An analytical method for studying DNA degradation by electrophoresis after cell lysis and visualization of DNA fragments with fluorescent dye, comet assay, was used to evaluate the viability of the endothelial layer of human arterial grafts with the aim of identifying the procedure that will least damage the tissue before cryopreservation. Four groups of samples were studied: cryopreserved arterial grafts that were thawed in two different ways, slowly lasting 2 hours or rapidly for approx. 7 minutes. Arterial grafts that were collected as part of multiorgan procurement with minimal warm ischemia time. Cadaveric grafts were taken as part of the autopsy, so they have a more extended period of warm ischemia. The HeadDNA (%) parameter and others commonly used parameters like TailDNA (%). TailMoment, TailLength, OliveMoment, TailMoment to characterize the comet were used to assess viability in this study. The ratio of non-decayed to decayed nuclei was determined from the values found. This ratio for cadaveric grafts was 0.63, for slowly thawed cryopreserved grafts 2.9, for rapidly thawed cryopreserved grafts 1.9, and for multi-organ procurement grafts 0.68. The results of the study confirmed the assumption that the allografts obtained from cadaveric donors are the least suitable. On the other hand, grafts obtained from multiorgan donors are better in terms of viability monitored by comet assay. Keywords: Arterial grafts, Cryopreservation, Cadaveric, Multiorgan procurement, Viability, Comet assay.

• MeSH
• arterie transplantace   MeSH
• kometový test * MeSH
• kryoprezervace * MeSH
• lidé MeSH
• mrtvola MeSH
• přežívání štěpu fyziologie   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH

Oxidative DNA damage markers (8OHdG, comet assay, gammaH2AX) are becoming widely used in clinical cardiology research. To conduct this review of DNA damage in relation to hypertension in humans, we used databases (e.g. PubMed, Web of Science) to search for English-language publications up to June 30, 2022 and the terms: DNA damage, comet assay, gammaH2AX, 8OHdG, strand breaks, and arterial hypertension. Exclusion criteria were: children, absence of relevant controls, extra-arterial hypertensive issues, animal, cell lines. From a total of 79526, 15 human studies were selected. A total of 902 hypertensive patients (pts): (comet: N=418 pts; 8OHdG: N=484 pts) and 587 controls (comet: N=203; 8OHdG: N=384) were included. DNA damage was significantly higher in hypertensive pts than healthy controls (comet 26.6±11.0 vs 11.7±4.07 arbitrary units /A.U./; P<0.05 and="" 8ohdg="" 13="" 1="" 4="" 12="" vs="" 6="" 97="" 2="" 67="" ng="" mg="" creatinine="" i=""> P<0.05) confirmed with meta-analysis for both. Greater DNA damage was observed in more adverse cases (concentric cardiac hypertrophy 43.4±15.4 vs 15.6±5.5; sustained/untreated hypertension 31.4±12.1 vs 14.2±5/35.0±5.0 vs 25.0 ±5.0; non-dippers 39.2±15.5 vs 29.4±11.1 A.U.; elderly 14.9±4.5 vs 9.3±4.1 ng/mg creatinine; without carvedilol 9.1±4.2 vs 5.7±3.9; with coronary heart disease 0.5±0.1 vs 0.2±0.1 ng/mL) (P<0.05) confirmed with meta-analysis. DNA damage correlated strongly positively with serum glycosylated haemoglobin (r=0.670; P<0.05) and negatively with total antioxidant status (r=-0.670 to -0.933; P<0.05). This is the first systematic review with meta-analysis showing that oxidative DNA damage was increased in humans with arterial hypertension compared to controls.

• Klíčová slova
• 8OHdG, DNA strand break damage, arterial hypertension, cardiovascular disease, comet assay, gammaH2AX,
• MeSH
• 8-hydroxy-2'-deoxyguanosin MeSH
• dítě MeSH
• hypertenze * MeSH
• kometový test MeSH
• kreatinin MeSH
• lidé MeSH
• poškození DNA * MeSH
• senioři MeSH
• zvířata MeSH
• Check Tag
• dítě MeSH
• lidé MeSH
• senioři MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• metaanalýza MeSH
• přehledy MeSH
• systematický přehled MeSH
• Názvy látek
• 8-hydroxy-2'-deoxyguanosin MeSH
• kreatinin MeSH

The proper course and reproducibility of diagnostic techniques depend on narrowly defined reaction conditions, including the reaction pH. Nevertheless, numerous assays are affected by an inaccurately defined reaction pH. Buffers are sometimes suggested for use outside their useful pH ranges, which complicates the reproducibility of results because the buffering capacity is insufficient to retain the disclosed pH. Here, we focus on the comet assay lysis buffer. Comet assay is broadly used for quantifying DNA breaks in eukaryotic cells. The most widespread comet assay protocols employ lysis of the cells before electrophoresis in a buffer containing Triton X-100, a high concentration of NaCl, sodium sarcosinate, EDTA, and Tris, with some modifications. However, nearly all researchers report that they use Tris buffer at pH 10, and some report the pH of the Tris additive alone. Alternatively, others report the pH of the final lysis buffer. However, the lysis solution used in the comet assay is buffered at a pH outside the useful range of Tris. Tris-based buffers have a useful pH range of 7.0 - 9.0. The buffer composed of 10 mM Tris has pKa 8.10 at 25°C and 8.69 at 4°C. The cell lysis conditions used in nearly all modifications of comet assay protocols remain imprecise and uncritically employed. Despite the pH of the lysis buffer likely has negligible effect on the detection of DNA breaks, precise lysis conditions are highly important for the use of comet assay in the detection of base modifications, which are often unstable and sensitive to pH.

• Klíčová slova
• Comet assay, DNA damage, DNA fragmentation, nucleoid, single-cell gel electrophoresis, single-strand breaks,
• MeSH
• DNA * MeSH
• kometový test metody   MeSH
• koncentrace vodíkových iontů MeSH
• lidé MeSH
• poškození DNA * MeSH
• reprodukovatelnost výsledků MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• DNA * MeSH

The comet assay is a versatile method to detect nuclear DNA damage in individual eukaryotic cells, from yeast to human. The types of damage detected encompass DNA strand breaks and alkali-labile sites (e.g., apurinic/apyrimidinic sites), alkylated and oxidized nucleobases, DNA-DNA crosslinks, UV-induced cyclobutane pyrimidine dimers and some chemically induced DNA adducts. Depending on the specimen type, there are important modifications to the comet assay protocol to avoid the formation of additional DNA damage during the processing of samples and to ensure sufficient sensitivity to detect differences in damage levels between sample groups. Various applications of the comet assay have been validated by research groups in academia, industry and regulatory agencies, and its strengths are highlighted by the adoption of the comet assay as an in vivo test for genotoxicity in animal organs by the Organisation for Economic Co-operation and Development. The present document includes a series of consensus protocols that describe the application of the comet assay to a wide variety of cell types, species and types of DNA damage, thereby demonstrating its versatility.

• MeSH
• DNA genetika   MeSH
• eukaryotické buňky MeSH
• kometový test metody   MeSH
• lidé MeSH
• poškození DNA * MeSH
• pyrimidinové dimery * MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• přehledy MeSH
• Research Support, N.I.H., Extramural MeSH
• Názvy látek
• DNA MeSH
• pyrimidinové dimery * MeSH

In the present review we addressed the determination of DNA damage induced by small-molecule carcinogens, considered their persistence in DNA and mutagenicity in in vitro and in vivo systems over a period of 30 years. The review spans from the investigation of the role of DNA damage in the cascade of chemical carcinogenesis. In the nineties, this concept evolved into the biomonitoring studies comprising multiple biomarkers that not only reflected DNA/chromosomal damage, but also the potential of the organism for biotransformation/elimination of various xenobiotics. Since first years of the new millennium, dynamic system of DNA repair and host susceptibility factors started to appear in studies and a considerable knowledge has been accumulated on carcinogens and their role in carcinogenesis. It was understood that the final biological links bridging the arising DNA damage and cancer onset remain to be elucidated. In further years the community of scientists learnt that cancer is a multifactorial disease evolving over several decades of individual´s life. Moreover, DNA damage and DNA repair are inseparable players also in treatment of malignant diseases, but affect substantially other processes, such as degeneration. Functional monitoring of DNA repair pathways and DNA damage response may cast some light on above aspects. Very little is currently known about the relationship between telomere homeostasis and DNA damage formation and repair. DNA damage/repair in genomic and mitochondrial DNA and crosstalk between these two entities emerge as a new interesting topic.

• Klíčová slova
• Butadiene, DNA damage, DNA repair, Formaldehyde, Occupational medicine, Styrene,
• MeSH
• DNA MeSH
• karcinogeneze genetika   MeSH
• karcinogeny MeSH
• kometový test MeSH
• lidé MeSH
• oprava DNA MeSH
• poškození DNA MeSH
• pracovní expozice * MeSH
• xenobiotika * toxicita   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• přehledy MeSH
• Názvy látek
• DNA MeSH
• karcinogeny MeSH
• xenobiotika * MeSH

Several trials have attempted to identify sources of inter-laboratory variability in comet assay results, aiming at achieving more equal responses. Ionising radiation induces a defined level of DNA single-strand breaks (per dose/base pairs) and is used as a reference when comparing comet results but relies on accurately determined radiation doses. In this ring test we studied the significance of dose calibrations and comet assay protocol differences, with the object of identifying causes of variability and how to deal with them. Eight participating laboratories, using either x-ray or gamma radiation units, measured dose rates using alanine pellet dosimeters that were subsequently sent to a specialised laboratory for analysis. We found substantial deviations between calibrated and nominal (uncalibrated) dose rates, with up to 46% difference comparing highest and lowest values. Three additional dosimetry systems were employed in some laboratories: thermoluminescence detectors and two aqueous chemical dosimeters. Fricke's and Benzoic Acid dosimetry solutions gave reliable quantitative dose estimations using local equipment. Mononuclear cells from fresh human blood or mammalian cell lines were irradiated locally with calibrated (alanine) radiation doses and analysed for DNA damage using a standardised comet assay protocol and a lab-specific protocol. The dose response of eight laboratories, calculated against calibrated radiation doses, was linear with slope variance CV= 29% with the lab-specific protocol, reduced to CV= 16% with the standard protocol. Variation between laboratories indicate post-irradiation repair differences. Intra-laboratory variation was very low judging from the dose response of 8 donors (CV=4%). Electrophoresis conditions were different in the lab-specific protocols explaining some dose response variations which were reduced by systematic corrections for electrophoresis conditions. The study shows that comet assay data obtained in different laboratories can be compared quantitatively using calibrated radiation doses and that systematic corrections for electrophoresis conditions are useful.

• Klíčová slova
• Calibration, Comet assay, Radiation, Ring-trial, Standardisation,
• MeSH
• ionizující záření * MeSH
• kalibrace MeSH
• kometový test metody   MeSH
• lidé MeSH
• poškození DNA * MeSH
• savci MeSH
• vztah dávky záření a odpovědi MeSH
• záření gama MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH

Levels of DNA damage represent the dynamics between damage formation and removal. Therefore, to better interpret human biomonitoring studies with DNA damage endpoints, an individual's ability to recognize and properly remove DNA damage should be characterized. Relatively few studies have included DNA repair as a biomarker and therefore, assembling and analyzing a pooled database of studies with data on base excision repair (BER) was one of the goals of hCOMET (EU-COST CA15132). A group of approximately 1911 individuals, was gathered from 8 laboratories which run population studies with the comet-based in vitro DNA repair assay. BER incision activity data were normalized and subsequently correlated with various host factors. BER was found to be significantly higher in women. Although it is generally accepted that age is inversely related to DNA repair, no overall effect of age was found, but sex differences were most pronounced in the oldest quartile (>61 years). No effect of smoking or occupational exposures was found. A body mass index (BMI) above 25 kg/m2 was related to higher levels of BER. However, when BMI exceeded 35 kg/m2, repair incision activity was significantly lower. Finally, higher BER incision activity was related to lower levels of DNA damage detected by the comet assay in combination with formamidopyrimidine DNA glycosylase (Fpg), which is in line with the fact that oxidatively damaged DNA is repaired by BER. These data indicate that BER plays a role in modulating the steady-state level of DNA damage that is detected in molecular epidemiological studies and should therefore be considered as a parallel endpoint in future studies.

• Klíčová slova
• BMI, Base excision repair, Biomarker, Comet assay, DNA repair, Oxidatively damaged DNA,
• MeSH
• DNA-formamidopyrimidinglykosylasa MeSH
• epidemiologické studie MeSH
• kometový test MeSH
• lidé středního věku MeSH
• lidé MeSH
• oprava DNA * genetika   MeSH
• poškození DNA * MeSH
• Check Tag
• lidé středního věku MeSH
• lidé MeSH
• mužské pohlaví MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• metaanalýza MeSH
• Názvy látek
• DNA-formamidopyrimidinglykosylasa MeSH

The alkaline comet assay, or single cell gel electrophoresis, is one of the most popular methods for assessing DNA damage in human population. One of the open issues concerning this assay is the identification of those factors that can explain the large inter-individual and inter-laboratory variation. International collaborative initiatives such as the hCOMET project - a COST Action launched in 2016 - represent a valuable tool to meet this challenge. The aims of hCOMET were to establish reference values for the level of DNA damage in humans, to investigate the effect of host factors, lifestyle and exposure to genotoxic agents, and to compare different sources of assay variability. A database of 19,320 subjects was generated, pooling data from 105 studies run by 44 laboratories in 26 countries between 1999 and 2019. A mixed random effect log-linear model, in parallel with a classic meta-analysis, was applied to take into account the extensive heterogeneity of data, due to descriptor, specimen and protocol variability. As a result of this analysis interquartile intervals of DNA strand breaks (which includes alkali-labile sites) were reported for tail intensity, tail length, and tail moment (comet assay descriptors). A small variation by age was reported in some datasets, suggesting higher DNA damage in oldest age-classes, while no effect could be shown for sex or smoking habit, although the lack of data on heavy smokers has still to be considered. Finally, highly significant differences in DNA damage were found for most exposures investigated in specific studies. In conclusion, these data, which confirm that DNA damage measured by the comet assay is an excellent biomarker of exposure in several conditions, may contribute to improving the quality of study design and to the standardization of results of the comet assay in human populations.

• Klíčová slova
• Biomarkers, Comet assay, DNA damage, Human biomonitoring, Pooled analysis,
• MeSH
• biologické markery krev   MeSH
• kometový test metody   MeSH
• lidé MeSH
• poškození DNA genetika   fyziologie   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• přehledy MeSH
• Názvy látek
• biologické markery MeSH

In the last decade, it has become evident that complex mixtures of cyanobacterial bioactive substances, simultaneously present in blooms, often exert adverse effects that are different from those of pure cyanotoxins, and awareness has been raised on the importance of studying complex mixtures and chemical interactions. We aimed to investigate cytotoxic and genotoxic effects of complex extracts from laboratory cultures of cyanobacterial species from different orders (Cylindrospermopsis raciborskii, Aphanizomenon gracile, Microcystis aeruginosa, M. viridis, M. ichtyoblabe, Planktothrix agardhii, Limnothrix redekei) and algae (Desmodesmus quadricauda), and examine possible relationships between the observed effects and toxin and retinoic acid (RA) content in the extracts. The cytotoxic and genotoxic effects of the extracts were studied in the human hepatocellular carcinoma HepG2 cell line, using the MTT assay, and the comet and cytokinesis-block micronucleus (cytome) assays, respectively. Liquid chromatography electrospray ionization mass spectrometry (LC/ESI-MS) was used to detect toxins (microcystins (MC-LR, MC-RR, MC-YR) and cylindrospermopsin) and RAs (ATRA and 9cis-RA) in the extracts. Six out of eight extracts were cytotoxic (0.04-2 mgDM/mL), and five induced DNA strand breaks at non-cytotoxic concentrations (0.2-2 mgDM/mL). The extracts with genotoxic activity also had the highest content of RAs and there was a linear association between RA content and genotoxicity, indicating their possible involvement; however further research is needed to identify and confirm the compounds involved and to elucidate possible genotoxic effects of RAs.

• Klíčová slova
• algae, chemical analysis, complex mixtures, cyanobacteria, cyanotoxins, cytotoxicity, extracts, genotoxicity, microcystins, retinoic acids,
• MeSH
• alkaloidy izolace a purifikace   toxicita   MeSH
• buňky Hep G2 MeSH
• Chlorophyta metabolismus   MeSH
• chromatografie kapalinová MeSH
• hmotnostní spektrometrie s elektrosprejovou ionizací MeSH
• kometový test MeSH
• lidé MeSH
• mikrocystiny izolace a purifikace   toxicita   MeSH
• mikrojaderné testy MeSH
• mikrojádra chromozomálně defektní chemicky indukované   MeSH
• poškození DNA * MeSH
• sinice metabolismus   MeSH
• tandemová hmotnostní spektrometrie MeSH
• toxiny kmene Cyanobacteria MeSH
• tretinoin izolace a purifikace   toxicita   MeSH
• viabilita buněk účinky léků   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• alkaloidy MeSH
• cylindrospermopsin MeSH Prohlížeč
• microcystin MeSH Prohlížeč
• mikrocystiny MeSH
• toxiny kmene Cyanobacteria MeSH
• tretinoin MeSH

Growing worldwide efforts to replace (reduce) animal testing and to improve alternative in vitro tests which may be more efficient in terms of both time, cost and scientific validity include also genotoxicity/mutagenicity endpoints. The aim of the review article was to summarize currently available in vitro testing approaches in this field, their regulatory acceptance and recommended combinations for classification of chemicals. A study using the combination of Comet Assay performed on two cell lines and the Chromosomal Aberration test on human peripheral lymphocytes was performed with the aim to predict the genotoxic potential of selected paraben esters, serving as a model chemical group. Parabens are widely used in consumer products as preservatives and have been reported to exhibit inconclusive results in numerous genotoxicity studies. The Comet Assay identified Ethylparaben and Benzylparaben as potentially genotoxic. The Chromosomal Aberration test revealed weak genotoxic potential in case of Ethylparaben and positive genotoxicity in case of Butylparaben, Propylparaben and Isopropylparaben. The main reasons for variability seem to be limited water solubility of parabens, determining their bioavailability at the cellular level, and absence of metabolic activation in the Comet Assay. The results confirmed that the Comet Assay should serve as a screening test and should not be used as a stand-alone method for classification of genotoxicity. The weight of evidence approach in risk assessment should be supported with data generated with the use of human relevant in vitro methods based on cells / tissues of human origin.

• MeSH
• alternativy testů na zvířatech * MeSH
• buněčné linie keratinocytů HaCaT MeSH
• chromozomální aberace chemicky indukované   MeSH
• hodnocení rizik MeSH
• kometový test MeSH
• lidé MeSH
• lymfocyty účinky léků   patologie   MeSH
• mikrojaderné testy MeSH
• mikrojádra chromozomálně defektní chemicky indukované   MeSH
• mutageneze účinky léků   MeSH
• parabeny toxicita   MeSH
• poškození DNA * MeSH
• testy genotoxicity * MeSH
• viabilita buněk účinky léků   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• přehledy MeSH
• srovnávací studie MeSH
• Názvy látek
• parabeny MeSH