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  • 1.
    Aasa, Jenny
    et al.
    Stockholm University, Faculty of Science, Department of Environmental Science and Analytical Chemistry.
    Vare, Daniel
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Motwani, Hitesh V.
    Stockholm University, Faculty of Science, Department of Environmental Science and Analytical Chemistry.
    Jenssen, Dag
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Törnqvist, Margareta
    Stockholm University, Faculty of Science, Department of Environmental Science and Analytical Chemistry.
    Quantification of the mutagenic potency and repair of glycidol-induced DNA lesions2016In: Mutation research. Genetic toxicology and environmental mutagenesis, ISSN 1383-5718, E-ISSN 1879-3592, Vol. 805, p. 38-45Article in journal (Refereed)
    Abstract [en]

    Glycidol (Gly) is an electrophilic low-molecular weight epoxide that is classified by IARC as probably carcinogenic to humans. Humans might be exposed to Gly from food, e.g. refined vegetable oils, where Gly has been found as a food process contaminant. It is therefore important to investigate and quantify the genotoxicity of Gly as a primary step towards cancer risk assessment of the human exposure. Here, quantification of the mutagenic potency expressed per dose (AUC: area under the concentration time curve) of Gly has been performed in Chinese hamster ovary (CHO) cells, using the HPRT assay. The dose of Gly was estimated in the cell exposure medium by trapping Gly with a strong nucleophile, cob(I)alamin, to form stable cobalamin adducts for analysis by LC-MS/MS. Gly was stable in the exposure medium during the time for cell treatment, and thus the dose in vitro is the initial concentration x cell treatment time. Gly induced mutations in the hprt-gene at ante of 0.08 +/- 0:01 mutations/10(5) cells/mMh. Through comparison with the effect of ionizing radiation in the same system a relative mutagenic potency of 9.5 rad-eq./mMh was obtained, which could be used for comparison of genotoxicity of chemicals and between test systems and also in procedures for quantitative cancer risk assessment. Gly was shown to induce strand breaks, that were repaired by base excision repair. Furthermore, Gly-induced lesions, present during replication, were found to delay the replication fork elongation. From experiments with repair deficient cells, homologous recombination repair and the ERCC1-XPF complex were indicated to be recruited to support in the repair of the damage related to the stalled replication elongation. The type of DNA damage responsible for the mutagenic effect of Gly could not be concluded from the present study.

  • 2. Al-Minawi, Ali Z.
    et al.
    Lee, Yin-Fai
    Håkansson, Daniel
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Johansson, Fredrik
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Lundin, Cecilia
    Saleh-Gohari, Nasrollah
    Schultz, Niklas
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Jenssen, Dag
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Bryant, Helen E.
    Meuth, Mark
    Hinz, John M.
    Helleday, Thomas
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    The ERCC1/XPF endonuclease is required for completion of homologous recombination at DNA replication forks stalled by inter-strand cross-links2009In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 37, no 19, p. 6400-6413Article in journal (Refereed)
    Abstract [en]

    Both the ERCC1-XPF complex and the proteins involved in homoIogous recombination (HR) have critical roles in inter-strand cross-link (ICL) repair. Here, we report that mitomycin C-induced lesions inhibit replication fork elongation. Furthermore, mitomycin C-induced DNA double-strand breaks (DSBs) are the result of the collapse of ICL-stalled replication forks. These are not formed through replication run off, as we show that mitomycin C or cisplatin-induced DNA lesions are not incised by global genome nucleotide excision repair (GGR). We also suggest that ICL-lesion repair is initiated either by replication or transcription, as the GGR does not incise ICL-lesions. Furthermore, we report that RAD51 foci are induced by cisplatin or mitomycin C independently of ERCC1, but that mitomycin C-induced HR measured in a reporter construct is impaired in ERCC1-defective cells. These data suggest that ERCC1-XPF plays a role in completion of HR in ICL repair. We also find no additional sensitivity to cisplatin by siRNA co-depletion of XRCC3 and ERCC1, showing that the two proteins act on the same pathway to promote survival.

  • 3.
    Carlsson, Henrik
    et al.
    Stockholm University, Faculty of Science, Department of Environmental Science and Analytical Chemistry.
    Aasa, Jenny
    Stockholm University, Faculty of Science, Department of Environmental Science and Analytical Chemistry.
    Abramsson-Zetterberg, Lilianne
    Vare, Daniel
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Kotova, Natalia
    Törnqvist, Margareta
    Stockholm University, Faculty of Science, Department of Environmental Science and Analytical Chemistry.
    Adductomic Screening of N-terminal Hemoglobin Adducts and Measurement of Micronuclei in Blood Samples from Swedish School ChildrenArticle in journal (Refereed)
  • 4.
    Kotova, Natalia
    et al.
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Hebert, N.
    Härnwall, Eva-Lena
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Vare, Daniel
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Mazurier, C.
    Douay, L.
    Jenssen, Dag
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Grawe, J.
    A novel micronucleus in vitro assay utilizing human hematopoietic stem cells2015In: Toxicology in Vitro, ISSN 0887-2333, E-ISSN 1879-3177, Vol. 29, no 7, p. 1897-1905Article in journal (Refereed)
    Abstract [en]

    The induction of micronucleated reticulocytes in the bone marrow is a sensitive indicator of chromosomal damage. Therefore, the micronucleus assay in rodents is widely used in genotoxicity and carcinogenicity testing. A test system based on cultured human primary cells could potentially provide better prediction compared to animal tests, increasing patient safety while also implementing the 3Rs principle, i.e. replace, reduce and refine. Hereby, we describe the development of an in vitro micronucleus assay based on animal-free ex vivo culture of human red blood cells from hematopoietic stem cells. To validate the method, five clastogens with direct action, three clastogens requiring metabolic activation, four aneugenic and three non-genotoxic compounds have been tested. Also, different metabolic systems have been applied. Flow cytometry was used for detection and enumeration of micronuclei. Altogether, the results were in agreement with the published data and indicated that a sensitive and cost effective in vitro assay to assess genotoxicity with a potential to high-throughput screening has been developed.

  • 5.
    Kotova, Natalia
    et al.
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Vare, Daniel
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Schultz, Niklas
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Gradecka Meesters, Dobrosława
    Stępnik, Maciej
    Grawé, Jan
    Helleday, Thomas
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Jenssen, Dag
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Genotoxicity of alcohol is linked to DNA replication-associated damage and homologous recombination repairIn: Carcinogenesis, ISSN 0143-3334, E-ISSN 1460-2180Article in journal (Refereed)
  • 6.
    Kotova, Natalia
    et al.
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Vare, Daniel
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Schultz, Niklas
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Meesters, Dobroslawa Gradecka
    Stepnik, Maciej
    Grawe, Jan
    Helleday, Thomas
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Jenssen, Dag
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Genotoxicity of alcohol is linked to DNA replication-associated damage and homologous recombination repair2013In: Carcinogenesis, ISSN 0143-3334, E-ISSN 1460-2180, Vol. 34, no 2, p. 325-330Article in journal (Refereed)
    Abstract [en]

    Although alcohol consumption is related to increased cancer risk, its molecular mechanism remains unclear. Here, we demonstrate that an intake of 10% alcohol for 4 weeks in rats is genotoxic due to induction of micronuclei. Acetaldehyde (AA), the first product of ethanol metabolism, is believed to be responsible for DNA damage induced by alcohol. Here, we observe that AA effectively blocks DNA replication elongation in mammalian cells, resulting in DNA double-strand breaks associated with replication. AA-induced DNA damage sites colocalize with the homologous recombination (HR) repair protein RAD51. HR measured in the hypoxhantineguaninefosforibosyltransferase (HPRT) gene is effectively induced by AA and recombination defective mammalian cells are hypersensitive to AA, clearly demonstrating that HR is essential in the repair of AA-induced DNA damage. Altogether, our data indicate that alcohol genotoxicity related to AA produces replication lesions on DNA triggering HR repair.

  • 7.
    Lagerqvist, Anne
    et al.
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Håkansson, Daniel
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Frank, Heinz
    Seidel, Albrecht
    Jenssen, Dag
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Structural requirements for mutation formation from polycyclic aromatic hydrocarbon dihydrodiol epoxides in their interaction with food chemopreventive compounds2011In: Food and Chemical Toxicology, ISSN 0278-6915, E-ISSN 1873-6351, Vol. 49, no 4, p. 879-886Article in journal (Refereed)
    Abstract [en]

    Chinese hamster V79 cells were used to investigate the protective effect of four known antimutagens present in food, chlorophyllin (CHL), ellagic acid (EA), epigallocathechingallate (EGCG) and benzylisothiocyanate (BITC), against potent mutagenic polycyclic aromatic hydrocarbon dial epoxides (PAH-DE) derived from benzo[a]pyrene (BP), dibenzo[a,h]anthracene (DBA), dibenzo[a,l]pyrene (DBP), and benzo[c]phenanthrene (BPh) known to be deposited on crops from polluted ambient air or formed during food processing. As fjord-region PAH-DE are more toxic and mutagenic than bay-region PAH-DE, we adjusted the concentrations of PAH-DE to induce approximately the same levels of adducts. The studies were performed using an assay indicating toxicity in terms of reduced cell proliferation together with the V79 Hprt assay for monitoring mutant frequencies. CHL significantly increased the survival and showed a protective effect against the mutagenicity of all PAH-DE. A significant protective effect of EA was found towards the mutagenicity of BPDE, DBPDE and BPhDE and with EGCG for BPDE and BPhDE. BITC had a slight positive effect on the mutagenicity of DBADE and BPhDE. Taken together, a novel and unexpected finding was that the antimutagenic activity could differ as much as by a factor of 7 towards four carcinogenic PAH metabolites being relatively similar in structure and genotoxic activity.

  • 8.
    Lagerqvist, Anne
    et al.
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Håkansson, Daniel
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Jenssen, Dag
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Prochaska, Gabriela
    Department of Biosciences and Nutrition, Novum, Karolinska Institute.
    Lundin, Cecilia
    Department of Biosciences and Nutrition, Novum, Karolinska Institute.
    Dreij, Kristian
    Segerbäck, Dan
    Department of Biosciences and Nutrition, Novum, Karolinska Institute.
    Jernström, Bengt
    The Institute for Environmental Medicine (IMM), Karolinska Institute.
    Törnqvist, Margareta
    Stockholm University, Faculty of Science, Department of Materials and Environmental Chemistry (MMK).
    Seidel, Albrecht
    Biochemical Institute for Environmental Carcinogens, Lurup .
    Erixon, Klaus
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Both replication bypass fidelity and repair efficiency influence the yield of mutations per target dose in intact mammalian cells induced by benzo(a)pyrene-diol-epoxide and dibenzo(a,l)-pyrene-diol-epoxide.2008In: DNA Repair, ISSN 1568-7864, E-ISSN 1568-7856, DNA repair, Vol. 7, no 8, p. 1202-1012Article in journal (Refereed)
    Abstract [en]

    Mutations induced by polycyclic aromatic hydrocarbons (PAH) are expected to be produced when error-prone DNA replication occurs across unrepaired DNA lesions formed by reactive PAH metabolites such as diol epoxides. The mutagenicity of the two PAH-diol epoxides (+)-anti-7,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE) and (±)-anti-11,12-dihydroxy-13,14-epoxy-11,12,13,14-tetrahydrodibenzo[a,l]pyrene (DBPDE) was compared in nucleotide excision repair (NER) proficient and deficient hamster cell lines. We applied the 32P-postlabelling assay to analyze adduct levels and the hprt gene mutation assay for monitoring mutations. It was found that the mutagenicity per target dose was 4 times higher for DBPDE compared to BPDE in NER proficient cells while in NER deficient cells, the mutagenicity per target dose was 1.4 times higher for BPDE. In order to investigate to what extent the mutagenicity of the different adducts in NER proficient cells was influenced by repair or replication bypass, we measured the overall NER incision rate, the rate of adduct removal, the rate of replication bypass and the frequency of induced recombination in the hprt gene. The results suggest that NER of BPDE lesions are 5 times more efficient than for DBPDE lesions, in NER proficient cells. However, DBPDE adducts block replication more efficiently and also induce 6 times more recombination events in the hprt gene than adducts of BPDE, suggesting that DBPDE adducts are, to a larger extent, bypassed by homologous recombination. The results obtained here indicate that the mutagenicity of PAH is influenced not only by NER, but also by replication bypass fidelity. This has been postulated earlier based on results using in vitro enzyme assays, but is now also being recognized in terms of forward mutations in intact mammalian cells.

  • 9.
    Lagerqvist, Anne
    et al.
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Håkansson, Daniel
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Lundin, Cecilia
    Prochazka, Gabriela
    Dreij, Kristian
    Segerbäck, Dan
    Jernström, Bengt
    Törnqvist, Margareta
    Stockholm University, Faculty of Science, Department of Materials and Environmental Chemistry (MMK).
    Franke, Heinz
    Seidel, Albrecht
    Erixon, Klaus
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Jenssen, Dag
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    DNA repair and replication influence the number of mutations per adduct of polycyclic aromatic hydrocarbons in mammalian cells2011In: DNA Repair, ISSN 1568-7864, E-ISSN 1568-7856, Vol. 10, no 8, p. 877-886Article in journal (Refereed)
    Abstract [en]

    Polycyclic aromatic hydrocarbons (PAH) are an important class of environmental contaminants many of which require metabolic activation to DNA-reactive bay or fjord region diolepoxides (DE) in order to exert their mutagenic and carcinogenic effects. In this study, the mutagenicity of the bay region diolepoxides (+)-anti-7,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE) and ()-anti-1,2-dihydroxy-3,4-epoxy-1,2,3,4-tetrahydrodibenzo[a,h]anthracene (DBADE) and the fjord region diolepoxides ()-anti-11,12-dihydroxy-13,14-epoxy-11,12,13,14-tetrahydrodibenzo[a,l]-pyrene (DBPDE) and (+/-)-anti-3,4-dihydroxy-1,2-epoxy-1,2,3,4-tetrahydrobenzo[c]-phenanthrene (BPhDE) was compared in nucleotide excision repair (NER) proficient and deficient hamster cell lines. The (32)P-postlabelling assay was applied to analyze DNA adduct levels and the Hprt gene mutation assay for monitoring mutations. Previously, we found that the mutagenicity per adduct was four times higher for DBPDE compared to BPDE in NER proficient cells. In these same cells, the mutagenicity of DBADE and BPhDE adducts was now found to be significantly lower compared to that of BPDE. In NER deficient cells the highest mutagenicity per adduct was found for BPDE and there was a tenfold and fivefold difference when comparing the BPDE data with the DBADE and BPhDE data, respectively. In order to investigate to what extent the mutagenicity of the different adducts in NER proficient cells was influenced by repair or replication bypass, we measured the overall NER incision rate, the rate of adduct removal, the rate of replication bypass and the frequency of induced recombination in the Hprt gene. Since NER turned out to be an important pathway for the yield of mutations, we further analyzed the role of transcription coupled NER versus global genome NER. However, our data demonstrate that neither of these pathways seems to be the sole factor determining the mutation frequency of the four PAH-DE and that the differences in the repair efficiency of these compounds could not be related to the presence of a bay or fjord region in the parent PAH.

  • 10.
    Latvala, Siiri
    et al.
    Stockholm University, Faculty of Science, Department of Environmental Science and Analytical Chemistry.
    Vare, Daniel
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Karlsson, Hanna
    Elihn, Karine
    Stockholm University, Faculty of Science, Department of Environmental Science and Analytical Chemistry.
    In vitro genotoxicity assessment of airborne nickel nanoparticles using air-liquid interface exposure2016Manuscript (preprint) (Other academic)
  • 11.
    Latvala, Siiri
    et al.
    Stockholm University, Faculty of Science, Department of Environmental Science and Analytical Chemistry.
    Vare, Daniel
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Karlsson, Hanna L.
    Elihn, Karine
    Stockholm University, Faculty of Science, Department of Environmental Science and Analytical Chemistry.
    In vitro genotoxicity of airborne Ni-NP in air-liquid interface2017In: Journal of Applied Toxicology, ISSN 0260-437X, E-ISSN 1099-1263, Vol. 37, no 12, p. 1420-1427Article in journal (Refereed)
    Abstract [en]

    Studies using advanced toxicological methods enabling in vitro conditions that are more realistic are currently needed for understanding the risks of pulmonary exposure to airborne nanoparticles. Owing to the carcinogenicity of certain nickel compounds, the increased production of nickel nanoparticles (Ni-NPs) raises occupational safety concerns. The aim of this study was to investigate the genotoxicity of airborne Ni-NPs using a recently developed air-liquid interface exposure system. The wild-type Chinese hamster lung fibroblast cell line (V79) was used and cytotoxicity, DNA damage and mutagenicity were studied by testing colony forming efficiency, alkaline DNA unwinding and HPRT mutation assays, respectively. Additionally, co-exposure to a PARP-1 inhibitor was performed to test possible involvement of base excision repair (BER) in repair of Ni-induced DNA damage. The results showed that cell viability was reduced significantly (to 45% and 46%) after 48hours Ni-NP exposure at concentrations of 0.15 and 0.32g cm(-2). DNA damage was significantly increased after Ni-NP exposure in the presence of the BER inhibitor indicating that Ni-NP-induced DNA damages are subsequently repaired by BER. Furthermore, there was no increased HPRT mutation frequency following Ni-NP exposure. In conclusion, this study shows that Ni-NP treatment of lung fibroblasts in an air-liquid interface system that mimics real-life exposure, results in increased DNA strand breaks and reduced cellular viability. These DNA lesions were repaired with BER in an error-free manner without resulting in mutations. This study also underlines the importance of appropriate quantification of the actual exposure concentrations during air-liquid interface exposure studies. The aim of this study was to investigate the genotoxicity of airborne Ni nanoparticles using a recently developed air-liquid interface exposure system that mimics real-life exposure. Cytotoxicity, DNA damage and mutagenicity were in the V79 cell line. Ni nanoparticle exposure of the cells in the air-liquid interface resulted in increased DNA strand breaks and reduced cellular viability at concentrations of 0.15 and 0.32 g cm (-2). These DNA lesions were repaired with BER in an error-free manner without resulting in mutations

  • 12.
    Vare, Daniel
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Interstrand Crosslinks - Induction and repair2012Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    DNA crosslinking agents exhibit a variety of DNA lesions, such as monoadducts, DNA-DNA interstrand or intrastrand crosslinks or DNA-protein crosslinks. Agents that produce interstrand crosslinks (ICLs) exist naturally and are widely used in chemotherapy. Therefore, it is important to understand how the lesions induced by these agents are repaired. In bacteria, the repair is mainly dependent on nucleotide excision repair (NER) together with homologous recombination (HR) or translesion synthesis (TLS). In human cells, it is not clear how these lesions are repaired, and it is believed to be a more complicated process in which NER does not play as important a role as in prokaryotes. Here, we investigated the repair mechanisms mainly after treatment with psoralen but also with acetaldehyde, cisplatin and mitomycin C in some studies. As expected from studies on plasmids and in bacteria, we used new techniques to confirm that various ICL-inducing agents block replication fork elongation in mammalian cells. We also found that the replication fork was unable to bypass these lesions. We confirmed that ERCC1/XPF and the HR proteins BRCA2 and XRCC2/3 are vital for protection against ICL treatments. These proteins were also found to be equally important for the repair of monoadducts. To better understand ICL repair in mammalian cells, we developed a method to study the induction and unhooking of ICL in human fibroblasts. We found that ICLs were repaired and that 50% of the induced ICLs were unhooked within 3 hours following exposure. Additionally, we determined that XPA, but not XPE, is involved in ICL unhooking, although not affecting lethality. A step in ICL repair is the formation of double-strand breaks (DSBs), and we identified a replication-dependent formation of DSBs following ICL treatment. Furthermore, ERCC1/XPF was not necessary for DSB formation. The repair of these DSBs was performed by HR and involved ERCC1/XPF. Additionally, we were able to quantify the ICL unhooking in human fibroblasts and found that they can unhook ~2500 ICL/h. We also determined that a dose of approximately 400 ICL/cell is lethal to 50% of the cells, indicating that ICL unhooking is not the most critical step during the repair process.

  • 13.
    Vare, Daniel
    et al.
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Erixon, Klaus
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Johansson, Fredrik
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Jenssen, Dag
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    XPA has a key role in the unhooking step of ICL repair in intact mammalian cellsManuscript (preprint) (Other academic)
  • 14.
    Vare, Daniel
    et al.
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Groth, Petra
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Carlsson, Rickard
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Johansson, Fredrik
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Erixon, Klaus
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Jenssen, Dag
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    DNA interstrand crosslinks induce a potent replication block followed by formation and repair of double strand breaks in intact mammalian cellsIn: DNA Repair, ISSN 1568-7864, E-ISSN 1568-7856Article in journal (Refereed)
  • 15.
    Vare, Daniel
    et al.
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Groth, Petra
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Carlsson, Rickard
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Johansson, Fredrik
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Erixon, Klaus
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Jenssen, Dag
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    DNA interstrand crosslinks induce a potent replication block followed by formation and repair of double strand breaks in intact mammalian cells2012In: DNA Repair, ISSN 1568-7864, E-ISSN 1568-7856, Vol. 11, no 12, p. 976-985Article in journal (Refereed)
    Abstract [en]

    DNA interstrand crosslinks (ICLs) are highly toxic lesions that covalently link both strands of DNA and distort the DNA helix. Crosslinking agents have been shown to stall DNA replication and failure to repair ICL lesions before encountered by replication forks may induce severe DNA damage. Most knowledge of the ICL repair process has been revealed from studies in bacteria and cell extracts. However, for mammalian cells the process of ICL repair is still unclear and conflicting data exist. In this study we have explored the fate of psoralen-induced ICLs during replication, by employing intact mammalian cells and novel techniques. By comparative studies distinguishing between effects by monoadducts versus ICLs, we have been able to link the block of replication to the ICLs induction. We found that the replication fork was equally blocked by ICLs in wild-type cells as in cells deficient in ERCC1/XPF and XRCC3. The formation of ICL induced double strand breaks (DSBs), detected by formation of 53PB1 foci, was equally induced in the three cell lines suggesting that these proteins are involved at a later step of the repair process. Furthermore, we found that forks blocked by ICLs were neither bypassed, restarted nor restored for several hours. We propose that this process is different from that taking place following monoadduct induction by UV-light treatment where replication bypass is taking place as an early step. Altogether our findings suggest that restoration of an ICL blocked replication fork, likely initiated by a DSB occurs relatively rapidly at a stalled fork, is followed by restoration, which seems to be a rather slow process in intact mammalian cells.

  • 16.
    Vare, Daniel
    et al.
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Johansson, Fredrik
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Persson, Jan-Olov
    Stockholm University, Faculty of Science, Department of Mathematics.
    Erixon, Klaus
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Jenssen, Dag
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Quantification and repair of psoralen-induced interstrand crosslinks in human cells2014In: Toxicology Letters, ISSN 0378-4274, E-ISSN 1879-3169, Vol. 226, no 3, p. 343-350Article in journal (Refereed)
    Abstract [en]

    Bi-functional alkylating agents that cause crosslinks are commonly used in chemotherapy. However, there is no conclusive knowledge for human cells regarding the number of induced interstrand crosslinks (ICLs) and the unhooking rate when the lesion is removed from one of the DNA strand. Using a newly developed method, we quantified the number of induced ICLs for the five furocoumarins; psoralen, 5-methoxypsoralen, 8-methoxypsoralen, tri-methoxypsoralen and angelicin. In quantitative terms, the results were in agreement with the values found by others. In kinetic studies using mammalian cells, we found that half of the psoralen-induced ICLs were unhooked within 2.5 h. The rate in normal human diploid fibroblasts was found to be 20,000 ICLs/h/cell. In comparison to survival, 2500 ICLs per cell led to 50% toxicity, indicating that the unhooking of the ICLs is not the crucial step for ICL tolerance. Surprisingly, only 3500 ICLs per cell corresponded to a significant delay in the replication fork elongation. The results indicate involvements of additional pathway(s) for the delay since the effect on replication elongation could be monitored when only 10% of the replication forks encounter an ICL.

  • 17.
    Vare, Daniel
    et al.
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Johansson, Fredrik
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Persson, Jan-Olov
    Erixon, Klaus
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Jenssen, Dag
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    The quantification and repair of psoralen-induced interstrand crosslinks in human cellsManuscript (preprint) (Other academic)
  • 18.
    Vielfort, Katarina
    et al.
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Söderholm, Niklas
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Weyler, Linda
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Vare, Daniel
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Löfmark, Sonja
    Aro, Helena
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Neisseria gonorrhoeae infection causes DNA damage and affects the expression of p21, p27 and p53 in non-tumor epithelial cells2013In: Journal of Cell Science, ISSN 0021-9533, E-ISSN 1477-9137, Vol. 126, no 1, p. 339-347Article in journal (Refereed)
    Abstract [en]

    The constant shedding and renewal of epithelial cells maintain the protection of epithelial barriers. Interference with the processes of host cell-cycle regulation and barrier integrity permits the bacterial pathogen Neisseria gonorrhoeae to effectively colonize and invade epithelial cells. Here, we show that a gonococcal infection causes DNA damage in human non-tumor vaginal VK2/E6E7 cells with an increase of 700 DNA strand breaks per cell per hour as detected by an alkaline DNA unwinding assay. Infected cells exhibited elevated levels of DNA double-strand breaks, as indicated by a more than 50% increase in cells expressing DNA damage-response protein 53BP1-positive foci that co-localized with phosphorylated histone H2AX (gamma H2AX). Furthermore, infected cells abolished their expression of the tumor protein p53 and induced an increase in the expression of cyclin-dependent kinase inhibitors p21 and p27 to 2.6-fold and 4.2-fold of controls, respectively. As shown by live-cell microscopy, flow cytometry assays, and BrdU incorporation assays, gonococcal infection slowed the host cell-cycle progression mainly by impairing progression through the G2 phase. Our findings show new cellular players that are involved in the control of the human cell cycle during gonococcal infection and the potential of bacteria to cause cellular abnormalities.

  • 19.
    Weyler, Linda
    et al.
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Engelbrecht, Mattias
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Mata Forsberg, Manuel
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Brehwens, Karl
    Vare, Daniel
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Vielfort, Katarina
    Wojcik, Andrzej
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Aro, Helena
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Restriction endonucleases from invasive Neisseria gonorrhoeae cause double-strand breaks and distort mitosis in epithelial cells during infectionManuscript (preprint) (Other academic)
  • 20.
    Weyler, Linda
    et al.
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Engelbrecht, Mattias
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Mata Forsberg, Manuel
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Brehwens, Karl
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Vare, Daniel
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Vielfort, Katarina
    Wojcik, Andrzej
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Aro, Helena
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Restriction Endonucleases from Invasive Neisseria gonorrhoeae Cause Double-Strand Breaks and Distort Mitosis in Epithelial Cells during Infection2014In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 9, no 12, p. e114208-Article in journal (Refereed)
    Abstract [en]

    The host epithelium is both a barrier against, and the target for microbial infections. Maintaining regulated cell growth ensures an intact protective layer towards microbial-induced cellular damage. Neisseria gonorrhoeae infections disrupt host cell cycle regulation machinery and the infection causes DNA double strand breaks that delay progression through the G2/M phase. We show that intracellular gonococci upregulate and release restriction endonucleases that enter the nucleus and damage human chromosomal DNA. Bacterial lysates containing restriction endonucleases were able to fragment genomic DNA as detected by PFGE. Lysates were also microinjected into the cytoplasm of cells in interphase and after 20 h, DNA double strand breaks were identified by 53BP1 staining. In addition, by using live-cell microscopy and NHS-ester stained live gonococci we visualized the subcellular location of the bacteria upon mitosis. Infected cells show dysregulation of the spindle assembly checkpoint proteins MAD1 and MAD2, impaired and prolonged M-phase, nuclear swelling, micronuclei formation and chromosomal instability. These data highlight basic molecular functions of how gonococcal infections affect host cell cycle regulation, cause DNA double strand breaks and predispose cellular malignancies.

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