ALBERT

Description: Among several factors affecting radiation sensitivity, genome size has received limited attention during the last 50 years since research at Brookhaven National Laboratory (USA) and other locations demonstrated substantial differences in radiation sensitivities, e.g. between tree species with large (e.g. conifers such as pines) versus small (e.g. dicots such as oaks) genome sizes. Taking advantage of the wide range of genome sizes among species, we investigated radiation sensitivity which we define in this study as DNA damage (break frequency) measured with the alkaline comet assay in isolated nuclei exposed to X-rays. As a starting point, we considered two possible explanations for the high radiation sensitivity of plants with large genome sizes: (i) inherently higher sensitivity of larger genomes and/or (ii) impaired DNA repair. In terms of genome size effects, experiments exposing isolated nuclei from six different plant species to X-rays, varying in genome sizes from 2.6 to 19.2 Gbp, showed that larger genomes are more sensitive to DNA damage by a relationship approximating the cube-root of the nuclear volume; e.g. a 10-fold increase in genome size increases sensitivity by about 2-fold. With regard to DNA repair, two conifer species, Sawara cypress ( Chamaecyparis pisifera , 8.9 Gbp genome size) and Scots pine ( Pinus sylvestris , 20 Gbp genome size), both effectively repaired DNA damage within 50 and 70 min, respectively, after acute X-ray exposures. Both species also showed delayed repair of double-strand DNA breaks, as we previously showed with Arabidopsis thaliana and Lolium multiflorum .

Description: Since there are several predicting factors associated with the comet assay parameters, we have decided to assess the impact of seasonal variations on the comet assay results. A total of 162 volunteers were retrospectively studied, based on the date when blood donations were made. The groups (winter, spring, summer and autumn) were matched in terms of age, gender, smoking status, body mass index and medical diagnostic exposure in order to minimise the impact of other possible predictors. Means and medians of the comet assay parameters were higher when blood was sampled in the warmer period of the year, the values of parameters being the highest during summer. Correlation of meteorological data (air temperature, sun radiation and sun insolation) was observed when data were presented as the median per person. Using multivariate analysis, sampling season and exposure to medical radiation were proved to be the most influential predictors for the comet assay parameters. Taken together, seasonal variation is another variable that needs to be accounted for when conducting a cohort study. Further studies are needed in order to improve the statistical power of the results related to the impact of sun radiation, air temperature and sun insolation on the comet assay parameters.

Description: The alkaline comet assay and a cell-free system were used to characterise DNA lesions induced by treatment with glycidamide (GA), a metabolite of the food contaminant acrylamide. DNA lesions induced by GA were sensitively detected when the formamidopyrimidine-DNA-glycosylase (Fpg) enzyme was included in the comet assay. We used LC-MS to characterise modified bases from GA-treated naked DNA with and without subsequent Fpg treatment. N7-GA-Guanine and N3-GA-Adenine aglycons were detected in the supernatant showing some depurination of adducted bases; treatment of naked DNA with Fpg revealed no further increase in the adduct yield nor occurrence of other adducted nucleobases. We treated human lymphocytes with GA and found large differences in DNA lesion levels detected with Fpg, depending on the duration and the pH of the lysis step. These lysis-dependent variations in GA-induced Fpg sensitive sites paralleled those observed after treatment of cells with methyl methane sulfonate (MMS). On the other hand, oxidative lesions (8-oxoGuanine) induced by a photoactive compound (Ro 12-9786) plus light, and also DNA strand breaks induced by X-rays, were detected largely independently of the lysis conditions. The results suggest that the GA-induced lesions are predominantly N7-GA-dG adducts slowly undergoing imidazole ring opening at pH 10 as in the standard lysis procedure; such structures are substrate for Fpg leading to strand breaks. The data suggest that the characteristic alkaline lysis dependence of some DNA lesions may be used to study specific types of DNA modifications. The comet assay is increasingly used in regulatory testing of chemicals; in this context, lysis-dependent variations represent a novel approach to obtain insight in the molecular nature of a genotoxic insult.

Description: Knowledge about the basal level of DNA damage in leucocytes of healthy control populations is essential before estimation of the effects of exposure to external agents in biomonitoring studies. The aim of this study was to analyse the effects of some lifestyle factors on baseline DNA damage in leucocytes of humans. The material consisted of the peripheral blood from 276 healthy volunteer blood donors. In addition to the standard blood donation questionnaire, they were asked about age, gender, occupation, radiological history, smoking habit, alcohol consumption, medicine use and pet ownership. The results showed marked intra-individual variability. Significant differences in DNA damage levels were observed between individuals in different age and sex groups, between smokers and non-smokers and between samples taken in different seasons of the year, with the highest DNA damage in those obtained in the summer. Significantly higher levels of DNA damage were noted in leucocytes of donors older than 29 years, in men compared with women and in male smokers. Significantly higher DNA strand breaks were observed in heavy smokers. A non-significantly higher level of DNA damage was observed in individuals subjected to radiological investigation and in those drinking alcohol, whereas lower levels were observed in leucocytes of pet owners and in donors taking medicines. Pet ownership influences the level of DNA damage and there is an interaction between this effect and that of smoking. The smoker/pet owners showed almost half the level of DNA damage of smokers without pets. The current results confirmed high intra-individual variability between the levels of DNA damage of individuals. The significant factors that influence the DNA damage in leucocytes are age, sex and smoking habit, especially in men and in heavy smokers. The finding of reduced DNA damage in the leucocytes of pet owners suggests the tendency towards a beneficial effect of such company.

Description: It is known that ceramic workers are potentially exposed to complex mixture of chemicals such as silica, inorganic lead, lime, beryllium and aluminum that can be associated with an increased risk of several diseases. All operations in the ceramic industries such as mixing, moulding, casting, shaking out and finishing jobs, have been associated with the higher exposure levels and in most of the silica-related industries, average overall exposure exceeded permissible exposure levels for respirable crystalline silica. The aim of this study was to evaluate the possible genotoxic damage in ceramic workers exposed to complex mixture of chemicals mainly crystalline silica. For this purpose, the blood and buccal epithelial cell samples were taken from the ceramic workers ( n = 99) and their controls ( n = 81). The genotoxicity was assessed by the alkaline comet assay in isolated lymphocytes and whole blood. Micronucleus (MN), binucleated (BN), pyknotic (PYC), condensed chromatin (CC), karyolytic (KYL), karyorrhectic (KHC) and nuclear bud (NBUD) frequencies in buccal epithelial cells and plasma 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodG) levels were also evaluated. In the study, 38 workers were diagnosed with silicosis, 9 workers were suspected to have silicosis, whereas 52 workers were found to be healthy. DNA damage in blood and lymphocytes; MN, CC + KHC, PYC frequencies in buccal epithelial cells and 8-oxodG levels in plasma were increased in workers compared to their controls. These results showed that occupational chemical mixture exposure in ceramic industry may cause genotoxic damage that can lead to important health problems in the workers.

Description: 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/10 6 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 (χ 2 = 7.14, P = 0.028). The levels of hOGG1-sensitive sites are lower (range: 0.04–0.18 lesions/10 6 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, KBrO 3 , 4-nitroquinoline-1-oxide, Na 2 Cr 2 O 7 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, KBrO 3 and 4-nitroquinoline-1-oxide provided suitable assay controls. We recommend these compounds as comet assay controls for oxidatively damaged DNA.

Description: During the last 30 years, the comet assay has become widely used for the measurement of DNA damage and repair in cells and tissues. A landmark achievement was reached in 2016 when the Organization for Economic Co-operation and Development adopted a comet assay guideline for in vivo testing of DNA strand breaks in animals. However, the comet assay has much more to offer than being an assay for testing DNA strand breaks in animal organs. The use of repair enzymes increases the range of DNA lesions that can be detected with the assay. It can also be modified to measure DNA repair activity. Still, despite the long-term use of the assay, there is a need for studies that assess the impact of variation in specific steps of the procedure. This is particularly important for the on-going efforts to decrease the variation between experiments and laboratories. The articles in this Special Issue of Mutagenesis cover important technical issues of the comet assay procedure, nanogenotoxicity and ionising radiation sensitivity on plant cells. The included biomonitoring studies have assessed seasonal variation and certain predictors for the basal level of DNA damage in white blood cells. Lastly, the comet assay has been used in studies on genotoxicity of environmental and occupational exposures in human biomonitoring studies and animal models. Overall, the articles in this Special Issue demonstrate the versatility of the comet assay and they hold promise that the assay is ready for the next 30 years.

Description: The alkaline comet assay, in vivo and in vitro , is currently used in several areas of research and in regulatory genotoxicity testing. Several efforts have been made in order to decrease the inter-experimental and inter-laboratory variability and increase the reliability of the assay. In this regard, lysis conditions are considered as one of the critical variables and need to be further studied. Here, we tested different times of lysis (from no lysis to 1 week) and two different lysis solutions in human lymphoblast (TK6) cells unexposed or exposed to X-rays. Similar % tail DNA values were obtained independently of the time of lysis employed for every X-ray dose tested and both lysis solutions. These results, taken together with our previous ones with methyl methanesulfonate and H 2 O 2 , which showed clear lysis-time dependence, support that the influence of the lysis time in the comet assay results depends on the type of lesion being detected; some DNA lesions may spontaneously give rise to apurinic or apyrimidinic (AP) sites during the lysis period, which can be converted into strand breaks detectable with the comet assay. Testing different times of lysis would be useful to increase the sensitivity of the comet assay and to ensure the detection of DNA lesions of an unknown compound, thereby providing some insight into the chemical nature of the lesions induced. However, the same lysis conditions (i.e. lysis time and lysis solution) should be used when comparing results between different experiments or laboratories.

Description: Obesity is associated with several detrimental health consequences, among them an increased risk for development of cancer, and an overall elevated mortality. Multiple factors like hyperinsulinemia, chronic microinflammation and oxidative stress may be involved. The comet assay has been proven to be very sensitive for detection of DNA damage and has been used to explore the relationship between overweight/obesity and DNA damage, but results are controversial. Very few investigations have been performed to correlate weight loss of obese individuals and possible reduction of DNA damage and these studies have not provided clear results. As currently, only surgical interventions (metabolic/bariatric surgery) enable substantial and sustained weight loss in the vast majority of morbidly obese patients, we analyzed whole blood samples of 56 subsequent patients prior, 6 and 12 months after bariatric surgery. No reduction of DNA damage was observed in comet assay analysis after 6 months despite efficient weight loss, but a significant reduction was observed 12 months after surgery. Concurrently, the ferric-reducing antioxidant power assay showed a significant reduction after 6 and 12 months. The level of oxidised glutathione and lipid peroxidation products were increased at 6 months but normalised at 12 months after surgery. As conclusion, a significant weight reduction in obese patients may help to diminish existing DNA damage besides improving many other health aspects in these patients.

Description: We would like to express our thanks to members of the Editorial Board and the reviewers listed below who have maintained the high standard of peer-review for manuscripts submitted to Mutagenesis.