| 1. |
- Lundqvist, Johan, et al.
(författare)
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In vitro bioanalytical evaluation of removal efficiency for bioactive chemicals in Swedish wastewater treatment plants
- 2019
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Ingår i: Scientific Reports. - : Nature Publishing Group. - 2045-2322. ; 9
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Tidskriftsartikel (refereegranskat)abstract
- Chemical contamination of wastewater is a problem of great environmental concern, as it poses a hazard to both the ecosystem and to human health. In this study, we have performed a bioanalytical evaluation of the presence and removal efficiency for bioactive chemicals in wastewater treatment plants (WWTPs), using in vitro assays for toxicity endpoints of high relevance for human health. Water samples were collected at the inlet and outlet of five Swedish WWTPs, all adopting a treatment technology including pretreatment, primary treatment (sedimenation), seconday treatment (biological processes), post-sedimentation, and sludge hand ling. The water samples were analyzed for cytotoxicity, estrogenicity, androgenicity, aryl hydrocarbon receptor (AhR) activity, oxidative stress response (Nrf2) and the ability to activate NF kappa B (nuclear factor kappa-light-chain-enhancer of activated B cells) signaling. We observed clear androgenic and estrogenic activities in all inlet samples. Androgenic and estrogenic activities were also observed in all outlet samples, but the activities were lower than the respective inlet sample. AhR activity was observed in all samples, with higher activities in the inlet samples compared to the outlet samples. The removal efficiency was found to be high for androgenic (>99% for two plants and 50-60%for two plants) and estrogenic (>90%for most plants) compounds, while the removal efficiency for AhR-inducing compounds was 50-60%for most plants and 16%for one plant.
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| 2. |
- Lungu-Mitea, Sebastian, et al.
(författare)
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Development of an oxidative stress in vitro assay in zebrafish (Danio rerio) cell lines
- 2018
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Ingår i: Scientific Reports. - : Nature Publishing Group. - 2045-2322. ; 8
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Tidskriftsartikel (refereegranskat)abstract
- The nuclear factor erythroid 2-related factor 2 (Nrf2) is a key regulator of cellular defense against oxidative stress and correlated with classical toxicological endpoints. In vitro methods using fish cell lines for the assessment of aquatic toxicity are needed for mechanistic studies and as an alternative to in vivo. We describe an in vitro assay to study oxidative stress using zebrafish cell lines. Transfection efficiency of twelve commercially available transfection reagents were tested in the zebrafish cell lines ZFL, ZF4, and Pac2. The most efficient reagent for each cell line was selected for further experiments. Cells were transiently transfected with an Nrf2-responsive luciferase plasmid. The assay was tested using the oxidative stress inducing chemicals tertbutylhydroquinone, hydrogen peroxide, and sulforaphane. Of the transfected cell lines, ZF4 and ZFL showed higher sensitivity. The latter were used to study potential oxidative stress induced by pesticides (diazinon, deltamethrin, atrazine, metazachlor, terbutylazine, diuron). Besides known inducers, Nrf2 activity was also significantly induced by diazinon, deltametrin, diuron, and metazachlor. Activation of Nrf2 by metazachlor is a novel finding. The described assay could be a valuable tool for research in toxicology to study the stress response of both pure chemicals and environmental water samples.
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| 3. |
- Lungu-Mitea, Sebastian, et al.
(författare)
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Development, scrutiny, and modulation of transient reporter gene assays of the xenobiotic metabolism pathway in zebrafish hepatocytes
- 2023
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Ingår i: Cell Biology and Toxicology. - : Springer Nature. - 0742-2091 .- 1573-6822. ; 39, s. 991-1013
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Tidskriftsartikel (refereegranskat)abstract
- The "toxicology in the twenty-first century" paradigm shift demands the development of alternative in vitro test systems. Especially in the field of ecotoxicology, coverage of aquatic species-specific assays is relatively scarce. Transient reporter gene assays could be a quick, economical, and reliable bridging technology. However, the user should be aware of potential pitfalls that are influenced by reporter vector geometry. Here, we report the development of an AhR-responsive transient reporter-gene assay in the permanent zebrafish hepatocytes cell line (ZFL). Additionally, we disclose how viral, constitutive promoters within reporter-gene assay cassettes induce squelching of the primary signal. To counter this, we designed a novel normalization vector, bearing an endogenous zebrafish-derived genomic promoter (zfEF1aPro), which rescues the squelching-delimited system, thus, giving new insights into the modulation of transient reporter systems under xenobiotic stress. Finally, we uncovered how the ubiquitously used ligand BNF promiscuously activates multiple toxicity pathways of the xenobiotic metabolism and cellular stress response in an orchestral manner, presumably leading to a concentration-related inhibition of the AhR/ARNT/XRE-toxicity pathway and non-monotonous concentration-response curves. We named such a multi-level inhibitory mechanism that might mask effects as "maisonette squelching."
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| 4. |
- Lungu-Mitea, Sebastian, et al.
(författare)
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Modeling Bioavailable Concentrations in Zebrafish Cell Lines and Embryos Increases the Correlation of Toxicity Potencies across Test Systems
- 2021
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Ingår i: Environmental Science and Technology. - : American Chemical Society (ACS). - 0013-936X .- 1520-5851. ; 55:1, s. 447-457
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Tidskriftsartikel (refereegranskat)abstract
- Linking cellular toxicity to low-tier animal toxicity and beyond is crucial within the adverse outcome pathway concept and the 3R framework. This study aimed to determine and compare the bioavailable effect concentrations in zebrafish cell lines and embryos. Acute, short-term toxicity (48 h) of eight veterinary pharmaceuticals was measured in two zebrafish cell lines (hepatocytes, fibroblasts) and zebrafish embryos. Seven endpoints of cytotoxicity were recorded. The fish embryo acute toxicity test was modified by adding sublethal endpoints. Chemical distribution modeling (mass balance) was applied to compute the bioavailable compound concentrations in cells (C-free) and embryos (C-int;aq) based on nominal effect concentrations (C-nom). Effect concentration ratios were calculated (cell effects/embryo effects). A low correlation was observed between cytotoxicity and embryo toxicity when nominal concentrations were used. Modeled bioavailable effect concentrations strongly increased correlations and placed regression lines close to the line of unity and axis origin. Cytotoxicity endpoints showed differences in sensitivity and predictability. The hepatocyte cell line depicted closer proximity to the embryo data. Conclusively, the high positive correlation between the cell- and embryo-based test systems emphasizes the appropriate modulation of toxicity when linked to bioavailable concentrations. Furthermore, it highlights the potential of fish cell lines to be utilized in integrated testing strategies.
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| 5. |
- Lungu-Mitea, Sebastian, et al.
(författare)
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Potentials and pitfalls of transient in vitro reporter bioassays : interference by vector geometry and cytotoxicity in recombinant zebrafish cell lines
- 2020
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Ingår i: Archives of Toxicology. - : Springer Nature. - 0340-5761 .- 1432-0738. ; 94:8, s. 2769-2784
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Tidskriftsartikel (refereegranskat)abstract
- The water framework directive re-evaluation proposes the integration of effect-based tools, increasing the need for alternative methods. Especially within aquatic toxicology, coverage of specific toxicity pathways is scarce, and most applications are based on mammalian or bacterial models, not reflecting realistic exposure scenarios. The use of transient reporter gene assays in cells from organisms of interest could be a quick and inexpensive solution. However, interference with cellular homeostasis may impact the system beyond the function of the manipulated gene and thus lead to non-specific results. We describe how varying vector geometry and different regulatory gene elements on plasmids used for transfection in zebrafish hepatocytes and embryonic fibroblasts may lead up to a tenfold difference in potency. Cells were transiently co-transfected with an Nrf2-responsive Firefly luciferase reporter plasmid and eight different Renilla luciferase normalization plasmids. Transfected cells were exposed to two different regimes (0.1-100 mu M and 7.8-250 mu M) of the oxidative stress-inducing compounds, sulforaphane, tertbutylhydroquinone, and metazachlor. Nrf2 activity was measured in dual-luciferase assays. In parallel, cytotoxicity was assessed for different endpoints (energy metabolism, protein amount, membrane stability, and cell proliferation) in non-transfected cells and cells co-transfected with constructs of increasing size, to be used for normalization. Transfected cells were more susceptible to cytotoxicity in a vector size-dependent manner. Conclusively, we report that vector geometries (size, backbones, gene-regulatory units), cell line (tissue origin), applied transfection methods, and signal normalization may alter the sensitivity of reporter bioassays in a synergistic manner. Further, we propose that thorough bioassay design is needed to ensure reliability and regulatory acceptance.
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| 6. |
- Lungu-Mitea, Sebastian
(författare)
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Toxicity pathways in zebrafish cell lines : an ecotoxicological perspective on "toxicity testing in the 21st century"
- 2020
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Standard toxicological in vivo testing has been challenged as the procedures are time-consuming, expensive, and require a large number of animals; given the number of problematic chemicals. Novel toxicological frameworks, such as "toxicity testing in the 21st century", proposed the use of "new approach methods" (in vitro and in silico techniques), that can be applied in high-throughput setups and would allow for the testing of a large number of compounds. However, such new approach methods need to be designed and evaluated first. Especially within ecotoxicology, the coverage of species-specific bioanalytical tools, e.g. for fish, is rather scarce. Currently, mainly in vitro assays of mammalian and bacterial origin are used. This thesis outlines how to design and scrutinise fish transient reporter gene assays. We have established transient reporter gene assays in permanent zebrafish fibroblasts and hepatocytes of the oxidative stress response and the xenobiotic metabolism toxicity pathways. We identified non-specific effects caused by transient transfection itself and suggested preventive strategies. Further, we identified toxicity pathways’ cross-talk as a significant driver of uncertainty in regards to the assessment of receptor-mediated toxicity. Additionally, we evaluated the correlation between cytotoxicity in cultured zebrafish cells and the acute toxicity observed in zebrafish embryos. When using chemical distribution models to derive bioavailable concentrations, we observed a good positive correlation between the two test systems. The results advocate an intensified use of fish in vitro assays in integrated testing strategies. Conclusively, new approach methods, as developed and applied in this thesis, show great potential in future toxicity testing and environmental monitoring.
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| 7. |
- Zhao, Haiyu, et al.
(författare)
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Modulation of DNA Repair Systems in Blind Cavefish during Evolution in Constant Darkness
- 2018
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Ingår i: Current Biology. - : Cell Press. - 0960-9822 .- 1879-0445. ; 28:20, s. 3229-3243
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Tidskriftsartikel (refereegranskat)abstract
- How the environment shapes the function and evolution of DNA repair systems is poorly understood. In a comparative study using zebrafish and the Somalian blind cavefish, Phreatichthys andruzzii, we reveal that during evolution for millions of years in continuous darkness, photoreactivation DNA repair function has been lost in P. andruzzii. We demonstrate that this loss results in part from loss-of-function mutations in pivotal DNA-repair genes. Specifically, C-terminal truncations in P. andruzzii DASH and 6-4 photolyase render these proteins predominantly cytoplasmic, with consequent loss in their functionality. In addition, we reveal a general absence of light-, UV-, and ROS-induced expression of P. andruzzii DNA-repair genes. This results from a loss of function of the D-box enhancer element, which coordinates and enhances DNA repair in response to sunlight. Our results point to P. andruzzii being the only species described, apart from placental mammals, that lacks the highly evolutionary conserved photoreactivation function. We predict that in the DNA repair systems of P. andruzzii, we may be witnessing the first stages in a process that previously occurred in the ancestors of placental mammals during the Mesozoic era.
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| 8. |
- Zhao, Haiyu, et al.
(författare)
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Regulation of ddb2 expression in blind cavefish and zebrafish reveals plasticity in the control of sunlight-induced DNA damage repair
- 2021
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Ingår i: PLOS Genetics. - : Public Library of Science (PLoS). - 1553-7390 .- 1553-7404. ; 17:2
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Tidskriftsartikel (refereegranskat)abstract
- We have gained considerable insight into the mechanisms which recognize and repair DNA damage, but how they adapt to extreme environmental challenges remains poorly understood. Cavefish have proven to be fascinating models for exploring the evolution of DNA repair in the complete absence of UV-induced DNA damage and light. We have previously revealed that the Somalian cavefish Phreatichthys andruzzii, lacks photoreactivation repair via the loss of light, UV and ROS-induced photolyase gene transcription mediated by D-box enhancer elements. Here, we explore whether other systems repairing UV-induced DNA damage have been similarly affected in this cavefish model. By performing a comparative study using P. andruzzii and the surface-dwelling zebrafish, we provide evidence for a conservation of sunlight-regulated Nucleotide Excision Repair (NER). Specifically, the expression of the ddb2 gene which encodes a key NER recognition factor is robustly induced following exposure to light, UV and oxidative stress in both species. As in the case of the photolyase genes, D-boxes in the ddb2 promoter are sufficient to induce transcription in zebrafish. Interestingly, despite the loss of D-box-regulated photolyase gene expression in P. andruzzii, the D-box is required for ddb2 induction by visible light and oxidative stress in cavefish. However, in the cavefish ddb2 gene this D-box-mediated induction requires cooperation with an adjacent, highly conserved E2F element. Furthermore, while in zebrafish UV-induced ddb2 expression results from transcriptional activation accompanied by stabilization of the ddb2 mRNA, in P. andruzzii UV induces ddb2 expression exclusively via an increase in mRNA stability. Thus, we reveal plasticity in the transcriptional and post transcriptional mechanisms regulating the repair of sunlight-induced DNA damage under long-term environmental challenges.Author summaryThe integrity of genetic information is frequently challenged by environmental factors such as sunlight which induce mutations in DNA. Therefore, DNA damage repair mechanisms are ubiquitous and highly conserved. While significant progress has been made in understanding how these mechanisms recognize and repair DNA damage, how they adapt to long-term environmental challenges remains poorly understood. Cavefish have proven to be fascinating models for exploring the function of DNA repair systems in extreme photic environments. We have previously shown that during evolution for millions of years in complete isolation from sunlight, the Somalian cavefish, Phreatichthys andruzzii has lost photoreactivation, a ubiquitous, light-dependent DNA repair system. This results in part from a loss of light, UV and ROS-induced gene transcription. Have other repair systems targeting UV-induced DNA damage been affected in cavefish? Here, we provide evidence that Nucleotide Excision Repair (NER) function is retained in cavefish and is upregulated upon sunlight exposure. Furthermore, we reveal complexity in the transcriptional and posttranscriptional mechanisms regulating the repair of UV-induced DNA damage.
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