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CHAPTER 1

The Comet Assay in Aquatic (Eco)genotoxicology Using Non-conventional Model Organisms: Relevance, Constraints and Prospects

MARTA MARTINS AND PEDRO M. COSTA

1.1 Introduction

The integrity of the genome is the very foundation of the organism and all the complex downstream events that mediate the various levels of biological organization, from gene to protein, then cell and tissue, and from there to individual, population and ecosystem. Not surprisingly, the "success of the fittest" involves the ability to cope with agents that may interfere with the genome and its transcription. When this ability is overwhelmed (or led into malfunction) by any given agent, such as chemical or radiation, the genetic material accumulates lesions that lead to metabolic dysfunction and then to cell death, or to the fixation of mutations if the cell does survive, thus propagating altered genetic material in somatic or germline cells. The latter case implies severe implications not only for the individual but also for the entire population since it may cause reproductive impairment, teratogenesis and, very importantly, tumorigenesis. Genotoxicity is therefore a phenomenon that affects all aspects of ecosystem functioning and may determine populational and species fitness in their changing habitat, rendering paramount the determination of its effects in ecologically relevant organisms outside the scope of the acknowledged laboratory model. The range of such "unconventional" models is increasingly wide, with particular respect to aquatic organisms, comprising many species of fish to molluscs (especially bivalves), to crustaceans, annelids and even echinoderms, cnidarians or macrophytes, whose exemplificative applications will be detailed in subsequent sections. On the contrary, the range of acknowledged model organisms holding some degree of ecological relevance is rather narrow. These include wild-type or genetically modified strains of the freshwater teleost zebrafish (Danio rerio) or the cladoceran crustacean Daphnia magna. In spite of their value in many fields of research (including biomedical, in the case of the former), these models are entirely laboratorial and cannot provide an entirely realistic insight into ecosystem function impairment by pollutants nor ensure the much needed long-term monitoring programs.

Not surprisingly, on account of the basic Paracelsian principle "it is only the dose that separates benefit from poison", toxicologists have long tried to understand, quantify and predict the effects of substances that may damage the genome of both humans and wildlife. As such, the first methods to detect and quantify DNA damage were adopted, adapted and improved from mammalian models (mostly in vitro) by environmental scientists from the start and successfully applied to a wide range of vertebrate and invertebrate organisms holding ecological and even economical relevance, thus giving birth to the domain of ecogenotoxicology. This implied, nonetheless, not only changes in protocols to harvest tissue and cells, for instance, but also in the interpretation of the findings per se, since genotoxicity is a complex biological phenomenon that depends on multiple pathways that likely differ between distinct taxa.

The methods to detect and quantify genotoxicity first focused on whole-chromosome changes, such as micronuclei and other nuclear abnormalities, which can be expeditiously scored, for instance, in whole-blood samples of non-mammalian vertebrates (since erythrocytes are nucleated), or the sister chromatid assay. These methods detect large-scale, irreparable, lesions that derive from clastogenic and aneugenic events. To this is added the widespread 32P-postlabelling method for detecting DNA–xenobiotic adducts. Other methods, such as the Ames test, address mutagenesis by itself by detecting the reversion of his-mutant Salmonella strains back to bacteria able to synthetize this amino acid, by the action of mutagens. Even though the adequacy and value of these methods is still beyond dispute, there was still a lack of a protocol that could efficiently detect alterations to the genome at the DNA strand level. A revolution thus took place when the single cell gel electrophoresis (SCGE) assay, or simply the "Comet" assay, was developed and rapidly incorporated within toxicological sciences, with emphasis on ecotoxicology and environmental toxicology. The common alkaline variant of the Comet assay, which stands as the workhorse of the protocol, originally settled by Singh et al. and based on the "neutral" version developed by Östling and Johanson, is nowadays little used. In fact, alkaline Comet assay or simply Comet assay are terms that are used almost interchangeably.

The principle of the assay is simple. Since DNA, like many organic molecules, is charged, when subjected to an electric field the smaller fragments will migrate faster towards one of the poles in a strong alkali environment, preceded by DNA denaturation in the same alkali buffer (~pH 13). Thus, the DNA of individual cells is exposed after embedding in an agarose matrix and the amount of fragment DNA migrating towards the positive electrode, i.e. the anode (since oligonucleotides are, essentially, anions), can be determined after staining and scoring using microscopy and imaging tools. The term "Comet" results from the typical shape of DNA after cell lysis and electrophoresis (the "nucleoid"), since large oligonucleotides, i.e., little or not at all fragmented, will be retained in the head whereas the smaller move toward the anode, forming the "tail". The migration of fragments, however, depends on several aspects that often tend to be overlooked. First, DNA is itself a supercoiled molecule formed by two oligonucleotide strands. Second, genotoxicants may or may not lead to direct strand breakage. In fact, one of the most critical factors of the assay is DNA denaturation under alkaline conditions since this will permit separation of the two strains and therefore allows the expression of single-strand breaks (if any), and the expression of double-strand breaks (if any) that were transformed into the single-strand after denaturation. To this is added the relaxation of altered DNA segments (loops) and expression of the so-called alkali-labile sites that consist essentially of altered nucleobases that, when DNA is loosened, may break upon electrophoresis (see for instance Tice et al.). The intensity of the staining between the head and tail can then be extrapolated as the relative proportion between fragmented and unfragmented DNA as a simple metric among the several direct or derived measures that can be retrieved from analysis and that will be debated further on.

In other words, in spite of the many types of DNA damage that might occur, the alkaline Comet assay indiscriminately detects damage that may either result in strand breakage or contribute to relaxing the DNA molecule to the point of favouring migration towards the anode. As such, the Comet assay has been used to provide a measure of "total strand breakage", which, in spite of some bias, is evidently more accurate than "total DNA damage". There are, however, variants of the alkaline Comet assay that permit some discrimination of damage by type, which will be addressed later on. By comparison, the scantly used "neutral" Comet assay follows the exact same principle. However, the denaturation/electrophoresis buffer has a lower pH (10), therefore failing to efficiently separate the DNA strands, favouring only the migration of double-strand fragments (Figure 1.1).

There is thus an important difference in the meaning of DNA strandbreakage when compared to other genotoxicity assessment methods, especially the micronucleus test and its variants, often referred to as nuclear abnormalities (NAs), since the latter refers to whole-chromosome damage such as aneugenesis (chromosomes that are not integrated within the nucleus of a daughter cell) or clastogenesis (chromosomal fragmentation), which is commonly associated with faulty cell division. Unlike DNA strand-breakage, lesions at the chromosome level are most unlikely to be repaired (Fenech et al.). Even though the relevance of scoring NAs in aquatic ecotoxicology is undisputable, it has been shown, even in studies with non-model marine fish like sole and bass, that the two measures may not necessarily be correlated. However, unlike assessing NAs, the Comet assay is not yet used regular biomonitoring approaches in aquatic ecotoxicology. It could be argued that the lack of standardization of protocols and its multiplicity could be hindering the value of the SCGE assay; however, in most cases, the logistics of field sampling greatly favour the high cost-effectiveness of preparing blood smears when compared to a molecular method whose accurateness greatly relies on avoiding accessory strand breakage. Still, the Comet assay has been widely employed in both in situ and ex situ (laboratory) bioassays and, although to a lesser extent, in passive biomonitoring of marine and freshwater ecosystems, thus involving surveys with a broad range of unconventional model organisms, as debated below. Still, there are many technical aspects that render aquatic ecogenotoxicity with these species particularly challenging. As previously highlighted in the few reviews specifically dedicated to the topic, the application of the Comet assay cannot be based on the same assumptions of biomedical research and human-oriented toxicology that, to date, still dictate most protocols and guidelines.

1.2 The Comet Assay in Aquatic Ecotoxicology: Role of Unconventional Models

1.2.1 Aquatic Ecosystems as the Ultimate Fate for Pollutants

When translated to ecotoxicology, the principle with which Paracelsus gave birth to toxicology simply stands as "contamination does not necessarily mean pollution". In other words, hazard and risk are two distinct concepts. Whereas some substances may be more hazardous than others (e.g., we can compare the metals cadmium and copper), risk is the probability of adverse effects occurring. This means that the dose or concentration can turn a scarcely hazardous agent into a high-risk pollutant. The ecotoxicologist must keep in mind that contamination occurs when the levels of one or more given agents surpass baseline environmental concentrations. If these concentrations cause deleterious effects to biota, then pollution is indeed occurring. The main challenges are, first, to detect deleterious effects and, second, to determine causality. In fact, ecosystem complexity is one of the major factors hindering the establishment of cause–effect relationships in this field of research. On the other hand, dealing with non-model organisms, quite often from "unconventional" taxa, poses additional challenges, albeit being crucial to understand how the ecosystem, and not just a species or a population, is affected by pollutants. Altogether, aquatic ecosystems hold many characteristics that render aquatic ecotoxicology as complex as it is important: (i) the aquatic environment is invariably the ultimate fate of environmental toxicants; (ii) areas adjacent to marine and freshwater ecosystems have always received the highest anthropogenic pressure; (iii) the sources of toxicants are multiple, natural or anthropogenic, and include aquatic transport, direct discharge, atmosphere, urban drainage and maritime/ fluvial transport; and (iv) aquatic ecosystems, especially those of transitional waters, have peculiar characteristics that render them ideal for accumulation, transformation and long-term storage of hazardous substances, particularly in sediments. Altogether, surveying the effects of pollutants on aquatic organisms is paramount as a tool for the diagnosis of ecological status and as a means to understand how a toxicant can affect the functioning of an entire ecosystem. In other words, surveying aquatic organisms plays an important role in Environmental Risk Assessment (ERA), whether as a measure of exposure (effects-oriented research) or as a means to understand why and how a substance becomes toxic to aquatic biota (mechanism-oriented research). In either case, model organisms, such as laboratory strains of the zebrafish or Daphnia, are mere surrogates and are not realistic representatives of wildlife. Even though clear advantages of these model strains, such as reduced intraspecific variability and high genomic annotation, permit important basic toxicological research, extrapolation towards wild organisms must be cautionary.

While mechanistic research in ecologically relevant organisms is far from being as developed as in human toxicology, effect-oriented studies are of upmost importance to quantify exposure in these "models" since, unless the concentrations of toxicants are either too high or too low, chemical analyses of sediments, waters and biota may be insufficient. Furthermore, it has long been acknowledged that ERA should not rely on a single Line-of-Evidence (LOE), such as a on a single biomarker or chemical determination of toxicants, but rather it should be an integrative approach comprising several LOEs, often referred to as the Weight-of-Evidence (WOE) approach. Determining genotoxicity has been proposed as an active component for these approaches as a biomarker of effect. The reader may refer to the excellent reviews by van der Oost et al., Martín-Díaz et al. and Chapman et al. for a definition of biomarker practices in Aquatic Ecotoxicology.

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Excerpted from "Ecotoxicology and Genotoxicology"
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