Mutation Research/Reviews in Mutation Research
ReviewThe Comet assay for the evaluation of genotoxic impact in aquatic environments
Introduction
The demand for a clean and safe supply of water for drinking, agriculture and recreation has rapidly increased over the last few decades. Receiving waters, such as lakes, rivers and marine coastal areas are the receptacles for huge amounts of wastes derived directly from industry, agriculture and urban settlements or indirectly from the atmospheric deposition of airborne emissions. Present amongst these waters are a complex environmental mixture of well-known toxicants along with an increasing number of emerging contaminants, which pose a threat to both aquatic ecosystems and the health and welfare of human populations [1]. It is known that a number of chemicals present are highly persistent and have mutagenic and/or clastogenic properties [2], [3]. The relevance of detecting the mutagenic/genotoxic risks associated with water pollution was firstly perceived in the late 1970s, when methods based on Salmonella bioassay [4] or sentinel species, such as mussels [5] and fish [6], [7] were set up for monitoring the presence of mutagens and genotoxicants in aquatic environments. Since that time several tests have been developed for evaluating DNA alterations in aquatic animals, these are based on potentially pre-mutagenic lesions such as, DNA adducts, base modifications, DNA–DNA and DNA–proteins cross-linking and DNA strand breaks [8].
The analysis of DNA alterations in aquatic organisms has been shown to be a highly suitable method for evaluating the genotoxic contamination of environments, being able to detect exposure to low concentrations of contaminants in a wide range of species. In general, these methods have the advantage of detecting and quantifying the genotoxic impact without requiring a detailed knowledge of the identity and the physical/chemical properties of the contaminants present. Tests directly assessing DNA strand breaks, or downstream alterations following DNA strand damage, are commonly used to assess genotoxic impact in aquatic animals. The early procedures for measuring DNA strand breaks were based on the separation of double-stranded DNA, assessed by centrifugation or filtration; or on the denaturation rate, under alkaline conditions, determined by the incorporation of a fluorescent dye by the double-stranded DNA. Alternative procedures based on cytogenetic investigations, such as the sister chromatid exchanges (SCE) assay and the micronucleus (MN) test have also been widely used. The SCE assay is capable of detecting the exchange of chromosomal fragments between sister chromatids which follow DNA strand breakage [9]. The micronucleus test is based on the assessment of chromosomal fragments or whole chromosomes, which are not incorporated into daughter nuclei after cell division. Such damage results in the production of a micronucleus outside the main nucleus [10], [11]. The MN test is widely used with fish or aquatic invertebrates, whose numerous and tiny chromosomes often inhibit the use of metaphase based assays (e.g. SCE). The Single Cell Gell Electrophoresis (SCGE) or Comet assay was first applied to ecotoxicology about 15 years ago, and it has become one of the most popular tests for detecting strand breaks in aquatic animals through in vitro, in vivo and in situ exposures [8] and has been review by Mitchelmore and Chipman [12], Cotelle and Fèrard [13], Lee and Steinert [14]. The advantages of the Comet assay include the following: (a) genotoxic damage is detected at the single cell level; (b) most eukaryotic cell types are suitable for the assay; (c) only a small number of cells are required; (d) it is generally faster to conduct and more sensitive than other available methods for the assessment of strand breaks; (e) DNA strand breaks form quickly following genotoxic exposure, allowing for an early response evaluation on biota.
In this review we will focus on a synopsis of the genotoxicity data based on the use of the Comet assay in aquatic animals, published between 2004 and 2007, 5 years after the publication of other detailed reviews on this topic [12], [13], [14]. Furthermore, general remarks regarding the requirement for suitable guidelines for standardizing Comet assay protocols in order to achieve a better comparability and interpretation of results are discussed.
The aquatic environment provides a sink for many natural and anthropogenically derived chemicals. The genotoxic effects on aquatic organisms have been reviewed by Lee and Steinert in 2003 [14]. In the last 4 years different in vivo, and in situ studies have been carried out on both invertebrates and vertebrates, as shown in Table 1, Table 2, Table 3.
Section snippets
Invertebrates
Hartl et al. [15] used clams as an indicator for the presence of potentially genotoxic substances in estuarine environments, demonstrating an increase of DNA damage in haemocytes, gill and digestive gland cells of animals exposed to contaminated sediments. These studies found gill and digestive cells to be the most sensitive tissue type in detecting genotoxic exposure. The same authors [16] also compared the effects of a 3 week exposure to sediment samples (marine and estuarine) in clams and
Invertebrates
The majority of freshwater studies using invertebrates have focused on the use of filter feeding organisms, such as bivalves as test species in both field and laboratory based experiments (see Table 1, Table 3). The freshwater mussel Unio tumidus has been used in a several in vitro studies investigating the genotoxicogical properties of tannins and polyphenols, common contaminants in the aquatic environment. In vivo studies exposing U. tumidus to polyphenols (60, 200, 500 μM) for 24 and 48 h
General remarks
In general, after 10 years of the Comet assay being almost exclusively applied to mammalian studies across numerous research fields [78], [79], [80], [81], [82], [83], SCGE started to be applied to environmental studies revolutioning the field of genetic ecotoxicology [12], [13], [14] and providing the opportunity to study DNA damage, repair and cell death (apoptosis) in different cell types without prior knowledge of their karyotype and their turn-over rate [84]. The Comet assay has proved to
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