Mutagenesis Advance Access originally published online on April 1, 2008
Mutagenesis 2008 23(3):207-221; doi:10.1093/mutage/gen014
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Ecotoxicological applications and significance of the comet assay
Ecotoxicology and Stress Biology Research Centre, School of Biological Sciences, University of Plymouth, Plymouth PL4 8AA, UK
Application of the single-cell gel electrophoresis or comet assay has revolutionized the field of genetic ecotoxicology or eco-genotoxicology. It is a rapid, sensitive and relatively inexpensive method providing the opportunity to study DNA damage (including oxidative damage), repair and cell death (apoptosis) in different cell types without prior knowledge of karyotype and cell turnover rate. The assay has, however, often attracted criticism for its lack of ecotoxicological relevance. In addition, in contrast to genetic toxicology where rapid technical progress has been made to improve cell- and tissue-specific adoption of the assay, only limited advancement has been made to transfer the methodologies to ecotoxicological studies. While reviewing the recent information available in the literature and underscoring the importance of induced genetic damage in natural species, the aims of this article are to (i) highlight and judiciously analyse the ecotoxicological relevance of the assay; (ii) attempt to correlate the comet response with other relevant biological responses or biomarkers; (iii) identify the technical challenges and various factors affecting its application in order to make it reliable, reproducible and robust; (iv) critically compare the technical developments in genetic toxicology and genetic ecotoxicology and (v) evaluate the future developments with respect to applications of the assay. It is suggested that while complementing other ecotoxicological parameters and further improving the methodologies, the comet assay will continue to play an important role in genetic ecotoxicology to determine induced genetic damage, which has significant consequences for short- and long-term survival of the natural or wild species. Information obtained through integrated studies using simultaneous applications of multiple biomarkers on different wild organisms could also provide an holistic dimension of toxicological impact of environmental contaminants for the protection of human health.
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Introduction |
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 Top Introduction Genetic toxicology, genetic...Linking the comet assay...Challenges for the application...Future perspective and...References |
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As a result of human population growth and changing life styles, production, consumption and disposal of man-made contaminants are on the increase. The aquatic environment is often the ultimate recipient of this increasing amount and range of contaminants, a large proportion of which could be potentially genotoxic and carcinogenic (1
). This poses a major challenge for the regulatory authorities and environmental managers to protect the quality of natural resources. This is particularly so as many of the hazardous substances could occur beyond the detection limit, despite substantial improvements in methodologies (either directly or through modelling) to determine the fate and concentrations of the contaminants in different compartments of the environment (viz. soil, water, sediment and biological samples). Even if these contaminants could be detected (either in the environment or in the biological samples), it is now being realized that their simple detection in abiotic and biotic compartments is not enough unless their biological or ecological effects are properly evaluated. Furthermore, contaminants in the environment, including genotoxic agents, could occur as complex mixtures, and the risk of such mixtures, the presence and potential interaction of unknown substances could lead to a discrepancy between the actual and the predicted risk posed by a contaminant using a substance-specific assessment. As biological systems are the target for the action of toxicants, they provide important information which is not readily available from chemical analyses of the environmental samples and thus could be used as diagnostic tools for integrated environmental management (2). It is therefore important that assessment of biological responses in the natural biota forms an integral part of hazard and risk assessment (3). It is to be remembered that in contrast to the human health arena, non-human species (except rare species or species with low reproductive output) are valued at the population level. The primary aim of ecotoxicological studies is therefore to determine the Darwinian fitness of populations (i.e. growth, fertility and fecundity) bearing in mind that long-term survival of natural populations is dependent on their ability to grow and reproduce, thus maintaining the population structure in a particular ecological niche or environment. Determining the qualitative and quantitative distribution of species in a particular environment has therefore been the main ecotoxicological parameter on the assumption that pollution incidents will have detrimental effect on population structure. However, this approach highlights the limitations of species distribution as an indicator of reproductive success as an early warning system to prevent any potential detrimental effects of contaminants at population or community levels.
If reproductive success is the main ecotoxicological parameter, it is also important to understand the biological and physical factors affecting the normal reproductive pattern of natural species. This is especially important for invertebrates which constitute >90% of extant species (4) and play important roles in ecosystem functioning. Despite their ecological significance, our knowledge pertaining to reproductive strategies and behaviour of invertebrates is very limited. One of the reasons for this limited knowledge is due to our inability to maintain natural species under routine laboratory culture conditions. It is therefore not surprising that only a handful of selected invertebrate species are being exploited for potential use for reproductive studies for ecotoxicological studies (5). However, reproductive success is not only influenced by seasonality and the physical factors but also by direct or indirect interactions with other species. For example, chemicals secreted by diatoms are known to produce a wide range of biologically active metabolites which may act as pheromone- or endocrine-disrupting agents. Although not applicable to all species, polyunsaturated aldehydes secreted by diatoms have been demonstrated to be broadly disruptive to reproductive processes in natural biota, affecting processes such as oocyte maturation, fertilization, sperm mobility, embryogenesis and larval fitness (6,7). Bearing in mind that reproduction is the most important characteristic feature of living organisms, the energy allocation in somatic and reproductive compartments is linked. This precedes in a balanced way to both somatic and reproductive compartments since it is the maturation of somatic compartments which leads to reproductive success (8; Figure 1). Any perturbations of energy assimilation in the somatic compartments of the body could lead to generation of biological responses which in turn could be used as an indicator or surrogate for potential impairment of reproductive success.
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Toxicants could challenge biological systems in a variety of possible ways. Given the inherent limitations in assessing the reproductive success in natural biota, which would be resource intensive, it is therefore logical to evaluate other sub-lethal compensatory responses in the somatic and if feasible in the germinal compartments of the body. Indeed, these sub-lethal compensatory responses have been the primary focus of ecotoxicological studies for the last two decades (9
, 10). Both laboratory and field studies have shown that sub-lethal and sub-organismal level effects (e.g. small turnover rate for proteins, DNA damage, etc.) influence energy metabolism, fitness and reproductive success. This leads to population level effects (11–15). Indeed, increase in genomic instability has been suggested to play important roles in decreased fitness of the populations not only in laboratory studies but also in field conditions (16,17).
While rapid progress has been made in the last few decades to determine the impact of environmental factors on the induction of genetic damage in human populations, there has been relatively limited progress to determine the impact of such factors in wild or natural species. This is despite the fact that Boveri (18) proposed the relationship between chromosomal changes and the origin of tumours as early as 1914 using developing echinoderm embryos as a model and use of a range of insect and plant species played a pivotal role in establishing genetic toxicology as an established field of toxicology in the last century (19). Several reasons could have contributed to this limited development including scientific priorities and consequently regulatory requirements with the assumption that as long as human beings are protected, the environment inhabited by natural species is also protected (20,21). Several other inherent limiting factors would include very limited knowledge and unsuitability of the chromosome complements of the species for cytogenetic studies, which has been one of the main methods to determine the induction of genetic damage since early period of genetic toxicology (19,22). However, although examples of changes in gene pools as a result of environmental pollutants (e.g. industrial melanism, tolerance to metals and pesticides) have been available in diverse literature, the need for evaluation of the potential impact of induced genetic damage in wild species has only been realized since last two decades. This has created an essential and well-deserved niche for genetic toxicology within the field of ecotoxicology, so-called genetic ecotoxicology or eco-genotoxicology, to evaluate the direct or indirect effects of pollutants on the genetic apparatus (23–27).
One of the other reasons for limited progress in this field has been lack of scientific initiative for proper implementation of up-and-coming molecular techniques, which evolved from the need to improve our ability to detect and study induced and inherited mutations in humans and which also gave rise to the idea of mapping the entire human genome (28). The exceptions to these would be the analyses of DNA adduct formation (29–31) and amplification of randomly amplified polymorphic DNA (for review see 32), both of which have some inherent technical limitations for their applications. In this context, application of the comet assay has significantly contributed for the determination of induced genetic damage in natural species.
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Genetic toxicology, genetic ecotoxicology (eco-genotoxicology) and the comet assay |
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TopIntroductionGenetic toxicology, genetic...Linking the comet assay...Challenges for the application...Future perspective and...References |
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In the absence of adequate applications of cytogenetic and molecular assays, the development of the alkaline version of the single-cell gel electrophoresis or comet assay (33
) has revolutionized the field of genetic ecotoxicology (27). It combines the simplicity of biochemical techniques for detecting DNA single-strand breaks (strand breaks and incomplete excision repair sites), alkali-labile sites and cross-linking, with the single-cell approach typical of cytogenetic assays (34,35). In common with its application for mammalian studies, this assay has provided the opportunity to study DNA damage, repair and cell death (programmed cell death including apoptosis) in different cell types of natural biota, without prior knowledge of karyotype and cell turnover rate (22,27). As mentioned earlier, this is particularly important when cytogenetic and molecular assays are either not available or difficult to adopt. It is not surprising therefore that in the last few years this assay has been successfully applied to a range of phylogenetically disparate group of organisms which include lower and higher plants, oligochaetes, polychaetes, planarians, crustaceans, insects, bivalves, gastropods, asteroids and echinoids, fishes, amphibians and mammals. These studies have used a variety of physical and chemical agents in different life stages and cell types, under laboratory and field conditions and have been effectively summarized along with procedural developments in the literature (22,36–38). With some exceptions, where exposure scenario and treatment with the chemicals have lead to the suggestion that a particular species is unsuitable for the detection of genetic damage (39), in general, these reviews conclude that in common with mammalian studies, the comet assay can be applied successfully as a non-specific, sensitive, rapid and economical biomarker for the detection of genetic damage in natural biota.
Despite its wide applications, it is being realized that the comet assay has not been able to establish itself as a robust ecotoxicological tool and has often attracted criticism for its lack of ecological relevance (40). Criticism of the application is often not because of the robustness of the assay itself but perhaps, as discussed later, due to results generated and lack of correlations among different molecular biomarkers in specific or individual experimental conditions (40). This is in contrast to genetic toxicology (i.e. mammalian studies), where despite having some concerns over the methodologies used, and the type and quality of data produced (35,41–43), for which the harmonization and inter-laboratory calibration are well underway (44); the importance of this assay is fully realized, not only in fundamental biosciences, diagnostic and translational research for cancer therapy (35,45) but also in regulatory genotoxicological studies. There is also even a move to replace some traditional, expensive genotoxicological assays which play a decisive role in regulatory studies (e.g. liver unscheduled DNA synthesis assay or UDS assay), with in vivo comet assay (43,44,46). It is somewhat surprising therefore that the relevance of the comet assay is not widely appreciated in ecotoxicological studies. The critical question is that if it is relevant for humans (or mammals), why is it not relevant for other natural species such as fish and mussels?
Relevance of malignancy in ecotoxicology
As mentioned above, it is apparent that there have been fundamental differences between the objectives of genetic toxicology and genetic ecotoxicology. Mammalian genotoxicology, in particular regulatory studies, endeavour to identify potential carcinogenic hazard. It is worth remembering that in addition to point mutations in critical genes, chromosomal loss and aberrations play a vital role for the onset of carcinogenic and teratogenic effects. In this context, it is the DNA strand breaks, presently most efficiently determined by the comet assay, that give rise to chromosomal aberrations (47,48). In contrast, sub-lethal biological responses (i.e. biomarkers such as DNA strand breaks or chromosomal aberrations) and incidence of malignancies in natural biota have not been considered relevant for ecotoxicological studies, especially for regulatory compliance. The main ecotoxicological end point, also modified and adapted from the human health arena, has been the determination of median lethal concentration (LC50) values of chemicals or effluents that will cause death in 50% of the exposed organisms under laboratory conditions. Appropriate safety factors are then estimated to ensure protection of key organisms under field conditions.
Unlike mammalian carcinogenic models (e.g. rats and mice), the failure to induce malignancies in many invertebrates (49) and a lack of clear aetiology (bearing in mind that in addition to radiation and chemicals, naturally occurring viruses could be responsible for the incidence of tumours), played down the significance of occurrence of malignancies in wild species. In addition, although some invertebrates and fish do get tumours in the natural environment and often such incidences have been correlated with environmental pollution (50–55), it was generally believed that natural invertebrates do not get malignancies. This notion even led to development of the concept of ‘genotoxic disease syndrome’ which suggested that if invertebrates do not get tumours or if it is very uncommon, genotoxins could impart detrimental impacts on the health of the organisms, ultimately leading to population extinction (56). However, it has been very difficult to provide experimental evidence to support the occurrence of genotoxic disease syndrome. In parallel, through a systematic investigation by registering tumours in lower organisms, it has, however, become increasingly clear that lower organisms which include molluscs, annelids, arthropods and other invertebrates do get tumours (49,57,58). Lack of evidence for wide spread occurrence of tumours in lower organisms perhaps reflects need to focus research resources in this area. It has, however, been argued that even if the lower animals get malignancies, it is not ecologically relevant since most of invertebrates have enormous reproductive surplus and such occurrence would have a very small impact at population level, apart from on a local scale when viewed against the enormous wastage that takes place naturally among the early reproductive stages of many invertebrates (22,23).
The significance of incidence of malignancies in wild species, however, needs to be considered in the light of emerging scientific priorities where humans are seen as part of the ecosystem. In this context, it has also emerged that increasing pollution could lead to higher incidence of cancer in the human population and concurrently contribute to loss of biodiversity (59), the main aim of ecotoxicology. As mentioned above, during the last two decades, there has been an increase in the incidence of malignancies in shell and fin fish and epidemiological investigations have found increased incidences of gonadal tumours in bivalves at different sampling sites in the USA. This has been correlated with higher mortality rate due to ovarian and other reproductive organ cancers in human females compared to the national average (49,60,61). These studies clearly suggest that not only fish but also many ecologically relevant or model invertebrates could be used as sentinel or surrogate species for human health risk for carcinogenesis and could also serve as models to elucidate fundamental mechanisms of occurrence of other life-threatening diseases (62,63).
Ecotoxicological relevance of DNA strand breaks
If the reproductive success (i.e. fertility and fecundity) is the most relevant and important ecotoxicological parameter (despite inherent difficulties in determining them in most cases), it is quite reasonable to accept that much less time and lower concentrations (or doses) of contaminants will be required to induce a response at lower levels of biological organization (i.e. molecular or cellular levels) than to impair the reproduction at the whole-organism level. Undoubtedly, the most important damage at the molecular level is the induction of various types of lesions induced in the DNA including strand breaks which are efficiently measured by the comet assay. Such breaks give rise to chromosomal aberrations if they are un- or misrepaired. Chromosomal aberrations can lead to cell death, which could lead to several pathophysiological conditions (64). The application of the comet assay has the advantage that not only repair kinetics but also such apoptotic cells could simultaneously be identified with DNA strand breaks (Figure 2). In the surviving cells, DNA strand breaks could lead to permanent heritable changes while in somatic cells, as mentioned earlier, could lead to carcinogenesis. During embryogenesis, in common with human studies where chromosomal aberrations have been clearly linked with abortions, perinatal deaths and teratogenic effects (65), several studies in aquatic ecotoxicology have linked the induced chromosomal aberrations with survival and developmental effects in invertebrate and fish species (2,66–71).
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In germ cells, induction of chromosomal aberrations may affect the progeny of exposed individuals. Using plant and insect models in the last century, classical cytogenetic studies have established that those induced aberrations in the germ cells which are balanced or stable (i.e. reciprocal, terminal or interstitial translocations; para- and pericentric inversions) would persist to be passed on to the next generation. These balanced changes have, however, a high probability of being removed, sooner or later, from the gene pool. For example, a balanced translocation or inversion in a gamete can, after fertilization, result in an individual, all of whose cells will contain the translocation or inversion. These individuals known as structural heterozygote (translocation or inversion) produce gametes of which only 50% are viable (making them semi-sterile) (72
–75).
Even for smaller chromosomal aberrations or mutations, the vast majority tend to be harmful and thus they are likely to be removed from the gene pool through reduced reproductive fitness and natural selection (21). This will have detrimental effects on long-term survival of the population. Furthermore, it has been shown that exposure of fish sperm and spermatids to ionizing radiation (which are very efficient in inducing DNA strand breaks) also induce mutations in the somatic cells of growing F1 embryos leading to lethal consequences potentially as a result of simultaneous induction of DNA strand breaks and hence chromosomal aberrations (76). Similar observations pertaining to untargeted mutation in maternal alleles at tandem repeat DNA sequences arising in mice born from irradiated spermatozoa have been reported (77). Ecotoxicological consequences of this induced genomic instability and its correlation with gross DNA breaks certainly needs attention and is an area where the comet assay could play an important role.
Clearly, in common with mammalian or human studies, the induction of DNA strand breaks in both somatic and germ cells of natural biota are of paramount importance. If unrepaired or misrepaired, such damage will have effects on the immediate fitness as well as on reproductive success of the exposed organisms. This will ultimately lead to adverse effects on long-term population survival and, hence, deterioration of the ecosystem quality (Figure 2). In contrast to the human health arena, however, where transmission of genetic damage to offspring is a primary concern for scientists and regulators and guidelines have been formulated which involve a weight-of-evidence approach to determine the heritable mutations hence identification of potential human mutagens (78), such approaches are non-existent for wild species. This is either due to technical limitations or due to lack of regulatory demands or a combination of both. These mammalian guidelines broadly take into account the intrinsic mutagenic potential of chemicals, their ability to reach the differentiating or differentiated germ cells through the blood–gonad barrier and their interaction with germ cell DNA. Given that in the majority of non-mammal wild species where the blood–gonad barrier does not exist and the turnover of germ cells will be faster due to shorter lifespans, the disappearance of populations from a particular ecological niche could be faster than anticipated. Such isolated effects, if combined or pooled together, could have global consequences with loss of intra- or interspecies diversity (23–25). It is, however, very challenging to prove this hypothesis experimentally under laboratory or natural conditions. Many of the published work adopting genetic and molecular approaches to evaluate the effects of contaminants at the population level are also not always carefully designed to maximize the utility offered by these approaches and therefore are often inconclusive (79). As summarized in their review article by Belfiore and Anderson (79), many studies, which attempted to evaluate genetic patterns using allozyme variations and changes in the DNA (e.g. randomly amplified polymorphic DNA), have demonstrated that exposure of contaminants including genotoxic agents can reduce genetic diversity and Darwinian fitness of the organisms. Recent studies also suggest that exposure to genotoxic agents such as arsenic and radionuclides reduce nucleotide diversity in the mitochondrial DNA of birds (80). Mathematical models have suggested that fixation of mildly deleterious mutations could significantly contribute to loss of Darwinian fitness and eventual extinction of small populations (81,82). Indeed, most species are not driven to extinction before genetic factors impact them (83). In the absence of any technical advancement to determine the germ cell mutations in non-mammalian species and the fact that DNA strand breaks are correlated with health outcome, application of the comet assay (either in somatic or germ cells) and their subsequent conversion to chromosomal aberrations could be an aid to the risk evaluation process bearing in mind that many germ cell mutagens have also been found to be somatic cell mutagens (84).
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Linking the comet assay with other biomarker or ecotoxicological responses |
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TopIntroductionGenetic toxicology, genetic...Linking the comet assay...Challenges for the application...Future perspective and...References |
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Both by definition and by scope, ecotoxicology is an interdisciplinary and multidisciplinary science combining the fields of ecology, chemistry and toxicology. In ecotoxicological studies, it is therefore important not only to link the observed biological responses with the fate and behaviour of the contaminants in the environment with potential ecological consequences but also how different toxicological responses are expressed and linked to each other. In other words, it is important to dissect out the levels of toxicological responses before they could be linked to potential ecological consequences. In this context, depending upon levels of exposure, target cells and the assays employed, it is becoming increasingly clear that toxicological properties of any substance could be manifested in a variety of ways. For example, an antifouling paint, tributyltin (TBT), in addition to being an endocrine-disrupting agent for some gastropods, could also be a cytotoxic, genotoxic, teratogenic and immunotoxic agent (2
,69–71). However, it should also be realized that the toxic potential of contaminants is a cell-, tissue- or organism-specific and exposure (time)-dependent phenomenon. Their expression using different biomarkers might not always be linked up or correlated as there could always be some attenuation or threshold values at different levels of biological organization. Lack of correlations among different toxic responses therefore should not be the basis of criticism of wider applications of biomarkers as suggested by some authors (40).
In the above context, linking comet assay response with other biological responses could be considered important to discriminate the biological responses (either of exposures or effects) with impairment of normal physiological, homoeostatic or phenotypic plasticity processes (40). It is obvious that this is in contrast to genetic toxicology where use of biomarkers is well established to evaluate genetic and cancer risk including the degree of susceptibility among the human populations (85). Establishing such linkages are particularly important when interspecies variability for toxic responses as a result of differences in uptake, accumulation, metabolism, excretion, sequestration and DNA repair efficiency is well accepted. This is one of the reasons why adoption of multiple biomarkers and in a range of species (i.e. multiple biomarkers and multiple species) is being advocated for holistic assessment of pollutant impact on the ecosystem (27,86).
It is important therefore that in order to be considered as a robust ecotoxicological tool, the comet assay shows some correlations or links with other well-established biological responses. Particular emphasis should be given to those responses which are well known to impair the health outcome in humans and fitness of natural biota. These biological responses could be divided into different levels or organizational units, each unit controlling or influencing the structure and function of subsequent units (Figure 3). This approach could be considered pragmatic and inevitable since detection of damage at lower levels of biological organization using newly developed molecular or biochemical techniques will have to show its potential ecological or ecotoxicological relevance to avoid any criticism. However, in common with other molecular approaches in the post-genomic era, we have to bear in mind that one of the major difficulties is to unequivocally link the molecular effects with ecological consequences (87). In this context, given that the comet assay reflects gross DNA damage; it is very likely to have knock-on effects at higher levels of biological organization (Figure 2). This could be considered more relevant compared to other relatively expensive genomic and proteomic approaches which measure single end points (i.e. RNA or protein expression) for a single gene or thousands of genes at a time (88). Indeed as discussed below, attempts have been made in several studies where comet assay observations have been very well correlated with other relevant parameters which would impair the Darwinian fitness of natural biota. In many cases, comet responses have been found to be more sensitive and reproducible than many established ecotoxicological parameters or assays.
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Oxyradical scavenging capacity, enzyme activities and oxidative stress
Oxidative stress, in particular oxidative damage to DNA, has been implicated in a variety of human diseases (89
) including reproductive success of both human males and females, which could lead to infertility, foetal dysmorphogenesis, abortions and intrauterine growth restriction (90). Given that induction of DNA strand breaks could be directly related with oxyradical scavenging capacity of the cells, it has also been shown that lower genetic integrity in mussels also correlates with a reduced efficiency in neutralizing potent cellular oxidizing species (i.e. peroxy radicals: ROO•, hydroxyl radicals: OH• and peroxynitrite: ONOO–). Therefore, adoption of an integrated approach using the comet and total oxyradical scavenging capacity assays can provide an indication of potential risk associated with environmental conditions (91). Using the modified comet assay, which uses bacterial enzymes such as formamidopyrimidine glycosylase (Fpg) or endonucleases III (Endo III), which recognize oxidized nitrogenous bases and convert them into strand breaks, a significant increase for DNA strand breaks in the erythrocytes of dab (Limanda limanda) collected from eastern English Channel was detected (92). This suggests that the comet assay is capable of detecting oxidized DNA bases in fish exposed to environmental contaminants. However, in a study carried out around Birmingham (UK), which had poor water quality as a result of polycyclic aromatic hydrocarbons (PAHs), polychlorinated biphenyls (PCBs) as well as heavy metals, insignificant differences in oxidative DNA damage were found in isolated nuclei from fish hepatocytes (i.e. following Fpg treatment), although the conventional comet assay showed enhanced levels of DNA damage as a function of pollution in the river (93). These differences could be attributed to different exposure and tissue-specific metabolic activities.
Induction of DNA strand breaks by sodium dichromate (pro-oxidant) in the gill cells of mussels has been correlated with glutathione (GSH) concentrations. The evidence for repair of oxidative DNA damage towards 8-oxo-dG using an oligonucleotide cutting assay was also demonstrated (94). In a subsequent study, it was shown that cadmium and chromium show distinct mechanisms involving both direct and indirect oxidative damage, as well as impairing different DNA repair capacities in the gill cells of mussels, further enhancing the utility of the assay to elucidate mechanisms of toxicity (95). Oxidative stress is also induced as a result of physical activities or exercise in human and mammals. In an interesting study with chub (Leuciscus cephalus), the conventional comet assay revealed significantly higher DNA damage in erythrocytes, hepatocytes and gill cells, following exhaustive exercise, the gill cells showing highest damage. The Fpg-modified comet assay also showed the highest level of damage in gill cells but the antioxidant defence as determined by the levels of superoxide dismutase and GSH did not show any significant differences in different cell types. This led the authors to conclude that either the degree of oxidative stress was not high enough to induce a response in terms of defence or the timescale of antioxidant defence response was somewhat different from the time between the application of stress and subsequent tissue sampling (96). These studies suggest that organisms living in stressed conditions, either natural or environmentally induced, are at increased risk of oxidative damage and associated risk to DNA damage. Needless to mention, chronic oxidative stress either as a result of physical activities or due to exposure of environmental stressors could influence cellular physiology, leading to impairment of the fitness of individual organisms. In common with humans, the effects of stress in fish and other organisms can, depending on severity, result in impaired growth, feeding, reproduction and increased susceptibility to disease and mortality (97).
Gene expression, point mutations, cell survival and death (apoptosis)
In fish (eel), Anguilla anguilla, following exposure to benzo(a)pyrene [B(a)P], an increase in DNA damage but no point mutation or changes in the ras oncogene expression levels were detected (98). While this is in contrast to previous reports where mutations in the ras oncogene in fish have been related with PAHs exposure and are thought to be an early event in the carcinogenic process (99,100), it supports some of the reports where no mutations in ras oncogene in fish liver were found (101,102). These studies therefore indicate that the comet assay, especially in cells with high turnover rate, could be applied as an early indicator of DNA damage, before changes in the structure and function (expression) of specific oncogenes could take place. While evaluating the genotoxic potential of two herbicides (viz. alachlor and atrazine) in a mesocosms study using common carp, Cyprinus carpio, a dose–response relationship for comet responses and an increase for cytochrome P4501A1 gene expression were found but no change in vitellogenin gene expression was evident (103).
In eel (A.anguilla), elevated comet response has been correlated with the induction of apoptosis (104). In another study, using eelpout (Zoarces viviparous) as a bioindicator species, induction of apoptosis correlated well with comet assay response in erythrocytes. In this study, while the induction of DNA damage recovered 5 months after the oil leak accident, the levels of apoptotic cells did not show any marked variation (105). In a study in fishes collected from rivers around Birmingham (UK) with poor water quality, the degree of apoptosis and DNA strand breaks using conventional comet assay in hepatocytes was compared. The Tunnel assay was employed to ensure that levels of DNA strand breaks were not due to apoptosis (as it labels exposed 3'-OH ends of DNA fragments), allowing the identification of fragmented DNA (93). Using Mytilus edulis, the cell viability [measured using neutral red retention (NRR) assay] in haemocytes has been found to be significantly correlated with comet response in laboratory and field studies (106,107). While comparing the relative sensitivity of bivalve mollusc (M.edulis) and fish (Symphodus melops) following exposure to styrene, it has been shown that sensitivity of mussel haemocytes is greater than fish erythrocytes and destabilization of the lysosomal membrane is significantly associated with comet response (108). Similar observations were reported in cells isolated from the digestive tract or with cell preparations from whole Daphnia magna (109). In brief, cellular viability correlates with DNA strand breaks and in common with humans, this could precipitate in other pathophysiological effects ultimately affecting the Darwinian fitness.
Other genotoxicological end points, histopathology and neoplasia
Attempts have also been made to correlate comet assay response with other genotoxicological and histopathological parameters. For example, positive correlations between comet and micronucleus (MN) formation (i.e. biomarkers of ‘exposure’ and ‘effects’, respectively) have in general been found in fish and mussels cells under in vitro and in vivo conditions, respectively (106,110–112). However, such a correlation using the erythrocytes of fish under in vivo conditions has not been found (103,113). In these studies, while the comet assay showed a positive response following exposure to environmental stressors (e.g. PCBs or herbicides), the MN assay was not found to be sensitive enough. The MN assay in fish erythrocytes has been used extensively (for review see 114), mainly because compared to the comet assay, it is cheaper to perform. However, the sensitivity of the MN assay in fish erythrocytes has always been debatable due to its low level of induction (103,113), except for some studies where the chemicals were injected intra-peritoneally and MN were analysed in hepatocytes and erythrocytes (113). Given that MN induction is a cell cycle-dependent phenomenon and there is relatively little known about the rate of hematopoiesis or erythropoiesis, which will differ in different fish species (114), it is not surprising that a correlation between MN induction and comet response under in vivo conditions in different fish species is not apparent. To find an effect in the comet assay, information pertaining to cell cycle is not required, which eliminates the problem encountered by MN assay. Therefore, the comet assay could be considered to be much more sensitive in eco-genotoxicological studies using fish erythrocytes either in laboratory or in field studies (113).
The observations mentioned above using fish erythrocytes are supported by several other laboratory and field studies. In a study, carried out in Goteborg harbour, Sweden, following an oil spill, enhanced DNA strand breaks in erythrocytes of eelpout (Z.viviparous) carried out using the comet assay correlated well with apoptotic cells paralleled by a peak in bile PAH metabolites in fish collected from the most impacted area 3 weeks after the oil spill. The observed damage showed some degree of recovery 5 months after the accident indicating the possibility of turnover of erythrocytes apart from DNA repair process (105). Overall, the study correlated well with other biological responses and showed that comet assay has sufficient sensitivity to detect DNA damage in fish erythrocytes in the field situation. This observation is further supported by a field study in the eastern English Channel where DNA adduct formation using post-labelling technique in liver cells was compared with the induction of DNA strand breaks in the erythrocytes of dab (L.limnda). While the comet assay revealed a significant interaction between age and sex on the extent of DNA damage, the post-labelling technique failed to reveal quantitative differences in adduct measurements. This lack of significance was attributed to limitations of the post-labelling technique which remains a semi-quantitative method of DNA adduct measurement (31). This study further confirmed earlier work carried out by the workers where no differences for the levels of total liver DNA adducts were found between the sites but the application of comet assay confirmed its potential value as a sensitive biomarker. This also correlated with the degree of contamination data provided by the French monitoring network (92). Although the mechanisms of formation of DNA adducts and strand breaks could be different (e.g. one through metabolic activation of contaminants and another through generation of reactive oxygen species, direct strand breaks or replicative gaps), the existing information further highlights the sensitivity and applications of comet assay under field conditions.
While evaluating the genotoxic potential of algal (Polysiphonia fucoides) extract using erythrocytes of rainbow trout (Oncorhynchus mykiss), it has been suggested that the positive response comparable to 20 mg/kg B(a)P effect was only found in the alkaline comet assay, but not for double-strand breaks, apoptosis and antioxidant response (7-ethoxyresorufin O-deethylase or EROD activities) in liver cells. The study suggested that the genotoxicity of an algal extract does not involve oxidative stress and the comet assay could serve as a sensitive assay even for those agents which do not involve oxidative stress (115). A study carried out using haemocytes of freshwater bivalves (Unio pictorum) and erythrocytes of common carp (C.carpio) collected from reference and polluted sites suggested that the sensitivity of the alkaline comet assay is very high but since it determines mostly repairable DNA lesions, it indicates only recent pollution status. On the other hand, since the MN assay determines a stable aberration which is a cell cycle-dependent phenomenon, it was suggested that combined use of these two assays should complement each other in a genotoxicity monitoring programme (116). Correlations between MN and comet assays have also been found in golden mussels (Limnoperna fortunei) following exposure to water and sediment samples collected from the Guaiba hydrographic region in Brazil (112). In contrast to fish erythrocytes, where no correlation between comet and MN assays have been found, use of bivalve haemocytes have shown significant correlations between the two assays following exposure to a range of contaminants (106,111). This indicates higher sensitivity of haemocytes. Overall, these studies suggest enhanced sensitivity of the comet assay compared to other available molecular assays.
In the study by Akcha et al. (92), carried out in the eastern English Channel on dab (L.limanda), where a positive response for the comet assay was observed, histopathological examination showed hepatic steatosis in most of the animals examined, although only one pre-cancerous lesion was detected (92). In contrast, a very high incidence of liver tumours relative to background levels was detected in dab (L.limanda) collected from Cardigan Bay, reportedly a pristine site on western coast of the UK. While the analysis of hepatic DNA adducts failed to distinguish differences between the sites showing normal and elevated levels of tumours, DNA strand breaks were shown to be significantly elevated in the erythrocytes from the site having high levels of tumours. Interestingly, analysis of the sediment failed to distinguish any differences in toxicity between the two sampling sites and hence the causative links could not be established for this elevated level of DNA damage and liver tumours (117). These interesting studies further indicate that fish erythrocytes could act as surrogate tissue to detect DNA strand breaks using the comet assay and this could potentially be used as a predictive tool for carcinogenic effects in fish. This notion is supported by an earlier study, where a short-term exposure of polluted water collected from Aveiro Lagoon, Portugal, induced early genotoxicity as DNA strand breaks in A.anguilla blood cells and a delayed genotoxicity in liver and kidney cells (118).
Immunotoxicity and neurotoxicity
In different integrated studies, attempts have been made to correlate the comet assay response with other sub-lethal parameters such as immunotoxicity and neurotoxicity. Needless to say, these important sub-lethal responses would influence the overall fitness including reproductive success of the organisms. In this context, in humans, significant correlations among comet response and immuno- and reproductive toxicities have been suggested (119). In parallel with human studies, for example, a significant correlation between comet response and T-cell proliferation in the blood cells of dolphins has been reported (38). However, in mussels, following exposure to an anti-fouling agent and a known immunotoxicant, TBT, while a positive correlation between cell viability and DNA strand breaks was found, no correlation between phagocytosis and the comet assay could be detected. This was suggested to be due to variability in the pre-existing immune status of the organisms (106). However, such sensitive assays need proper validation before they are applied for robust comparison. Inhibition of acetylcholinesterase, an enzyme involved in the synaptic transmission of nerve impulses, is suggested to be a very useful biomarker of neurotoxicity following exposure to a variety of environmental stressors including organic and metallic contaminations (120). The inhibition of this enzyme has been correlated with a positive comet response in gill cells of native and transplanted mussels (M.edulis) in coastal areas of Western Denmark, potentially impacted by anthropogenic pollutants originating from a chemical dumping site (107). Correlations of comet response with immunotoxic and neurotoxic responses further support the hypothesis that toxic compounds may manifest their toxicity in a variety of ways (70).
Growth, development and reproductive success
DNA damage if not repaired during embryogenesis could lead to pronounced effects on the organism. Embryotoxicity could be considered as one of the important effects of contaminant exposure which could directly influence recruitment rate and hence the population dynamics (121). As mentioned earlier (Ecotoxicological relevance of DNA strand breaks), relationships between genotoxicity and embryotoxicity have been demonstrated in several studies using early life stages of aquatic organisms (2,69–71). In this context, a positive correlation was demonstrated between embryotoxicity and genotoxicity as determined by the comet assay in embryo–larval stages of oysters (Crassostrea gigas) following exposure to a polycyclic aromatic hydrocarbon [i.e. B(a)P] and the organochlorine pesticide, endosulfan (121). Increasing concentrations of a reference genotoxic agent, 4-nitroquinoline-N-oxide caused a proportional decrease in the percentage of grass shrimp (Palaemonetes pugio) embryos hatching from their eggs and a proportional increase in DNA damage as measured by the comet assay. A study of grass shrimp populations, from a reference and chromium-contaminated site, showed that the contaminated site had low density, low embryo production and high DNA damage as determined by the comet assay compared to shrimps from the reference site (38). In addition, the DNA damage has also been associated with effects on growth and reproduction. For example, mussels (M.edulis) collected from polluted sites in San Diego Bay, CA (USA), showed DNA damage as detected by the comet assay and reduction in growth rate, which was correlated with the levels of contamination (122,123). Another group of mussels in San Diego Bay, had sperm with damaged DNA that correlated with reduction in fertilization success (38). In these studies, highest levels of DNA damage were correlated with various contaminants in water and sediments, results of bioassays and benthic community degradation (38). Because genotoxic agents were present at many contaminated sites, these limited studies suggested that the comet assay could be used to determine if there are linkages between DNA damage and effects at the population and community levels (109). The embryo development has been correlated with comet assay response in the embryo–larval cells of grass shrimp (P.pugio) following exposure with a range of reference chemicals. In an interesting study, neutral red retention and the comet assays were performed following exposure to five reference toxicants with fresh preparations of pieces of tissues from the digestive tract or with cell preparations from whole freshwater fleas, D.magna. Preliminary results of long-term exposure suggested that these biomarker responses (i.e. NRR and comet assays) can be related to chronic effects on survival and/or reproduction of D.magna (109). In general, it has been suggested that comet response can be linked to effects on growth, development and reproduction in a variety of organisms and could be used as an early warning biomarker for toxicant exposure on the populations (109,124).
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Challenges for the application of comet assay in ecotoxicological studies |
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Due to its simplicity, sensitivity and relative economy, the comet assay was rapidly adopted to determine genotoxic potential of chemicals and as a tool in human biomonitoring programme. As mentioned earlier, the assay has also been modified by including digestion with lesion-specific repair endonucleases to get more specific information about oxidative DNA damage and its repair in human or mammalian cells (41
,125). This methodology has also been successfully transferred to aquatic organisms (92–96). Subsequently, the importance of this assay has also been realized in regulatory genetic toxicology. In this context, under the banner of International Workshops on Genotoxicity Test Procedures, the ‘expert panels’ are developing guidelines for conducting the assay under in vitro and in vivo conditions. The main goal of these workshops is to identify minimal standards for obtaining reproducible and reliable data deemed suitable for regulatory submission for chemicals. These workshops also aim to identify sources of variability influencing the results of the assay (42,43). International collaborations, led by the Japanese Committee for the Validation of Alternative Methods, aim to develop an Organisation for Economic Co-Operation and Development (OECD) guideline for the in vivo comet assay to address the regulatory requirements (44). Compared to regulatory genetic toxicology, where substantial progress has been made in the last few years to harmonize the protocols to develop the assay as a robust tool, there has been rather slow development with respect to optimization and harmonization of methodologies in ecotoxicological studies. Considering the potential amount of variations (e.g. varieties of organisms, cell types, etc.) which will influence the outcome of the assay under laboratory and field studies, the challenges are enormous and have been summarized below.
Optimization of basic procedures and generation of historical control data
It has long been realized that lack of banding pattern, as observed in mammalian chromosomes, indicate that there is some fundamental differences the way in which the chromatin is packaged in lower animals (22,126). As a result, the basic comet assay procedures (viz. lysis, unwinding and electrophoresis) could differ in different group of species. Indeed, interspecies differences in alkaline elution profiles have been observed and attributed to different lengths of DNA from different sources and to differences in the number of strand breaks present during normal cellular events in different phyla. This interspecies variation could also influence species-specific susceptibility to environmental agents (127). Similar observations have been made while evaluating the outcome of comet response in various experimental conditions on cells from a range of species. Elevated concentrations of ethylenediamine tetra acetic acid (EDTA) and the use of proteinase K (PK) after the lysis procedure reduced the background levels of DNA strand breaks in mussels. Elevated levels of EDTA were also needed for isolation of intact DNA in mussels but not in terrestrial snails. The basal levels of DNA damage in snails (Helix aspersa) and sea bass (Dicentrarchus labrax) were lower than mussels (Mytilus galloprovincialis) and it was suggested that internal fluids in mussels may increase the basal levels of DNA breaks through oxidative and/or enzyme-mediated pathways (128).
Dimethylsulphoxide (DMSO) is used as a component of lysis solution for mammalian studies mainly to scavenge iron particles released by haemoglobin or haemoglobin-like substances. Since there is limited information in the literature about the occurrence of haemocytes (red blood cells) or iron-containing substances in the haemocytes or coelomocytes of different animal phyla (129), it is important to carry out some optimization studies to evaluate the impact of such organic solvents, given that they could be toxic in their own right to natural organisms (130). In this context, a study using somatic cells of Drosophila melanogaster, removal of DMSO resulted in the appearance of scorable comets, which was otherwise found to be toxic (131). Size of the cells is also an important factor while using the low melting point agarose (LMA). It has been suggested that smaller cell size would need increased concentration of LMA to increase the frequency of scorable cells (131). Given that the cell size would vary considerably in different tissue of different organisms, this could be an important contributory factor for obtaining sufficient numbers of scorable cells. In addition, unwinding and electrophoresis time might require adjusting to improve performance of the assay as used in cells from D.melanogaster (131). Likewise, the standard enzyme (i.e. Fpg)-based comet assay generated unacceptably high amounts of DNA damage in haemocytes of bivalve molluscs, necessitating modification of existing methods and the use of sodium chloride-based buffers (132). Furthermore, in common with mammalian sperm DNA (133), fish erythrocytes have been suggested to have high levels of alkali-labile sites. It has therefore been recommended that the comet assay should be performed at pH 12.1 in order to detect only single-strand breaks while using fish erythrocytes (134,135).
Proper optimization and validation is therefore important before the assay is applied in cells collected from different tissues and species. Given that seasonal variations have been shown to influence the induction of DNA damage (31,93,112,127,136), optimization and standardization of procedures are also important for generating historical control and positive control data while identifying the factors influencing the outcome of the assay in field or biomonitoring studies. These should include both biological (e.g. age, life stages, sex, reproductive stage, inter-individual variability, etc.) in addition to physico-chemical (e.g. temperature, salinity, pH, etc.) factors. Needless to mention, identification of these factors are important to produce reproducible and reliable results which would reduce variability for the hazard and risk assessment.
Tissue- or organ-specific assay
It is well established that the genotoxic potential of chemicals depends on the properties of cell or tissue and contaminants accumulate differentially in the body (110,111). Tissue-specific accumulation has implications for the biomonitoring process so as to target appropriate tissues. Although in principle, the comet assay could be applied to any cell type, in mammals, because of their metabolic activation capacity, hepatocytes are considered to be most important. However, depending upon the nature of the chemical to be tested, in genetic toxicology, liver, bladder or intestinal cells are considered to be relevant (42). While in genetic toxicology, tissue- or organ-specific comet assays have been carried out successfully (137,138), except for limited studies in fish (96,118), insects like D.melanogaster (131) and cells isolated from embryos (139) or whole animals (109), there has been virtually no attempt to determine tissue- or organ-specific DNA damage in natural species. In common with mammalian studies, the technique associated with isolation of a single-cell suspension, which often involves use of enzymes (e.g. dispase, collagenase, etc.), has not been optimized properly for natural species. Although in mammalian studies, tissue homogenization techniques to isolate nuclei for the in vivo comet assay has been suggested to be acceptable for mouse organs (140), such standardization has not been carried out for natural species. For enzymatic processes, a loss of DNA integrity has been reported for cells isolated from digestive glands of mussels that have been treated with trypsin (141), which is most frequently used in the enzymatic dissociation protocols. Different enzymatic and mechanical techniques were applied to obtain single-cell suspensions from 16-h post-fertilized embryo–larvae of oyster, C.gigas for use in the comet assay (121). These techniques included use of three different enzymes (viz. trypsin, collagenase and accutase). Larvae digestion by collagenase was selected for the study due to a high dissociation efficiency, low background for different comet parameters and good viability as determined by the trypan blue exclusion test (121). Similarly, when using a non-enzymatic mechanical method to isolate mussel gill cells, increased amount of DNA damage as a function of dissociation time was found (142). Even if a single-cell suspension is obtained, for example from haemolymph or gill tissues of mussels, often the homogeneity or heterogeneity of the cells have not been worked out for the majority of the organisms. For example, despite extensive uses, it is still not exactly clear how many cell types are present in the gill and blood cells of mussels (143; see ref. 22 for more details) or other invertebrates (129). Therefore, not only the technical aspects of the isolation procedure requires attention but also proper characterization of cell types deserves due consideration to avoid variability while analysing cell- or tissue-specific comet response.
Determination of cytotoxicity or cellular viability
Cell death could lead to degradation of DNA, hence all tests that evaluate primary DNA damage, including comet assay, have the potential to detect agents that are cytotoxic rather than genotoxic (43). In genetic toxicology, it has therefore become customary to measure viability of cells prior to performing the comet assay as a prerequisite. For in vivo assays, cell viability in the target tissue that is <70–80% of that in the control animals may be considered excessive (42,144). It is, however, also suggested that viability per se is not necessary for sound comets and that the best test of whether cells are in a satisfactory condition for the assay could be considered when untreated or control cells give a comet with 
10% of DNA in the tail or class 0 in the visual scoring system (125). Although no specific recommendation for the procedure, either for simple or fluorochrome-based stains (e.g. 5-6 carboxyfluorescein diacetate and ethidium bromide), has been made in genetic toxicology (42), cellular viability is routinely checked using trypan blue dye exclusion techniques, which rather than indicating real cellular viability, might just indicate the integrity of the cellular membrane. With the exception of some fish cell lines maintained under routine in vitro culture conditions, for most of the natural species cells for the assay are procured from either coelomic cavity or isolated from solid tissues (e.g. gill, liver, digestive glands, etc.). In this situation, this approach could be non-informative if mincing or homogenization is used to obtain single-cell or nuclei populations (42). Application of other cytotoxicity tests such as histopathological tests (137) or neutral diffusion assay (145,146) to identify necrotic or apoptotic cells are also technically difficult to apply. Application of simple viability tests in a variety of cell types, most often heterogeneous in nature, collected from a range of tissues becomes very challenging. This is particularly so when these staining procedures are pH, temperature and salinity dependent. It is because of these challenges, while some of the studies apply trypan blue (this dye can be endocytosed) and Eosin Y stains for freshwater and marine species, respectively (86,111), many of the studies using cells collected from invertebrate and plant species ignore the determination of cellular viability or survival assays. In common with genetic toxicology, where only limited information is available whether cytotoxicity results in increased DNA migration under in vivo conditions (43), it is important that sound cell-specific survival or viability assays are adopted in genetic ecotoxicology.
Natural and biological factors as sources of variation
Compared to humans, natural organisms are distributed and restricted to well-defined occupancy. This is particularly so for the aquatic or marine environment where the organisms occupy distinct ecological niches, along the vertical and horizontal habitats of the ecosystem. In these ecological niches, it is hard to find a single species, representative of all. In addition to seasonal variation in different organisms as mentioned earlier (31,93,112,127,136), there are several other physical and biological factors which could influence the comet assay results. For example, large numbers of naturally occurring toxins have been found in the marine environment (147,148) which could influence the genotoxic responses in the natural organisms (115). Similarly, in common with human studies, presence of vitamins (e.g. vitamin E) or micronutrients (e.g. selenium) have also been found to influence the comet assay response in natural species (149,150). Natural availability of these factors could therefore be an important aspect to consider when sentinel organisms from different contaminated and reference sites are collected or compared to evaluate the health status of an ecosystem (150). The variation in response to seasons, as mentioned above, could partly be due to temporal variations affecting the mixed function oxygenase levels in the tissues, as suggested for the induction of MN in mussels (151). It could therefore be important to include the determination of enzyme levels in the experimental plan to complement the comet assay as carried out by Tran et al. (150), especially under field conditions. Furthermore, since physiological condition varies with reproductive phase and hormonal level, comet response could vary considerably due to age and reproductive status of the organisms, although in human population monitoring studies such correlations with age and sex have not been established (152). Likewise, the reproductive timing in natural species could vary due to water temperature differences (153). In this context, an elevated level of spontaneous and induced levels of comet damage have been reported in the erythrocytes of mullet and sea catfish (154). It is therefore likely that even when samples are collected in the same season from different areas of the country, baseline and induced comet response may differ significantly. These variables need to be taken into account if the robustness of the assay is to be established.
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Future perspective and conclusions |
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Despite wide applications in ecotoxicology, there are many areas where the application of the assay has been quite limited. The assay therefore needs to be extended to elucidate many fundamental aspects of induction of genetic damage. These are summarized below.
Application to a wide range of ecologically relevant organisms
The assay is presently applied successfully to only a very limited number of species, especially for environmental monitoring. It is often seen that at contaminated sites, these species are either absent or if present, their responses might not be indicative of other species in the community (86). It will therefore be important to extend the application of the assay in different species representing different functional groups (e.g. predators or carnivores, omnivores, grazers, filter feeders, detritus feeders, etc.) inhabiting different ecological niche in the biomonitoring programme. This should also provide the relative sensitivity of different species to take necessary preventive or precautionary action. Furthermore, the study, after proper optimization (for example to obtain suspensions of nuclei or cells), should also extend to different plant and phytoplanktonic species. At the moment, the limited information on comet responses in plants is restricted to laboratory-based studies (155–159), which will warrant due consideration given their role in ecosystem functioning. The study at different trophic levels in plants and animals should also attempt to discriminate the importance of genotypic differences in determining the susceptibility of the organisms. This is important given that even in parthogenetically reproducing organisms (viz. D.magna), sensitivity towards reference toxicants could vary as much as three orders of magnitude and that different clones could be sensitive to different toxicants (160).
Application in the germ cells
In recent years, the comet assay has been applied to assess the quality of sperm DNA for different purposes which include diagnosis of male infertility, occupational, pharmacological, epidemiological and toxicological studies (119,162,163). Nevertheless, compared to mammalian studies, its application in ecotoxicological studies has been rather non-existent. Some of the available studies using sperm from wild species reflect aquacultural rather than toxicological applications of the assay (164,165). Given that direct release of gametes into the environment is the principal mode of reproduction for the majority of natural species, in particular for aquatic organisms, where they are exposed to contaminants, this could lead to detrimental consequences for the reproductive success of the organisms. It is therefore important that the application of the assay is extended to the gametes of organisms. Such studies will also allow investigators (i) to determine the mechanisms by which induced damage in the paternal DNA could potentially cause disruption of early developmental stages and (ii) to discriminate between induced and spontaneous wastage of early life stages which occurs abundantly in many natural species. However, given the unique structure of DNA in the germ cells, especially in sperm (e.g. 85% histone replaced by protamine), the assay needs to be modified and optimized, which would include prolonged treatment of proteinase K (PK) to decondense highly compact DNA (162,163), in addition to some specific treatments such as removal of jelly coats, perivitelline membrane and mucopolysaccharides for eggs of some aquatic organisms. It will also be important to consider the differences in the gametogenesis processes in different species and sexes, which would affect the susceptibility of environmental agents for the induction of genetic damage including DNA breaks (166). Other factors such as chemical composition (e.g. polyunsaturated fatty acids being substrates for reactive oxygen species) and lack of DNA repair mechanisms in the sperm will also need to be accounted for while applying the assay to germ cells.
DNA repair and gene/sequence-specific studies
It is needless to mention that the consequences of genotoxicity are dependent upon the efficiency and fidelity of different repair mechanisms since the fundamental processes of DNA replication and repair in different prokaryotic and eukaryotic groups are similar. The possibility for most of the induced DNA damage to be repaired in wild species is one of the silent and unsubstantiated arguments in some quarters. This could limit the significance of genotoxicological studies in ecotoxicology. The advantage of the application of the comet assay is that it could provide an estimation of heterogeneity of cellular response to toxicants but following modifications, it is also able to detect a wide variety of additional types of damage in the DNA including DNA inter-strand cross-links and base damages (as endonuclease sensitive sites). The versatility of the comet assay is unlikely to be easily duplicated (167). The versatile applications of the comet assay to elucidate intercellular, intragenomic heterogeneity and lesion-specific (i.e cross-links and base damages) repair capacity in mammalian or human studies is likely to be extensively adopted in ecotoxicological studies.
In human and mammalian studies, the combination of the comet assay with the fluorescence in situ hybridization (FISH) technique has provided the insights of the role and fate of specific DNA sequences at the whole-genome level (168–171). Despite the fact that whole or partial genomes of many fish (e.g. Danio rerio and Gasterosteus aculeatus), invertebrates (e.g. Caenorhabditis elegans) and plants (e.g. Arabidopsis thaliana) have been known and conservation of many sequences (viz. oncogenes, tumour suppressor genes and telomeric sequences) across the phyla have been established (98,172–175), there has been virtually no studies to use these methodologies to elucidate the role of specific DNA sequences in the induction and repair of DNA damage in ecotoxicology. In fact, very little is known about DNA repair efficiency in different wild organisms. This is particularly because of the lack of mutant cell lines or identification of repair-deficient individuals and there has been virtually no use of DNA repair inhibitors (e.g. aphidicolin and 3-aminobenzamide) either alone or in conjunction with the comet assay. Future studies will therefore explore the potential differential sensitivity and elucidation of repair efficiency of apparently homogenous genomes from different cell types of the natural organisms which will include use of combination of comet and FISH techniques. Repair kinetics or removal of lesions or strands breaks, as carried out by Tran et al. (150), need to be further extended in different species and cell types. Cell turnover rate, which has not been elucidated in the majority of the cases, could also influence the interpretation of repair kinetics results. Since contaminants in the natural environment could occur in all probable combinations, interactive effects of different components will also need to be elucidated as carried out in gill cells of mussels (176).
In conclusion, in the absence of other convenient or practical methods, the comet assay will continue to play an important role in assessing the induction of genetic damage in natural biota. The assay does, however, require proper optimization, validation and inter-laboratory harmonization, using different ecologically relevant species inhabiting different niches with different feeding habits and reproductive strategies. It is also important to link the assay with other relevant end points if it has to be accepted as a robust ecotoxicological tool. Information obtained through these integrated studies on wild organisms could also be translated to the human health arena, where simultaneous applications of other assays are logistically difficult to perform. Finally, in the 21st century, with rapid developments in molecular techniques in the post-genomic era, we need to look at the ecotoxicological objectives through a different lens, and that is, preservation of the integrity of DNA, the blueprint of life. When the issues of biodiversity and climate change become top of the scientific agenda, the preservation of genetic entity is bound to get its due importance in ecotoxicological studies.
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Acknowledgments |
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I would like to express my sincere thanks to Prof. Michael Moore (Plymouth Marine Laboratory, Plymouth, UK) for going through the earlier draft of the manuscript, Dr T. S. Kumararvel [Advanced Technologies (Cambridge) Ltd, Cambridge, UK] for helpful discussion, colleagues in Stress Biology and Ecotoxicology Research Centre, University of Plymouth, in particular, Dr John Moody, Dr Murray Brown, Dr Awantha Dissanayake, Mr Chris Pook and Mr Martin Canty for their help in various ways during the preparation of the manuscript. This paper is dedicated to Prof. A. T. Natarajan on the occasion of his 80th birthday.
Conflict of interest statement: None declared.
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Notes |
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* To whom correspondence should be addressed. Tel: +44 1752 232 978; Fax: +44 1752 232 970; Email: a.jha{at}plymouth.ac.uk
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References |
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Received on December 3, 2007; revised on January 31, 2008; accepted on February 11, 2008.
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