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well with biliary fluorescent metabolites in fish exposed to PAHs. Mussels are also useful
sentinel species in PAH-contaminated areas, provided that adducts are measured in spe-
cific tissues (gills or digestive gland) instead of whole tissues, which may dilute the adduct
pool and weaken the signal. Yet, abnormal DNA adducts detected by the
32
P-postlabeling
method remain generally nonidentified, which may be considered a limitation, but a limi-
tation that is not specific to this biomarker. Another confusing result is the presence of
abnormal DNA adducts on autoradiograms of controls or specimens from reference sites.
13.2.2 Cytogenetic Assays and MN Test
A wide range of
in vitro
and
in vivo
cytogenetic assays have been standardized to detect
genotoxic agents in mammals for regulatory and sanitary purposes. Criteria are structural
or numerical chromosomal aberrations, sister chromatid exchanges, and micronuclei,
measured in cells or in dividing tissues, using microscopic examination during mito-
sis, or using flow cytometry. These criteria have rapidly been applied to environmental
biomonitoring. Anaphase aberrations were proposed for use to survey fish populations
(Kocan et al. 1982). Their measurement was applied to study pollution and developmental
abnormalities of Atlantic fish (Longwell et al. 1992). Planktonic eggs provided evidence
that pollution was associated with mortality, malformation, and abnormal chromosomal
division of fish embryos developing near the surface water off Atlantic coasts. Correlations
were obtained between mitotic abnormalities, lethality and pollution by PAHs, metals and
embryo abnormalities, chlorinated hydrocarbons and defects in late stages of embryogen-
esis in mackerel (
Scomber scombrus
) on the east coast of New Jersey, USA (Hose 1994).
These markers have also been applied to invertebrates, especially to sea urchins
(
Strongylocentrus purpuratus
) to assess the effects of polluted sediments in San Francisco
Bay (Long et al. 1990).
In the Baltic Sea, karyotypic changes in gill epithelial cells were described in the bivalve
Macoma balthica
from the Gulf of Gdansk, and have been associated with gill neopla-
sia (Thiriot-Quiévreux and Wolowicz 2001; Smolarz et al. 2003). Cytogenetic damage in
somatic cells and in gonads of the gastropod
Lymnaea ovata
, the bivalve
Mytilus edulis
(micronuclei), and the crustacean
Balanus improvisus
were described on Lithuanian coasts
from 1995 to 1999 (Baršienė 2002).
Today, the MN test is one of the most widely used bioassays for genotoxicity in field
studies. It was first carried out on hemocytes in invertebrates or erythrocytes in fish and
is now applied to a wide range of aquatic organisms including sea urchins, oysters, crabs,
and worms, and in both wild and transplanted species (Bolognesi and Hayashi 2011).
It was applied to larvae of amphibians, the newt
Pleurodeles waltl
(Gauthier et al. 1989)
and the toad
Xenopus laevis
. The amphibian MN assay has been standardized (ISO 2006)
and used for the biomonitoring of surface waters, sampled, and tested in the laboratory
(Gauthier et al. 2004) or with caged amphibians
in situ
(Wirz et al. 2005). Besides assessing
chemicals and recently nanomaterials (Mouchet et al. 2011), micronuclei measurement is
also used for offshore biomonitoring using mussels (Gorbi et al. 2008; Sundt et al. 2011) or
fish (Rybakovas et al. 2009).
The measurement of MN is easier and more rapid than karyotype analysis (Vasseur et
al. 1995). Indeed, cytogenetic studies are difficult because of the relatively large number
of chromosomes of most fish species and the small chromosome size of many aquatic
organisms. The MN assay targets interphase cells of any proliferating cell population
regardless of its karyotype, which is an advantage in environmental monitoring pro-
grams. Micronuclei (MN) originate from chromosome fragments, or whole chromosomes
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