Pollutant Responses in Marine Organisms Meeting (PRIMO 17)

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.

Developmental Toxicology - Society of Toxicology 52cd Annual Meeting

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.

Although cadmium (Cd) exerts its toxic effects through the generation of oxidative stress, many of the cellular mechanisms involved in Cd toxicity are not fully established. Utilizing zebrafish embryos as a model, we undertook a transcriptomic-based approach to further investigate the role of Cd as a developmental toxicant and to assist in the identification of novel mechanisms of cellular dysfunction. Zebrafish embryos (24, 48, 72 and 96 hpf) were continuously exposed to Cd (25, 50, 100, 150 µM) for 24 hours prior to mortality assessment. No significant mortality was observed at any of the Cd concentrations prior to 72 hpf. However, at 96 and 120 hpf the 50 µM Cd dose resulted in 20% and 40% mortality, respectively, while the 100 and 150 µM Cd doses resulted in an average of 75% and >95% mortality, respectively. To investigate global changes in gene expression, zebrafish embryos (72, 96 and 120 hpf) were exposed to 50 µM Cd for 4 or 8 hours prior to sampling. Global gene expression profiling was performed using the Agilent 4x44K Zebrafish Oligo Microarray. Overall, 788 probes were significantly upregulated, while 679 probes were downregulated in one or more treatments. Principle component analysis was employed to find trends among the various treatments. The first principle component separated out the 96 and 120 hpf Cd-treated embryos, consistent with the mortality curves suggesting that zebrafish embryos become most sensitive to Cd toxicity around 96 hpf. The majority of genes altered by Cd exposure resulted in the upregulation of a large subset of genes responsive to oxidative stress, genes involved in glutathione synthesis and heme/iron homeostasis, mitochondrial uncoupling proteins, and various solute carriers with roles in zinc transport/homeostasis or mitochondrial oxidative phosphorylation. The majority of genes downregulated by Cd were involved in cell cycle control and DNA replication. A thorough analysis of genes differentially regulated by Cd during zebrafish development will be presented.

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.

Nrf2 is a transcription factor that regulates antioxidant defenses in response to oxidative stress. Embryonic development is highly susceptible to disruption by exposure to chemicals, including those that alter redox balance. The role of Nrf2 in the oxidative stress response (OSR) during embryonic development remains unclear. Our previous work identified a novel Nrf2 paralog, nrf2b, in zebrafish (Danio rerio). This study builds upon that work to elucidate the roles of nrf2a and nrf2b in regulating the OSR during vertebrate embryonic development. Zebrafish embryos were micro-injected with antisense morpholino oligonucleotides (MO) to knock down translation of either nrf2a or nrf2b, or a standard morpholino control (co-MO). At 48 hours post fertilization, embryos were exposed to the pro-oxidant and Nrf2 activator, tertbutylhydroquinone (tBHQ) or vehicle (DMSO) for 4 hours, and preserved for RNA isolation. Microarrays were conducted using Agilent’s V3 44k array, and selected genes validated by QPCR. In response to tBHQ, 71 probes were up-regulated in the co-MO group, including gstp1, gclc, ferritin, peroxiredoxin1, hsp70, sod1, and other genes typically found as part of the OSR. Interestingly, we found that an important and often overlooked part of the OSR is the down-regulation of genes, including cathespin, various complement components, and apolipoprotein Ea. Knockdown of Nrf2a or Nrf2b blocked some but not all of the tBHQ-induced changes in OSR gene expression and the effects of Nrf2a-MO and Nrf2b-MO were distinct. The results show that Nrf2 paralogs primarily regulate distinct gene sets, with some overlapping targets, in response to oxidative stress in embryos. This study also highlights the importance of gene down-regulation as a component of the OSR during embryonic development. 

Molecular typing of Mycobacterium tuberculosis isolates from Yaoundé reveals RIF resistance markers, clonal relatedness and mutation patterns

Authors: PV Tedom, R Kieft, AG McArthur, W Mbacham, D Mark Welch, SL Hajduk, VPK Titanji

Asian Pacific Journal of Tropical Disease, 2012, 2(5): 342-347.

Objective: To understand the spread of drug resistance and identifying diagnostic probes among the local tuberculosis (TB) strains in order to design rational control tools for tuberculosis controls. Methods: TA cloning and sequencing were used to characterize mutation associated with RIF resistance in 69 bp region of the gene, rpoB. The analysis identified two regions of mutations but no unusual insertion and deletion. No mutation was observed in RIF sensitive strains. Results: We employed Random Amplified Polymorphic DNA (RAPD) analysis for typing strains of M. tuberculosis to determine whether new strains were present among M. tuberculosis isolates circulating in Yaounde. Three groups (I to III) of M. tuberculosis were identified among 93 isolates randomly selected. RAPD analysis provided a rapid and easy means of identifying polymorphism in M. tuberculosis isolates, and it was found to be a valuable alternative epidemiological tool. RAPD was used to select the new site of diagnostic by PCR. Also single nucleotide polymorphisms between M. tuberculosis and M. bovis were found, suggesting that RAPD can be a useful technique for distinguishing between species. Conclusions: Molecular typing is defined as the integration of conventional epidemiological approach to track specific strains of pathogens in order to understand the distribution of disease in populations.

Göteborgs Universitet PhD Course in Ecotoxicogenomics and Mechanisms of Toxicity

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. See more information here.

During the course, I will be providing both lectures and computer labs related to an "Introduction to Microarray Approaches in Ecotoxicogenomics".

Sweden 2012-2013

McArthur Bioinformatics will be moving to Gothenburg, Sweden for 13 months starting July, 2012.  During that time, I will be a Guest Investigator in the Department of Biological & Environmental Sciences, University of Gothenburg in addition to my continuing consultancy work.  All business matters will continue via the company's Canadian address.  My contact information remains unchanged.

Society of Toxicology 51st Annual Meeting

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.

A small molecule discrimination map of the antibiotic resistance kinome

Authors: Shakya T, Stogios PJ, Waglechner N, Evdokimova E, Ejim L, Blanchard JE, McArthur AG, Savchenko A, Wright GD

Chemistry & Biology 2011 Dec 23;18(12):1591-601

Kinase-mediated resistance to antibiotics is a significant clinical challenge. These enzymes share a common protein fold characteristic of Ser/Thr/Tyr protein kinases. We screened 14 antibiotic resistance kinases against 80 chemically diverse protein kinase inhibitors to map resistance kinase chemical space. The screens identified molecules with both broad and narrow inhibition profiles, proving that protein kinase inhibitors offer privileged chemical matter with the potential to block antibiotic resistance. One example is the flavonol quercetin, which inhibited a number of resistance kinases in vitro and in vivo. This activity was rationalized by determination of the crystal structure of the aminoglycoside kinase APH(2″)-IVa in complex with quercetin and its antibiotic substrate kanamycin. Our data demonstrate that protein kinase inhibitors offer chemical scaffolds that can block antibiotic resistance, providing leads for co-drug design.

Nrf2b: novel zebrafish paralog of the oxidant-responsive transcription factor NF-E2-related factor 2 (NRF2)

Authors: Timme-Laragy AR, Karchner SI, Franks DG, Jenny MJ, Harbeitner RC, Goldstone JV, McArthur AG, Hahn ME

Journal of Biological Chemistry 2012 Feb 10;287(7):4609-27.

NF-E2-related factor 2 (NRF2, also called NFE2L2) and related NRF family members regulate antioxidant defenses by activating gene expression via antioxidant response elements (AREs), but their roles in embryonic development are not well understood. We report here that zebrafish (Danio rerio), an important developmental model species, possesses six nrf genes, including duplicated nrf1 and nrf2 genes. We cloned a novel zebrafish nrf2 paralog, nrf2b. The predicted Nrf2b protein sequence shares several domains with the original Nrf2 (now Nrf2a), but lacks the Neh4 transactivation domain. Zebrafish-human comparisons demonstrate conserved synteny involving nrf2 and hox genes, indicating that nrf2a and nrf2b are co-orthologs of human NRF2. nrf2a and nrf2b displayed distinct patterns of expression during embryonic development; nrf2b was more highly expressed at all stages. Embryos in which Nrf2a expression had been knocked down with morpholino oligonucleotides were more sensitive to tert-butylhydroperoxide (tBOOH) but not tert-butyl hydroquinone (tBHQ), whereas knockdown of Nrf2b did not affect sensitivity of embryos to either chemical. Gene expression profiling by microarray identified a specific role for Nrf2b as a negative regulator of several genes including p53, cyclin g1, and heme oxygenase 1 in embryos. Nrf2a and Nrf2b exhibited different mechanisms of crosstalk with the Ahr2 signaling pathway. Together, these results demonstrate distinct roles for nrf2a and nrf2b, consistent with subfunction partitioning, and identify a novel negative regulatory role for Nrf2b during development. The identification of zebrafish nrf2 co-orthologs will facilitate new understanding of the multiple roles of NRF2 in protecting vertebrate embryos from oxidative damage.

Generic Model Organism Database Meeting, Ontario Institute for Cancer Research, Toronto, Canada

A.G. McArthur, N. Waglechner, F. Nizam, M.A. Azad, K. Bhullar, M.J. Canova, G. De Pascale, L. Ejim, L. Kalan, A.M. King, K. Koteva, M. Morar, J.S. O’Brien, A.C. Pawlowski, P. Spanogiannopoulos, A.D. Sutherland, I. Tang, P.L. Taylor, M. Thaker, W. Wang, M. Yan, T. Yu, & G.D. Wright. 2011. Towards a Comprehensive Antibiotic Resistance Database (CARD). Oral presentation at the Generic Model Organism Database Meeting, Ontario Institute for Cancer Research, Toronto, Canada.

Addressing the challenge of antibiotic resistance requires the combined efforts of researchers that span inquiry at the molecular, patient, and population levels. Yet, despite the common objective of finding solutions to drug resistance, practitioners of these research areas do not often integrate their research findings. This reality reflects the disparate investigative tools, timelines and specific project aims of these disciplines. Nevertheless, resistance genes and their products are the common elements that bridge antibiotic resistance research from bench to clinic to population. What is lacking in the field is a comprehensive database of resistance genes that includes molecular, clinical, and surveillance data that can serve to unify research. The Comprehensive Antibiotic Resistance Database (CARD) is a bioinformatic database of resistance genes, their products and associated phenotypes designed to address this need by combining a Drupal front-end, an ontological-centric design, GMOD's Chado schema, and the Gbrowse genome browser.

http://arpcard.mcmaster.ca

16th International Symposium on Pollutant Response in Marine Organisms

Smith, E.M., A.G. McArthur, M. Galus, S. Higgins, N. Kirischian, J. Jeyaranjaan, & J.Y. Wilson. 2011. Transcriptional responses of zebrafish to chronic, low-dose pharmaceutical exposure. Oral presentation at the 16th International Symposium on Pollutant Response in Marine Organisms, Long Beach, California.

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) and assessed hepatic transcriptional responses using a commercial microarray. These four pharmaceuticals have been frequently found in wastewater effluent and receiving waters. For all compounds, reproduction was significantly reduced and histopathological changes induced in kidney with at least the 10 μg L-1 exposure. Livers were pooled to provide sufficient RNA for microarray and qPCR analyses. Gene expression was 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 (LIST) had 100 fold up- or down- regulated. Venlafaxine responses were few in males and carbamazepine impacted hepatic transcription the least in both sexes. There was a strong sex dependant response to both venlafaxine and carbamazepine. Unique probes were identified for all exposures suggesting a unique transcriptional response may occur for each pharmaceutical. The biological relevance of these exposures is being explored through analysis of enriched GO terms, metabolic pathways and interactome networks.