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Neurodegenerative diseases show an increase in prevalence and incidence, with the most prominent example being Alzheimer's disease. DNA damage has been suggested to play a role in the pathogenesis, but the exact mechanisms remain elusive. We enrolled 425 participants with and without neurodegenerative diseases and analyzed DNA damage in the form of micronuclei in buccal mucosa samples. In addition, other parameters such as binucleated cells, karyolytic cells, and karyorrhectic cells were quantified. No relevant differences in DNA damage and cytotoxicity markers were observed in patients compared to healthy participants. Furthermore, other parameters such as lifestyle factors and diseases were also investigated. Overall, this study could not identify a direct link between changes in buccal cells and neurogenerative diseases, but highlights the influence of lifestyle factors and diseases on the human buccal cytome.
Long-term fate of etoposide-induced micronuclei and micronucleated cells in Hela-H2B-GFP cells
(2020)
Micronuclei are small nuclear cellular structures containing whole chromosomes or chromosomal fragments. While there is a lot of information available about the origin and formation of micronuclei, less is known about the fate of micronuclei and micronucleated cells. Possible fates include extrusion, degradation, reincorporation and persistence. Live cell imaging was performed to quantitatively analyse the fates of micronuclei and micronucleated cells occurring in vitro. Imaging was conducted for up to 96 h in HeLa-H2B-GFP cells treated with 0.5, 1 and 2 µg/ml etoposide. While a minority of micronuclei was reincorporated into the main nucleus during mitosis, the majority of micronuclei persisted without any alterations. Degradation and extrusion were observed rarely or never. The presence of micronuclei affected the proliferation of the daughter cells and also had an influence on cell death rates. Mitotic errors were found to be clearly increased in micronucleus-containing cells. The results show that micronuclei and micronucleated cells can, although delayed in cell cycle, sustain for multiple divisions.
Investigation of processes that contribute to the maintenance of genomic stability is one crucial factor in the attempt to understand mechanisms that facilitate ageing. The DNA damage response (DDR) and DNA repair mechanisms are crucial to safeguard the integrity of DNA and to prevent accumulation of persistent DNA damage. Among them, base excision repair (BER) plays a decisive role. BER is the major repair pathway for small oxidative base modifications and apurinic/apyrimidinic (AP) sites. We established a highly sensitive non-radioactive assay to measure BER incision activity in murine liver samples. Incision activity can be assessed towards the three DNA lesions 8-oxo-2'-deoxyguanosine (8-oxodG), 5-hydroxy-2'-deoxyuracil (5-OHdU), and an AP site analogue. We applied the established assay to murine livers of adult and old mice of both sexes. Furthermore, poly(ADP-ribosyl)ation (PARylation) was assessed, which is an important determinant in DDR and BER. Additionally, DNA damage levels were measured to examine the overall damage levels. No impact of ageing on the investigated endpoints in liver tissue were found. However, animal sex seems to be a significant impact factor, as evident by sex-dependent alterations in all endpoints investigated. Moreover, our results revealed interrelationships between the investigated endpoints indicative for the synergetic mode of action of the cellular DNA integrity maintaining machinery.
The comet assay is a commonly used method to determine DNA damage and repair activity in many types of samples. In recent years, the use of the comet assay in human biomonitoring became highly attractive due to its various modified versions, which may be useful to determine individual susceptibility in blood samples. However, in human biomonitoring studies, working with large sample numbers that are acquired over an extended time period requires some additional considerations. One of the most important issues is the storage of samples and its effect on the outcome of the comet assay. Another important question is the suitability of different blood preparations. In this study, we analysed the effect of cryopreservation on DNA damage and repair activity in human blood samples. In addition, we investigated the suitability of different blood preparations. The alkaline and FPG as well as two different types of repair comet assay and an in vitro hydrogen peroxide challenge were applied. Our results confirmed that cryopreserved blood preparations are suitable for investigating DNA damage in the alkaline and FPG comet assay in whole blood, buffy coat and PBMCs. Ex vivo hydrogen peroxide challenge yielded its optimal effect in isolated PBMCs. The utilised repair comet assay with either UVC or hydrogen peroxide-induced lesions and an aphidicolin block worked well in fresh PBMCs. Cryopreserved PBMCs could not be used immediately after thawing. However, a 16-h recovery with or without mitotic stimulation enabled the application of the repair comet assay, albeit only in a surviving cell fraction.
The comet assay is widely used in basic research, genotoxicity testing, and human biomonitoring. However, interpretation of the comet assay data might benefit from a better understanding of the future fate of a cell with DNA damage. DNA damage is in principle repairable, or if extensive, can lead to cell death. Here, we have correlated the maximally induced DNA damage with three test substances in TK6 cells with the survival of the cells. For this, we selected hydrogen peroxide (H\(_{2}\)O\(_{2}\)) as an oxidizing agent, methyl methanesulfonate (MMS) as an alkylating agent and etoposide as a topoisomerase II inhibitor. We measured cell viability, cell proliferation, apoptosis, and micronucleus frequency on the following day, in the same cell culture, which had been analyzed in the comet assay. After treatment, a concentration dependent increase in DNA damage and in the percentage of non-vital and apoptotic cells was found for each substance. Values greater than 20-30% DNA in tail caused the death of more than 50% of the cells, with etoposide causing slightly more cell death than H\(_{2}\)O\(_{2}\) or MMS. Despite that, cells seemed to repair of at least some DNA damage within few hours after substance removal. Overall, the reduction of DNA damage over time is due to both DNA repair and death of heavily damaged cells. We recommend that in experiments with induction of DNA damage of more than 20% DNA in tail, survival data for the cells are provided.
Einfluss des Gewichtsverlusts auf den oxidativen Stress und den DNS-Schaden in adipösen Patient*innen nach bariatrischer Chirurgie
Adipositas ist eine Erkrankung, die durch ein erhöhtes Krebsrisiko neben zahlreichen anderen Komorbiditäten mit weitreichenden Folgen für die Gesundheit adipöser Patient*innen einhergeht. In der Pathogenese der adipositas-assoziierten Krebsarten sind dabei ein erhöhter oxidativer Stress sowie die damit einhergehende Schädigung der DNS maßgeblich beteiligt. Im Umkehrschluss wurde in der vorliegenden Arbeit der Einfluss eines durch bariatrische Chirurgie induzierten Gewichtsverlusts auf den oxidativen Stress und DNS-Schaden in adipösen Patient*innen anhand von Blutproben präoperativ sowie 6 und 12 Monate postoperativ untersucht. In einer Subpopulation der Patient*innen konnte eine tendenzielle Verringerung des DNS-Schadens anhand des Comet-Assays in peripheren Lymphozyten beobachtet werden. Im Hinblick auf den oxidativen Stress wurde im Plasma die Eisenreduktionsfähigkeit als Maß für antioxidative Kapazität sowie Malondialdehyd als Surrogatmarker für das Ausmaß an Lipidperoxidation bestimmt. Weiterhin wurde in Erythrozyten das Gesamtglutathion und oxidierte Glutathion bestimmt. Die oxidativen Stressparameter zeigten insgesamt nach einer initialen Zunahme im oxidativen Stress 6 Monate postoperativ eine rückläufige Tendenz im oxidativen Stress am Studienende. Somit geben die Beobachtungen dieser Arbeit Anlass zur Hoffnung, dass adipöse Patient*innen durch einen bariatrisch induzierten Gewichtsverlust von einer Verringerung des Krebsrisikos profitieren könnten.
Diabetes, a chronic group of medical disorders characterized byhyperglycemia, has become a global pandemic. Some hormones may influence the course and outcome of diabetes, especially if they potentiate the formation of reactive oxygen species (ROS). There is a close relationship between thyroid disorders and diabetes. The main objective of this investigation was to find out whether peripheral blood mononuclear cells (PBMCs) are more prone to DNA damage by triiodothyronine (T\(_3\)) (0.1, 1 and 10 μM) at various stages of progression through diabetes (obese, prediabetics, and type 2 diabetes mellitus—T2DM persons). In addition, some biochemical parameters of oxidative stress (catalase-CAT, thiobarbituric acid reactive substances—TBARS) and lactate dehydrogenase (LDH) were evaluated. PBMCs from prediabetic and diabetic patients exhibited increased sensitivity for T\(_3\) regarding elevated level of DNA damage, inhibition of catalase, and increase of TBARS and LDH. PBMCs from obese patients reacted in the same manner, except for DNA damage. The results of this study should contribute to a better understanding of the role of thyroid hormones in the progression of T2DM.
Adipositas ist eine Erkrankung, die durch ein erhöhtes Krebsrisiko neben zahlreichen anderen Komorbiditäten mit weitreichenden Folgen für die Gesundheit adipöser Patient*innen einhergeht. In der Pathogenese der adipositas-assoziierten Krebsarten sind dabei ein erhöhter oxidativer Stress sowie die damit einhergehende Schädigung der DNS maßgeblich beteiligt. Im Umkehrschluss wurde in der vorliegenden Arbeit der Einfluss eines durch bariatrische Chirurgie induzierten Gewichtsverlusts auf den oxidativen Stress und DNS-Schaden in adipösen Patient*innen anhand von Blutproben präoperativ sowie 6 und 12 Monate postoperativ untersucht. In einer Subpopulation der Patient*innen konnte eine tendenzielle Verringerung des DNS-Schadens anhand des Comet-Assays in peripheren Lymphozyten beobachtet werden. Im Hinblick auf den oxidativen Stress wurde im Plasma die Eisenreduktionsfähigkeit als Maß für die antioxidative Kapazität sowie Malondialdehyd als Surrogatmarker für das Ausmaß an Lipidperoxidation bestimmt. Weiterhin wurde in Erythrozyten das Gesamtglutathion und das oxidierte Glutathion bestimmt. Die oxidativen Stressparameter zeigten insgesamt nach einer initialen Zunahme im oxidativen Stress 6 Monate postoperativ eine rückläufige Tendenz im oxidativen Stress am Studienende. Somit geben die Beobachtungen dieser Arbeit Anlass zur Hoffnung, dass adipöse Patient*innen durch einen bariatrisch induzierten Gewichtsverlust von einer Verringerung des Krebsrisikos profitieren könnten.