Nuestros investigadores

Amaya Azqueta Oscoz

Farmacología y Toxicología
Facultad de Farmacia y Nutrición. Universidad de Navarra
Líneas de investigación
Biomonitorización humana, Daño y reparación de ADN, Nanotoxicología, Genotoxicología
Índice H
27, (WoS, 26/09/2017)

Publicaciones científicas más recientes (desde 2010)

Autores: Koppen, G., ; Azqueta Oscoz, Amaya; Pourrut, B., ; et al.
ISSN 0267-8357  Vol. 32  Nº 3  2017  págs. 397 - 408
The International Comet Assay Workshops are a series of scientific conferences dealing with practical and theoretical aspects of the Comet Assay (single-cell gel electrophoresis)-a simple method for detecting DNA strand breaks. The first paper describing such an assay was published over 30 years ago in 1984 by Swedish researchers O. Ostling and K. J. Johanson. Appropriately, the theme for the 2015 meeting was looking to the future: 'The Next 3 Decades of the Comet Assay'. The programme included 25 oral and 43 poster presentations depicting the latest advances in technical developments as well as applications of the comet assay in genotoxicity testing (in vitro and in vivo) and biomonitoring of both humans and the environment. Open discussion sessions based on questions from the participants allowed exchange of practical details on current comet assay protocols. This report summarises technical issues of high importance which were discussed during the sessions. We provide information on ways to improve the assay performance, by testing for cytotoxicity, by using reference samples to reduce or allow for inter-experimental variation, and by standardising quantification of the damage, including replicates and scoring enough comets to ensure statistical validity. After 30 years of experimentation with the comet assay, we are in a position to control the important experimental parameters and make the comet assay a truly reliable method with a wealth of possible applications.
Autores: Iglesias Alonso, Tamara; Dusinska, M., ; El Yamani, N., ; et al.
ISSN 0378-5173  Vol. 523  Nº 1  2017  págs. 418 - 426
In the last years, the development of nanomaterials has significantly increased due to the immense variety of potential applications in technological sectors, such as medicine, pharmacy and food safety. Focusing on the nanodevices for oral drug delivery, poly(anhydride) nanoparticles have received extensive attention due to their unique properties, such as their capability to develop intense adhesive interactions within the gut mucosa, their modifiable surface and their biodegradable and easy-to produce profile. However, current knowledge of the possible adverse health effects as well as, toxicological information, is still exceedingly limited. Thus, we investigated the capacity of two poly(anhydride) nanoparticles, Gantrez (R) AN 119 -NP (GN-NP) and Gantrez (R) AN 119 covered with mannosamine (GN-MA-NP), and their main bulk material (Gantrez (R) AN 119-Polymer), to induce DNA damage and thymidine kinase (TK+/-) mutations in L5178Y TK+/- mouse lymphoma cells after 24 h of exposure. The results showed that GN-NP, GN-MA-NP and their polymer did not induce DNA strand breaks or oxidative damage at concentrations ranging from 7.4 to 600 mu g/mL. Besides, the mutagenic potential of these nanoparticles and their polymer revealed no significant or biologically relevant gene mutation induction at concentrations up to 600 mu g/mL under our experimental settings. Considering the non-genotoxic effects of GN-NP and GN-MA-NP, as well as their exceptional properties, these nanoparticles are promising nanocarriers for oral medical administrations. (C) 2017 Elsevier B.V. All rights reserved.
Autores: Iglesias Alonso, Tamara; López de Cerain Salsamendi, Adela María; Irache Garreta, Juan Manuel; et al.
ISSN 0378-5173  Vol. 517  Nº 1 - 2  2017  págs. 67 - 79
he main concerns with drugs designed for oral administration are their inactivation or degradation in the harsh conditions of the gastrointestinal tract, their poor solubility through the gastrointestinal mucus gel layer, the poor intestinal epithelium permeability that limits their absorption, and their toxicity. In this context, poly(anhydride) nanoparticles are capable of protecting the drug from the harsh environment, reduce the drug's toxicity and, by virtue of surface modification, to enhance or reduce their mucus permeability and the bioadhesion to specific target cells. The copolymer between methyl vinyl ether and maleic anhydride (commercialized as Gantrez® AN 119) are part of the poly(anhydride) nanoparticles. These biocompatible and biodegradable nanoparticles (NPs) can be modified by using different ligands. Their usefulness as drug carriers and their bioadhesion with components of the intestinal mucosa have been described. However, their toxicity, genotoxicity and mucus permeation capacity has not been thoroughly studied. The aim of this work was to evaluate and compare the in vitro toxicity, cell viability and in vitro genotoxicity of the bioadhesive empty Gantrez® AN 119 NPs modified with dextran, aminodextran, 2-hydroxypropyl-ß-cyclodextrin, mannosamine and poly-ethylene glycol of different molecular weights. Results showed that, in general, coated NPs exhibit better mucus permeability than the bare ones, those coated with mannosamine being the most permeable ones. The NPs studied did not affect cell metabolism, membrane integrity or viability of Caco-2 cells at the different conditions tested. Moreover, they did not induce a relevant level of DNA strand breaks and FPG-sensitive sites (as detected with the comet assay).
Autores: Azqueta Oscoz, Amaya; Collins, A. R.,
ISSN 2072-6643  Vol. 8  Nº 12  2016  págs. 785
Polyphenols are a very broad group of chemicals, widely distributed in plant foods, and endowed with antioxidant activity by virtue of their numerous phenol groups. They are widely studied as putative cancer-protective agents, potentially contributing to the cancer preventive properties of fruits and vegetables. We review recent publications relating to human trials, animal experiments and cell culture, grouping them according to whether polyphenols are investigated in whole foods and drinks, in plant extracts, or as individual compounds. A variety of assays are in use to study genetic damage endpoints. Human trials, of which there are rather few, tend to show decreases in endogenous DNA damage and protection against DNA damage induced ex vivo in blood cells. Most animal experiments have investigated the effects of polyphenols (often at high doses) in combination with known DNA-damaging agents, and generally they show protection. High concentrations can themselves induce DNA damage, as demonstrated in numerous cell culture experiments; low concentrations, on the other hand, tend to decrease DNA damage.
Autores: Pérez Silanes, Silvia; Torres Eguia, Eduardo; Arbillaga Lacunza, Leire; et al.
ISSN 0960-894X  Vol. 26  Nº 3  2016  págs. 903 - 906
We report the synthesis and in vitro activity against Trypanosoma cruzi epimastigotes of 15 novel quinoxaline derivatives. Ten of the derivatives presented IC50 values lower than the reference drugs Nfx and Bzn; four of them standed out with IC50 values lower than 1.5 ¿M. Moreover, unspecific cytotoxicity and genotoxicity studies are also reported. Compound 14 showed a SI higher than 24, whereas compound 10 was the only one that was negative in the genotoxicity screening.
Autores: Moreno de Viguri, Elsa; Jiménez-Montes, C., ; Martín-Escolano, R., ; et al.
ISSN 0022-2623  Vol. 59  Nº 24  2016  págs. 10929 - 10945
Chagas disease is a neglected tropical disease with 6-7 million people infected worldwide, and there is no effective treatment. Therefore, there is an urgent need to continue researching in order to discover novel therapeutic alternatives. We present a series of arylaminoketone derivatives as means of identifying new drugs to treat Chagas disease in the acute phase with greater activity, less toxicity, and a larger spectrum of action than that corresponding to the reference drug benznidazole. Indexes of high selectivity found in vitro formed the basis for later in vivo assays in BALB/c mice. Murine model results show that compounds 3, 4, 7, and 10 induced a remarkable decrease in parasitemia levels in acute phase and the parasitemia reactivation following immunosuppression, and curative rates were higher than with benznidazole. These high antiparasitic activities encourage us to propose these compounds as promising molecules for developing an easy to synthesize anti-Chagas agent.
Autores: Bengoetxea Bausela, Xabier; López de Cerain Salsamendi, Adela María; Azqueta Oscoz, Amaya; et al.
ISSN 1387-2877  Vol. 54  Nº 3  2016  págs. 1085 - 1094
Alzheimer's disease (AD) is a complex neurodegenerative disorder characterized by the presence of aggregates of the amyloid-ß peptide (Aß) that are believed to be neurotoxic. One of the purposed damaging mechanisms of Aß is oxidative insult, which eventually could damage the cellular genome. Stress and associated increases in glucocorticoids (GCs) have been described as a risk factor for the development of AD, although the purported genotoxic effects of GCs have not been fully characterized. Therefore, it is possible to speculate about purported synergistic effects of GCs on the Aß-driven genotoxic damage. This in vitro study addresses the single and combined cyto/genotoxic effects of Aß and GCs in SH-SY5Y cells. Cytotoxicity was determined by the MTT assay, and the genotoxic effects were studied using the comet assay. A comet assay derivation allows for measuring the presence of the FPG-sensitive sites (mainly 8-oxoguanines) in the DNA, apart from the DNA strand breaks. Treatment with Aß (10 ¿M, 72¿h) induced cytotoxicity (35% decrease in cell viability) and DNA strand breaks, but had no significant effect on oxidative DNA damage (FPG sites). Corticosterone showed no effect on cell viability, genotoxicity, or reparation processes. Corticosterone was unable to neither reverse nor potentiate Aß driven effects. The present results suggest the existence of alternative mechanisms for the Aß driven damage, not involving oxidative damage of DNA. In addition, could be suggested that the interaction between Aß and GCs in AD does not seem to involve DNA damage.
Autores: Ojer Ojer, Patricia; Iglesias Alonso, Tamara; Azqueta Oscoz, Amaya; et al.
ISSN 0939-6411  Vol. 97  Nº A  2015  págs. 206 - 217
Oral administration is the most commonly used and accepted route for drug administration. However, two of the main concerns are the poor intestinal epithelium permeability and rapid degradation, which limit absorption of drugs. In this context, nanocarriers have shown great potential for oral drug delivery. Nevertheless, special importance should be given to the possible toxic effect of these nanocarriers, such as their bioaccumulation in different tissues of the body, as well as, the different physicochemical parameters influencing their properties and so their potential toxic effect. This review describes first some aspects related to the behavior of nanosystems within the gastrointestinal tract and then some aspects of nanotoxicology and its evaluation, including the most popular techniques and approaches used for in vitro and in vivo toxicity studies. It also reviews the physicochemical characteristics of polymeric nanoparticles that may influence the development of toxicological effects, and finally it summarizes the toxicity results that have been published regarding polymeric nanocarriers.
Autores: Azqueta Oscoz, Amaya; Dusinska, M.,
ISSN 1664-8021  Vol. 6  2015  págs. 239
Autores: Martín-Cameán, A., ; Puerto, M., ; Jos, A., ; et al.
ISSN 1537-6516  Vol. 25  Nº 6  2015  págs. 487 - 493
Miniscrew implants are widely used nowadays in orthodontic treatments due to their good results in clinical practice. However, data regarding the biocompatibility of commercially available orthodontic miniscrews and temporary devices are very scarce, and their role as genotoxicity inducers has been not previously evaluated with the alkaline comet assay. The aim of this study was to investigate the DNA damage in buccal cells of patients subjected to orthodontic treatments. The alkaline comet assay has been applied in oral mucosa cells from patients treated with conventional orthodontic treatment in comparison to patients treated additionally with miniscrews, non-treated volunteers (control) and smoking volunteers (positive control). The application of orthodontic appliances and miniscrews induced significant and similar (2-fold) increases of %DNA in tail in comparison to control group. Females experienced a significant increase in %DNA in all the treatments in comparison to the control group, whereas males showed significant damage only with the combined orthodontic and miniscrew treatment. In conclusion, conventional orthodontic appliances induced genotoxicity, and the incorporation of miniscrews assayed did not imply any additional increase of DNA damage.
Autores: Enciso, J. M., ; Sánchez, O., ; López de Cerain Salsamendi, Adela María; et al.
ISSN 0267-8357  Vol. 30  Nº 1  2015  págs. 21 - 28
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.
Autores: Huk, A., ; Collins, A. R., ; El Yamani, N., ; et al.
ISSN 0267-8357  Vol. 30  Nº 1  2015  págs. 85 - 88
The comet assay is widely used to test the genotoxicity of engineered nanomaterials (ENMs) but outcomes may vary when results from different laboratories, or even within one laboratory, are compared. We address some basic methodological considerations, such as the importance of carrying out physico-chemical characterisation of the ENMs in test-medium, performing uptake and cytotoxicity tests, and testing several genotoxicity-related endpoints. In this commentary, we discuss the different ways in which concentration of ENMs can be expressed, and stress the need to include appropriate controls and reference standards to monitor variation and avoid interference. Treatment conditions, including cell number, cell culture plate format and volume of treatment medium on the plate are crucial factors that may impact on results and thus should be kept constant within the study.
Autores: Ibero Baraibar, Idoya; Azqueta Oscoz, Amaya; López de Cerain Salsamendi, Adela María; et al.
ISSN 0267-8357  Vol. 30  Nº 1  2015  págs. 139 - 146
Nutrient excess and unbalanced diets can result in overproduction of reactive oxygen species (ROS), which are associated with oxidative stress. Cocoa extract contains antioxidants that inhibit the harmful effects of ROS. This trial analysed the effect of cocoa extract consumption integrated as a bioactive compound into ready-to-eat meals, on oxidative stress at the level of DNA in overweight/obese subjects. Fifty volunteers [57.26(5.24) years, 30.59(2.33)kg/m(2)] participated in a 4-week double-blind, randomised, placebo-controlled parallel nutritional intervention. Half of the volunteers received meals supplemented with 1.4 g/day cocoa extract, while the other half received control meals, both within a 15% energy restriction diet. Lymphocytes were isolated and endogenous strand breaks, oxidised bases and resistance to H2O2-induced damage were measured by the comet assay. The intake of ready-to-eat meals supplemented with cocoa extract did not show relevant changes in the oxidative status of DNA. However, in the cocoa group, oxidised bases negatively correlated with methyl epicatechin-O-sulphate (r = -0.76; P = -0.007) and epicatechin sulphate (r = -0.61; P = -0.046). When volunteers of both groups were analysed together, a marginal decrease (P = 0.072) in oxidised bases was observed, which attributed to weight loss. Subjects who started the intervention with higher levels of damage showed a greater reduction in oxidised bases after 4 weeks (P = 0.040) compared to those who had lower baseline levels. In conclusion, even if 1.4 g of cocoa supplementation for 4 weeks did not show notable changes in terms of antioxidant status of DNA, the energy restriction showed a slightly decrease in oxidised bases and this was seen to a greater extent in subjects who started the intervention with higher levels of damage. On the other hand, the inverse associations found between oxidised bases and some cocoa-derived metabolites suggest that a protective effect might be seen in a longer period of time or in subjects with higher baseline DNA damage.
Autores: Langie, S. A. S., ; Azqueta Oscoz, Amaya; Collins, A. R.,
ISSN 1664-8021  Vol. 6  2015  págs. 266
Autores: Corcuera Martínez, Laura Ana; Vettorazzi Armental, Ariane Renata; Arbillaga Lacunza, Leire; et al.
ISSN 0278-6915  Vol. 76  2015  págs. 116 - 124
Aflatoxin B1 (AFB1) and Ochratoxin A (OTA) are genotoxic mycotoxins that can contaminate a variety of foodstuffs, the liver and the kidney being their target organs, respectively. The micronucleus (MN) assay (bone marrow) and the comet assay (liver and kidney) were performed simultaneously in F344 rats, treated with AFB1 (0.25 mg/kg b.w.), OTA (0.5 mg/kg b.w.) or both mycotoxins. After AFB1 treatment, histopathology and biochemistry analysis showed liver necrosis, focal inflammation and an increase in Alanine Aminotransferase and Aspartate Aminotransferase. OTA alone did not cause any alteration. The acute hepatotmdc effects caused by AFB1 were less pronounced in animals treated with both mycotoxins. With regard to the MN assay, after 24 h, positive results were obtained for AFB1 and negative results were obtained for OTA, although both toxins caused bone marrow toxicity. In the combined treatment, OTA reduced the toxicity and the number of MN produced by AFB1. In the comet assay, after 3 h, positive results were obtained for AFB1 in the liver and for OTA in the kidney. The combined treatment reduced DNA damage in the liver and had no influence in the kidney. Altogether, these results may be indicative of an antagonistic relationship regarding the genotoxicity of both mycotoxins
Autores: Brunborg, G., ; Jackson, P., ; Shaposhnikov, S., ; et al.
ISSN 1664-8021  Vol. 5  2014  págs. 373
The comet assay is a sensitive and versatile method for assessing DNA damage in cells. In the traditional version of the assay, there are many manual steps involved and few samples can be treated in one experiment. High throughput (HT) modifications have been developed during recent years, and they are reviewed and discussed. These modifications include accelerated scoring of comets; other important elements that have been studied and adapted to HT are cultivation and manipulation of cells or tissues before and after exposure, and freezing of treated samples until comet analysis and scoring. HT methods save time and money but they are useful also for other reasons: large-scale experiments may be performed which are otherwise not practicable (e.g., analysis of many organs from exposed animals, and human biomonitoring studies), and automation gives more uniform sample treatment and less dependence on operator performance. The HT modifications now available vary largely in their versatility, capacity, complexity, and costs. The bottleneck for further increase of throughput appears to be the scoring.
Autores: Azqueta Oscoz, Amaya; Slyskova, J., ; Langie, S. A. S., ; et al.
ISSN 1664-8021  Vol. 5  2014  págs. 288
Cellular repair enzymes remove virtually all DNA damage before it is fixed; repair therefore plays a crucial role in preventing cancer. Repair studied at the level of transcription correlates poorly with enzyme activity, and so assays of phenotype are needed. In a biochemical approach, substrate nucleoids containing specific DNA lesions are incubated with cell extract; repair enzymes in the extract induce breaks at damage sites; and the breaks are measured with the comet assay. The nature of the substrate lesions defines the repair pathway to be studied. This in vitro DNA repair assay has been modified for use in animal tissues, specifically to study the effects of aging and nutritional intervention on repair. Recently, the assay was applied to different strains of Drosophila melanogaster proficient and deficient in DNA repair. Most applications of the repair assay have been in human biomonitoring. Individual DNA repair activity may be a marker of cancer susceptibility; alternatively, high repair activity may result from induction of repair enzymes by exposure to DNA-damaging agents. Studies to date have examined effects of environment, nutrition, lifestyle, and occupation, in addition to clinical investigations.
Autores: Collins, A. R., ; El-Yamani, N., ; Lorenzo, Y., ; et al.
ISSN 1664-8021  Vol. 5  2014  págs. 359
Variability of the comet assay is a serious issue, whether it occurs from experiment to experiment in the same laboratory, or between different laboratories analysing identical samples. Do we have to live with high variability, just because the comet assay is a biological assay rather than analytical chemistry? Numerous attempts have been made to limit variability by standardizing the assay protocol, and the critical steps in the assay have been identified; agarose concentration, duration of alkaline incubation, and electrophoresis conditions (time, temperature, and voltage gradient) are particularly important. Even when these are controlled, variation seems to be inevitable. It is helpful to include in experiments reference standards, i.e., cells with a known amount of specific damage to the DNA. They can be aliquots frozen from a single large batch of cells, either untreated (negative controls) or treated with, for example, H2O2 or X-rays to induce strand breaks (positive control for the basic assay), or photosensitiser plus light to oxidize guanine (positive control for Fpg- or OGG1-sensitive sites). Reference standards are especially valuable when performing a series of experiments over a long period-for example, analysing samples of white blood cells from a large human biomonitoring trial-to check that the assay is performing consistently, and to identify anomalous results necessitating a repeat experiment. The reference values of tail intensity can also be used to iron out small variations occurring from day to day. We present examples of the use of reference standards in human trials, both within one laboratory and between different laboratories, and describe procedures that can be used to control variation.
Autores: Stoyanova, E., ; Pastor, S., ; Coll, E., ; et al.
ISSN 0263-6484  Vol. 32  Nº 2  2014  págs. 177 - 182
The aim of this study was to determine if the differences observed in the levels of DNA damage in a group of patients suffering from chronic renal failure are due to differences in the repair capability. DNA damage was initially measured with the comet assay in 106 hemodialysis patients. A selected group of 21 patients representing high (ten patients) and low (11 patients) levels of DNA damage were obtained for determination of base excision repair capacity. This was measured in an in vitro assay where protein extracts from lymphocytes were incubated with a substrate of DNA containing 8-oxoguanine, and the rate of incision was measured with the comet assay. Patients with high levels of genomic damage showed, as an average, significantly lower repair capacity (1273 +/- 184) in comparison with patients with low levels of genomic damage (1813 +/- 113). Nevertheless, the correlation coefficient between repair ability and levels of genomic damage was found to be only close to the significance value (r:-0423, p: 0056). Although DNA damage was clearly related to time on hemodialysis, base excision repair capacity was not. This is one of the few studies providing information on the repair capacity of chronic renal failure patients undergoing hemodialysis. As a summary, our results would indicate that DNA damage levels are in part associated to the repair capacity of the patients, and this repair capacity is not associated with the duration of hemodialysis treatment.
Autores: Godschalk, R.W.L., ; Ersson, C., ; Stepnik, M., ; et al.
ISSN 0267-8357  Vol. 29  Nº 4  2014  págs. 241 - 249
This study investigated the levels of DNA strand breaks and formamidopyrimidine DNA glycosylase (FPG) sensitive sites, as assessed by the comet assay, in peripheral blood mononuclear cells (PBMC) from healthy women from five different countries in Europe. The laboratory in each country (referred to as 'centre') collected and cryopreserved PBMC samples from three donors, using a standardised cell isolation protocol. The samples were analysed in 13 different laboratories for DNA damage, which is measured by the comet assay. The study aim was to assess variation in DNA damage in PBMC samples that were collected in the same way and processed using the same blood isolation procedure. The inter-laboratory variation was the prominent contributor to the overall variation. The inter-laboratory coefficient of variation decreased for both DNA strand breaks (from 68 to 26%) and FPG sensitive sites (from 57 to 12%) by standardisation of the primary comet assay endpoint with calibration curve samples. The level of DNA strand breaks in the samples from two of the centres (0.56-0.61 lesions/10(6) bp) was significantly higher compared with the other three centres (0.41-0.45 lesions/10(6) bp). In contrast, there was no difference between the levels of FPG sensitive sites in PBMC samples from healthy donors in the different centres (0.41-0.52 lesion/10(6) bp).
Autores: Godschalk, R. W., ; Ersson, C., ; Stepnik, M., ; et al.
ISSN 0267-8357  Vol. 29  Nº 4  2014  págs. 241 - 249
This study investigated the levels of DNA strand breaks and formamidopyrimidine DNA glycosylase (FPG) sensitive sites, as assessed by the comet assay, in peripheral blood mononuclear cells (PBMC) from healthy women from five different countries in Europe. The laboratory in each country (referred to as 'centre') collected and cryopreserved PBMC samples from three donors, using a standardised cell isolation protocol. The samples were analysed in 13 different laboratories for DNA damage, which is measured by the comet assay. The study aim was to assess variation in DNA damage in PBMC samples that were collected in the same way and processed using the same blood isolation procedure. The inter-laboratory variation was the prominent contributor to the overall variation. The inter-laboratory coefficient of variation decreased for both DNA strand breaks (from 68 to 26%) and FPG sensitive sites (from 57 to 12%) by standardisation of the primary comet assay endpoint with calibration curve samples. The level of DNA strand breaks in the samples from two of the centres (0.56-0.61 lesions/10(6) bp) was significantly higher compared with the other three centres (0.41-0.45 lesions/10(6) bp). In contrast, there was no difference between the levels of FPG sensitive sites in PBMC samples from healthy donors in the different centres (0.41-0.52 lesion/10(6) bp).
Autores: Forchhammer, L., ; Ersson, E., ; Loft, S., ; et al.
ISSN 0267-8357  Vol. 28  Nº 2  2013  págs. 241
Correction to Forchhammer et al. 27 (6): 665
Autores: Azqueta Oscoz, Amaya; Langie, S. A., ; Slyskova, J., ; et al.
ISSN 1568-7864  Vol. 12  Nº 11  2013  págs. 1007 - 1010
There is an increasing demand for phenotyping assays in the field of human functional genetics. DNA repair activity is representative of this functional approach, being seen as a valuable biomarker related to cancer risk. Repair activity is evaluated by incubating a cell extract with a DNA substrate containing lesions specific for the DNA repair pathway of interest. Enzymic incision at the lesion sites can be measured by means of the comet assay (single cell gel electrophoresis). The assay is particularly applicable for evaluation of base and nucleotide excision repair pathways (BER and NER). Substrate DNA containing oxidised purines gives a measure of BER, while UV-induced photolesions are the substrate for NER. While applications of comet-based DNA repair assays continue to increase, there are no commonly accepted standard protocols, which complicates inter-laboratory comparisons of results. Here we provide a comprehensive summary of protocols for the comet-based BER- and NER-specific in vitro DNA repair assays that can be applied to a wide spectrum of biological material - cultured cell lines, blood cells, animal tissue samples and human biopsies. Our intention is to provide a detailed and user-friendly account of the assays, including practical tips and recommendations to help in setting them up. By proposing standard protocols, we hope to facilitate comparison of results obtained in different laboratories.
Autores: Azqueta Oscoz, Amaya; Costa, S., ; Lorenzo, Y., ; et al.
ISSN 2072-6643  Vol. 5  Nº 4  2013  págs. 1200 - 1217
Aims: Dietary antioxidants, including vitamin C, may be in part responsible for the cancer-preventive effects of fruits and vegetables. Human intervention trials with clinical endpoints have failed to confirm their protective effects, and mechanistic studies have given inconsistent results. Our aim was to investigate antioxidant/pro-oxidant effects of vitamin C at the cellular level. Experimental approach: We have used the comet assay to investigate effects of vitamin C on DNA damage, antioxidant status, and DNA repair, in HeLa (human tumor) cells, and HPLC to measure uptake of vitamin C into cells. Results: Even at concentrations in the medium as high as 200 mu M, vitamin C did not increase the background level of strand breaks or of oxidized purines in nuclear DNA. Vitamin C is taken up by HeLa cells and accumulates to mM levels. Preincubation of cells with vitamin C did not render them resistant to strand breakage induced by H2O2 or to purine oxidation by photosensitizer plus light. Vitamin C had no effect on the rate of repair of strand breaks or oxidized bases by HeLa cells. However, vitamin C at a concentration of less than 1 mu M, or extract from cells preincubated for 6 h with vitamin C, was able to induce damage (strand breaks) in lysed, histone-depleted nuclei (nucleoids). Conclusion: In these cultured human cells, vitamin C displays neither antioxidant nor pro-oxidant properties; nor does it affect DNA strand break or base excision repair.
Autores: Torres Pastor, Enrique; Moreno de Viguri, Elsa; Galiano Ruiz, Silvia; et al.
ISSN 0223-5234  Vol. 66  2013  págs. 324 - 334
As a continuation of our research and with the aim of obtaining new agents against Chagas disease, an extremely neglected disease which threatens 100 million people, eighteen new quinoxaline 1,4-di-N-oxide derivatives have been synthesized following the Beirut reaction. The synthesis of the new derivatives was optimized through the use of a new and more efficient microwave-assisted organic synthetic method. The new derivatives showed excellent in vitro biological activity against Trypanosoma cruzi. Compound 17, which was substituted with fluoro groups at the 6- and 7-positions of the quinoxaline ring, was the most active and selective in the cytotoxicity assay. The electrochemical study showed that the most active compounds, which were substituted by electron-withdrawing groups, possessed a greater ease of reduction of the N-oxide groups.
Autores: Lorenzo, Y., ; Costa, S., ; Collins, A. R., ; et al.
ISSN 0267-8357  Vol. 28  Nº 4  2013  págs. 427 - 432
DNA damage is commonly measured at the level of individual cells using the so-called comet assay (single-cell gel electrophoresis). As the frequency of DNA breaks increases, so does the fraction of the DNA extending towards the anode, forming the comet tail. Comets with almost all DNA in the tail are often referred to as 'hedgehog' comets and are widely assumed to represent apoptotic cells. We review the literature and present theoretical and empirical arguments against this interpretation. The level of DNA damage in these comets is far less than the massive fragmentation that occurs in apoptosis. 'Hedgehog' comets are formed after moderate exposure of cells to, for example, H2O2, but if the cells are incubated for a short period, 'hedgehogs' are no longer seen. We confirm that this is not because DNA has degraded further and been lost from the gel, but because the DNA is repaired. The comet assay may detect the earliest stages of apoptosis, but as it proceeds, comets disappear in a smear of unattached DNA. It is clear that 'hedgehogs' can correspond to one level on a continuum of genotoxic damage, are not diagnostic of apoptosis and should not be regarded as an indicator of cytotoxicity.
Autores: Azqueta Oscoz, Amaya; Gutzkow, K. B., ; Priestley, C. C., ; et al.
ISSN 0887-2333  Vol. 27  Nº 2  2013  págs. 768 - 773
A serious limitation of the conventional comet assay (single cell gel electrophoresis) is the restriction on the number of samples that can be processed in one experiment, imposed by the size of the electrophoresis platform. One approach to increasing throughput is to reduce the size of gels. We here compare the conventional system of two large gels on a microscope slide, with two recent developments, namely 12 minigels per slide, and a format with 96 minigels on GelBond (R) film. We used cells treated with X-rays or methylmethanesulphonate (MMS). The level of damage detected (% tail DNA) in X-irradiated or MMS-treated cells was not affected by the format used. Parallel experiments, using all three formats, were performed with MMS-treated cells in two independent laboratories; the difference in results between the two laboratories was of borderline significance. The potential problem of anomalous comets seen at the border of the gel, the so-called 'edge effects', has been addressed. A reliable, high throughput comet assay has applications in genotoxicity testing (particularly for in vivo studies with samples from different organs) as well as ecogenotoxicology and human biomonitoring, where the numbers of samples collected can be considerable.
Autores: Osnes-Ringen, O., ; Azqueta Oscoz, Amaya; Moe, M. C., ; et al.
ISSN 1755-375X  Vol. 91  Nº 7  2013  págs. 652 - 656
PURPOSE: DNA damage has been described in the human cataractous lens epithelium, and oxidative stress generated by UV radiation and endogenous metabolic processes has been suggested to play a significant role in the pathogenesis of cataract. In this study, the aim was to explore the quality and relative quantity of DNA damage in lens epithelium of cataract patients in vivo and after incubation in a cell culture system. METHODS: Capsulotomy specimens were analysed, before and after 1 week of ex vivo cultivation, using the comet assay to measure DNA strand breaks, oxidized purine and pyrimidine bases and UV-induced cyclobutane pyrimidine dimers. RESULTS: DNA strand breaks were barely detectable, oxidized pyrimidines and pyrimidine dimers were present at low levels, whereas there was a relatively high level of oxidized purines, which further increased after cultivation. CONCLUSION: The observed levels of oxidized purines in cataractous lens epithelium may support a theory consistent with light damage and oxidative stress as mediators of molecular damage to the human lens epithelium. Damage commonly associated with UV-B irradiation was relatively low. The levels of oxidized purines increased further in a commonly used culture system. This is of interest considering the importance and versatility of ex vivo systems in studies exploring the pathogenesis of cataract.
Autores: Azqueta Oscoz, Amaya; Arbillaga Lacunza, Leire; López de Cerain Salsamendi, Adela María; et al.
ISSN 0267-8357  Vol. 28  Nº 3  2013  págs. 271 - 277
The alkaline comet assay, when employed as a genotoxicity test, has relatively low sensitivity because it fails to detectuat non-cytotoxic concentrationsuknown genotoxins that do not induce breaks or alkali-labile sites. We demonstrate that this limitation is overcome by incorporating in the assay the DNA repair enzyme formamidopyrimidine DNA glycosylase (FPG) to convert damaged bases to breaks. We tested 11 chemicals in human TK-6 cells: three non-cytotoxicud-mannitol, Tris and EDTA; two cytotoxicuTriton X-100 and fluometuron; and six genotoxicumethylmethanesulphonate (MMS), methylnitrosourea (MNU), cyclophosphamide, benzo(a)pyrene, 4-nitroquinoline-1-oxide (4NQO) and etoposide. At concentrations of MMS, MNU, benzo(a)pyrene or 4NQO causing little or no cytotoxicity and few if any DNA breaks, FPG substantially enhanced the cellular response. Etoposide increased breaks but not FPG-sensitive sites. Cyclophosphamide, a DNA cross linker, gave a response without FPG at 1 M, but there was no increase with FPG. Triton X-100-induced breaks were secondary to cytotoxicity. The remaining compounds induced no damage. Thus, FPG enhances sensitivity of the comet assay without compromising selectivity.
Autores: Ersson, C., ; Moller, P., ; Forchhammer, L., ; et al.
ISSN 0267-8357  Vol. 28  Nº 3  2013  págs. 279 - 286
The alkaline comet assay is an established, sensitive method extensively used in biomonitoring studies. This method can be modified to measure a range of different types of DNA damage. However, considerable differences in the protocols used by different research groups affect the inter-laboratory comparisons of results. The aim of this study was to assess the inter-laboratory, intra-laboratory, sample and residual (unexplained) variations in DNA strand breaks and formamidopyrimidine DNA glycosylase (FPG)-sensitive sites measured by the comet assay by using a balanced Latin square design. Fourteen participating laboratories used their own comet assay protocols to measure the level of DNA strand breaks and FPG-sensitive sites in coded samples containing peripheral blood mononuclear cells (PBMC) and the level of DNA strand breaks in coded calibration curve samples (cells exposed to different doses of ionising radiation) on three different days of analysis. Eleven laboratories found doseresponse relationships in the coded calibration curve samples on two or three days of analysis, whereas three laboratories had technical problems in their assay. In the coded calibration curve samples, the dose of ionising radiation, inter-laboratory variation, intra-laboratory variation and residual variation contributed to 60.9, 19.4, 0.1 and 19.5%, respectively, of the total variation. In the coded PBMC samples, the inter-laboratory variation explained the largest fraction of the overall variation of DNA strand breaks (79.2%) and the residual variation (19.9%) was much larger than the intra-laboratory (0.3%) and inter-subject (0.5%) variation. The same partitioning of the overall variation of FPG-sensitive sites in the PBMC samples indicated that the inter-laboratory variation was the strongest contributor (56.7%), whereas the residual (42.9%), intra-laboratory (0.2%) and inter-subject (0.3%) variations again contributed less to the overall variation. The results suggest that the variation in DNA damage, measured by comet assay, in PBMC from healthy subjects is assay variation rather than variation between subjects.
Autores: Godschalk, R. W., ; Ersson, C., ; Riso, P., ; et al.
ISSN 1383-5718  Vol. 757  Nº 1  2013  págs. 60 - 67
The measurement of DNA-repair activity by extracts from cells or tissues by means of the single-cell gel electrophoresis (comet) assay has a high potential to become widely used in biomonitoring studies. We assessed the inter-laboratory variation in reported values of DNA-repair activity on substrate cells that had been incubated with Ro19-8022 plus light to generate oxidatively damaged DNA. Eight laboratories assessed the DNA-repair activity of three cell lines (i.e. one epithelial and two fibroblast cell lines), starting with cell pellets or with cell extracts provided by the coordinating laboratory. There was a large inter-laboratory variation, as evidenced by the range in the mean level of repair incisions between the laboratory with the lowest (0.002 incisions/10(6) bp) and highest (0.988 incisions/10(6) bp) incision activity. Nevertheless, six out of eight laboratories reported the same cell line as having the highest level of DNA-repair activity. The two laboratories that reported discordant results (with another cell line having the highest level of DNA-repair activity) were those that reported to have little experience with the modified comet assay to assess DNA repair. The laboratories were also less consistent in ordering the repair activity of the other two cell lines, probably because the DNA-repair activity by extracts from these cell lines were very similar (on average approximately 60-65% of the cell line with the highest repair capacity). A significant correlation was observed between the repair activity found in the provided and the self-made cell extracts (r = 0.71, P<0.001), which indicates that the predominant source for inter-laboratory variation is derived from the incubation of the extract with substrate cells embedded in the gel. Overall, we conclude that the incubation step of cell extracts with the substrate cells can be identified as a major source of inter-laboratory variation in the modified comet assay for base-excision repair.
Autores: Haug, K., ; Azqueta Oscoz, Amaya; Johnsen-Soriano, S., ; et al.
ISSN 1755-375X  Vol. 91  Nº 3  2013  págs. 219 - 225
PURPOSE: Storage time for donor corneas in Optisol GS is limited compared to Eye Bank Organ Culture (EBOC). We here examine the epithelium on donor corneoscleral rims after primary storage in Optisol GS and subsequent incubation in EBOC. METHODS: Morphology was monitored by light and electron microscopy, expression of phenotypic and genotypic markers by immunohistochemistry and RT-PCR and changes in oxidative lipid and DNA damage by ELISA and COMET assay. RESULTS: A prominent loss of cells was observed after storage in Optisol GS. After maintenance in EBOC, spreading apical cells were Occludin(+) , while the staining for E-cadherin and Connexin-43 was less intense. There were an upregulation of Occludin and a downregulation of E-cadherin and Connexin-43. Eye Bank Organ Culture was associated with an ongoing proliferative activity and a downregulation of putative progenitor/stem cell marker ABCG2 and p63. Staining for 8-OHdG and Caspase-3 did not increase, while levels of malondialdehyde and number of DNA strand breaks and oxidized bases increased. CONCLUSIONS: This dual procedure should be pursued as an option to increase the storage time and the pool of available donor corneas. The observed downregulation of markers associated with stemness during EBOC is relevant considering the potential use of donor epithelium in the treatment of ocular surface disorders.
Autores: Collins, A. R., ; Azqueta Oscoz, Amaya; Langie, S. A. S.,
ISSN 1436-6207  Vol. 51  Nº 3  2012  págs. 261 - 279
DNA repair is an essential cellular function, which, by removing DNA damage before it can cause mutations, contributes crucially to the prevention of cancer. Interest in the influence of micronutrients on DNA repair activity is prompted by the possibility that the protective effects of fruits and vegetables might thus be explained. Two approaches to measuring repair-monitoring cellular removal of DNA damage and incubating cell extract with specifically damaged DNA in an in vitro assay-have been applied in cell culture, whole animal studies, and human trials. In addition, there are numerous investigations at the level of expression of DNA repair-related genes. Depending on the pathway studied and the phytochemical or food tested, there are varied reports of stimulation, inhibition or no effect on DNA repair. The clearest findings are from human supplementation trials in which lymphocytes are assessed for their repair capacity ex vivo. Studying cellular repair of strand breaks is complicated by the fact that lymphocytes appear to repair them very slowly. Applying the in vitro repair assay to human lymphocytes has revealed stimulatory effects on repair of oxidised bases by various micronutrients or a fruit- and vegetable-rich diet, while other studies have failed to demonstrate effects. Despite varied results from different studies, it seems clear that micronutrients can influence DNA repair, usually but not always enhancing activity. Different modes of DNA repair are likely to be subject to different regulatory mechanisms. Measures of gene expression tend to be a poor guide to repair activity, and there is no substitute for phenotypic assays.
Autores: Collins, A. R., ; Azqueta Oscoz, Amaya
ISSN 0027-5107  Vol. 736  Nº 1 - 2  2012  págs. 122 - 129
DNA repair plays a major role in maintaining genetic stability, and so measurement of individual DNA repair capacity should be a valued tool in molecular epidemiology studies. The comet assay (single cell gel electrophoresis), in different versions, is commonly used to measure the repair pathways represented by strand break rejoining, removal of 8-oxoguanine, and repair of bulky adducts or UV-induced damage. Repair enzyme activity generally does not reflect the level of gene expression; but there is evidence albeit piecemeal - that it is affected by polymorphisms in repair genes. There are mixed reports concerning the regulation of repair by environmental factors; several nutritional supplementation trials with phytochemical-rich foods have demonstrated increases in base excision repair of oxidation damage, while others have shown no effect. Exposure to genotoxic agents has in general not been found to stimulate repair. Crucial questions concerning the factors regulating repair and the causes of individual variation are as yet unanswered.
Autores: Azqueta Oscoz, Amaya; Collins, A. R.,
ISSN 0027-5107  Vol. 733  Nº 1 - 2  2012  págs. 4 - 13
Carotenoids are among the best known antioxidant phytochemicals, and are widely believed to contribute to the health-promoting properties of fruits and vegetables. Investigations of the effects of carotenoids have been carried out at different levels: in cultured cells, in experimental animals, and in humans. Studying reports from the last 5 years, we find a clear distinction between effects of vitamin A and provitamin A carotenoids (the carotenes and beta-cryptoxanthin), and effects of non-vitamin A carotenoids (lycopene, lutein, astaxanthin and zeaxanthin). Whereas the latter group are almost invariably reported to protect against DNA damage, whether endogenous or induced by exogenous agents, the provitamin A carotenoids show a more varied spectrum of effects. sometimes protecting and sometimes enhancing DNA damage. The tendency to exacerbate damage is seen mainly at high concentrations, and might be accounted for by pro-oxidant actions of these carotenoids.
Autores: Forchhammer, L., ; Ersson, C., ; Loft, S., ; et al.
ISSN 0267-8357  Vol. 27  Nº 6  2012  págs. 665 - 672
There are substantial inter-laboratory variations in the levels of DNA damage measured by the comet assay. The aim of this study was to investigate whether adherence to a standard comet assay protocol would reduce inter-laboratory variation in reported values of DNA damage. Fourteen laboratories determined the baseline level of DNA strand breaks (SBs)/alkaline labile sites and formamidopyrimidine DNA glycosylase (FPG)-sensitive sites in coded samples of mononuclear blood cells (MNBCs) from healthy volunteers. There were technical problems in seven laboratories in adopting the standard protocol, which were not related to the level of experience. Therefore, the inter-laboratory variation in DNA damage was only analysed using the results from laboratories that had obtained complete data with the standard comet assay protocol. This analysis showed that the differences between reported levels of DNA SBs/alkaline labile sites in MNBCs were not reduced by applying the standard assay protocol as compared with the laboratory's own protocol. There was large inter-laboratory variation in FPG-sensitive sites by the laboratory-specific protocol and the variation was reduced when the samples were analysed by the standard protocol. The SBs and FPG-sensitive sites were measured in the same experiment, indicating that the large spread in the latter lesions was the main reason for the reduced inter-laboratory variation. However, it remains worrying that half of the participating laboratories obtained poor results using the standard procedure. This study indicates that future comet assay validation trials should take steps to evaluate the implementation of standard procedures in participating laboratories.
Autores: Azqueta Oscoz, Amaya; Meier, S., ; Priestley, C. C., ; et al.
ISSN 0267-8357  Vol. 26  Nº 3  2011  págs. 393 - 399
As part of a project to develop high throughput versions of the comet assay (single cell gel electrophoresis), with a consequent need for more efficient scoring, we have compared the performance of visual scoring, automated and semi-automated image analysis when assessing comets in the same set of gels from dose-response experiments with typical DNA-damaging agents. Human lymphoblastoid TK-6 cells were treated with concentrations of methylmethanesulphonate between 0.04 and 0.6 mM, and peripheral human lymphocytes were incubated, after embedding in agarose, with H(2)O(2) concentrations from 2.5 to 160 mu M. All three scoring methods proved capable of detecting a significant level of damage at the lowest concentration of each agent. Visual scoring systematically overestimates low levels of damage compared with computerised image analysis; on the other hand, heavily damaged comets are less efficiently detected with image analysis. Overall, the degree of agreement between the scoring methods is within acceptable limits according to a Bland-Altman analysis.
Autores: Brevik, A., ; Karlsen, A., ; Azqueta Oscoz, Amaya; et al.
ISSN 0263-6484  Vol. 29  Nº 1  2011  págs. 36 - 42
Lack of reliable assays for DNA repair has largely prevented measurements of DNA repair from being included in human biomonitoring studies. Using newly developed modifications of the comet assay we tested whether a fruit- and antioxidant-rich plant-based intervention could affect base excision repair (BER) and nucleotide excision repair (NER) in a group of 102 male volunteers. BER and NER repair capacities were measured in lymphocytes before and after a dietary intervention lasting 8 weeks. The study had one control group, one group consuming three kiwifruits per day and one group consuming a variety of antioxidant-rich fruits and plant products in addition to their normal diet. DNA strand breaks were reduced following consumption of both kiwifruits (13%, p=0.05) and antioxidant-rich plant products (20%, p=0.02). Increased BER (55%, p=0.01) and reduced NER (-39%, p<0.01) were observed in the group consuming a wide variety of plant products. Reduced NER was also observed in the kiwifruit group (-38%, p=0.05), but BER was not affected in this group. Here we have demonstrated that DNA repair is affected by diet and that modified versions of the comet assay can be used to assess activity of different DNA repair pathways in human biomonitoring studies.
Autores: Piasek, A., ; Kusznierewicz, B., ; Grzybowska, I., ; et al.
ISSN 0889-1575  Vol. 24  Nº 6  2011  págs. 880 - 888
In this study, fruit juices that are rich sources of anthocyanins, obtained from aronia (Am. chokeberry, Aronia melanocarpa) and blue-berried honeysuckle (Lonicera caerulea L. var. edulis) were used to examine the preservation of plant phytochemicals and bioactivity upon sterilization either thermal, or with an EnbioJet (R) Microwave Flow Pasteurizer. The chemical properties verified included determinations of anthocyanins and other polyphenols by HPLC, total antioxidant activity, and profiles of antioxidants by post-column derivatization. Compared to heat treatment, the higher stability of aronia and blue-berried honeysuckle phytocomplexes during processing with the EnbioJet (R) device for temperatures ensuring microbial purity (range investigated 90-130 degrees C) was demonstrated. In the same batches of juices submitted to heating at 100 degrees C, the rapid decline of anthocyanin content accompanied by lowered antioxidant activity was observed. The changes in chemical composition were reflected in altered biological activity. Both cytotoxicity and protection of DNA against oxidative damage were higher for microwaved juices than for juices processed by the heating method that caused degradation of bioactive phytochemicals.
Autores: Corcuera Martínez, Laura Ana; Arbillaga Lacunza, Leire; Vettorazzi Armental, Ariane Renata; et al.
ISSN 0278-6915  Vol. 49  Nº 11  2011  págs. 2883 - 2889
Mycotoxins aflatoxin B1 (AFB1) and ochratoxin A (OTA) can be present together in food commodities. These food contaminants are considered to be genotoxins, acting by different mechanisms. The aim of this work was to characterize combined genotoxic in vitro effects of both mycotoxins in Hep G2 cells. For this purpose, cytotoxicity was first determined in isolated and combined treatments in order to determine the dose range of genotoxicity studies. Co-exposure of cells to OTA + AFB1 for 24 h resulted in additive effects. Genotoxicity was determined in Hep G2 cells by the modified comet assay with restriction enzymes (endo ill and FPG). Significant reactive oxygen species formation was detected in both single and combined treatments. AFB1 was genotoxic after 3 h with external metabolic activation (S9 mix) and after 24 h without metabolic activation. Co-exposure to OTA significantly decreased DNA damage induced by AFB1, not only in breaks and apurinic sites but also in FPG-sensitive sites. The apparent contradiction between additive cytotoxic effects and antagonic genotoxic effects may be explained if AFB1 and OTA compete for the same CYPs, yielding more ROS but less AFB1 adducts.
Autores: Azqueta Oscoz, Amaya; Gutzkow, K. B., ; Brunborg, G., ; et al.
ISSN 1383-5718  Vol. 724  Nº 1 - 2  2011  págs. 41 - 45
The comet assay is now the method of choice for measuring most kinds of DNA damage in cells. However, due to the lack of a standardised protocol inter-laboratory comparisons are of limited value. The aim of this paper is to demonstrate how small changes in comet-assay variables may significantly affect the results. We examined the effect of varying agarose concentrations, alkaline unwinding time, electrophoresis time, voltage and current, by use of two cell types, viz, human peripheral blood lymphocytes and the lymphoblastoid cell line TK-6. All these variables have marked effects on assay performance and, therefore, on the determination of DNA damage. Here we identify factors of particular importance.
Autores: Azqueta Oscoz, Amaya; Bentzen, T. G., ; Collins, A. R., ; et al.
ISSN 0801-6828  Vol. 5  2010  págs. 6 - 11
Autores: Shaposhnikov, S., ; Azqueta Oscoz, Amaya; Henriksson, S., ; et al.
ISSN 0378-4274  Vol. 195  Nº 1  2010  págs. 31 - 34
The comet assay is widely used to measure DNA damage and repair in basic research, genotoxicity testing and human biomonitoring. The conventional format has 1 or 2 gels on a microscope slide, 1 sample per slide. To increase throughput, we have designed and tested a system with 12 smaller gels on one slide, allowing incubation of individual gels with different reagents or enzymes. Thus several times more samples can be analysed with one electrophoresis run, and fewer cells and smaller volumes of test solutions are required. Applications of the modified method include treatment with genotoxic agents at different concentrations; simultaneous analysis of different lesions using a range of enzymes; analysis of cell extracts for DNA repair activity; and fluorescent in situ hybridisation (FISH) to comet DNA with specific labelled probes.
Autores: Johansson, C., ; Moller, P., ; Forchhammer, L., ; et al.
ISSN 0267-8357  Vol. 25  Nº 2  2010  págs. 125 - 132
The increasing use of single cell gel electrophoresis (the comet assay) highlights its popularity as a method for detecting DNA damage, including the use of enzymes for assessment of oxidatively damaged DNA. However, comparison of DNA damage levels between laboratories can be difficult due to differences in assay protocols (e.g. lysis conditions, enzyme treatment, the duration of the alkaline treatment and electrophoresis) and in the end points used for reporting results (e.g. %DNA in tail, arbitrary units, tail moment and tail length). One way to facilitate comparisons is to convert primary comet assay end points to number of lesions/10(6) bp by calibration with ionizing radiation. The aim of this study was to investigate the inter-laboratory variation in assessment of oxidatively damaged DNA by the comet assay in terms of oxidized purines converted to strand breaks with formamidopyrimidine DNA glycosylase (FPG). Coded samples with DNA oxidation damage induced by treatment with different concentrations of photosensitizer (Ro 19-8022) plus light and calibration samples irradiated with ionizing radiation were distributed to the 10 participating laboratories to measure DNA damage using their own comet assay protocols. Nine of 10 laboratories reported the same ranking of the level of damage in the coded samples. The variation in assessment of oxidatively damaged DNA was largely due to differences in protocols. After conversion of the data to lesions/10(6) bp using laboratory-specific calibration curves, the variation between the laboratories was reduced. The contribution of the concentration of photosensitizer to the variation in net FPG-sensitive sites increased from 49 to 73%, whereas the inter-laboratory variation decreased. The participating laboratories were successful in finding a dose-response of oxidatively damaged DNA in coded samples, but there remains a need to standardize the protocols to enable direct comparisons between laboratories.
Autores: Ramos, A. A., ; Azqueta Oscoz, Amaya; Pereira-Wilson, C., ; et al.
ISSN 0021-8561  Vol. 58  Nº 12  2010  págs. 7465 - 7471
DNA damage can lead to carcinogenesis if replication proceeds without proper repair. This study evaluated the effects of the water extracts of three Salvia sp., Salvia officinalis (SO), Salvia fruticosa (SF), and Salvia lavandulifolia (SL), and of the major phenolic constituents, rosmarinic acid (RA) and luteolin-7-glucoside (L-7-G), on DNA protection in Caco-2 and HeLa cells exposed to oxidative agents and on DNA repair in Caco-2 cells. The comet assay was used to measure DNA damage and repair capacity. The final concentration of each sage extract was 50 mu g/mL, and concentrations of RA and L-7-G were 50 and 20 mu M, respectively. After a short incubation (2 h), L-7-G protected DNA in Caco-2 cells from damage induced by H(2)O(2) (75 mu M); also, after a long incubation (24 h), SF, RA, and L-7-G had protective effects in Caco-2 cells. In HeLa cells, SO, SF, and RA protected against damage induced by H(2)O(2) after 24 h of incubation. Assays of DNA repair show that SO, SF, and L-7-G increased the rate of DNA repair (rejoining of strand breaks) in Caco-2 cells treated with H(2)O(2). The incision activity of a Caco-2 cell extract on a DNA substrate containing specific damage (8-oxoGua) was also measured to evaluate effects on base excision repair (BER) activity. Preincubation for 24 h with SO and L-7-G had a BER inductive effect, increasing incision activity in Caco-2 cells. In conclusion, SO, SF, and the isolated compounds (RA and L-7-G) demonstrated chemopreventive activity by protecting cells against oxidative DNA damage and stimulating DNA repair (SO, SF, and L-7-G).
Autores: Forchhammer, L., ; Johansson, C., ; Loft, S., ; et al.
ISSN 0267-8357  Vol. 25  Nº 2  2010  págs. 113 - 123
The comet assay has become a popular method for the assessment of DNA damage in biomonitoring studies and genetic toxicology. However, few studies have addressed the issue of the noted inter-laboratory variability of DNA damage measured by the comet assay. In this study, 12 laboratories analysed the level of DNA damage in monocyte-derived THP-1 cells by either visual classification or computer-aided image analysis of pre-made slides, coded cryopreserved samples of cells and reference standard cells (calibration curve samples). The reference standard samples were irradiated with ionizing radiation (0-10 Gy) and used to construct a calibration curve to calculate the number of lesions per 10(6) base pair. All laboratories detected dose-response relationships in the coded samples irradiated with ionizing radiation (1.5-7 Gy), but there were overt differences in the level of DNA damage reported by the different laboratories as evidenced by an inter-laboratory coefficient of variation (CV) of 47%. Adjustment of the primary comet assay end points by a calibration curve prepared in each laboratory reduced the CV to 28%, a statistically significant reduction (P < 0.05, Levene's test). A large fraction of the inter-laboratory variation originated from differences in image analysis, whereas the intra-laboratory variation was considerably smaller than the variation between laboratories. In summary, adjustment of primary comet assay results by reference standards reduces inter-laboratory variation in the level of DNA damage measured by the alkaline version of the comet assay.
Autores: Irache Garreta, Juan Manuel; Martín Arbella, Nekane; Ojer Ojer, Patricia; et al.
Libro:  Polymer nanoparticles for nanomedicines: a guide for their design, preparation and development
2016  págs. 521 - 550
Autores: Azqueta Oscoz, Amaya; Campos Costa-Amaral, I., ; Collins, A. R.,
Libro:  The comet assay in toxicology
2016  págs. 67 - 92
Autores: Azqueta Oscoz, Amaya; Arbillaga Lacunza, Leire; López de Cerain Salsamendi, Adela María
Libro:  Biointeractions of nanomaterials
2015  págs. 353 - 363
Autores: Azqueta Oscoz, Amaya; Collins, A. R.,
Libro:  Genotoxicity and DNA repair: a practical approach
2014  págs. 199 - 217
The alkaline comet assay in its standard form is well established as a genotoxicity testing assay, widely used in screening novel chemicals and pharmaceuticals for potentially carcinogenic effects. Incorporation of a digestion of DNA with lesion-specific enzymes is an accepted modification which has allowed, for example, the quantitative assessment of levels of 8-oxoguanine in DNA as a measure of oxidative damage, using the enzyme formamidopyrimidine DNA glycosylase (FPG). However, FPG is not restricted to the measurement of oxidized bases, and we describe here its use in a wider context to detect various kinds of DNA damage. A limitation of the standard assay is the relatively low number of samples that can be run in one experiment (restricted by the number of microscope slides fitting in the electrophoresis tank). Recent developments of high throughput versions of the comet assay have alleviated this problem, and we describe a modification based on the use of 12 minigels on each slide. We provide a detailed protocol for running 12 minigels per slide, with the inclusion of FPG to obtain enhanced sensitivity. We emphasize the conditions of the comet assay that are most critical for reproducibility and accuracy.
Autores: Slyskova, J., ; Langie, S. A. S., ; Gaivao, I., ; et al.
Libro:  Genotoxicity and DNA repair: a practical approach
2014  págs. 377 - 395
DNA repair is regarded as an important biomarker to be measured alongside DNA damage when considering the risk of cancer from environmental or genetic causes. Efficient repair deals with DNA lesions before they can disrupt replication and create mutations. Repair capacity can be readily assessed using an in vitro comet-based DNA repair assay, which is particularly useful in human biomonitoring studies where many samples are collected over an extended period, stored frozen, and analyzed at a later date. In this assay, a protein lysate is extracted from studied cells or tissues and is incubated with damage-containing substrate DNA. Repair proteins in extract are able to recognize and incise DNA lesions and cumulate DNA breaks, which are quantified with the comet assay. Here we provide detailed protocols for the in vitro estimation of base excision repair (on a substrate containing 8-oxoguanine induced by visible light in the presence of a photosensitizer) and nucleotide excision repair (with UV-induced pyrimidine dimers and 6-4 photoproducts as substrate). We describe the preparation of extracts from different kinds of source material (cultured cells, peripheral blood mononuclear cells, animal tissues, human biopsies) and emphasize the need for careful control of the extract concentration. Furthermore, we discuss not only conventional comet assay format (2 gels on microscope slide), but also a medium-throughput version (12 minigels in microscope slide), which is recommended for reduction of experimental variability.
Autores: Collins, A. R., ; Azqueta Oscoz, Amaya
Libro:  Genotoxicity and DNA repair: a practical approach
2014  págs. 365 - 376
DNA repair pathways provide a critically important cellular defence system, effectively protecting us from mutations and cancer. Distinct pathways deal with various classes of damage: single- and double-strand breaks, oxidized and alkylated bases, bulky adducts, intra- and inter-strand cross-links. A simple approach to measuring the DNA repair capacity of cell lines, or samples of blood cells, for instance, is the cellular repair assay, or challenge assay, in which cells are treated with a specific DNA-damaging agent, and incubated; at intervals, samples are taken and the residual damage is measured. The comet assay is well suited for measuring strand break rejoining, excision repair of oxidized or alkylated bases (with a lesion-specific endonuclease to convert the altered bases into breaks), and nucleotide excision repair of UV-induced lesions (again, using an appropriate enzyme to detect the damage). An alternative way to assess nucleotide excision repair capability is the incision assay: after UV irradiation, cells are incubated with inhibitors of repair DNA synthesis, so that incomplete repair sites accumulate as DNA breaks. We provide protocols for these DNA repair assays, and discuss their applications¿and their limitations. We also raise some important, so far unanswered questions concerning the regulation of repair, and the factors that might account for the wide variations seen in individual repair capacities.
Autores: Collins, A. R., ; Azqueta Oscoz, Amaya
Libro:  Laboratory Methods in cell Biology: biochemistry and cell culture
Vol. 112  2012  págs. 69 - 92
The comet assay (single-cell gel electrophoresis) is a simple, sensitive method for measuring DNA strand breaks, widely used in genotoxicity testing, human biomonitoring, ecogenotoxicology and fundamental research into mechanisms of DNA damage and repair. Cells embedded in agarose on a glass slide are lysed, leaving supercoiled DNA loops attached to the nuclear matrix as "nucleoids". Electophoresis attracts DNA to the anode, but only those loops with breaks migrate, forming a comet-like image on fluorescence microscopy. The relative intensity of the comet tail reflects the frequency of DNA breaks, with a detection range up to a few thousand breaks per cell. DNA breaks, being produced by many diverse agents and as intermediates in DNA repair, are an unspecific marker of damage. More detailed information is obtained by incorporating a digestion with a lesion-specific endonuclease, after the lysis step. Here, we concentrate on the detection of strand breaks and oxidized bases in human peripheral blood mononuclear cells or cultured mammalian cells. We cover preparation of cells, precoating of slides, embedding the cells in agarose, lysis, enzyme digestion, alkaline electrophoresis, fixation and staining, and scoring of comets both visually and with computerized image analysis.
Autores: Azqueta Oscoz, Amaya; Collins, A. R.,
Libro:  Encyclopedia of analytical chemistry: applications, theory and instrumentation
2011  págs. 1 - 19
The comet assay (single-cell gel electrophoresis) is a simple method for measuring DNA strand breaks in cells that are embedded in agarose and lysed to remove membranes and soluble cell constituents (including most histones). The assay depends on the ability of breaks to relax supercoiling, which allows DNA still attached to a nuclear matrix to move toward the anode under electrophoresis, forming a `comet tail¿ when viewed by fluorescence microscopy. The percentage of DNA in the tail indicates the frequency of DNA breaks. An additional step ¿ digestion with lesion-specific endonucleases ¿ is introduced after lysis in order to detect different kinds of DNA damage. The assay can be calibrated to give quantitative measures of DNA damage. It has been widely used in genotoxicity testing and human biomonitoring, and also in ecogenotoxicology, as well as in basic research. We here discuss the development of the method, its principles, applications, and limitations, and attempt to dispel some fallacies that trouble the assay. We provide a detailed protocol, including a recent modification that increases the assay's throughput.
Autores: Azqueta Oscoz, Amaya; Shaposhnikov, S., ; Collins, A. R.,
Libro:  DNA repair
2011  págs. 615 - 636
Autores: López de Cerain Salsamendi, Adela María; Azqueta Oscoz, Amaya; Vettorazzi Armental, Ariane Renata
Libro:  Olives and olive oil in health and disease prevention
2010  págs. 989 - 996
Autores: López de Cerain Salsamendi, Adela María; Azqueta Oscoz, Amaya; Vettorazzi Armental, Ariane Renata
Libro:  Olives and olive oil in health and disease prevention
2010  págs. 989 - 996
Meat composition and temperature are the most important factors that determine the generation of mutagens in fried meat. At moderate temperatures (below 200°C), a high content of proteins and creatine is necessary for generating, by Maillard reaction, the heterocyclic amines (HCAs) that are responsible for mutagenic activity. For this reason, the browner the external crust of fried meat, the more potent the mutagenic activity. The presence of fat or oil has two opposite effects: it can facilitate heat transfer and thus increase the generation of mutagens or it may act as a dilutant for the mutagenic compounds. Deep-frying of meat in vegetable oils for long times (approximately 10 min) gives rise to a significant quantity of mutagens; olive oil yields slightly more mutagens than sunflower oil does, probably because the smoking temperature for olive oil is higher (180°C versus 170°C). Very long frying times (20¿30 min) significantly increase the mutagenic activity in meat fried in sunflower oil, probably due to the degradation of polyunsaturated fatty acids (PUFA). Thus, a general recommendation for reducing the presence of mutagens in fried meat would be to cook during short times and at moderate temperatures (below 170 °C).