| 1. |
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| 2. |
- Bluhm, Kerstin, et al.
(författare)
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Impacts of Different Exposure Scenarios on Transcript Abundances in Danio rerio Embryos when Investigating the Toxicological Burden of Riverine Sediments
- 2014
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Ingår i: PLOS ONE. - San francisco, USA : Public Library of Science. - 1932-6203. ; 9:9
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Tidskriftsartikel (refereegranskat)abstract
- Purpose: Recently, a proof-of-concept study revealed the suitability of transcriptome analyses to obtain and assess changes in the abundance of transcripts in zebrafish (Danio rerio) embryos after exposure to organic sediment extracts. The present study investigated changes in the transcript abundance in zebrafish embryos exposed to whole sediment samples and corresponding organic extracts in order to identify the impact of different exposure pathways on sediment toxicity.Materials and Methods: Danio rerio embryos were exposed to sublethal concentrations of three sediment samples from the Danube River, Germany. The sediment samples were investigated both as freeze-dried samples and as organic extracts. Silica dust and a process control of the extraction procedure were used as references. After exposure, mRNA was isolated and changes in profiles of gene expression levels were examined by an oligonucleotide microarray. The microarray results were compared with bioassays, chemical analysis of the sediments and profiles of gene expression levels induced by several single substances.Results and Discussion: The microarray approach elucidated significant changes in the abundance of transcripts in exposed zebrafish embryos compared to the references. Generally, results could be related to Ah-receptor-mediated effects asconfirmed by bioassays and chemical analysis of dioxin-like contaminants, as well as to exposure to stress-inducing compounds. Furthermore, the results indicated that mixtures of chemicals, as present in sediment and extract samples, result in complex changes of gene expression level profiles difficult to compare with profiles induced by single chemical substances. Specifically, patterns of transcript abundances were less influenced by the chemical composition at the sampling site compared t the method of exposure (sediment/extract). This effect might be related to different bioavailability of chemicals.Conclusions: The apparent difference between the exposure scenarios is an important aspect that needs to be addressed when conducting analyses of alterations in the expression level of mRNA.
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| 3. |
- Böttcher, Melanie, et al.
(författare)
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Comparison of in vitro and in situ genotoxicity in the Danube River by means of the comet assay and the micronucleus test
- 2010
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Ingår i: Mutation research. Genetic toxicology and environmental mutagenesis. - Amsterdam, Neteherlands : Elsevier. - 1383-5718 .- 1879-3592. ; 700:1-2, s. 11-17
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Tidskriftsartikel (refereegranskat)abstract
- Genotoxicity can be correlated with adverse reproductive effects or may even result in elevated extinction risk for particular species of an ecosystem. It may thus be a valuable tool for screening of pollution and potential environmental harm. Since many genotoxicants tend to adsorb on to particulate matter, sediments and suspended materials are of particular interest for genotoxicity screening under field conditions. In order to correlate the genotoxic potential of sediments with genetic damage in fish, rainbow-trout liver (RTL-W1) cells were exposed in vitro to acetone extracts of sediments collected at 10 selected sites along the upper Danube River and analyzed in the comet and micronucleus assays. These in vitro results were compared with micronucleus formation in erythrocytes of the European barbel (Barbus barbus) caught int he field. The two in vitro bioassays showed excellent correlation, indicating comparability of genotoxic potentials in vitro. Sampling sites could be clearly differentiated with respect to severity of effects, with Rottenacker as the most heavily contaminated site, Ehingen and Schwarzach as moderately genotoxic,and with the weakest effects in the tributary Lauchert. All other sediment extracts showed intermediate genotoxic or clastogenic effects. In situ, micronucleus formation in barbel erythrocytes indicated severe genotoxicity at Rottenacker, moderate effects at Ehingen, but minor contamination at Riedlingen and Sigmaringen. In situ observations thus showed excellent correlation with corresponding in vitro tests and document the ecological relevance of in vitro studies with sediment extracts. With respect to the ecological status of the Danube River, the results overall indicate a moderate to severe genotoxic potential with a highly differential localization.
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| 4. |
- Escher, Sylvia E., et al.
(författare)
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Integrate mechanistic evidence from new approach methodologies (NAMs) into a read-across assessment to characterise trends in shared mode of action
- 2022
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Ingår i: Toxicology in Vitro. - : Elsevier. - 0887-2333 .- 1879-3177. ; 79
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Tidskriftsartikel (refereegranskat)abstract
- Read-across approaches often remain inconclusive as they do not provide sufficient evidence on a common mode of action across the category members. This read-across case study on thirteen, structurally similar, branched aliphatic carboxylic acids investigates the concept of using human-based new approach methods, such as in vitro and in silico models, to demonstrate biological similarity.Five out of the thirteen analogues have preclinical in vivo studies. Three out of them induced lipid accumulation or hypertrophy in preclinical studies with repeated exposure, which leads to the read-across hypothesis that the analogues can potentially induce hepatic steatosis.To confirm the selection of analogues, the expression patterns of the induced differentially expressed genes (DEGs) were analysed in a human liver model. With increasing dose, the expression pattern within the tested analogues got more similar, which serves as a first indication of a common mode of action and suggests differences in the potency of the analogues.Hepatic steatosis is a well-known adverse outcome, for which over 55 adverse outcome pathways have been identified. The resulting adverse outcome pathway (AOP) network, comprised a total 43 MIEs/KEs and enabled the design of an in vitro testing battery. From the AOP network, ten MIEs, early and late KEs were tested to systematically investigate a common mode of action among the grouped compounds.The targeted testing of AOP specific MIE/KEs shows that biological activity in the category decreases with side chain length. A similar trend was evident in measuring liver alterations in zebra fish embryos. However, activation of single MIEs or early KEs at in vivo relevant doses did not necessarily progress to the late KE “lipid accumulation”. KEs not related to the read-across hypothesis, testing for example general mitochondrial stress responses in liver cells, showed no trend or biological similarity.Testing scope is a key issue in the design of in vitro test batteries. The Dempster-Shafer decision theory predicted those analogues with in vivo reference data correctly using one human liver model or the CALUX reporter assays.The case study shows that the read-across hypothesis is the key element to designing the testing strategy. In the case of a good mechanistic understanding, an AOP facilitates the selection of reliable human in vitro models to demonstrate a common mode of action. Testing DEGs, MIEs and early KEs served to show biological similarity, whereas the late KEs become important for confirmation, as progression from MIEs to AO is not always guaranteed.
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| 5. |
- Keiter, Steffen, 1971-, et al.
(författare)
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Ecotoxicological assessment of sediment, suspended matter andwater samples in the upper danube river : a pilot study in search for the causes for the decline of fish catches
- 2006
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Ingår i: Environmental Science and Pollution Research. - : Springer Science and Business Media LLC. - 0944-1344 .- 1614-7499. ; 13:5, s. 308-319
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Tidskriftsartikel (refereegranskat)abstract
- Goals, Scope and Background. Fish populations, especially thoseof the grayling (Thymallus thymallus), have declined over thelast two decades in the upper Danube River between Sigmaringenand Ulm, despite intensive and continuous stocking and improvementof water quality since the 1970s. Similar problems havebeen reported for other rivers, e.g. in Switzerland, Great Britain,the United States and Canada. In order to assess if ecotoxicologicaleffects might be related to the decline in fish catchat the upper Danube River, sediment, suspended matter andwaste water samples from sewage treatment plants were collectedat selected locations and analyzed in a bioanalytical approachusing a battery of bioassays. The results of this pilotstudy will be used to decide if a comprehensive weight-of-evidencestudy is needed.Methods. Freeze-dried sediments and suspended particulate matterswere extracted with acetone in a Soxhlet apparatus. Organicpollutants from sewage water were concentrated usingXAD-resins. In order to investigate the ecotoxicological burden,the following bioassays were used: (1) neutral red assaywith RTL-W1 cells (cytotoxicity), (2) comet assay with RTLW1cells (genotoxicity), (3) Arthrobacter globiformis dehydrogenaseassay (toxicity to bacteria), (4) yeast estrogen screen assay(endocrine disruption), (5) fish egg assay with the zebrafish(Danio rerio; embryo toxicity) and (6) Ames test with TA98(mutagenicity).Results and Discussion. The results of the in vitro tests elucidateda considerable genotoxic, cytotoxic, mutagenic, bacteriotoxic,embryotoxic and estrogenic burden in the upper DanubeRiver, although with a very inhomogeneous distribution of effects.The samples taken from Riedlingen, for example, inducedlow embryo toxicity, but the second highest 17β-estradiol equivalentconcentration (1.8 ng/L). Using the fish egg assay with nativesediments, a broad range of embryotoxic effects could beelucidated, with clear-cut dose-response relationships for theembryotoxic effects of contaminated sediments. With nativesediments, embryotoxicity was clearly higher than with correspondingpore waters, thus corroborating the view that – atleast for fish eggs – the bioavailability of particle-bound lipophilicsubstances in native sediments is higher than generally assumed. The effect observed most frequently in the fish egg assay was a developmental delay. A comparison of our own results with locationsalong the rivers Rhine and Neckar demonstrated similaror even higher ranges of ecotoxicological burdens in theDanube River.Conclusions. The complex pattern of ecotoxicological effectscaused by environmental samples from the Danube River, whenassessed in an in vitro biotest battery using both acute and morespecific endpoints, showed that integration of different endpointsis essential for appropriate hazard assessment. Overall, theecotoxicological hazard potential shown has indeed to be consideredas one potential reason for the decline in fish catches atthe upper Danube River. However, based on the results of thispilot study, it is not possible to elucidate that chemically inducedalterations are responsible for the fish decline.Recommendations and Perspectives. In order to confirm the ecologicalrelevance of the in vitro results for the situation in thefield and especially for the decline of the grayling and otherfishes, further integrated investigations are required. For linkingthe weight of evidence obtained by in vitro assays and fishpopulation investigations, the application of additional, morespecific biomarkers (e.g. vitellogenin induction, EROD and micronucleusassay) has been initiated in fish taken from the fieldas well as in situ investigations.
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| 6. |
- Klee, Nina, et al.
(författare)
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Changes in toxicity and genotoxicity of industrial sewage sludge samples containing nitro- and amino-aromatic compounds following treatment in bioreactors with different oxygen regimes
- 2004
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Ingår i: Environmental Science and Pollution Research. - 0944-1344 .- 1614-7499. ; 11:5, s. 313-320
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Tidskriftsartikel (refereegranskat)abstract
- GOALS, SCOPE AND BACKGROUND: From 2005, deposition of organic waste will be banned in Sweden. Likewise, in Germany and Austria, similar bans are being planned, and further countries will probably follow. Thus, there is a need to develop new methods and to refine established techniques for sludge management in the whole of the European Union. For this end, there is also an urgent need for appropriate ecotoxicological approaches to elucidate and assess the hazard potential of sewage sludge. Therefore, the present study was designed to assess the capacity of various established sludge treatment methods using different oxygen regimes to degrade recalcitrant nitro-substituted organic compounds and reduce their toxicity. Sewage sludge samples from a wastewater treatment plant in Sweden (Cambrex Karlskoga AB, industrial area Björkborn) receiving wastewater from industries manufacturing pharmaceutical substances, chemical intermediates and explosives were processed with different sludge treatment methods. Among other treatment methods, bioreactors (for anaerobic and aerobic sludge treatment) were used. In the present investigation, a battery of in vitro bioassays was employed to compare the cytotoxic and genotoxic potentials of different fractions of sludge samples in order to elucidate whether the treatments were suitable to reduce the toxicity of the sludge. METHODS: In order to investigate the cytotoxicity of the extracts of treated and untreated sludge samples, the acute cytotoxicity test with the permanent cell line RTL-W1 was used. Genotoxicity was tested by means of the comet assay (single cell gel electrophoresis) with RTL-W1 cells, and mutagenicity was assessed with the Ames test using the Salmonella typhimurium strains TA98, TA98NR and TA100. Sludge toxicity was tested in different fractions of organic extracts produced by acetone and hexane extractions. The subsequent clean-up procedure (silica gel chromatography and elution with hexane and dichloromethane) resulted in two fractions, a lipophilic hexane-fraction and a semi-lipophilic dichloromethane-fraction. For the genotoxicity and mutagenicity tests, these fractions were reunited at equal ratios. RESULTS AND DISCUSSION: The acute cytotoxicity test with RTL-W1 cells revealed a high cytotoxic potential for the semi-lipophilic DM-fractions of all sludge samples with NR50 values (= effective concentration for 50% cell death in the neutral red test) from 8.9 up to 20 mg sludge d.w./ml medium. A low cytotoxic potential for the hexane fractions of the untreated sludge samples (NR50 400 to > 400 mg sludge d.w./ml medium) was observed, whereas the hexane fractions of the treated sludge samples showed elevated cytotoxicity increasing further with treatment in the bioreactors. The comet assay indicated that three out of eight of the reunited fractions had a significant genotoxic potential. Whereas the genotoxic potential of one sample treated anaerobically was very high with an induction factor of 11.6, a similar sample (taken from the same anaerobic reactor four months later) and one untreated sample showed lower potentials. The samples treated in another anaerobic bioreactor as well as the samples treated aerobically showed no genotoxic potential. Results indicate that aerobic treatment was basically adequate for reducing the genotoxicity of the sludge, whereas anaerobic treatment was only partly useful for reduction of genotoxicity. The Ames test revealed a very high mutagenic potential for the reunited fractions of the untreated sludge samples with strain TA98 (maximum induction factors (IFmax) up to 45) and a relatively high potential for one of the samples treated aerobically (S2, IFmax = 18 (TA98, S9-)), thus documenting the suitability of both anaerobic and aerobic treatments to reduce the mutagenicity of the samples, however, with the aerobic treatment being less effective. CONCLUSIONS: Overall, none of the microbiological treatments for wastewater sludge in bioreactors was found to be ideal for general toxicity reduction of the sludge samples. Whereas cytotoxicity of the sludge increased or levelled off in most cases following either treatment, genotoxicity both increased or decreased after anaerobic treatment, depending on the specific sample. However, mutagenicity could generally be reduced by anaerobic treatment and, to a lesser degree, by aerobic treatment. RECOMMENDATIONS AND PERSPECTIVES: The complex modification of the diverse damage potentials of sludge sample extracts by use of an in vitro biotest battery following treatment for toxicity reduction in bioreactors showed that considerations of different toxicological endpoints is essential for an adequate hazard assessment. Whereas in the case of cytotoxicity reduction, the reactors proved ineffective, mutagenicity could be reduced significantly at least in some cases in this case study.
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| 7. |
- Kosmehl, Thomas, et al.
(författare)
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A combined DNA-microarray and mechanism-specific toxicity approach with zebrafish embryos to investigate the pollution of river sediments
- 2012
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Ingår i: Reproductive Toxicology. - Oxford, United Kingdom : Elsevier. - 0890-6238 .- 1873-1708. ; 33:2, s. 245-253
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Tidskriftsartikel (refereegranskat)abstract
- The zebrafish embryo has repeatedly proved to be a useful model for the analysis of effects by environmental toxicants. This proof-of-concept study was performed to investigate if an approach combining mechanism-specific bioassays with microarray techniques can obtain more in-depth insights into theecotoxicity of complex pollutant mixtures as present, e.g., in sediment extracts. For this end, altered gene expression was compared to data from established bioassays as well as to results from chemical analysis. Mechanism-specific biotests indicated a defined hazard potential of the sediment extracts, and microarray analysis revealed several classes of significantly regulated genes which could be related to the hazard potential. Results indicate that potential classes of contaminants can be assigned to sediment extracts by both classical biomarker genes and corresponding expression profile analyses of known substances. However, it is difficult to distinguish between specific responses and more universal detoxification of the organism.
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| 8. |
- Krebs, Alice, et al.
(författare)
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The EU-ToxRisk method documentation, data processing and chemical testing pipeline for the regulatory use of new approach methods
- 2020
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Ingår i: Archives of Toxicology. - : Springer Science and Business Media LLC. - 0340-5761 .- 1432-0738. ; 94:7, s. 2435-2461
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Tidskriftsartikel (refereegranskat)abstract
- Hazard assessment, based on new approach methods (NAM), requires the use of batteries of assays, where individual tests may be contributed by different laboratories. A unified strategy for such collaborative testing is presented. It details all procedures required to allow test information to be usable for integrated hazard assessment, strategic project decisions and/or for regulatory purposes. The EU-ToxRisk project developed a strategy to provide regulatorily valid data, and exemplified this using a panel of > 20 assays (with > 50 individual endpoints), each exposed to 19 well-known test compounds (e.g. rotenone, colchicine, mercury, paracetamol, rifampicine, paraquat, taxol). Examples of strategy implementation are provided for all aspects required to ensure data validity: (i) documentation of test methods in a publicly accessible database; (ii) deposition of standard operating procedures (SOP) at the European Union DB-ALM repository; (iii) test readiness scoring accoding to defined criteria; (iv) disclosure of the pipeline for data processing; (v) link of uncertainty measures and metadata to the data; (vi) definition of test chemicals, their handling and their behavior in test media; (vii) specification of the test purpose and overall evaluation plans. Moreover, data generation was exemplified by providing results from 25 reporter assays. A complete evaluation of the entire test battery will be described elsewhere. A major learning from the retrospective analysis of this large testing project was the need for thorough definitions of the above strategy aspects, ideally in form of a study pre-registration, to allow adequate interpretation of the data and to ensure overall scientific/toxicological validity.
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| 9. |
- Leist, Marcel, et al.
(författare)
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Adverse outcome pathways : opportunities, limitations and open questions
- 2017
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Ingår i: Archives of Toxicology. - : Springer Science and Business Media LLC. - 0340-5761 .- 1432-0738. ; 91:11, s. 3477-3505
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Tidskriftsartikel (refereegranskat)abstract
- Adverse outcome pathways (AOPs) are a recent toxicological construct that connects, in a formalized, transparent and quality-controlled way, mechanistic information to apical endpoints for regulatory purposes. AOP links a molecular initiating event (MIE) to the adverse outcome (AO) via key events (KE), in a way specified by key event erelationships (KER). Although this approach to formalize mechanistic toxicological information only started in 2010, over 200 AOPs have already been established. At this stage, new requirements arise, such as the need for harmonization and re-assessment, for continuous updating, as well as for alerting about pitfalls, misuses and limits of applicability. In this review, the history of the AOP concept and its most prominent strengths are discussed, including the advantages of a formalized approach, the systematic collection of weight of evidence, the linkage of mechanisms to apical end points, the examination of the plausibility of epidemiological data, the identification of critical knowledge gaps and the design of mechanistic test methods. To prepare the ground for a broadened and appropriate use of AOPs, some widespread misconceptions are explained. Moreover, potential weaknesses and shortcomings of the current AOP rule set are addressed (1) to facilitate the discussion on its further evolution and (2) to better define appropriate vs. less suitable application areas. Exemplary toxicological studies are presented to discuss the linearity assumptions of AOP, the management of event modifiers and compensatory mechanisms, and whether a separation of toxicodynamics from toxicokinetics including metabolism is possible in the framework of pathway plasticity. Suggestions on how to compromise between different needs of AOP stakeholders have been added. A clear definition of open questions and limitations is provided to encourage further progress in the field.
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| 10. |
- Seitz, Nadja, et al.
(författare)
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A novel statistical approach for the evaluation of comet assay data
- 2008
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Ingår i: Mutation Research. - : Elsevier. - 1383-5742 .- 1388-2139. ; 652:1, s. 38-45
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Tidskriftsartikel (refereegranskat)abstract
- The present study forms part of a weight-of-evidence framework including genotoxicological studies in the upper Danube River basin, which aim at elucidating the reasons for the decline in fish catch. The major focus of this paper is the assessment of genotoxicity of sediments from the Danube River basin by use of the comet assay with RTL-W1 cells and with embryos of zebrafish (Danio rerio). A frequently discussed question in this type of approach is how to aggregate and compare the data obtained from genotoxicity testing. There is a need to develop mathematical method combining the information from dose–response curves and level of effectiveness (maximum genotoxic effect). For comparison and ranking of the genotoxic potential of samples from different locations along the Danube River, several methods based on EC50, Lowest Observed Effect Concentration (LOEC), and maximum induction factor were compared with respect to their validity. An evaluation system termed the “3-step, analysis” was developed to facilitate consideration of a maximum number of aspects of the raw data. The so-called “concentration-dependent induction factor” (CDI) introduces an index for a straightforward, precise and realistic assessment of the genotoxic potential of any kind of field sample or genotoxic agent.
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