Modified Alkaline Comet Assay Research Articles

Ym155 Induces Oxidative Stress-Mediated DNA Damage and Cell Cycle Arrest, and Causes Programmed Cell Death in Anaplastic Thyroid Cancer Cells.

Anaplastic thyroid cancer (ATC) is one of the most lethal malignancies with a median survival time of about 4 months. Currently, there is no effective treatment, and the development of new therapies is an important and urgent issue for ATC patients. YM155 is a small molecule that was identified as the top candidate in a high-throughput screen of small molecule inhibitors performed against a panel of ATC cell lines by the National Cancer Institute. However, there were no follow-up studies investigating YM155 in ATC. Here, we determined the effects of YM155 on ATC and human primary benign thyroid cell (PBTC) survival with alamarBlue assay. Our data show that YM155 inhibited proliferation of ATC cell lines while sparing normal thyroid cells, suggesting a high therapeutic window. YM155-induced DNA damage was detected by measuring phosphorylation of γ-H2AX as a marker for DNA double-strand breaks. The formamidopyrimidine-DNA glycosylase (FPG)-modified alkaline comet assay in conjunction with reactive oxygen species (ROS) assay and glutathione (GSH)/glutathione (GSSG) assay suggests that YM155-mediated oxidative stress contributes to DNA damage. In addition, we provide evidence that YM155 causes cell cycle arrest in S phase and in the G2/M transition and causes apoptosis, as seen with flow cytometry. In this study, we show for the first time the multiple effects of YM155 in ATC cells, furthering a potential therapeutic approach for ATC.

Genotoxic and mutagenic potential of camphorquinone in L5178/TK+/− mouse lymphoma cells

ObjectivesCamphorquinone (CQ) is the most important photoinitiator used in dental composite resins. Sparse data indicate a mutagenic potential of CQ. Therefore, it was aim of this study to evaluate the cytotoxicity, genotoxicity, and mutagenicity of CQ in L5178Y TK+/− mouse lymphoma cells. MethodsL5178Y/TK+/− cells were exposed to different concentrations of non-irradiated CQ (0.25–2.5mM). Cytotoxicity was evaluated by propidium iodide assay, determination of suspension growth rate, relative total growth and the mitotic index. Intracellular levels of reactive oxygen/nitrogen species (ROS/RNS) were quantified by 2′,7′-dichlorofluoresceine diacetate (DCFH-DA). Early induction of DNA strand breaks and oxidative DNA base lesions was assessed using the 8-hydroxyguanine DNA-glycosylase 1 (hOGG1)-modified alkaline comet assay, whereas mutagenicity of CQ was determined in the mouse lymphoma TK assay (MLA), according to OECD Guideline No. 490. ResultsCQ (0.5–2.5mM) induced concentration- and time-dependent inhibition of cell growth associated with increased ROS/RNS production, amounting to 2342%±1108% of controls after 90min at 2.5mM. Additionally, CQ concentration-dependently caused direct DNA-damage, i.e. formation of DNA strand breaks and 8-hydroxy-2′-deoxyguanosine. Whereas the MLA indicated lack of mutagenicity of CQ after a 4h of treatment, CQ concentration-dependently increased total mutant frequency (MF) after 24h (about 2-fold at 2.5mM). But, based on the global evaluation factor concept, increase in MF did not reach biologically relevance. SignificanceCQ induced concentration-dependent, cytotoxic and genotoxic effects in L5178Y/TK+/− cells, most likely due to oxidative stress, but without mediating obvious biological relevant mutagenicity.

3D-human small intestinal tissue to study ligand-induced acute and chronic inflammation in the gastrointestinal tract

Camphorquinone (CQ) is the most important photoinitiator used in dental composite resins. Sparse data indicate a mutagenic potential of CQ. Therefore, it was aim of this study to evaluate the cytotoxicity, genotoxicity, and mutagenicity of CQ in L5178Y TK+/− mouse lymphoma cells.L5178Y/TK+/− cells were exposed to different concentrations of non-irradiated CQ (0.25–2.5 mM). Cytotoxicity was evaluated by propidium iodide assay, determination of suspension growth rate, relative total growth and the mitotic index. Intracellular levels of reactive oxygen/nitrogen species (ROS/RNS) were quantified by 2′,7′-dichlorofluoresceine diacetate (DCFH-DA). Early induction of DNA strand breaks and oxidative DNA base lesions was assessed using the 8-hydroxyguanine DNA-glycosylase 1 (hOGG1)-modified alkaline comet assay, whereas mutagenicity of CQ was determined in the mouse lymphoma TK assay (MLA), according to OECD Guideline No. 490.CQ (0.5–2.5 mM) induced concentration- and time-dependent inhibition of cell growth associated with increased ROS/RNS production, amounting to 2342% ± 1108% of controls after 90 min at 2.5 mM. Additionally, CQ concentration-dependently caused direct DNA-damage, i.e. formation of DNA strand breaks and 8-hydroxy-2′-deoxyguanosine. Whereas the MLA indicated lack of mutagenicity of CQ after a 4 h of treatment, CQ concentration-dependently increased total mutant frequency (MF) after 24 h (about 2-fold at 2.5 mM). But, based on the global evaluation factor concept, increase in MF did not reach biologically relevance.CQ induced concentration-dependent, cytotoxic and genotoxic effects in L5178Y/TK+/− cells, most likely due to oxidative stress, but without mediating obvious biological relevant mutagenicity.

The development and validation of EpiComet-Chip, a modified high-throughput comet assay for the assessment of DNA methylation status.

DNA damage and alterations in global DNA methylation status are associated with multiple human diseases and are frequently correlated with clinically relevant information. Therefore, assessing DNA damage and epigenetic modifications, including DNA methylation, is critical for predicting human exposure risk of pharmacological and biological agents. We previously developed a higher-throughput platform for the single cell gel electrophoresis (comet) assay, CometChip, to assess DNA damage and genotoxic potential. Here, we utilized the methylation-dependent endonuclease, McrBC, to develop a modified alkaline comet assay, "EpiComet," which allows single platform evaluation of genotoxicity and global DNA methylation [5-methylcytosine (5-mC)] status of single-cell populations under user-defined conditions. Further, we leveraged the CometChip platform to create an EpiComet-Chip system capable of performing quantification across simultaneous exposure protocols to enable unprecedented speed and simplicity. This system detected global methylation alterations in response to exposures which included chemotherapeutic and environmental agents. Using EpiComet-Chip on 63 matched samples, we correctly identified single-sample hypermethylation (≥1.5-fold) at 87% (20/23), hypomethylation (≥1.25-fold) at 100% (9/9), with a 4% (2/54) false-negative rate (FNR), and 10% (4/40) false-positive rate (FPR). Using a more stringent threshold to define hypermethylation (≥1.75-fold) allowed us to correctly identify 94% of hypermethylation (17/18), but increased our FPR to 16% (7/45). The successful application of this novel technology will aid hazard identification and risk characterization of FDA-regulated products, while providing utility for investigating epigenetic modes of action of agents in target organs, as the assay is amenable to cultured cells or nucleated cells from any tissue. Environ. Mol. Mutagen. 58:508-521, 2017. © 2017 Wiley Periodicals, Inc.

Proteomics study revealed altered proteome of Dichogaster curgensis upon exposure to fly ash

Fly ash is toxic and its escalating use as a soil amendment and disposal by dumping into environment is receiving alarming attention due to its impact on environment. Proteomics technology is being used for environmental studies since proteins respond rapidly when an organism is exposed to a toxicant, and hence soil engineers such as earthworms are used as model organisms to assess the toxic effects of soil toxicants. This study adopted proteomics technology and profiled proteome of earthworm Dichogaster curgensis that was exposed to fly ash, with main aim to elucidate fly ash effects on cellular and metabolic pathways. The functional classification of identified proteins revealed carbohydrate metabolism (14.36%), genetic information processing (15.02%), folding, sorting and degradation (10.83%), replication and repair (3.95%); environmental information processing (2.19%), signal transduction (9.61%), transport and catabolism (17.27%), energy metabolism (6.69%), etc. in the proteome. Proteomics data and functional assays revealed that the exposure of earthworm to fly ash induced protein synthesis, up-regulation of gluconeogenesis, disturbed energy metabolism, oxidative and cellular stress, and mis-folding of proteins. The regulation of ubiquitination, proteasome and modified alkaline comet assay in earthworm coelomocytes suggested DNA-protein cross link affecting chromatin remodeling and protein folding.

Single-Cell-Arrayed Agarose Chip for in Situ Analysis of Cytotoxicity and Genotoxicity of DNA Cross-Linking Agents.

Development of approach or device to allow continuous multiple measurements, such as integrating cytotoxic and genotoxic analysis, is quite appealing for study of the drug's activity and mechanism of action or resistance. In this study, a single-cell-arrayed agarose chip system was developed to combine cell cultivation with subsequent in situ analysis of cytotoxicity and genotoxicity of the chemotherapeutic agent. The modified alkaline comet assay coupled with the Live/Dead assay was used to monitor the interstrand cross-links (ICLs) formation and the cytotoxic effects in different glioma cell lines. In addition, the ICL-induced double strand breaks (DSBs) was measured on the chip to reflect the level of ICLs indirectly. Compared with the traditional methods, the microarray agarose device offers higher throughput, reproducibility, and robustness, exhibiting good potential for high-content drug screening.

A clickable psoralen to directly quantify DNA interstrand crosslinking and repair

DNA interstrand crosslinks (ICLs) represent physical obstacles to advancing replication forks and transcription complexes. A range of ICL-inducing agents have successfully been incorporated into cancer therapeutics. While studies have adopted UVA-activated psoralens as model ICL-inducing agents for investigating ICL repair, direct detection of the lesion has often been tempered by tagging the psoralen scaffold with a relatively large reporter group that may perturb the biological activity of the parent psoralen. Here a minimally-modified psoralen probe was prepared featuring a small alkyne handle suitable for click chemistry. The psoralen probe, designated 8-propargyloxypsoralen (8-POP), can be activated by UVA in vitro to generate ICLs that are susceptible to post-labeling with an azide-tagged fluorescent reporter via a copper-catalyzed reaction. A modified alkaline comet assay demonstrated that UVA-activated 8-POP proficiently generated ICLs in cells. Cellular 8-POP–DNA lesions were amenable to click-mediated ligation to fluorescent reporters in situ, which permitted their detection and quantitation by fluorescence microscopy and flow cytometry. Small molecule DNA repair inhibitors to 8-POP-treated cells attenuated the removal of 8-POP–DNA lesions, validating 8-POP as an appropriate probe for investigating cellular ICL repair. The post-labeling strategy applied in this study is inexpensive, rapid and highly modular in nature with the potential for multiple applications in DNA repair studies.

Harmful Effects of the Dermal Intake of Commercial Formulations Containing Chlorpyrifos, 2,4-D, and Glyphosate on the Common Toad Rhinella arenarum (Anura: Bufonidae)

Amphibians have complex life cycles with aquatic and terrestrial life and uncovered skins; therefore, they are exposed to chemical contamination, where dermal exposure is a significant route for pesticide uptake in both habitats. In this study, measurements in blood samples such as levels of butyrylcholinesterase (BChE), carboxylesterase (CbE), glutathione S-transferases (GST), thiobarbituric acid reactive substances (TBARS), modified alkaline comet assay (ACA) for detection of oxidized bases (FPG and Endo III sites), as well as the ratio of heterophils and lymphocytes (H/L), were evaluated as non-destructive biomarkers to monitor dermal pesticide exposure in male toads of Rhinella arenarum. Toads were exposed to a solution containing a nominal concentration of commercial formulations of the insecticide chlorpyrifos (CPF, 10 mg/L), and herbicides 2,4-D and glyphosate (GLY) (20 mg/L, respectively). After 48 h of exposure, the levels of plasma B-sterases (BChE and CbE) were inhibited (55 and 43 %, respectively) in toads exposed to CPF. Also, the activity of GST was inducted for dermal exposure to 2,4-D, as well as the levels of TBARS due to CPF exposure. Besides this, CPF and 2,4-D exposure induced oxidative DNA damage, and the H/L ratio decreased for the both herbicide exposures. Our results showed that exposure via dermal uptake to CPF, 2,4-D, and GLY in the common toad R. arenarum induced neurotoxicity, oxidative stress, and immunological depression. Thus, some blood biomarkers employed in our study (B-esterases, GST, levels of TBARS, ACA, and H/L ratio) might be used as predictors in health and ecological risk assessment of amphibian populations exposed to OP insecticides and herbicides.

The role of base excision repair in pathogenesis of breast cancer in the Polish population.

Breast cancer (BC) is leading type of cancer among group of women, which determines almost 23% of invasive cancers. It has been reported repeatedly that the level of oxidative stress is higher for BC in comparison to cancer-free woman. The goal of the present study was to evaluate the role of base excision repair (BER) pathway in the development of BC. One-hundred seventy-one women with confirmed BC and 222 healthy controls were enrolled in presented study. The level of oxidative DNA damage and the kinetic of their repair were analyzed by the modified alkaline comet assay. The efficiency of BER pathway was evaluated by BER assay. The presence of the 326Cys/Cys genotype and 326Cys allele of OGG1 gene and the 324His/His of MUTYH gene are associated with increased risk of BC development. Moreover, correlation between clinical parameter with selected genes has shown increased risk of BC progression. The survival analysis has shown a significant lower DFS for individuals with the 762Ala/Ala genotype compared to 762Val/Vla carriers and the 762Val/Ala genotype in relation to concomitant chemotherapy and radiotherapy. In subgroup of patients with alone chemotherapy and alone radiotherapy, the 762Val/Val genotype was significantly associated with lower overall survival. Furthermore, we also elevated the level of basal and oxidative DNA damage in a group of patients with BC in relation to healthy controls. We also observed the difference in effectiveness of DNA damage repair. The results of present studies suggested the important role of BER pathway in BC development. © 2015 Wiley Periodicals, Inc.

In vitro and in vivo assessment of the genotoxicity and antigenotoxicity of the Filipendula hexapetala and Filipendula ulmaria methanol extracts

Ethnopharmacological relevanceThe two species of Filipendula genus, Filipendula hexapetala Gilib. and Filipendula ulmaria (L.) Maxim are a traditional herbal medicine widely used to treat haemorrhoids, diarrhoea, fever, rheumatism and arthritic pain, kidney problems, to stop bleeding, and the common cold, as well as food supplements. However, no scientific study has been performed to validate genotoxic and/or antigenotoxic potentials of these two Filipendula species. Aim of the studyThe aim of the present study was to examine the genotoxic and possible in vitro and in vivo DNA protection potential of methanol extracts of F. hexapetala and F. ulmaria. Materials and methodsThe genotoxicity of different concentrations of F. hexapetala and F. ulmaria methanol extracts from roots and aerial parts (20, 40 and 80mg/ml), mixed with standard food for Drosophila, was evaluated in vivo in the anterior midgut of Drosophila melanogaster using a modified alkaline comet assay. The protective effects of the highest dose of extracts were observed in somatic cells of third-instar larvae against ethyl methanesulphonate (EMS)-induced genotoxicity. Also, DNA protection activity of methanol extracts from F. hexapetala and F. ulmaria (100, 200, and 400μg/ml) against hydroxyl radical-induced DNA damage was determined under in vitro conditions. ResultsThe results showed that methanol extracts from the root and aerial part of F. hexapetala at a concentration of 20mg/ml indicated the absence of genotoxicity. Also, there were no statistically significant differences in total scores between any of the groups treated with F. ulmaria root extract and the negative control group, while F. ulmaria aerial part extract possess weak genotoxic effects depending on the concentrations. The percentage reduction in DNA damage was more evident in the group of larvae simultaneously treated with EMS and the highest dose of F. hexapetala root or aerial part extracts and F. ulmaria root extract (91.02, 80.21, and 87.5%, respectively) and less expressive in the group simultaneously treated with F. ulmaria aerial part extract (54.7%). F. hexapetala root and aerial part extracts and F. ulmaria root extract possess strong capabilities to protect DNA from being damaged by hydroxyl radicals. ConclusionsIt can be concluded that F. hexapetala root and aerial part extracts and F. ulmaria root extract demonstrated the absence of genotoxic activity. The extracts appeared to have antigenotoxic effect, reducing the levels of DNA damage induced by EMS by more than 80%. Also, F. hexapetala root and aerial part extracts and F. ulmaria root extracts could effectively protect against hydroxyl radical-induced DNA damage.

Comparison of the genotoxic effects induced by 50 Hz extremely low-frequency electromagnetic fields and 1800 MHz radiofrequency electromagnetic fields in GC-2 cells.

Extremely low-frequency electromagnetic fields (ELF-EMF) and radiofrequency electromagnetic fields (RF-EMF) have been considered to be possibly carcinogenic to humans. However, their genotoxic effects remain controversial. To make experiments controllable and results comparable, we standardized exposure conditions and explored the potential genotoxicity of 50 Hz ELF-EMF and 1800 MHz RF-EMF. A mouse spermatocyte-derived GC-2 cell line was intermittently (5 min on and 10 min off) exposed to 50 Hz ELF-EMF at an intensity of 1, 2 or 3 mT or to RF-EMF in GSM-Talk mode at the specific absorption rates (SAR) of 1, 2 or 4 W/kg. After exposure for 24 h, we found that neither ELF-EMF nor RF-EMF affected cell viability using Cell Counting Kit-8. Through the use of an alkaline comet assay and immunofluorescence against γ-H2AX foci, we found that ELF-EMF exposure resulted in a significant increase of DNA strand breaks at 3 mT, whereas RF-EMF exposure had insufficient energy to induce such effects. Using a formamidopyrimidine DNA glycosylase (FPG)-modified alkaline comet assay, we observed that RF-EMF exposure significantly induced oxidative DNA base damage at a SAR value of 4 W/kg, whereas ELF-EMF exposure did not. Our results suggest that both ELF-EMF and RF-EMF under the same experimental conditions may produce genotoxicity at relative high intensities, but they create different patterns of DNA damage. Therefore, the potential mechanisms underlying the genotoxicity of different frequency electromagnetic fields may be different.

The alkaline comet assay as a method to investigate the DNA strand breaking effect of phenylpropanoids in mammalian cells

Event Abstract Back to Event The alkaline comet assay as a method to investigate the DNA strand breaking effect of phenylpropanoids in mammalian cells Sabrina Haupenthal1, Sabrina Vallicotti2, Melanie Hemgesberg2 and Melanie Esselen1* 1 University of Münster, Institute of Food Chemistry, Germany 2 University of Kaiserslautern, Department of Food Chemistry and Toxicology, Germany The propenylic α-asarone (aA) and β-asarone (bA) as well as the allylic γ-asarone (gA) are naturally occurring constituents in several herbs and spices such as Acorus calamus (aA and bA) or Aniba hostmanniana (gA). Alcoholic beverages, teas, herbal medicine and cosmetics exhibit the most important route for human exposure [1]. aA and bA are classified as carcinogenic in rodents, whereas the data for gA is very limited so far [2]. For risk assessment further cellular studies about genotoxicity are required, so the question was addressed, whether the asarone isomers induce DNA strand breaks. Therefore, a modified alkaline comet assay to investigate DNA strand breaking effects of the asarone isomers in hamster lung fibroblasts (V79 cells and with human cytochrome P450 1A2 and human sulfotransferase 1C2 transfected V79 cells) was used. Furthermore, the DNA repair enzyme formamidopyrimidine DNA glycosylase (FPG) as a marker for oxidative DNA damage was also included in our test system. aA, bA as well as gA significantly induce DNA damage in V79 cells at concentrations > 10 µM. However, no enhanced DNA damage was observed after FPG-incubation of V79 cells. In contrast, in metabolic competent transfected V79 cells both propenylic asarone derivatives aA and bA significantly increase the amount of FPG-sensitive sites, whereas the allylic compound gA was not effective. This study assumes that an impact on the cellular redox status of the propenylic asarone isomers and their respective metabolites might contribute to their genotoxic properties. In conclusion, further in vitro studies on DNA damage and repair to elucidate the mechanism of genotoxicity and mutagenicity of these carcinogenic plant constituents are still under investigation.

Comet assay as a procedure for detecting possible genotoxicity induced by non-ionizing radiation

Comet assay as a procedure for detecting possible genotoxicity induced by non-ionizing radiation

The eucalyptus oil ingredient 1,8-cineol induces oxidative DNA damage.

The natural compound 1,8-cineol, also known as eucalyptol, is a major constituent of eucalyptus oil. This epoxy-monoterpene is used as flavor and fragrance in consumer goods as well as medical therapies. Due to its anti-inflammatory properties, 1,8-cineol is also applied to treat upper and lower airway diseases. Despite its widespread use, only little is known about the genotoxicity of 1,8-cineol in mammalian cells. This study investigates the genotoxicity and cytotoxicity of 1,8-cineol in human and hamster cells. First, we observed a significant and concentration-dependent increase in oxidative DNA damage in human colon cancer cells, as detected by the Formamidopyrimidine-DNA glycosylase (Fpg)-modified alkaline comet assay. Pre-treatment of cells with the antioxidant N-acetylcysteine prevented the formation of Fpg-sensitive sites after 1,8-cineol treatment, supporting the notion that 1,8-cineol induces oxidative DNA damage. In the dose range of DNA damage induction, 1,8-cineol did neither reduce the viability of colon cancer cells nor affected their cell cycle distribution, suggesting that cells tolerate 1,8-cineol-induced oxidative DNA damage by engaging DNA repair. To test this hypothesis, hamster cell lines with defects in BRCA2 and Rad51, which are essentials players of homologous recombination (HR)-mediated repair, were treated with 1,8-cineol. The monoterpene induced oxidative DNA damage and subsequent DNA double-strand breaks in the hamster cell lines tested. Intriguingly, we detected a significant concentration-dependent decrease in viability of the HR-defective cells, whereas the corresponding wild-type cell lines with functional HR were not affected. Based on these findings, we conclude that 1,8-cineol is weakly genotoxic, inducing primarily oxidative DNA damage, which is most likely tolerated in DNA repair proficient cells without resulting in cell cycle arrest and cell death. However, cells with deficiency in HR were compromised after 1,8-cineol treatment, suggesting a protective role of HR in response to high doses of 1,8-cineol.

In vitrostudy of cypermethrin on human spermatozoa and the possible protective role of vitamins C and E

Cypermethrin, a type II synthetic pyrethroid pesticide, is widely used in pest control programmes in agriculture and public health. This study aimed to assess the potential effect of cypermethrin on human spermatozoa and the possible ameliorative effects of vitamins C and E. Semen samples of 20 healthy normozoospermic men were divided into six aliquots at room temperature. The first aliquot served as control not exposed to treatments, and the second was incubated with 20mm vit. C and 2mm vit. E where the third one was exposed to 10μm cypermethrin for 6h. The other three aliquots were incubated with vit. C, vit. E and both vitamins for 30min before cypermethrin exposure. Semen aliquots were analysed for sperm motility, sperm viability, hypo-osmotic swelling test and modified alkaline comet assay. The results demonstrated a significant decrease in sperm motion, sperm function and increased sperm DNA damage in the cypermethrin group. Addition of vitamins C and E alone/combined led to significant improvement in sperm motion, sperm function and DNA damage, being maximal with both vitamins together. It is concluded that in vitro cypermethrin can alter sperm function and induce DNA damage in spermatozoa, which is improved after using vitamins C and E.

Determination of TiO2, ZrO2, and Al2O3 Nanoparticles on Genotoxic Responses in Human Peripheral Blood Lymphocytes and Cultured Embyronic Kidney Cells

In this study a genotoxic evaluation of titanium dioxide (TiO2, 2.3 nm), zirconium oxide (ZrO2, 6 nm), aluminum oxide (Al2O3, 16.7 nm) nanoparticles (NP) and their ionic forms was conducted using human peripheral blood lymphocytes and cultured human embryonic kidney (HEK293) cells by means of a modified alkaline comet assay with/without the formamidopyrimidine-DNA glycosylase (Fpg) and endonuclease III (Endo III) enzymes. Modifications to the comet assay by using lesion-specific endonucleases, such as Endo III and Fpg, detect DNA bases with oxidative damage. Both human peripheral blood lymphocytes and cultured embryonic kidney cells were incubated with TiO2, ZrO2, or Al2O3 NP at concentrations of 1, 10, or 100 μg/ml. Our results showed no significant induction in DNA damage by the comet assay with/without the Endo III and Fpg enzymes at all concentrations of ZrO2 and Al2O3. In the case of TiO2 NP only the highest concentration of 100 μg/ml significantly induced a genotoxic response. Data thus indicate that both ZrO2 and Al2O3 NP were not genotoxic in our system and in the case of TiO2 the lowest-observed-adverse-effect level (LOAEL) for genotoxicity was 100 μg/ml. Evidence indicates that these metallic NP are considered safe in light of the fact that no genotoxicity was noted with ZrO2 and Al2O3 and that the highest TiO2 concentration is not environmentally relevant.

Methylglyoxal induces DNA crosslinks in ECV304 cells via a reactive oxygen species-independent protein carbonylation pathway

Methylglyoxal (MG) is a reactive dicarbonyl compound that is produced endogenously from glycolytic intermediates and via gluconeogenesis. Elevated MG levels in diabetes patients are believed to cause diabetic complications. MG-induced crosslinking products from the covalent binding of DNA or protein alone or together could be relevant to carcinogenesis and multiple complications in diabetes. However, the mechanisms governing DNA crosslink formation by MG are unclear. We investigated whether MG could induce DNA crosslinks in human ECV304 cells and the possible mechanism of this action. The level of DNA crosslinks and reactive oxygen species production were assayed by a modified alkaline Comet assay and a 2′,7′ dichlorofluorescin diacetate (DCHF-DA) assay, respectively. MG caused a time- and dose-dependent increase in DNA crosslinks and a dose-dependent increase in protein carbonylation in ECV304 cells. Addition of 2mM MG resulted in a transient increase in protein carbonylation, and this increase peaked within 2h and then rapidly decreased. Most notably, MG did not cause significantly enhanced ROS generation in ECV304 cells. Co-treatment with carbonyl-scavenging drugs, such as aminoguanidine, N-acetyl-l-cysteine, and glutathione, significantly inhibited the formation of DNA crosslinks by MG, whereas co-treatment with the antioxidant ascorbic acid did not. In conclusion, our results imply that MG induces DNA crosslink formation in ECV304 cells via a reactive oxygen species-independent protein carbonylation pathway. Our findings also suggest that non-toxic aminothiol antioxidants with carbonyl scavenging capabilities are potential therapeutic agent for MG-related diseases, such as diabetes and neurodegeneration. Furthermore, our findings also imply that DNA nonbinding proteins, bovine serum albumin might be able to crosslink calf thymus DNA in the presence of MG.

Role of base-excision repair in the treatment of childhood acute lymphoblastic leukaemia with 6-mercaptopurine and high doses of methotrexate

Methotrexate (MTX) and 6-mercaptopurine (6MP) are the most commonly used drugs in the therapy of childhood acute lymphoblastic leukaemia (ALL). The main genotoxic effect of MTX resulting from inhibition of thymidylate synthase is mis-incorporation of uracil into DNA, which is considered essential for the effectiveness of the Protocol M in ALL IC BFM 2002/EURO LB 2002 regimens. In this study, we investigated the level of basal and induced DNA damage as well as the effectiveness of DNA repair in lymphocytes of children with ALL at four time-points during therapy with MTX and 6MP. To assess DNA damage and the efficacy of DNA repair we used the modified alkaline comet assay with uracil DNA glycosylase (Udg) and endonuclease III (EndoIII). In addition, we examined the induction of apoptosis in the lymphocytes of the patients during treatment. Finally, we compared the activity of base-excision repair (BER), involved in removal of both uracil and oxidized bases from DNA in lymphocytes of children with ALL and lymphocytes of healthy children. BER efficiency was estimated in an in vitro assay with cellular extracts and plasmid substrates of heteroduplex DNA with an AP-site. Our results indicate that there is a significant decrease in the efficacy of DNA repair associated with an increased level of uracil in DNA and induction of apoptosis during therapy. Moreover, it was found that the BER capacity was decreased in the lymphocytes of ALL patients in contrast to that in lymphocytes of healthy children. Thus, we suggest that an impairment of the BER pathway may play a role in the pathogenesis and therapy of childhood ALL.

A modified alkaline Comet assay for in vivo detection of oxidative DNA damage in Drosophila melanogaster

Modifications to the alkaline Comet assay by using lesion-specific endonucleases, such as formamidopyrimidine-DNA glycosylase (FPG) and endonuclease III (ENDOIII, also known as Nth), can detect DNA bases with oxidative damage. This modified assay can be used to assess the genotoxic/carcinogenic potential of environmental chemicals. The goal of this study was to validate the ability of this modified assay to detect oxidative stress-induced genotoxicity in Drosophila melanogaster (Oregon R+). In this study, we used three well known chemical oxidative stress inducers: hydrogen peroxide (H2O2), cadmium chloride (CdCl2) and copper sulfate (CuSO4). Third instar larvae of D. melanogaster were fed various concentrations of the test chemicals (50–200μM) mixed with a standard Drosophila food for 24h. Alkaline Comet assays with and without the FPG and ENDOIII enzymes were performed with midgut cells that were isolated from the control and treated larvae. Our results show a concentration-dependent increase (p<0.05–0.001) in the migration of DNA from the treated larvae. ENDOIII treatment detected more oxidative DNA damage (specifically pyrimidine damage) in the H2O2 exposed larvae compared to FPG or no enzyme treatment (buffer only). In contrast, FPG treatment detected more oxidative DNA damage (specifically purine damage) in CuSO4 exposed larvae compared to ENDOIII. Although previously reported to be a potent genotoxic agent, CdCl2 did not induce more oxidative DNA damage than the other test chemicals. Our results show that the modified alkaline Comet assay can be used to detect oxidative stress-induced DNA damage in D. melanogaster and thus may be applicable for in vivo genotoxic assessments of environmental chemicals.

Evaluation of DNA damage after tourniquet-induced ischaemia/reperfusion injury during lower extremity surgery

Ischemia/reperfusion (I/R) injury represents a source of substantial morbidity and mortality in various statuses that is, coronary bypass, myocardial infarction, and so on. Oxygen free radicals, formed during I/R, have been proposed as one of the main causes of tissue injury and play important role in I/R injury. Leucocytes have been shown to play an important role in the development of tissue injury after I/R. Accordingly, numerous studies have shown that even short-time I/R-induced DNA damage can be investigated in human peripheral leucocytes using the alkaline single-cell gel-electrophoresis assay (comet assay). After ischaemia, the genotoxic damage detected in human peripheral leucocytes locally in the reperfused tissue results in numerical, morphologic and biochemical alterations of all circulating white blood cells in the human organism. It leads particularly to the release of substantial amounts of oxygen radicals and other reactive agents. Simultaneously, local ischaemia in the reperfused tissue is extended to the whole body systemically through these activated inflammatory cells and, possibly, results in secondary detectable tissue damage in endothelial cells of the systemic circulation inducing prolonged DNA damage even in the early reperfusion period. Thus, we aimed to investigate whether the I/R during the routinely practised operation causes DNA damage, since other published studies were on animal and in vitro models and did not exactly reflect the operation procedure in the clinic. We measured DNA damage (single-strand breaks, oxidised purines and pyrimidines) by modified alkaline comet assay using two bacterial enzymes (formamidopyrimidine glycosylase (Fpg), endonuclease III (EndoIII)), which recognise oxidised purines and pyrimidine bases, in patients who had lower extremity surgery. There was no statistically significant difference in DNA damage between time periods (before surgery, after I/R; T1, T2 and T3). According to our findings, we indicate that at the molecular level DNA is damaged due to the I/R during the routinely practised operation; however, this effect could not be determined phenotypically. Thus, we may suggest that the surgeons and anaesthetist/operation room personnel could be informed about that fact and encouraged to use antioxidants and/or apply prophylaxis. Our findings showed that I/R injury did not induce DNA damage. Further studies of this approach are needed.