considered in earthworm studies. Markers indicative of an oxidative stress condition,
potentially enhanced as a consequence of xenobiotic P450 metabolism, include the
production of reactive oxygen species (ROS), the total antioxidant capacity (TAC)
as well as the responses of antioxidant defences such as the activity of the so-called
antioxidant enzymes such as catalase (CAT; transform H 2 O 2 in H 2 O and O 2 ),
superoxide dismutase (SOD) involved in scavenging superoxide (O
2-Á
) radicals,
glutathione peroxidases (GPX) and guaiacol peroxidase (POD) involved in the
reduction of a broad range of organic peroxides to the corresponding alcohols.
Other enzymes with a dual role (antioxidant and phase II conjugation reaction) are
the activities of glutathione S-transferase (GST) mainly involved in detoxification
reactions and glutathione reductase (GR) engaged in maintaining the levels of
reduced glutathione (GSH) from its oxidised disulphide form (GSSG) with a relevant antioxidant role. Endogenous molecules such as GSH and other molecular
scavengers such as β-carotene and vitamins C and E also have a described
antioxidant role.
Constitutive stress proteins, and in particular the heat shock proteins
(e.g. HSP70), are also inducible and involved in the protection and repair of these
cell biomolecules’ integrity under multiple stress conditions, including heat. When
the cell’s natural defence mechanisms are overwhelmed, oxidation of biological
molecules such as lipids occurs. This is known as lipid peroxidation (LPO), and the
analytical assay (thiobarbituric acid reactive species or TBARS) to measure LPO
quantifies the malondialdehyde (MDA) levels. In the case that oxidised molecules
are proteins, its oxidation is measured as protein carbonyl (PC) formation. If the
genetic material is that affected, then DNA damage occurs and can be measured in
several ways, by the electrophoretic comet assay or single gel electrophoresis (SGE),
by the formation of the oxidised nucleoside 8-hydroxy-2-deoxyguanosine (8-OHdG)
using ELISA techniques and by measuring DNA degradation in the late stages of
apoptosis by the TUNEL assay, amongst others. In earthworms, this effect biomarker (DNA damage) is especially relevant since it can be easily measured in
coelomocytes as a validated non-destructive technique. Another effect marker
widely used to measure damage occurrence in biological membranes is the lysosomal membrane stability (LMS) and the neutral red retention (NRR) assay a
frequent method of evaluation. Immunology assessment is considered to be a general
stress marker and has the advantage of being easily measured in biological fluids
such as coelomocytes and can thus also be classed as a conservative technique.
Histological alterations are morphometric measures considered signs of unequivocal
damage consequences to higher hierarchy levels of biological organisation.
Although damage evaluation is the most common measure included in many
ecotoxicity studies with earthworms, this chapter places more emphasis on early
warning biomarker responses such as those experienced by biochemical and molecular parameters. Some reviews have comprehensively addressed each particular
biomarker in earthworms: ecogenotoxicity [14], genotoxicity, immune system,
histology and antioxidant protective defences were addressed by Roubalova et al.
[15] and esterases by Sanchez-Hernandez [6]. Other novel biomarkers applied to
Biomarkers in Earthworms
315
potentially enhanced as a consequence of xenobiotic P450 metabolism, include the
production of reactive oxygen species (ROS), the total antioxidant capacity (TAC)
as well as the responses of antioxidant defences such as the activity of the so-called
antioxidant enzymes such as catalase (CAT; transform H 2 O 2 in H 2 O and O 2 ),
superoxide dismutase (SOD) involved in scavenging superoxide (O
2-Á
) radicals,
glutathione peroxidases (GPX) and guaiacol peroxidase (POD) involved in the
reduction of a broad range of organic peroxides to the corresponding alcohols.
Other enzymes with a dual role (antioxidant and phase II conjugation reaction) are
the activities of glutathione S-transferase (GST) mainly involved in detoxification
reactions and glutathione reductase (GR) engaged in maintaining the levels of
reduced glutathione (GSH) from its oxidised disulphide form (GSSG) with a relevant antioxidant role. Endogenous molecules such as GSH and other molecular
scavengers such as β-carotene and vitamins C and E also have a described
antioxidant role.
Constitutive stress proteins, and in particular the heat shock proteins
(e.g. HSP70), are also inducible and involved in the protection and repair of these
cell biomolecules’ integrity under multiple stress conditions, including heat. When
the cell’s natural defence mechanisms are overwhelmed, oxidation of biological
molecules such as lipids occurs. This is known as lipid peroxidation (LPO), and the
analytical assay (thiobarbituric acid reactive species or TBARS) to measure LPO
quantifies the malondialdehyde (MDA) levels. In the case that oxidised molecules
are proteins, its oxidation is measured as protein carbonyl (PC) formation. If the
genetic material is that affected, then DNA damage occurs and can be measured in
several ways, by the electrophoretic comet assay or single gel electrophoresis (SGE),
by the formation of the oxidised nucleoside 8-hydroxy-2-deoxyguanosine (8-OHdG)
using ELISA techniques and by measuring DNA degradation in the late stages of
apoptosis by the TUNEL assay, amongst others. In earthworms, this effect biomarker (DNA damage) is especially relevant since it can be easily measured in
coelomocytes as a validated non-destructive technique. Another effect marker
widely used to measure damage occurrence in biological membranes is the lysosomal membrane stability (LMS) and the neutral red retention (NRR) assay a
frequent method of evaluation. Immunology assessment is considered to be a general
stress marker and has the advantage of being easily measured in biological fluids
such as coelomocytes and can thus also be classed as a conservative technique.
Histological alterations are morphometric measures considered signs of unequivocal
damage consequences to higher hierarchy levels of biological organisation.
Although damage evaluation is the most common measure included in many
ecotoxicity studies with earthworms, this chapter places more emphasis on early
warning biomarker responses such as those experienced by biochemical and molecular parameters. Some reviews have comprehensively addressed each particular
biomarker in earthworms: ecogenotoxicity [14], genotoxicity, immune system,
histology and antioxidant protective defences were addressed by Roubalova et al.
[15] and esterases by Sanchez-Hernandez [6]. Other novel biomarkers applied to
Biomarkers in Earthworms
315