The comet assay is a rapid, sensitive and inexpensive method for measuring DNA strand breaks. The comet assay has advantages over other DNA damage methods, such as sister chromatid exchange, alkali elution and micronucleus assay, because of its high sensitivity and that DNA strand breaks are determined in individual cells. This review describes a number of studies that used the comet assay to determine DNA strand breaks in aquatic animals exposed to genotoxicants both in vitro and in vivo, including assessment of DNA damage in aquatic animals collected from contaminated sites. One difficulty of using the comet assay in environmental work is that of comparing results from studies that used different methods, such as empirical scoring or comet tail lengths. There seems to be a consensus in more recent studies to use both the intensity of the tail and the length of the tail, i.e. DNA tail moment, percentage of DNA in the tail. The comet assay has been used to assess DNA repair and apoptosis in aquatic animals and modifications of the comet assay have allowed the detection of specific DNA lesions. There have been some recent studies to link DNA strand breaks in aquatic animals to effects on the immune system, reproduction, growth, and population dynamics. Further work is required before the comet assay can be used as a standard bio-indicator in aquatic environments, including standardization of methods (such as ASTM method E2186-02a) and measurements.

Use of the single cell gel electrophoresis/comet assay for detecting DNA damage in aquatic (marine and freshwater) animals.

New chemicals are being added each year to the existing burden of toxic substances in the environment. This has led to increased pollution of ecosystems as well as deterioration of the air, water, and soil quality. Excessive agricultural and industrial activities adversely affect biodiversity, threatening the survival of species in a particular habitat as well as posing disease risks to humans. Some of the chemicals, e.g., pesticides and heavy metals, may be genotoxic to the sentinel species and/or to non-target species, causing deleterious effects in somatic or germ cells. Test systems which help in hazard prediction and risk assessment are important to assess the genotoxic potential of chemicals before their release into the environment or commercial use as well as DNA damage in flora and fauna affected by contaminated/polluted habitats. The Comet assay has been widely accepted as a simple, sensitive, and rapid tool for assessing DNA damage and repair in individual eukaryotic as well as some prokaryotic cells, and has increasingly found application in diverse fields ranging from genetic toxicology to human epidemiology. This review is an attempt to comprehensively encase the use of Comet assay in different models from bacteria to man, employing diverse cell types to assess the DNA-damaging potential of chemicals and/or environmental conditions. Sentinel species are the first to be affected by adverse changes in their environment. Determination of DNA damage using the Comet assay in these indicator organisms would thus provide information about the genotoxic potential of their habitat at an early stage. This would allow for intervention strategies to be implemented for prevention or reduction of deleterious health effects in the sentinel species as well as in humans.

Comet assay: a reliable tool for the assessment of DNA damage in different models

High power, nanosecond pulsed electric field (nsPEF) effects have been focused on bacterial decontamination, but the impact on mammalian cells is now being revealed. During nsPEF applications, electrical pulses of 10, 60 or 300 ns durations were applied to cells using electric field amplitudes as high as 300 kV/cm. Because of the ultra-short pulse durations, the energy transferred to cells is negligible, and only non-thermal effects are observed. We investigated the genotoxicity of nsPEF on adherent and non-adherent cell lines including 10 human lines and one mouse cell line with different origin and growth characteristics. We present data examining the effects of nsPEF exposure on cell survival assessed by clonogenic formation or live cell count; DNA damage determined by the comet assay and chromosome aberrations; and cell cycle parameters by measuring the mitotic indices of exposed cells. Using each of these indicators, we observed differential effects among cell types with non-adherent cells being more sensitive to the genotoxic effects of nsPEF exposures than adherent cells. Non-adherent cultures showed a rapid decrease in cell viability (90%), induction of DNA damage, and a decrease in the number of cells reaching mitosis after one 60 ns pulse with an electric field intensity of 60 kV/cm. These effects were not observed in cells grown as adherent cultures, with the exception of the mouse 3T3 cell line, which showed survival characteristics similar to non-adherent cultures. These data suggest that nsPEF genotoxicity may be cell type specific, and therefore have potential applications in the selective removal of one cell type from another, for example, in diseased states.

Differential effects in cells exposed to ultra-short, high intensity electric fields: cell survival, DNA damage, and cell cycle analysis.

Application of the micronucleus and comet assays to mussel Dreissena polymorpha haemocytes for genotoxicity monitoring of freshwater environments.

The development of methodologies for biomonitoring freshwater ecosystems is of particular relevance in view of the serious problem of aquatic environmental pollution. The mussel species Limnoperna fortunei (golden mussel) was chosen to be tested as a biomonitor organism based on its population data and distribution. L. fortunei individuals were exposed to UV radiation in vitro, and in vivo to pentachlorophenol (PCP) and copper sulphate (CuSO(4)), with the aim of standardizing comet assay and micronucleus test methodologies and evaluating the potential of this organism as a biomonitor. Haemolymph cells immobilized in agarose on slides exposed to UV radiation showed a dose-response relationship with maximum damage at 4.2 J/m(2). For the chemical tests, individuals were exposed for 2h for the comet assay and 24 and 48 h for the micronucleus test. A dose-response relationship was observed for both chemicals. 3x10(-5) M CuSO(4) induced high genotoxicity, also producing some toxicity after 48 h of exposure. PCP induced maximum damage in both assays at 150 μg/L. Individuals exposed to PCP showed 100% repair 2 h after the exposure period, as assessed by the comet assay. Exposure to an environmental sample over 7 days confirmed the mussel sensitivity to water contaminants, detected both by the comet assay and the micronucleus test. The results allow us to suggest the golden mussel as a potential biomonitor organism.

DNA damage and repair in haemolymph cells of golden mussel (Limnoperna fortunei) exposed to environmental contaminants.

The aim of the study was to use the comet assay on haemocytes of freshwater mussel, Dreissena polymorpha Pallas, for detection of possible DNA damage after exposure to pentachlorophenol (PCP) and to evaluate the potential application of the comet assay on mussel haemocytes for genotoxicity monitoring of freshwater environment. Zebra mussels were exposed for seven days to different concentrations (10, 80, 100, 150 μg/l) of PCP and in the river Sava downstream from Zagreb municipal wastewater outlet. Significant increase in DNA damage was observed after exposure to PCP at doses of 80 μg/l and higher and after in situ exposure in the river Sava as well. This study confirmed that the comet assay applied on zebra mussel haemocytes may be a useful tool in determining the potential genotoxicity of water pollutants.

Nina Mojaš

Papers

Detection of DNA damage in haemocytes of zebra mussel using comet assay.