The Comet Assay and its applications in the field of ecotoxicology: a mature tool that continues to expand its perspectives

Abstract

Since Singh and colleagues, in 1988, launched to the scientific community the alkaline Single Cell Gel Electrophoresis (SCGE) protocol, or Comet Assay, its uses and applications has been increasing. The thematic areas of its current employment in the evaluation of genetic toxicity are vast, either in vitro or in vivo, both in the laboratory and in the environment, terrestrial or aquatic. It has been applied to a wide range of experimental models: bacteria, fungi, cells culture, arthropods, fishes, amphibians, reptiles, mammals, and humans. This document is intended to be a comprehensive review of what has been published to date on the field of ecotoxicology, aiming at the following main aspects: (i) to show the most relevant experimental models used as bioindicators both in the laboratory and in the field. Fishes are clearly the most adopted group, reflecting their popularity as bioindicator models, as well as a primary concern over the aquatic environment health. Amphibians are among the most sensitive organisms to environmental changes, mainly due to an early aquatic-dependent development stage and a highly permeable skin. Moreover, in the terrestrial approach, earthworms, plants or mammalians are excellent organisms to be used as experimental models for genotoxic evaluation of pollutants, complex mix of pollutants and chemicals, in both laboratory and natural environment. (ii) To review the development and modifications of the protocols used and the cell types (or tissues) used. The most recent developments concern the adoption of the enzyme linked assay (digestion with lesion-specific repair endonucleases) and prediction of the ability to repair of oxidative DNA damage, which is becoming a widespread approach, albeit challenging. For practical/technical reasons, blood is the most common choice but tissues/cells like gills, sperm cells, early larval stages, coelomocytes, liver or kidney have been also used. (iii) To highlight correlations with other biomarkers. (iv) To build a constructive criticism and summarize the needs for protocol improvements for future test applications within the field of ecotoxicology. The Comet Assay is still developing and its potential is yet underexploited in experimental models, mesocosmos or natural ecosystems.

Water browning mediates predatory decimation of the Arctic fairy shrimp Branchinecta paludosa

“…DNA strand breaks are among the more direct and easily detectable effects which can be assessed by means of comet assays, quantifying the degree of DNA fragmentation of single nuclei (Singh et al, 1988) by single-cell gel electrophoresis. While long acknowledged as a routine tool in human toxicology (Faust et al, 2004;Collins et al, 2014a), the comet assay has also gained interest in ecotoxicology (Jha, 2008;de Lapuente et al, 2015), where fields of application range from DNA damage analysis in immortalised cell cultures, to field-sampled mammalian blood. Chaoborus nyblaei larvae were sampled from one clear water (3.9 mg TOC L À1 ) and one brownwater pond (10.5 mg TOC L À1 ).…”

Potential Role of Mosquito Larvae <i>Culex Pipiens</i> as a Biological Indicator of Environmental Water Pollution in Egypt

“…Water pollution may influence DNA of fish and actuate hereditary modifications that can be utilized as markers of DNA changes in ecological contamination (Jha, 2008). Different aquatic and terrestrial invertebrates have been utilized for genotoxicity examines utilizing the Comet assay which have likewise been reviewed (Mitchelmore and Chipman, 1998;Lee and Steinert, 2003;Martins et al, 2013;de Lapuente et al, 2015). Cells from haemolymph and different tissues have been utilized for ecogenotoxicity considers utilizing the Comet assay.…”

The Impact of UV Radiation on Paramecium Populations from Alpine Lakes

“…To analyze the extent of DNA damage, we applied a modified alkaline comet assay that allows detecting and quantifying DNA damage of the macronucleus by measuring the migration of DNA from immobilized nuclear DNA (De Lapuente et al. ; Lee and Steinert ). Additionally, ciliate mortality and their swimming behavior were assessed before and after UVR exposure.…”

“…Under the assumption that less DNA damage and lower mortality occurred at a higher temperature due to the presence of possible temperaturedependent repair mechanisms, both populations were cultivated and experimentally tested at 8°C (i.e., mean lake temperature) and at 15°C (i.e., close to the lake maximum temperature). To analyze the extent of DNA damage, we applied a modified alkaline comet assay that allows detecting and quantifying DNA damage of the macronucleus by measuring the migration of DNA from immobilized nuclear DNA (De Lapuente et al 2015;Lee and Steinert 2003). Additionally, ciliate mortality and their swimming behavior were assessed before and after UVR exposure.…”