levels of enzymes responsible for DA metabolism, namely - monoamine oxidase (maoa and maob) and catechol-O methyltransferase (comt). We detected increased transcript level for DA receptor 2 (dr2) in PFAS exposed females, while expression of DA receptor 1 (dr1), DA transporter (dat) and vesicular monoamine transporter (vmat) were not affected by PFAS exposure. Regarding the steroid hormones, plasma and muscle testosterone (T), 11-ketotestosterone (11-KT) and 17ß-estradiol (E2) levels, as well as transcripts for estrogen receptors (esr1 and esr2), gonadotropin releasing hormone (gnrh) and aromatase (cyp19) were unaltered by the PFAS treatment. These results indicate that exposure to PFAS doses, comparable to previous observation in earthworms at a Norwegian skiing area, may alter the dopaminergic system of mice with overt consequences for health, general physiology, cognitive behavior, reproduction and metabolism.

Pre-validation of choriogenin H transgenic medaka eleutheroembryos as a quantitative estrogenic activity test method.

The choriogenin H - EGFP transgenic medaka (Oryzias melastigma) has been used to test estrogenic substances and quantify estrogenic activity into 17ß-estradiol (E2) equivalency (EEQ). The method uses 8 eleutheroembryos in 2 ml solution per well and 3 wells per treatment in 24-well plates at 26 ± 1 °C for 24 ± 2 h, with subsequent measurements of induced GFP signal intensity. EEQ measurements are calculated using a E2 probit regression model with a coefficient of determination (R2) > 0.90. The selectivity was confirmed evaluating 27 known estrogenic and 5 known non-estrogenic compounds. Limit of quantitation (LOQ), recovery rate and bias were calculated to be 1 ng/ml EEQ, 104% and 4% respectively. Robustness analysis revealed exposure temperature is a sensitive parameter that should be kept at 26 ± 1 °C. The repeatability of intra- and inter-laboratories achieved CV < 30% for most tested food and cosmetics samples. The lot-lot stability was confirmed by the stable EEQ qualitative control (QC, 1 ng/mL E2) and calibration curve results. The stability of standard reagents, samples and sample extracts was also investigated. These data demonstrated this method to be an accurate indicator of estrogenic activity for both chemicals and extracts.

miR133b Microinjection during Early Development Targets Transcripts of Cardiomyocyte Ion Channels and Induces Oil-like Cardiotoxicity in Zebrafish (Danio rerio) Embryos.

Previous studies have shown that altered expression of a family of small noncoding RNAs (microRNAs, or miRs) regulates the expression of downstream mRNAs and is associated with diseases and developmental disorders. miR133b is highly expressed in mammalian cardiac and skeletal muscle, and aberrant expression is associated with cardiac disorders and electrophysiological changes in cardiomyocytes. Similarly, cardiac dysfunction has been observed in early life-stage mahi-mahi (Coryphaena hippurus) exposed to crude oil, a phenotype that has been associated with an upregulation of miR133b as well as subsequent downregulation of a delayed rectifier potassium channel (IKr) and calcium signaling genes that are important for proper heart development during embryogenesis. To examine the potential role of miR133b in oil-induced early life-stage cardiotoxicity in fish, cleavage-stage zebrafish (Danio rerio) embryos were either (1) microinjected with ∼3 nL of negative control miR (75 µM) or miR133b (75 µM) or (2) exposed to a treatment solution containing 5 µM benzo(a)pyrene (BaP), a model polycyclic aromatic hydrocarbon, as a positive control. At 72 h post fertilization (hpf), miR133b-injected fish exhibited BaP-like cardiovascular malformations, including a significantly increased pericardial area relative to negative control miR-injected embryos, as well as a significantly reduced eye area. qPCR revealed that miR133b microinjection decreased the abundance of cardiac-specific IKr kcnh6 at 5 hpf, which may contribute to action potential elongation in oil-exposed cardiomyocytes. Additionally, ryanodine receptor 2, a crucial calcium receptor in the sarcoplasmic reticulum, was also downregulated by miR133b. These results indicate that an oil-induced increase in miR133b may contribute to cardiac abnormalities in oil-exposed fish by targeting cardiac-specific genes essential for proper heart development.

Dietary Seleno-l-methionine Alters the Microbial Communities and Causes Damage in the Gastrointestinal Tract of Japanese Medaka Oryzias latipes.

Excess dietary seleno-l-methionine (Se-Met) induces various adverse effects in fish inhabiting the Se-contaminated environments. However, there is an extreme paucity of data on the effects of excess dietary Se-Met on the microbiota in the gastrointestinal (GI) tract in fish. In this study, Japanese medaka Oryzias latipes (three months old) were fed the Se-Met enriched diets at environmentally relevant concentrations: 2.90 (Control: (C), 6.69 (L), 11.89 (M), and 27.05 (H) µg Se/g dw) for 60 d. Histopathological, high throughput sequencing, and biochemical approaches were used to investigate the alterations in histology and microbial communities of the GI tract, enzymatic activity, and transcripts of closely related genes. The results showed that the fish weight was reduced at ∼13% from the L and H treatments. Decreased height and thickness of villus in the GI tract were observed in the H treatment. Meanwhile, the level of D-lactate and activity of diamine oxidase (DAO), protease, and lipase were inhibited in the H treatment. The transcripts of the genes related to the inflammation (i.e., IL-1ß and IL-8) were elevated, while those of the genes related to the intestinal barrier (i.e., cdh1, ZO-1, ocln, and cldn7) were inhibited in the H treatment. In addition, alpha diversity at the genus level was higher in the L treatment than the control, and the composition of the microbial community was altered by dietary Se-Met. Furthermore, 5 genera (Rhodobacter, Cloacibacterium, Bdellovibrio, Shinella, and Aeromonas) exhibited the largest variation in abundance among treatments. This study has demonstrated that excess dietary Se-Met inhibits growth, causes hispathological damage to the GI tract, and alters the composition of the microbial community in Oryzias latipes.