Publications by authors named "Markus Hecker"

261 Publications

Demonstration of an aggregated biomarker response approach to assess the impact of point and diffuse contaminant sources in feral fish in a small river case study.

Sci Total Environ 2021 Sep 1;804:150020. Epub 2021 Sep 1.

Department for Evolutionary Ecology and Environmental Toxicology, Goethe University, Max-von-Laue Straße 13, 60438 Frankfurt am Main, Germany; LOEWE Centre for Translational Biodiversity Genomics (LOEWE-TBG), 60325 Frankfurt am Main, Germany. Electronic address:

The assessment of the exposure of aquatic wildlife to complex environmental mixtures of chemicals originating from both point and diffuse sources and evaluating the potential impact thereof constitutes a significant step towards mitigating toxic pressure and the improvement of ecological status. In the current proof-of-concept study, we demonstrate the potential of a novel Aggregated Biomarker Response (ABR) approach involving a comprehensive set of biomarkers to identify complex exposure and impacts on wild brown trout (Salmo trutta fario). Our scenario used a small lowland river in Germany (Holtemme river in the Elbe river catchment) impacted by two wastewater treatment plants (WWTP) and diffuse agricultural runoff as a case study. The trout were collected along a pollution gradient (characterised in a parallel study) in the river. Compared to fish from the reference site upstream of the first WWTP, the trout collected downstream of the WWTPs showed a significant increase in micronucleus formation, phase I and II enzyme activities, and oxidative stress parameters in agreement with increasing exposure to various chemicals. By integrating single biomarker responses into an aggregated biomarker response, the two WWTPs' contribution to the observed toxicity could be clearly differentiated. The ABR results were supported by chemical analyses and whole transcriptome data, which revealed alterations of steroid biosynthesis and associated pathways, including an anti-androgenic effect, as some of the key drivers of the observed toxicity. Overall, this combined approach of in situ biomarker responses complemented with molecular pathway analysis allowed for a comprehensive ecotoxicological assessment of fish along the river. This study provides evidence for specific hazard potentials caused by mixtures of agricultural and WWTP derived chemicals at sublethal concentrations. Using aggregated biomarker responses combined with chemical analyses enabled an evidence-based ranking of sites with different degrees of pollution according to toxic stress and observed effects.
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http://dx.doi.org/10.1016/j.scitotenv.2021.150020DOI Listing
September 2021

A Multi-Life Stage Comparison of Silver Nanoparticles Toxicity on the Early Development of Three Canadian Fish Species.

Environ Toxicol Chem 2021 Sep 10. Epub 2021 Sep 10.

Toxicology Centre, University of Saskatchewan, Saskatoon, SK, Canada.

Information on the effects of silver nanoparticles (AgNPs) in fish have mostly been generated from standard laboratory species and short-term toxicity tests. However, there is significant uncertainty regarding AgNP toxicity to native species of concern in North America, particularly in northern freshwater ecosystems. Here, we assessed the chronic toxicity of AgNPs in early life stages of three North American fish species: rainbow trout (Oncorhynchus mykiss), lake trout (Salvelinus namaycush) and northern pike (Esox lucius). Newly fertilized embryos were exposed to nominal aqueous concentrations of 0.1, 0.3, 1.0, 3.0, 10.0, or 30.0 nM AgNPs for 126 (rainbow trout), 210 (lake trout), and 25 (northern pike) days. Endpoints included cumulative developmental time ( C x day or degree-days to 50% life stage transition), mortality, fork length, embryonic malformations, cumulative survival, and histopathology of gill and liver in larvae/alevins. Results showed life stage-specific differences in responses, with endpoints during the embryonic stage occurring more often and at lower concentrations, compared to larval/alevin and juvenile stages. Sensitivities among species were highly dependent upon the endpoints measured although developmental time appeared to be the most consistent endpoint across species. At embryonic and larval/alevin stages, northern pike was the most sensitive species (lowest observable effect concentration of 0.1 nM using developmental time). Rainbow trout displayed similar responses to lake trout across multiple endpoints and therefore seems to be an adequate surrogate for trout species in ecotoxicology studies. Moreover, while mortality during individual life stages was not generally affected, the cumulative mortality across life stages was significantly affected, which highlights the importance of chronic, multi-life stage studies. This article is protected by copyright. All rights reserved.
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http://dx.doi.org/10.1002/etc.5210DOI Listing
September 2021

Substrate Stiffness Influences Structural and Functional Remodeling in Induced Pluripotent Stem Cell-Derived Cardiomyocytes.

Front Physiol 2021 19;12:710619. Epub 2021 Aug 19.

Division of Cardiovascular Physiology, Institute of Physiology and Pathophysiology, Heidelberg University, Heidelberg, Germany.

Novel treatment strategies for cardiac tissue regeneration are heading for the use of engineered cardiac tissue made from induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs). Despite the proven cardiogenic phenotype of these cells, a significant lack of structural and functional properties of mature myocytes prevents safe integration into the diseased heart. To date, maturation processes of cardiomyocytes remain largely unknown but may comprise biophysical cues from the immediate cell environment. Mechanosensing is one critical ability of cells to react to environmental changes. Accordingly, the surrounding substrate stiffness, comprised of extracellular matrix (ECM), cells, and growth surface, critically influences the myocyte's physiology, as known from deleterious remodeling processes in fibrotic hearts. Conversely, the mechanical properties during culture of iPSC-CMs may impact on their structural and functional maturation. Here, we tested the hypothesis that the environmental stiffness influences structural and functional properties of iPSC-CMs and investigated the effect of different substrate stiffnesses on cell contractility, excitation-contraction (EC) coupling, and intercellular coupling. Culture surfaces with defined stiffnesses ranging from rigid glass with 25GPa to PDMS of physiological softness were coated with ECM proteins and seeded with murine iPSC-CMs. Using confocal imaging, cardiac protein expression was assessed. Ca handling and contractile properties were analyzed on different substrate stiffnesses. Intercellular coupling gap junctions was investigated by fluorescence recovery after photobleaching (FRAP). Our data revealed greater organization of L-type Ca channels and ryanodine receptors and increased EC-coupling gain, demonstrating structural and functional maturation in cells grown on soft surfaces. In addition, increased shortening and altered contraction dynamics revealed increased myofilament Ca sensitivity in phase-plane loops. Moreover, connexin 43 expression was significantly increased in iPSC-CMs grown on soft surfaces leading to improved intercellular coupling. Taken together, our results demonstrate that soft surfaces with stiffnesses in the physiological range improve the expression pattern and interaction of cardiac proteins relevant for EC-coupling. In parallel, soft substrates influence contractile properties and improve intercellular coupling in iPSC-CMs. We conclude that the mechanical stiffness of the cell environment plays an important role in driving iPSC-CMs toward further maturation by inducing adaptive responses.
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http://dx.doi.org/10.3389/fphys.2021.710619DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8416903PMC
August 2021

Loss of Nfat5 promotes lipid accumulation in vascular smooth muscle cells.

FASEB J 2021 09;35(9):e21831

Department of Cardiovascular Physiology, Institute of Physiology and Pathophysiology, Heidelberg University, Heidelberg, Germany.

The nuclear factor of activated T-cells 5 (NFAT5) is a transcriptional regulator of macrophage activation and T-cell development, which controls stabilizing responses of cells to hypertonic and biomechanical stress. In this study, we detected NFAT5 in the media layer of arteries adjacent to human arteriosclerotic plaques and analyzed its role in vascular smooth muscle cells (VSMCs) known to contribute to arteriosclerosis through the uptake of lipids and transformation into foam cells. Exposure of both human and mouse VSMCs to cholesterol stimulated the nuclear translocation of NFAT5 and increased the expression of the ATP-binding cassette transporter Abca1, required to regulate cholesterol efflux from cells. Loss of Nfat5 promoted cholesterol accumulation in these cells and inhibited the expression of genes involved in the management of oxidative stress or lipid handling, such as Sod1, Plin2, Fabp3, and Ppard. The functional relevance of these observations was subsequently investigated in mice fed a high-fat diet upon induction of a smooth muscle cell-specific genetic ablation of Nfat5 (Nfat5 ). Under these conditions, Nfat5 but not Nfat5 mice developed small, focal lipid-rich lesions in the aorta after 14 and 25 weeks, which were formed by intracellular lipid droplets deposited in the sub-intimal VSMCs layer. While known for being activated by external stimuli, NFAT5 was found to mediate the expression of VSMC genes associated with the handling of lipids in response to a cholesterol-rich environment. Failure of this protective function may promote the formation of lipid-laden arterial VSMCs and pro-atherogenic vascular responses.
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http://dx.doi.org/10.1096/fj.202100682RDOI Listing
September 2021

Toxicokinetic Models for Bioconcentration of Organic Contaminants in Two Life Stages of White Sturgeon ().

Environ Sci Technol 2021 09 12;55(17):11590-11600. Epub 2021 Aug 12.

Toxicology Centre, University of Saskatchewan, Saskatoon, Saskatchewan S7N 5B3, Canada.

The white sturgeon () is an endangered ancient fish species that is known to be particularly sensitive to certain environmental contaminants, partly because of the uptake and subsequent toxicity of lipophilic pollutants prone to bioconcentration as a result of their high lipid content. To better understand the bioconcentration of organic contaminants in this species, toxicokinetic (TK) models were developed for the embryo-larval and subadult life stages. The embryo-larval model was designed as a one-compartment model and validated using whole-body measurements of benzo[]pyrene (B[]P) metabolites from a waterborne exposure to B[]P. A physiologically based TK (PBTK) model was used for the subadult model. The predictive power of the subadult model was validated with an experimental data set of four chemicals. Results showed that the TK models could accurately predict the bioconcentration of organic contaminants for both life stages of white sturgeon within 1 order of magnitude of measured values. These models provide a tool to better understand the impact of environmental contaminants on the health and the survival of endangered white sturgeon populations.
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http://dx.doi.org/10.1021/acs.est.0c06867DOI Listing
September 2021

RGS5 Attenuates Baseline Activity of ERK1/2 and Promotes Growth Arrest of Vascular Smooth Muscle Cells.

Cells 2021 Jul 11;10(7). Epub 2021 Jul 11.

Department of Cardiovascular Physiology, Institute of Physiology and Pathophysiology, Heidelberg University, 69120 Heidelberg, Germany.

The regulator of G-protein signaling 5 (RGS5) acts as an inhibitor of Gα and Gα activity in vascular smooth muscle cells (VSMCs), which regulate arterial tone and blood pressure. While RGS5 has been described as a crucial determinant regulating the VSMC responses during various vascular remodeling processes, its regulatory features in resting VSMCs and its impact on their phenotype are still under debate and were subject of this study. While shows a variable expression in mouse arteries, neither global nor SMC-specific genetic ablation of affected the baseline blood pressure yet elevated the phosphorylation level of the MAP kinase ERK1/2. Comparable results were obtained with 3D cultured resting VSMCs. In contrast, overexpression of RGS5 in 2D-cultured proliferating VSMCs promoted their resting state as evidenced by microarray-based expression profiling and attenuated the activity of Akt- and MAP kinase-related signaling cascades. Moreover, overexpression attenuated ERK1/2 phosphorylation, VSMC proliferation, and migration, which was mimicked by selectively inhibiting Gα but not Gα activity. Collectively, the heterogeneous expression of suggests arterial blood vessel type-specific functions in mouse VSMCs. This comprises inhibition of acute agonist-induced Gα/calcium release as well as the support of a resting VSMC phenotype with low ERK1/2 activity by suppressing the activity of Gα.
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http://dx.doi.org/10.3390/cells10071748DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8306326PMC
July 2021

Inverse Regulation of Confluence-Dependent ADAMTS13 and von Willebrand Factor Expression in Human Endothelial Cells.

Thromb Haemost 2021 Aug 5. Epub 2021 Aug 5.

Division of Cardiovascular Physiology, Institute of Physiology and Pathophysiology, Heidelberg University, Heidelberg, Germany.

ADAMTS13 (a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13) is a zinc-containing metalloprotease also known as von Willebrand factor (vWF)-cleaving protease. Low ADAMTS13 plasma levels are associated with an increased risk of arterial thrombosis, including myocardial infarction and cerebrovascular disease. The expression and regulation of this metalloprotease in human endothelial cells have not been systematically investigated. In this study, we demonstrate that ADAMTS13 expression is inhibited by proinflammatory cytokines tumor necrosis factor-α and interferon-γ as well as by CD40 ligand, which was hitherto unknown. Factors protecting against atherosclerosis such as exposure to continuous unidirectional shear stress, interleukin-10, or different HMG-CoA reductase inhibitors like, e.g., simvastatin, atorvastatin, or rosuvastatin, did not influence ADAMTS13 expression. Unidirectional periodic orbital shear stress, mimicking oscillatory flow conditions found at atherosclerosis-prone arterial bifurcations, had also no effect. In contrast, a reciprocal correlation between ADAMTS13 and vWF expression in endothelial cells depending on the differentiation state was noted. ADAMTS13 abundance significantly rose on both the mRNA and intracellular protein level and also tethered to the endothelial glycocalyx with the degree of confluency while vWF protein levels were highest in proliferating cells but significantly decreased upon reaching confluence. This finding could explain the anti-inflammatory and antithrombotic phenotype of dormant endothelial cells mediated by contact inhibition.
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http://dx.doi.org/10.1055/s-0041-1733800DOI Listing
August 2021

Remobilization of pollutants during extreme flood events poses severe risks to human and environmental health.

J Hazard Mater 2021 Jul 19;421:126691. Epub 2021 Jul 19.

Department of Evolutionary Ecology and Environmental Toxicology, Institute of Ecology, Evolution and Diversity, Goethe University Frankfurt, Frankfurt am Main, Germany; LOEWE Centre for Translational Biodiversity Genomics (LOEWE-TBG), Frankfurt am Main, Germany. Electronic address:

While it is well recognized that the frequency and intensity of flood events are increasing worldwide, the environmental, economic, and societal consequences of remobilization and distribution of pollutants during flood events are not widely recognized. Loss of life, damage to infrastructure, and monetary cleanup costs associated with floods are important direct effects. However, there is a lack of attention towards the indirect effects of pollutants that are remobilized and redistributed during such catastrophic flood events, particularly considering the known toxic effects of substances present in flood-prone areas. The global examination of floods caused by a range of extreme events (e.g., heavy rainfall, tsunamis, extra- and tropical storms) and subsequent distribution of sediment-bound pollutants are needed to improve interdisciplinary investigations. Such examinations will aid in the remediation and management action plans necessary to tackle issues of environmental pollution from flooding. River basin-wide and coastal lowland action plans need to balance the opposing goals of flood retention, catchment conservation, and economical use of water.
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http://dx.doi.org/10.1016/j.jhazmat.2021.126691DOI Listing
July 2021

Using Transcriptomics and Metabolomics to Understand Species Differences in Sensitivity to Chlorpyrifos in Japanese Quail and Double-Crested Cormorant Embryos.

Environ Toxicol Chem 2021 Jul 22. Epub 2021 Jul 22.

Ecotoxicology and Wildlife Health Division, Environment Canada, National Wildlife Research Centre, Carleton University, Ottawa, Ontario, Canada.

Modern 21st-century toxicity testing makes use of omics technologies to address critical questions in toxicology and chemical management. Of interest are questions relating to chemical mechanisms of toxicity, differences in species sensitivity, and translation of molecular effects to observable apical endpoints. Our study addressed these questions by comparing apical outcomes and multiple omics responses in early-life stage exposure studies with Japanese quail (Coturnix japonica) and double-crested cormorant (Phalacrocorax auritus), representing a model and ecological species, respectively. Specifically, we investigated the dose-dependent response of apical outcomes as well as transcriptomics and metabolomics in the liver of each species exposed to chlorpyrifos, a widely used organophosphate pesticide. Our results revealed a clear pattern of dose-dependent disruption of gene expression and metabolic profiles in Japanese quail but not double-crested cormorant at similar chlorpyrifos exposure concentrations. The difference in sensitivity between species was likely due to higher metabolic transformation of chlorpyrifos in Japanese quail compared to double-crested cormorant. The most impacted biological pathways after chlorpyrifos exposure in Japanese quail included hepatic metabolism, oxidative stress, endocrine disruption (steroid and nonsteroid hormones), and metabolic disease (lipid and fatty acid metabolism). Importantly, we show consistent responses across biological scales, suggesting that significant disruption at the level of gene expression and metabolite profiles leads to observable apical responses at the organism level. Our study demonstrates the utility of evaluating effects at multiple biological levels of organization to understand how modern toxicity testing relates to outcomes of regulatory relevance, while also highlighting important, yet poorly understood, species differences in sensitivity to chemical exposure. Environ Toxicol Chem 2021;00:1-15. © 2021 SETAC.
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http://dx.doi.org/10.1002/etc.5174DOI Listing
July 2021

Assessing the Toxicity of 17α-Ethinylestradiol in Rainbow Trout Using a 4-Day Transcriptomics Benchmark Dose (BMD) Embryo Assay.

Environ Sci Technol 2021 08 22;55(15):10608-10618. Epub 2021 Jul 22.

Toxicology Centre, University of Saskatchewan, Saskatoon, Saskatchewan, Canada S7N 5B3.

There is an urgent demand for more efficient and ethical approaches in ecological risk assessment. Using 17α-ethinylestradiol (EE2) as a model compound, this study established an embryo benchmark dose (BMD) assay for rainbow trout (RBT; ) to derive transcriptomic points-of-departure (tPODs) as an alternative to live-animal tests. Embryos were exposed to graded concentrations of EE2 (measured: 0, 1.13, 1.57, 6.22, 16.3, 55.1, and 169 ng/L) from hatch to 4 and up to 60 days post-hatch (dph) to assess molecular and apical responses, respectively. Whole proteome analyses of alevins did not show clear estrogenic effects. In contrast, transcriptomics revealed responses that were in agreement with apical effects, including excessive accumulation of intravascular and hepatic proteinaceous fluid and significant increases in mortality at 55.1 and 169 ng/L EE2 at later time points. Transcriptomic BMD analysis estimated the median of the 20th lowest geneBMD to be 0.18 ng/L, the most sensitive tPOD. Other estimates (0.78, 3.64, and 1.63 ng/L for the 10th percentile geneBMD, first peak geneBMD distribution, and median geneBMD of the most sensitive over-represented pathway, respectively) were within the same order of magnitude as empirically derived apical PODs for EE2 in the literature. This 4-day alternative RBT embryonic assay was effective in deriving tPODs that are protective of chronic effects of EE2.
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http://dx.doi.org/10.1021/acs.est.1c02401DOI Listing
August 2021

A Novel Multispecies Toxicokinetic Modeling Approach in Support of Chemical Risk Assessment.

Environ Sci Technol 2021 07 24;55(13):9109-9118. Epub 2021 Jun 24.

Toxicology Centre, University of Saskatchewan, Saskatoon S7N 5B3, Canada.

Standardized laboratory tests with a limited number of model species are a key component of chemical risk assessments. These surrogate species cannot represent the entire diversity of native species, but there are practical and ethical objections against testing chemicals in a large variety of species. In previous research, we have developed a multispecies toxicokinetic model to extrapolate chemical bioconcentration across species by combining single-species physiologically based toxicokinetic (PBTK) models. This "top-down" approach was limited, however, by the availability of fully parameterized single-species models. Here, we present a "bottom-up" multispecies PBTK model based on available data from 69 freshwater fishes found in Canada. Monte Carlo-like simulations were performed using statistical distributions of model parameters derived from these data to predict steady-state bioconcentration factors (BCFs) for a set of well-studied chemicals. The distributions of predicted BCFs for 1,4-dichlorobenzene and dichlorodiphenyltrichloroethane largely overlapped those of empirical data, although a tendency existed toward overestimation of measured values. When expressed as means, predicted BCFs for 26 of 34 chemicals (82%) deviated by less than 10-fold from measured data, indicating an accuracy similar to that of previously published single-species models. This new model potentially enables more environmentally relevant predictions of bioconcentration in support of chemical risk assessments.
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http://dx.doi.org/10.1021/acs.est.1c02055DOI Listing
July 2021

AAV-mediated expression of NFAT decoy oligonucleotides protects from cardiac hypertrophy and heart failure.

Basic Res Cardiol 2021 06 4;116(1):38. Epub 2021 Jun 4.

Department of Internal Medicine III, University Hospital Schleswig-Holstein and University of Kiel , Arnold-Heller-Str. 3 , Kiel, Germany.

Previous studies have underlined the substantial role of nuclear factor of activated T cells (NFAT) in hypertension-induced myocardial hypertrophy ultimately leading to heart failure. Here, we aimed at neutralizing four members of the NFAT family of transcription factors as a therapeutic strategy for myocardial hypertrophy transiting to heart failure through AAV-mediated cardiac expression of a RNA-based decoy oligonucleotide (dON) targeting NFATc1-c4. AAV-mediated dON expression markedly decreased endothelin-1 induced cardiomyocyte hypertrophy in vitro and resulted in efficient expression of these dONs in the heart of adult mice as evidenced by fluorescent in situ hybridization. Cardiomyocyte-specific dON expression both before and after induction of transverse aortic constriction protected mice from development of cardiac hypertrophy, cardiac remodeling, and heart failure. Singular systemic administration of AAVs enabling a cell-specific expression of dONs for selective neutralization of a given transcription factor may thus represent a novel and powerful therapeutic approach.
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http://dx.doi.org/10.1007/s00395-021-00880-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8178147PMC
June 2021

Trophic dynamics of selenium in a boreal lake food web.

Environ Pollut 2021 Jul 17;280:116956. Epub 2021 Mar 17.

Toxicology Centre, University of Saskatchewan, Saskatoon, Saskatchewan, Canada; Department of Veterinary Biomedical Sciences, University of Saskatchewan, Saskatoon, Saskatchewan, Canada.

Selenium (Se) is both an essential micronutrient and a contaminant of concern that is of particular interest in mining-influenced waterbodies in Canada. The objective of this research was to characterize the trophic dynamics of selenium along a gradient of exposure concentrations in a Canadian boreal lake ecosystem. From June 20 to August 22, 2018, six limnocorrals (littoral, ∼3000 L enclosures) were spiked with mean measured concentrations of 0.4, 0.8, 1.6, 3.4, 5.6 and 7.9 μg Se/L as selenite, and three limnocorrals served as untreated controls (background aqueous Se = 0.08-0.09 μg/L). Total Se (TSe) concentrations in water, periphyton, phytoplankton, sediment, benthic macroinvertebrates, zooplankton and female finescale dace (Phoxinus neogaeus; added on day 21 of the experiment) were measured throughout and at the end of the experiment. Total Se bioaccumulation by organisms was generally non-linear. Greater uptake by phytoplankton than periphyton was observed. Taxonomic differences in accumulation of TSe by invertebrates (Heptageniidae = Chironomidae > zooplankton) were observed as well. Fish muscle and ovary tissue TSe bioaccumulation was more variable than that at lower trophic levels and uptake patterns indicated that fish did not reach steady state concentrations. This research provides field-derived models for the uptake of Se by algae and invertebrates, and contributes to a better understanding of the dynamics of TSe bioaccumulation over a gradient of exposure concentrations in cold-water lentic systems.
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http://dx.doi.org/10.1016/j.envpol.2021.116956DOI Listing
July 2021

Alginate hydrogel polymers enable efficient delivery of a vascular-targeted AAV vector into aortic tissue.

Mol Ther Methods Clin Dev 2021 Jun 24;21:83-93. Epub 2021 Feb 24.

Department of Internal Medicine III, University of Kiel, Kiel, Germany.

Gene therapeutic approaches to aortic diseases require efficient vectors and delivery systems for transduction of endothelial cells (ECs) and smooth muscle cells (SMCs). Here, we developed a novel strategy to efficiently deliver a previously described vascular-specific adeno-associated viral (AAV) vector to the abdominal aorta by application of alginate hydrogels. To efficiently transduce ECs and SMCs, we used AAV9 vectors with a modified capsid (AAV9SLR) encoding enhanced green fluorescent protein (EGFP), as wild-type AAV vectors do not transduce ECs and SMCs well. AAV9SLR vectors were embedded into a solution containing sodium alginate and polymerized into hydrogels. Gels were surgically implanted around the adventitia of the infrarenal abdominal aorta of adult mice. Three weeks after surgery, an almost complete transduction of both the endothelium and tunica media adjacent to the gel was demonstrated in tissue sections. Hydrogel-mediated delivery resulted in induction of neutralizing antibodies but did not cause inflammatory responses in serum or the aortic wall. To further determine the translational potential, aortic tissue from patients was embedded into AAV9SLR-containing hydrogel, and efficient transduction could be confirmed. These findings demonstrate that alginate hydrogel harboring a vascular-targeting AAV9SLR vector allows efficient local transduction of the aortic wall.
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http://dx.doi.org/10.1016/j.omtm.2021.02.017DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7973147PMC
June 2021

Development of a Comprehensive Toxicity Pathway Model for 17α-Ethinylestradiol in Early Life Stage Fathead Minnows ().

Environ Sci Technol 2021 04 23;55(8):5024-5036. Epub 2021 Mar 23.

Toxicology Centre, University of Saskatchewan, Saskatoon, Saskatchewan S7N 5B3, Canada.

There is increasing pressure to develop alternative ecotoxicological risk assessment approaches that do not rely on expensive, time-consuming, and ethically questionable live animal testing. This study aimed to develop a comprehensive early life stage toxicity pathway model for the exposure of fish to estrogenic chemicals that is rooted in mechanistic toxicology. Embryo-larval fathead minnows (FHM; ) were exposed to graded concentrations of 17α-ethinylestradiol (water control, 0.01% DMSO, 4, 20, and 100 ng/L) for 32 days. Fish were assessed for transcriptomic and proteomic responses at 4 days post-hatch (dph), and for histological and apical end points at 28 dph. Molecular analyses revealed core responses that were indicative of observed apical outcomes, including biological processes resulting in overproduction of vitellogenin and impairment of visual development. Histological observations indicated accumulation of proteinaceous fluid in liver and kidney tissues, energy depletion, and delayed or suppressed gonad development. Additionally, fish in the 100 ng/L treatment group were smaller than controls. Integration of omics data improved the interpretation of perturbations in early life stage FHM, providing evidence of conservation of toxicity pathways across levels of biological organization. Overall, the mechanism-based embryo-larval FHM model showed promise as a replacement for standard adult live animal tests.
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http://dx.doi.org/10.1021/acs.est.0c05942DOI Listing
April 2021

AAV-mediated AP-1 decoy oligonucleotide expression inhibits aortic elastolysis in a mouse model of marfan syndrome.

Cardiovasc Res 2021 Jan 20. Epub 2021 Jan 20.

Institute of Physiology and Pathophysiology, Heidelberg University, Germany.

Aims: Marfan syndrome is one of the most common inherited disorders of connective tissue caused by fibrillin-1 mutations, characterized by enhanced transcription factor AP-1 DNA binding activity and subsequently abnormally increased expression and activity of matrix-metalloproteinases (MMPs). We aimed to establish a novel adeno-associated virus (AAV)-based strategy for long-term expression of an AP-1 neutralising RNA hairpin (hp) decoy oligonucleotide (dON) in the aorta to prevent aortic elastolysis in a murine model of Marfan syndrome.

Methods And Results: Using fibrillin-1 hypomorphic mice (mgR/mgR), aortic grafts from young (9 weeks old) donor mgR/mgR mice were transduced ex vivo with AAV vectors and implanted as infrarenal aortic interposition grafts in mgR/mgR mice. Grafts were explanted after 30 days. For in vitro studies isolated primary aortic smooth muscle cells from mgR/mgR mice were used. Elastica-van-Giesson staining visualized elastolysis, ROS production was assessed using DHE staining. RNA F.I.S.H. verified AP-1 hp dON generation in the ex vivo transduced aortic tissue. MMP expression and activity were assessed by western blotting and immunoprecipitation combined with zymography.Transduction resulted in stable therapeutic dON expression in endothelial and smooth muscle cells. MMP expression and activity, ROS formation as well as expression of monocyte chemoattractant protein-1 were significantly reduced. Monocyte graft infiltration declined and the integrity of the elastin architecture was maintained. RNAseq analyzis confirmed the beneficial effect of AP-1 neutralisation on the pro-inflammatory environment in smooth muscle cells.

Conclusions: This novel approach protects from deterioration of aortic stability by sustained delivery of nucleic acids-based therapeutics and further elucidated how to interfere with the mechanism of elastolysis.

Translational Perspective: This study provides a novel single treatment option to achieve long-term expression of a transcription factor AP-1 neutralising decoy oligonucleotide in the aorta of mgR/mgR mice with the potential to prevent life-threatening elastolysis and aortic complications.
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http://dx.doi.org/10.1093/cvr/cvab012DOI Listing
January 2021

Endothelial cells control vascular smooth muscle cell cholesterol levels by regulating 24-dehydrocholesterol reductase expression.

Exp Cell Res 2021 02 7;399(2):112446. Epub 2021 Jan 7.

Institute of Physiology and Pathophysiology, Department of Cardiovascular Physiology, Heidelberg University, Germany; European Center for Angioscience (ECAS), Medical Faculty Mannheim, Heidelberg University, Germany. Electronic address:

Communication of vascular cells is essential for the control of organotypic functions of blood vessels. In this context, vascular endothelial cells (EC) act as potent regulators of vascular smooth muscle cell (VSMC) functions such as contraction and relaxation. However, the impact of ECs on the gene expression pattern of VSMCs is largely unknown. Here, we investigated changes of the VSMC transcriptome by utilizing 3D human vascular organoids organized as a core of VSMCs enclosed by a monolayer of ECs. Microarray-based analyses indicated that interaction with ECs for 48 h down-regulates expression of genes in VSMCs controlling rate-limiting steps of the cholesterol biosynthesis such as HMGCR, HMGCS1, DHCR24 and DHCR7. Protein analyses revealed a decrease in the abundance of DHCR24 (24-dehydrocholesterol reductase) and lower cholesterol levels in VSMCs co-cultured with ECs. On the functional level, the blockade of the DHCR24 activity impaired adhesion, migration and proliferation of VSMCs. Collectively, these findings indicate that ECs have the capacity to instruct VSMCs to shut down the expression of DHCR24 thereby limiting their cholesterol biosynthesis, which may support their functional steady state.
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http://dx.doi.org/10.1016/j.yexcr.2020.112446DOI Listing
February 2021

Identification of molecular toxicity pathways across early life-stages of zebrafish exposed to PCB126 using a whole transcriptomics approach.

Ecotoxicol Environ Saf 2021 Jan 11;208:111716. Epub 2020 Dec 11.

Institute for Environmental Research, RWTH Aachen University, Worringerweg 1, 52074 Aachen, Germany; Department of Evolutionary Ecology and Environmental Toxicology, Goethe University Frankfurt, Max-von-Laue-Str. 13, 60438 Frankfurt am Main, Germany.

Although withdrawn from the market in the 1980s, polychlorinated biphenyls (PCBs) are still found ubiquitously in the aquatic environment and pose a serious risk to biota due to their teratogenic potential. In fish, early life-stages are often considered most sensitive with regard to their exposure to PCBs and other dioxin-like compounds. However, little is known about the molecular drivers of the frequently observed teratogenic effects. Therefore, the aims of our study were to: (1) characterize the baseline transcriptome profiles at different embryonic life-stages in zebrafish (Danio rerio); and (2) to identify the molecular response to PCB exposure and life-stage specific-effects of the chemical on associated processes. For both objectives, embryos were sampled at 12, 48, and 96 h post-fertilization (hpf) and subjected to Illumina sequence-by-synthesis and RNAseq analysis. Results revealed that with increasing age more genes and related pathways were upregulated both in terms of number and magnitude. Yet, other transcripts followed an opposite pattern with greater transcript abundance at the earlier time points. Additionally, embryos were exposed to PCB126, a potent agonist of the aryl hydrocarbon receptor (AHR). ClueGO network analysis revealed significant enrichment of genes associated with basic cell metabolism, communication, and homeostasis as well as eye development, muscle formation, and skeletal formation. We selected eight genes involved in the affected pathways for an in-depth characterization of their regulation throughout normal embryogenesis and after exposure to PCB126 by quantification of transcript abundances every 12 h until 118 hpf. Among these, fgf7 and c9 stood out because of their strong upregulation by PCB126 exposure at 48 and 96 hpf, respectively. Cyp2aa12 was upregulated from 84 hpf on. Fabp10ab, myhz1.1, col8a1a, sulf1, and opn1sw1 displayed specific regulation depending on the developmental stage. Overall, we demonstrate that (1) the developmental transcriptome of zebrafish is highly dynamic, and (2) dysregulation of gene expression by exposure to PCB126 was significant and in several cases not directly connected to AHR-signaling. Hence, this study improves the understanding of linkages between molecular events and apical outcomes that are of regulatory relevance.
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http://dx.doi.org/10.1016/j.ecoenv.2020.111716DOI Listing
January 2021

Differences in the subcellular distribution of cadmium and copper in the gills and liver of white sturgeon (Acipenser transmontanus) and rainbow trout (Oncorhynchus mykiss).

Chemosphere 2021 Feb 1;265:129142. Epub 2020 Dec 1.

Toxicology Centre, University of Saskatchewan, Saskatoon, SK, S7N 5B3, Canada; Department of Biology, University of Saskatchewan, Saskatoon, SK, S7N 5E2, Canada.

Recent studies have shown that white sturgeon (Acipenser transmontanus) are more resistant to cadmium (Cd) compared to rainbow trout (Oncorhynchus mykiss), whereas they are more sensitive than rainbow trout when exposed to copper (Cu). Differences in the subcellular distribution of metals among species could be one of the factors responsible for the differences in the sensitivity to metals. Although, subcellular distribution has been studied extensively in many species with many metals, its direct role in species-specific differences in the sensitivity has not been well studied. The objective of this study was to evaluate the role of subcellular distribution of metals in species-specific differences in the sensitivity to metals between sturgeon and trout. We compared the subcellular distribution of metals Cd and Cu in the cellular debris, heat-stable proteins, heat-denatured fraction, metal-rich granules, and organelles fractions from the gills and liver after exposure of juveniles of both species to 1.25 and 20 μg/L Cd and Cu for 8 days, respectively. Sturgeon diverted a higher amount of Cd towards biologically inactive metal pool (BIM) and a lower amount towards the biologically active metal pool (BAM) compared to trout in both tissues. This explained why sturgeon are able to tolerate a relatively higher exposure level to Cd compared to trout. For Cu, there was no statistically significant species-specific differences in the amounts diverted towards either BAM or BIM; hence, white sturgeon's greater sensitivity to Cu was not explained by its subcellular distribution strategies.
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http://dx.doi.org/10.1016/j.chemosphere.2020.129142DOI Listing
February 2021

Effects on Apical Outcomes of Regulatory Relevance of Early-Life Stage Exposure of Double-Crested Cormorant Embryos to 4 Environmental Chemicals.

Environ Toxicol Chem 2021 02 18;40(2):390-401. Epub 2020 Dec 18.

Department of Natural Resource Sciences, McGill University, Montreal, Quebec, Canada.

Environmental risk assessment is often challenged by a lack of toxicity data for ecological species. The overall goal of the present study was to employ an avian early-life stage toxicity test to determine the effects of 4 chemicals (benzo[a]pyrene [BaP], chlorpyrifos, fluoxetine hydrochloride [FLX], and ethinyl estradiol [EE2]) on an ecologically relevant avian species, the double-crested cormorant (Phalacrocorax auritus), and to compare our results with those we previously reported for a laboratory model species, Japanese quail. Chemicals were dissolved in dimethyl sulfoxide and administered via air cell injection to fertilized, unincubated double-crested cormorant eggs at 3 nominal concentrations, the highest selected to approximate the 20% lethal dose. Of the 4 chemicals, only chlorpyrifos and FLX were detected in liver tissue of embryos at midincubation (day 14) and termination (day 26; 1-2 d prior to hatch); EE2 and BaP were not detectable, suggesting embryonic clearance/metabolism. No apical effects were observed in double-crested cormorant embryos up to the highest concentrations of chlorpyrifos (no-observed-effect level [NOEL] = 25 µg/g) or FLX (NOEL = 18 µg/g). Exposure to EE2 reduced embryonic viability and increased deformities at a concentration of 2.3 µg/g (NOEL = 0.18 µg/g), and BaP decreased embryonic viability (median lethal dose = 0.015 µg/g; NOEL = 0.0027 µg/g). Compared with Japanese quail, double-crested cormorant were more sensitive with regard to embryolethality and deformities for EE2 and embryolethality for BaP, whereas they were less sensitive to embryonic deformities associated with chlorpyrifos exposure. These data reinforce the idea that standardized toxicity tests using a laboratory model species may not always be protective of wild birds, and thus they stress the importance of developing such alternative testing strategies (e.g., the EcoToxChip Project) for ecologically relevant species to augment risk assessment efforts. Environ Toxicol Chem 2021;40:390-401. © 2020 SETAC.
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http://dx.doi.org/10.1002/etc.4922DOI Listing
February 2021

Drivers of and Obstacles to the Adoption of Toxicogenomics for Chemical Risk Assessment: Insights from Social Science Perspectives.

Environ Health Perspect 2020 10 28;128(10):105002. Epub 2020 Oct 28.

University of Sydney Business School and University of Sydney Nano Institute, Sydney, New South Wales, Australia; Department of Chemistry, Faculty of Science, McGill University, Montreal, Quebec, Canada.

Background: Some 20 y ago, scientific and regulatory communities identified the potential of omics sciences (genomics, transcriptomics, proteomics, metabolomics) to improve chemical risk assessment through development of toxicogenomics. Recognizing that regulators adopt new scientific methods cautiously given accountability to diverse stakeholders, the scope and pace of adoption of toxicogenomics tools and data have nonetheless not met the ambitious, early expectations of omics proponents.

Objective: Our objective was, therefore, to inventory, investigate, and derive insights into drivers of and obstacles to adoption of toxicogenomics in chemical risk assessment. By invoking established social science frameworks conceptualizing innovation adoption, we also aimed to develop recommendations for proponents of toxicogenomics and other new approach methodologies (NAMs).

Methods: We report findings from an analysis of 56 scientific and regulatory publications from 1998 through 2017 that address the adoption of toxicogenomics for chemical risk assessment. From this purposeful sample of toxicogenomics discourse, we identified major categories of drivers of and obstacles to adoption of toxicogenomics tools and data sets. We then mapped these categories onto social science frameworks for conceptualizing innovation adoption to generate actionable insights for proponents of toxicogenomics.

Discussion: We identify the most salient drivers and obstacles. From 1998 through 2017, adoption of toxicogenomics was understood to be helped by drivers such as those we labeled , , and but hindered by obstacles such as those we labeled , , and . Leveraging social science frameworks, we find that arguments for adoption that draw on the most salient drivers, which emphasize superior and novel functionality of omics as rationales, overlook potential adopters' key concerns: simplicity of use and compatibility with existing practices. We also identify two perspectives-innovation-centric and adopter-centric-on omics adoption and explain how overreliance on the former may be undermining efforts to promote toxicogenomics. https://doi.org/10.1289/EHP6500.
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http://dx.doi.org/10.1289/EHP6500DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7592882PMC
October 2020

In vitro-in vivo and cross-life stage extrapolation of uptake and biotransformation of benzo[a]pyrene in the fathead minnow (Pimephales promelas).

Aquat Toxicol 2020 Nov 15;228:105616. Epub 2020 Sep 15.

Toxicology Centre, University of Saskatchewan, Saskatoon, Saskatchewan, Canada; School of Environment and Sustainability, University of Saskatchewan, Saskatoon, Saskatchewan, Canada; Global Institute for Water Security, University of Saskatchewan, Saskatoon, Saskatchewan, Canada.

Understanding internal dose metrics is integral to adequately assess effects environmental contaminants might have on aquatic wildlife, including fish. In silico toxicokinetic (TK) models are a leading approach for quantifying internal exposure metrics for fishes; however, they often do not adequately consider chemicals that are actively biotransformed and have not been validated against early-life stages (ELS) that are often considered the most sensitive to the exposure to contaminants. To address these uncertainties, TK models were parameterized for the rapidly biotransformed chemical benzo[a]pyrene (B[a]P) in embryo-larval and adult life stages of fathead minnows. Biotransformation of B[a]P was determined through measurements of in vitro clearance. Using in vitro-in vivo extrapolation, in vitro clearance was integrated into a multi-compartment TK model for adult fish and a one-compartment model for ELS. Model predictions were validated using measurements of B[a]P metabolites from in vivo flow-through exposures to graded concentrations of water-borne B[a]P. Significantly greater amounts of B[a]P metabolites were observed with exposure to greater concentrations of parent compound in both life stages. However, when assessing biotransformation capacity, no differences in phase I or phase II biotransformation were observed with greater exposures to B[a]P. Results of modelling suggested that biotransformation of B[a]P can be successfully implemented into in silico models to accurately predict life stage-specific abundances of B[a]P metabolites in either whole-body larvae or the bile of adult fish. Models developed increase the scope of applications in which TK models can be used to support environmental risk assessments.
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http://dx.doi.org/10.1016/j.aquatox.2020.105616DOI Listing
November 2020

A Global Cndp1-Knock-Out Selectively Increases Renal Carnosine and Anserine Concentrations in an Age- and Gender-Specific Manner in Mice.

Int J Mol Sci 2020 Jul 10;21(14). Epub 2020 Jul 10.

Centre for Paediatric and Adolescent Medicine, University of Heidelberg, 69120 Heidelberg, Germany.

Carnosinase 1 (CN1) is encoded by the gene and degrades carnosine and anserine, two natural histidine-containing dipeptides. In vitro and in vivo studies suggest carnosine- and anserine-mediated protection against long-term sequelae of reactive metabolites accumulating, e.g., in diabetes mellitus. We have characterized the metabolic impact of CN1 in 11- and 55-week-old -knockout (-KO) mice and litter-matched wildtypes (WT). In -KO mice, renal carnosine and anserine concentrations were gender-specifically increased 2- to 9-fold, respectively in the kidney and both most abundant in the renal cortex, but remained unchanged in all other organs and in serum. Renal oxidized/reduced glutathione concentrations, renal morphology and function were unaltered. In -KO mice at week 11, renal asparagine, serine and glutamine levels and at week 55, renal arginine concentration were reduced. Renal heat-shock-protein 70 () mRNA declined with age in WT but not in -KO mice, transcription factor heat-shock-factor 1 was higher in 55-week-old KO mice. Fasting blood glucose concentrations decreased with age in WT mice, but were unchanged in mice. Blood glucose response to intraperitoneal insulin was gender- but not genotype-dependent, the response to intraperitoneal glucose injection was similar in all groups. A global -KO selectively, age- and gender-specifically, increases renal carnosine and anserine concentrations, alters renal amino acid- and HSP70 profile and modifies systemic glucose homeostasis. Increase of the natural occurring carnosine and anserine levels in the kidney by modulation of CN1 represents a promising therapeutic approach to mitigate or prevent chronic kidney diseases such as diabetic nephropathy.
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http://dx.doi.org/10.3390/ijms21144887DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7402351PMC
July 2020

Mechanisms of pH-Dependent Uptake of Ionizable Organic Chemicals by Fish from Oil Sands Process-Affected Water (OSPW).

Environ Sci Technol 2020 08 19;54(15):9547-9555. Epub 2020 Jul 19.

School of Environment and Sustainability (SENS), University of Saskatchewan, 44 Campus Drive, Saskatoon S7N 5C8, Canada.

Uptake and effects of ionizable organic chemicals (IOCs) that are weak acids in aqueous solution by fish can differ as a function of pH. While the pH-dependent behavior of select IOCs is well-understood, complex mixtures of IOCs, e.g., from oil sands process-affected water (OSPW), have not yet been studied systematically. Here, we established an in vitro screening method using the rainbow trout gill cell line, RTgill-W1, to investigate pH-dependent cytotoxicity and permeation of IOCs across cultured epithelia using ultra-high-performance liquid chromatography with high-resolution mass spectrometry (UPLC-HRMS). The assay was benchmarked using model chemicals and technical mixtures, and then used to characterize fractions and reconstituted extracts of field-collected OSPW. Significant pH-dependent cytotoxicity of individual IOCs, acidic fractions, and reconstituted extracts of OSPW was observed. In vitro data were in good agreement with data from a 96 h in vivo exposure experiment with juvenile rainbow trout. Permeation of some IOCs from OSPW was mediated by active transport, as revealed by studies in which inhibitors of these active transport mechanisms were applied. We conclude that the RTgill-W1 in vitro assay is useful for the screening of pH-dependent uptake of IOCs in fish, and has applications for in vitroin vivo extrapolation, and prioritization of chemicals in nontarget screenings.
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http://dx.doi.org/10.1021/acs.est.0c02522DOI Listing
August 2020

Differential responses of gut microbiota of male and female fathead minnow (Pimephales promelas) to a short-term environmentally-relevant, aqueous exposure to benzo[a]pyrene.

Chemosphere 2020 Aug 12;252:126461. Epub 2020 Mar 12.

Toxicology Centre, University of Saskatchewan, Saskatoon, Saskatchewan, Canada; Department of Veterinary Biomedical Sciences, University of Saskatchewan, Saskatoon, Saskatchewan, Canada; Department of Environmental Sciences, Baylor University, Waco, TX, USA.

In addition to aiding in digestion of food and uptake of nutrients, microbiota in guts of vertebrates are responsible for regulating several beneficial functions, including development of an organism and maintaining homeostasis. However, little is known about effects of exposures to chemicals on structure and function of gut microbiota of fishes. To assess effects of exposure to polycyclic aromatic hydrocarbons (PAHs) on gut microbiota, male and female fathead minnows (Pimephales promelas) were exposed to environmentally-relevant concentrations of the legacy PAH benzo[a]pyrene (BaP) in water. Measured concentrations of BaP ranged from 2.3 × 10 to 1.3 μg L. The community of microbiota in the gut were assessed by use of 16S rRNA metagenetics. Exposure to environmentally-relevant aqueous concentrations of BaP did not alter expression levels of mRNA for cyp1a1, a "classic" biomarker of exposure to BaP, but resulted in shifts in relative compositions of gut microbiota in females rather than males. Results presented here illustrate that in addition to effects on more well-studied molecular endpoints, relative compositions of the microbiota in guts of fish can also quickly respond to exposure to chemicals, which can provide additional mechanisms for adverse effects on individuals.
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http://dx.doi.org/10.1016/j.chemosphere.2020.126461DOI Listing
August 2020

Metals and PFAS in stormwater and surface runoff in a semi-arid Canadian city subject to large variations in temperature among seasons.

Environ Sci Pollut Res Int 2020 May 16;27(15):18232-18241. Epub 2020 Mar 16.

Toxicology Centre, University of Saskatchewan, Saskatoon, SK, S7N 5B3, Canada.

Because compounds accumulate through dry periods and enter aquatic systems in just a few seasonal events such as snowmelt and summer storms, surface waters in semi-arid, cold regions, such as the Canadian Prairies, are particularly vulnerable to loading of contaminant from runoff events from surfaces. This study assessed concentrations of metals and selected trace organics entering a river via surface runoff from an urban region and how these semi-arid regions with large seasonal variations in temperature might differ from more temperate regions. Selected potentially harmful elements (PHEs) including, Mn with Cr, Cu, Zn, Ba and U all exceeded guideline discharge values set by the Canadian Council of the Ministers of the Environment (CCME) by as much as 16-fold. Variation among discharges during spring, summer and winter was observed. For example, across the whole city, an estimated 6 kg of zinc was discharged in a spring storm, 36 kg in a summer storm and 17 tonnes in snowmelt. The mass of Zn discharged is similar to the annual loading estimated for Stockholm, Sweden, but in Saskatoon, Saskatchewan, Canada, the bulk of runoff was during snowmelt. The mean sum of poly- and per-fluoroalkyl substances (PFAS) in stormwater was 9.0 ng L, which is consistent with concentrations observed in other Canadian cities (6.5-16 ng L). These concentrations of PFAS are likely due to dispersed sources and orders of magnitude less than thresholds for toxicity to fish and aquatic invertebrates.
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http://dx.doi.org/10.1007/s11356-020-08070-2DOI Listing
May 2020

Endothelial CD40 Mediates Microvascular von Willebrand Factor-Dependent Platelet Adhesion Inducing Inflammatory Venothrombosis in ADAMTS13 Knockout Mice.

Thromb Haemost 2020 Mar 5;120(3):466-476. Epub 2020 Mar 5.

Walter Brendel Centre of Experimental Medicine and Biomedical Center, Ludwig-Maximilians-University of Munich, Germany.

Background:  von Willebrand factor (vWF) plays an important role in platelet activation. CD40-CD40 ligand (CD40L) induced vWF release has been described in large vessels and cultured endothelium, but its role in the microcirculation is not known. Here, we studied whether CD40 is expressed in murine microvessels , whether CD40L induces platelet adhesion and leukocyte activation, and how deficiency of the vWF cleaving enzyme ADAMTS13 affects these processes.

Methods And Results:  The role of CD40L in the formation of beaded platelet strings reflecting their adhesion to ultralarge vWF fibers (ULVWF) was analyzed in the murine cremaster microcirculation . Expression of CD40 and vWF was studied by immunohistochemistry in isolated and fixed cremasters. Microvascular CD40 was only expressed under inflammatory conditions and exclusively in venous endothelium. We demonstrate that CD40L treatment augmented the number of platelet strings, reflecting ULVWF multimer formation exclusively in venules and small veins. In ADAMTS13 knockout mice, the number of platelet strings further increased to a significant extent. As a consequence extensive thrombus formation was induced in venules of ADAMTS13 knockout mice. In addition, circulating leukocytes showed primary and rapid adherence to these platelet strings followed by preferential extravasation in these areas.

Conclusion:  CD40L is an important stimulus of microvascular endothelial ULVWF release, subsequent platelet string formation and leukocyte extravasation but only in venous vessels under inflammatory conditions. Here, the lack of ADAMTS13 leads to severe thrombus formation. The results identify CD40 expression and ADAMTS13 activity as important targets to prevent microvascular inflammatory thrombosis.
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http://dx.doi.org/10.1055/s-0040-1702228DOI Listing
March 2020

EcoToxModules: Custom Gene Sets to Organize and Analyze Toxicogenomics Data from Ecological Species.

Environ Sci Technol 2020 04 10;54(7):4376-4387. Epub 2020 Mar 10.

Faculty of Agricultural and Environmental Sciences, McGill University, Sainte-Anne-de-Bellevue H9X 3V9, Canada.

Traditional results from toxicogenomics studies are complex lists of significantly impacted genes or gene sets, which are challenging to synthesize down to actionable results with a clear interpretation. Here, we defined two sets of 21 custom gene sets, called the functional and statistical EcoToxModules, in fathead minnow () to (1) re-cast predefined molecular pathways into a toxicological framework and (2) provide a data-driven, unsupervised grouping of genes impacted by exposure to environmental contaminants. The functional EcoToxModules were identified by re-organizing KEGG pathways into biological processes that are more relevant to ecotoxicology based on the input from expert scientists and regulators. The statistical EcoToxModules were identified using co-expression analysis of publicly available microarray data ( = 303 profiles) measured in livers of fathead minnows after exposure to 38 different conditions. Potential applications of the EcoToxModules were demonstrated with two case studies that represent exposure to a pure chemical and to environmental wastewater samples. In comparisons to differential expression and gene set analysis, we found that EcoToxModule responses were consistent with these traditional results. Additionally, they were easier to visualize and quantitatively compare across different conditions, which facilitated drawing conclusions about the relative toxicity of the exposures within each case study.
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http://dx.doi.org/10.1021/acs.est.9b06607DOI Listing
April 2020

Aryl hydrocarbon receptor nuclear translocators (ARNT1, ARNT2, and ARNT3) of white sturgeon (Acipenser transmontanus): Sequences, tissue-specific expressions, and response to β-naphthoflavone.

Comp Biochem Physiol C Toxicol Pharmacol 2020 May 17;231:108726. Epub 2020 Feb 17.

Department of Biological Sciences, University of Lethbridge, Lethbridge, Alberta T1K 3M4, Canada.

Sturgeons (Acipenseridae) are ancient fishes that have tissue-specific profiles of transcriptional responses to dioxin-like compounds (DLCs) that are unique from those generally measured in teleost fishes. Because DLCs exert their critical toxicities through activation of the aryl hydrocarbon receptor (AHR), this transcription factor has been the subject of intensive study. However, less attention has focused on the aryl hydrocarbon receptor nuclear translocator (ARNT), which is the dimerization partner of the AHR and required for AHR-mediated transcription. The present study sequenced ARNT1, ARNT2, and ARNT3 in a representative species of sturgeon, the white sturgeon (Acipenser transmontanus), and quantified tissue-specific basal transcript abundance for each ARNT and the response following exposure to the model agonist of the AHR, β-naphthoflavone. In common with other proteins in sturgeons, the amino acid sequences of ARNTs are more similar to those of tetrapods than are ARNTs of other fishes. Transcripts of ARNT1, ARNT2, and ARNT3 were detected in all tissues investigated. Expression of ARNTs are tightly regulated in vertebrates, but β-naphthoflavone caused down-regulation in liver and up-regulation in gill, while an upward trend was measured in intestine. ARNTs are dimeric partners for multiple proteins, including the hypoxia inducible factor 1α (HIF1α), which mediates response to hypoxia. A downward trend in abundance of HIF1α transcript was measured in liver of white sturgeon exposed to β-naphthoflavone. Altered expression of ARNTs and HIF1α caused by activation of the AHR might affect the ability of certain tissues in sturgeons to respond to hypoxia when co-exposed to DLCs or other agonists.
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http://dx.doi.org/10.1016/j.cbpc.2020.108726DOI Listing
May 2020

Comparative analyses of oxidative stress response and metallothionein induction in white sturgeon and rainbow trout during acute waterborne copper exposure.

Comp Biochem Physiol C Toxicol Pharmacol 2020 May 7;231:108723. Epub 2020 Feb 7.

Toxicology Centre, University of Saskatchewan, Saskatoon, SK S7N 5B3, Canada; School of Environment and Sustainability, University of Saskatchewan, Saskatoon, SK S7N 5C8, Canada.

Early life-stages of the endangered white sturgeon (Acipenser transmontanus) have been shown to be among the most sensitive fishes to aqueous copper (Cu) exposure. In a recent analogous study, we examined the role of whole-body Cu accumulation and Na homeostasis in species-specific differences between the sensitivity of white sturgeon and a common laboratory fish model, rainbow trout, to Cu. However, the potential roles of important mechanisms such as Cu-induced oxidative stress and/or metallothionein (MT) induction as potential drivers of sensitivity of white sturgeon to Cu have not been investigated to date. Here, rainbow trout and white sturgeon from three different early life-stages were exposed to waterborne Cu for 96 h, following which major antioxidant parameters, lipid peroxidation and MT gene expression were evaluated. Results indicated that during larval and swim-up life-stages, Cu induced oxidative damage in white sturgeon was greater than in rainbow trout. Moreover, baseline glutathione (GSH) was significantly greater in rainbow trout than white sturgeon. Observations also suggested that trout exceedingly relied on GSH to combat Cu-induced oxidative stress as they grew older. In contrast, sturgeon recruited an increasing level of MT to neutralize Cu-induced oxidative stress and/or Cu loading. In our recent study, we demonstrated that Na homeostasis is more susceptible to Cu in white sturgeon than in rainbow trout. Collectively, these findings indicate that the greater degree of oxidative damage in early life-stages, in addition to the higher magnitude of the disruption of Na homeostasis, contributes to the higher sensitivity of white sturgeon to Cu exposure.
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http://dx.doi.org/10.1016/j.cbpc.2020.108723DOI Listing
May 2020
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