Comparative analysis between zebrafish and an automated live-cell assay to classify developmental neurotoxicant chemicals.

Modern toxicology's throughput has dramatically increased due to alternative models, laboratory automation, and machine learning. This has enabled comparative studies across species and assays to prioritize chemical hazard potential and to understand how different model systems might complement one another. However, such comparative studies of high-throughput data are still in their infancy, with more groundwork needed to firmly establish the approach. Therefore, this study aimed to compare the bioactivity of the NIEHS Division of Translational Toxicology's (DTT) 87-compound developmental neurotoxicant (DNT) library in zebrafish and an in vitro high-throughput cell culture system. The early life-stage zebrafish provided a whole animal approach to developmental toxicity assessment. Chemical hits for abnormalities in embryonic zebrafish morphology, mortality, and behavior (ZBEscreen™) were compared with chemicals classified as high-risk by the Cell Health Index (CHI™), which is an outcome class probability from a machine learning classifier using 12 parameters from the SYSTEMETRIC® Cell Health Screen (CHS). The CHS was developed to assess human toxicity risk using supervised machine learning to classify acute cell stress phenotypes in a human leukemia cell line (HL60 cells) following a 4-h exposure to a chemical of interest. Due to the design of the screen, the zebrafish assays were more exhaustive, yielding 86 total bioactive hits, whereas the SYSTEMETRIC® CHS focusing on acute toxicity identified 20 chemicals as potentially toxic. The zebrafish embryonic and larval photomotor response assays (EPR and LPR, respectively) detected 40 of the 47 chemicals not found by the zebrafish morphological screen and CHS. Collectively, these results illustrate the advantages of using two alternative models in tandem for rapid hazard assessment and chemical prioritization and the effectiveness of CHI™ in identifying toxicity within a single multiparametric assay.

Leveraging high-throughput screening data, deep neural networks, and conditional generative adversarial networks to advance predictive toxicology.

There are currently 85,000 chemicals registered with the Environmental Protection Agency (EPA) under the Toxic Substances Control Act, but only a small fraction have measured toxicological data. To address this gap, high-throughput screening (HTS) and computational methods are vital. As part of one such HTS effort, embryonic zebrafish were used to examine a suite of morphological and mortality endpoints at six concentrations from over 1,000 unique chemicals found in the ToxCast library (phase 1 and 2). We hypothesized that by using a conditional generative adversarial network (cGAN) or deep neural networks (DNN), and leveraging this large set of toxicity data we could efficiently predict toxic outcomes of untested chemicals. Utilizing a novel method in this space, we converted the 3D structural information into a weighted set of points while retaining all information about the structure. In vivo toxicity and chemical data were used to train two neural network generators. The first was a DNN (Go-ZT) while the second utilized cGAN architecture (GAN-ZT) to train generators to produce toxicity data. Our results showed that Go-ZT significantly outperformed the cGAN, support vector machine, random forest and multilayer perceptron models in cross-validation, and when tested against an external test dataset. By combining both Go-ZT and GAN-ZT, our consensus model improved the SE, SP, PPV, and Kappa, to 71.4%, 95.9%, 71.4% and 0.673, respectively, resulting in an area under the receiver operating characteristic (AUROC) of 0.837. Considering their potential use as prescreening tools, these models could provide in vivo toxicity predictions and insight into the hundreds of thousands of untested chemicals to prioritize compounds for HT testing.

Behavior Effects of Structurally Diverse Per- and Polyfluoroalkyl Substances in Zebrafish.

Per- and polyfluoroalkyl substances (PFAS) are ubiquitously detected in the environment, and some pose significant human and environmental health concerns globally. While some PFAS induce adverse health effects, relatively few toxicological studies adequately address the broad structural diversity of this chemical class. In the current study, we evaluated 58 individual PFAS spanning 14 structural subclasses and 2 mixtures at single concentrations for developmental toxicity in zebrafish using highly sensitive behavior endpoints. Following developmental exposure to PFAS, zebrafish were assessed for mortality and challenged with an embryonic photomotor response (EPR) assay at 24 h postfertilization (hpf) and with larval photomotor response (LPR) and larval startle response assays at 120 hpf. We found that none of the tested PFAS exposures elicited significant mortality or aberrant EPR; however, exposure to 21 individual PFAS from multiple structural subclasses and 1 mixture induced aberrant larval behavior. We then evaluated developmental toxicity across a concentration range of 0-100 µM for 10 perfluoroalkyl carboxylic acids (PFCAs; 4-carbon perfluorobutanoic acid through the 13-carbon perfluorotridecanoic acid). Exposure to the PFCAs did not cause significant mortality or morphological effects, with the exception of perfluorooctanoic acid and perfluorononanoic acid, and did not induce aberrant EPR. All PFCAs, except for longer-chain perfluorododecanoic acid caused abnormal LPR following exposure to at least one concentration. In this study, we evaluated a broad set of PFAS not previously assessed for in vivo sublethal behavior endpoints and confirmed previous findings that exposure to some PFAS induces abnormal behavior in developing zebrafish. The data from this study will guide the selection of PFAS for which to investigate modes of toxic action.

Zinc oxide-induced changes to sunscreen ingredient efficacy and toxicity under UV irradiation.

Sunscreen safety and efficacy is generally evaluated based upon the properties of the individual chemicals in a formulation. However, the photostability of sunscreens has been shown to be highly dependent on the mixture of chemicals present. To better understand how sunscreen formulation influences stability, and to establish a foundation for probing the influence of zinc oxide additives, we formulated five different small-molecule based ultraviolet-filter (UV-filter) mixtures with a Sun Protection Factor (SPF) of 15. These mixtures contained active ingredients approved in either the United States or European Union and were designed to represent formulations of actual products on the market. We evaluated the photostability and toxicity of these mixtures in the absence and presence of zinc oxide after UV exposure for two hours. Changes in UV absorbance were minimal for all five small-molecule-based mixtures without zinc oxide. The presence of either micro- or nano-sized zinc oxide caused significant small-molecule photodegradation and the degraded mixtures exhibited higher levels of toxicity in embryonic zebrafish assays. This study suggests that caution must be taken when formulating sunscreens containing both zinc oxide and small-molecule UV-filters to avoid unintended consequences during use.

The Incidence and Etiology of Previously Undiagnosed Hypoglycemic Disorders in the Emergency Department.

OBJECTIVE: The aim of this study was to determine the incidence and etiology of previously undiagnosed hypoglycemia in children (<18 years of age) seen in a hospital emergency department (ED). METHODS: A retrospective review of all emergency room visits over a 2-year period was conducted to identify patients younger than 18 years who had hypoglycemia (<50 mg/dL) not associated with a previously known cause. Evaluation of hypoglycemia was conducted during a spontaneous hypoglycemic event or during hypoglycemia induced by a fasting study. Insulin and counter-regulatory hormones were measured simultaneously when the blood glucose was less than 50 mg/dL. RESULTS: Of 224,125 children seen in the ED during the study, 160 (1:1400) were documented to have hypoglycemia not caused by a previously known condition. Eighty-five (53%) of the 160 hypoglycemic subjects underwent a diagnostic evaluation. Seventeen (20%) of the 85 were classified as having a high-risk disorder causing hypoglycemia, whereas 63 (74%) had a low-risk disorder. Seventy-five patients (47%) did not undergo a diagnostic evaluation during the ED visit or hospital admission. CONCLUSIONS: Hypoglycemia of unknown etiology occurs in 1:1400 (0.07%) children who attended the ED during the study. Assuming that none of the children who failed to undergo a diagnostic evaluation had a high-risk disorder, 10.6% of the subjects with hypoglycemia were found to have a high-risk disorder. Because of the increased incidence of high-risk disorders causing hypoglycemia and the long-term health risk associated with hypoglycemia, we recommend that all children with hypoglycemia of unknown etiology have a critical blood sample drawn at the time of hypoglycemia (blood glucose <50 mg/dL) or be admitted for a diagnostic evaluation.

Congenital hyperinsulinism as the presenting feature of Kabuki syndrome: clinical and molecular characterization of 9 affected individuals.

PURPOSE: Describe the clinical and molecular findings of patients with Kabuki syndrome (KS) who present with hypoglycemia due to congenital hyperinsulinism (HI), and assess the incidence of KS in patients with HI. METHODS: We documented the clinical features and molecular diagnoses of 9 infants with persistent HI and KS via a combination of sequencing and copy-number profiling methodologies. Subsequently, we retrospectively evaluated 100 infants with HI lacking a genetic diagnosis, for causative variants in KS genes. RESULTS: Molecular diagnoses of KS were established by identification of pathogenic variants in KMT2D (n = 5) and KDM6A (n = 4). Among the 100 infants with HI of unknown genetic etiology, a KS diagnosis was uncovered in one patient. CONCLUSIONS: The incidence of HI among patients with KS may be higher than previously reported, and KS may account for as much as 1% of patients diagnosed with HI. As the recognition of dysmorphic features associated with KS is challenging in the neonatal period, we propose KS should be considered in the differential diagnosis of HI. Since HI in patients with KS is well managed medically, a timely recognition of hyperinsulinemic episodes will improve outcomes, and prevent aggravation of the preexisting mild to moderate intellectual disability in KS.

Correction: "Congenital hyperinsulinism as the presenting feature of Kabuki syndrome: clinical and molecular characterization of 10 affected individuals".

The author Diva D. De Leon was incorrectly listed as instead of Diva D. De Leó-Critchlow in the original version of this paper.

Treatment with Nitrate, but Not Nitrite, Lowers the Oxygen Cost of Exercise and Decreases Glycolytic Intermediates While Increasing Fatty Acid Metabolites in Exercised Zebrafish.

BACKGROUND: Dietary nitrate improves exercise performance by reducing the oxygen cost of exercise, although the mechanisms responsible are not fully understood. OBJECTIVES: We tested the hypothesis that nitrate and nitrite treatment would lower the oxygen cost of exercise by improving mitochondrial function and stimulating changes in the availability of metabolic fuels for energy production. METHODS: We treated 9-mo-old zebrafish with nitrate (sodium nitrate, 606.9 mg/L), nitrite (sodium nitrite, 19.5 mg/L), or control (no treatment) water for 21 d. We measured oxygen consumption during a 2-h, strenuous exercise test; assessed the respiration of skeletal muscle mitochondria; and performed untargeted metabolomics on treated fish, with and without exercise. RESULTS: Nitrate and nitrite treatment increased blood nitrate and nitrite levels. Nitrate treatment significantly lowered the oxygen cost of exercise, as compared with pretreatment values. In contrast, nitrite treatment significantly increased oxygen consumption with exercise. Nitrate and nitrite treatments did not change mitochondrial function measured ex vivo, but significantly increased the abundances of ATP, ADP, lactate, glycolytic intermediates (e.g., fructose 1,6-bisphosphate), tricarboxylic acid (TCA) cycle intermediates (e.g., succinate), and ketone bodies (e.g., ß-hydroxybutyrate) by 1.8- to 3.8-fold, relative to controls. Exercise significantly depleted glycolytic and TCA intermediates in nitrate- and nitrite-treated fish, as compared with their rested counterparts, while exercise did not change, or increased, these metabolites in control fish. There was a significant net depletion of fatty acids, acyl carnitines, and ketone bodies in exercised, nitrite-treated fish (2- to 4-fold), while exercise increased net fatty acids and acyl carnitines in nitrate-treated fish (1.5- to 12-fold), relative to their treated and rested counterparts. CONCLUSIONS: Nitrate and nitrite treatment increased the availability of metabolic fuels (ATP, glycolytic and TCA intermediates, lactate, and ketone bodies) in rested zebrafish. Nitrate treatment may improve exercise performance, in part, by stimulating the preferential use of fuels that require less oxygen for energy production.

Formation of PAH Derivatives and Increased Developmental Toxicity during Steam Enhanced Extraction Remediation of Creosote Contaminated Superfund Soil.

Steam enhanced extraction (SEE) is an in situ thermal remediation technique used to remove and recover polycyclic aromatic hydrocarbons (PAHs) from contaminated soils. However, limited studies have been conducted on the formation of PAH derivatives during and after SEE of PAH contaminated soils. Creosote contaminated soil samples collected from the Wyckoff-Eagle Harbor Superfund site were remediated with laboratory scale SEE. The samples were quantified for unsubstituted PAHs and their derivatives and assessed for developmental toxicity, pre- and post-SEE. Following SEE, unsubstituted PAH concentrations decreased, while oxygenated PAH concentrations increased in soil and aqueous extracts. Differences in developmental toxicity were also measured and linked to the formation of PAH derivatives. Additive toxicity was measured when comparing unfractionated extracts to fractionated extracts in pre- and post-SEE samples. SEE is effective in removing unsubstituted PAHs from contaminated soil, but other, potentially more toxic, PAH derivatives are formed.

Coupling Genome-wide Transcriptomics and Developmental Toxicity Profiles in Zebrafish to Characterize Polycyclic Aromatic Hydrocarbon (PAH) Hazard.

Polycyclic Aromatic Hydrocarbons (PAHs) are diverse environmental pollutants associated with adverse human health effects. Many studies focus on the carcinogenic effects of a limited number of PAHs and there is an increasing need to understand mechanisms of developmental toxicity of more varied yet environmentally relevant PAHs. A previous study characterized the developmental toxicity of 123 PAHs in zebrafish. Based on phenotypic responses ranging from complete inactivity to acute mortality, we classified these PAHs into eight bins, selected 16 representative PAHs, and exposed developing zebrafish to the concentration of each PAH that induced 80% phenotypic effect. We conducted RNA sequencing at 48 h post fertilization to identify gene expression changes as a result of PAH exposure. Using the Context Likelihood of Relatedness algorithm, we inferred a network that links the PAHs based on coordinated gene responses to PAH exposure. The 16 PAHs formed two broad clusters: Cluster A was transcriptionally more similar to the controls, while Cluster B consisted of PAHs that were generally more developmentally toxic, significantly elevated cyp1a transcript levels, and induced Ahr2-dependent Cyp1a protein expression in the skin confirmed by gene-silencing studies. We found that cyp1a transcript levels were associated with transcriptomic response, but not with PAH developmental toxicity. While all cluster B PAHs predominantly activated Ahr2, they also each enriched unique pathways like ion transport signaling, which likely points to differing molecular events between the PAHs downstream of Ahr2. Thus, using a systems biology approach, we have begun to evaluate, classify, and define mechanisms of PAH toxicity.

Population genetic diversity in zebrafish lines.

Toxicological and pharmacological researchers have seized upon the many benefits of zebrafish, including the short generation time, well-characterized development, and early maturation as clear embryos. A major difference from many model organisms is that standard husbandry practices in zebrafish are designed to maintain population diversity. While this diversity is attractive for translational applications in human and ecological health, it raises critical questions on how interindividual genetic variation might contribute to chemical exposure or disease susceptibility differences. Findings from pooled samples of zebrafish support this supposition of diversity yet cannot directly measure allele frequencies for reference versus alternate alleles. Using the Tanguay lab Tropical 5D zebrafish line (T5D), we performed whole genome sequencing on a large group (n = 276) of individual zebrafish embryos. Paired-end reads were collected on an Illumina 3000HT, then aligned to the most recent zebrafish reference genome (GRCz10). These data were used to compare observed population genetic variation across species (humans, mice, zebrafish), then across lines within zebrafish. We found more single nucleotide polymorphisms (SNPs) in T5D than have been reported in SNP databases for any of the WIK, TU, TL, or AB lines. We theorize that some subset of the novel SNPs may be shared with other zebrafish lines but have not been identified in other studies due to the limitations of capturing population diversity in pooled sequencing strategies. We establish T5D as a model that is representative of diversity levels within laboratory zebrafish lines and demonstrate that experimental design and analysis can exert major effects when characterizing genetic diversity in heterogeneous populations.

Systematic developmental neurotoxicity assessment of a representative PAH Superfund mixture using zebrafish.

Superfund sites often consist of complex mixtures of polycyclic aromatic hydrocarbons (PAHs). It is widely recognized that PAHs pose risks to human and environmental health, but the risks posed by exposure to PAH mixtures are unclear. We constructed an environmentally relevant PAH mixture with the top 10 most prevalent PAHs (SM10) from a Superfund site derived from environmental passive sampling data. Using the zebrafish model, we measured body burden at 48 hours post fertilization (hpf) and evaluated the developmental and neurotoxicity of SM10 and the 10 individual constituents at 24 hours post fertilization (hpf) and 5 days post fertilization (dpf). Zebrafish embryos were exposed from 6 to 120 hpf to (1) the SM10 mixture, (2) a variety of individual PAHs: pyrene, fluoranthene, retene, benzo[a]anthracene, chrysene, naphthalene, acenaphthene, phenanthrene, fluorene, and 2-methylnaphthalene. We demonstrated that SM10 and only 3 of the individual PAHs were developmentally toxic. Subsequently, we constructed and exposed developing zebrafish to two sub-mixtures: SM3 (comprised of 3 of the developmentally toxicity PAHs) and SM7 (7 non-developmentally toxic PAHs). We found that the SM3 toxicity profile was similar to SM10, and SM7 unexpectedly elicited developmental toxicity unlike that seen with its individual components. The results demonstrated that the overall developmental toxicity in the mixtures could be explained using the general concentration addition model. To determine if exposures activated the AHR pathway, spatial expression of CYP1A was evaluated in the 10 individual PAHs and the 3 mixtures at 5 dpf. Results showed activation of AHR in the liver and vasculature for the mixtures and some individual PAHs. Embryos exposed to SM10 during development and raised in chemical-free water into adulthood exhibited decreased learning and responses to startle stimulus indicating that developmental SM10 exposures affect neurobehavior. Collectively, these results exemplify the utility of zebrafish to investigate the developmental and neurotoxicity of complex mixtures.

Comparative developmental toxicity of a comprehensive suite of polycyclic aromatic hydrocarbons.

Polycyclic aromatic hydrocarbons (PAHs) are widespread environmental contaminants that occur in complex mixtures. Several PAHs are known or suspected mutagens and/or carcinogens, but developmental toxicity data is lacking for PAHs, particularly their oxygenated and nitrated derivatives. Such data are necessary to understand and predict the toxicity of environmental mixtures. 123 PAHs were assessed for morphological and neurobehavioral effects for a range of concentrations between 0.1 and 50 µM, using a high throughput early-life stage zebrafish assay, including 33 parent, 22 nitrated, 17 oxygenated, 19 hydroxylated, 14 methylated, 16 heterocyclic, and 2 aminated PAHs. Additionally, each PAH was evaluated for AHR activation, by assessing CYP1A protein expression using whole animal immunohistochemistry (IHC). Responses to PAHs varied in a structurally dependent manner. High-molecular weight PAHs were significantly more developmentally toxic than the low-molecular weight PAHs, and CYP1A expression was detected in five distinct tissues, including vasculature, liver, skin, neuromasts and yolk.

Predicting in vivo effect levels for repeat-dose systemic toxicity using chemical, biological, kinetic and study covariates.

In an effort to address a major challenge in chemical safety assessment, alternative approaches for characterizing systemic effect levels, a predictive model was developed. Systemic effect levels were curated from ToxRefDB, HESS-DB and COSMOS-DB from numerous study types totaling 4379 in vivo studies for 1247 chemicals. Observed systemic effects in mammalian models are a complex function of chemical dynamics, kinetics, and inter- and intra-individual variability. To address this complex problem, systemic effect levels were modeled at the study-level by leveraging study covariates (e.g., study type, strain, administration route) in addition to multiple descriptor sets, including chemical (ToxPrint, PaDEL, and Physchem), biological (ToxCast), and kinetic descriptors. Using random forest modeling with cross-validation and external validation procedures, study-level covariates alone accounted for approximately 15% of the variance reducing the root mean squared error (RMSE) from 0.96 log10 to 0.85 log10 mg/kg/day, providing a baseline performance metric (lower expectation of model performance). A consensus model developed using a combination of study-level covariates, chemical, biological, and kinetic descriptors explained a total of 43% of the variance with an RMSE of 0.69 log10 mg/kg/day. A benchmark model (upper expectation of model performance) was also developed with an RMSE of 0.5 log10 mg/kg/day by incorporating study-level covariates and the mean effect level per chemical. To achieve a representative chemical-level prediction, the minimum study-level predicted and observed effect level per chemical were compared reducing the RMSE from 1.0 to 0.73 log10 mg/kg/day, equivalent to 87% of predictions falling within an order-of-magnitude of the observed value. Although biological descriptors did not improve model performance, the final model was enriched for biological descriptors that indicated xenobiotic metabolism gene expression, oxidative stress, and cytotoxicity, demonstrating the importance of accounting for kinetics and non-specific bioactivity in predicting systemic effect levels. Herein, we generated an externally predictive model of systemic effect levels for use as a safety assessment tool and have generated forward predictions for over 30,000 chemicals.

A data-driven weighting scheme for multivariate phenotypic endpoints recapitulates zebrafish developmental cascades.

Zebrafish have become a key alternative model for studying health effects of environmental stressors, partly due to their genetic similarity to humans, fast generation time, and the efficiency of generating high-dimensional systematic data. Studies aiming to characterize adverse health effects in zebrafish typically include several phenotypic measurements (endpoints). While there is a solid biomedical basis for capturing a comprehensive set of endpoints, making summary judgments regarding health effects requires thoughtful integration across endpoints. Here, we introduce a Bayesian method to quantify the informativeness of 17 distinct zebrafish endpoints as a data-driven weighting scheme for a multi-endpoint summary measure, called weighted Aggregate Entropy (wAggE). We implement wAggE using high-throughput screening (HTS) data from zebrafish exposed to five concentrations of all 1060 ToxCast chemicals. Our results show that our empirical weighting scheme provides better performance in terms of the Receiver Operating Characteristic (ROC) curve for identifying significant morphological effects and improves robustness over traditional curve-fitting approaches. From a biological perspective, our results suggest that developmental cascade effects triggered by chemical exposure can be recapitulated by analyzing the relationships among endpoints. Thus, wAggE offers a powerful approach for analysis of multivariate phenotypes that can reveal underlying etiological processes.

Transgenerational inheritance of neurobehavioral and physiological deficits from developmental exposure to benzo[a]pyrene in zebrafish.

Benzo[a]pyrene (B[a]P) is a well-known genotoxic polycylic aromatic compound whose toxicity is dependent on signaling via the aryl hydrocarbon receptor (AHR). It is unclear to what extent detrimental effects of B[a]P exposures might impact future generations and whether transgenerational effects might be AHR-dependent. This study examined the effects of developmental B[a]P exposure on 3 generations of zebrafish. Zebrafish embryos were exposed from 6 to 120h post fertilization (hpf) to 5 and 10µM B[a]P and raised in chemical-free water until adulthood (F0). Two generations were raised from F0 fish to evaluate transgenerational inheritance. Morphological, physiological and neurobehavioral parameters were measured at two life stages. Juveniles of the F0 and F2 exhibited hyper locomotor activity, decreased heartbeat and mitochondrial function. B[a]P exposure during development resulted in decreased global DNA methylation levels and generally reduced expression of DNA methyltransferases in wild type zebrafish, with the latter effect largely reversed in an AHR2-null background. Adults from the F0 B[a]P exposed lineage displayed social anxiety-like behavior. Adults in the F2 transgeneration manifested gender-specific increased body mass index (BMI), increased oxygen consumption and hyper-avoidance behavior. Exposure to benzo[a]pyrene during development resulted in transgenerational inheritance of neurobehavioral and physiological deficiencies. Indirect evidence suggested the potential for an AHR2-dependent epigenetic route.

From smooth to rough, from water to air: the intertidal habitat of Northern clingfish (Gobiesox maeandricus).

The Northern clingfish is a small, Eastern North Pacific fish that can attach to rough, fouled rocks in the intertidal. Their ability to attach to surfaces has been measured previously in the laboratory, and in this study, we show the roughness and fouling of the natural habitat of these fish. We introduce a new method for measuring surface roughness of natural substrates with time-limited accessibility. We expect this method to be broadly applicable in studies of animal/substrate surface interactions in habitats difficult to characterize. Our roughness measurements demonstrate that the fish's ability to attach to very coarse roughness is required in its natural environment. Some of the rocks showed even coarser roughness than the fish could attach to in the lab setting. We also characterized the clingfish's preference for other habitat descriptors such as the size of the rocks, biofilm, and Aufwuchs (macroalgae, encrusting invertebrates) cover, as well as grain size of underlying substrate. Northern clingfish seek shelter under rocks of 15-45 cm in size. These rocks have variable Aufwuchs cover, and gravel is the main underlying substrate type. In the intertidal, environmental conditions change with the tides, and for clingfish, the daily time under water (DTUW%) was a key parameter explaining distribution. Rather than location being determined by intertidal zonation, an 80% DTUW, a finer scale concept of tidal inundation, was required by the fish. We expect that this is likely because the mobility of the fish allows them to more closely track the ideal inundation in the marine intertidal.

Formation of Developmentally Toxic Phenanthrene Metabolite Mixtures by Mycobacterium sp. ELW1.

Mycobacterium sp. ELW1 co-metabolically degraded up to 1.8 µmol of phenanthrene (PHE) in ∼48 h, and hydroxyphenanthrene (OHPHE) metabolites, including 1-hydroxyphenanthrene (1-OHPHE), 3-hydroxyphenanthrene (3-OHPHE), 4-hydroxyphenanthrene (4-OHPHE), 9-hydroxyphenanthrene (9-OHPHE), 9,10-dihydroxyphenanthrene (1,9-OHPHE), and trans-9,10-dihydroxy-9,10-dihydrophenanthrene (trans-9,10-OHPHE), were identified and quantified over time. The monooxygenase responsible for co-metabolic transformation of PHE was inhibited by 1-octyne. First-order PHE transformation rates, kPHE, and half-lives, t1/2, for PHE-exposed cells were 0.16-0.51 h-1 and 1.4-4.3 h, respectively, and the 1-octyne controls ranged from 0.015-0.10 h-1 to 7.0-47 h, respectively. While single compound standards of PHE and trans-9,10-OHPHE, the major OHPHE metabolite formed by ELW1, were not toxic to embryonic zebrafish (Danio rerio), single compound standards of minor OHPHE metabolites, 1-OHPHE, 3-OHPHE, 4-OHPHE, 9-OHPHE, and 1,9-OHPHE, were toxic, with effective concentrations (EC50's) ranging from 0.5 to 5.5 µM. The metabolite mixtures formed by ELW1, and the reconstructed standard mixtures of the identified OHPHE metabolites, elicited a toxic response in zebrafish for the same three time points. EC50s for the metabolite mixtures formed by ELW1 were lower (more toxic) than those for the reconstructed standard mixtures of the identified OHPHE metabolites. Ten unidentified hydroxy PHE metabolites were measured in the derivatized mixtures formed by ELW1 and may explain the increased toxicity of the ELW1 metabolites mixture relative to the reconstructed standard mixtures of the identified OHPHE metabolites.

Zebrafish embryo toxicity of 15 chlorinated, brominated, and iodinated disinfection by-products.

Disinfection to protect human health occurs at drinking water and wastewater facilities through application of non-selective oxidants including chlorine. Oxidants also transform organic material and form disinfection by-products (DBPs), many of which are halogenated and cyto- and genotoxic. Only a handful of assays have been used to compare DBP toxicity, and researchers are unsure which DBP(s) drive the increased cancer risk associated with drinking chlorinated water. The most extensive data set employs an in vitro model cell, Chinese hamster ovary cells. Traditionally, most DBP research focuses on the threat to human health, but the effects on aquatic species exposed to DBPs in wastewater effluents remain ill defined. We present the developmental toxicity for 15 DBPs and a chlorinated wastewater to a model aquatic vertebrate, zebrafish. Mono-halogenated DBPs followed the in vivo toxicity rank order: acetamides>acetic acids>acetonitriles~nitrosamines, which agrees well with previously published mammalian in vitro data. Di- and tri-halogenated acetonitriles were more toxic than their mono-halogenated analogues, and bromine- and iodine-substituted DBPs tended to be more toxic than chlorinated analogues. No zebrafish development effects were observed after exposure to undiluted or non-concentrated, chlorinated wastewater. We find zebrafish development to be a viable in vivo alternative or confirmatory assay to mammalian in vitro cell assays.

High-throughput characterization of chemical-associated embryonic behavioral changes predicts teratogenic outcomes.

New strategies are needed to address the data gap between the bioactivity of chemicals in the environment versus existing hazard information. We address whether a high-throughput screening (HTS) system using a vertebrate organism (embryonic zebrafish) can characterize chemical-elicited behavioral responses at an early, 24 hours post-fertilization (hpf) stage that predict teratogenic consequences at a later developmental stage. The system was used to generate full concentration-response behavioral profiles at 24 hpf across 1060 ToxCast™ chemicals. Detailed, morphological evaluation of all individuals was performed as experimental follow-up at 5 days post-fertilization (dpf). Chemicals eliciting behavioral responses were also mapped against external HTS in vitro results to identify specific molecular targets and neurosignalling pathways. We found that, as an integrative measure of normal development, significant alterations in movement highlighted active chemicals representing several modes of action. These early behavioral responses were predictive for 17 specific developmental abnormalities and mortality measured at 5 dpf, often at lower (i.e., more potent) concentrations than those at which morphological effects were observed. Therefore, this system can provide rapid characterization of chemical-elicited behavioral responses at an early developmental stage that are predictive of observable adverse effects later in life.