Catherine W. McCollum

Developmental toxicity screening in zebrafish.

Given the ever-increasing toxic exposure ubiquitously present in our environment as well as emerging evidence that these exposures are hazardous to human health, the current rodent-based regulations are proving inadequate. In the process of overhauling risk assessment methodology, a nonrodent test organism, the zebrafish, is emerging as tractable for medium- and high-throughput assessments, which may help to accelerate the restructuring of standards. Zebrafish have high developmental similarity to mammals in most aspects of embryo development, including early embryonic processes, and on cardiovascular, somite, muscular, skeletal, and neuronal systems. Here, we briefly describe the development of these systems and then chronicle the toxic impacts assessed following chemical exposure. We also compare the available data in zebrafish toxicity assays with two databases containing mammalian toxicity data. Finally, we identify gaps in our collective knowledge that are ripe for future studies.

ERβ1 represses basal-like breast cancer epithelial to mesenchymal transition by destabilizing EGFR

IntroductionEpithelial to mesenchymal transition (EMT) is associated with the basal-like breast cancer phenotypes. Sixty percent of basal-like cancers have been shown to express wild-type estrogen receptor beta (ERβ1). However, it is still unclear whether the ERβ expression is related to EMT, invasion and metastasis in breast cancer. In the present study, we examined whether ERβ1 through regulating EMT can influence invasion and metastasis in basal-like cancers.MethodsBasal-like breast cancer cells (MDA-MB-231 and Hs578T), in which ERβ1 was either overexpressed or down-regulated were analyzed for their ability to migrate and invade (wound-healing assay, matrigel-coated Transwell assay) as well as for the expression of EMT markers and components of the EGFR pathway (immunoblotting, RT-PCR). Co-immunoprecipitation and ubiquitylation assays were employed to examine whether ERβ1 alters epidermal growth factor receptor (EGFR) protein degradation and the interaction between EGFR and the ubiquitin ligase c-Cbl. The metastatic potential of the ERβ1-expressing MDA-MB-231 cells was evaluated in vivo in a zebrafish xenotransplantation model and the correlation between ERβ1 and E-cadherin expression was examined in 208 clinical breast cancer specimens by immunohistochemistry.ResultsHere we show that ERβ1 inhibits EMT and invasion in basal-like breast cancer cells when they grow either in vitro or in vivo in zebrafish. The inhibition of EMT correlates with an ERβ1-mediated up-regulation of miR-200a/b/429 and the subsequent repression of ZEB1 and SIP1, which results in increased expression of E-cadherin. The positive correlation of ERβ1 and E-cadherin expression was additionally observed in breast tumor samples. Down-regulation of the basal marker EGFR through stabilization of the ubiquitin ligase c-Cbl complexes and subsequent ubiquitylation and degradation of the activated receptor is involved in the ERβ1-mediated repression of EMT and induction of EGFR signaling abolished the ability of ERβ1 to sustain the epithelial phenotype.ConclusionsTaken together, the results of our study strengthen the association of ERβ1 with the regulation of EMT and propose the receptor as a potential crucial marker in predicting metastasis in breast cancer.

Identification of Estrogen Target Genes during Zebrafish Embryonic Development through Transcriptomic Analysis

Estrogen signaling is important for vertebrate embryonic development. Here we have used zebrafish (Danio rerio) as a vertebrate model to analyze estrogen signaling during development. Zebrafish embryos were exposed to 1 µM 17β-estradiol (E2) or vehicle from 3 hours to 4 days post fertilization (dpf), harvested at 1, 2, 3 and 4 dpf, and subjected to RNA extraction for transcriptome analysis using microarrays. Differentially expressed genes by E2-treatment were analyzed with hierarchical clustering followed by biological process and tissue enrichment analysis. Markedly distinct sets of genes were up and down-regulated by E2 at the four different time points. Among these genes, only the well-known estrogenic marker vtg1 was co-regulated at all time points. Despite this, the biological functional categories targeted by E2 were relatively similar throughout zebrafish development. According to knowledge-based tissue enrichment, estrogen responsive genes were clustered mainly in the liver, pancreas and brain. This was in line with the developmental dynamics of estrogen-target tissues that were visualized using transgenic zebrafish containing estrogen responsive elements driving the expression of GFP (Tg(5xERE:GFP)). Finally, the identified embryonic estrogen-responsive genes were compared to already published estrogen-responsive genes identified in male adult zebrafish (Gene Expression Omnibus database). The expressions of a few genes were co-regulated by E2 in both embryonic and adult zebrafish. These could potentially be used as estrogenic biomarkers for exposure to estrogens or estrogenic endocrine disruptors in zebrafish. In conclusion, our data suggests that estrogen effects on early embryonic zebrafish development are stage- and tissue- specific.

Meta-analysis of toxicity and teratogenicity of 133 chemicals from zebrafish developmental toxicity studies

Zebrafish developmental toxicity testing is an emerging field, which faces considerable challenges regarding data meta-analysis and the establishment of standardized test protocols. Here, we present an initial correlation study on toxicity of 133 chemicals based on data in the literature to ascertain predictive developmental toxicity endpoints. We found that the physical properties of chemicals (BCF or logP) did not fully predict lethality or developmental outcomes. Instead, individual outcomes such as pericardial edema and yolk sac edema were more reliable indicators of developmental toxicity. In addition, we ranked the chemicals based on toxicity with the Toxicological Priority Index (ToxPi) program and via a teratogenic ratio, and found that perfluorooctane sulfonate (PFOS) had the highest ToxPi score, triphenyltin acetate had the highest average ToxPi score (corrected for missing data and having more than 4 outcomes), and N-methyl-dithiocarbamate had the highest teratogenic ratio.

Immediate and long-term consequences of vascular toxicity during zebrafish development

Proper formation of the vascular system is necessary for embryogenesis, and chemical disruption of vascular development may be a key event driving developmental toxicity. In order to test the effect of environmental chemicals on this critical process, we evaluated a quantitative assay in transgenic zebrafish using angiogenesis inhibitors that target VEGFR2 (PTK787) or EGFR (AG1478). Both PTK787 and AG1478 exposure impaired intersegmental vessel (ISV) sprouting, while AG1478 also produced caudal and pectoral fin defects at concentrations below those necessary to blunt ISV morphogenesis. The functional consequences of vessel toxicity during early development included decreased body length and survival in juvenile cohorts developmentally exposed to inhibitor concentrations sufficient to completely block ISV sprouting angiogenesis. These data show that concentration-dependent disruption of the presumed targets for these inhibitors produce adverse outcomes at advanced life stages.

Embryonic exposure to sodium arsenite perturbs vascular development in zebrafish.

Exposure to arsenic in its inorganic form, arsenite, causes adverse effects to many different organs and tissues. Here, we have investigated arsenite-induced adverse effects on vascular tissues in the model organism zebrafish, Danio rerio. Zebrafish embryos were exposed to arsenite at different exposure windows and the susceptibility to vascular tissue damage was recorded at 72hours post fertilization (hpf). Intersegmental vessel sprouting and growth was most perturbed by exposure to arsenite during the 24-48hpf window, while disruption in the condensation of the caudal vein plexus was more often observed at the 48-72hpf exposure window, reflecting when these structures develop during normal embryogenesis. The vascular growth rate was decreased by arsenite exposure, and deviated from that of control embryos at around 24-26.5hpf. We further mapped changes in expression of key regulators of angiogenesis and vasculogenesis. Downregulation of vascular endothelial growth factor receptor 1/fms-related tyrosine kinase 1 (vegfr1/flt1) expression was evident already at 24hpf, coinciding with the decreased vascular growth rate. At later time points, matrix metalloproteinase 9 (mmp9) expression was upregulated, suggesting that arsenite affects the composition of the extracellular matrix. In total, the expression of eight key factors involved in different aspects of vascularization was significantly altered by arsenic exposure. In conclusion, our results show that arsenite is a potent vascular disruptor in the developing zebrafish embryo, a finding that calls for an evaluation of arsenite as a developmental vascular toxicant in mammalian model systems.

Screening for angiogenic inhibitors in zebrafish to evaluate a predictive model for developmental vascular toxicity

Chemically-induced vascular toxicity during embryonic development may cause a wide range of adverse effects. To identify putative vascular disrupting chemicals (pVDCs), a predictive pVDC signature was constructed from 124 U.S. EPA ToxCast high-throughput screening (HTS) assays and used to rank 1060 chemicals for their potential to disrupt vascular development. Thirty-seven compounds were selected for targeted testing in transgenic Tg(kdrl:EGFP) and Tg(fli1:EGFP) zebrafish embryos to identify chemicals that impair developmental angiogenesis. We hypothesized that zebrafish angiogenesis toxicity data would correlate with human cell-based and cell-free in vitro HTS ToxCast data. Univariate statistical associations used to filter HTS data based on correlations with zebrafish angiogenic inhibition in vivo revealed 132 total significant associations, 33 of which were already captured in the pVDC signature, and 689 non-significant assay associations. Correlated assays were enriched in cytokine and extracellular matrix pathways. Taken together, the findings indicate the utility of zebrafish assays to evaluate an HTS-based predictive toxicity signature and also provide an experimental basis for expansion of the pVDC signature with novel HTS assays.

Estrogen receptor beta reduces colon cancer metastasis through a novel miR-205 - PROX1 mechanism

Colon cancer is a common cause of cancer death in the Western world. Accumulating evidence supports a protective role of estrogen via estrogen receptor beta (ERβ) but the mechanism of action is not known. Here, we elucidate a molecular mechanism whereby ERβ represses the oncogenic prospero homebox 1 (PROX1) through the upregulation of miR-205. We show that PROX1 is a potential target of miR-205 and that in clinical specimens from The Cancer Genome Atlas data, ERβ and miR-205 are decreased in colorectal cancer tissue compared to non-tumorous colon, while PROX1 levels are increased. Through mechanistic studies in multiple colorectal cancer cell lines, we show that ERβ upregulates miR-205, and that miR-205 targets and represses PROX1 through direct interaction with its 3′UTR. Through the generation of intestine-specific ERβ knockout mice, we establish that this pathway is correspondingly regulated in normal intestinal epithelial cells in vivo. Functionally, we demonstrate that miR-205 decreases cell proliferation and decreases migratory and invasive potential of colon cancer cells, leading to a reduction of micrometastasis in vivo. In conclusion, ERβ in both normal and cancerous colon epithelial cells upregulates miRNA-205, which subsequently reduces PROX1 through direct interaction with its 3′UTR. This results in reduced proliferative and metastatic potential of the cells. Our study proposes a novel pathway that may be exploited using ERβ-selective agonists and/or miR-205-replacement therapy in order to improve preventive and therapeutic approaches against colon cancer.

Lxr regulates lipid metabolic and visual perception pathways during zebrafish development

The Liver X Receptors (LXRs) play important roles in multiple metabolic pathways, including fatty acid, cholesterol, carbohydrate and energy metabolism. To expand the knowledge of the functions of LXR signaling during embryonic development, we performed a whole-genome microarray analysis of Lxr target genes in zebrafish larvae treated with either one of the synthetic LXR ligands T0901317 or GW3965. Assessment of the biological processes enriched by differentially expressed genes revealed a prime role for Lxr in regulating lipid metabolic processes, similarly to the function of LXR in mammals. In addition, exposure to the Lxr ligands induced changes in expression of genes in the neural retina and lens of the zebrafish eye, including the photoreceptor guanylate cyclase activators and lens gamma crystallins, suggesting a potential novel role for Lxr in modulating the transcription of genes associated with visual function in zebrafish. The regulation of expression of metabolic genes was phenotypically reflected in an increased absorption of yolk in the zebrafish larvae, and changes in the expression of genes involved in visual perception were associated with morphological alterations in the retina and lens of the developing zebrafish eye. The regulation of expression of both lipid metabolic and eye specific genes was sustained in 1 month old fish. The transcriptional networks demonstrated several conserved effects of LXR activation between zebrafish and mammals, and also identified potential novel functions of Lxr, supporting zebrafish as a promising model for investigating the role of Lxr during development.

Automated analysis of zebrafish images for screening toxicants

An important factor facilitating the application of zebrafish in biomedical research is high throughput screening of vertebrate animal models. For example, being able to model the growth of blood vessel in the vasculature system is interesting for understanding both the circulatory system in humans, and for facilitating large scale screening of the influence of various chemicals on vascular development. Compared to other models, the zebrafish embryo is an attractive alternative for environmental risk assessment of chemicals since it offers the possibility to perform high-throughput analyses in vivo. However the lack of an automated image analysis framework restricts high throughput screening. In this paper, we provide a method for quantitative measurements of zebrafish blood vessel morphology since it is difficult to assess changes in vessel structure by visual inspection. The method presented is generalized, i.e. it is not restricted to any specific chemically treated zebrafish, and can be used with wide variety of chemicals.