History of Biomarkers - Ecological Biomarkers: Indicators of Ecotoxicological Effects - page 29

environmental pollutants belonging to different classes of a different nature (Cossu et al.
2000; Giguère et al. 2003; Roméo et al. 2003; Aït Alla et al. 2006; Damiens et al. 2007).
2.4.5 DNA Damage
As reported above, ROS continuously produced in aerobic organisms when not neutral-
ized may cause deleterious cellular effects such as lipid peroxidation described in the
previous paragraph, protein breakdown, or DNA base oxidation (Figure 2.2). The pre-
servation of DNA molecule integrity is critical for all living organisms, and they possess
efficient protective systems for their genetic material.
Between the first contact of a xenobiotic with the DNA molecule and a potential muta-
tion, an event sequence is produced beginning with the direct or indirect formation of
DNA adducts. The secondary modifications of DNA produced can be induced by an
oxidative stress and correspond to a single- or double-strand breakdown, an increase of
its repair level or base oxidation. When DNA disturbances become permanent, they can
induce an alteration of cellular functions and uncontrolled proliferation leading to carci-
nogenesis. Finally, when the contaminant impact is observed during cell division, it can
produce a mutation transmitted to future generations (Møller and Wallin 1998; Burcham
1999; Valavanidis et al. 2006; Almeida et al. 2007; Hwang and Kim 2007; Monserrat et al.
2007 and references quoted by these authors).
The detection and quantification of DNA damage allow its use as a biomarker of geno-
toxicity under acute or chronic conditions (Chapter 13). Usually, stress conditions induce
cellular disturbances in organisms and an increase in DNA damage. Most of the recent
published studies are focused on DNA damage induced by oxidative stress.
DNA oxidation generates different modified bases of which 8-oxo-7,8-dihydro-2ʹ-
deoxiguanosine (8-oxodGuo), produced by the reaction between oxygen and guanine,
are the most measured in aquatic organisms by high-performance liquid chromatogra-
phy. Other oxidized bases can be studied such as thymine glycol, 5-hydroxymethyluracil,
formylamidopyrimidine, and 8-hydroxydeoxyadenine (Martinez et al. 2003; Hwang and
Kim 2007).
The Comet test (SCG or single cell gel electrophoresis) is a quantitative technique,
quick and visual, to measure DNA strand breakdown in eukaryote cells (Devaux et al.
1997; Burlinson et al. 2007). The method is based on migration during electrophoresis of
damaged DNA from the nucleus, forming an impression of a comet, the head of which
corresponds to the cell nucleus with intact DNA, whereas the tail is formed by the cut
DNA strands. Recent modifications of this test specifically reveal the oxidized DNA bases
(Hwang and Kim 2007).
Other DNA damages assessed as genotoxicity biomarkers involve the DNA adducts
formed by the nucleotides on which the chemical mutagens are fixed ( 32 P postlabeling)
and the mutation quantified at the chromosomal level by the micronucleus test (Monserrat
et al. 2007).
More recent molecular biology techniques of DNA amplification (random amplified poly-
morphic DNA) or polymerase chain reaction have been used to assess the direct effects of
xenobiotics on DNA, and also the genetic diversity of studied populations. Actually, these
techniques still lack reproducibility and only with difficulty allow the separation of the
two mechanisms (Atienzar and Jha 2006).
An increasing number of aquatic and terrestrial ecotoxicological studies include the
measurement of different forms of DNA damage in order to evaluate the genotoxicity of
physical and chemical environmental stress on plants or animals, whether vertebrates or