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.

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.

9,10-Dihydro-5-hydroxy-2,3,6-trimethoxyphenanthrene-1,4-dione: a new dihydrophenanthrene from commercial cannabis and its effect on zebrafish larval behaviour.

A new dihydrophenanthrene derivative namely 9,10-dihydro-5-hydroxy-2, 3,6-trimethoxyphenanthrene-1,4-dione (1) was isolated from commercial cannabis product together with 4,5-dihydroxy-2,3,6-trimethoxy-9,10-dihydrophenanthrene (2), 4-hydroxy-2,3,6,7-tetramethoxy-9,10-dihydrophenanthrene (3), combretastatin B-2 (4) and isocannbispiradienone (5). Structure elucidation of the isolated compounds were done based on the interpretation of the mass spectrometry (MS) and nuclear magnetic resonance (NMR) data. New dihydrophenanthrene derivative (1) was tested for its effect on zebrafish larval behaviour. Preliminary results suggested that the new dihydrophenanthrene derivative (1) exhibits similar effect on zebrafish larval behaviour as cannabidiol (CBD), a biologically active component of Cannabis.

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.

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.

Identification, growth and toxicity assessment of Coolia Meunier (Dinophyceae) from Nova Scotia, Canada.

Benthic dinoflagellates of the toxigenic genus Coolia Meunier (Dinophyceae) are known to have a global distribution in both tropical and temperate waters. The type species, C. monotis, has been reported from the Mediterranean Sea, the NE Atlantic and from Rhode Island, USA in the NW Atlantic, whereas other species in the genus have been reported from tropical locations. Coolia cells were observed in algal drift samples collected at seven sites in Nova Scotia, Canada. Clonal isolates were established from four of these locations and identified with light and scanning electron microscopy, then confirmed with genetic sequencing to be C. monotis. This is the first record of this species in Nova Scotia. The isolates were established and incubated at 18 °C under a 14:10 L:D photoperiod with an approximate photon flux density of 50-60 µmol m-2 s-1. Growth experiments using an isolate from Johnston Harbour (CMJH) were carried out at temperatures ranging from 5 to 30 °C under the same photoperiod with an approximate photon flux density of 45-50 µmol m-2 s-1. Cells tolerated temperatures from 5 to 25 °C with optimum growth and mucilage aggregate production between 15 and 20 °C. Methanol extracts of this isolate examined by Liquid Chromatography-Mass Spectrometry (LC-MS) did not show the presence of the previously reported cooliatoxin. Toxic effects were assayed using two zebrafish bioassays, the Fish Embryo Toxicity (FET) assay and the General Behaviour and Toxicity (GBT) assay. The results of this study demonstrate a lack of toxicity in C. monotis from Nova Scotia, as has been reported for other genetically-confirmed isolates of this species. Conditions in which cell growth that could potentially degrade water quality and provide substrate and dispersal mechanisms for other harmful microorganisms via mucilage production are indicated.

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.

A thermostable d-polymerase for mirror-image PCR.

Biological evolution resulted in a homochiral world in which nucleic acids consist exclusively of d-nucleotides and proteins made by ribosomal translation of l-amino acids. From the perspective of synthetic biology, however, particularly anabolic enzymes that could build the mirror-image counterparts of biological macromolecules such as l-DNA or l-RNA are lacking. Based on a convergent synthesis strategy, we have chemically produced and characterized a thermostable mirror-image polymerase that efficiently replicates and amplifies mirror-image (l)-DNA. This artificial enzyme, dubbed d-Dpo4-3C, is a mutant of Sulfolobus solfataricus DNA polymerase IV consisting of 352 d-amino acids. d-Dpo4-3C was reliably deployed in classical polymerase chain reactions (PCR) and it was used to assemble a first mirror-image gene coding for the protein Sso7d. We believe that this d-polymerase provides a valuable tool to further investigate the mysteries of biological (homo)chirality and to pave the way for potential novel life forms running on a mirror-image genome.

Outwitting EF-Tu and the ribosome: translation with d-amino acids.

Key components of the translational apparatus, i.e. ribosomes, elongation factor EF-Tu and most aminoacyl-tRNA synthetases, are stereoselective and prevent incorporation of d-amino acids (d-aa) into polypeptides. The rare appearance of d-aa in natural polypeptides arises from post-translational modifications or non-ribosomal synthesis. We introduce an in vitro translation system that enables single incorporation of 17 out of 18 tested d-aa into a polypeptide; incorporation of two or three successive d-aa was also observed in several cases. The system consists of wild-type components and d-aa are introduced via artificially charged, unmodified tRNA(Gly) that was selected according to the rules of 'thermodynamic compensation'. The results reveal an unexpected plasticity of the ribosomal peptidyltransferase center and thus shed new light on the mechanism of chiral discrimination during translation. Furthermore, ribosomal incorporation of d-aa into polypeptides may greatly expand the armamentarium of in vitro translation towards the identification of peptides and proteins with new properties and functions.

Crystal structure of a mirror-image L-RNA aptamer (Spiegelmer) in complex with the natural L-protein target CCL2.

We report the crystal structure of a 40 mer mirror-image RNA oligonucleotide completely built from nucleotides of the non-natural L-chirality in complex with the pro-inflammatory chemokine L-CLL2 (monocyte chemoattractant protein-1), a natural protein composed of regular L-amino acids. The L-oligonucleotide is an L-aptamer (a Spiegelmer) identified to bind L-CCL2 with high affinity, thereby neutralizing the chemokine's activity. CCL2 plays a key role in attracting and positioning monocytes; its overexpression in several inflammatory diseases makes CCL2 an interesting pharmacological target. The PEGylated form of the L-aptamer, NOX-E36 (emapticap pegol), already showed promising efficacy in clinical Phase II studies conducted in diabetic nephropathy patients. The structure of the L-oligonucleotide[Symbol: see text]L-protein complex was solved and refined to 2.05 Å. It unveils the L-aptamer's intramolecular contacts and permits a detailed analysis of its structure-function relationship. Furthermore, the analysis of the intermolecular drug-target interactions reveals insight into the selectivity of the L-aptamer for certain related chemokines.

The mechanics of ribosomal translocation.

The ribosome translates the sequence of codons of an mRNA into the corresponding sequence of amino acids as it moves along the mRNA with a codon-step width of about 10 Å. The movement of the million-dalton complex ribosome is triggered by the universal elongation factor G (EF2 in archaea and eukaryotes) and is termed translocation. Unraveling the molecular details of translocation is one of the most challenging tasks of current ribosome research. In the last two years, enormous progress has been obtained by highly-resolved X-ray and cryo-electron microscopic structures as well as by sophisticated biochemical approaches concerning the trigger and control of the movement of the tRNA2·mRNA complex inside the ribosome during translocation. This review inspects and surveys these achievements.

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.

EF-G catalyzes tRNA translocation by disrupting interactions between decoding center and codon-anticodon duplex.

During translation, elongation factor G (EF-G) catalyzes the translocation of tRNA2-mRNA inside the ribosome. Translocation is coupled to a cycle of conformational rearrangements of the ribosomal machinery, and how EF-G initiates translocation remains unresolved. Here we performed systematic mutagenesis of Escherichia coli EF-G and analyzed inhibitory single-site mutants of EF-G that preserved pretranslocation (Pre)-state ribosomes with tRNAs in A/P and P/E sites (Pre-EF-G). Our results suggest that the interactions between the decoding center and the codon-anticodon duplex constitute the barrier for translocation. Catalysis of translocation by EF-G involves the factor's highly conserved loops I and II at the tip of domain IV, which disrupt the hydrogen bonds between the decoding center and the duplex to release the latter, hence inducing subsequent translocation events, namely 30S head swiveling and tRNA2-mRNA movement on the 30S subunit.

EF-G and EF4: translocation and back-translocation on the bacterial ribosome.

Ribosomes translate the codon sequence of an mRNA into the amino acid sequence of the corresponding protein. One of the most crucial events is the translocation reaction, which involves movement of both the mRNA and the attached tRNAs by one codon length and is catalysed by the GTPase elongation factor G (EF-G). Interestingly, recent studies have identified a structurally related GTPase, EF4, that catalyses movement of the tRNA2-mRNA complex in the opposite direction when the ribosome stalls, which is known as back-translocation. In this Review, we describe recent insights into the mechanistic basis of both translocation and back-translocation.

Molecular characterization of zebrafish embryogenesis via DNA microarrays and multiplatform time course metabolomics studies.

One of the greatest strengths of "-omics" technologies is their ability to capture a molecular snapshot of multiple cellular processes simultaneously. Transcriptomics, proteomics, and metabolomics have, individually, been used in wide-ranging studies involving cell lines, tissues, model organisms, and human subjects. Nonetheless, despite the fact that their power lies in the global acquisition of parallel data streams, these methods continue to be employed separately. We highlight work done to merge transcriptomics and metabolomics technologies to study zebrafish (Danio rerio) embryogenesis. We combine information from three bioanalytical platforms, that is, DNA microarrays, (1)H nuclear magnetic resonance ((1)H NMR), and mass spectrometry (MS)-based metabolomics, to identify and provide insights into the organism's developmental regulators. We apply a customized approach to the analysis of such time-ordered measurements to provide temporal profiles that depict the modulation of metabolites and gene transcription. Initially, the three data sets were analyzed individually but later they were fused to highlight the advantages gained through such an integrated approach. Unique challenges posed by fusion of such data are discussed given differences in the measurement error structures, the wide dynamic range for the molecular species, and the analytical platforms used to measure them (i.e., fluorescence ratios, NMR, and MS intensities). Our data analysis reveals that changes in transcript levels at specific developmental stages correlate with previously published data with over 90% accuracy. In addition, transcript profiles exhibited trends that were similar to the accumulation of metabolites over time. Profiles for metabolites such as choline-like compounds (Trimethylamine-N-oxide, phosphocholine, betaine), creatinine/creatine, and other metabolites involved in energy metabolism exhibited a steady increase from 15 hours post fertilization (hpf) to 48 hpf. Other metabolite and transcript profiles were transiently rising and then falling back to baseline. The "house keeping" metabolites such as branched chain amino acids exhibited a steady presence throughout embryogenesis. Although the transcript profiling corresponds to only 16 384 genes, a subset of the total number of genes in the zebrafish genome, we identified examples where gene transcript and metabolite profiles correlate with one another, reflective of a relationship between gene and metabolite regulation over the course of embryogenesis.

Electrical microarrays for highly sensitive detection of multiplex PCR products from biological agents.

For the sensitive detection of amplicons derived from diagnostic PCR, a novel electrical low-density microarray is applied and compared to state-of-the-art quantitative real-time PCR. The principle of the electrochemical method and the effective use for analysis are described. Interdigitated array gold electrodes (IDA-E) embedded into a silicon chip are the core technology of the fully automated compact biosensor system, basing on enzyme coupled electrochemical detection. The biointerface is built up with thiol-modified capture oligonucleotides on gold and mediates the specific recognition of hybridised target DNA amplified with uniplex or multiplex PCR. In here we show the potential of the designed electrical microarray to function as an advanced screening method for the parallel detection of a panel of the four pathogens Bacillus anthracis, Yersinia pestis, Francisella tularensis and ortho pox viruses (genus), which are among the most relevant biowarfare agents. PCR products, generated from 10 to 50 gene equivalents, have been detected reproducibly. The experiments with varying pathogen amounts showed the good reliability and the high sensitivity of the method, equivalent to optical real-time PCR detection systems. Without PCR the total assay time amounts to 27 min. The advantage of the combination of multiplex-PCR with electrical microarray detection avoiding intensive PCR probe labelling strategies is illustrated.

Seasonal expressed sequence tags of rainbow smelt (Osmerus mordax) revealed by subtractive hybridization and the identification of two genes up-regulated during winter.

The rainbow smelt (Osmerus mordax) is freeze-resistant and maintains swimming and feeding activity during winter. In order to identify genes differentially expressed in smelt liver response to winter water temperatures, a large-scale analysis of gene expression using suppression subtractive hybridization was carried out using samples obtained in fall and winter. Forward and reverse subtractions were performed, subtraction-enriched products were cloned, and clones were sequenced from both of the resulting libraries. When 27 of these genes were screened by semi-quantitative RT-PCR to identify candidates for differential expression based generally on 2-fold changes in expression, one encoding FK506-binding protein 5 was classified as up-regulated in response to seasonal change, another encoding the mitochondrial solute carrier 25 member 25 (ATP-Mg/Pi carrier) was similarly classified with seasonal change and low temperature shift, and the one encoding the 78 kDa glucose-regulated protein was provisionally classified as down-regulated with low temperature shift. Analysis of fall (warm) and winter (cold) seasonal samples by quantitative PCR (qPCR) revealed significant up-regulation of genes encoding FK506-binding protein 51 and the mitochondrial solute carrier, whereas the gene encoding the glucose-regulated protein showed no significant change in expression. The mitochondrial solute carrier and FK506-binding protein results may relate to changes in cortisol action, as both are regulated by cortisol in other species.