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• W1524208657 abstract "The increasing use of appliances, which generate electromagnetic fields (EMFs), hasprovoked public concern about their safety. Scientific research into possible health effectshowever produced conflicting results. One of the open questions is whether or not EMFexposure has genotoxic effects. Therefore, the main objective of my thesis was toinvestigate DNA damage formation and repair, cell cycle progression, apoptosis and DNAdamage signalling in cultured human cells under EMF exposure. In particular, the nature ofpossible genotoxic effects and the mechanisms underlying the cellular responses were to beaddressed.As in the past, genotoxic effects of EMF exposure often could not be reproduced inindependent studies, I first aimed at the validation of results of previous studies [1-3]. Inthese studies, different genotoxicity tests revealed increased DNA damage after exposure ofhuman fibroblast cells to EMFs in the low frequency range, as used in power lines, as well asin the radiofrequency range, as applied by mobile phones and wireless technologies. I couldshow that genotoxic effects of 50Hz EMFs can be reproduced independently. Effects ofradiofrequency EMF exposure, however, were detectable only in one particular cell line (HR-1d), but not in the cell line used in the original study (ES-1). Because the visual scoringmethod of DNA fragmentation analysis (comet assay) used in the previous studies wascriticized in the scientific community, I compared this method with an automatedcomputerized comet analysis. This established that increases of DNA fragmentation following EMF exposure are detectable in both types of analyses.Expanding the study to other cell lines, I was able to show, that 50Hz EMF exposure in twodifferent fibroblast cell lines but not in the cancer cell line HeLa lead to comet assay effects.Furthermore, I showed that DNA fragmentation is not found in G1 blocked cells, suggestingreplicating cells to be involved in EMF directed effects. This indicated, that the DNAfragmentation detected following EMF exposure might not reflect direct induction of DNAdamage but rather an EMF dependent alteration of the S-phase population. Furthermoreapoptosis was suggested as confounder for comet assay effects before. Addressing thisquestion, I found decreased replication efficiency and an increased apoptotic fraction after50Hz EMF exposure in the fibroblast cell line showing the higher comet assay effect.Therefore, I conclude that these cells encounter problems in entering S-phase or progression through S-phase, which could lead to apoptosis and, hence, apoptotic DNA fragmentation, ina subpopulation. These effects, however, cannot entirely explain the genotoxicity observed,as the fraction of cells with increased DNA fragmentation was higher than the proportion ofapoptotic cells.I then addressed the type of possible DNA damage generated by EMF exposure. An inhibitorof the DNA single strand break (SSB) sensor poly-ADP-ribosylation polymerase was used toexamine an engagement of DNA single strand break repair following EMF exposure. Theresults showed that the increase of DNA fragmentation did not change further by applyingboth inhibitor and EMF exposure compared to inhibitor or EMF exposure alone. Thereforethe effects appear to be epistatic, indicating that EMF exposure may affect DNA SSB repairrather than inducing DNA damage itself. To address the occurrence of DNA double strandbreaks or stalled replication forks, I made use of phosphorylated H2AX (γH2AX) as a markerin EMF exposed cells. This revealed no difference between non-exposed and exposed cells,suggesting that the increase in DNA fragmentation is unlikely due to such lesions.Biological effects of EMF exposure were hypothesized to reflect an influence on the freeradical pool of cells and, thus oxidative stress. I examined the steady state levels of oxidativeDNA damage after EMF exposure and found no indications for increased generation ofindicator lesions. This result fails to support the hypothesis of EMF induced oxidative stress,although I cannot completely rule out small changes of a sub-detectable level. DNA baseexcision repair (BER) is the system specifically repairing small lesions including oxidative DNAbase damage. To examine, if this pathway is activated during EMF exposure, I examined theformation and levels of nuclear XRCC1 foci. XRCC1 is a central component of the BER systemand can be seen to localize to sites of DNA damage and repair. However, immunostaining ofXRCC1 revealed no difference in numbers and distribution of foci following EMF exposure.Adding a DNA Polymerase β (the BER polymerase) inhibitor, however, the subG1 fraction ofcells increased synergistically with ELF-EMF exposure. This could indicate, that either BERprotects cells from entering apoptosis following EMF exposure or that the DNA damagegenerated by inhibiting DNA Polymerase β is less efficiently processed under EMF exposure.Taken together, these results suggest, that the small increase in DNA fragmentationobserved in human fibroblasts exposed to 50Hz EMFs can be accounted for by a combinationof effects including impaired repair of endogenously arising DNA damage, disturbance of Sphaseprogression and apoptosis in a small fraction of cells, rather than by directly induced DNA damage.In a second part of my thesis, I used the highly sensitive comet assay, cell cycle analysis andimmunofluorescence staining technologies established for the EMF studies to contribute todifferent projects addressing regulatory aspects of DNA BER. In a first study, we showed thatThymine DNA Glycosylase (TDG) levels were cell cycle regulated and TDG is absent in Sphasein biochemical assays. Regulation occurs at the protein level, as mRNA levels remainconstant throughout the cell cycle. The protein is ubiquitinated and degraded by theproteasome. To provide biological evidence for such a regulation in vivo, I stained cells withantibodies for TDG and the S-phase marker PCNA by immunofluorescence and counted cellnumbers of double and single stained cells. PCNA positive cells did not stain for TDG and viceversa. As PCNA is a marker for S-phase, this shows, that TDG is absent in S-phase cells.In a second study we provided evidence for a regulation of DNA Polymerase β (DNA Pol β) byprotein arginine methylation. This methylation has impact on its in vitro performance likeDNA binding and processivity, but an in vivo relevance of this modification remained to beshown. I showed that DNA Pol β knock out cells complemented with a mutated form of DNAPol β, not able to be methylated, showed a higher level of DNA fragmentation upon inducedDNA damage than cell complemented with wild type DNA Pol β. Together with reducedsurvival rates and an increased subG1 fraction in cells challenged with a DNA damagingagent, this established the in vivo relevance of DNA Pol β methylation. Arginine methylation therefore might represent a novel regulatory protein modification in DNA BER.In a third study, I contributed to the investigation of the toxicity mechanism of thechemotherapeutic drug 5-fluorouracil (5-FU), which is not fully understood so far. Aninvolvement of the BER enzyme TDG was suggested by biochemical evidence, leading to thequestion, if TDG wild type and knock out cells respond differently to 5-FU. TDG knock outcells displayed hypersensitivity to 5-FU, which suggested a deleterious repair mechanismthrough TDG, probably leading to the induction of DNA SSBs. I indeed found increased DNAstrand breaks in TDG wild type cells compared to knock out cells, while XRCC1, a marker forBER, was more activated in knock out cells. In cell cycle analyses 5-FU induced accumulationon S-phase of TDG deficient cells was less pronounced than in wild type cells. This suggeststhat TDG contributes to 5-FU mediated cytotoxicity, probably by inducing DNA SSBs due toits slow turnover rate and the resulting saturation of BER, leading then to checkpointactivation and S-phase accumulation." @default.
• W1524208657 created "2016-06-24" @default.
• W1524208657 creator A5038126681 @default.
• W1524208657 date "2008-01-01" @default.
• W1524208657 modified "2023-09-24" @default.
• W1524208657 title "Insights into genotoxic effects of electromagnetic fields" @default.
• W1524208657 doi "https://doi.org/10.5451/unibas-004702469" @default.
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