Comparative toxicological assessment of two bisphenols using a systems approach: Evaluation of the behavioral and transcriptomic responses of Danio rerio to Bisphenol A and Tetrabromobisphenol A.

The zebrafish (Danio rerio) is becoming a critical component of New Approach Methods (NAMs) in chemical risk assessment. As a whole organism in vitro NAM, the zebrafish model offers significant advantages over individual cell-line testing, including toxicokinetic and toxicodynamic competencies. A transcriptomic approach not only allows for insight into mechanism of action for both apical endpoints and unobservable adverse outcomes, but also changes in gene expression induced by lower, environmentally relevant concentrations. In this study, we used a larval zebrafish model to assess the behavioral and transcriptomic alterations caused by sub-phenotypic concentrations of two chemicals with the same structural backbone, the endocrine disrupting chemicals: Bisphenol A and Tetrabromobisphenol A. Following assessment of behavioral toxicity, we used a transcriptomic approach to identify molecular pathways associated with previously described phenotypes. We also determined the transcriptomic Point of Departure (POD) for each chemical by modelling gene expression changes as continuous systems which allows for the identification of a single concentration at which toxic effects can be predicted. This can then be investigated with confirmatory cell-based testing in an integrated approach to testing and assessment (IATA) to determine risk to human health and the environment with greater confidence. This paper demonstrates the impact of using a multi-faceted approach for evaluating the physiological and neurotoxic effects of exposure to structurally related chemicals. By comparing phenotypic effects with transcriptomic outcomes, we were able to differentiate, characterize and rank the toxicities of related bisphenols, which demonstrates methodological advantages unique to the larval zebrafish NAM.

Long and Short Duration Exposures to the Selective Serotonin Reuptake Inhibitors (SSRIs) Fluoxetine, Paroxetine and Sertraline at Environmentally Relevant Concentrations Lead to Adverse Effects on Zebrafish Behaviour and Reproduction.

Selective serotonin reuptake inhibitors (SSRIs) are currently the most prescribed class of psychotropic medications. Their increased global manufacture and use have become growing concerns for aquatic toxicologists and environmental biologists, who assess both the direct and indirect effects of substances on the environment and on human health. In order to assess the potential impact of environmentally relevant levels of SSRIs on fish development, behaviour and reproduction, we exposed juvenile and adult zebrafish to a select group of SSRIs using two separate exposure paradigms. In the first paradigm, juvenile zebrafish were exposed to Fluoxetine (Prozac), Paroxetine (Paxil), Sertraline (Zoloft) or a mixture of the three beginning at environmentally relevant levels (10 µg/L) for 135 days (long-term exposure) beginning at 5 days post fertilization (dpf). In the second paradigm, adult zebrafish were exposed to matching concentrations of the same SSRIs for 35 days (short-term exposure). The long-term exposure paradigm proved to have little to no overt effect on growth or development at sub-lethal concentrations (10 and 100 µg/L). However, both the stress/anxiety response (novel tank tests) and reproduction (fecundity and fertility) were dramatically reduced. Importantly, the short-term exposure of reproductively mature fish led to similar adverse effects on both the stress response and reproduction. Following both the short and long duration exposure paradigms, a 2-week washout period led to a small reduction in the adverse effects. These findings highlight the potential for SSRIs to negatively impact population dynamics in zebrafish and may be of particular value should they be found in other fish species in the environment.

The contribution of larval zebrafish transcriptomics to chemical risk assessment.

In Canada, the Canadian Environmental Protection Act (1999) requires human health and environmental risk assessments be conducted for new substances prior to their manufacture or import. While this toxicity data is historically obtained using rodents, in response to the international effort to eliminate animal testing, Health Canada is collaborating with the National Research Council (NRC) of Canada to develop a New Approach Method by refining existing NRC zebrafish models. The embryo/larval zebrafish model evaluates systemic (whole body) general toxicity which is currently unachievable with cell-based testing. The model is strengthened using behavioral, toxicokinetic and transcriptomic responses to assess non-visible indicators of toxicity following chemical exposure at sub-phenotypic concentrations. In this paper, the predictive power of zebrafish transcriptomics is demonstrated using two chemicals; Raloxifene and Resorcinol. Raloxifene exposure produced darkening of the liver and malformation of the nose/mandible, while Resorcinol exposure produced increased locomotor activity. Transcriptomic analysis correlated differentially expressed genes with the phenotypic effects and benchmark dose calculations determined that the transcriptomic Point of Departure (POD) occurred at subphenotypic concentrations. Correlating gene expression with apical (phenotypic) effects strengthens confidence in evaluation of chemical toxicity, thereby demonstrating the significant advancement that the larval zebrafish transcriptomics model represents in chemical risk assessment.

Identification of minimum essential therapeutic mixtures from cannabis plant extracts by screening in cell and animal models of Parkinson's disease.

Medicinal cannabis has shown promise for the symptomatic treatment of Parkinson's disease (PD), but patient exposure to whole plant mixtures may be undesirable due to concerns around safety, consistency, regulatory issues, and psychoactivity. Identification of a subset of components responsible for the potential therapeutic effects within cannabis represents a direct path forward for the generation of anti-PD drugs. Using an in silico database, literature reviews, and cell based assays, GB Sciences previously identified and patented a subset of five cannabinoids and five terpenes that could potentially recapitulate the anti-PD attributes of cannabis. While this work represents a critical step towards harnessing the anti-PD capabilities of cannabis, polypharmaceutical drugs of this complexity may not be feasible as therapeutics. In this paper, we utilize a reductionist approach to identify minimal essential mixtures (MEMs) of these components that are amenable to pharmacological formulation. In the first phase, cell-based models revealed that the cannabinoids had the most significant positive effects on neuroprotection and dopamine secretion. We then evaluated the ability of combinations of these cannabinoids to ameliorate a 6-hydroxydopmamine (OHDA)-induced change in locomotion in larval zebrafish, which has become a well-established PD disease model. Equimolar mixtures that each contained three cannabinoids were able to significantly reverse the OHDA mediated changes in locomotion and other advanced metrics of behavior. Additional screening of sixty-three variations of the original cannabinoid mixtures identified five highly efficacious mixtures that outperformed the original equimolar cannabinoid MEMs and represent the most attractive candidates for therapeutic development. This work highlights the strength of the reductionist approach for the development of ratio-controlled, cannabis mixture-based therapeutics for the treatment of Parkinson's disease.

Evaluation of the Uptake, Metabolism, and Secretion of Toxicants by Zebrafish Larvae.

Zebrafish larvae have classically been used as a high-throughput model with which to test both the bioactivity and toxicity of known and novel compounds, making them a promising whole organism New Approach Method in the context of the international momentum to eliminate animal testing. Larvae are generally exposed to the chemicals being tested in a static environment and the concentration-response patterns are calculated based on the initial bath concentrations of the compounds. This approach rarely takes into account the absorption, distribution, metabolism, and excretion of the compounds being tested, which can have a significant effect on the toxicokinetic profiles of the compounds and thus impact the predictive ability of the model. In this study, we have evaluated the toxicokinetic profile of 5 known toxicants, 3 phenolic compounds, along with thiabendazole and 3,4-dicholoronalanine, at 6, 8, 24, 72, and 120 h postfertilization in order to match the exposure timelines of a standard in vitro fish embryo toxicity test. It was revealed that in addition to bioaccumulation effects, the compounds were all actively metabolized and excreted by the larvae. Importantly, comparisons between the toxicants revealed that the patterns of uptake and metabolism were varied and could often partially explain the differences in their concentration-response patterns. The findings of this study are significant as they highlight the requirement for an assessment of the stability and toxicokinetic profile of chemicals tested using standard zebrafish larval toxicity assays in order to better understand and compare their toxicity profiles.

Assessing the Morphological and Behavioral Toxicity of Catechol Using Larval Zebrafish.

Catechol is a ubiquitous chemical used in the manufacturing of fragrances, pharmaceuticals and flavorants. Environmental exposure occurs in a variety of ways through industrial processes, during pyrolysis and in effluent, yet despite its prevalence, there is limited information regarding its toxicity. While the genotoxicity and gastric carcinogenicity of catechol have been described in depth, toxicological studies have potentially overlooked a number of other effects relevant to humans. Here, we have made use of a general and behavioral larval zebrafish toxicity assay to describe previously unknown catechol-based toxicological phenomena. Behavioral testing revealed catechol-induced hypoactivity at concentrations an order of magnitude lower than observable endpoints. Catechol exposure also resulted in punctate melanocytes with concomitant decreases in the expression of pigment production and regulation markers mitfa, mc1r and tyr. Because catechol is converted into a number of toxic metabolites by tyrosinase, an enzyme found almost exclusively in melanocytes, an evaluation of the effects of catechol on these cells is critical to evaluating the safety of this chemical. This work provides insights into the toxic nature of catechol and highlights the benefits of the zebrafish larval testing platform in being able to dissect multiple aspects of toxicity with one model.

Comparison of the Zebrafish Embryo Toxicity Assay and the General and Behavioral Embryo Toxicity Assay as New Approach Methods for Chemical Screening.

The movement away from mammalian testing of potential toxicants and new chemical entities has primarily led to cell line testing and protein-based assays. However, these assays may not yet be sufficient to properly characterize the toxic potential of a chemical. The zebrafish embryo model is widely recognized as a potential new approach method for chemical testing that may provide a bridge between cell and protein-based assays and mammalian testing. The Zebrafish Embryo Toxicity (ZET) model is increasingly recognized as a valuable toxicity testing platform. The ZET assay focuses on the early stages of embryo development and is considered a more humane model compared to adult zebrafish testing. A complementary model has been developed that exposes larvae to toxicants at a later time point during development where body patterning has already been established. Here we compare the toxicity profiles of 20 compounds for this General and Behavioral Toxicity (GBT) assay to the ZET assay. The results show partially overlapping toxicity profiles along with unique information provided by each assay. It appears from this work that these two assays applied together can strengthen the use of zebrafish embryos/larvae as standard toxicity testing models.

Anti-Cancer Potential of Cannabinoids, Terpenes, and Flavonoids Present in Cannabis.

In recent years, and even more since its legalization in several jurisdictions, cannabis and the endocannabinoid system have received an increasing amount of interest related to their potential exploitation in clinical settings. Cannabinoids have been suggested and shown to be effective in the treatment of various conditions. In cancer, the endocannabinoid system is altered in numerous types of tumours and can relate to cancer prognosis and disease outcome. Additionally, cannabinoids display anticancer effects in several models by suppressing the proliferation, migration and/or invasion of cancer cells, as well as tumour angiogenesis. However, the therapeutic use of cannabinoids is currently limited to the treatment of symptoms and pain associated with chemotherapy, while their potential use as cytotoxic drugs in chemotherapy still requires validation in patients. Along with cannabinoids, cannabis contains several other compounds that have also been shown to exert anti-tumorigenic actions. The potential anti-cancer effects of cannabinoids, terpenes and flavonoids, present in cannabis, are explored in this literature review.

Antitumor Activity of Abnormal Cannabidiol and Its Analog O-1602 in Taxol-Resistant Preclinical Models of Breast Cancer.

Cannabinoids exhibit anti-inflammatory and antitumorigenic properties. Contrary to most cannabinoids present in the Cannabis plant, some, such as O-1602 and abnormal cannabidiol, have no or only little affinity to the CB1 or CB2 cannabinoid receptors and instead exert their effects through other receptors. Here, we investigated whether the synthetic regioisomers of cannabidiol, abnormal cannabidiol, and a closely related compound, O-1602, display antitumorigenic effects in cellular models of breast cancer and whether it could reduce tumorigenesis in vivo. Several studies have shown the effects of cannabinoids on chemotherapy-sensitive breast cancer cell lines, but less is known about the antitumorigenic effects of cannabinoids in chemotherapy-resistant cell lines. Paclitaxel-resistant MDA-MB-231 and MCF-7 breast cancer cell lines were used to study the effect of O-1602 and abnormal cannabidiol on viability, apoptosis, and migration. The effects of O-1602 and abnormal cannabidiol on cell viability were completely blocked by the combination of GPR55 and GPR18-specific siRNAs. Both O-1602 and abnormal cannabidiol decreased viability in paclitaxel-resistant breast cancer cells in a concentration-dependent manner through induction of apoptosis. The effect of these cannabinoids on tumor growth in vivo was studied in a zebrafish xenograft model. In this model, treatment with O-1602 and abnormal cannabidiol (2 µM) significantly reduced tumor growth. Our results suggest that atypical cannabinoids, like O-1602 and abnormal cannabidiol, exert antitumorigenic effects on paclitaxel-resistant breast cancer cells. Due to their lack of central sedation and psychoactive effects, these atypical cannabinoids could represent new leads for the development of additional anticancer treatments when resistance to conventional chemotherapy occurs during the treatment of breast and possibly other cancers.

Structure Elucidation and Relative Toxicity of (24R)-24-Hydroxyyessotoxin from a Namibian Isolate of Gonyaulax spinifera.

Liquid chromatography-high-resolution mass spectrometry (LC-HRMS) analysis of a Namibian strain of Gonyaulax spinifera showed the presence of a number of yessotoxins (YTXs). Principal among these were YTX (1), homoYTX (2), and a tentative hydroxylated analogue that did not correspond to any previously confirmed YTX structures. Culturing the G. spinifera strain afforded sufficient biomass for purification of the new analogue through a series of solvent partitioning and chromatographic steps, yielding ∼0.9 mg as a solid. NMR spectroscopy, ion-trap mass spectrometry, and HRMS identified the new analogue as 24-hydroxyYTX (7). Purified 24-hydroxyYTX was quantitated by NMR, and its relative toxicity evaluated using two embryonic zebrafish toxicity assays. 24-HydroxyYTX demonstrated reduced toxicity compared to YTX.

Single and Synergistic Effects of Cannabidiol and Δ-9-Tetrahydrocannabinol on Zebrafish Models of Neuro-Hyperactivity.

In this study, we aimed to investigate the effect of the two main active cannabinoids extracted from cannabis: Δ-9-tetrahydrocannabinol (THC) and cannabidiol (CBD) on two distinct behavioral models of induced neuro-hyperactivity. We have taken advantage of two previously developed zebrafish models of neuro-hyperactivity: a chemically induced pentylenetetrazole model and a genetic model caused by loss-of-function mutations in the GABA receptor subunit alpha 1 (GABRA1-/-). Both CBD and THC have a significant effect on the behavioral changes induced by both models. Importantly, we have also shown that when applied together at different ratios of THC to CBD (1:1, 1:5, and 1:10), there was a synergistic effect at a ratio of 1:1. This was particularly important for the genetically induced neuro-hyperactivity as it brought the concentrations of THC and CBD required to oppose the induced behavioral changes to levels that had much less of an effect on baseline larval behavior. The results of this study help to validate the ability of THC and CBD to oppose neuro-hyperactivity linked to seizure modalities. Additionally, it appears that individually, each cannabinoid may be more effective against the chemically induced model than against the GABRA1-/- transgenic model. However, when applied together, the concentration of each compound required to oppose the GABRA1-/- light-induced activity was lowered. This is of particular interest since the use of cannabinoids as therapeutics can be dampened by their side-effect profile. Reducing the level of each cannabinoid required may help to prevent off target effects that lead to side effects. Additionally, this study provides a validation of the complimentary nature of the two zebrafish models and sets a platform for future work with cannabinoids, particularly in the context of neuro-hyperactivity disorders such as epilepsy.

Analysis of the Uptake, Metabolism, and Behavioral Effects of Cannabinoids on Zebrafish Larvae.

The Cannabis sativa plant contains numerous phytocannabinoids and terpenes with known or potential biological activity. For decades, plant breeders have been breeding the Cannabis plant to control for a desired ratio of the major cannabinoids. A high-throughput in vivo model to understand the relationship between the chemical composition of different strains and their therapeutic potential then becomes of value. Measuring changes in the behavioral patterns of zebrafish larvae is an established model with which to test the biological activity of neuroactive compounds. However, there is currently little information regarding the uptake kinetics and metabolism of compounds by larvae. In this study, we chose to compare the uptake kinetics and metabolism of Δ9-tetrahydrocannabinol (THC) and cannabidiol (CBD) alone or in combination with their effects on larval behavior. We have shown that both compounds have distinct behavioral patterns and concentration response profiles. Additionally, the uptake kinetics observed for each compound appears to correlate with the change in behavior observed in the behavioral assays. When combinations of THC and CBD were tested there were shifts in both the behavioral activity and the uptake kinetics of each compound compared with when they were tested alone. Finally, the THC/CBD-derived metabolites detected in the larvae are similar to those found in mammalian systems. This study thus provides a model for further testing of additional cannabinoids and potentially plant extracts.

Use of the zebrafish larvae as a model to study cigarette smoke condensate toxicity.

The smoking of tobacco continues to be the leading cause of premature death worldwide and is linked to the development of a number of serious illnesses including heart disease, respiratory diseases, stroke and cancer. Currently, cell line based toxicity assays are typically used to gain information on the general toxicity of cigarettes and other tobacco products. However, they provide little information regarding the complex disease-related changes that have been linked to smoking. The ethical concerns and high cost associated with mammalian studies have limited their widespread use for in vivo toxicological studies of tobacco. The zebrafish has emerged as a low-cost, high-throughput, in vivo model in the study of toxicology. In this study, smoke condensates from 2 reference cigarettes and 6 Canadian brands of cigarettes with different design features were assessed for acute, developmental, cardiac, and behavioural toxicity (neurotoxicity) in zebrafish larvae. By making use of this multifaceted approach we have developed an in vivo model with which to compare the toxicity profiles of smoke condensates from cigarettes with different design features. This model system may provide insights into the development of smoking related disease and could provide a cost-effective, high-throughput platform for the future evaluation of tobacco products.

Ionic and neuromodulatory regulation of burst discharge controls frequency tuning.

Sensory neurons encode natural stimuli by changes in firing rate or by generating specific firing patterns, such as bursts. Many neural computations rely on the fact that neurons can be tuned to specific stimulus frequencies. It is thus important to understand the mechanisms underlying frequency tuning. In the electrosensory system of the weakly electric fish, Apteronotus leptorhynchus, the primary processing of behaviourally relevant sensory signals occurs in pyramidal neurons of the electrosensory lateral line lobe (ELL). These cells encode low frequency prey stimuli with bursts of spikes and high frequency communication signals with single spikes. We describe here how bursting in pyramidal neurons can be regulated by intrinsic conductances in a cell subtype specific fashion across the sensory maps found within the ELL, thereby regulating their frequency tuning. Further, the neuromodulatory regulation of such conductances within individual cells and the consequences to frequency tuning are highlighted. Such alterations in the tuning of the pyramidal neurons may allow weakly electric fish to preferentially select for certain stimuli under various behaviourally relevant circumstances.

Differential distribution of SK channel subtypes in the brain of the weakly electric fish Apteronotus leptorhynchus.

Calcium signals in vertebrate neurons can induce hyperpolarizing membrane responses through the activation of Ca(2+)-activated potassium channels. Of these, small conductance (SK) channels regulate neuronal responses through the generation of the medium after-hyperpolarization (mAHP). We have previously shown that an SK channel (AptSK2) contributes to signal processing in the electrosensory system of Apteronotus leptorhynchus. It was shown that for pyramidal neurons in the electrosensory lateral line lobe (ELL), AptSK2 expression selectively decreases responses to low-frequency signals. The localization of all the SK subunits throughout the brain of Apteronotus then became of substantial interest. We have now cloned two additional SK channel subunits from Apteronotus and determined the expression patterns of all three AptSK subunits throughout the brain. In situ hybridization experiments have revealed that, as in mammalian systems, the AptSK1 and 2 channels showed a partially overlapping expression pattern, whereas the AptSK3 channel was expressed in different brain areas. The AptSK1 and 2 channels were the primary subunits found in the major electrosensory processing areas. Immunohistochemistry further revealed distinct compartmentalization of the AptSK1 and 2 channels in the ELL. AptSK1 was localized to the apical dendrites of pyramidal neurons, whereas AptSK2 channels are primarily somatic. The distinct expression patterns of all three AptSK channels may reflect subtype-specific contributions to neuronal function, and the high homology between subtypes from a number of species suggests that the functional roles for each channel subtype are conserved from early vertebrate evolution.

SK channels provide a novel mechanism for the control of frequency tuning in electrosensory neurons.

One important characteristic of sensory input is frequency, with sensory neurons often tuned to narrow stimulus frequency ranges. Although vital for many neural computations, the cellular basis of such frequency tuning remains mostly unknown. In the electrosensory system of Apteronotus leptorhynchus, the primary processing of important environmental and communication signals occurs in pyramidal neurons of the electrosensory lateral line lobe. Spike trains transmitted by these cells can encode low-frequency prey stimuli with bursts of spikes and high-frequency communication signals with single spikes. Here, we demonstrate that the selective expression of SK2 channels in a subset of pyramidal neurons reduces their response to low-frequency stimuli by opposing their burst responses. Apamin block of the SK2 current in this subset of cells induced bursting and increased their response to low-frequency inputs. SK channel expression thus provides an intrinsic mechanism that predisposes a neuron to respond to higher frequencies and thus specific, behaviorally relevant stimuli.

Muscarinic receptors control frequency tuning through the downregulation of an A-type potassium current.

The functional role of cholinergic input in the modulation of sensory responses was studied using a combination of in vivo and in vitro electrophysiology supplemented by mathematical modeling. The electrosensory system of weakly electric fish recognizes different environmental stimuli by their unique alteration of a self-generated electric field. Variations in the patterns of stimuli are primarily distinguished based on their frequency. Pyramidal neurons in the electrosensory lateral line lobe (ELL) are often tuned to respond to specific input frequencies. Alterations in the tuning of the pyramidal neurons may allow weakly electric fish to preferentially select for certain stimuli. Here we show that muscarinic receptor activation in vivo enhances the excitability, burst firing, and subsequently the response of pyramidal cells to naturalistic sensory input. Through a combination of in vitro electrophysiology and mathematical modeling, we reveal that this enhanced excitability and bursting likely results from the down-regulation of an A-type potassium current. Further, we provide an explanation of the mechanism by which these currents can mediate frequency tuning.