Amos Raphenya and Pearl Guo have joined the McArthur Lab! Amos graduated from McMaster with Bachelor of Computer Engineering in 2008 and joins the lab as a core software engineer, for both our drug resistance and ecotoxicogenomics projects. Pearl just finished her second year at the University of Waterloo’s Computer Science co-op program, with a minor in Bioinformatics. Pearl will be performing a 3 month co-op position in the lab, with a focus on algorithms for prediction of glycopeptide resistance.
Oxidative stress is an important mechanism of chemical toxicity, contributing to teratogenesis and to cardiovascular and neurodegenerative diseases. Developing animals may be especially sensitive to chemicals causing oxidative stress. The developmental expression and inducibility of anti-oxidant defenses through activation of NF-E2-related factor 2 (NRF2) affect susceptibility to oxidants, but the embryonic response to oxidants is not well understood. To assess the response to chemically mediated oxidative stress and how it may vary during development, zebrafish embryos, eleutheroembryos, or larvae at 1, 2, 3, 4, 5, and 6 days post fertilization (dpf) were exposed to DMSO (0.1%), tert-butylhydroquinone (tBHQ; 10 µM) or 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD; 2 nM) for 6 hr. Transcript abundance was assessed by real-time qRT-PCR and microarray. qRT-PCR showed strong (4- to 5-fold) induction of gstp1 by tBHQ as early as 1 dpf. tBHQ also induced gclc (2 dpf), but not sod1, nqo1, or cyp1a. TCDD induced cyp1a but none of the other genes. Microarray analysis showed that 1477 probes were significantly different among the DMSO-, tBHQ-, and TCDD-treated eleutheroembryos at 4 dpf. There was substantial overlap between genes induced in developing zebrafish and a set of marker genes induced by oxidative stress in mammals. Genes induced by tBHQ in 4-dpf zebrafish included those involved in glutathione synthesis and utilization, signal transduction, and DNA damage/stress response. The strong induction of hsp70 determined by microarray was confirmed by qRT-PCR and by use of transgenic zebrafish expressing enhanced green fluorescent protein (EGFP) under control of the hsp70 promoter. Genes strongly down-regulated by tBHQ included mitfa, providing a molecular explanation for the loss of pigmentation in tBHQ-exposed embryos. These data show that zebrafish embryos are responsive to oxidative stress as early as 1 dpf, that responsiveness varies with development in a gene-specific manner, and that the oxidative stress response is substantially conserved in vertebrate animals.
Smith, E.M., A.G. McArthur, M. Galus, S. Higgins, N. Kirischian, J. Jeyaranjaan, & J.Y. Wilson. 2014. Transcriptional responses of zebrafish to pharmaceutical and wastewater exposure: are single compound exposures predictive of mixtures? Keynote presentation at the Aquatic Toxicology Workshop 2014, Ottawa, Canada.
Human pharmaceuticals have been well documented in receiving waters yet their impacts on aquatic species are not clear. We have exposed adult zebrafish for 6 weeks to waterborne acetaminophen, gemfibrozil, venlafaxine, and carbamazepine at two doses (0.5 and 10 μg L-1). Fish were then exposed to a mixture of all four pharmaceuticals or wastewater effluent (5 and 25%) to assess whether transcriptional responses are similar with mixtures.. For all exposures, reproduction was significantly reduced and histopathological changes induced in kidney with at least the high dose exposure. Livers were pooled to provide sufficient RNA for microarray analyses. Hepatic transcriptional responses were determined with a modified Agilent 44K zebrafish microarry using a single channel approach. Significantly different probes were identified with a 2-way ANOVA (sex and treatment) and rank product analyses with a 10% false discovery rate. Transcriptional responses were particularly marked with acetaminophen exposure and there was broad overlap in the significant probes found between doses and across gender for this compound. 52 probes were at least 20 fold up- or down- regulated in acetaminophen exposed fish; 3 probes were 100 fold up-regulated (apolipoprotein Eb precursor, cdc73, and a hypothetical protein). Unique probes were identified for all exposures suggesting a unique transcriptional response may occur for each pharmaceutical, the pharmaceutical mixture, and wastewater effluent. Interestingly, there was almost no overlap in the transcriptional response found with single pharmaceutical exposure and either the mixture or wastewater effluent exposure. Indeed, the large transcriptional response from acetaminophen exposure was largely absent in fish exposed to the pharmaceutical mixture and wastewater effluent. This suggests that identifying individual or clusters of genes that may be useful in effects based monitoring may be difficult for pharmaceutical compounds.
The metal responsive element-binding transcription factor-1 (MTF-1) responds to changes in cellular zinc levels caused by zinc exposure or disruption of endogenous zinc homeostasis by heavy metals or oxygen-related stress. Here we report the functional characterization of a complete zebrafish MTF-1 in comparison with the previously identified isoform lacking the highly conserved cysteine-rich motif (Cys-X-Cys-Cys-X-Cys) found in all other vertebrate MTF-1 orthologs. In an effort to develop novel molecular tools, a constitutively nuclear dominant-negative MTF-1 (dnMTF-1) was generated as tool for inhibiting endogenous MTF-1 signaling. The in vivo efficacy of the dnMTF-1 was determined by microinjecting in vitro transcribed dnMTF-1 mRNA into zebrafish embryos (1-2 cell stage) followed by transcriptomic profiling using an Agilent 4x44K array on 28- and 36-hpf embryos. A total of 594 and 560 probes were identified as differentially expressed at 28 hpf and 36 hpf, respectively, with interesting overlaps between timepoints. The main categories of genes affected by the inhibition of MTF-1 signaling were: nuclear receptors and genes involved in stress signaling, neurogenesis, muscle development and contraction, eye development, and metal homeostasis, including novel observations in iron and heme homeostasis. Finally, we investigate both the transcriptional activator and transcriptional repressor role of MTF-1 in potential novel target genes identified by transcriptomic profiling during early zebrafish development.
Transcription factors in the CNC-bZIP family (NFE2, NRF1, NRF2 and NRF3) regulate genes with a wide range of functions in response to both physiological and exogenous signals, including those indicating changes in cellular redox status. Given their role in helping to maintain cellular homeostasis, it is imperative to understand the expression, regulation, and function of CNC-bZIP genes during embryonic development. We explored the expression and function of six nrf genes (nfe2, nrf1a, nrf1b, nrf2a, nrf2b, and nrf3) using zebrafish embryos as a model system. Analysis by microarray and quantitative RT-PCR showed that genes in the nrf family were expressed throughout development from oocytes to larvae. The spatial expression of nrf3 suggested a role in regulating the development of the brain, brachia and pectoral fins. Knock-down by morpholino anti-sense oligonucleotides suggested that none of the genes were necessary for embryonic viability, but nfe2 was required for proper cellular organization in the pneumatic duct and subsequent swim bladder function, as well as for proper formation of the otic vesicles. nrf genes were induced by the oxidant tert-butylhydroperoxide, and some of this response was regulated through family members Nrf2a and Nrf2b. Our results provide a foundation for understanding the role of nrf genes in normal development and in regulating the response to oxidative stress in vertebrate embryos.
Karchner, S.I., D.G. Franks, A.R. Timme-Laragy, A.G. McArthur, & M.E. Hahn. 2013. Chemical-specific oxidative stress response in zebrafish embryos. Presentation at the Pollutant Responses in Marine Organisms (PRIMO 17) Meeting, Faro, Portugal. Exposure to natural and anthropogenic stressors often leads to oxidative stress—a disruption in the regulation of intracellular redox conditions. Animals have evolved protective responses to mitigate damage caused by oxidative stress. However, the mechanisms by which the oxidative stress response is regulated during development are poorly understood. Oxidants, electrophiles, and some phenolic anti-oxidants initiate this response by activating NF-E2-related factor 2 (NRF2) and related cap’n’collar (CNC)- basic-leucine zipper (bZIP) family proteins, which bind to the anti-oxidant response element (ARE) and activate transcription of genes such as glutathione S-transferases (GST), NAD(P)H-quinone oxidoreductase (NQO1), glutamate-cysteine ligase (GCL), and superoxide dismutase (SOD). In order to determine the genes that are induced or repressed in response to oxidative stress during development, and whether there is a “core” set of oxidant responsive genes that is induced by structurally distinct activators of NRF2, zebrafish (Danio rerio) larvae (96 hours post-fertilization) were exposed to model oxidants (tert-butylhydroquinone (tBHQ), tert-butylhydroperoxide (tBOOH), diquat (DQ) or sulforaphane (SFN)) and gene expression was measured 6 hr later by microarray and Q-RT-PCR. There was a robust response to oxidative stress by all chemicals, with a total of 1281 probes significantly altered in expression. The compounds caused overlapping but distinct patterns of altered gene expression. A core set of genes responded to all oxidants. However, other genes exhibited oxidant-specific changes in expression. Principal components analysis revealed that the changes in gene expression caused by SFN, a sulfhydryl-reactive agent, were distinct from those produced by the other oxidants. The results demonstrate that the oxidative stress response in developing animals is dependent upon the nature of the oxidative stress.
Jenny, M.J., E.E. Linney, B. O’Shields, A. Holowiecki, A.G. McArthur. 2013. Global transcriptional analysis of zebrafish embryos following acute exposure to cadmium. Presentation at the Society of Toxicology 52cd Annual Meeting, San Antonio, Texas.
A.R Timme-Laragy, S.I. Karchner, R.C. Harbeitner, A.G. McArthur, M.E. Hahn. 2013. Nrf2 gene regulation during oxidative stress in embryonic development. Presentation at the Society of Toxicology 52cd Annual Meeting, San Antonio, Texas.
A 5-day PhD course in Ecotoxicology with focus on Ecotoxicogenomics and Mechanism of Toxicity in aquatic as well as terrestrial ecosystems. The course will be held at the Sven Loven Center for marine research at Kristineberg, Fiskebäckskil located on the Swedish Westcoast. During the course, Dr. McArthur will be providing both lectures and computer labs related to an “Introduction to Microarray Approaches in Ecotoxicogenomics”.
Holowiecki, A., B. O’Shields, A.G McArthur, & M.J. Jenny. 2012. Inhibition of endogenous MTF-1 signaling in zebrafish embryos identifies novel role for MTF-1 in iron and heme homeostasis. Presentation at the Society of Toxicology 51st Annual Meeting, San Francisco, California.
The MTF-1 transcription factor is considered to be a master regulator of zinc homoestasis. In an effort to develop novel molecular tools, we previously characterized a constitutively nuclear, dominant-negative zebrafish MTF-1/eGFP fusion protein (dnMTF-1). To test the in vivo effectiveness of the dnMTF-1, in vitro transcribed dnMTF-1 mRNA was microinjected into zebrafish embryos (2-cell stage) and transcriptomic profiling was performed using an Agilent 4 x 44K array on 28- and 36-hpf embryos. A total of 594 and 560 probes were identified as differentially expressed at 28 hpf and 36 hpf, respectively, with interesting overlaps between timepoints. There were several main categories of genes affected by the inhibition of MTF-1 signaling including novel observations in iron and heme homeostasis. Hepcidin, a peptide hormone that regulates iron homestasis, was not affected at 28 hpf but was significantly downregulated 2-fold at 36 hpf by inhibition of MTF-1 signaling. In contrast the major heme-binding protein, hemopexin, was significantly upregulated (3.7 and 2.6 fold) at each timepoint. To investigate the responsiveness of these two genes to an MTF-1 activator, 72 hpf zebrafish embryos were exposed to concentrations of Cd (50, 100, 150 µM) for 4 or 24 hours and gene expression was determined by real-time PCR. Interestingly, hemopexin was significantly downregulated by all Cd doses at both timepoints while hepcidin was significantly upregulated only after 24 hours of Cd exposure. 72 hpf zebrafish embryos were further exposed to hemin concentrations (50, 100, 150 µM) for 4 and 24 hours and gene expression was determined by real-time PCR. Both hepcidin and metallothionein were significantly upregulated after 4 hours of hemin exposure at the highest concentration, while hemopexin expression remained consistent. However after 24 hours of hemin exposure, hemopexin was significantly downregulated at the highest hemin concentration while hepcidin and metallothionein appeared unaffected. A search of the 3kb upstream of the transcription start site identified multiple MTF-1 binding sites within the proximal promoter of the hemopexin gene. The current observations suggest a novel role for MTF-1 as a transcriptional repressor of hemopexin in response to Cd or hemin exposure.
Increasing use of zebrafish in drug discovery and mechanistic toxicology demands knowledge of cytochrome P450 (CYP) gene regulation and function. CYP enzymes catalyze oxidative transformation leading to activation or inactivation of many endogenous and exogenous chemicals, with consequences for normal physiology and disease processes. Many CYPs potentially have roles in developmental specification, and many chemicals that cause developmental abnormalities are substrates for CYPs. Here we identify and annotate the full suite of CYP genes in zebrafish, compare these to the human CYP gene complement, and determine the expression of CYP genes during normal development. Zebrafish have a total of 94 CYP genes, distributed among 18 gene families found also in mammals. There are 32 genes in CYP families 5 to 51, most of which are direct orthologs of human CYPs that are involved in endogenous functions including synthesis or inactivation of regulatory molecules. The high degree of sequence similarity suggests conservation of enzyme activities for these CYPs, confirmed in reports for some steroidogenic enzymes (e.g. CYP19, aromatase; CYP11A, P450scc; CYP17, steroid 17a-hydroxylase), and the CYP26 retinoic acid hydroxylases. Complexity is much greater in gene families 1, 2, and 3, which include CYPs prominent in metabolism of drugs and pollutants, as well as of endogenous substrates. There are orthologous relationships for some CYP1 s and some CYP3 s between zebrafish and human. In contrast, zebrafish have 47 CYP2 genes, compared to 16 in human, with only two (CYP2R1 and CYP2U1) recognized as orthologous based on sequence. Analysis of shared synteny identified CYP2 gene clusters evolutionarily related to mammalian CYP2 s, as well as unique clusters. Transcript profiling by microarray and quantitative PCR revealed that the majority of zebrafish CYP genes are expressed in embryos, with waves of expression of different sets of genes over the course of development. Transcripts of some CYP occur also in oocytes. The results provide a foundation for the use of zebrafish as a model in toxicological, pharmacological and chemical disease research.