| 1. |
- Arnaudeau, Catherine, 1975-
(författare)
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Mitotic recombination in mammalian cells
- 2000
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Recombination is usually defined as the exchange of genetic material between two strands or regions of nucleic acids. This process occurs in all known organisms and is highly conserved, especially among higher eukaryotes. Various types of recombination, involving homologous or non-homologous nucleic acid sequences, are known to exist. Recombination is a double-edged sword that may be beneficial or harmful for the cell. On one hand, it fulfills essential functions in connection with, e.g., repair of DNA double- strand breaks and maintenance of genomic stability; but, at the same time, this process is also partly responsible for, among other things, error prone repair and genomic instability, which can lead to cancer.The aim of the present study has been to investigate molecular mechanisms underlying spontaneous and induced mitotic recombination in mammalian cells and, in particular, to characterize the role of the RAD51 protein in these processes. For this purpose, V79 Chinese hamster cell lines containing spontaneous partial duplications of the hprt gene were employed. A new approach to investigate homologous recombination, which offers the unique possibility of determining the type of homologous recombination involved, was developed. This assay procedure was compared to other systems used previously for detection of induced recombination. Use of this newly developed method to characterize mechanisms underlying induction of homologous recombination revealed that inhibition of DNA synthesis is a potent pathway for such induction.Subsequently, the effect of overexpressing RAD51 on two different assays for recombination was determined. Our findings suggest that the RAD51 protein supports spontaneous homologous recombination via an exchange mechanism, as well as being involved in spontaneous non-homologous recombination, possibly with respect to class switch recombination. However, RAD51 was found not to affect induced non-homologous recombination, suggesting that this protein might not be involved in repairing DNA damage via non-homologous end-joining.Finally, the repair of DNA double-strand breaks induced in the S phase of the cell cycle was examined. Our observations in this case suggest that homologous recombination by strand invasion, employing an exchange mechanism, is a major feature of such repair and, furthermore, that a functional pathway for recombination is essential for the survival of cells in which DNA double-strand breaks have occurred.In summary, the work described here improves our understanding of the molecular mechanisms underlying spontaneous and induced recombination, as well as the repair of DNA double-strand breaks in mammalian cells.
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| 2. |
- Fotouhi, Asal, 1985-
(författare)
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The role of MTH1 in ultraviolet radiation-induced mutagenesis
- 2015
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Ultraviolet radiation (UVR) is known to be highly mutagenic. What types of DNA lesions that are induced by different UVR wavelengths are still a matter of debate. UVR induces mutagenesis mostly by the formation of photoproducts and the induction of reactive oxygen species (ROS). ROS can give rise to mutations via oxidation of nucleotides in the DNA or the nucleotide pool. Oxidized nucleotides in the nucleotide pool can thereby be incorporated into the DNA during replication and ultimately give rise to mutations. MTH1 however, dephosphorylates oxidized nucleotides in the nucleotide pool, in particular 8-oxo-dGTP and 2-OH-dATP, and inhibits their incorporation into the DNA.The aim of the present study was to investigate the role of MTH1 in mutagenesis and cytogenetic damage induced by UVR in a human lymphoblastoid TK6 cell line. The clonogenic survival, mutant frequency and micronucleus frequency were measured following exposure to UVA, UVB and UVC in MTH1-knockdown and wild-type TK6 cells. As a biomarker for oxidative damage the level of intracellular and extracellular 8-oxo-dG was measured in TK6 cells exposed to UVA. The mutational spectra of UVA-induced mutations at the thymidine kinase gene in MTH1-knockdown and wild-type TK6 cells were investigated.The results show that MTH1 protects against UVA and UVB mutagenesis significantly. MTH1, however, has been shown to offer no protection against UVR-induced cytogenetic damage and is therefore suggested to mainly inhibit mutagenesis. The mutational spectra show that GC>AT and AT>GC transitions are the dominant mutation types in cells exposed to UVA.In conclusion, MTH1 protects TK6 cells against mutagenesis induced by longer wavelengths of UVR. This indicates that the nucleotide pool is a significant target in mutagenesis for longer wavelengths of UVR. The type of mutations induced by UVA, GC>AT and AT>GC, can be formed by the incorporation of 2-OH-dATP from nucleotide pool into the DNA. UVA is therefore suggested to induce mutations by induction of oxidized nucleotides such as 2-OH-dATP.
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| 3. |
- Johansson, Fredrik, 1970-
(författare)
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Interplay between DNA repair pathways at replication forks in mammalian cells
- 2005
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Cellular DNA is continuously being damaged by various agents of both endogenous and exogenous origin including a wide range of chemical compounds, UV-light and ionizing radiation. In order to remove lesions from the DNA and maintain genomic stability, different repair processes have evolved. If not repaired prior to DNA replication, such lesions may block the replication machinery and the capacity to rescue replication stalled at the site of a DNA lesion is thus also important for cellular survival and the maintenance of genome integrity. To date, the interplay between pathways of DNA repair and mechanisms of replication bypass in mammalian cells has not been fully elucidated.The present thesis focuses on the roles played by base- and nucleotide-excision repair and by homologous recombination in reducing errors in connection with replication of damaged DNA in mammalian cells. We were also interested in the effects of caffeine on cellular mechanisms that facilitate replication bypass. Furthermore, we have investigated the mutagenicity of glycidamide and attempted to identify the DNA repair pathway responsible for removing lesions induced by this genotoxic metabolite of acrylamide. In all of these studies Chinese hamster cell lines harbouring mutations in genes encoding proteins associated with DNA repair were employed as the model system. Moreover, we explored the possibility of using these cell lines to develop a system for high-throughput, rapid detection of genotoxic agents.Our results indicates that both nucleotide excision repair and homologous recombination are involved in maintaining replication fork progression on DNA damaged by UV-light or benzo(a)pyrene-7,8-diol-9,10-epoxide. In this connection homologous recombination appears to be a time-consuming process. We also found that the replication fork delay observed following exposure to UV-light is prolonged even further by subsequent treatment with caffeine, both in the case of wild-type cells and cells deficient in homologous recombination or nucleotide-excision repair. In addition, the frequency of mutations induced by UV-irradiation was attenuated, while the level of recombination was enhanced by caffeine. These results indicate that caffeine inhibits translesion DNA synthesis, thereby favouring the use of homologous recombination to bypass lesions that stall replication.On the basis of our findings with methyl methanesulfonate (MMS) and N-methyl-N´-nitro-N-nitrosoguanidine (MNNG), we propose that O6-methylguanine is the major substrate for homologous recombination following treatment with alkylating agents. Both of these agents also stall replication forks, probably due to the presence of unrepaired intermediates in base-excision repair. Moreover, we discuss the possibility of a Rad51-mediated pathway for homologous recombination that is dependent on XRCC1.Furthermore, glycidamide was shown to cause mutations in the hprt gene of wild-type Chinese hamster ovary cells. Our results also suggest that the DNA lesions induced by this compound are repaired by short-patch BER. However, we were unable to identify the lesion responsible for the mutations.Finally, the Chinese hamster ovary cell lines employed here provide a useful tool for the screening of genotoxic compounds and, in addition, for obtaining mechanistic information concerning the pathways involved in the repair of DNA lesions induced by various agents.
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| 4. |
- Karlsson, Hanna L., et al.
(författare)
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Mechanism-based genotoxicity screening of metal oxide nanoparticles using the ToxTracker panel of reporter cell lines
- 2014
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Ingår i: Particle and Fibre Toxicology. - : Springer Science and Business Media LLC. - 1743-8977. ; 11, s. 41-
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Tidskriftsartikel (refereegranskat)abstract
- Background: The rapid expansion of manufacturing and use of nano-sized materials fuels the demand for fast and reliable assays to identify their potential hazardous properties and underlying mechanisms. The ToxTracker assay is a recently developed mechanism-based reporter assay based on mouse embryonic stem (mES) cells that uses GFP-tagged biomarkers for detection of DNA damage, oxidative stress and general cellular stress upon exposure. Here, we evaluated the ability of the ToxTracker assay to identify the hazardous properties and underlying mechanisms of a panel of metal oxide-and silver nanoparticles (NPs) as well as additional non-metallic materials (diesel, carbon nanotubes and quartz). Methods: The metal oxide-and silver nanoparticles were characterized in terms of agglomeration and ion release in cell medium (using photon cross correlation spectroscopy and inductively coupled plasma with optical emission spectroscopy, respectively) as well as acellular ROS production (DCFH-DA assay). Cellular uptake was investigated by means of transmission electron microscopy. GFP reporter induction and cytotoxicity of the NPs was simultaneously determined using flow cytometry, and genotoxicity was further tested using conventional assays (comet assay, gamma-H(2)AX and RAD51 foci formation). Results: We show that the reporter cells were able to take up nanoparticles and, furthermore, that exposure to CuO, NiO and ZnO nanoparticles as well as to quartz resulted in activation of the oxidative stress reporter, although only at high cytotoxicity for ZnO. NiO NPs activated additionally a p53-associated cellular stress response, indicating additional reactive properties. Conventional assays for genotoxicity assessment confirmed the response observed in the ToxTracker assay. We show for CuO NPs that the induction of oxidative stress is likely the consequence of released Cu ions whereas the effect by NiO was related to the particles per se. The DNA replication stress-induced reporter, which is most strongly associated with carcinogenicity, was not activated by any of the tested nanoparticles. Conclusions: We conclude that the ToxTracker reporter system can be used as a rapid mechanism-based tool for the identification of hazardous properties of metal oxide NPs. Furthermore, genotoxicity of metal oxide NPs seems to occur mainly via oxidative stress rather than direct DNA binding with subsequent replication stress.
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| 5. |
- 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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| 6. |
- 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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| 7. |
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| 8. |
- van Bezooijen, Rutger L., et al.
(författare)
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Wnt but not BMP signaling is involved in the inhibitory action of sclerostin on BMP-stimulated bone formation
- 2007
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Ingår i: Journal of Bone and Mineral Research. - 0884-0431 .- 1523-4681. ; 22:1, s. 19-28
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Tidskriftsartikel (refereegranskat)abstract
- Sclerostin is an osteocyte-derived negative regulator of bone formation. It inhibits BMP-stimulated bone formation both in vitro and in vivo but has no direct effect on BMP signaling. Instead, sclerostin inhibits Wnt signaling that is required for BMP-stimulated osteoblastic differentiation. Introduction: Sclerostin is a member of the Dan family of glycoproteins of which many members have been reported to antagonize BMP activity. Sclerostin has been shown to inhibit BMP-stimulated bone formation, but its mechanism of action seems to be different from classical BMP antagonists. In this study, we investigated the mechanism by which sclerostin inhibits BMP-stimulated bone formation. Materials and Methods: DNA electroporation of calf muscle of mice using expression plasmids for BMP and sclerostin was used to study the effect of sclerostin on BMP-induced bone formation in vivo. Transcriptional profiling using microarrays of osteoblastic cells treated with BMP in the absence or presence of sclerostin was used to find specific growth factor signaling pathways affected by sclerostin. The affected pathways were further studied using growth factor-specific reporter constructs. Results: BMP-induced ectopic bone formation in calf muscle of mice was prevented by co-expression of sclerostin in vivo. Transcriptional profiling analysis of osteoblastic cultures indicated that sclerostin specifically affects BMP and Wnt signaling out of many other growth signaling pathways. Sclerostin, however, did not inhibit stimulation of direct BMP target genes. Furthermore, we did not obtain any evidence for sclerostin acting as a direct BMP antagonist using a BMP-specific reporter construct. In contrast, sclerostin shared many characteristics with the Wnt antagonist dickkopf-1 in antagonizing BMP-stimulated bone formation and BMP- and Wnt-induced Wnt reporter construct activation. Conclusions: Sclerostin inhibits BMP-stimulated bone formation but does not affect BMP signaling. Instead, it antagonizes Wnt signaling in osteoblastic cells. High bone mass in sclerosteosis and van Buchem disease may, therefore, result from increased Wnt signaling.
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