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Yet, a higher sensitivity of the MN test compared to the comet assay has been found
in
D. polymorpha
(gill cells) after 2 months' caging in the Orge river in the Seine Basin
(Bourgeault et al. 2010) and in
Mytilus galloprovincialis
caged for 4 weeks for offshore mon-
itoring in the Adriatic sea (Gorbi et al. 2008). Higher sensitivity of the MN response has
also been reported by Bombail et al. (2001) in the fish
Pholis gunnellus
from the Firth of
Forth, Scotland.
A MN represents irreversible damage that lasts until cell death (Kirsch-Volders et
al. 2003). In contrast, increased DNA fragmentation may occur rapidly but transiently,
being subjected to repair (Collins 2004). The MN level is an index of accumulated
genetic damage during the life span of the cells, and is therefore one of the most suit-
able techniques to identify integrated responses to complex mixtures of contaminants.
In addition to clastogens, the MN assay detects aneugens, responsible for aneuploïdy
(loss or gain of one chromosome), which cannot be identified using the comet assay.
Aneugens can be discriminated from clastogens by visualizing the centromere on a
whole chromosome in the fluorescence
in situ
hybridization test (Eastmond et al. 1995),
whereas an acentric chromosome fragment (a fragment without a centromere) will not
be probed. The technique is widely developed in medical diagnostics, but not yet in
aquatic ecotoxicology.
13.2.3 Comet Assay
The single cell gel electrophoresis assay, also known as the comet assay, is based on the
fact that the DNA organization and its association with matrix protein in the nucleus are
disrupted when damaged. The individual strands of DNA lose their compact supercoiled
structure and relax, allowing DNA extension. This occurs regardless of the pH, neutral
or alkaline (Collins et al. 2008). The assay involves embedding of cells in agarose, mem-
brane lysis with detergents, and high salt concentrations to solubilize histones. Under
an electric field, damaged DNA is drawn to the anode, whereas undamaged DNA does
not move. The halo of relaxed DNA looks like a comet and is visualized after staining,
generally using fluorescent dyes. The level of damage is related to the intensity of DNA
staining in the tail of the comet as stressed by Collins et al. (2008). Fragments in the tail
will result if two breaks occur within one strand or if DNA detaches from the matrix at
high pH.
The assay originated from Ostling and Johänson (1984), who first used microelectropho-
resis, lysis with detergents, and pH 9.5 to detect breaks induced by ionizing radiation. A
few years later, Singh et al. (1988) proposed a similar method, but using alkaline condi-
tions (pH >13), to enlarge detection of damage, especially to alkaline labile sites. The latter
include apurinic and apyrimidic (AP) sites, which arise from the loss of a damaged base,
leaving a base-less sugar in the backbone. AP sites occur as intermediates during base
excision repair (BER) and may also arise spontaneously owing to altered chemical stabil-
ity resulting from changes in bases or sugars (Collins et al. 2008). Alkaline pH > 13 (usual
protocol) converts AP sites into breaks, whereas pH ca. 12 (less commonly used) does not.
Performing the assay in parallel at these two pH levels distinguishes agents that induce
just strand breaks from those that produce AP sites. Using the alkaline version at pH > 13
alone integrates breaks resulting from both exposure and repair.
Protocols have been optimized and applications defined by several authors successively
(Mitchelmore and Chipman 1998; Tice et al. 2000; Devaux and Bony 2012). Results are pref-
erably expressed as the percentage of DNA in the tail that covers the widest range of dam-
age and is linearly related to break frequency (Collins et al. 2008).
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