Biomarkers of Genotoxicity for In Situ Studies at Individual and Population Levels - Ecological Biomarkers: Indicators of Ecotoxicological Effects - page 339

The assay provides the opportunity to study DNA damage, including oxidative dam-
age, repair, and cell death in different cell types, without prior knowledge of karyo-
type and cell turnover (Jha 2008). Ecotoxicological applications and significance of the
comet assay have recently been reviewed by Jha (2008) and Frenzilli et al. (2009), who
underlined the potential ecological relevance of comet assay-derived genotoxicity data.
A wide range of phylogenetically disparate groups of organisms from oligochaetes to
fishes, amphibians, and mammals have been successfully investigated in aquatic toxicol-
ogy (Jha 2008; Dhawan et al. 2009). A number of advantages explain the popularity of the
comet assay in environmental biomonitoring. The assay is relatively simple and sensi-
tive, and requires a small number of cells. It is applicable to cell suspensions and dissoci-
ated tissues of many eukaryotes. Hemocytes and erythrocytes are often analyzed given
their practical and noninvasive sampling. Yet, cells of solid tissues such as gills, liver,
and digestive gland are also examined, although tissue dissociation is a critical step
that requires careful handling to avoid artifactual DNA breakage. Although mechanical
dissociation was preferred for the bivalve Unio tumidus (Lemière et al. 2005a), enzymatic
digestion of gill epithelium with dispase or collagenase was more efficient in another
freshwater mussel, D. polymorpha (Vincent-Hubert et al. 2011). Comparison of cell type
sensitivity indicates that circulating cells appear less sensitive than gill cells (Rank et
al. 2005; Bourgeault et al. 2010; Vincent-Hubert et al. 2011) or hepatocytes (Frenzilli et al.
2009). Gill cells and hepatocytes are involved in the uptake of environmental contami-
nants from water and food, and they are at the first line of exposure to pollutants and
their active metabolites. Approaches for the preservation and storage of cell samples
from remote marine sites and from large experiments have also been developed (Hartl
et al. 2010).
The comet assay is used to assess the genotoxic potential of chemicals, environmental
samples (Lee and Steinert 2003; Kilemade et al. 2004), and more recently, nanomaterials
(Galloway et al. 2010; Bernardeschi et al. 2010). Aquatic organisms or cells (Vincent-Hubert
et al. 2012) are used in laboratory testing facilities.
The assay has been widely applied to evaluate the impact of urban, agricultural, and
industrial activities on native fish species in river and estuarine biomonitoring. Generally,
DNA damage in erythrocytes parallels the degree of pollution (Larno et al. 2001;
Flammarion et al. 2002; Roy et al. 2003; Akcha et al. 2003; Bony et al. 2008; Nahrgang et al.
2010). The comet assay in fish embryos is now being developed to assess the genotoxicity
of chemicals (Pereira et al. 2012) and environmental samples, to study the long-term conse-
quences of DNA damage and embryo abnormalities (Vicquelin et al. 2011).
In the context of linking genotoxicity in individuals to population effects, the impact
of paternal genotoxin exposure was explored on the reproductive biology of two marine
invertebrates: the polychaete Arenicola marina and the mussel M. edulis (Lewis and Galloway
2009). A 72-h in vivo exposure of males to methylmethane sulfonate (MMS) and benzo[ a ]
pyrene at high concentrations resulted in DNA damage in somatic cells and sperm. The
fertilization success of DNA-damaged sperm was unaffected, but abnormal development
was recorded in the two species, suggesting potential long-term consequences for popu-
lation success. In M. edulis allowed to recover for 72 h after exposure, hemocytes showed
rapid and better recovery from DNA damage than sperm.
An analogous experiment, but a long-term survey, was performed on two fish species,
the brown trout Salmo trutta and the Arctic charr Salvelinus alpinus injected with a high
dose of MMS (50 mg kg -1 body weight) (Devaux et al. 2011). DNA damage increased in
sperm collected 3 weeks after the injection. It had no impact on fertilization and hatching
survival, but induced embryo abnormalities (that persisted 1 year after breeding in brown