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.

Gene co-expression network analysis in zebrafish reveals chemical class specific modules.

BACKGROUND: Zebrafish is a popular animal model used for high-throughput screening of chemical hazards, however, investigations of transcriptomic mechanisms of toxicity are still needed. Here, our goal was to identify genes and biological pathways that Aryl Hydrocarbon Receptor 2 (AHR2) Activators and flame retardant chemicals (FRCs) alter in developing zebrafish. Taking advantage of a compendium of phenotypically-anchored RNA sequencing data collected from 48-h post fertilization (hpf) zebrafish, we inferred a co-expression network that grouped genes based on their transcriptional response. RESULTS: Genes responding to the FRCs and AHR2 Activators localized to distinct regions of the network, with FRCs inducing a broader response related to neurobehavior. AHR2 Activators centered in one region related to chemical stress responses. We also discovered several highly co-expressed genes in this module, including cyp1a, and we subsequently show that these genes are definitively within the AHR2 signaling pathway. Systematic removal of the two chemical types from the data, and analysis of network changes identified neurogenesis associated with FRCs, and regulation of vascular development associated with both chemical classes. We also identified highly connected genes responding specifically to each class that are potential biomarkers of exposure. CONCLUSIONS: Overall, we created the first zebrafish chemical-specific gene co-expression network illuminating how chemicals alter the transcriptome relative to each other. In addition to our conclusions regarding FRCs and AHR2 Activators, our network can be leveraged by other studies investigating chemical mechanisms of toxicity.

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.

Developmental co-exposure of TBBPA and titanium dioxide nanoparticle induced behavioral deficits in larval zebrafish.

Both tetrabromobisphenol A (TBBPA) and titanium dioxide nanoparticle (TiO2 NP) have widespread commercial applications, resulting in their ubiquitous co-presence in the environment and biota. Although environmental chemicals exist as mixtures, toxicity studies are nearly always conducted with single chemicals. Few studies explore potential interactions of different chemical mixtures. In this study, we employ the sensitive developing nerve system in zebrafish to assess the neurotoxicity of TBBPA/TiO2 NP mixtures. Specifically, zebrafish embryos were exposed to solvent control (0.1% DMSO), 2 µM TBBPA, 0.1 mg/L TiO2 NP, and their mixture from 8 to 120 h post fertilization (hpf), and motor/social behavioral assessments were conducted on embryos/larvae at different developmental stages. Our results showed that TBBPA/TiO2 NP single or co-exposures increased spontaneous movement, decreased touch response and swim speed, and affected social behaviors of light/dark preference, shoaling, mirror attack and social contact. In particular, many of these phenotypes were manifested with higher magnitude of changes from the mixture exposure. These behavioral deficits were also accompanied with increased cell death in olfactory region and neuromasts in the lateral line system, increased ROS in gallbladder, pancreas, liver, and intestine, as well as increased lipid peroxidation and decreased ATP levels in whole larval tissue homogenates. Further, genes coding for key cell apoptosis marker and antioxidant enzyme were significantly upregulated by these two chemicals, in particular to their mixture. Interestingly, the co-presence of TBBPA also increased the mean particle size of TiO2 NP in the exposure solutions and the TiO2 NP content in larval tissue. Together, our analysis suggests that TBBPA/TiO2 NP induced behavioral changes may be due to physical accumulation of these two chemicals in the target organs, and TiO2 NP may serve as carriers for increased accumulation of TBBPA. To conclude, we demonstrated that TBBPA/TiO2 NP together cause increased bioaccumulation of TiO2, and heightened responses in behavior, cell apoptosis and oxidative stress. Our findings also highlight the importance of toxicity assessment using chemical mixtures.

Workflow for Comparison of Chemical and Biological Metrics of Filter Collected PM2.5.

There is limited understanding of adverse health effect associations with chemical constituents of fine particulate matter (PM2.5) as well as the underlying mechanisms. We outlined a workflow to assess metrics, beyond concentration, using household and personal PM2.5 filter samples collected in India as a proof of concept for future large-scale studies. Oxidative potential, chemical composition (polycyclic aromatic hydrocarbons and elements), and bioactivity (developmental exposures in zebrafish) were determined. Significant differences were observed in all metrics between personal and household PM2.5 samples. This work established methods to characterize multiple metrics of PM2.5 to ultimately support the identification of more health-relevant metrics than concentration.

The addition of mammalian cell culture medium impacts nanoparticle toxicity in zebrafish.

Engineered nanomaterials (ENMs) are ubiquitous in contemporary applications, yet their environmental and human health impacts remain inadequately understood. This study addresses the challenge of identifying potential risks associated with ENM exposure by highlighting the significant variability in existing research methodologies. Without a systematic collection of toxicological data that encompasses standardized materials, relevant platforms, and assays, the task of identifying potential risks linked to ENM exposure becomes an intricate challenge. In vitro assessments often use media rich in ionic species, such as RPMI and fetal bovine serum (FBS). Zebrafish embryos, known to develop normally in low-ionic environments, were exposed to Cerium Oxide, Zinc Oxide, and Graphene Oxides in different media at varying concentrations. Here, we discovered that zebrafish embryos tolerated a mix of 80 % RPMI, 2 % FBS, and 1 % antibiotic cocktail. The results revealed that adverse effects observed in zebrafish with certain nanomaterials in Ultra-Pure (UP) water were mitigated in cell culture medium, emphasizing the importance of revisiting previously considered non-toxic materials in vitro. The zebrafish results underscore the importance of utilizing a multidimensional in vivo platform to gauge the biological activity of nanomaterials accurately.

The zebrafish gut microbiome influences benzo[a]pyrene developmental neurobehavioral toxicity.

Early-life exposure to environmental toxicants like Benzo[a]pyrene (BaP) is associated with several health consequences in vertebrates (i.e., impaired or altered neurophysiological and behavioral development). Although toxicant impacts were initially studied relative to host physiology, recent studies suggest that the gut microbiome is a possible target and/or mediator of behavioral responses to chemical exposure in organisms, via the gut-brain axis. However, the connection between BaP exposure, gut microbiota, and developmental neurotoxicity remains understudied. Using a zebrafish model, we determined whether the gut microbiome influences BaP impacts on behavior development. Embryonic zebrafish were treated with increasing concentrations of BaP and allowed to grow to the larval life stage, during which they underwent behavioral testing and intestinal dissection for gut microbiome profiling via high-throughput sequencing. We found that exposure affected larval zebrafish microbiome diversity and composition in a manner tied to behavioral development: increasing concentrations of BaP were associated with increased taxonomic diversity, exposure was associated with unweighted UniFrac distance, and microbiome diversity and exposure predicted larval behavior. Further, a gnotobiotic zebrafish experiment clarified whether microbiome presence was associated with BaP exposure response and behavioral changes. We found that gut microbiome state altered the relationship between BaP exposure concentration and behavioral response. These results support the idea that the zebrafish gut microbiome is a determinant of the developmental neurotoxicity that results from chemical exposure.

PAH bioremediation with Rhodococcus rhodochrous ATCC 21198: Impact of cell immobilization and surfactant use on PAH treatment and post-remediation toxicity.

Polycyclic aromatic hydrocarbons (PAHs) are prevalent environmental contaminants that are harmful to ecological and human health. Bioremediation is a promising technique for remediating PAHs in the environment, however bioremediation often results in the accumulation of toxic PAH metabolites. The objectives of this research were to demonstrate the cometabolic treatment of a mixture of PAHs by a pure bacterial culture, Rhodococcus rhodochrous ATCC 21198, and investigate PAH metabolites and toxicity. Additionally, the surfactant Tween ® 80 and cell immobilization techniques were used to enhance bioremediation. Total PAH removal ranged from 70-95% for fluorene, 44-89% for phenanthrene, 86-97% for anthracene, and 6.5-78% for pyrene. Maximum removal was achieved with immobilized cells in the presence of Tween ® 80. Investigation of PAH metabolites produced by 21198 revealed a complex mixture of hydroxylated compounds, quinones, and ring-fission products. Toxicity appeared to increase after bioremediation, manifesting as mortality and developmental effects in embryonic zebrafish. 21198's ability to rapidly transform PAHs of a variety of molecular structures and sizes suggests that 21198 can be a valuable microorganism for catalyzing PAH remediation. However, implementing further treatment processes to address toxic PAH metabolites should be pursued to help lower post-remediation toxicity in future studies.

Interlaboratory Study on Zebrafish in Toxicology: Systematic Evaluation of the Application of Zebrafish in Toxicology's (SEAZIT's) Evaluation of Developmental Toxicity.

Embryonic zebrafish represent a useful test system to screen substances for their ability to perturb development. The exposure scenarios, endpoints captured, and data analysis vary among the laboratories who conduct screening. A lack of harmonization impedes the comparison of the substance potency and toxicity outcomes across laboratories and may hinder the broader adoption of this model for regulatory use. The Systematic Evaluation of the Application of Zebrafish in Toxicology (SEAZIT) initiative was developed to investigate the sources of variability in toxicity testing. This initiative involved an interlaboratory study to determine whether experimental parameters altered the developmental toxicity of a set of 42 substances (3 tested in duplicate) in three diverse laboratories. An initial dose-range-finding study using in-house protocols was followed by a definitive study using four experimental conditions: chorion-on and chorion-off using both static and static renewal exposures. We observed reasonable agreement across the three laboratories as 33 of 42 test substances (78.6%) had the same activity call. However, the differences in potency seen using variable in-house protocols emphasizes the importance of harmonization of the exposure variables under evaluation in the second phase of this study. The outcome of the Def will facilitate future practical discussions on harmonization within the zebrafish research community.

Defining the environmental determinants of dysbiosis at scale with zebrafish.

The gut microbiome, critical to maintaining vertebrate homeostasis, is susceptible to a various exposures. In some cases, these exposures induce dysbiosis, wherein the microbiome changes into a state conducive to disease progression. To better prevent, manage, and treat health disorders, we need to define which exposures induce dysbiosis. Contemporary methods face challenges due to the immense diversity of the exposome and the restricted throughput of conventional experimental tools used for dysbiosis evaluation. We propose integrating high-throughput model systems as an augment to traditional techniques for rapid identification of dysbiosis-inducing agents. Although high-throughput screening tools revolutionized areas such as pharmacology and toxicology, their incorporation in gut microbiome research remains limited. One particularly powerful high-throughput model system is the zebrafish, which affords access to scalable in vivo experimentation involving a complex gut microbiome. Numerous studies have employed this model to identify potential dysbiosis triggers. However, its potential could be further harnessed via innovative study designs, such as evaluation of synergistic effects from combined exposures, expansions to the methodological toolkit to discern causal effects of microbiota, and efforts to assess and improve the translational relevance of the model. Ultimately, this burgeoning experimental resource can accelerate the discovery of agents that underlie dysbiotic disorders.

Advances in PAH mixture toxicology enabled by zebrafish.

Polycyclic aromatic hydrocarbons (PAHs) are a class of organic compounds produced by a variety of petrogenic and pyrogenic sources. PAHs inherently occur in the environment in complex mixtures. The early life-stage zebrafish model is a valuable tool for high-throughput screening (HTS) for toxicity of complex chemical mixtures due to its rapid development, high fecundity, and superb sensitivity to chemical insult. Zebrafish are amenable to exposure to surrogate mixtures as well as extracts of environmental samples and effect-directed analysis. In addition to its utility to HTS, the zebrafish has proven an excellent model for assessing chemical modes of action and identifying molecular initiating and other key events in an Adverse Outcome Pathway framework. Traditional methods of assessing PAH mixture toxicity prioritize carcinogenic potential and lack consideration of non-carcinogenic modes of action, assuming a similar molecular initiating event for all PAHs. Recent work in zebrafish has made it clear that while PAHs belong to the same chemical class, their modes of action can be divergent. Future research should use zebrafish to better classify PAHs by their bioactivity and modes of action to better understand mixture hazards.

Review of the zebrafish as a model to investigate per- and polyfluoroalkyl substance toxicity.

The existence of thousands of per- and polyfluoroalkyl substances (PFAS) and evidence that some cause adverse health effects has created immense need to better understand PFAS toxicity and to move beyond one-chemical-at-a-time approaches to hazard assessment for this chemical class. The zebrafish model enables rapid assessment of large libraries of PFAS, powerful comparison of compounds in a single in vivo system, and evaluation across life stages and generations, and has led to significant advances in PFAS research in recent years. The focus of this review is to assess contemporary findings regarding PFAS toxicokinetics, toxicity and apical adverse health outcomes, and potential modes of action using the zebrafish model. Much of the peer-reviewed literature has focused on a small subset of PFAS structural subclasses, such as the perfluoroalkyl sulfonic acids and perfluoroalkyl carboxylic acids. However, recent data on more diverse PFAS structures are enabling prioritization of compounds of concern. Structure-activity comparisons and the utilization of modeling and 'omics technologies in zebrafish have greatly contributed to our understanding of the hazard potential for a growing number of PFAS and will surely inform our understanding and predictive capabilities for many more PFAS in the future.

3' RNA-seq is superior to standard RNA-seq in cases of sparse data but inferior at identifying toxicity pathways in a model organism.

Introduction: The application of RNA-sequencing has led to numerous breakthroughs related to investigating gene expression levels in complex biological systems. Among these are knowledge of how organisms, such as the vertebrate model organism zebrafish (Danio rerio), respond to toxicant exposure. Recently, the development of 3' RNA-seq has allowed for the determination of gene expression levels with a fraction of the required reads compared to standard RNA-seq. While 3' RNA-seq has many advantages, a comparison to standard RNA-seq has not been performed in the context of whole organism toxicity and sparse data. Methods and results: Here, we examined samples from zebrafish exposed to perfluorobutane sulfonamide (FBSA) with either 3' or standard RNA-seq to determine the advantages of each with regards to the identification of functionally enriched pathways. We found that 3' and standard RNA-seq showed specific advantages when focusing on annotated or unannotated regions of the genome. We also found that standard RNA-seq identified more differentially expressed genes (DEGs), but that this advantage disappeared under conditions of sparse data. We also found that standard RNA-seq had a significant advantage in identifying functionally enriched pathways via analysis of DEG lists but that this advantage was minimal when identifying pathways via gene set enrichment analysis of all genes. Conclusions: These results show that each approach has experimental conditions where they may be advantageous. Our observations can help guide others in the choice of 3' RNA-seq vs standard RNA sequencing to query gene expression levels in a range of biological systems.

A CRISPR-Cas9 mutation in sox9b long intergenic noncoding RNA (slincR) affects zebrafish development, behavior, and regeneration.

The role of long noncoding RNAs (lncRNAs) regulators of toxicological responses to environmental chemicals is gaining prominence. Previously, our laboratory discovered an lncRNA, sox9b long intergenic noncoding RNA (slincR), that is activated by multiple ligands of aryl hydrocarbon receptor (AHR). Within this study, we designed a CRISPR-Cas9-mediated slincR zebrafish mutant line to better understand its biological function in presence or absence of a model AHR ligand, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). The slincRosu3 line contains an 18 bp insertion within the slincR sequence that changes its predicted mRNA secondary structure. Toxicological profiling showed that slincRosu3 is equally or more sensitive to TCDD for morphological and behavioral phenotypes. Embryonic mRNA-sequencing showed differential responses of 499 or 908 genes in slincRosu3 in absence or presence of TCDD Specifically, unexposed slincRosu3 embryos showed disruptions in metabolic pathways, suggesting an endogenous role for slincR. slincRosu3 embryos also had repressed mRNA levels of sox9b-a transcription factor that slincR is known to negatively regulate. Hence, we studied cartilage development and regenerative capacity-both processes partially regulated by sox9b. Cartilage development was disrupted in slincRosu3 embryos both in presence and absence of TCDD. slincRosu3 embryos also displayed a lack of regenerative capacity of amputated tail fins, accompanied by a lack of cell proliferation. In summary, using a novel slincR mutant line, we show that a mutation in slincR can have widespread impacts on gene expression and structural development endogenously and limited, but significant impacts in presence of AHR induction that further highlights its importance in the developmental process.

Chronic Vitamin E Deficiency Dysregulates Purine, Phospholipid, and Amino Acid Metabolism in Aging Zebrafish Skeletal Muscle.

Muscle wasting occurs with aging and may be a result of oxidative stress damage and potentially inadequate protection by lipophilic antioxidants, such as vitamin E. Previous studies have shown muscular abnormalities and behavioral defects in vitamin E-deficient adult zebrafish. To test the hypothesis that there is an interaction between muscle degeneration caused by aging and oxidative damage caused by vitamin E deficiency, we evaluated long-term vitamin E deficiency in the skeletal muscle of aging zebrafish using metabolomics. Zebrafish (55 days old) were fed E+ and E- diets for 12 or 18 months. Then, skeletal muscle samples were analyzed using UPLC-MS/MS. Data were analyzed to highlight metabolite and pathway changes seen with either aging or vitamin E status or both. We found that aging altered purines, various amino acids, and DHA-containing phospholipids. Vitamin E deficiency at 18 months was associated with changes in amino acid metabolism, specifically tryptophan pathways, systemic changes in the regulation of purine metabolism, and DHA-containing phospholipids. In sum, while both aging and induced vitamin E deficiency did have some overlap in altered and potentially dysregulated metabolic pathways, each factor also presented unique alterations, which require further study with more confirmatory approaches.

Coupling Environmental Whole Mixture Toxicity Screening with Unbiased RNA-Seq Reveals Site-Specific Biological Responses in Zebrafish.

Passive sampling device (PSD) extracts paired with developmental toxicity assays in Danio Rerio (zebrafish) are excellent sensors for whole mixture toxicity associated with the bioavailable non-polar organics at environmental sites. We expand this concept by incorporating RNA-Seq in 48-h post fertilization zebrafish statically exposed to PSD extracts from two Portland Harbor Superfund Site locations: river mile 6.5W (RM 6.5W) and river mile 7W (RM 7W). RM 6.5W contained higher concentrations of polycyclic aromatic hydrocarbons (PAHs), but the diagnostic ratios of both extracts indicated similar PAH sourcing and composition. Developmental screens determined RM 6.5W to be more toxic with the most sensitive endpoint being a "wavy" notochord malformation. Differential gene expression from exposure to both extracts was largely parallel, although more pronounced for RM 6.5W. When compared to the gene expression associated with individual chemical exposures, PSD extracts produced some gene signatures parallel to PAHs but were more closely matched by oxygenated-PAHs. Additionally, differential expression, reminiscent of the wavy notochord phenotype, was not accounted for by either class of chemical, indicating the potential of other contaminants driving mixture toxicity. These techniques offer a compelling method for non-targeted hazard characterization of whole mixtures in an in vivo vertebrate system without requiring complete chemical characterization.

Deep autoencoder-based behavioral pattern recognition outperforms standard statistical methods in high-dimensional zebrafish studies.

Zebrafish have become an essential tool in screening for developmental neurotoxic chemicals and their molecular targets. The success of zebrafish as a screening model is partially due to their physical characteristics including their relatively simple nervous system, rapid development, experimental tractability, and genetic diversity combined with technical advantages that allow for the generation of large amounts of high-dimensional behavioral data. These data are complex and require advanced machine learning and statistical techniques to comprehensively analyze and capture spatiotemporal responses. To accomplish this goal, we have trained semi-supervised deep autoencoders using behavior data from unexposed larval zebrafish to extract quintessential "normal" behavior. Following training, our network was evaluated using data from larvae shown to have significant changes in behavior (using a traditional statistical framework) following exposure to toxicants that include nanomaterials, aromatics, per- and polyfluoroalkyl substances (PFAS), and other environmental contaminants. Further, our model identified new chemicals (Perfluoro-n-octadecanoic acid, 8-Chloroperfluorooctylphosphonic acid, and Nonafluoropentanamide) as capable of inducing abnormal behavior at multiple chemical-concentrations pairs not captured using distance moved alone. Leveraging this deep learning model will allow for better characterization of the different exposure-induced behavioral phenotypes, facilitate improved genetic and neurobehavioral analysis in mechanistic determination studies and provide a robust framework for analyzing complex behaviors found in higher-order model systems.

11-Cl-BBQ, a select modulator of AhR-regulated transcription, suppresses lung cancer cell growth via activation of p53 and p27Kip1.

Aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor and functions as a tumour suppressor in different cancer models. In the present study, we report detailed characterization of 11-chloro-7H-benzimidazo[2,1-a]benzo[de]iso-quinolin-7-one (11-Cl-BBQ) as a select modulator of AhR-regulated transcription (SMAhRT) with anti-cancer actions. Treatment of lung cancer cells with 11-Cl-BBQ induced potent and sustained AhR-dependent anti-proliferative effects by promoting G1 phase cell cycle arrest. Investigation of 11-Cl-BBQ-induced transcription in H460 cells with or without the AhR expression by RNA-sequencing revealed activation of p53 signalling. In addition, 11-Cl-BBQ suppressed multiple pathways involved in DNA replication and increased expression of cyclin-dependent kinase inhibitors, including p27Kip1 , in an AhR-dependent manner. CRISPR/Cas9 knockout of individual genes revealed the requirement for both p53 and p27Kip1 for the AhR-mediated anti-proliferative effects. Our results identify 11-Cl-BBQ as a potential lung cancer therapeutic, highlight the feasibility of targeting AhR and provide important mechanistic insights into AhR-mediated-anticancer actions.