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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 subphenotypic concentrations of 2 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 for each chemical by modeling 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 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., (© The Author(s) 2024. Published by Oxford University Press on behalf of the Society of Toxicology.)

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.

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., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Crown Copyright © 2023. Published by Elsevier Inc. All rights reserved.)

With the advent of high-throughput DNA sequencing technology, the genomic sequence of many disparate species has led to the relatively new discipline of genomics, the study of genome structure, function and evolution. Much work has been focused on the role of whole genome duplications (WGD) in the architecture of extant vertebrate genomes, particularly those of teleost fishes which underwent a WGD early in the teleost radiation >230 million years ago (mya). Our past work has focused on the fate of duplicated copies of a multigene family coding for the intracellular lipid-binding protein (iLBP) genes in the teleost fishes. To define the evolutionary processes that determined the fate of duplicated genes and generated the structure of extant fish genomes, however, requires comparative genomic analysis with a fish lineage that diverged before the teleost WGD, such as the spotted gar (Lepisosteus oculatus), an ancient, air-breathing, ray-finned fish. Here, we describe the genomic organization, chromosomal location and tissue-specific expression of a subfamily of the iLBP genes that code for fatty acid-binding proteins (Fabps) in spotted gar. Based on this work, we have defined the minimum suite of fabp genes prior to their duplication in the teleost lineages ~230-400 mya. Spotted gar, therefore, serves as an appropriate outgroup, or ancestral/ancient fish, that did not undergo the teleost-specific WGD. As such, analyses of the spatio-temporal regulation of spotted gar genes provides a foundation to determine whether the duplicated fabp genes have been retained in teleost genomes owing to either sub- or neofunctionalization., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Increasing organismal complexity during the evolution of life has been attributed to the duplication of genes and entire genomes. More recently, theoretical models have been proposed that postulate the fate of duplicated genes, among them the duplication-degeneration-complementation (DDC) model. In the DDC model, the common fate of a duplicated gene is lost from the genome owing to nonfunctionalization. Duplicated genes are retained in the genome either by subfunctionalization, where the functions of the ancestral gene are sub-divided between the sister duplicate genes, or by neofunctionalization, where one of the duplicate genes acquires a new function. Both processes occur either by loss or gain of regulatory elements in the promoters of duplicated genes. Here, we review the genomic organization, evolution, and transcriptional regulation of the multigene family of intracellular lipid-binding protein (iLBP) genes from teleost fishes. Teleost fishes possess many copies of iLBP genes owing to a whole genome duplication (WGD) early in the teleost fish radiation. Moreover, the retention of duplicated iLBP genes is substantially higher than the retention of all other genes duplicated in the teleost genome. The fatty acid-binding protein genes, a subfamily of the iLBP multigene family in zebrafish, are differentially regulated by peroxisome proliferator-activated receptor (PPAR) isoforms, which may account for the retention of iLBP genes in the zebrafish genome by the process of subfunctionalization of cis-acting regulatory elements in iLBP gene promoters.

Background/purpose: Liver transplantation is the treatment of choice in patients with end-stage liver disease. During liver transplantation, ischemia-reperfusion injury (IRI) occurs, which is an inevitable consequence of the transplantation process. To reduce the extent of cellular injury, one of the proteins that have been extensively investigated is heme oxygenase 1 (HO-1), which plays an important role in protecting the organs against IRI. The aim of this study was to introduce an active and functional HO-1 protein conjugated to a cell-penetrating peptide (CPP) in vitro and ex vivo into liver cells in hypothermic and anoxic conditions and to assert its cytoprotective effects., Methods: We generated an enzymatically active soluble (s)HO-1-CPP recombinant protein. The ability of the sHO-1-CPP protein to penetrate McA-RH7777, Clone 9, and Hep G2 cells, primary hepatocytes, and Kupffer and human umbilical vein endothelial cells in vitro, as well as its ability to penetrate a whole liver ex vivo under hypothermic and anoxic conditions, was assessed. An in vitro hypoxia-reoxygenation (HR) model was used to determine the cytoprotective effect of the sHO-1-CPP protein., Results: We showed that our recombinant protein sHO-1-CPP can cross cell membranes into rodent and human liver cells in vitro, and the results were further validated ex vivo, where rodent livers were perfused with an organ preservation solution supplemented with sHO-1-CPP under anoxic and hypothermic conditions. Immunohistochemistry revealed an intracellular localization of sHO-1-CPP in zones 1-3 of the perfused livers. The CPP did not exert any significant toxicity on the cells. Treating cells with sHO-1-CPP showed significant cytoprotection in the in vitro HR model., Conclusions: Our findings show that the recombinant protein sHO-1-CPP can be successfully delivered to cells of a whole organ in an ex vivo hypothermic and anoxic perfusion model and that it provides cytoprotection to hepatocytes in an in vitro HR model. These results hold great potential for future repair and protection of donor organs. Future experiments are planned to confirm these data in in vivo models of IRI., (© 2016 S. Karger AG, Basel.)

Purpose of the Study: Postsurgical adhesions can occur after laparotomy and can cause morbidity. Local delivery of sirolimus prevented adhesion formation in various experiments. We analyzed the impact of orally dosed mammalian target of rapamycin inhibitors on abdominal adhesion formation and wound tensile strength in an experimental model., Materials and Methods: Wistar albino rats were divided into sirolimus, everolimus, and control groups (n = 6 per group). Experimental animals underwent midline laparotomy and adhesion induction procedure which included cecum abrasion and mesh implantation. Animals were administered oral sirolimus (4 mg/kg), everolimus (3 mg/kg), or placebo starting on postoperative day 1. Treatments were given until postoperative day 7. At postoperative day 21, adhesions were scored. Meshes were resected with the attached abdominal wall and cecal segment and stained with Sirius red for collagen density analysis. Midline scars were excised for tensile strength measurement. Effects of sirolimus and everolimus on fibroblast proliferation were also assessed., Results: Mean adhesion score of the everolimus group (7.83 ± 1.17) was significantly lower compared to sirolimus (11.00 ± 0.63) and control (11.66 ± 0.51) groups. Mean collagen density of the everolimus group (33.5 ± 7.8) was significantly lower compared to sirolimus (50.7 ± 9.69) and control (53.8 ± 12.4) groups. Mean tensile strength of the control group (26.41 ± 2.10) was significantly higher compared to sirolimus (17.89 ± 1.9) and everolimus (21.37 ± 1.25) groups. It was significantly lower in sirolimus group than everolimus group. Both sirolimus and everolimus treated media inhibited fibroblast proliferation significantly compared to media alone., Conclusions: Everolimus effectively reduced adhesions. Nevertheless, it also reduced wound tensile strength: an effect which seemed to be due to inhibition of fibroblast proliferation.

Ischemia / reperfusion injury (IRI) during the course of liver transplantation enhances the immunogenicity of allografts and thus impacts overall graft outcome. This sterile inflammatory insult is known to activate innate immunity and propagate organ damage through the recognition of damage-associate molecular pattern (DAMP) molecules. The purpose of the present study was to investigate the role of mitochondrial DAMPs (MTDs) in the pathogenesis of hepatic IRI. Using in vitro models we observed that levels of MTDs were significantly higher in both transplantation-associated and warm IR, and that co-culture of MTDs with human and rat hepatocytes significantly increased cell death. MTDs were also released in an in vivo rat model of hepatic IRI and associated with increased secretion of inflammatory cytokines (TNF-α, IL-6, and IL-10) and increased liver injury compared to the sham group. Our results suggest that hepatic IR results in a significant increase of MTDs both in vitro and in vivo suggesting that MTDs may serve as a novel marker in hepatic IRI. Co-culture of MTDs with hepatocytes showed a decrease in cell viability in a concentration dependent manner, which indicates that MTDs is a toxic mediator participating in the pathogenesis of liver IR injury.

All fabp genes, except fabp2, fabp3 and fabp6, exist as duplicates in the zebrafish genome owing to a whole genome duplication event ~230-400 million years ago. Transcription of some duplicated fabp genes is modulated by fatty acids (FAs) and/or clofibrate, a peroxisome proliferator-activated receptor (PPAR) agonist. We had also shown previously that the steady-state level of acyl-CoA oxidase 1 (acox1) mRNA, a marker of PPARα activation, was elevated in liver, intestine, heart and muscle of fish fed clofibrate demonstrating that zebrafish, unlike some fishes, is responsive to this drug. acox1 transcripts were not induced in the brain of fish fed clofibrate, which suggests this drug may not cross the blood brain barrier. Here, we investigated the effect of dietary FAs and clofibrate on the transcription of single copy fabp genes, fabp2, fabp3 and fabp6, in five tissues of inbred zebrafish. The steady-state level of fabp2 transcripts increased in intestine, while fabp3 mRNA increased in liver of fish fed diets differing in FA content. In fish fed clofibrate, fabp3 mRNA in intestine, and fabp6 mRNA in intestine and heart, were elevated. Based on these findings, modulation of fabp2, fabp3 and fabp6 transcription by FAs and/or clofibrate in zebrafish implicates control of these genes by PPAR interaction with peroxisome proliferator response elements (PPRE) most likely in fabp promoters. Moreover, transcriptional induction of these fabp genes by dietary FAs and/or clofibrate is over-ridden by a tissue-specific mechanism(s), e.g., transcriptional activator or repressor proteins., (Copyright © 2013 Elsevier B.V. All rights reserved.)

We describe the evolutionary diversification of the duplicated ileal fatty acid-binding protein genes (fabp6a and fabp6b) from Japanese ricefish (Oryzias latipes; medaka) and three-spined stickleback (Gasterosteus aculeatus). The fabp6a and fabp6b genes from medaka and three-spined stickleback encode polypeptides of 125-127 amino acids, which share highest sequence identity with their orthologs in teleost fishes and tetrapods. All Fabp6a and Fabp6b from different species cluster together in a distinct clade in phylogenetic analysis and the topology of the tree suggests that fabp6a and fabp6b from medaka and three-spined stickleback are most likely duplicated genes of an ancestral FABP6 owing to teleost-specific whole-genome duplication. However, the topology of an alternate phylogenetic tree revealed that the duplication of the ancestral FABP6 that gave rise to the extant fabp6a and fabp6b possibly occurred before the divergence of tetrapods and fishes. Conserved gene synteny was evident between the teleost fabp6a and fabp6b genes and the human FABP6 gene. The tissue-specific distribution of fabp6a transcripts suggests the retention of ancestral function(s) of the fabp6a gene in medaka and three-spined stickleback with acquisition of new function(s) in different tissues. However, the tissue-specific regulation of the fabp6b gene has diverged markedly in medaka and three-spined stickleback since the duplication of the fabp6 gene., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Background: Force, Lynch and Conery proposed the duplication-degeneration-complementation (DDC) model in which partitioning of ancestral functions (subfunctionalization) and acquisition of novel functions (neofunctionalization) were the two primary mechanisms for the retention of duplicated genes. The DDC model was tested by analyzing the transcriptional induction of the duplicated fatty acid-binding protein (fabp) genes by clofibrate in zebrafish. Clofibrate is a specific ligand of the peroxisome proliferator-activated receptor (PPAR); it activates PPAR which then binds to a peroxisome proliferator response element (PPRE) to induce the transcriptional initiation of genes primarily involved in lipid homeostasis. Zebrafish was chosen as our model organism as it has many duplicated genes owing to a whole genome duplication (WGD) event that occurred ~230-400 million years ago in the teleost fish lineage. We assayed the steady-state levels of fabp mRNA and heterogeneous nuclear RNA (hnRNA) transcripts in liver, intestine, muscle, brain and heart for four sets of duplicated fabp genes, fabp1a/fabp1b.1/fabp1b.2, fabp7a/fabp7b, fabp10a/fabp10b and fabp11a/fabp11b in zebrafish fed different concentrations of clofibrate., Result: Electron microscopy showed an increase in the number of peroxisomes and mitochondria in liver and heart, respectively, in zebrafish fed clofibrate. Clofibrate also increased the steady-state level of acox1 mRNA and hnRNA transcripts in different tissues, a gene with a functional PPRE. These results demonstrate that zebrafish is responsive to clofibrate, unlike some other fishes. The levels of fabp mRNA and hnRNA transcripts for the four sets of duplicated fabp genes was determined by reverse transcription, quantitative polymerase chain reaction (RT-qPCR). The level of hnRNA coded by a gene is an indirect estimate of the rate of transcriptional initiation of that gene. Clofibrate increased the steady-state level of fabp mRNAs and hnRNAs for both the duplicated copies of fabp1a/fabp1b.1, and fabp7a/fabp7b, but in different tissues. Clofibrate also increased the steady-state level of fabp10a and fabp11a mRNAs and hnRNAs in liver, but not for fabp10b and fabp11b., Conclusion: Some duplicated fabp genes have, most likely, retained PPREs, but induction by clofibrate is over-ridden by an, as yet, unknown tissue-specific mechanism(s). Regardless of the tissue-specific mechanism(s), transcriptional control of duplicated zebrafish fabp genes by clofibrate has markedly diverged since the WGD event.

Here we describe the structure of the fatty acid-binding protein 11a and 11b genes (fabp11a and fabp11b) in medaka, and their evolutionary relationship to fabp11 genes from other teleost fishes. Initial studies indicated that the medaka fabp11a gene is intronless, but the fabp11b gene consists of four exons separated by three introns, a genomic organization that is characteristic of most members of the intracellular lipid-binding protein family. Based on genomic sequence, we conclude that the intronless fabp11a gene most likely arose as a result of reverse transcription of its mRNA transcript into cDNA followed by integration into chromosomal DNA. The ancestral intron-containing fabp11a gene was presumably lost from the medaka genome. The duplicated fabp11 genes extant in medaka encode polypeptides of 134 amino acids, which share highest sequence identity and similarity, and cluster in a distinct phylogenetic clade, with their orthologs in other teleost fishes. The fabp11a and fabp11b genes in medaka are therefore orthologs of the fabp11a and fabp11b genes, respectively, of other teleost fishes. No conserved gene synteny was found between medaka fabp11a and fabp11a genes from other teleost fishes, supporting our suggestion as to how this intronless gene arose. However, conserved gene synteny was evident between medaka fabp11b and fabp11b genes from other teleost fishes. The tissue-specific distribution of transcripts for medaka and zebrafish fabp11a and fabp11b genes revealed acquisition of a new function(s) in various tissues by the medaka fabp11b gene, which may explain the retention of sister duplicates of fabp11 in the medaka genome., (© 2012 The Authors Journal compilation © 2012 FEBS.)

Genomic and cDNA sequences coding for two cellular retinol-binding proteins (rbp) in zebrafish were retrieved from DNA sequence databases. Phylogenetic analysis revealed that these proteins were most similar to mammalian RBP7/Rbp7 proteins. Hence, the genes coding for these proteins were named rbp7a and rbp7b. Using a radiation hybrid panel, rbp7a and rbp7b were mapped to the zebrafish chromosomes 23 and 6, respectively. Conserved gene synteny indicated that these genes most likely arose as a result of a fish-specific whole-genome duplication event that had occurred 230-400 million years ago. Whole-mount in situ hybridization to zebrafish embryos detected rbp7a transcripts from the sphere stage (4h post-fertilization (hpf)) in the forerunner cells and the yolk syncytial layer, as well as in Kuppfer's vesicle and the periderm at 12 hpf. The transcripts of rbp7b were seen primarily in the somite stages (10-24 hpf) of zebrafish embryos, but also in the floor plate and hypochord, and did not overlap with the distribution of rbp7a transcripts in embryos. The hybridization signal for rbp7a and rbp7b transcripts was not detected in embryos after 12 hpf and 24 hpf, respectively. While transcripts for both rbp7a and rbp7b were found in all adult tissues assayed by RT-qPCR, the steady-state level of rbp7a transcripts were significantly higher than that of rbp7b transcripts in gill and ovary, whereas rbp7b transcripts were significantly higher than rbp7a transcripts in muscle and brain. The distribution of rbp7a and rbp7b transcripts in embryos and adult zebrafish indicate that the cis-elements that control the transcriptional regulation of the rbp7a and rbp7b genes have diverged considerably since their duplication., (Copyright 2010 Elsevier B.V. All rights reserved.)

Genomic and cDNA sequences coding for a fatty acid-binding protein (FABP) in zebrafish were retrieved from DNA sequence databases. The cDNA codes for a protein of 14.7 kDa (pI = 5.94), and the gene consists of four exons, properties characteristic of most vertebrate FABP genes. Phylogenetic analyses using vertebrate FABPs indicated that this protein is most similar to zebrafish Fabp10. Currently, only one fabp10 gene is annotated in the zebrafish genome. In this article, the notations 'fabp10a' and 'fabp10b' are used to refer to the duplicate copies of fabp10. The zebrafish fabp10a and fabp10b genes were assigned by radiation hybrid mapping to chromosomes 16 and 19, respectively. On the basis of conserved gene synteny with chicken FABP10 on chromosome 23, zebrafish fabp10a and fabp10b are duplicates resulting from a whole-genome duplication event early in the ray-finned fish lineage some 230-400 million years ago. Whole-mount in situ hybridization detected fabp10b transcripts only in the olfactory vesicles of embryos and larvae, whereas fabp10a transcripts have been shown previously to be present only in the liver of embryos and larvae. In adults, RT-PCR detected fabp10b transcripts in all tissues assayed. By contrast, fabp10a transcripts were detected only in adult liver, intestine and testis. This differential tissue distribution of transcripts for the duplicated fabp10 genes suggests considerable divergence of their cis-acting regulatory elements since their duplication.