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that lag behind at anaphase during nuclear division. In environmental studies, micro-
nuclei are counted after cell harvesting, fixation, and staining with chromatin reagents,
and expressed per 1000 scored cells. Nuclear abnormalities, which include nuclear buds,
binucleated cells, nucleoplasmic bridges, and apoptotic nuclei, may be also counted and
reported, but separately to MN (Fenech 2007). An MN requires cell division after dam-
age induction to be observed in the cytoplasm of the daughter cells. Therefore, the divi-
sion kinetics of the tissue studied governs the sensitivity of the response and needs to be
known for correct interpretation of MN frequencies. A low mitotic index of the cell popu-
lation may explain negative results, even at polluted sites, either as a result of exposure to
cytotoxic pollutants that impair growth and cell division, or of physiologically low divi-
sion rates of the analyzed tissue. The age of the animals or their developmental stage is not
a trivial factor in the response.
The results of the micronuclei assays in a number of field studies carried out using wild
fish and mussels have been reviewed by Bolognesi and Hayashi (2011), and by Mouchet
and Gauthier (2012) regarding MN responses in amphibians.
In fish, micronuclei are measured in erythrocytes, gills, and fins of native species in the
field (Baršienė et al. 2012) or in laboratory studies (Goanvec et al. 2008; Hoshina et al. 2008)
to assess the impact of anthropogenic contaminants such as petroleum refinery effluents.
In the Baltic Sea, a large survey of genotoxicity in native species, flounder
P. l e su s
, herring
Clupea harengus
, and eelpout
Zoarces viviparus
, over the past decade was carried out using
the MN assay in erythrocytes. Background responses of MN incidences determined using
data from reference sites obtained in 2001-2011 were below 0.39 MN/1000 erythrocytes in
the native species, with offshore values lower than those in coastal zones. Long-term envi-
ronmental genotoxicity studies (2001-2011) in different zones of the Baltic Sea showed clear
increases in the level of environmental genotoxicity in 2009-2011, compared to lower MN
levels in fish collected in 2001-2007 (Hylland et al. 2008; Baršienė et al. 2012). Contamination
in zones close to estuaries or industrial activities that were measured within the HELCOM
Baltic Sea Action Plan (HELCOM 2010), and accidental spills of contaminants have been
suggested as possible causes for this increase.
In bivalves, hemolymph is easily collectable by noninvasive sampling, but gills are pre-
ferred for MN examination, although their preparation is not adequately standardized.
Gill cells are proliferative and more sensitive than hemocytes. Persistence of MN frequen-
cies in gill cells of
Mytilus
sp. was shown to be >1 month in short-term experiments using
genotoxic agents. Under laboratory conditions, higher baseline and induced MN frequen-
cies have been found in gill cells compared to hemocytes. Because of the influence of tem-
perature on the mitotic rate studied in controlled experimental conditions, seasonal effects
have often been taken into consideration in field studies (Baršienė et al. 2006a). This also
explains the higher background levels in warm southern countries compared to north-
ern ones, for instance, at Mediterranean sites compared to the Baltic Sea (Fernandez et al.
2011). At a reference site in the Baltic Sea, the lowest MN incidence (0.37 MN/1000 gill cells)
occurred in
M. edulis
collected at a temperature of 5°C in autumn, whereas a higher level
(1.16 MN/1000 cells) was detected at 13.6°C (Baršienė et al. 2006b).
The MN frequency in caged mussels requires several weeks of deployment, and com-
parison shows lower induction levels than in native mussels (Bolognesi and Hayashi 2011).
In field studies, the MN test is generally applied in a multibiomarker approach among
other genotoxicity markers and indicators of cytotoxicity, oxidative stress, and bioaccu-
mulation of contaminants. Correlations between response of the comet assay, apoptosis,
and micronuclei measurements have been found in
Dreissena
polymorpha
exposed
ex situ
(in the laboratory) to river samples from the Lambro/Po confluence (Binelli et al. 2010).
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