| 1. |
- Fotouhi, Asal, et al.
(author)
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Reduction of 8-oxodGTP in the nucleotide pool by hMTH1 leads to reduction in mutations in the human lymphoblastoid cell line TK6 exposed to UVA
- 2011
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In: Mutation research. - : Elsevier BV. - 0027-5107 .- 1873-135X. ; 715:1-2, s. 13-18
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Journal article (peer-reviewed)abstract
- UVA has been suggested to play an important role in UV-induced mutagenesis. The mechanisms by which UVA induces mutations are still a matter of debate. Our aim was to investigate the protective capacity of hMTH1, a nucleotide pool sanitization enzyme with 8-oxodGTPase activity. Human B lymphoblastoid cells were stably transfected with shRNA directed against hMTH1. Clonogenic survival, mutations, intracellular and extracellular levels of 8-oxodG (8-oxo-7, 8-dihydro-2'-deoxyguanosine) and dG in the nucleotide pool of UVA-irradiated transfected and non-transfected cells were investigated. Mutations were determined in the thymidine kinase locus. Intracellular 8-oxodG and dG were measured using a modified ELISA and HPLC, respectively, after extraction of the nucleotide pool and conversion of nucleotides to their corresponding nucleosides. 8-oxodG in the medium was measured using ELISA. UVA-induced mutations were significantly higher while the survival was slightly lower in transfected compared to non-transfected cells. The increased mutation rate in transfected cells at increased exposure correlated with enhanced levels of 8-oxodG in the nucleotide pool, and a somewhat reduced level of 8-oxodG in the medium. The results indicate that the nucleotide pool is a significant target for UVA-induced mutations and implicates that hMTH1 plays an important role in protecting cells from UVA-induced oxidative stress.
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| 2. |
- Dang, Li, et al.
(author)
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Radioprotective effect of hypothermia on cells - a multiparametric approach to delineate the mechanisms
- 2012
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In: International Journal of Radiation Biology. - : Informa UK Limited. - 0955-3002 .- 1362-3095. ; 88:7, s. 507-514
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Journal article (peer-reviewed)abstract
- Purpose: Low temperature (hypothermia) during irradiation of cells has been reported to have a radioprotective effect. The mechanisms are not fully understood. This study further investigates the possible mechanisms behind hypothermia-mediated radioprotection. Materials and methods: Human lymphoblastoid TK6 cells were incubated for 20 min at 0.8 or 37 degrees C and subsequently exposed to 1 Gy of gamma- or X-rays. The influence of ataxia telangiectasia mutated (ATM)-mediated double-strand break signalling and histone deacetylase-dependent chromatin condensation was investigated using the micronucleus assay. Furthermore, the effect of hypothermia was investigated at the level of phosphorylated histone 2AX (gamma H2AX) foci, clonogenic cell survival and micronuclei in sequentially-harvested cells. Results: The radioprotective effect of hypothermia (called the temperature effect [TE]) was evident only at the level of micronuclei at a single fixation time, was not influenced by the inhibition of ATM kinase activity and completely abolished by the histone deacetylase inhibition. No TE was seen at the level of gamma H2AX foci and cell survival. Conclusions: We suggest that low temperature during irradiation can induce a temporary cell cycle shift, which could lead to a reduced micronucleus frequency. Future experiments focused on cell cycle progression are needed to confirm this hypothesis.
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| 3. |
- Shakeri Manesh, Sara, 1982-
(author)
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Adaptive response induced by different dose rates of γ-radiation in MCF-10A cells
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Other publication (other academic/artistic)abstract
- A phenomenon in which exposure to a low adapting dose of radiation makes cells more resistant to the effects of a subsequent high dose exposure is termed radio-adaptive response. Adaptive response could hypothetically reduce the risk of late adverse effects of chronic or acute radiation exposures in humans. Understanding the underlying mechanisms of such responses are of relevance for radiation protection as well as for the clinical applications of radiation in medicine. However, due to the variability of responses depending on the model system and radiation condition, there is a need to further study under what conditions adaptive response can be induced. In this study, we analyzed if there is a dose rate dependence for the adapting dose, assuming that the adapting dose induces DNA response/repair pathways that are dose rate dependent. MCF-10A cells were exposed to a 50 mGy adapting dose administered acutely (24 Gy/h) or chronically (1.4 or 4.1 mGy/h). After 2 hours incubation time cells were exposed to a challenging dose of 1 to 5 Gy. Adaptive response was absent at the level of clonogenic survival and present at the level of mutations only at 1.4 mGy/h administration of adapting dose. Overall, no dose rate effect of the adapting dose was observed at the level of clonogenic survival, while it was seen at the frequency of mutants. On the other hand, a dose rate effect was absent at the level of mutant frequency.
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| 4. |
- Shakeri Manesh, Sara, 1982-, et al.
(author)
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Cooperation of MTH1 and MYH proteins in response to oxidative stress induced by chronic γ-radiation
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Other publication (other academic/artistic)abstract
- Mutation in the MYH gene has been suggested as a risk factor for colorectal cancer. MYH plays an important role in mutation avoidance induced by 8-oxo-G:A mispairs which may occur during oxidative stress. Oxidative stress has been suggested to be involved in several human disorders. In the present study we have exposed human lymphoblastoid TK6 cells to very low dose rates of γ-radiation to induce chronic oxidative stress. The aim of the study is to investigate the protective role of MYH and MTH1 against mutagenicity and cytotoxicity induced by chronic radiation in the cells with knockdown MYH or knockdown MYH/MTH1. The levels of MYH and/or MTH1 were knockdown permanently in cells using shRNA. Wild type and knockdown cells were exposed to chronic γ-radiation with the dose rates ranged from 1.4 to 30 mGy/h during growth. The cells were also subjected to an acute high dose rate. Growth rate, clonogenic survival and mutant frequency were analyzed in all cell types. A reduced level of cell growth and survival as well as an increased mutant frequency were observed in cells lacking both MYH and MTH1 proteins as compared to cells lacking only MYH and wild type cells. In conclusion, our results suggest that MYH and MTH1 cooperatively respond to oxidative stress induced by chronic radiation in human cells.
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| 5. |
- Shakeri Manesh, Sara, et al.
(author)
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MTH1, an 8-oxo-2'-deoxyguanosine triphosphatase, and MYH, a DNA glycosylase, cooperate to inhibit mutations induced by chronic exposure to oxidative stress of ionising radiation
- 2017
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In: Mutagenesis. - : Oxford University Press (OUP). - 0267-8357 .- 1464-3804. ; 32:3, s. 389-396
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Journal article (peer-reviewed)abstract
- Our previous results showed that in addition to the immediate interaction of ionising radiation with DNA (direct and indirect effect), low-dose and chronic low-dose rate of irradiation induce endogenous oxidative stress. During oxidative stress, free radicals react with DNA, nucleoside triphosphates (dNTPs), proteins and lipids, and modify their structures. The MYH and MTH1 genes play important roles in preventing mutations induced by 8-hydroxy-guanine, which is an oxidised product of guanine. In this study, we used short-hairpin RNA to permanently knockdown MYH and MTH1 proteins in human lymphoblastoid TK6 cells. Knockdown and wild-type cells were chronically exposed to low dose rates of gamma-radiation (between 1.4 and 30 mGy/h). The cells were also subjected to acute doses delivered at a high-dose rate. Growth rate, extracellular 8-hydroxy-2'-deoxyguanosine, clonogenic cell survival and mutant frequencies were analysed in all cell types. A reduced level of cell growth and survival as well as increased mutant frequencies were observed in cells lacking both MYH and MTH1 proteins as compared to cells lacking only MYH and wild-type cells. To sum up, our results suggest that low-dose rates elevate oxidative stress. MTH1 together with MYH plays an important role in protection against mutations induced by modified dNTPs during chronic oxidative stress. In addition, we found no dose-rate effect at the level of mutations in the wild-type TK6 and MYH-KD cells. Our data interestingly indicate a dose-rate threshold for mutation induction in MTH1/MYH double knockdown cells.
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| 6. |
- Shakeri Manesh, Sara, et al.
(author)
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Mutations and chromosomal aberrations in hMTH1-transfected and non-transfected TK6 cells after exposure to low dose rates of gamma radiation
- 2014
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In: Radiation and Environmental Biophysics. - : Springer Science and Business Media LLC. - 0301-634X .- 1432-2099. ; 53:2, s. 417-425
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Journal article (peer-reviewed)abstract
- The aim of the present study was to analyse the dose rate effect of gamma radiation at the level of mutations, chromosomal aberrations, and cell growth in TK6 cells with normal as well as reduced levels of hMTH1 protein. TK6 cells were exposed to gamma radiation at dose rates ranging from 1.4 to 30.0 mGy/h (chronic exposure) as well as 24 Gy/h (acute exposure). Cell growth, frequency of thymidine kinase mutants, and of chromosomal aberrations in painted chromosomes 2, 8, and 14 were analysed. A decline in cell growth and an increase in unstable-type chromosomal aberrations with increasing dose rate were observed in both cell lines. A dose rate effect was not seen on mutations or stable-type chromosomal aberrations in any of the two cell lines. Reduction in the hMTH1 protein does not influence the sensitivity of TK6 cells to gamma radiation. This result fits well with data of others generated with the same cell line.
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| 7. |
- Shakeri Manesh, Sara, 1982-
(author)
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Role of MTH1 and MYH proteins in genotoxic effects of radiation
- 2015
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Doctoral thesis (other academic/artistic)abstract
- Humans are constantly exposed to different types of radiations. It has been suggested that low dose and low dose rate of γ-radiation as well as ultra violet A (UVA) induce oxidative stress in cells that may promote mutations. The mechanisms behind radiation-induced oxidative stress and its relation to genotoxicity and cancer induction are not well understood. In the majority of investigations, the DNA molecule has been studied as the target for mutations, however the results obtained in our group point out that DNA bases in the cytoplasm could also be a significant target. MTH1 and MYH are two of the key proteins of the repair pathway that prevent mutations arising from oxidized DNA bases. In this thesis, we studied the role of MTH1 and MYH in genotoxicity of UVA and γ-radiation. The adaptive response to low dose rates of γ-radiation was also investigated. MTH1 and/or MYH were knockdown in human lymphoblastoid TK6 cells. The clonogenic survival, mutant frequency and chromosomal aberration assays were performed following UVA or γ-radiation exposure. Our results indicated that acute exposure to UVA or γ-radiation affects cell survival and also increases the mutant frequency above the background. The mutant frequency in MTH1 deficient cells was higher than that in wild types after UVA exposure. Following γ-radiation exposure, a higher mutant frequency was observed in the MYH and MTH1 deficient cells, in comparison to either MYH or MTH1 deficient or wild type cells. No dose rate effect of γ-radiation for mutations was observed. An adaptive response to γ-radiation was observed at the mutation level in MCF-10A cells but not at the survival level. In summary, our results suggest that; a) MYH and MTH1 cooperatively protect cells against genotoxic effects of γ-radiation; b) MTH1 protects cells from UVA-induced mutations; c) low dose rates of γ-radiation may induce an adaptive response at the mutation level; d) there is no dose rate effect for γ-radiation at the mutation level.
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| 8. |
- Shakeri Manesh, Sara, et al.
(author)
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Studies of adaptive response and mutation induction in MCF-10A cells following exposure to chronic or acute ionizing radiation
- 2015
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In: Mutation research. - : Elsevier BV. - 0027-5107 .- 1873-135X. ; 780, s. 55-59
-
Journal article (peer-reviewed)abstract
- A phenomenon in which exposure to a low adapting dose of radiation makes cells more resistant to the effects of a subsequent high dose exposure is termed radio-adaptive response. Adaptive response could hypothetically reduce the risk of late adverse effects of chronic or acute radiation exposures in humans. Understanding the underlying mechanisms of such responses is of relevance for radiation protection as well as for the clinical applications of radiation in medicine. However, due to the variability of responses depending on the model system and radiation condition, there is a need to further study under what conditions adaptive response can be induced. In this study, we analyzed if there is a dose rate dependence for the adapting dose, assuming that the adapting dose induces DNA response/repair pathways that are dose rate dependent. MCF-10A cells were exposed to a 50 mGy adapting dose administered acutely (0.40 Gy/min) or chronically (1.4 mGy/h or 4.1 mGy/h) and then irradiated by high acute challenging doses. The endpoints of study include clonogenic cell survival and mutation frequency at X-linked hprt locus. In another series of experiment, cells were exposed to 100 mGy and 1 Gy at different dose rates (acutely and chronically) and then the mutation frequencies were studied. Adaptive response was absent at the level of clonogenic survival. The mutation frequencies were significantly decreased in the cells pre-exposed to 50 mGy at 1.4 mGy/h followed by 1 Gy acute exposure as challenging dose. Importantly, at single dose exposures (1 Gy or 100 mGy), no differences at the level of mutation were found comparing different dose rates.
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| 9. |
- Sollazzo, Alice, et al.
(author)
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Interaction of low and high LET radiation in TK6 cells-mechanistic aspects and significance for radiation protection
- 2016
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In: Journal of Radiological Protection. - : IOP Publishing. - 0952-4746 .- 1361-6498. ; 36:4, s. 721-735
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Journal article (peer-reviewed)abstract
- Most environmental, occupational and medical exposures to ionising radiation are associated with a simultaneous action of different radiation types. An open question remains whether radiations of different qualities interact with each other to yield effects stronger than expected based on the assumption of additivity. It is possible that DNA damage induced by high linear energy transfer (LET) radiation will lead to an opening of the chromatin structure making the DNA more susceptible to attack by reactive oxygen species (ROS) generated by the low LET radiation. In such case, the effect of mixed beams should be strongly expressed in cells that are sensitive to ROS. The present investigation was carried out to test if cells with an impaired capacity to handle oxidative stress are particularly sensitive to the effect of mixed beams of alpha particles and x-rays. Clonogenic cell survival curves and mutant frequencies were analysed in TK6 wild type (wt) cells and in TK6 cells with a knocked down hMYH glycosylase. The results showed a synergistic effect of mixed beams on clonogenic cell survival of TK6(wt) but not TK6(MYH)-cells. The frequencies of mutants showed a high degree of interexperimental variability without any indications for synergistic effects of mixed beams. TK6(MYH)-cells were generally more tolerant to radiation exposure with respect to clonogenic cell survival but showed a strong increase in mutant frequency. The results demonstrate that exposure of wt cells to a mixed beam of alpha particles and x-rays leads to a detrimental effect which is stronger than expected based on the assumption of additivity. The role of oxidative stress in the reaction of cells to mixed beams remains unclear.
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