Maria Dusinska

The comet assay: what can it really tell us?

A range of applications of the alkaline comet assay is covered, from investigations of the physicochemical behaviour of DNA, through studies of cellular responses to DNA damage, to biomonitoring of human populations. The underlying principles of this assay are discussed, and new evidence presented which supports the concept of relaxation of supercoiled loops, rather than alkaline unwinding, as the primary reason for comet tail formation. DNA-damaging agents that do not induce strand breaks directly can be detected when cellular repair processes convert lesions to transient strand breaks; an approach is outlined here which maximises this effect and thus widens the scope of the assay. Purified repair enzymes, applied to DNA during the course of the comet assay procedure, greatly increase the sensitivity and specificity of the assay; recent developments with formamidopyrimidine glycosylase (recognising 8-OH-gua and other damaged purines) and uvrABC (for bulky lesions) are presented. The kinetics of cellular repair after low doses of oxidative damage have been followed with this modified comet assay. Finally, the successful measurement of biomarkers of oxidative damage in human populations establishes the comet assay as a valuable tool in molecular epidemiology.

Comet assay in human biomonitoring studies: Reliability, validation, and applications

The comet assay (single‐cell gel electrophoresis), which measures DNA strand breaks at the level of single cells, is very easily applied to human lymphocytes, and therefore lends itself to human biomonitoring studies. For the examination of DNA base oxidation (a specific marker of oxidative damage), the assay is modified by including a stage at which the DNA is incubated with a suitable lesion‐specific endonuclease. Here we report on the reliability and reproducibility of this approach, from the level of comparing results from duplicate gels prepared from the same sample of cells, up to an assessment of the natural intra‐ and interindividual variability in lymphocyte DNA damage measured in groups of normal, healthy human volunteers. We applied the assay in investigations of human disease and occupational exposure of factory workers. Environ. Mol. Mutagen. 30:139–146, 1997.

Nanomaterials for environmental studies: Classification, reference material issues, and strategies for physico-chemical characterisation

NanoImpactNet is a European Commission Framework Programme 7 (FP7) funded project that provides a forum for the discussion of current opinions on nanomaterials in relation to human and environmental issues. In September 2008, in Zurich, a NanoImpactNet environmental workshop focused on three key questions: 1. What properties should be characterised for nanomaterials used in environmental and ecotoxicology studies? 2. What reference materials should be developed for use in environmental and ecotoxicological studies? 3. Is it possible to group different nanomaterials into categories for consideration in environmental studies? Such questions have been, at least partially, addressed by other projects/workshops especially in relation to human health effects. Such projects provide a useful basis on which this workshop was based, but in this particular case these questions were reformulated in order to focus specifically on environmental studies. The workshop participants, through a series of discussion and reflection sessions, generated the conclusions listed below. The physicochemical characterisation information identified as important for environmental studies included measures of aggregation/agglomeration/dispersability, size, dissolution (solubility), surface area, surface charge, surface chemistry/composition, with the assumption that chemical composition would already be known. There is a need to have test materials for ecotoxicology, and several substances are potentially useful, including TiO(2) nanoparticles, polystyrene beads labelled with fluorescent dyes, and silver nanoparticles. Some of these test materials could then be developed into certified reference materials over time. No clear consensus was reached regarding the classification of nanomaterials into categories to aid environmental studies, except that a chemistry-based classification system was a reasonable starting point, with some modifications. It was suggested, that additional work may be required to derive criteria that can be used to generate such categories, that would also include aspects of the material structure and physical behaviour.

Mechanisms of genotoxicity. A review of in vitro and in vivo studies with engineered nanoparticles

Abstract Engineered nanoparticles (NPs) are widely used in different technologies but their unique properties might also cause adverse health effects. In reviewing recent in vitro and in vivo genotoxicity studies we discuss potential mechanisms of genotoxicity induced by NPs. Various factors that may influence genotoxic response, including physico-chemical properties and experimental conditions, are highlighted. From 4346 articles on NP toxicity, 112 describe genotoxicity studies (94 in vitro, 22 in vivo). The most used assays are the comet assay (58 in vitro, 9 in vivo), the micronucleus assay (31 in vitro, 14 in vivo), the chromosome aberrations test (10 in vitro, 1 in vivo) and the bacterial reverse mutation assay (13 studies). We describe advantages and potential problems with different methods and suggest the need for appropriate methodologies to be used for investigation of genotoxic effects of NPs, in vitro and in vivo.

The comet assay in human biomonitoring: gene-environment interactions.

The comet assay is the method of choice for measuring DNA damage, of various sorts, in human cells such as lymphocytes obtained in the course of population-based studies of environmental and occupational exposure to different genotoxic agents, including radiation, chemicals and oxidative stress. It is noted for its versatility and the breadth of its possible applications. In terms of simplicity, cost, small amount of material required, sensitivity and reliability, the comet assay in its various modifications has few serious competitors. When standardized and validated, the comet assay can provide invaluable information in the areas of hazard identification and risk assessment of environmental and occupational exposure, diseases linked with oxidative stress (e.g. diabetes and cardiovascular disease), nutrition, monitoring the effectiveness of medical treatment and investigating individual variation in response to DNA damage that may reflect genetic or environmental influences. The information obtained could lead to individual advice on lifestyle changes to promote health and especially on relative risks of genotoxic exposure to environmental pollution.

DNA DAMAGE IN DIABETES : CORRELATION WITH A CLINICAL MARKER

Levels of DNA damage in groups of 10 patients with insulin-dependent diabetes mellitus and 10 matched controls were compared using the comet assay; DNA strand breaks, oxidized pyrimidines (endonuclease III-sensitive sites) and altered purines (sites sensitive to formamidopyrimidine glycosylase) were measured. Mean values of strand breaks and oxidized pyrimidines were significantly higher in diabetics. Strand breaks correlated with body mass index in the diabetic group. A strong correlation was seen between formamidopyrimidine glycosylase-sensitive sites and serum glucose concentrations. When three patients with normal glucose levels were excluded from the statistical analysis, the mean value of formamidopyrimidine glycosylase-sensitive sites was very significantly elevated compared with normal. DNA damage in lymphocytes is thus a useful marker of oxidative stress, and in particular formamidopyrimidine glycosylase-sensitive sites seem to represent changes specifically related to hyperglycemia.

The importance of life cycle concepts for the development of safe nanoproducts

Whilst the global players in industry are rapidly moving forward to take advantage of the new opportunities and prospects offered by nanotechnologies, it is imperative that such developments take place in a safe and sustainable manner. The increasing use of engineered nanomaterials (ENMs) in consumer products has raised certain concerns over their safety to human health and the environment. There are currently a number of major uncertainties and knowledge gaps in regard to behavior, chemical and biological interactions and toxicological properties of ENMs. As dealing with these uncertainties will require the generation of new basic knowledge, it is unlikely that they will be resolved in the immediate future. One has to consider the whole life cycle of nanoproducts to ensure that possible impacts can be systematically discovered. For example, life cycle assessment (LCA) - a formalized life cycle concept - may be used to assess the relative environmental sustainability performance of nanoproducts in comparison with their conventional equivalents. Other less formalized life cycle concepts in the framework of prospective technology assessment may uncover further detailed and prospective knowledge for human and environmental exposure to ENMs during the life cycle of nanoproducts. They systematically reveal impacts such as cross product contamination or dissipation of scarce materials among others. The combination of different life cycle concepts with the evolving knowledge from toxicology and risk assessment can mitigate uncertainties and can provide an early basis for informed decision making by the industry and regulators.

Practical considerations for conducting ecotoxicity test methods with manufactured nanomaterials: what have we learnt so far?

This review paper reports the consensus of a technical workshop hosted by the European network, NanoImpactNet (NIN). The workshop aimed to review the collective experience of working at the bench with manufactured nanomaterials (MNMs), and to recommend modifications to existing experimental methods and OECD protocols. Current procedures for cleaning glassware are appropriate for most MNMs, although interference with electrodes may occur. Maintaining exposure is more difficult with MNMs compared to conventional chemicals. A metal salt control is recommended for experiments with metallic MNMs that may release free metal ions. Dispersing agents should be avoided, but if they must be used, then natural or synthetic dispersing agents are possible, and dispersion controls essential. Time constraints and technology gaps indicate that full characterisation of test media during ecotoxicity tests is currently not practical. Details of electron microscopy, dark-field microscopy, a range of spectroscopic methods (EDX, XRD, XANES, EXAFS), light scattering techniques (DLS, SLS) and chromatography are discussed. The development of user-friendly software to predict particle behaviour in test media according to DLVO theory is in progress, and simple optical methods are available to estimate the settling behaviour of suspensions during experiments. However, for soil matrices such simple approaches may not be applicable. Alternatively, a Critical Body Residue approach may be taken in which body concentrations in organisms are related to effects, and toxicity thresholds derived. For microbial assays, the cell wall is a formidable barrier to MNMs and end points that rely on the test substance penetrating the cell may be insensitive. Instead assays based on the cell envelope should be developed for MNMs. In algal growth tests, the abiotic factors that promote particle aggregation in the media (e.g. ionic strength) are also important in providing nutrients, and manipulation of the media to control the dispersion may also inhibit growth. Controls to quantify shading effects, and precise details of lighting regimes, shaking or mixing should be reported in algal tests. Photosynthesis may be more sensitive than traditional growth end points for algae and plants. Tests with invertebrates should consider non-chemical toxicity from particle adherence to the organisms. The use of semi-static exposure methods with fish can reduce the logistical issues of waste water disposal and facilitate aspects of animal husbandry relevant to MMNs. There are concerns that the existing bioaccumulation tests are conceptually flawed for MNMs and that new test(s) are required. In vitro testing strategies, as exemplified by genotoxicity assays, can be modified for MNMs, but the risk of false negatives in some assays is highlighted. In conclusion, most protocols will require some modifications and recommendations are made to aid the researcher at the bench.

Single cell gel electrophoresis: Detection of DNA damage at different levels of sensitivity

Single cell gel electrophoresis, also known as the comet assay, is widely used for the detection and measurement of DNA strand breaks. With the addition of a step in which DNA is incubated with specific endonucleases recognising damaged bases, these lesions can be measured, too. In the standard protocol, electrophoresis is carried out at high pH. If, instead, electrophoresis is in neutral buffer, the effect of DNA damage seems to be much reduced — either because alkaline conditions are needed to reveal certain lesions, or because the effect of the same number of breaks on DNA migration is greater at high pH. A lower sensitivity can be useful in some circumstances, as it extends the range of DNA damage levels over which the assay can be used. Here we compare the performance of standard and modified techniques with a variety of DNA‐damaging agents and offer possible explanations for the differences in behaviour of DNA under alternative electrophoretic conditions.

Glutathione S-transferase polymorphisms influence the level of oxidative DNA damage and antioxidant protection in humans

Glutathione S-transferase genotypes GSTT1, GSTM1, GSTP1 were characterised in 155 middle-aged men and compared with parameters of oxidative stress at the level of DNA and lipids, with antioxidant enzymes, and with plasma antioxidants in smokers and non-smokers. Smokers had on average significantly lower levels of Vitamin C, beta-carotene and beta-cryptoxanthin and higher amounts of oxidised purines and pyrimidines in lymphocyte DNA. The GSTM1 null genotype was associated with elevated glutathione as well as with higher Vitamin C concentration in plasma. Vitamin C was higher in GSTT1+ compared with GSTT1 null--as was glucose-6-phosphate dehydrogenase activity. The homozygous GSTP1 a/a genotype was associated with significantly higher levels of GST activity measured in lymphocytes, in comparison with the b/b genotype. Using multifactorial statistical analysis we found significant associations between smoking, GSTP1 genotype, plasma Vitamin C, and purine base damage in lymphocyte DNA. The difference in Vitamin C plasma levels between smokers and non-smokers was seen only with the GSTP1 b/b genotype. This group accounted also for most of the increase in purine oxidation in smokers. In contrast, the link between smoking and oxidised pyrimidines in DNA was seen only in the GSTT1 null group. It seems that polymorphisms in the phase II metabolising enzyme glutathione S-transferase may be important determinants of commonly measured biomarkers.