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

Abstract

Assessment of DNA damage is of primary concern when determining the pollution-related stress in living organisms. To monitor genotoxicity of the freshwater environments we used micronucleus (MN) and comet assay on Dreissena polymorpha haemocytes. Caged mussels, collected from the river Drava, were transplanted to four monitoring sites of different pollution intensity in the river Sava. Exposition lasted for a month. The baseline level of MN frequencies in the haemocytes of mussels from reference site (river Drava) was 0.5‰. No increase in MN frequency was found in mussels from the medium-polluted site (Zagreb) in the river Sava while other, more polluted sites showed higher MN frequencies ranging from 2.7‰ (Lukavec) and 3.1‰ (Oborovo) to 5.2‰ (Sisak). Results from comet assay showed concordance with MN assay in indicating intensity of DNA damage. The use of haemocytes from caged, non-indigenous mussels in MN and comet assay proved to be a sensitive tool for the freshwater genotoxicity monitoring.

Introduction

Assessment of DNA damage is of primary concern when determining the pollution-related stress in living organisms. Genotoxic substances can cause effects such as carcinogenesis, teratogenesis, embryotoxicity as well as a suite of health disorders referred to as genotoxic disease syndrome (Kurelec, 1993) that can have adverse effects on stability of ecosystems (Nacci et al., 1996, Mitchelmore and Chipman, 1998). Furthermore, genotoxic agents rarely damage only DNA. Most chemicals exert their effects via both genotoxic and metabolically toxic mechanisms operating simultaneously (Depledge, 1998, Henderson et al., 2000). Therefore, there is an increasing need for sensitive assays to monitor the genotoxicity of the environment. For that purpose we examined zebra mussel (Dreissena polymorpha Pallas) haemocytes using the micronucleus (MN) and comet (single cell gel electrophoresis) assays. Zebra mussel is a sedentary filter feeder residing in freshwaters of Europe and North America. A number of studies have demonstrated that bivalves are suitable for the detection of genotoxic agents (Majone et al., 1987, Mersch and Beauvais, 1997, Bolognesi et al., 1999, Steinert, 1999, Pavlica et al., 2001). They are mostly sessile organisms, prone to accumulate high concentrations of many pollutants, widely distributed and easy to collect. Although present on many locations D. polymorpha is not found in all freshwater ecosystems. Therefore, indigenous mussels from clean site were used on all investigated sites. By using transplanted animals we wanted to reduce the influence of adaptive mechanisms that inevitably occur in native mussels from polluted sites, as well as to have rather uniform sample of a common genetic background for better comparison of the results (Ralph and Petras, 1998). Haemocytes are easily obtainable and demand very little manipulation for preparation of slides levelling the possibility of damaging to a minimum. They are closely exposed to environmental agents through their physiological roles in the transport of toxicants and in various defence mechanisms (Mersch et al., 1996).

MN assay has been successfully used for genotoxicity testing of environmental pollutants and different chemicals since late eighties. MN are small intracytoplasmic masses of chromatin resulting from either chromosomal breakages during cell division or chromosomes that are lagging in anaphase (Kirsch-Volders et al., 2000). MN assay was applied to many marine (Majone et al., 1988, Burgeot et al., 1995, Dopp et al., 1996, Venier et al., 1997, Bolognesi et al., 1999) and freshwater organisms (Jaylet et al., 1986, van Hummelen et al., 1989, Al-Sabti, 1991, Belpaeme et al., 1996, Mersch et al., 1996, Grisolia and Starling, 2001). In freshwater environments MN assay has been mainly applied to vertebrates (Jaylet et al., 1986, van Hummelen et al., 1989, Al-Sabti, 1991, Belpaeme et al., 1996, Sánchez-Galán et al., 1998, Grisolia and Starling, 2001). Only recently has freshwater bivalve, zebra mussel (D. polymorpha Pallas), been used for genotoxicity testing of different chemicals and environmental pollutants by MN test (Mersch et al., 1996, Mersch and Beauvais, 1997, Pavlica et al., 2000).

The comet assay is a simple, sensitive and rapid technique for detection of DNA damage (single-, double-strand breaks, alkali labile sites or DNA–DNA and DNA–protein crosslinks) in individual cells (Fairbairn et al., 1995) and therefore can be very useful in studies of genetic toxicology, especially ecogenotoxicology (Cotelle and Férard, 1999). So far, this technique was mostly used on mammalian cells (for review, see Fairbairn et al., 1995, Rojas et al., 1999) but other organisms were used as well (for review, see Cotelle and Férard, 1999). Since the comet assay has been used to assess the ability of potential aquatic contaminants to induce DNA damage, investigations of its applicability in environmental biomonitoring have come into focus (Pandrangi et al., 1995, Ralph et al., 1996, Šalagovič et al., 1996, Ralph and Petras, 1997, Ralph and Petras, 1998, Steinert et al., 1998, Steinert, 1999, Frenzilli et al., 2001). Despite increased interest, surprisingly little research has been done using the comet assay on freshwater species (Pandrangi et al., 1995, Ralph et al., 1996, Ralph and Petras, 1997, Devaux et al., 1998, Ralph and Petras, 1998) and almost none on freshwater invertebrates (Guecheva et al., 2001, Pavlica et al., 2001).

Unlike the measurement of MN frequency, which has been successfully utilised in many field studies of genetic damage (Burgeot et al., 1995, Mersch and Beauvais, 1997, Bombail et al., 2001, Grisolia and Starling, 2001), comet assay has not been used in field research until recently (Pandrangi et al., 1995, Ralph and Petras, 1998, Devaux et al., 1998, Bombail et al., 2001, Frenzilli et al., 2001).

The aim of this study was to assess the genotoxicity of freshwaters using the MN and comet assay on D. polymorpha haemocytes. Sites with different level of pollution were chosen in order to get better insight in the sensitivity of the both assays in the field. The rationale for the use of the above-mentioned assays is that they are both non-specific biomarkers of genotoxicity that can reflect different forms of environmental stress (Burgeot et al., 1995, Mitchelmore and Chipman, 1998).