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Rośliny wytworzyły szereg mechanizmów odpowiadających za naprawę uszkodzeń oksydacyjnych wywołanych reaktywnymi formami tlenu. System naprawy poprzez wycinanie zasad wydaje się głównym system naprawczym prowadzącym do zniwelowania zmian wytworzonych przez reaktywne formy tlenu. UUważa się, że dwoma ważnymi enzymami odpowiadającymi za usuwanie 8-oksoguaniny są glikozylaza 8-oksoguaniny DNA i glikozylaza DNA formamidopirimidyny. Wielu badaczy zauważa zależność pomiędzy nagromadzeniem zmian oksydacyjnych w komórce, a aktywnością glikozylazy 8-oksoguaniny DNA i glikozylazy DNA formamidopirimidyny, oraz starzeniem się nasion. Potwierdzono, że poziom FPG oraz OGG1 wzrasta podczas imbibicji nasion. Niniejsza praca ma na celu przybliżenie działania systemu naprawczego BER w procesie napraw uszkodzeń oksydacyjnych oddziaływujących na starzenie się nasion. Celem pracy jest przedstawienie obecnego stanu na temat działania systemu BER (ang Base Excision Repair) w naprawie uszkodzeń oksydacyjnych wpływających na starzenie się nasion.

The comet assay is a simple technique for measurements of low levels of DNA damage and repair in single cells. However, there is variation in background levels of DNA damage in peripheral blood mononuclear cells (PBMCs). This variation has been documented by inter-laboratory ring-trials where identical samples have been analysed in different laboratories using the formamidopyrimidine DNA glycosylase (Fpg)-modified comet assay. The coefficient of variation of background levels of Fpg-sensitive sites was 128 % in the first inter-laboratory validation trial called European Standards Committee on Oxidative DNA Damage. The variation was reduced to 44 % by the end of the project. Subsequent ring-trials by the European Comet Assay Validation Group showed similar inter-laboratory variation in Fpg-sensitive sites in PBMCs (45 %). The lowest inter-laboratory variation in Fpg-sensitive sites in PBMCs was 12 % when using calibration to standardize comet assay descriptors. Introduction of standard comet assay procedures was surprisingly unsuccessful as certain laboratories experienced technical problems using unaccustomed assay conditions. This problem was alleviated by using flexible assay standard conditions rather than a standard protocol in a ring-trial by the hCOMET group. The approach reduced technical problems, but the inter-laboratory variation in Fpg-sensitive sites was not reduced. The ring-trials have not pinpointed specific assay steps as major determinants of the variation in DNA damage levels. It is likely that small differences in several steps cause inter-laboratory variation. Although this variation in reported DNA damage levels causes concern, ring-trials have also shown that the comet assay is a reliable tool in biomonitoring studies.

DNA glycosylases, the enzymes that initiate base excision DNA repair, recognize damaged bases through a series of precisely orchestrated movements. Most glycosylases sharply kink the DNA axis at the lesion site and extrude the target base from the DNA double helix into the enzyme's active site. Little attention has been paid so far to the role of the physical continuity of the DNA backbone in allowing the required conformational distortion. Here, we analyze base excision by formamidopyrimidine-DNA glycosylase (Fpg) from substrates keeping all phosphates but containing a nick within three nucleotides of the lesion in either DNA strand. Four phosphoester linkages at the damaged nucleotide and two nucleotides 3' to it were essential for Fpg activity, while the breakage of the others, even at the same critical phosphates, had no effect or even stimulated the reaction. Reduction of the likelihood of hydrogen bonding at the nicks by using dideoxynucleotides as their 3'-terminal groups was more detrimental for the activity. All phosphoester bonds in the complementary strand were dispensable for base excision, but nicks close to the orphaned nucleotide caused early termination of damaged strand cleavage. Elastic network analysis of Fpg-DNA structures showed that the vibrational motions of the critical phosphates are strongly correlated, in part due to the presence of the protein. Overall, our results suggest that mechanical forces propagating along the DNA backbone play a critical role in the correct conformational distortion of DNA by Fpg and possibly by other target base-everting DNA glycosylases.

Base excision repair and active DNA demethylation produce repair intermediates with DNA molecules blocked at the 3′-OH end by an aldehyde or phosphate group. However, both the physiological consequences of these accumulated single-strand DNAs break with 3′-blocked ends (DNA 3′-blocks) and the signaling pathways responding to unrepaired DNA 3′-blocks remain unclear in plants. Here, we investigated the effects of DNA 3′-blocks on plant development using the zinc finger DNA 3'-phosphoesterase (zdp) AP endonuclease2 (ape2) double mutant, in which 3′-blocking residues are poorly repaired. The accumulation of DNA 3′-blocked triggered diverse developmental defects that were dependent on the ATM and RAD3-related (ATR)-suppressor of gamma response 1 (SOG1) signaling module. SOG1 mutation rescued the developmental defects of zdp ape2 leaves by preventing cell endoreplication and promoting cell proliferation. However, SOG1 mutation caused intensive meristematic cell death in the radicle of zdp ape2 following germination, resulting in rapid termination of radicle growth. Notably, mutating FORMAMIDOPYRIMIDINE DNA GLYCOSYLASE (FPG) in zdp ape2 sog1 partially recovered its radicle growth, demonstrating that DNA 3′-blocks generated by FPG caused the meristematic defects. Surprisingly, despite lacking a functional radicle, zdp ape2 sog1 mutants compensated the lack of root growth by generating anchor roots having low levels of DNA damage response. Our results reveal dual roles of SOG1 in regulating root establishment when seeds germinate with excess DNA 3′-blocks.

Mechanistic toxicology is gaining weight for human health risk assessment. Different mechanistic assays are available, such as the comet assay, which detects DNA damage at the level of individual cells. However, the conventional alkaline version only detects strand breaks and alkali-labile sites. We have validated two modifications of the in vitro assay to generate mechanistic information: (1) use of DNA-repair enzymes (i.e., formamidopyrimidine DNA glycosylase, endonuclease III, human 8-oxoguanine DNA glycosylase I and human alkyladenine DNA glycosylase) for detection of oxidized and alkylated bases as well as (2) a modification for detecting cross-links. Seven genotoxicants with different mechanisms of action (potassium bromate, methyl methanesulfonate, ethyl methanesulfonate, hydrogen peroxide, cisplatin, mitomycin C, and benzo[a]pyrene diol epoxide), as well as a non-genotoxic compound (dimethyl sulfoxide) and a cytotoxic compound (Triton X-100) were tested on TK-6 cells. We were able to detect with high sensitivity and clearly differentiate oxidizing, alkylating and cross-linking agents. These modifications of the comet assay significantly increase its sensitivity and its specificity towards DNA lesions, providing mechanistic information regarding the type of damage.

The enzyme-modified comet assay was developed in order to detect DNA lesions other than those detected by the standard version (single and double strand breaks and alkali-labile sites). Various lesion-specific enzymes, from the DNA repair machinery of bacteria and humans, have been combined with the comet assay, allowing detection of different oxidized and alkylated bases as well as cyclobutane pyrimidine dimers, mis-incorporated uracil and apurinic/apyrimidinic sites. The enzyme-modified comet assay has been applied in different fields - human biomonitoring, environmental toxicology, and genotoxicity testing (both in vitro and in vivo) - as well as in basic research. Up to now, twelve enzymes have been employed; here we describe the enzymes and give examples of studies in which they have been applied. The bacterial formamidopyrimidine DNA glycosylase (Fpg) and endonuclease III (EndoIII) have been extensively used while others have been used only rarely. Adding further enzymes to the comet assay toolbox could potentially increase the variety of DNA lesions that can be detected. The enzyme-modified comet assay can play a crucial role in the elucidation of the mechanism of action of both direct and indirect genotoxins, thus increasing the value of the assay in the regulatory context.

The enzyme-modified comet assay is widely used for the detection of oxidized DNA lesions. Here we describe for the first time the use of the human alkyladenine DNA glycosylase (hAAG) for the detection of alkylated bases. hAAG was titrated using untreated and methyl methanesulfonate (MMS)-treated TK-6 cells. The hAAG-modified comet assay was compared to the formamidopyrimidine DNA glycosylase (Fpg)-modified comet assay, widely used to detect oxidized lesions but that also detects ring-opened purines derived from some alkylated lesions, using cells treated with potassium bromate (oxidizing agent) or MMS. Moreover, neutral and alkaline lysis conditions were used to determine the nature of detected lesions. When alkaline lysis was employed (condition normally used), the level of hAAG-sensitive sites was higher than the Fpg-sensitive sites in MMS-treated cells and hAAG, unlike Fpg, did not detect oxidized bases. After neutral lysis, Fpg did not detect MMS-induced lesions; however, results obtained with hAAG remained unchanged. As expected, Fpg detected oxidized purines and imidazole ring-opened purines, derived from N7-methylguanines under alkaline conditions. It seems that hAAG detected N7-methylguanines, the ring-opened purines derived at high pH, and 3-methlyladenines. Specificity of hAAG towards different DNA lesions was evaluated using a multiplex oligonucleotide-cleavage assay, confirming the ability of hAAG to detect ethenoadenines and hypoxanthine. The hAAG-modified comet assay is a new tool for the detection of alkylated bases.

In protein evolution, functionally important intramolecular interactions, such as polar bridges or hydrophobic interfaces, tend to be conserved. We have analyzed coevolution of physicochemical properties in pairs of amino acid residues in the formamidopyrimidine–DNA glycosylase (Fpg) protein family, identified three conserved polar bridges (Arg54–Glu131, Gln234–Arg244, and Tyr170–Ser208 in the E. coli protein) located in known functional regions of the protein, and analyzed their roles by site-directed mutagenesis. The structure and molecular dynamic modeling showed that the coevolving pairs do not form isolated bridges but rather participate in tight local clusters of hydrogen bonds. The Arg54–Glu131 bridge, connecting the N- and C-terminal domains, was important for DNA binding, as its abolishment or even ion pair reversal inactivated Fpg and greatly decreased the enzyme’s affinity for DNA. Mutations of the Gln234–Arg244 bridge, located at the base of the single Fpg β-hairpin zinc finger, did not affect the activity but sharply decreased the melting temperature of the protein, with the bridge reversal partially restoring the thermal stability. Finally, Tyr170 mutation to Phe decreased Fpg binding but did not fully inactivate the protein, whereas Ser208 replacement with Ala had no effect; molecular dynamics showed that in both wild-type and S208 A Fpg, Tyr170 quickly re-orients to form an alternative set of hydrogen bonds. Thus, the coevolution analysis approach, combined with biochemical and computational studies, provides a powerful tool for understanding intramolecular interactions important for the function of DNA repair enzymes.

Even if the comet assay has been widely used for decades, there is still a need for controlled studies and good mathematical models to assess the variability of the different versions of this assay and in particular to assess potential intra-experimental variability of the high-throughput comet assay. To address this point, we further validate a high-throughput comet assay that uses hydrophilic polyester film (Gelbond®). Experiments were performed using human peripheral blood mononuclear cells (PBMC) either untreated or treated with different concentration of MMS (methyl methanesulfonate). A positive control for the Fpg (Formamidopyrimidine DNA glycosylase)-modified comet assay (Ro 19-8022 with light) was also included. To quantify the sources of variability of the assay, including intradeposit variability, instead of summarizing DNA damage on 50 cells from a deposit by the mean or median of their percentage DNA tail, we analyzed all logit-transformed data with a linear mixed model. The main source of variation in our experimental data is between cells within the same deposit, suggesting genuine variability in the response of the cells rather than variation caused by technical treatment of cell samples. The second source of variation is the inter-experimental variation (day-to-day experiment); the coefficient of this variation for the control was 13.6%. The variation between deposits in the same experiment is negligible. Moreover, there is no systematic bias because of the position of samples on the Gelbond® film nor the position of the films in the electrophoresis tank. This high-throughput comet assay is thus reliable for various applications. Environ. Mol. Mutagen. 59:595-602, 2018. © 2018 Wiley Periodicals, Inc.

The widespread use of titanium dioxide nanoparticles (TiO2 NPs) in commercial food products makes intestinal cells a suitable target. Accordingly, we have used the human colon adenocarcinoma Caco-2 cells to detect their potential harmful effects. Caco-2 cells can differentiate in to enterocytic-like cells, forming consistent cell monolayers and are used as a model of the intestinal barrier. Using both undifferentiated and differentiated Caco-2 cells, we have explored a set of biomarkers, aiming to evaluate undesirable effects associated to TiO2 NP exposure. Results indicate non-toxic effects in exposures ranging 1-200 μg ml-1 . Significant differences were observed in cell uptake, with a higher amount of incorporated TiO2 NPs in undifferentiated cells, as visualized using confocal microscopy. In well-established monolayers, translocation was detected using both confocal microscopy and transmission electron microscopy with energy-dispersive X-ray spectroscopy. In spite of the observed uptake and translocation, TiO2 NP exposures did not modify the integrity of the monolayer, as measured using the transepithelial electrical resistance and Lucifer yellow methods. The potential genotoxic effects in differentiated cells were evaluated in the comet assay, with and without formamidopyrimidine DNA glycosylase enzyme to detect oxidatively the damaged DNA bases. Although some changes were detected at the lower dose (10 μg ml-1 ), no effects were observed at higher doses.

The 8-oxoguanine (8-oxoG) represents the most common DNA damage type, and it has been regarded as the oxidative stress biomarker, but the reported 8-oxoguanine assays are limited by poor specificity and low sensitivity. Herein, we demonstrate the construction of damage site-specific fluorescent biosensor for 8-oxoG assay by integrating single-molecule detection with hyperbranched signal amplification. In this assay, the 8-oxoG damages in DNA can generate free 3′ OH with the assistance of formamidopyrimidine DNA glycosylase (Fpg) and polynucleotide kinase (PNK), which subsequently triggers the incorporation of abundant Cy5-labeled dUTPs via terminal deoxynucleotidyl transferase (TDT)-mediated site-specific hyperbranched nucleic acid amplification. After digestion of amplification products with nuclease treatment, abundant mononucleotide Cy5-dUTPs are produced, which will be easily monitored via single-molecule imaging and detection. The introduction of hyperbranched nucleic acid amplification and single-molecule detection can greatly improve the sensitivity to achieve a detection limit of 7.62 × 10−18 M. This biosensor is highly specific with the capability of discriminating 0.001% 8-oxoG target from the DNA mixture. Moreover, it can be applied for quantitative detection of 8-oxoG damage in genomic DNAs with a detection limit of 0.0017 ng, and even accurately quantifies the absolute number (7025 – 8506) of 8-oxoG damage base in single HeLa cell treated with 150 μM H2O2. Importantly, this biosensor can measure the 8-oxoG damage level in different cancer cell lines, facilitating the oxidative damage-associated biomedical researches and clinical diagnosis.

We developed a novel approach to determine formamidopyrimidine DNA glycosylase (FPG) activity by taking advantage of target-induced self-primed rolling circle amplification (RCA) and magnetic nanoprobes. Herein, a unique nick (8-oxoguanine, 8-oxoG) was positioned in duplex DNA containing P-circle and P1, which together serve as a FPG substrate, RCA template, and RCA primer probe. The presence of FPG specifically binds 8-oxoG and cleaves the P-circle into two parts, producing 5'-phosphoryl termini. A phosphodiester bond between the 5'-phosphoryl and 3'-hydroxyl termini was formed with the addition of T4 DNA ligase, producing an unnicked circular strand. Using the unnicked strand as the RCA template, the P1 hybridized with the circle probe as a primer will trigger the RCA process. The RCA reaction produces amounts of long tandem-repeat DNA tiles with multiple recognizing regions for the FAM modified DNA probes (FP) and biotin-modified DNA probes (BP). With the streptavidin-biotin interaction, the BPs and FPs can be easily immobilized on the surface of streptavidin-modified magnetic microbeads (MBs). Due to the RCA enhanced and highly-concentrated fluorescence accumulation on the MBs, an ultralow detection limit of 1.033 U mL-1 for FPG was obtained. Combined with the high tolerance capability of human blood serum owing to magnetic isolation, the FPG assays in human blood serum were also obtained using fluorescence and confocal laser scanning microscopy. These results indicate that this robust self-primed RCA combined with magnetic nanoprobes is an excellent candidate for quantitatively monitoring the FPG activity responsible for DNA oxidative damage-related clinical diagnosis and therapy.

The formamidopyrimidine DNA glycosylase (Fpg) and human 8-oxoguanine DNA glycosylase (hOGG1)-modified comet assays have been widely used in human biomonitoring studies. The purpose of this article is to assess differences in reported levels of Fpg- and hOGG1-sensitive sites in leukocytes and suggest suitable assay controls for the measurement of oxidatively damaged DNA. An assessment of the literature showed a large variation in the reported levels of Fpg-sensitive sites (range 0.05-1.31 lesions/106 bp). The levels of Fpg-sensitive sites are lower in studies where Fpg has been obtained from commercial suppliers or unknown sources as compared to Fpg from one particular non-commercial source (χ2 = 7.14, P = 0.028). The levels of hOGG1-sensitive sites are lower (range: 0.04-0.18 lesions/106 bp in leukocytes) compared to the Fpg-sensitive sites. Surprisingly, few publications have reported the use of oxidising agents as assay controls, with the exception of hydrogen peroxide. This may be due to a lack of consensus about suitable controls for the Fpg- and hOGG1-modified comet assay. A major challenge is to find an oxidising agent that only oxidises nucleobases and does not generate DNA strand breaks because this reduces the dynamic range of Fpg- and hOGG1-sensitive sites in the comet assay. Based on a literature search we selected the photosensitiser Ro19-8022 plus light, KBrO3, 4-nitroquinoline-1-oxide, Na2Cr2O7 and ferric nitrilotriacetate as possible assay controls. A subsequent assessment of these compounds for generating cryopreserved assay controls in mononuclear blood cells showed that Ro19-8022 plus light, KBrO3 and 4-nitroquinoline-1-oxide provided suitable assay controls. We recommend these compounds as comet assay controls for oxidatively damaged DNA.

From a toxicological point of view, nanomaterials are of interest; because – on account of their great surface area relative to mass - they tend to be more reactive than the bulk chemicals from which they are derived. They might in some cases have the potential to damage DNA directly, or could act via the induction of oxidative stress. The comet assay (single cell gel electrophoresis) is widely used to measure DNA strand breaks and also oxidised bases, by including in the procedure digestion with lesion-specific enzymes such as formamidopyrimidine DNA glycosylase (which converts oxidised purines to breaks) or endonuclease III (recognising oxidised pyrimidines). We summarise reports in which these enzymes have been used to study a variety of nanomaterials in diverse cell types. We also stress that it is important to carry out tests of cell viability alongside the genotoxicity assay, since cytotoxicity can lead to adventitious DNA damage. Different concentrations of nanomaterials should be investigated, concentrating on a non-cytotoxic range; and incubating for short and longer periods can give valuable information about the mode of damage induction. The use of lesion-specific enzymes can substantially enhance the sensitivity of the comet assay in detecting genotoxic effects.

The class of carbazoles includes compounds with high biological activities and broad spectra of action. PLX01107 and PLX01008 are xenomycins, a new subclass of antimicrobial carbazole derivatives demonstrating strong antifungal activity in vitro. We performed three tests, a bacterial reverse mutation assay (Ames test), in vitro cytokinesis-block micronucleus assay, and chromosome aberration test in mouse bone marrow cells, to investigate the possible genotoxicity of these compounds. Despite their structural similarity, the two compounds had different genotoxicity profiles. PLX01008 showed positive effects in all assays. PLX01107 showed no mutagenicity in the Ames test but demonstrated strong cytogenetic activity in vitro and in vivo. PLX01107 was also tested in the in vivo alkaline comet assay, where a weak but statistically significant increase in DNA damage was seen in liver cells 24h after treatment. Significantly increased levels of formamidopyrimidine DNA glycosylase (FPG)-sensitive sites were found in bone marrow cells of PLX01107-treated mice (FPG-modified comet assay), suggesting induction of oxidative or alkylation damage to DNA.

The in vivo comet assay is an established genotoxicity test, with an OECD test guideline, but in its standard form it measures only DNA strand breaks. Including in the assay an additional step, in which the DNA is incubated with a lesion-specific enzyme, can provide important information about the nature of the DNA damage. Formamidopyrimidine DNA glycosylase, 8-oxoguanine DNA glycosylase or endonuclease III are commonly used in the in vitro genotoxicity test and in human biomonitoring to detect oxidised bases, but in vivo applications are rarer. A systematic literature search has identified a total of 60 papers that report such in vivo experiments, testing a variety of agents. In many cases, strand breaks were not seen, but significant levels of enzyme-sensitive sites were induced - indicating a mechanism of action involving oxidative stress. Compounds such as methyl methanesulfonate (MMS) or ethyl methanesulfonate (EMS) could be used as positive controls in both the standard and the enzyme-modified in vivo comet assays.

Molds of the genus Alternaria produce several mycotoxins, some of which may pose a threat for health due to their genotoxicity. Due to the lack of adequate toxicological and occurrence data, they are currently not regulated. Interactions between mycotoxins, gut microbiota and food constituents might occur after food ingestion, modifying the bioavailability and, therefore, overall toxicity of mycotoxins. The present work aimed to investigate the impact of in vitro short-term fecal incubation on the in vitro DNA-damaging effects exerted by 5 µg/mL of an Alternaria alternata extract, containing, among others, 15 nM alternariol, 12 nM alternariol monomethyl ether, 241 nM altertoxin II and 301 nM stemphyltoxin III, all of which are known as genotoxic. The involvement of microorganisms, undigested food constituents and soluble substances of human fecal samples in modifying the composition and the genotoxicity of the extract was investigated through the application of LC–MS/MS analysis and comet assays in HT-29 cells. Results showed that the potential of the mycotoxins to induce DNA strand breaks was almost completely quenched, even before anaerobic incubation, by contact with the different fractions of the fecal samples, while the potency to induce formamidopyrimidine DNA glycosylase (FPG)-sensitive sites was only slightly reduced. These effects were in line with a reduction of mycotoxin concentrations found in samples analyzed by LC–MS/MS. Although a direct correlation between the metabolic activity of the gut microbiota and modifications in mycotoxin contents was not clearly observed, adsorptive phenomena to bacterial cells and to undigested food constituents might explain the observed modifications. Electronic supplementary material The online version of this article (10.1007/s00204-020-02831-1) contains supplementary material, which is available to authorized users.

To date, only a limited number of toxicological studies have focused on the establishment and validation of in vitro genotoxicity screening systems using primary hepatocytes, and the results of these studies have been inconsistent. Therefore, the aim of this study was to develop an effective co-culture model of mouse-derived primary hepatocytes and splenocytes for screening chemicals for genotoxicity using the medium-throughput Comet assay. This cocultured model was constructed and verified using known genotoxic and non-genotoxic compounds as positive and negative controls, respectively. Cytotoxicity was measured using Cell Counting Kit-8 and lactate dehydrogenase methods. DNA damage was detected using both alkaline and formamidopyrimidine DNA glycosylase (FPG) Comet assays. Compared with the controls, DNA strand breaks and FPG-sensitive sites showed significant concentration-dependent increases in genotoxic-agent-treated groups. In contrast, DNA damage remained unchanged in non-genotoxic-agent-treated groups. In addition, different types of genotoxic agents resulted in different time-dependent DNA lesions. Our results indicated that the % tail DNA indicating both DNA strand breaks and FPG-sensitive sites might be effective markers for predicting chemical-induced DNA damage and oxidative DNA damage using the cocultured model of hepatocytes and splenocytes. Collectively, these findings provide reliable experimental data for the establishment of in vitro genotoxicity screening methods.

Applying the single-cell gel electrophoresis (comet) assay, we show that the widely used organophosphorus pesticide malathion is cytotoxic, genotoxic, and induces oxidative stress in human lymphocytes.

The formulation Mancozan®, containing mancozeb as active ingredient, is among the most widely used fungicides. Although mancozeb has been detected in surface waters, studies addressing the genotoxic risk to fish arising from the use of this formulation, testing environmentally realistic concentrations, are absent from the literature. Hence, this work aimed to investigate the DNA and chromosome damaging potential of Mancozan® (0.29 and 2.9μgL-1) in the European eel (Anguilla anguilla L.), after a short-term exposure (3days), through the adoption of the comet and the erythrocytic nuclear abnormality (ENA) assays. In addition, it was intended to elucidate the subjacent damage mechanisms, improving the comet assay with the adoption of the endonucleases formamidopyrimidine DNA glycosylase (FPG) and endonuclease III (EndoIII), which detect oxidized bases. The highest Mancozan® concentration was able to affect the DNA integrity (comet assay), while the adoption of endonucleases pointed out an oxidative cause to the damage. Regarding the chromosomal damage (ENA assay), both concentrations displayed significant effects, revealing the clastogenic and/or aneugenic properties of Mancozan®. Furthermore, the two genotoxic endpoints were significantly correlated. Overall, the results revealed a genetic hazard to fish inhabiting aquatic systems contaminated by Mancozan® and strongly recommend the development of biomonitoring and regulatory policies regarding the utilization of this agrochemical.

Grapes are one of the most commonly consumed fruit, in both fresh and processed forms; however, a significant amount is disposed of in the environment. Searching for a use of this waste, the antigenotoxic, antimutagenic, and antioxidant activities of aqueous extracts from organic and conventional Vitis labrusca leaves were determined using V79 cells as model. The antigenotoxic activity was analyzed by the alkaline comet assay using endonuclease III and formamidopyrimidine DNA glycosylase enzymes. The antimutagenic property was assessed through the micronucleus (MN) formation, and antioxidant activities were assessed using 2',7'-dichlorodihydrofluorescin diacetate (DCFH-DA) assay and 2,2-diphenyl-1-picrylhydrazyl (DPPH(●)) radical scavenging, as well as with superoxide dismutase (SOD) and catalase (CAT) activity assays. In addition, phenolic content and ascorbic acid levels of both extracts were determined. Data showed that both organic and conventional grapevine leaves extracts possessed antigenotoxic and antimutagenic properties. The extract of organic leaves significantly reduced intracellular reactive oxygen species (ROS) levels in V79 cells, and displayed greater ability for DPPH(●) scavenging and higher SOD and CAT activities than extract from conventional leaves. Further, the extract from organic leaves contained higher phenolic and ascorbic acid concentrations. In summary, extracts from organic and conventional grape leaves induced important in vitro biological effects.

Due to the significant role of formamidopyrimidine DNA glycosylase (Fpg) in physiological processes and DNA oxidative damage-related diseases, it is essential to establish sensitive methods for monitoring the Fpg activity in vitro and in vivo so as to illustrate its concrete role in these events. In this work, a sensitive, simple and reliable fluorescence assay was developed by taking the advantages of DNAzyme assisted cascade signal amplification and ultra-high fluorescence quenching efficiency of reduced graphene oxide (rGO). This detection system consisted of DNAzyme, rGO and fluorescence probe allows the activity of Fpg to be detected in a linear range from 0 to 80 U/mL with a detection limit of 0.66 U/mL. With the help of this method, 11 natural compounds were screened, and 7 compounds were identified as activators of Fpg. More importantly, the developed assay was used to monitor the activity of Fpg through fluorescence imaging in living Escherichia coli for the first time. The imaging results visually demonstrated the dynamic activation effect of natural compound Ginsenoside Re on the Fpg of Escherichia coli. In summary, these results indicated that this DNAzyme and rGO based fluorescence assay provides a potent strategy for Fpg quantitative assay in vitro and real-time monitoring in living bacteria, which holds great potential for applying on biological study and Fpg-targeted drug screening.

Mitochondrial oxidative stress accumulates with aging and age-related diseases and induces alterations in mitochondrial DNA (mtDNA) content. Since mtDNA qualitative alterations are also associated with aging, repair of mtDNA damage is of great importance. The most relevant form of DNA repair in this context is base excision repair (BER), which removes oxidized bases such as 8-oxoguanine (8-oxoG) and thymine glycol through the action of the mitochondrial isoform of the specific 8-oxoG DNA glycosylase/apurinic or apyrimidinic (AP) lyase (OGG1) or the endonuclease III homolog (NTH1). Mouse strains lacking OGG1 (OGG1−/−) or NTH1 (NTH1−/−) were analyzed for mtDNA alterations. Interestingly, both knockout strains presented a significant increase in mtDNA content, suggestive of a compensatory mtDNA replication. The mtDNA “common deletion” was not detected in either knockout mouse strain, likely because of the young age of the mice. Formamidopyrimidine DNA glycosylase (Fpg)-sensitive sites accumulated in mtDNA from OGG1−/− but not from NTH1−/− mice. Interestingly, the D-loop region was most severely affected by the absence of OGG1, suggesting that this region may be a hotspot for oxidative damage. Thus, we speculate that mtDNA alterations may send a stress message to evoke cell changes through a retrograde mitochondrial−nucleus communication.

This study investigated associations between levels of oxidatively damaged DNA, measured by the formamidopyrimidine DNA glycosylase (Fpg)-modified comet assay and intake of fish, salad, fruits, vegetables, wholegrain items, and potatoes in a cross-sectional study of 382 men and 591 women between 18 and 93 years. Intake of dietary items was obtained from questionnaires, and stratified into less than once per week, weekly or daily consumption. Intake of fish as main course was inversely associated with levels of Fpg-sensitive sites in peripheral blood mononuclear cells (PBMCs) in especially women (P < 0.001 multivariate linear regression). Intake of fish was also inversely associated with lower levels of Fpg-sensitive sites in men (P < 0.05, univariate analysis), although it was not statistically significant in analysis adjusted for lifestyle and other dietary factors. Intake of salad was inversely associated with levels of Fpg-sensitive sites in men (P < 0.001, multivariate linear regression). Statistically significant associations were also observed for intake of vegetables and potatoes in men, although these were weak and not robust in all statistical models. The sum the six individual dietary items was inversely associated with levels of Fpg-sensitive sites in the strata of men (P < 0.001, multivariate linear regression). Finally, levels of DNA repair incision activity were not associated with individual food categories or the total dietary food score. In summary, consumption of health-promoting foods is associated with lower levels of Fpg-sensitive sites in human PBMCs and strongest effects in the present population were ingestions of fish and salad.

The comet assay is often applied in human biomonitoring. Most of the time the assay is performed with isolated peripheral blood mononuclear cells (PBMC). However, using whole blood instead of isolated cells reduces processing time, and only 20 µl is sufficient for analysis. In this study, a cryopreservation protocol for human whole blood for application in the comet assay was optimised by removing excess plasma before adding freezing medium. Cryopreservation of whole blood samples (n = 30) did not increase the detected level of strand breaks and formamidopyrimidine DNA glycosylase (FPG)-sensitive sites. Although there was no significant correlation with breaks measured in fresh whole blood, strand breaks detected in frozen whole blood were significantly correlated with breaks measured in frozen PBMC (Pearson correlation r = 0.54, P < 0.01). This correlation was however not observed for FPG-sensitive sites. Since we do not yet know the full extent to which cryopreservation might influence the blood cell population, care should be taken to ensure a similar cell type and storage conditions for all samples in one study.

The GO-system is a DNA repair mechanism that prevents and corrects oxidative DNA damage. Formamidopyrimidine-DNA glycosylase (FPG/MutM) participates in this system, avoiding the mutagenic effects of 8-oxoguanine lesion into DNA. Corynebacterium pseudotuberculosis, the etiological agent of caseous lymphadenitis, is a facultative intracellular microorganism vulnerable to oxidative DNA damage. Since inefficiencies in the DNA damage repair system can lead to death, the characterization of repair genes may provide valuable molecular targets for caseous lymphadenitis therapy. The purposes of this study were to functionally characterize MutM1 and MutM2 proteins from C. pseudotuberculosis in silico, in vivo, and in vitro and to examine their role in the repair of 8-oxoguanine damage. In silico investigation revealed that both proteins have conserved domains typical of DNA glycosylases, such as DNA binding domains and DNA glycosylase/AP lyase catalytic domain. In comparison with the MutM protein of Escherichia coli, however, CpMutM2 was found to lack residues that are essential for recognizing and excising 8-oxoguanine damage. Molecular docking calculations have shown a native-like orientation of 8-oxoguanine at the CpMutM1 active site, while the same is not observed for CpMutM2, which seems to poorly interact with DNA. Surface charge analyses have corroborated this finding. Overexpression of CpMutM1 or CpMutM2 has toxic effects on E. coli strain BH20 (mutM-), as shown by growth curves obtained in the presence of hydrogen peroxide and cell viability assays. This cytotoxicity can be attributed to an imbalance in the repair pathway, resulting from hyperactivity of DNA glycosylases, leading to formation of AP sites and DNA strand breakage at levels that exceed the processing capacity of other enzymes in the BER pathway. In order to demonstrate the involvement of these enzymes in the recognition and excision of 8-oxoguanine lesion, glycosylase activity was evaluated in vitro. Only the CpMutM1 protein was proven to be capable of recognizing and excising 8-oxoguanine. Taken together, these results suggest that although the formamidopyrimidine-DNA glycosylase domain is conserved in both proteins, only one proved to be functional in recognizing and excising 8-oxoguanine lesion.

In vitro and in vivo genotoxic effects of differently sized amorphous SiO2 nanomaterials were investigated. In the alkaline Comet assay (with V79 cells), non-cytotoxic concentrations of 300 and 100-300μg/mL 15nm-SiO2 and 55nm-SiO2, respectively, relevant (at least 2-fold relative to the negative control) DNA damage. In the Alkaline unwinding assay (with V79 cells), only 15nm-SiO2 significantly increased DNA strand breaks (and only at 100μg/mL), whereas neither nanomaterial (up to 300μg/mL) increased Fpg (Formamidopyrimidine DNA glycosylase)-sensitive sites reflecting oxidative DNA base modifications. In the Comet assay using rat precision-cut lung slices, 15nm-SiO2 and 55nm-SiO2 induced significant DNA damage at ≥100μg/mL. In the Alkaline unwinding assay (with A549 cells), 30nm-SiO2 and 55nm-SiO2 (with larger primary particle size (PPS)) induced significant increases in DNA strand breaks at ≥50μg/mL, whereas 9nm-SiO2 and 15nm-SiO2 (with smaller PPS) induced significant DNA damage at higher concentrations. These two amorphous SiO2 also increased Fpg-sensitive sites (significant at 100μg/mL). In vivo, within 3 days after single intratracheal instillation of 360μg, neither 15nm-SiO2 nor 55nm-SiO2 caused genotoxic effects in the rat lung or in the bone marrow. However, pulmonary inflammation was observed in both test groups with findings being more pronounced upon treatment with 15nm-SiO2 than with 55nm-SiO2. Taken together, the study shows that colloidal amorphous SiO2 with different particle sizes may induce genotoxic effects in lung cells in vitro at comparatively high concentrations. However, the same materials elicited no genotoxic effects in the rat lung even though pronounced pulmonary inflammation evolved. This may be explained by the fact that a considerably lower dose reached the target cells in vivo than in vitro. Additionally, the different time points of investigation may provide more time for DNA damage repair after instillation.

Background/Aims: The purpose of this study was to explore the in vitro putative genotoxicity during exposure of Neuro-2a cells to radiofrequency electromagnetic fields (RF-EMFs) with or without silencing of 8-oxoG DNA glycosylase-1 (OGG1). Methods: Neuro-2a cells treated with or without OGG1 siRNA were exposed to 900 MHz Global System for Mobile Communication (GSM) Talk signals continuously at a specific absorption rate (SAR) of 0, 0.5, 1 or 2 W/kg for 24 h. DNA strand breakage and DNA base damage were measured by the alkaline comet assay and a modified comet assay using formamidopyrimidine DNA glycosylase (FPG), respectively. Reactive oxygen species (ROS) levels and cell viability were monitored using the non-fluorescent probe 2, 7-dichlorofluorescein diacetate (DCFH-DA) and CCK-8 assay. Results: Exposure to 900 MHz RF-EMFs with insufficient energy could induce oxidative DNA base damage in Neuro-2a cells. These increases were concomitant with similar increases in the generation of reactive oxygen species (ROS). Without OGG1 siRNA, 2 W/kg RF-EMFs induced oxidative DNA base damage in Neuro-2a cells. Interestingly, with OGG1 siRNA, RF-EMFs could cause DNA base damage in Neuro-2a cells as low as 1 W/kg. However, neither DNA strand breakage nor altered cell viability was observed. Conclusion: Even if further studies remain conducted we support the hypothesis that OGG1 is involved in the process of DNA base repair and may play a pivotal role in protecting DNA bases from RF-EMF induced oxidative damage.

Background and Objective In regenerative medicine, there are increasing applications of low-level lasers in therapeutic protocols for treatment of diseases in soft and in bone tissues. However, there are doubts about effects on DNA, and an adequate dosimetry could improve the safety of clinical applications of these lasers. This work aimed to evaluate DNA damage in peripheral blood cells of Wistar rats induced by low-level red and infrared lasers at different fluences, powers, and emission modes according to therapeutic protocols. Material and Methods Peripheral blood samples were exposed to lasers and DNA damage was accessed by comet assay. In other experiments, DNA damage was accessed in blood cells by modified comet assay using formamidopyrimidine DNA glycosylase (Fpg) and endonuclease III enzymes. Results Data show that exposure to low-level red and infrared lasers induce DNA damage depending on fluence, power and emission mode, which are targeted by Fpg and endonuclease III. Conclusion Oxidative DNA damage should be considered for therapeutic efficacy and patient safety in clinical applications based on low-level red and infrared lasers. Lasers Surg. Med. 47:361–368, 2015. © 2015 Wiley Periodicals, Inc.

We showed previously that tungsten carbide–cobalt (WC–Co) nanoparticles (NP) can be used as a nanoparticulate positive control in some in vitro mammalian genotoxicity assays. Here, we investigate the mechanisms of action involved in WC–Co NP genotoxicity in L5178Y mouse lymphoma cells and primary human lymphocytes, in vitro. Data from the micronucleus assay coupled with centromere staining and from the chromosome-aberration assay show the involvement of both clastogenic and aneugenic events. Experiments with the formamidopyrimidine DNA glycosylase (FPG)-modified comet assay showed a slight (non-significant) increase in FPG-sensitive sites in the L5178Y mouse lymphoma cells but not in the human lymphocytes. Electron paramagnetic resonance spin-trapping results showed the presence of hydroxyl radicals (radical dotOH) in WC–Co NP suspensions, with or without cells, but with time-dependent production in the presence of cells. However, a significant difference in radical dotOH production was observed between human lymphocytes from two different donors. Using H2O2, we showed that WC–Co NP can participate in Fenton-like reactions. Thus, radical dotOH might be produced either via intrinsic generation by WC–Co NP or through a Fenton-like reaction in the presence of cells.

hOgg1 and FPG are the primary DNA repair enzymes responsible for removing the major guanine (G) oxidative product, namely, 7,8-dihydro-8-oxoguanine (OG), in humans and bacteria, respectively. While natural G adopts the anti conformation and forms a Watson-Crick pair with cytosine (C), OG can also adopt the syn conformation and form a Hoogsteen pair with adenine (A). hOgg1 removes OG paired with C but is inactive toward the OG:A pair. In contrast, FPG removes OG from OG:C pairs and also exhibits appreciable (although diminished) activity toward OG:A pairs. As a first step toward understanding this difference in activity, we have employed molecular dynamics simulations to examine how the anti and syn conformers of OG are accommodated in the hOgg1 and FPG active sites. When anti-OG is bound, hOgg1 active site residues are properly aligned to initiate catalytic base departure, while geometrical parameters required for the catalytic reaction are not conserved for syn-OG. On the other hand, the FPG catalytic residues are suitably aligned for both OG conformers, with anti-OG being more favorably bound. Thus, our data suggests that the differential ability of hOgg1 and FPG to accommodate the anti- and syn-OG glycosidic conformations is an important factor that contributes to the relative experimental excision rates. Nevertheless, the positions of the nucleophiles with respect to the lesion in the active sites suggest that the reactant complex is poised to initiate catalysis through a similar mechanism for both repair enzymes and supports a recently proposed mechanism in which sugar-ring opening precedes nucleoside deglycosylation.

Rapid, cost-effective and sensitive detection of nucleic acids has the ability to improve upon current practices employed for pathogen detection in diagnosis of infectious disease and food testing. Furthermore, if assay complexity can be reduced, nucleic acid amplification tests could be deployed in resource-limited and home use scenarios. In this study, we developed a novel Fpg (Formamidopyrimidine DNA glycosylase) probe chemistry, which allows lateral flow detection of amplification in undiluted recombinase polymerase amplification (RPA) reactions. The prototype nucleic acid lateral flow chemistry was applied to a human genomic target (rs1207445), Campylobacter jejuni 16S rDNA and two genetic markers of the important food pathogen E. coli O157:H7. All four assays have an analytical sensitivity between 10 and 100 copies DNA per amplification. Furthermore, the assay is performed with fewer hands-on steps than using the current RPA Nfo lateral flow method as dilution of amplicon is not required for lateral flow analysis. Due to the simplicity of the workflow, we believe that the lateral flow chemistry for direct detection could be readily adapted to a cost-effective single-use consumable, ideal for use in non-laboratory settings.

Knowledge of biological properties of natural compounds allows to understand their therapeutic value, efficacy and security. We investigated: composition of Lavandula angustifolia (LA) and Rosmarinus officinalis (RO) extracts, their antioxidant capacity, cytotoxicity and genotoxicity, their DNA-protective potential against DNA damage induced in hamster V79 cells by several genotoxins or in plasmid DNA by Fe2+ ions and activity of antioxidant enzymes in cells treated with these extracts. Higher cytotoxicity, observed at higher concentrations of extracts, was accompanied by the increased level of single-strand (ss) DNA breaks as well as formamidopyrimidine DNA glycosylase (Fpg) sensitive sites. LA and RO extracts were able to protect DNA of hamster cells as well as plasmid DNA against ss DNA breaks induced by genotoxins and Fe2+. LA extract mildly increased the activity of superoxide dismutase (SOD), glutathione peroxidase (GPx) and catalase (CAT), while RO extract decreased the activity of SOD, but increased the activity of CAT and GPx. Cell-free tests confirmed antioxidant activity of both extracts. The biological properties of LA and RO extracts showed that they could have a positive impact on human health.

Anthropogenic pollutants produce oxidative stress in marine organisms, directly or following generation of reactive oxygen species (ROS), potentially resulting in increased accumulation of DNA strand breaks quantified. The aim of this study is to quantify baseline levels of DNA strand breaks in marine species from four phyla and to assess relative sensitivity to oxidative stress as well as ability to recover. DNA strand breaks were determined using a formamidopyrimidine DNA glycosylase (Fpg)-amended comet assay in circulating cells from blue mussel (Mytilus edulis), shore crab (Carcinus maenas), sea star (Asterias rubens), and vase tunicate (Ciona intestinalis). Lymphocytes from Atlantic cod (Gadus morhua) were used as a reference. In addition to immediate analysis, cells from all species were exposed ex vivo to two concentrations of hydrogen peroxide (H2O2) at 25 or 250 μM prior to assay. Mean baseline DNA strand breaks were highest for cells from sea star (34%) followed by crab (25%), mussel (22%), tunicate (17%), and cod (14%). Circulating cells from invertebrates were markedly more sensitive to oxidative stress compared to cod lymphocytes. DNA strand breaks exceeded 80% for sea star, crab, and mussel cells following exposure to the lowest H2O2 concentration. There was no recovery for cells from any species following 1 hr in buffer. This study provides an in-depth analysis of DNA integrity for ecologically important species representing 4 phyla. Data indicate that circulating cells from invertebrates are more sensitive to oxidative stress than cells from fish as evidenced by DNA strand breaks. Future studies need to address the extent to which DNA strand breaks may exert consequences for body maintenance costs in marine invertebrates. The final version of this research has been published in Journal of Toxicology and Environmental Health, Part A: Current Issues. © 2017 Taylor & Francis

Purpose To examine levels of oxidative DNA base damage and expression of selected genes and proteins related to DNA damage repair in human limbal epithelium engineered ex vivo. Methods Cells were expanded from limbal tissue on cell culture‐treated inserts in medium containing fetal bovine serum, recombinant growth factors, hormones and cholera toxin (COM) and in medium with human serum as the single growth‐promoting additive (HS). Cells were analysed after two, three and four weeks in culture for DNA strand breaks and oxidized purine bases (Comet assay using the enzyme formamidopyrimidine DNA glycosylase, Fpg) and for expression of DNA repair enzymes APE1, OGG1 and Polβ by in situ hybridization (ISH) and by immunohistochemistry (IHC). Results Levels of strand breaks were substantial while levels of net Fpg‐sensitive sites (8‐oxoguanine and ring‐opened FaPy bases) were relatively low in cells engineered in COM and in HS. Both types of medium were found to support expression of base excision repair (BER) enzymes APE1, OGG1 and Polβ at the gene level. At the protein level, expression of APE1 and OGG1 was noticeable in both conditions while expression of Polβ was low. Conclusion Our findings indicate low levels of oxidative stress and/or efficient DNA purine base damage repair in human limbal epithelium engineered in a medium with human serum as the single growth‐promoting additive as well as in traditional medium with xenobiotics.

Gantrez ® AN 119-based NPs have been developed as oral drug carriers due to their strong bioadhesive interaction with components of the gastrointestinal mucosa and to their adaptable surface. The use of mannosamine to coat Gantrez ® AN 119-based NPs results in a high mucus-permeable carrier, able to reach the gastrointestinal epithelium. Although their efficacy to transport a therapeutic agent has been demonstrated, their safety has not yet been thoroughly studied. They have proved to be non-cytotoxic, non-genotoxic and non-mutagenic in vitro; however, the in vivo toxicity profile has not yet been determined. In this study, the in vivo genotoxic potential of Gantrez ® AN 119 NPs coated with mannosamine (GN-MA-NP) has been assessed using the in vivo comet assay in combination with the enzyme formamidopyrimidine DNA glycosylase in mice, following the OECD test guideline 489. To determine the relevant organs to analyse and the sampling times, an in vivo biodistribution study was also carried out. Results showed a statistically significant induction of DNA strand breaks and oxidized bases in the duodenum of animals exposed to 2000 mg/kg bw. However, this effect was not observed at lower doses (i.e. 500 and 1000 mg/kg which are closer to the potential therapeutic doses) or in other organs. In conclusion, GN-MA-NP are promising nanocarriers as oral drug delivery systems.

Background Formamidopyrimidine-DNA glycosylase (Fpg) removes abundant pre-mutagenic 8-oxoguanine (oxoG) bases from DNA through nucleophilic attack of its N-terminal proline at C1′ of the damaged nucleotide. Since oxoG efficiently pairs with both C and A, Fpg must excise oxoG from pairs with C but not with A, otherwise a mutation occurs. The crystal structures of several Fpg–DNA complexes have been solved, yet no structure with A opposite the lesion is available. Results Here we use molecular dynamic simulation to model interactions in the pre-catalytic complex of Lactococcus lactis Fpg with DNA containing oxoG opposite C or A, the latter in either syn or anti conformation. The catalytic dyad, Pro1–Glu2, was modeled in all four possible protonation states. Only one transition was observed in the experimental reaction rate pH dependence plots, and Glu2 kept the same set of interactions regardless of its protonation state, suggesting that it does not limit the reaction rate. The adenine base opposite oxoG was highly distorting for the adjacent nucleotides: in the more stable syn models it formed non-canonical bonds with out-of-register nucleotides in both the damaged and the complementary strand, whereas in the anti models the adenine either formed non-canonical bonds or was expelled into the major groove. The side chains of Arg109 and Phe111 that Fpg inserts into DNA to maintain its kinked conformation tended to withdraw from their positions if A was opposite to the lesion. The region showing the largest differences in the dynamics between oxoG:C and oxoG:A substrates was unexpectedly remote from the active site, located near the linker joining the two domains of Fpg. This region was also highly conserved among 124 analyzed Fpg sequences. Three sites trapping water molecules through multiple bonds were identified on the protein–DNA interface, apparently helping to maintain enzyme-induced DNA distortion and participating in oxoG recognition. Conclusion Overall, the discrimination against A opposite to the lesion seems to be due to incorrect DNA distortion around the lesion-containing base pair and, possibly, to gross movement of protein domains connected by the linker. Electronic supplementary material The online version of this article (doi:10.1186/s12900-017-0075-y) contains supplementary material, which is available to authorized users.

The alkaline comet assay is now the method of choice for measuring different kinds of DNA damage in cells. Several attempts have been made to identify and evaluate the critical points affecting the comet assay outcome, highlighting the requirement of arriving at a standardised protocol in order to be able to compare the results obtained in different laboratories. However, reports on the effect of modifying the time of lysis are lacking. Here we tested different times of lysis (from no lysis to 1 week) in control HeLa cells and HeLa cells treated with different concentrations of methyl methanesulfonate (MMS) or H2O2. We also tested different times of lysis in the comet assay combined with formamidopyrimidine DNA glycosylase (FPG) in untreated and Ro 19-8022 plus light-treated HeLa cells. The same DNA damage levels were detected in the absence of lysis or after 1h of lysis when the standard comet assay was used to detect the MMS- and H2O2-induced lesions; the response increased when longer lysis was used, up to at least 1 week. When FPG was used, a minimum lysis period of 5 min was necessary to allow the enzyme to reach the DNA; the same DNA damage levels were detected after 5 min or 1h of lysis and the response increased up to 24h. In conclusion, the time of lysis can be varied depending on the sensitivity needed in both versions of the assay, and a constant time of lysis should be used if results from different experiments or laboratories are to be compared.