Publications by authors named "Lucia Vergauwen"

37 Publications

Sublethal Effect Concentrations for Non-Polar Narcosis in the Zebrafish Embryo.

Environ Toxicol Chem 2021 Jul 20. Epub 2021 Jul 20.

Zebrafishlab, Veterinary, Department of Veterinary Sciences, University of Antwerp, Universiteitsplein 1, 2610, Wilrijk, Belgium.

Non-polar narcosis, also known as baseline toxicity, has been described as the minimal toxicity that an organic chemical may elicit based on its lipophilicity. While lethal effects of narcosis-inducing chemicals (NICs) have been thoroughly investigated, knowledge of sublethal effects is still very limited. We investigated the effects of three well-known NICs (phenanthrene, 1,3,5-trichlorobenzene and pentachlorobenzene) on a variety of organismal endpoints (malformations, swim bladder inflation, respiration, heart rate, swimming activity and turning angles), which can be plausibly linked to narcosis in zebrafish embryos. Baseline toxicity recorded as mortality is typically observed in similar exposure ranges in a wide variety of species including fish, corresponding to a chemical activity range between 0.01 and 0.1. In the present study we found that sublethal effects occurred at concentrations around 5 times below lethal concentrations. Altered swimming activity and impaired swim bladder inflation were the most sensitive endpoints occurring at exposure levels below the generally accepted threshold for baseline toxicity for two out of three compounds. Overall, most effective exposure levels across the sublethal endpoints and compounds did fall within the range typically associated with baseline toxicity, and deviations were generally limited to a factor 10. While there could be benefit in adding sublethal endpoints to toxicity tests, such as the Fish Embryo Acute Toxicity (FET) test, based on the present sublethal endpoints and available evidence from our and other studies, the underestimation of toxicity due to the sole assessment of mortality as an endpoint in a FET test may be limited for narcosis. This article is protected by copyright. All rights reserved.
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http://dx.doi.org/10.1002/etc.5170DOI Listing
July 2021

Toward an AOP Network-Based Tiered Testing Strategy for the Assessment of Thyroid Hormone Disruption.

Environ Sci Technol 2020 07 9;54(14):8491-8499. Epub 2020 Jul 9.

Zebrafishlab, Veterinary Physiology and Biochemistry, Department of Veterinary Sciences, University of Antwerp, Universiteitsplein 1, 2610 Wilrijk, Belgium.

A growing number of environmental pollutants are known to adversely affect the thyroid hormone system, and major gaps have been identified in the tools available for the identification, and the hazard and risk assessment of these thyroid hormone disrupting chemicals. We provide an example of how the adverse outcome pathway (AOP) framework and associated data generation can address current testing challenges in the context of fish early life stage tests, and fish tests in general. We demonstrate how a suite of assays covering biological processes involved in the underlying toxicological pathways can be implemented in a tiered screening and testing approach for thyroid hormone disruption, using the levels of assessment of the OECD's Conceptual Framework for the Testing and Assessment of Endocrine Disrupting Chemicals as a guide.
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http://dx.doi.org/10.1021/acs.est.9b07205DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7477622PMC
July 2020

Effect of Thyroperoxidase and Deiodinase Inhibition on Anterior Swim Bladder Inflation in the Zebrafish.

Environ Sci Technol 2020 05 29;54(10):6213-6223. Epub 2020 Apr 29.

Zebrafishlab, Veterinary Physiology and Biochemistry, Department of Veterinary Sciences, University of Antwerp, Universiteitsplein 1, 2610 Wilrijk, Belgium.

A set of adverse outcome pathways (AOPs) linking inhibition of thyroperoxidase and deiodinase to impaired swim bladder inflation in fish has recently been developed. These AOPs help to establish links between these thyroid hormone (TH) disrupting molecular events and adverse outcomes relevant to aquatic ecological risk assessment. Until now, very little data on the effects of TH disruption on inflation of the anterior chamber (AC) of the swim bladder were available. The present study used zebrafish exposure experiments with three model compounds with distinct thyroperoxidase and deiodinase inhibition potencies (methimazole, iopanoic acid, and propylthiouracil) to evaluate this linkage. Exposure to all three chemicals decreased whole body triiodothyronine (T3) concentrations, either through inhibition of thyroxine (T4) synthesis or through inhibition of Dio mediated conversion of T4 to T3. A quantitative relationship between reduced T3 and reduced AC inflation was established, a critical key event relationship linking impaired swim bladder inflation to TH disruption. Reduced inflation of the AC was directly linked to reductions in swimming distance compared to controls as well as to chemical-exposed fish whose ACs inflated. Together the data provide compelling support for AOPs linking TH disruption to impaired AC inflation in fish.
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http://dx.doi.org/10.1021/acs.est.9b07204DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7477623PMC
May 2020

Thyroid Hormone Disruptors Interfere with Molecular Pathways of Eye Development and Function in Zebrafish.

Int J Mol Sci 2019 Mar 27;20(7). Epub 2019 Mar 27.

Department of Veterinary Sciences, Veterinary Physiology and Biochemistry, Zebrafishlab, University of Antwerp, Universiteitsplein 1, 2160 Wilrijk, Belgium.

The effects of thyroid hormone disrupting chemicals (THDCs) on eye development of zebrafish were investigated. We expected THDC exposure to cause transcriptional changes of vision-related genes, which find their phenotypic anchoring in eye malformations and dysfunction, as observed in our previous studies. Zebrafish were exposed from 0 to 5 days post fertilization (dpf) to either propylthiouracil (PTU), a thyroid hormone synthesis inhibitor, or tetrabromobisphenol-A (TBBPA), which interacts with thyroid hormone receptors. Full genome microarray analyses of RNA isolated from eye tissue revealed that the number of affected transcripts was substantially higher in PTU- than in TBBPA-treated larvae. However, multiple components of phototransduction (e.g., phosphodiesterase, opsins) were responsive to both THDC exposures. Yet, the response pattern for the gene ontology (GO)-class "sensory perception" differed between treatments, with over 90% down-regulation in PTU-exposed fish, compared to over 80% up-regulation in TBBPA-exposed fish. Additionally, the reversibility of effects after recovery in clean water for three days was investigated. Transcriptional patterns in the eyes were still altered and partly overlapped between 5 and 8 dpf, showing that no full recovery occurred within the time period investigated. However, pathways involved in repair mechanisms were significantly upregulated, which indicates activation of regeneration processes.
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http://dx.doi.org/10.3390/ijms20071543DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6479403PMC
March 2019

Optimizing the Use of Zebrafish Feeding Trials for the Safety Evaluation of Genetically Modified Crops.

Int J Mol Sci 2019 Mar 23;20(6). Epub 2019 Mar 23.

Zebrafishlab, Veterinary Physiology and Biochemistry, Department of Veterinary Sciences, University of Antwerp, Universiteitsplein 1, 2610 Wilrijk, Belgium.

In Europe, the toxicological safety of genetically modified (GM) crops is routinely evaluated using rodent feeding trials, originally designed for testing oral toxicity of chemical compounds. We aimed to develop and optimize methods for advancing the use of zebrafish feeding trials for the safety evaluation of GM crops, using maize as a case study. In a first step, we evaluated the effect of different maize substitution levels. Our results demonstrate the need for preliminary testing to assess potential feed component-related effects on the overall nutritional balance. Next, since a potential effect of a GM crop should ideally be interpreted relative to the natural response variation (i.e., the range of biological values that is considered normal for a particular endpoint) in order to assess the toxicological relevance, we established natural response variation datasets for various zebrafish endpoints. We applied equivalence testing to calculate threshold equivalence limits (ELs) based on the natural response variation as a method for quantifying the range within which a GM crop and its control are considered equivalent. Finally, our results illustrate that the use of commercial control diets (CCDs) and null segregant (NS) controls (helpful for assessing potential effects of the transformation process) would be valuable additions to GM safety assessment strategies.
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http://dx.doi.org/10.3390/ijms20061472DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6471220PMC
March 2019

Advancing the Zebrafish embryo test for endocrine disruptor screening using micro-injection: Ethinyl estradiol as a case study.

Environ Toxicol Chem 2019 03 11;38(3):533-547. Epub 2019 Feb 11.

Zebrafishlab, Veterinary Physiology and Biochemistry, Department of Veterinary Sciences, University of Antwerp, Wilrijk, Belgium.

Fish (embryo) toxicity test guidelines are mostly based on aquatic exposures. However, in some cases, other exposure routes can be more practical and relevant. Micro-injection into the yolk of fish embryos could offer a particular advantage for administering hydrophobic compounds, such as many endocrine disruptors. Single-dose micro-injection was compared with continuous aquatic exposure in terms of compound accumulation and biological responses. 17α-Ethinyl estradiol (EE2) was used as a model compound. First, the optimal solvent and droplet size were optimized, and needle variation was assessed. Next, biological endpoints were evaluated. The accumulated internal dose of EE2 decreased over time in both exposure scenarios. Estrogen receptor activation was concentration/injected dose dependent, increased daily, and was related to esr2b transcription. Transcription of vitellogenin 1 (vtg1) and brain aromatase (cyp19a1b) was induced in both scenarios, but the cyp19a1b transcription pattern differed between routes. Injection caused an increase in cyp19a1b transcripts from 48 hours post fertilization (hpf) onward, whereas after aquatic exposure the main increase occurred between 96 and 120 hpf. Some malformations only occurred after injection, whereas others were present for both scenarios. We conclude that responses can differ between exposure routes and therefore micro-injection is not a direct substitute for, but can be complementary to aquatic exposure. Nevertheless, vtg1and cyp19a1b transcription and estrogen receptor activation are suitable biomarkers for endocrine disruptor screening in both scenarios. Environ Toxicol Chem 2019;38:533-547. © 2018 SETAC.
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http://dx.doi.org/10.1002/etc.4343DOI Listing
March 2019

From mRNA Expression of Drug Disposition Genes to In Vivo Assessment of CYP-Mediated Biotransformation during Zebrafish Embryonic and Larval Development.

Int J Mol Sci 2018 Dec 10;19(12). Epub 2018 Dec 10.

Applied Veterinary Morphology, Department of Veterinary Sciences, University of Antwerp, Wilrijk 2610, Antwerp, Belgium.

The zebrafish () embryo is currently explored as an alternative for developmental toxicity testing. As maternal metabolism is lacking in this model, knowledge of the disposition of xenobiotics during zebrafish organogenesis is pivotal in order to correctly interpret the outcome of teratogenicity assays. Therefore, the aim of this study was to assess cytochrome P450 (CYP) activity in zebrafish embryos and larvae until 14 d post-fertilization (dpf) by using a non-specific CYP substrate, i.e., benzyloxy-methyl-resorufin (BOMR) and a CYP1-specific substrate, i.e., 7-ethoxyresorufin (ER). Moreover, the constitutive mRNA expression of , , , , , , , phase II enzymes uridine diphosphate glucuronosyltransferase 1A1 () and sulfotransferase 1st1 (), and an ATP-binding cassette (ABC) drug transporter, i.e., , was assessed during zebrafish development until 32 dpf by means of quantitative PCR (qPCR). The present study showed that trancripts and/or the activity of these proteins involved in disposition of xenobiotics are generally low to undetectable before 72 h post-fertilization (hpf), which has to be taken into account in teratogenicity testing. Full capacity appears to be reached by the end of organogenesis (i.e., 120 hpf), although -except -and were shown to be already mature in early embryonic development.
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http://dx.doi.org/10.3390/ijms19123976DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6321216PMC
December 2018

Gene transcription ontogeny of hypothalamic-pituitary-thyroid axis development in early-life stage fathead minnow and zebrafish.

Gen Comp Endocrinol 2018 09 4;266:87-100. Epub 2018 May 4.

University of Antwerp, Zebrafishlab, Veterinary Physiology and Biochemistry, Dept. Veterinary Sciences, Universiteitsplein 1, 2610 Wilrijk, Belgium. Electronic address:

The hypothalamic-pituitary-thyroid (HPT) axis is known to play a crucial role in the development of teleost fish. However, knowledge of endogenous transcription profiles of thyroid-related genes in developing teleosts remains fragmented. We selected two model teleost species, the fathead minnow (Pimephales promelas) and the zebrafish (Danio rerio), to compare the gene transcription ontogeny of the HPT axis. Control organisms were sampled at several time points during embryonic and larval development until 33 days post-fertilization. Total RNA was extracted from pooled, whole fish, and thyroid-related mRNA expression was evaluated using quantitative polymerase chain reaction. Gene transcripts examined included: thyrotropin-releasing hormone receptor (trhr), thyroid-stimulating hormone receptor (tshr), sodium-iodide symporter (nis), thyroid peroxidase (tpo), thyroglobulin (tg), transthyretin (ttr), deiodinases 1, 2, 3a, and 3b (dio1, dio2, dio3a and 3b), and thyroid hormone receptors alpha and beta (thrα and β). A loess regression method was successful in identifying maxima and minima of transcriptional expression during early development of both species. Overall, we observed great similarities between the species, including maternal transfer, at least to some extent, of almost all transcripts (confirmed in unfertilized eggs), increasing expression of most transcripts during hatching and embryo-larval transition, and indications of a fully functional HPT axis in larvae. These data will aid in the development of hypotheses on the role of certain genes and pathways during development. Furthermore, this provides a background reference dataset for designing and interpreting targeted transcriptional expression studies both for fundamental research and for applications such as toxicology.
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http://dx.doi.org/10.1016/j.ygcen.2018.05.001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6540109PMC
September 2018

An AOP-based alternative testing strategy to predict the impact of thyroid hormone disruption on swim bladder inflation in zebrafish.

Aquat Toxicol 2018 Jul 21;200:1-12. Epub 2018 Apr 21.

Zebrafishlab, Veterinary Physiology and Biochemistry, Department of Veterinary Sciences, University of Antwerp, Universiteitsplein 1, 2610 Wilrijk, Belgium. Electronic address:

The adverse outcome pathway (AOP) framework can be used to help support the development of alternative testing strategies aimed at predicting adverse outcomes caused by triggering specific toxicity pathways. In this paper, we present a case-study demonstrating the selection of alternative in chemico assays targeting the molecular initiating events of established AOPs, and evaluate use of the resulting data to predict higher level biological endpoints. Based on two AOPs linking inhibition of the deiodinase (DIO) enzymes to impaired posterior swim bladder inflation in fish, we used in chemico enzyme inhibition assays to measure the molecular initiating events for an array of 51 chemicals. Zebrafish embryos were then exposed to 14 compounds with different measured inhibition potentials. Effects on posterior swim bladder inflation, predicted based on the information captured by the AOPs, were evaluated. By linking the two datasets and setting thresholds, we were able to demonstrate that the in chemico dataset can be used to predict biological effects on posterior chamber inflation, with only two outliers out of the 14 tested compounds. Our results show how information organized using the AOP framework can be employed to develop or select alternative assays, and successfully forecast downstream key events along the AOP. In general, such in chemico assays could serve as a first-tier high-throughput system to screen and prioritize chemicals for subsequent acute and chronic fish testing, potentially reducing the need for long-term and costly toxicity tests requiring large numbers of animals.
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http://dx.doi.org/10.1016/j.aquatox.2018.04.009DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6002951PMC
July 2018

Adverse outcome pathways: a concise introduction for toxicologists.

Arch Toxicol 2017 Nov 28;91(11):3697-3707. Epub 2017 Jun 28.

European Commission, Joint Research Centre (JRC), Ispra, Italy.

Adverse outcome pathways (AOPs) are designed to provide a clear-cut mechanistic representation of critical toxicological effects that propagate over different layers of biological organization from the initial interaction of a chemical with a molecular target to an adverse outcome at the individual or population level. Adverse outcome pathways are currently gaining momentum, especially in view of their many potential applications as pragmatic tools in the fields of human toxicology, ecotoxicology, and risk assessment. A number of guidance documents, issued by the Organization for Economic Cooperation and Development, as well as landmark papers, outlining best practices to develop, assess and use AOPs, have been published in the last few years. The present paper provides a synopsis of the main principles related to the AOP framework for the toxicologist less familiar with this area, followed by two case studies relevant for human toxicology and ecotoxicology.
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http://dx.doi.org/10.1007/s00204-017-2020-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5805086PMC
November 2017

Impaired swim bladder inflation in early life stage fathead minnows exposed to a deiodinase inhibitor, iopanoic acid.

Environ Toxicol Chem 2017 Nov 28;36(11):2942-2952. Epub 2017 Jun 28.

Mid-Continent Ecology Division, US Environmental Protection Agency, Duluth, Minnesota, USA.

Inflation of the posterior and/or anterior swim bladder is a process previously demonstrated to be regulated by thyroid hormones. We investigated whether inhibition of deiodinases, which convert thyroxine (T4) to the more biologically active form, 3,5,3'-triiodothyronine (T3), would impact swim bladder inflation. Two experiments were conducted using a model deiodinase inhibitor, iopanoic acid (IOP). First, fathead minnow embryos were exposed to 0.6, 1.9, or 6.0 mg/L or control water until 6 d postfertilization (dpf), at which time posterior swim bladder inflation was assessed. To examine anterior swim bladder inflation, a second study was conducted with 6-dpf larvae exposed to the same IOP concentrations until 21 dpf. Fish from both studies were sampled for T4/T3 measurements and gene transcription analyses. Incidence and length of inflated posterior swim bladders were significantly reduced in the 6.0 mg/L treatment at 6 dpf. Incidence of inflation and length of anterior swim bladder were significantly reduced in all IOP treatments at 14 dpf, but inflation recovered by 18 dpf. Throughout the larval study, whole-body T4 concentrations increased and T3 concentrations decreased in all IOP treatments. Consistent with hypothesized compensatory responses, deiodinase-2 messenger ribonucleic acid (mRNA) was up-regulated in the larval study, and thyroperoxidase mRNA was down-regulated in all IOP treatments in both studies. These results support the hypothesized adverse outcome pathways linking inhibition of deiodinase activity to impaired swim bladder inflation. Environ Toxicol Chem 2017;36:2942-2952. Published 2017 Wiley Periodicals Inc. on behalf of SETAC. This article is a US government work and, as such, is in the public domain in the United States of America.
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http://dx.doi.org/10.1002/etc.3855DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5733732PMC
November 2017

Evaluating Complex Mixtures in the Zebrafish Embryo by Reconstituting Field Water Samples: A Metal Pollution Case Study.

Int J Mol Sci 2017 Mar 2;18(3). Epub 2017 Mar 2.

Zebrafishlab, Veterinary Physiology and Biochemistry, Department of Veterinary Sciences, University of Antwerp, Universiteitsplein 1, 2610 Wilrijk, Belgium.

Accurately assessing the toxicity of complex, environmentally relevant mixtures remains an important challenge in ecotoxicology. The goal was to identify biological effects after exposure to environmental water samples and to determine whether the observed effects could be explained by the waterborne metal mixture found in the samples. Zebrafish embryos were exposed to water samples of five different sites originating from two Flemish (Mol and Olen, Belgium) metal contaminated streams: "Scheppelijke Nete" (SN) and "Kneutersloop" (K), and a ditch (D), which is the contamination source of SN. Trace metal concentrations, and Na, K, Mg and Ca concentrations were measured using ICP-MS and were used to reconstitute site-specific water samples. We assessed whether the effects that were observed after exposure to environmental samples could be explained by metal mixture toxicity under standardized laboratory conditions. Exposure to "D" or "reconstituted D" water caused 100% mortality. SN and reconstituted SN water caused similar effects on hatching, swim bladder inflation, growth and swimming activity. A canonical discriminant analysis confirmed a high similarity between both exposure scenarios, indicating that the observed toxicity was indeed primarily caused by metals. The applied workflow could be a valuable approach to evaluate mixture toxicity that limits time and costs while maintaining biological relevance.
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http://dx.doi.org/10.3390/ijms18030539DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5372555PMC
March 2017

Transcriptional Analysis of The Adaptive Digestive System of The Migratory Locust in Response to Plant Defensive Protease Inhibitors.

Sci Rep 2016 09 1;6:32460. Epub 2016 Sep 1.

Department of Animal Physiology and Neurobiology, Zoological Institute KU Leuven, Naamsestraat 59, B-3000 Leuven, Belgium.

Herbivorous insects evolved adaptive mechanisms to compensate for the presence of plant defensive protease inhibitors (PI) in their food. The underlying regulatory mechanisms of these compensatory responses remain largely elusive. In the current study, we investigated the initiation of this adaptive response in the migratory locust, Locusta migratoria, via microarray analysis of gut tissues. Four hours after dietary uptake of PIs, 114 and 150 transcripts were respectively found up- or downregulated. The results suggest a quick trade-off between compensating for potential loss of digestive activity on the one hand, and stress tolerance, defense, and structural integrity of the gut on the other hand. We additionally addressed the role of a group of related upregulated hexamerin-like proteins in the PI-induced response. Simultaneous knockdown of corresponding transcripts by means of RNA interference resulted in a reduced capacity of the locust nymphs to cope with the effects of PI. Moreover, since insect hexamerins have been shown to bind Juvenile Hormone (JH), we also investigated the effect of JH on the proteolytic digestion in L. migratoria. Our results indicate that JH has a stimulatory effect on the expression of three homologous chymotrypsin genes, while knocking down the JH receptor (methoprene tolerant) led to opposite effects.
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http://dx.doi.org/10.1038/srep32460DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5007527PMC
September 2016

Prioritization of contaminated watercourses using an integrated biomarker approach in caged carp.

Water Res 2016 08 27;99:129-139. Epub 2016 Apr 27.

Zebrafishlab, Veterinary Physiology and Biochemistry, Department of Veterinary Sciences, University of Antwerp, Universiteitsplein 1, 2610 Wilrijk, Belgium; Systemic Physiological and Ecotoxicological Research (SPHERE), Department of Biology, University of Antwerp, Groenenborgerlaan 171, 2020 Antwerpen, Belgium.

Because of the ever increasing complexity of environmental contamination profiles, there are limitations to the use of analytical pollutant measurements for monitoring and prioritization of watercourses. The potential of biomarkers has been debated for many years, especially in laboratory settings, but there is a need for studies evaluating these approaches in the field. We evaluated the usefulness of a selection of biomarkers, mostly indicators of general physiological status and common stress responses such as oxidative stress, to discriminate among environmental pollution profiles, with the aim of prioritizing contaminated watercourses for targeted remediation efforts. To this end, juvenile common carp (Cyprinus carpio Lin.) were exposed in cages in the field to Flemish watercourses with varying pollution profiles. After six weeks of exposure, the bioaccumulation of key pollutants was measured, and a set of organismal, biochemical and transcriptional endpoints was determined in several tissue types. After data integration a discrete set of 14 parameters was identified, that could successfully distinguish all watercourses from each other. We show that an integrated biomarker approach, mainly targeting common stress responses, can offer the resolving power to discriminate among environmentally relevant exposure scenarios, and a means to prioritize watercourses for targeted remediation.
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http://dx.doi.org/10.1016/j.watres.2016.04.059DOI Listing
August 2016

Impaired anterior swim bladder inflation following exposure to the thyroid peroxidase inhibitor 2-mercaptobenzothiazole part I: Fathead minnow.

Aquat Toxicol 2016 Apr 7;173:192-203. Epub 2016 Jan 7.

U.S. Environmental Protection Agency, Office of Research and Development, National Health and Environmental Effects Research Laboratory, Mid-Continent Ecology Division, 6201Congdon Blvd., Duluth, MN 55804, USA.

In the present study, a hypothesized adverse outcome pathway linking inhibition of thyroid peroxidase (TPO) activity to impaired swim bladder inflation was investigated in two experiments in which fathead minnows (Pimephales promelas) were exposed to 2-mercaptobenzothiazole (MBT). Continuous exposure to 1mg MBT/L for up to 22 days had no effect on inflation of the posterior chamber of the swim bladder, which typically inflates around 6 days post fertilization (dpf), a period during which maternally-derived thyroid hormone is presumed to be present. In contrast, inflation of the anterior swim bladder, which occurs around 14dpf, was impacted. Specifically, at 14dpf, approximately 50% of fish exposed to 1mg MBT/L did not have an inflated anterior swim bladder. In fish exposed to MBT through 21 or 22dpf, the anterior swim bladder was able to inflate, but the ratio of the anterior/posterior chamber length was significantly reduced compared to controls. Both abundance of thyroid peroxidase mRNA and thyroid follicle histology suggest that fathead minnows mounted a compensatory response to the presumed inhibition of TPO activity by MBT. Time-course characterization showed that fish exposed to MBT for at least 4 days prior to normal anterior swim bladder inflation had significant reductions in anterior swim bladder size, relative to the posterior chamber, compared to controls. These results, along with similar results observed in zebrafish (see part II, this issue) are consistent with the hypothesis that thyroid hormone signaling plays a significant role in mediating anterior swim bladder inflation and development in cyprinids, and that role can be disrupted by exposure to thyroid hormone synthesis inhibitors. Nonetheless, possible thyroid-independent actions of MBT on anterior swim bladder inflation cannot be ruled out based on the present results. Overall, although anterior swim bladder inflation has not been directly linked to survival as posterior swim bladder inflation has, potential links to adverse ecological outcomes are plausible given involvement of the anterior chamber in sound production and detection.
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http://dx.doi.org/10.1016/j.aquatox.2015.12.024DOI Listing
April 2016

Impaired anterior swim bladder inflation following exposure to the thyroid peroxidase inhibitor 2-mercaptobenzothiazole part II: Zebrafish.

Aquat Toxicol 2016 Apr 18;173:204-217. Epub 2016 Jan 18.

Zebrafishlab, Veterinary Physiology and Biochemistry, Department of Veterinary Sciences, University of Antwerp, Universiteitsplein 1, 2610 Wilrijk, Belgium. Electronic address:

Disruption of the thyroid hormone (TH) system, an important mode of action, can lead to ecologically relevant adverse outcomes, especially during embryonic development. The present study characterizes the effects of disruption of TH synthesis on swim bladder inflation during zebrafish early-life stages using 2-mercaptobenzothiazole (MBT), a thyroid peroxidase (TPO) inhibitor. Zebrafish were exposed to different MBT concentrations until 120/168h post fertilization (hpf) and 32days post fertilization (dpf), in two sets of experiments, to investigate the effects of TPO inhibition on posterior and anterior swim bladder inflation respectively, as well as whole body thyroid hormone concentrations (triiodothyronine (T3) and its prohormone, thyroxine (T4)). At 120hpf, MBT did not directly impair posterior chamber inflation or size, while anterior chamber inflation and size was impaired at 32dpf. As previously shown in amphibians and mammals, we confirmed that MBT inhibits TPO in fish. Whole-body T4 decreased after MBT exposure at both time points, while T3 levels were unaltered. There was a significant relationship between T4 levels and the anterior chamber surface at 32dpf. The absence of effects on posterior chamber inflation can possibly be explained by maternal transfer of T4 into the eggs. These maternally derived THs are depleted at 32dpf and cannot offset TPO inhibition, resulting in impaired anterior chamber inflation. Therefore, we hypothesize that TPO inhibition only inhibits swim bladder inflation during late development, after depletion of maternally derived T4. In a previous study, we showed that iodothyronine deiodinase (ID) knockdown impaired posterior chamber inflation during early development. Our findings, in parallel with similar effects observed in fathead minnow (see part I, this issue) suggest that thyroid disruption impacts swim bladder inflation, and imply an important distinction among specific subtypes of TH disrupting chemicals. However, the existence of another - yet unknown - mode of action of MBT impacting swim bladder inflation cannot be excluded. These results can be helpful for delineating adverse outcome pathways (AOPs) linking TPO inhibition, ID inhibition and other TH related molecular initiating events, to impaired swim bladder inflation in fish during early life stages. Such AOPs can support the use of in vitro enzyme inhibition assays for predicting reduced survival due to impaired posterior and anterior chamber inflation.
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http://dx.doi.org/10.1016/j.aquatox.2015.12.023DOI Listing
April 2016

Deiodinase knockdown affects zebrafish eye development at the level of gene expression, morphology and function.

Mol Cell Endocrinol 2016 Mar 21;424:81-93. Epub 2016 Jan 21.

Laboratory of Comparative Endocrinology, Department of Biology, Division of Animal Physiology and Neurobiology, KU Leuven, B-3000, Leuven, Belgium. Electronic address:

Retinal development in vertebrates relies extensively on thyroid hormones. Their local availability is tightly controlled by several regulators, including deiodinases (Ds). Here we used morpholino technology to explore the roles of Ds during eye development in zebrafish. Transcriptome analysis at 3 days post fertilization (dpf) revealed a pronounced effect of knockdown of both T4-activating Ds (D1D2MO) or knockdown of T3-inactivating D3 (D3bMO) on phototransduction and retinoid recycling. This was accompanied by morphological defects (studied from 1 to 7 dpf) including reduced eye size, disturbed retinal lamination and strong reduction in rods and all four cone types. Defects were more prominent and persistent in D3-deficient fish. Finally, D3-deficient zebrafish larvae had disrupted visual function at 4 dpf and were less sensitive to a light stimulus at 5 dpf. These data demonstrate the importance of TH-activating and -inactivating Ds for correct zebrafish eye development, and point to D3b as a central player.
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http://dx.doi.org/10.1016/j.mce.2016.01.018DOI Listing
March 2016

Drought Induces Distinct Growth Response, Protection, and Recovery Mechanisms in the Maize Leaf Growth Zone.

Plant Physiol 2015 Oct 21;169(2):1382-96. Epub 2015 Aug 21.

Department of Biology, University of Antwerp, 2020 Antwerp, Belgium (V.A., H.Ab., L.V., Y.G., H.As., G.T.S.B.);Department of Botany, Faculty of Science, University of Beni-Suef, Beni-Suef 62511, Egypt (H.Ab.);Hubei Key Laboratory of Genetic Regulation and Integrative Biology, School of Life Sciences, Central China Normal University, Wuhan 430079, China (Z.Z.);Institute of Animal Reproduction and Food Research, 10-748 Olsztyn, Poland (B.F.);Centro de Estudios Fotosintéticos y Bioquímicos, Consejo Nacional de Investigaciones Científicas y Técnicas, Universidad Nacional de Rosario, S2002 LRK Rosario, Argentina (R.C.);Department of Veterinary Sciences, University of Antwerp, Campus Drie Eiken, 2610 Wilrijk, Belgium (D.K.); andConsejo Nacional de Investigaciones Científicas y Técnicas, Instituto de Fisiología y Recursos Genéticos Vegetales, Centro de Investigaciones Agropecuarias (CIAP), Instituto Nacional de Tecnología Agropecuaria (INTA), X5020ICA Cordoba, Argentina (E.T.)

Drought is the most important crop yield-limiting factor, and detailed knowledge of its impact on plant growth regulation is crucial. The maize (Zea mays) leaf growth zone offers unique possibilities for studying the spatiotemporal regulation of developmental processes by transcriptional analyses and methods that require more material, such as metabolite and enzyme activity measurements. By means of a kinematic analysis, we show that drought inhibits maize leaf growth by inhibiting cell division in the meristem and cell expansion in the elongation zone. Through a microarray study, we observed the down-regulation of 32 of the 54 cell cycle genes, providing a basis for the inhibited cell division. We also found evidence for an up-regulation of the photosynthetic machinery and the antioxidant and redox systems. This was confirmed by increased chlorophyll content in mature cells and increased activity of antioxidant enzymes and metabolite levels across the growth zone, respectively. We demonstrate the functional significance of the identified transcriptional reprogramming by showing that increasing the antioxidant capacity in the proliferation zone, by overexpression of the Arabidopsis (Arabidopsis thaliana) iron-superoxide dismutase gene, increases leaf growth rate by stimulating cell division. We also show that the increased photosynthetic capacity leads to enhanced photosynthesis upon rewatering, facilitating the often-observed growth compensation.
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http://dx.doi.org/10.1104/pp.15.00276DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4587441PMC
October 2015

A high throughput passive dosing format for the Fish Embryo Acute Toxicity test.

Chemosphere 2015 Nov 27;139:9-17. Epub 2015 May 27.

Zebrafishlab, Veterinary Physiology and Biochemistry, Department of Veterinary Sciences, University of Antwerp, Universiteitsplein 1, 2610 Wilrijk, Belgium.

High throughput testing according to the Fish Embryo Acute Toxicity (FET) test (OECD Testing Guideline 236) is usually conducted in well plates. In the case of hydrophobic test substances, sorptive and evaporative losses often result in declining and poorly controlled exposure conditions. Therefore, our objective was to improve exposure conditions in FET tests by evaluating a passive dosing format using silicone O-rings in standard 24-well polystyrene plates. We exposed zebrafish embryos to a series of phenanthrene concentrations until 120h post fertilization (hpf), and obtained a linear dilution series. We report effect values for both mortality and sublethal morphological effects based on (1) measured exposure concentrations, (2) (lipid normalized) body residues and (3) chemical activity. The LC50 for 120hpf was 310μg/L, CBR50 (critical body residue) was 2.72mmol/kg fresh wt and La50 (lethal chemical activity) was 0.047. All values were within ranges expected for baseline toxicity. Impaired swim bladder inflation was the most pronounced morphological effect and swimming activity was reduced in all exposure concentrations. Further analysis showed that the effect on swimming activity was not attributed to impaired swim bladder inflation, but rather to baseline toxicity. We conclude that silicone O-rings (1) produce a linear dilution series of phenanthrene in the 120hpf FET test, (2) generate and maintain aqueous concentrations for reliable determination of effect concentrations, and allow for obtaining mechanistic toxicity information, and (3) cause no toxicity, demonstrating its potential as an extension of the FET test when testing hydrophobic chemicals.
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http://dx.doi.org/10.1016/j.chemosphere.2015.05.041DOI Listing
November 2015

Optimisation of the bovine whole in vitro embryo system as a sentinel for toxicity screening: a cadmium challenge.

Altern Lab Anim 2015 May;43(2):89-100

University of Antwerp, Laboratory of Veterinary Physiology and Biochemistry, Department of Veterinary Sciences, Antwerp, Belgium.

Developmental toxicity testing could greatly benefit from the availability of an in vitro alternative model based on the use of animal embryos that have better human-like physiology than the currently-used alternative models. These current models are insufficient, as extrapolation of the results can be challenging. Therefore, an in vitro bovine embryo culture system was used to expose individual morulae to test substances, and to study developmental characteristics up to the blastocyst stage. Cadmium was chosen as the reference toxicant to investigate the sensitivity of the bovine morulae to various concentrations and exposure times. Oocytes from slaughterhouse-obtained bovine ovaries, were maturated, fertilised and cultured up until the morula stage. Morulae were exposed to different cadmium concentrations for 18 or 70 hours, and developmental competence, embryo quality and the expression of cadmium exposure-related genes were evaluated. Cadmium exposure hampered embryonic developmental competence and quality. Compared with the 18-hour exposure, the 70-hour exposure induced a 20-fold higher toxic response with regard to developmental competence and a more 'cadmium-typical' transcript expression. The bovine morula might be a promising tool for toxicity testing as, following exposure, the embryos reacted in a sensitive and 'cadmium-typical' manner to our reference toxicant.
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http://dx.doi.org/10.1177/026119291504300204DOI Listing
May 2015

The potential of AOP networks for reproductive and developmental toxicity assay development.

Reprod Toxicol 2015 Aug 15;56:52-5. Epub 2015 Apr 15.

Mid-Continent Ecology Division, Office of Research and Development, US Environmental Protection Agency, 6201 Congdon Blvd, Duluth, MN 55804, USA.

Historically, the prediction of reproductive and developmental toxicity has largely relied on the use of animals. The adverse outcome pathway (AOP) framework forms a basis for the development of new non-animal test methods. It also provides biological context for mechanistic information from existing assays. However, a single AOP may not capture all events that contribute to any relevant toxic effect, even in single chemical exposure scenarios. AOP networks, defined as sets of AOPs sharing at least one common element, are capable of more realistically representing potential chemical effects. They provide information on interactions between AOPs and have the potential to reveal previously unknown links between biological pathways. Analysis of these AOP networks can aid the prioritization of assay development, whether the goal is to develop a single assay with predictive utility of multiple outcomes, or development of assays that are highly specific for a particular mode of action. This paper provides a brief overview of the AOPs related to reproductive and developmental toxicity currently available in the AOP Wiki (http://aopwiki.org), and gives an example of an AOP network based on five reproductive and developmental toxicity-related AOPs for fish to illustrate how AOP networks can be used for assay development and refinement.
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http://dx.doi.org/10.1016/j.reprotox.2015.04.003DOI Listing
August 2015

Deiodinase knockdown during early zebrafish development affects growth, development, energy metabolism, motility and phototransduction.

PLoS One 2015 9;10(4):e0123285. Epub 2015 Apr 9.

Zebrafishlab, Veterinary Physiology and Biochemistry, Department of Veterinary Sciences, University of Antwerp, B-2160 Wilrijk, Belgium; Systemic Physiological and Ecotoxicological Research (SPHERE), Department of Biology, University of Antwerp, B-2020 Antwerpen, Belgium.

Thyroid hormone (TH) balance is essential for vertebrate development. Deiodinase type 1 (D1) and type 2 (D2) increase and deiodinase type 3 (D3) decreases local intracellular levels of T3, the most important active TH. The role of deiodinase-mediated TH effects in early vertebrate development is only partially understood. Therefore, we investigated the role of deiodinases during early development of zebrafish until 96 hours post fertilization at the level of the transcriptome (microarray), biochemistry, morphology and physiology using morpholino (MO) knockdown. Knockdown of D1+D2 (D1D2MO) and knockdown of D3 (D3MO) both resulted in transcriptional regulation of energy metabolism and (muscle) development in abdomen and tail, together with reduced growth, impaired swim bladder inflation, reduced protein content and reduced motility. The reduced growth and impaired swim bladder inflation in D1D2MO could be due to lower levels of T3 which is known to drive growth and development. The pronounced upregulation of a large number of transcripts coding for key proteins in ATP-producing pathways in D1D2MO could reflect a compensatory response to a decreased metabolic rate, also typically linked to hypothyroidism. Compared to D1D2MO, the effects were more pronounced or more frequent in D3MO, in which hyperthyroidism is expected. More specifically, increased heart rate, delayed hatching and increased carbohydrate content were observed only in D3MO. An increase of the metabolic rate, a decrease of the metabolic efficiency and a stimulation of gluconeogenesis using amino acids as substrates may have been involved in the observed reduced protein content, growth and motility in D3MO larvae. Furthermore, expression of transcripts involved in purine metabolism coupled to vision was decreased in both knockdown conditions, suggesting that both may impair vision. This study provides new insights, not only into the role of deiodinases, but also into the importance of a correct TH balance during vertebrate embryonic development.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0123285PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4391947PMC
December 2015

Gene transcription patterns and energy reserves in Daphnia magna show no nanoparticle specific toxicity when exposed to ZnO and CuO nanoparticles.

Environ Res 2015 Apr 19;138:82-92. Epub 2015 Feb 19.

Zebrafishlab, Physiology and Biochemistry of Domestic Animals, Department of Veterinary Sciences, University of Antwerp. Universiteitslaan 1, 2610 Wilrijk, Belgium. Electronic address:

There is still a lot of contradiction on whether metal ions are solely responsible for the observed toxicity of ZnO and CuO nanoparticles to aquatic species. While most experiments have studied nanoparticle effects at organismal levels (e.g. mortality, reproduction), effects at lower levels of biological organization may clarify the role of metal ions, nanoparticles and nanoparticle aggregates. In this study, the effect of ZnO and CuO nanoparticles was tested at two lower levels: energy reserves and gene transcription and compared with zinc and copper salts. Daphnia magna was exposed during 96h to 10% immobilization concentrations of all chemicals, after which daphnids were sampled for determination of glycogen, lipid and protein concentration and for a differential gene transcription analysis using microarray. The dissolved, nanoparticle and aggregated fraction in the medium was characterized. The results showed that ZnO nanoparticles had largely dissolved directly after addition to the test medium. The CuO nanoparticles mostly formed aggregates, while only a small fraction dissolved. The exposure to zinc (both nano and metal salt) had no effect on the available energy reserves. However, in the copper exposure, the glycogen, lipid and protein concentration in the exposed daphnids was lower than in the unexposed ones. When comparing the nanoparticle (ZnO or CuO) exposed daphnids to the metal salt (zinc or copper salt) exposed daphnids, the microarray results showed no significantly differentially transcribed gene fragments. The results indicate that under the current exposure conditions the toxicity of ZnO and CuO nanoparticles to D. magna is solely caused by toxic metal ions.
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http://dx.doi.org/10.1016/j.envres.2015.02.014DOI Listing
April 2015

Adverse outcome pathway development II: best practices.

Toxicol Sci 2014 Dec;142(2):321-30

*US EPA Mid-Continent Ecology Division, 6201 Congdon Blvd, Duluth, MN 55804, Environment Canada, Ecotoxicology and Wildlife Health Division, Ottawa, Ontario, Canada K1A 0H3, Institute for Genomics, Biocomputing and Biotechnology, Mississippi State University, Starkville, MS 39762, School of the Environment and Sustainability and Toxicology Centre, University of Saskatchewan, Saskatoon, Saskatchewan, Canada SK S7N 5B3, School of Biological Sciences, University of Plymouth, Plymouth, Devon, PL4 8AA, UK, Water Resources Center, University of Minnesota, St. Paul, MN 55108, European Commission, Joint Research Centre, Via E. Fermi 2749, 21027 Ispra, Italy, Department of Biology and Biochemistry, University of Houston, Houston, TX 77004, Zebrafishlab, Veterinary Physiology and Biochemistry, Department of Veterinary Sciences, University of Antwerp, Universiteitsplein 1, 2610 Wilrijk, Belgium *US EPA Mid-Continent Ecology Division, 6201 Congdon Blvd, Duluth, MN 55804, Environment Canada, Ecotoxicology and Wildlife Health Division, Ottawa, Ontario, Canada K1A 0H3, Institute for Genomics, Biocomputing and Biotechnology, Mississippi State University, Starkville, MS 39762, School of the Environment and Sustainability and Toxicology Centre, University of Saskatchewan, Saskatoon, Saskatchewan, Canada SK S7N 5B3, School of Biological Sciences, University of Plymouth, Plymouth, Devon, PL4 8AA, UK, Water Resources Center, University of Minnesota, St. Paul, MN 55108, European Commission, Joint Research Centre, Via E. Fermi 2749, 21027 Ispra, Italy, Department of Biology and Biochemistry, University of Houston, Houston, TX 77004, Zebrafishlab, Veterinary Physiology and Biochemistry, Department of Veterinary Sciences, University of Antwerp, Universiteitsplein 1, 2610 Wilrijk, Belgium *US EPA Mid-Continent Ecology Division, 6201 Congdon Blvd, Duluth, MN 55804, Environment Canada, Ecotoxicology and Wildlife Health Division, Ottawa, Ontario, Canada K1A 0H3, Institute for Genomics, Biocompu

Organization of existing and emerging toxicological knowledge into adverse outcome pathway (AOP) descriptions can facilitate greater application of mechanistic data, including those derived through high-throughput in vitro, high content omics and imaging, and biomarker approaches, in risk-based decision making. The previously ad hoc process of AOP development is being formalized through development of internationally harmonized guidance and principles. The goal of this article was to outline the information content desired for formal AOP description and some rules of thumb and best practices intended to facilitate reuse and connectivity of elements of an AOP description in a knowledgebase and network context. For example, key events (KEs) are measurements of change in biological state that are indicative of progression of a perturbation toward a specified adverse outcome. Best practices for KE description suggest that each KE should be defined as an independent measurement made at a particular level of biological organization. The concept of "functional equivalence" can help guide both decisions about how many KEs to include in an AOP and the specificity with which they are defined. Likewise, in describing both KEs and evidence that supports a causal linkage or statistical association between them (ie, a key event relationship; KER), best practice is to build from and contribute to existing KE or KER descriptions in the AOP knowledgebase rather than creating redundant descriptions. The best practices proposed address many of the challenges and uncertainties related to AOP development and help promote a consistent and reliable, yet flexible approach.
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http://dx.doi.org/10.1093/toxsci/kfu200DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4318924PMC
December 2014

Adverse outcome pathway (AOP) development I: strategies and principles.

Toxicol Sci 2014 Dec;142(2):312-20

*US EPA Mid-Continent Ecology Division, 6201 Congdon Blvd, Duluth, Minnesota 55804, Environment Canada, Ecotoxicology and Wildlife Health Division, Ottawa, Ontario, K1A 0H3 Canada, Mississippi State University, Institute for Genomics, Biocomputing and Biotechnology, Starkville, Mississippi 39762, University of Saskatchewan, School of the Environment and Sustainability and Toxicology Centre, Saskatoon, Saskatchewan, SK S7N 5B3, Canada, University of Plymouth, School of Biological Sciences, Plymouth, Devon, PL4 8AA, UK, University of Minnesota, Water Resources Center, St. Paul, Minnesota 55108, European Commission, Joint Research Centre, Via E. Fermi 2749, 21027 Ispra, Italy, Department of Biology and Biochemistry, University of Houston, Houston, Texas, 77004, Zebrafishlab, Veterinary Physiology and Biochemistry, Department of Veterinary Sciences, University of Antwerp, Universiteitsplein 1, 2610 Wilrijk, Belgium.

An adverse outcome pathway (AOP) is a conceptual framework that organizes existing knowledge concerning biologically plausible, and empirically supported, links between molecular-level perturbation of a biological system and an adverse outcome at a level of biological organization of regulatory relevance. Systematic organization of information into AOP frameworks has potential to improve regulatory decision-making through greater integration and more meaningful use of mechanistic data. However, for the scientific community to collectively develop a useful AOP knowledgebase that encompasses toxicological contexts of concern to human health and ecological risk assessment, it is critical that AOPs be developed in accordance with a consistent set of core principles. Based on the experiences and scientific discourse among a group of AOP practitioners, we propose a set of five fundamental principles that guide AOP development: (1) AOPs are not chemical specific; (2) AOPs are modular and composed of reusable components-notably key events (KEs) and key event relationships (KERs); (3) an individual AOP, composed of a single sequence of KEs and KERs, is a pragmatic unit of AOP development and evaluation; (4) networks composed of multiple AOPs that share common KEs and KERs are likely to be the functional unit of prediction for most real-world scenarios; and (5) AOPs are living documents that will evolve over time as new knowledge is generated. The goal of the present article was to introduce some strategies for AOP development and detail the rationale behind these 5 key principles. Consideration of these principles addresses many of the current uncertainties regarding the AOP framework and its application and is intended to foster greater consistency in AOP development.
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http://dx.doi.org/10.1093/toxsci/kfu199DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4318923PMC
December 2014

Toxicogenomics in the 3T3-L1 cell line, a new approach for screening of obesogenic compounds.

Toxicol Sci 2014 Aug 20;140(2):352-63. Epub 2014 May 20.

Systemic Physiological and Ecotoxicological Research (SPHERE), Department of Biology, University of Antwerp, Groenenborgerlaan 171, 2020 Antwerpen, Belgium.

The obesogen hypothesis states that together with an energy imbalance between calories consumed and calories expended, exposure to environmental compounds early in life or throughout lifetime might have an influence on obesity development. In this work, we propose a new approach for obesogen screening, i.e., the use of transcriptomics in the 3T3-L1 pre-adipocyte cell line. Based on the data from a previous study of our group using a lipid accumulation based adipocyte differentiation assay, several human-relevant obesogenic compounds were selected: reference obesogens (Rosiglitazone, Tributyltin), test obesogens (Butylbenzyl phthalate, butylparaben, propylparaben, Bisphenol A), and non-obesogens (Ethylene Brassylate, Bis (2-ethylhexyl)phthalate). The high stability and reproducibility of the 3T3-L1 gene transcription patterns over different experiments and cell batches is demonstrated by this study. Obesogens and non-obesogen gene transcription profiles were clearly distinguished using hierarchical clustering. Furthermore, a gradual distinction corresponding to differences in induction of lipid accumulation could be made between test and reference obesogens based on transcription patterns, indicating the potential use of this strategy for classification of obesogens. Marker genes that are able to distinguish between non, test, and reference obesogens were identified. Well-known genes involved in adipocyte differentiation as well as genes with unknown functions were selected, implying a potential adipocyte-related function of the latter. Cell-physiological lipid accumulation was well estimated based on transcription levels of the marker genes, indicating the biological relevance of omics data. In conclusion, this study shows the high relevance and reproducibility of this 3T3-L1 based in vitro toxicogenomics tool for classification of obesogens and biomarker discovery. Although the results presented here are promising, further confirmation of the predictive value of the set of candidate biomarkers identified as well as the validation of their clinical role will be needed.
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http://dx.doi.org/10.1093/toxsci/kfu092DOI Listing
August 2014

Physiological, biochemical, and genome-wide transcriptional analysis reveals that elevated CO2 mitigates the impact of combined heat wave and drought stress in Arabidopsis thaliana at multiple organizational levels.

Glob Chang Biol 2014 Dec 4;20(12):3670-85. Epub 2014 Jun 4.

Research Group of Plant and Vegetation Ecology, Department of Biology, University of Antwerp, Universiteitsplein 1, Antwerp, Wilrijk, B-2610, Belgium; Laboratory for Molecular Plant Physiology and Biotechnology, Department of Biology, University of Antwerp, Groenenborgerlaan 171, Antwerp, B-2020, Belgium.

Climate changes increasingly threaten plant growth and productivity. Such changes are complex and involve multiple environmental factors, including rising CO2 levels and climate extreme events. As the molecular and physiological mechanisms underlying plant responses to realistic future climate extreme conditions are still poorly understood, a multiple organizational level analysis (i.e. eco-physiological, biochemical, and transcriptional) was performed, using Arabidopsis exposed to incremental heat wave and water deficit under ambient and elevated CO2 . The climate extreme resulted in biomass reduction, photosynthesis inhibition, and considerable increases in stress parameters. Photosynthesis was a major target as demonstrated at the physiological and transcriptional levels. In contrast, the climate extreme treatment induced a protective effect on oxidative membrane damage, most likely as a result of strongly increased lipophilic antioxidants and membrane-protecting enzymes. Elevated CO2 significantly mitigated the negative impact of a combined heat and drought, as apparent in biomass reduction, photosynthesis inhibition, chlorophyll fluorescence decline, H2 O2 production, and protein oxidation. Analysis of enzymatic and molecular antioxidants revealed that the stress-mitigating CO2 effect operates through up-regulation of antioxidant defense metabolism, as well as by reduced photorespiration resulting in lowered oxidative pressure. Therefore, exposure to future climate extreme episodes will negatively impact plant growth and production, but elevated CO2 is likely to mitigate this effect.
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http://dx.doi.org/10.1111/gcb.12626DOI Listing
December 2014

Gene expression profiling of three different stressors in the water flea Daphnia magna.

Ecotoxicology 2013 Jul 6;22(5):900-14. Epub 2013 Apr 6.

Laboratory of Aquatic Ecology, Evolution and Conservation, Catholic University of Leuven, Charles Deberiotstraat 32, 3000 Leuven, Belgium.

Microarrays are an ideal tool to screen for differences in gene expression of thousands of genes simultaneously. However, often commercial arrays are not available. In this study, we performed microarray analyses to evaluate patterns of gene transcription following exposure to two natural and one anthropogenic stressor. cDNA microarrays compiled of three life stage specific and three stressor-specific EST libraries, yielding 1734 different EST sequences, were used. We exposed juveniles of the water flea Daphnia magna for 48, 96 and 144 h to three stressors known to exert strong selection in natural populations of this species i.e. a sublethal concentration of the pesticide carbaryl, infective spores of the endoparasite Pasteuria ramosa, and fish predation risk mimicked by exposure to fish kairomones. A total of 148 gene fragments were differentially expressed compared to the control. Based on a PCA, the exposure treatments were separated into two main groups based on the extent of the transcriptional response: a low and a high (144 h of fish or carbaryl exposure and 96 h of parasite exposure) stress group. Firstly, we observed a general stress-related transcriptional expression profile independent of the treatment characterized by repression of transcripts involved in transcription, translation, signal transduction and energy metabolism. Secondly, we observed treatment-specific responses including signs of migration to deeper water layers in response to fish predation, structural challenge of the cuticle in response to carbaryl exposure, and disturbance of the ATP production in parasite exposure. A third important conclusion is that transcription expression patterns exhibit stress-specific changes over time. Parasite exposure shows the most differentially expressed gene fragments after 96 h. The peak of differentially expressed transcripts came only after 144 h of fish exposure, while carbaryl exposure induced a more stable number of differently expressed gene fragments over time.
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http://dx.doi.org/10.1007/s10646-013-1072-yDOI Listing
July 2013

Unraveling the mode of action of an obesogen: mechanistic analysis of the model obesogen tributyltin in the 3T3-L1 cell line.

Mol Cell Endocrinol 2013 May 18;370(1-2):52-64. Epub 2013 Feb 18.

Systemic Physiological and Ecotoxicological Research (SPHERE), Department of Biology, University of Antwerp, Groenenborgerlaan 171, 2020 Antwerpen, Belgium.

Obesogenic compounds are chemicals that have an influence on obesity development. This study was designed to unravel the molecular mechanisms of the model obesogen TBT, using microarray analysis in the 3T3-L1 in vitro system, and to evaluate the use of toxicogenomics for obesogen screening. The microarray results revealed enrichment of Gene Ontology terms involved in energy and fat metabolism after 10 days of TBT exposure. Pathway analysis unveiled PPAR signalling pathway as the sole pathway significantly enriched after 1 day and the most significantly enriched pathway after 10 days of exposure. To our knowledge, this is the first study delivering an in depth mechanistic outline of the mode of action of TBT as an obesogen, combining effects on both cell physiological and gene expression level. Furthermore, our results show that combining transcriptomics with 3T3-L1 cells is a promising tool for screening of potential obesogenic compounds.
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http://dx.doi.org/10.1016/j.mce.2013.02.011DOI Listing
May 2013

Hypothermal and hyperthermal acclimation differentially modulate cadmium accumulation and toxicity in the zebrafish.

Chemosphere 2013 Apr 18;91(4):521-9. Epub 2013 Jan 18.

Systemic Physiological and Ecotoxicological Research (SPHERE), Department of Biology, University of Antwerp, Groenenborgerlaan 171, 2020 Antwerpen, Belgium.

Despite the fact that aquatic organisms are mostly poikilothermic and environmental temperature variations can have considerable impact on chemical toxicity, toxicity studies are mainly performed at the species' specific standard or optimal temperature. Since the zebrafish is a recommended test species for use in toxicity tests, we investigated the temperature dependence of 96 h cadmium accumulation and toxicity in zebrafish acclimated to 18, 26, 30 or 34°C. Zebrafish showed high cadmium tolerance with acute 96 h LC50 values of 121.5, 102.4, 124.6 and 126.7 μM at 18, 26, 30 and 34°C respectively. Differences in cadmium toxicity at the different temperatures were small and toxicity did not increase with increasing temperature as is often suggested. We did however observe an interesting concentration dependent crossover pattern in which the temperature dependence at the highest exposure concentrations was exactly opposite to the pattern at the lower concentrations. At the highest concentrations the following order of toxicity was observed: 26°C>18°C>30°C>34°C. Possibly, either the warm acclimation provoked a general stress response which protected organisms against future severe stress situations, or resulted in specific defence mechanisms which also provided protection against cadmium exposure. Although at 18°C cadmium accumulation decreased more than would be expected based on the metabolic rate, cadmium toxicity was not proportionately decreased. This increased cadmium sensitivity in the cold was likely due to the combined effect of low temperature and cadmium exposure on sodium loss. This study shows that the temperature dependence of cadmium toxicity results from the combination of altered cadmium accumulation and sensitivity. Inclusion of the temperature effect in the calculation of environmental quality standards may have to be considered to ensure that more sensitive species are also protected at suboptimal temperatures.
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http://dx.doi.org/10.1016/j.chemosphere.2012.12.028DOI Listing
April 2013
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