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coupled with electrochemical detection described by Shigenaga et al. (1994). In spite of its
sensitivity, this method suffers from several drawbacks: oxidative artifacts during DNA
extraction and sample preparation. To avoid artifactual oxidation, extraction using chao-
tropic reagents such as NaI, and addition of desferrioxamine as an iron chelator to inhibit
ROS production were recommended (Helbock et al. 1998; Ravanat et al. 2002). Basal lev-
els measured in aquatic species were in the range 0.6-5 8-oxodGuo/10
5
dGuo in bivalves
(Lemière et al. 2005a; Almeida et al. 2007) and fish (Rodríguez-Ariza et al. 1999). Higher
values have been found in the digestive glands of
M. galloprovincialis
(Akcha et al. 2000a,
2000b) and in the gills of
Unio tumidus
(Charissou et al. 2004). This method is now replaced
by the comet assay with enzymatic detection.
The modified comet assay relies on the addition of endonucleases after cell lysis and
DNA digestion with the lesion-specific endocucleases (Figure 13.1). The method is detailed
by Friedman-Angeli et al. (2012). Two bacterial DNA repair endonucleases were first used
to recognize oxidized purines and pyrimidines and convert them into strand breaks:
formamidopyrimidine DNA glycosylase (Fpg), which recognizes the oxidized purine
8-oxoGua, and also ring-opened purines, or formamidopyrimidines; endonuclease III,
which recognizes oxidized pyrimidines (Collins 2004; Collins et al. 2008). Fpg has been
used to study oxidative damage in
Limanda limanda
from the Seine and Somme estuaries
in France (Akcha et al. 2003), in isolated hemocytes of oyster and clam exposed to oxidant
(Gielazyn et al. 2003) and in gill cells of
M. edulis
from the Mersey estuary in the United
Kingdom (Emmanouil et al. 2008).
The human hOGG1, as 8-oxoguanine DNA glycosylase involved in the BER pathway
of 8-oxodG, has been recommended by Smart et al. (2006) and Smith et al. (2006) owing
to its better sensitivity and specificity. Its better specificity was confirmed by Michel and
Vincent-Hubert (2012), who compared the performances of Fpg and hOGG1 in gill cells of
D. polymorpha
exposed to contaminants.
13.2.5 Mutations
DNA damage generally induces DNA repair mechanisms to protect genome integrity. It is
of primary importance that DNA repair occurs before DNA replication takes place; other-
wise, mutations will be fixed. The rapid DNA repair mechanisms primarily target genes
that are being transcribed. Despite the high efficiency of DNA repair, DNA lesions may
persist particularly in case of many lesions in both DNA strands (coding and noncoding
regions). Genotoxins can also directly or indirectly inhibit DNA repair enzymes. Mutated
DNA is not stable and is more prone to rearrangements such as the loss, migration, and
translocation of DNA sequences into another chromosome. This may affect somatic cells
as well as germinal cells, and consequently, mutations could be transmitted to future
generations.
Genes controlling cell cycle and differentiation are widely studied in medicine because
their mutation may initiate cancer. Mutations activating the protooncogene ras have been
found in flounder
P. l e su s
, in relation to hepatic neoplasms in contaminated sites (Peck-
Miller et al. 1998; Vincent-Hubert 2000). High frequency at mutational hotspots in K-ras
genes was measured in pink salmon
Oncorhynchus gorbuscha
experimentally exposed to
Exxon Valdez
oil (Roy et al. 1999). The so-called “tumor suppressor”
P53
gene has been
sequenced in a number of fish species including the flounder, since its human homologue
is often inactivated by mutation in mammals and human carcinogenesis (Cachot et al.
2004). In contrast to human, the fish
P53
gene is infrequently mutated in tumors, but a
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