Optimization of the alkaline comet assay for easy repair capacity quantification of oxidative DNA damage in PBMC from human volunteers using aphidicolin block

Assessment of DNA repair capacity (DRC) upon ex vivo challenge of peripheral blood mononuclear cells (PBMC) with oxidative damage inducing agents, as evaluated by the comet assay, is widely used as biomarker to assess the antioxidant status in human studies. Here, the alkaline comet assay was now optimized for easy and time saving detection of repair capacity upon oxidative stress-induced DNA damage using the DNA polymerase inhibitor aphidicolin (APC) to block repair of hydrogen peroxide (H₂O₂) induced DNA damage. Addition of a DMSO-containing DNA damage stop solution was found suitable to replace washing steps for H₂O₂ removal before APC block. Cell treatment with APC at 6 μM did not impact baseline DNA damage but could reliably block DNA repair after H₂O₂ challenge in both fresh and cryopreserved samples thus omitting the use of a starting time point control. Under the conditions used, frozen cells, with or without an additional 4 h rest, showed the same repair capacity as their fresh counterpart. The intra assay coefficient of variation (CV) was 3.3%. To provide proof of principle, the modified assay was applied to cryopreserved PBMC from 19 participants of a short-term Brassica diet intervention study investigating potential health promoting effects of the food intervention. Then, a 33% increase in DRC (p ≤ 0.01) could be shown in samples after intervention (mean ± SD: 5.82 ± 1) as compared to baseline (mean ± SD: 4.38 ± 1.21). Individual samples from baseline and intervention showed an inter-individual CV of 27.65% (baseline) and 17.26% (intervention). Taken together this modified comet assay protocol allows the facilitated detection of DNA repair in fresh or cryopreserved human PBMC samples with a good sensitivity and reliability and could be useful in human studies addressing the antioxidant status and repair capacity of PBMC.     

DNA damage in peripheral blood lymphocytes in patients during combined chemotherapy for breast cancer

Combined chemotherapy is used for the treatment of a number of malignancies such as breast cancer. The target of these antineoplastic agents is nuclear DNA, although it is not restricted to malignant cells. The aim of the present study was to assess DNA damage in peripheral blood lymphocytes (PBLs) of breast cancer patients subjected to combined adjuvant chemotherapy (5-fluorouracil, epirubicin and cyclophosphamide, FEC), using a modified comet assay to detect DNA single-strand breaks (SSB) and double-strand breaks (DSB).

Forty-one female patients with advanced breast cancer before and after chemotherapy and 60 healthy females participated in the study. Alkaline and neutral comet assays were performed in PBLs according to a standard protocol, and DNA tail moment was measured by a computer-based image analysis system.

Breast cancer patients before treatment had higher increased background levels of SSB and DSB as compared to healthy women. During treatment, a significant increase in DNA damage was observed after the 2nd cycle, which persisted until the end of treatment. Eighty days after the end of treatment the percentage of PBLs with SSB and DSB remained elevated, but the magnitude of DNA damage (tail moment) returned to baseline levels. There was no correlation between PBL DNA damage and response to chemotherapy.

DNA-SSB and DSB in PBLs are present in cancer patients before treatment and increase significantly after combined chemotherapy. No correlation with response to adjuvant chemotherapy was found. Biomonitoring DNA damage in PBLs of cancer patients could help prevent secondary effects and the potential risks of developing secondary cancers.     

Measuring oxidative damage to DNA and its repair with the comet assay

Background

Single cell gel electrophoresis, or the comet assay, was devised as a sensitive method for detecting DNA strand breaks, at the level of individual cells. A simple modification, incorporating a digestion of DNA with a lesion-specific endonuclease, makes it possible to measure oxidised bases.

Scope of review

With the inclusion of formamidopyrimidine DNA glycosylase to recognise oxidised purines, or Nth (endonuclease III) to detect oxidised pyrimidines, the comet assay has been used extensively in human biomonitoring to monitor oxidative stress, usually in peripheral blood mononuclear cells.

Major conclusions

There is evidence to suggest that the enzymic approach is more accurate than chromatographic methods, when applied to low background levels of base oxidation. However, there are potential problems of over-estimation (because the enzymes are not completely specific) or under-estimation (failure to detect lesions that are close together). Attempts have been made to improve the inter-laboratory reproducibility of the comet assay.

General significance

In addition to measuring DNA damage, the assay can be used to monitor the cellular or in vitro repair of strand breaks or oxidised bases. It also has applications in assessing the antioxidant status of cells. In its various forms, the comet assay is now an invaluable tool in human biomonitoring and genotoxicity testing. This article is part of a Special Issue entitled Current methods to study reactive oxygen species - pros and cons and biophysics of membrane proteins. Guest Editor: Christine Winterbourn.     

The comet assay for DNA damage and repair

The comet assay (single-cell gel electrophoresis) is a simple method for measuring deoxyribonucleic acid (DNA) strand breaks in eukaryotic cells. Cells embedded in agarose on a microscope slide are lysed with detergent and high salt to form nucleoids containing supercoiled loops of DNA linked to the nuclear matrix. Electrophoresis at high pH results in structures resembling comets, observed by fluorescence microscopy; the intensity of the comet tail relative to the head reflects the number of DNA breaks. The likely basis for this is that loops containing a break lose their supercoiling and become free to extend toward the anode. The assay has applications in testing novel chemicals for genotoxicity, monitoring environmental contamination with genotoxins, human biomonitoring and molecular epidemiology, and fundamental research in DNA damage and repair. The sensitivity and specificity of the assay are greatly enhanced if the nucleoids are incubated with bacterial repair endonucleases that recognize specific kinds of damage in the DNA and convert lesions to DNA breaks, increasing the amount of DNA in the comet tail. DNA repair can be monitored by incubating cells after treatment with damaging agent and measuring the damage remaining at intervals. Alternatively, the repair activity in a cell extract can be measured by incubating it with nucleoids containing specific damage.     

Novel nitrogen compounds enhance protection and repair of oxidative DNA damage in a neuronal cell model: Comparison with quercetin

Oxidative DNA damage has been described as an important type of damage that occurs in neuronal cells, with severe implications in many neurodegenerative diseases and in aging. We have previously reported the protection of four new synthetic nitrogen compounds (FMA4, FMA7, FMA762 and FMA796) against oxidative stress conditions. In this work, we studied their effects on oxidative DNA damage induced in rat pheochromocytoma (PC12) cells, using the Comet assay, and compared them with a natural antioxidant, quercetin. Among the compounds tested, FMA762 and FMA796 were the most effective in preventing tert-butylhydroperoxide (t-BHP)-induced formation of DNA strand breaks and in improving the cells’ capacity to repair this kind of damage. These effects were similar to the ones of quercetin, a flavonoid with known antioxidant activity. Moreover, contrarily to quercetin, they increased the repair capacity of oxidised bases induced with the photosensitiser Ro 19-8022. This effect seems to be mediated by an increase in DNA repair enzymes activity, assessed by the in vitro BER assay, but no regulation at the expression of OGG1 and APE1 genes was detected. In addition to other properties previously found for the nitrogen compounds, they now prove their effectiveness against oxidative stress-induced DNA damage in the neuronal cell model used.     

Chronic exposure to sublethal doses of radiation mimetic Zeocin selects for clones deficient in homologous recombination

Research indicates that exposure to hypoxia is associated with oxidative stress. In this investigation, healthy subjects were exposed to hypoxia by inhalation of 10% oxygen for 2 h (corresponding to 5500 m above sea level). The levels of strand breaks and oxidatively damaged purine bases, measured by the comet assay, and the expression of genes involved in DNA repair of oxidatively damaged DNA were investigated in mononuclear blood cells (MNBC) at baseline, after 2 h of hypoxia, 2 h of reoxygenation, and 1 day and 8 days after the exposure. The level of strand breaks and oxidized purine bases in MNBC increased following both the 2 h of hypoxia and the 2 h reoxygenation period, whereas this effect was not observed in unexposed subjects. The expressions of oxoguanine DNA glycosylase 1 (OGG1), nucleoside diphosphate linked moiety X-type motif 1 (NUDT1), nei endonuclease VIII-like 1 (NEIL1), and mutY homolog (MUTYH) were unaltered throughout the experiment in both groups of subjects, indicating that DNA repair genes are not up-regulated by the hypoxia and reoxygenation treatment. Taken together, this report shows that inhalation of 10% oxygen for 2 h is associated with increased number of oxidized DNA lesions in MNBC, but acute hypoxia may not inflict upon the regulation of genes involved in repair of oxidized DNA.     

DNA damage response and repair in ovarian cancer: Potential targets for therapeutic strategies

Ovarian cancer is among the most lethal gynecologic malignancies with a poor survival prognosis. The current therapeutic strategies involve surgery and chemotherapy. Research is now focused on novel agents especially those targeting DNA damage response (DDR) pathways. Understanding the DDR process in ovarian cancer necessitates having a detailed knowledge on a series of signaling mediators at the cellular and molecular levels. The complexity of the DDR process in ovarian cancer and how this process works in metastatic conditions is comprehensively reviewed. For evaluating the efficacy of therapeutic agents targeting DNA damage in ovarian cancer, we will discuss the components of this system including DDR sensors, DDR transducers, DDR mediators, and DDR effectors. The constituent pathways include DNA repair machinery, cell cycle checkpoints, and apoptotic pathways. We also will assess the potential of active mediators involved in the DDR process such as therapeutic and prognostic candidates that may facilitate future studies.     

DNA damage and repair in Helicobacter pylori-infected gastric mucosa cells

Helicobacter pylori is a common human pathogen and its infection is believed to contribute to gastric cancer. Impaired DNA repair may fuel up cancer transformation by the accumulation of mutation and increased susceptibility to exogenous carcinogens. To evaluate the role of infection of H. pylori in DNA damage and repair we determined: (1) the level of endogenous basal, oxidative and alkylative DNA damage, and (2) the efficacy of removal of DNA damage induced by hydrogen peroxide and the antibiotic amoxicillin in the H. pylori-infected and non-infected GMCs. DNA damage and the efficacy of DNA repair were evaluated by the alkaline single cell gel electrophoresis (comet assay). Specific damage to the DNA bases were assayed with the DNA repair enzymes formamidopyrimidine-DNA glycosylase (Fpg) recognizing oxidized DNA bases and 3-methyladenine-DNA glycosylase II (AlkA) recognizing alkylated bases. The level of basal and oxidative DNA in the infected GMCs was higher than non-infected cells. H. pylori-infected GMCs displayed enhanced susceptibility to hydrogen peroxide than control cells. There was no difference between the efficacy of DNA repair in the infected and non-infected cells after treatment with hydrogen peroxide and amoxicillin. Our results indicate that H. pylori infection may be correlated with oxidative DNA damage in GMCs. Therefore, these features can be considered as a risk marker for gastric cancer associated with H. pylori infection and the comet assay may be applied to evaluate this marker.     

Increased oxidative DNA damage in mononuclear leukocytes in vitiligo

Vitiligo is an acquired pigmentary disorder of the skin of unknown aetiology. The autocytotoxic hypothesis suggests that melanocyte impairment could be related to increased oxidative stress. Evidences have been reported that in vitiligo oxidative stress might also be present systemically. We used the comet assay (single cell alkaline gel electrophoresis) to evaluate DNA strand breaks and DNA base oxidation, measured as formamidopyrimidine DNA glycosylase (FPG)-sensitive sites, in peripheral blood cells from patients with active vitiligo and healthy controls. The basal level of oxidative DNA damage in mononuclear leukocytes was increased in vitiligo compared to normal subjects, whereas DNA strand breaks (SBs) were not changed. This alteration was not accompanied by a different capability to respond to in vitro oxidative challenge. No differences in the basal levels of DNA damage in polymorphonuclear leukocytes were found between patients and healthy subjects. Thus, this study supports the hypothesis that in vitiligo a systemic oxidative stress exists, and demonstrates for the first time the presence of oxidative alterations at the nuclear level. The increase in oxidative DNA damage shown in the mononuclear component of peripheral blood leukocytes from vitiligo patients was not particularly severe. However, these findings support an adjuvant role of antioxidant treatment in vitiligo.     

Population-based retrospective study to investigate preexisting and new depression diagnosis among head and neck cancer patients

This study aimed to estimate the pre-cancer prevalence and post-cancer incidence of depression in older adult head and neck cancer patients. Using SEER-Medicare files, cancer was identified from SEER data and depression diagnosis was identified using Medicare claims. Of 3533 head and neck cancer patients, 10.6% were diagnosed with depression during the two years prior to cancer diagnosis, and an additional 8.9% developed depression in the year following cancer diagnosis. This study supports the critical need of screening for depression throughout cancer diagnosis and treatment, as well as a preventative approach in depression development in the older head and neck cancer patient population.     

Predicting toxicity for head and neck cancer patients undergoing radiation therapy: an independent and external validation of MDASI-HN based nomogram

AimWe conducted a study to validate the MDASI-HN based nomogram, which is used to predict the acute toxicities in head and neck cancer patients undergoing radiation therapy with or without chemotherapy.BackgroundTolerance to radiation varies from patient to patient and also depends on various other factors like tumor volume, dose of radiation, chemotherapy. Predicting the toxicities allow us to identify potential candidates who are likely to have a higher toxicity and, in addition, evaluates the nomogram when done on an independent group of patients.Materials and MethodsSixty biopsy confirmed head and neck cancer patients undergoing radiation were the subjects of the study. The patients completed patient reported outcome instrument (PRO) MDASI-HN questionnaire at the beginning and at the fifth week of radiation. The baseline score obtained was used to obtain the predicted score using nomogram. The nomogram was also externally validated as per the TRIPOD guidelines.ResultsThe mean baseline, predicted and score at the fifth week were 27.28 ± 11.04, 73.33 ± 15.51 and 82.62 ± 17.67, respectively, for all sub-sites. A positive, significant correlation (p < 0.01) between the predicted score and the score at the fifth week was seen across all sub sites such as Oral cavity (p = 0.05), Oropharynx (p = 0.02), Hypo pharynx (p = 0.02) and Larynx (p = 0.02).ConclusionThe MDASI-HN questionnaire based nomogram is simple, easily doable and takes into consideration the initial symptoms as well the treatment details; thereby, it is able to predict the toxicities accurately.     

Inflammation-induced DNA damage,mutations and cancer

The relationships between inflammation and cancer are varied and complex. An important connection linking inflammation to cancer development is DNA damage. During inflammation reactive oxygen and nitrogen species (RONS) are created to combat pathogens and to stimulate tissue repair and regeneration, but these chemicals can also damage DNA, which in turn can promote mutations that initiate and promote cancer. DNA repair pathways are essential for preventing DNA damage from causing mutations and cytotoxicity, but RONS can interfere with repair mechanisms, reducing their efficacy. Further, cellular responses to DNA damage, such as damage signaling and cytotoxicity, can promote inflammation, creating a positive feedback loop. Despite coordination of DNA repair and oxidative stress responses, there are nevertheless examples whereby inflammation has been shown to promote mutagenesis, tissue damage, and ultimately carcinogenesis. Here, we discuss the DNA damage-mediated associations between inflammation, mutagenesis and cancer.     

The use of comet assay in measuring DNA damage and repair efficiency in child,adult, and old age populations

In the present study, we used the Comet assay to estimate basal DNA damage in three distinct populations aged 5–10, 40–50, and 60–70 years old. The DNA damage induced by hydrogen peroxide and γ-irradiation in the lymphocytes of these populations, as well as their repair activity, was also studied. Finally, we measured apoptosis and necrosis after the effect of these agents. Our results indicate that the older population (60–70 years old) showed higher basal levels of DNA damage and was more sensitive to the effects of the DNA-damaging agents than the adult one (40–50 years old), who, in turn, was more sensitive than the younger population (5–10 years old). A decline of the repair efficiency with age to the DNA damage induced by the two agents was also observed. Apoptosis and necrosis were also affected by age.     

Smoking during radiotherapy for head and neck cancer and acute mucosal reaction

AimWe compared the incidence of RTOG/EORTC grade III and higher acute mucositis in patients with head and neck cancer who continued to smoke during radiotherapy with those who quit smoking.BackgroundThere are conflicting data on the relationship between smoking during radiotherapy and the severity of acute mucosal reaction. More studies dealing with this issue are needed.Materials and methodsAmong 136 patients receiving curative radio(chemo)therapy, 37 (27%) declared that they had not quit smoking during radiotherapy. The intensity of mucositis was scored daily by a nurse and weekly by a physician using the RTOG/EORTC scale. The main end-point of the study was the highest observed RTOG/EORTC grade of mucositis.ResultsPatients who smoked during radiotherapy (smokers) were younger than their counterparts who quit smoking (non-smokers), p = 0.06. There were no other differences in the baseline characteristics between smokers and non-smokers. Grade III/IV acute mucositis was observed in 43.5% of all patients. The percentage of patients with grade III/IV acute mucositis was similar in smokers and non-smokers (46% vs. 42%, p = 0.71). Nine patients (smokers [13.5%]; non-smokers [4%], p = 0.05) required prolonged hospitalization to heal mucositis.ConclusionsIn the whole group, smoking during radiotherapy was not related to acute mucosal toxicity evaluated as the rate of the highest observed grade of mucositis.     

Bufalin influences the repair of X-ray-induced DNA breaks in Chinese hamster cells

The bufadienolide bufalin, a component of the Chinese medicine chan'su, has been reported to selectively inhibit the growth of various lines of human cancer cells, due at least in part to its specific effect on topoisomerase (topo) II. We have treated Chinese hamster ovary (CHO) cells with doses of bufalin that result in a dramatic reduction in both the level and catalytic activity of topo II without any concomitant induction of DNA damage, as assessed by the comet assay. When cells were pre-treated with bufalin and then irradiated with X-rays, a follow-up study revealed that the kinetics of DNA repair was clearly affected, with a general delay in the restoration of DNA to the situation observed in non-irradiated controls. The possible involvement of topo II in radiation damage repair is discussed.     

Malnutrition and cachexia in patients with head and neck cancer treated with (chemo)radiotherapy

AimTo highlight the problems associated with nutrition that occur in patients with squamous cell carcinoma of the head and neck (SCCHN).BackgroundSCCHN is associated with weight loss before, during and after radiotherapy or concurrent chemoradiotherapy. Because of serious consequences of malnutrition and cachexia on treatment outcome, mortality, morbidity, and quality of life, it is important to identify SCCHN patients with increased risk for the development of malnutrition and cachexia.Materials and methodsCritical review of the literature.ResultsThis review describes pathogenesis, diagnosis and treatment of malnutrition and cancer cachexia. Treatment of malnutrition and cancer cachexia includes nutritional interventions and pharmacological therapy. Advantages and disadvantages of different nutritional interventions and their effect on the nutritional status, quality of life and specific oncological treatment are presented.ConclusionsNutritional management is an essential part of care of these patients, including early screening, assessment of nutritional status and appropriate intervention.     

Aldehyde dehydrogenase 1 is a putative marker for cancer stem cells in head and neck squamous cancer

Aldehyde dehydrogenase 1 (ALDH1) has been considered to be a marker for cancer stem cells. However, the role of ALDH1 in head and neck squamous cell carcinoma (HNSCC) has yet to be determined. In this study, we isolated ALDH1-positive cells from HNSCC patients and showed that these HNSCC-ALDH1⁺ cells displayed radioresistance and represented a reservoir for generating tumors. Based on microarray findings, the results of Western blotting and immunofluorescent assays further confirmed that ALDH1⁺-lineage cells showed evidence of having epithelial-mesenchymal transition (EMT) shifting and endogenously co-expressed Snail. Furthermore, the knockdown of Snail expression significantly decreased the expression of ALDH1, inhibited cancer stem-like properties, and blocked the tumorigenic abilities of CD44⁺CD24⁻ALDH1⁺ cells. Finally, in a xenotransplanted tumorigenicity study, we confirmed that the treatment effect of chemoradiotherapy for ALDH1⁺ could be improved by Snail siRNA. In summary, it is likely that ALDH1 is a specific marker for the cancer stem-like cells of HNSCC.     

Immunotherapy for head and neck cancer

Head and neck cancer represents a challenging disease. Despite recent treatment advances, which have improved functional outcomes, the long-term survival of head and neck cancer patients has remained unchanged for the past 25 years. One of the goals of adjuvant cancer therapy is to eradicate local regional microscopic and micrometastatic disease with minimal toxicity to surrounding normal cells. In this respect, antigen-specific immunotherapy is an attractive therapeutic approach. With the advances in molecular genetics and fundamental immunology, antigen-specific immunotherapy is being actively explored using DNA, bacterial vector, viral vector, peptide, protein, dendritic cell, and tumor-cell based vaccines. Early phase clinical trials have demonstrated the safety and feasibility of these novel therapies and the emphasis is now shifting towards the development of strategies, which can increase the potency of these vaccines. As the field of immunotherapy matures and as our understanding of the complex interaction between tumor and host develops, we get closer to realizing the potential of immunotherapy as an adjunctive method to control head and neck cancer and improve long-term survival in this patient population.     

Assessment of DNA damage in multiple organs of mice after whole body X-irradiation using the comet assay

The comet assay was performed to elucidate the linearity of calibration curves and detection limits for DNA damage in multiple organs of whole body X-irradiated mice, and rates of reduction in DNA damage by DNA repair during the irradiation period were estimated in the respective organs by comparing the rates of increase in DNA damage at different absorbed dose rates of X-rays. Of the assay parameters, tail length and the percentage DNA in the tail showed a higher sensitivity to DNA damage in most organs than Olive tail moment. Data at the higher absorbed dose rates (2.22 or 1.44 Gy/min) showed good correlations between absorbed doses and these two parameters, with correlation coefficients of more than 0.7 in many organs. However, this assay had difficulty detecting DNA damage at the lower absorption dose rate (0.72 Gy/min). The estimated rates of increase in DNA damage and those of DNA repair during the irradiation period in the respective organs suggested differences in the radiosensitivity of nuclear DNA and DNA repair capacity among organs. Our results indicated that absorbed dose rates of 1.0–1.3 Gy/min or greater were needed to induce detectable DNA damages by the comet assay in many organs.