Comparative Biochemistry and Physiology, Part C 140 (2005) 175 – 186

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Antioxidant enzyme activities and lipid peroxidation in the freshwater

cladoceran Daphnia magna exposed to redox cycling compounds
Carlos Barataa,T, Inma Varob, Juan Carlos Navarrob, Solayan Arunc, Cinta Portec
a
Laboratory of Environmental Toxicology, INTEXTER-UPC, CN 150, Km 14.5, 08220 Terrassa, Barcelona, Spain
b
Instituto de Acuicultura Torre de la Sal (CSIC), Ribera de Cabanes 12595, Castellón, Spain
c
Department of Environmental Chemistry, IIQAB, (CSIC), Jordi Girona 18, Barcelona 08034, Spain

Received 21 September 2004; received in revised form 18 January 2005; accepted 20 January 2005

Abstract

Contaminant-related changes in antioxidative processes in the freshwater crustacea Daphnia magna exposed to model redox cycling
contaminant were assessed. Activities of key antioxidant enzymes including catalase, superoxide dismutase, glutathione peroxidase and
glutathione S-transferases and levels of lipid peroxidation measured as thiobarbituric acid-reactive substances (TBARS) and lipofucsin
pigment content were determined in D. magna juveniles after being exposed to sublethal levels of menadione, paraquat, endosulfan,
cadmium and copper for 48 h. Results denoted different patterns of antioxidant enzyme responses, suggesting that different toxicants may
induce different antioxidant/prooxidant responses depending on their ability to produce reactive oxygen species and antioxidant enzymes to
detoxify them. Low responses of antioxidant enzyme activities for menadione and endosulfan, associated with increasing levels of lipid
peroxidation and enhanced levels of antioxidant enzyme activities for paraquat, seemed to prevent lipid peroxidation, whereas high levels of
both antioxidant enzyme activities and lipid peroxidation were found for copper. For cadmium, low antioxidant enzyme responses coupled
with negligible increases in lipid peroxidation indicated low potential for cadmium to alter the antioxidant/prooxidant status in Daphnia.
Among the studied enzymes, total glutathione peroxidase, catalase and glutathione S-transferase appeared to be the most responsive
biomarkers of oxidative stress.
D 2005 Elsevier Inc. All rights reserved.

Keywords: Oxidative stress; Daphnia; Paraquat; Endosulfan; Menadione; Cadmium; Copper; Antioxidant enzymes

1. Introduction carbon quinones, nitro-aromatics, biphenyls, Livingstone,

Aquatic organisms are currently being exposed to multi-
ple chemical contaminants with different mechanisms of
toxicity, each contributing to a final overall adverse effect.
Consequently, in ecological quality monitoring programs,
the integration of chemical data with biological responses
(biomarkers) is strongly recommended to characterize
effects of contaminants to organisms. At the biochemical
level, biomarkers include studies on enhanced production of
reactive oxygen species (ROS) as a general pathway of
toxicity induced by many redox cycling chemicals (hydro-
T Corresponding author. Tel.: +34 93 7398396; fax: +34 93 7398392.
E-mail address: barata@intexter.upc.es (C. Barata).
1532-0456/$ - see front matter D 2005 Elsevier Inc. All rights reserved.
doi:10.1016/j.cca.2005.01.013
1991; transition metals, Stohs and Bagghi, 1995), and many
other compounds leading to a condition of oxidative stress.
S
These ROS include superoxide anion radical (O2!), hydro-
gen peroxide (H2O2) and the highly reactive hydroxyl
radical (OHS).
To minimize oxidative damage to cellular components,
organisms have developed antioxidant defenses. Important
antioxidant enzymes are the enzymes superoxide dismutase
S
(SOD, EC 1.15.1.1 converts O2! to H2O2), catalase (CAT,
EC 1.11.1.6 reduces H2O2 to water) and glutathione
peroxidase (GPX, EC 1.11.1.9 detoxifies H2O2 or organic
hydroperoxides produced, for example, by lipid peroxida-
tion; Di Giulio et al., 1995; Halliwell and Gutteridge, 1999).
Glutathione S-transferases (GST, EC 2.5.1.18) catalyses the
conjugation of glutathione (GSH) with various electrophilic
176 C. Barata et al. / Comparative Biochemistry and Physiology, Part C 140 (2005) 175–186
substances and plays a role preventing oxidative damage by
conjugating breakdown products of lipid peroxides to GSH
(Ketterer et al., 1983). Some GST isozymes display
peroxidase activity (Ketterer et al., 1983; Hayes and
Pulford, 1995).
Organisms can adapt to increasing ROS production by
up-regulating antioxidant defences, such as the activities of
antioxidant enzymes (Livingstone, 2003). Failure of anti-
oxidant defences to detoxify excess ROS production can
lead to significant oxidative damage including enzyme
inactivation, protein degradation, DNA damage and lipid
peroxidation (Halliwell and Gutteridge, 1999). In particular,
lipid peroxidation is considered to be a major mechanisms,
by which oxyradicals can cause tissue damage, leading to
impaired cellular function and alterations in physico-
chemical properties of cell membranes, which in turn
disrupt vital functions (Rikans and Hornbrook, 1997). Lipid
peroxides are known to decompose and produce a variety of
substances, the most important of which is malondialdehyde
(MDA; Leibovitz and Siegel, 1980). MDA is incorporated
into various large fluorescent biomolecules, which accumu-
late in the cells. These lipid soluble fluorescent products
also known as lipofucsin are regarded as good biomarkers of
age and of the degree of oxidative stress in animals (Sohal,
1981).
Most studies on ROS production and effects have been
conducted at the sub-organismal level and limited to
vertebrate species. Studies that have demonstrated potential
for ROS production, antioxidant defense responses and
oxidative damage in aquatic invertebrates are restricted to
few species, and information relating biochemical effects
with individual level responses is scarce or absent (see
reviews of Di Giulio et al., 1995; Livingstone, 2001).
Therefore, validation of prooxidant and antioxidant bio-
chemical biomarkers as early indicators of toxic stress to
aquatic biota require those systems to be characterized in
more invertebrate species and their ecological relevance to
be established.
Daphnia magna is among the most sensitive studied
organism to toxic chemicals, it is widely used in aquatic
risk assessment and it is broadly distributed within
freshwater habitats (Baird and Barata, 1998). It offers
therefore an unique opportunity to assess the relationship
between antioxidant defences and toxicity responses,
which may help to understand the physiological role of
these systems in related species. Antioxidant enzymes are
an important protective mechanisms against ROS and, like
many other biochemical systems, their responses and
effectiveness may vary across contaminants and species
(Halliwell and Gutteridge, 1999; Livingstone, 2001). In
Daphnia species, recent studies have reported antioxidant
enzyme responses including CAT, SOD, GST and oxida-
tive tissue damage (lipid peroxidation) to UV radiation and
varying oxygen concentration (Vega and Pizarro, 2000;
Borgeraas and Hessen, 2000, 2002a,b), but information on
antioxidant defensive systems against prooxidant contam-
inants is absent. In molluscs and other crustacean species,
however, oxyradical production as a polluted-mediated
mechanism of toxicity and induction of antioxidant
enzyme defences and lipid peroxidation have been
observed in individuals exposed to many contaminants
(i.e. copper, menadione and paraquat; Livingstone et al.,
1990, 1992; Livingstone, 2001; Brouwner and Brouwner,
1998; Correia et al., 2002).
The development of biomarkers of oxidative stress as a
polluted-mediated mechanism of toxicity require knowl-
edge of how antioxidant biochemical systems and target
molecules are influences by model pollutants. The aim of
this study is to use the Cladoceran crustacea D. magna as
a model organism to study biochemical responses of
oxidative stress against redox cycling chemicals and their
toxicity consequences. For this purpose, we undertook the
study of the variation in whole body antioxidant enzyme
activities and lipid peroxidation of D. magna juveniles
exposed to five model redox cycling compounds including
the metals cadmium and copper, the pesticides endosulfan
and paraquat and the quinone menadione. Copper may act
as a catalyst for the Fenton reaction, facilitating the
conversion of superoxide anion and hydrogen peroxide
to hydroxyl radical, a species frequently proposed to
initiate lipid peroxidation (Stohs and Bagghi, 1995).
Cadmium does not appear to generate free radicals, but
does elevate lipid peroxidation in tissues soon after
exposure (Stohs and Bagghi, 1995). Research into the
mechanisms of toxicity of paraquat has identified several
toxic outcomes of redox cycling reaction including the
generation of superoxide anion, which can lead to the
formation of more toxic ROS such as hydrogen peroxide
or hydroxyl radical and the formation of lipid peroxides
(Suntres, 2002). Cyclodiene class of pesticides, including
endosulfan, are known to induce oxidative stress tissue
damage resulting from the release of ROS (Hincal et al.,
1995). Menadione (2-methyl-1,4-naphthoquinone, vitamin
K3) is a quinone-containing compound that like other
quinones may undergo one electron reduction leading to
the formation of very unstable free radical substances that
react rapidly with oxygen, with formation of ROS
(Livingstone et al., 1989).
2. Materials and methods
2.1. Chemicals
Chemicals used for toxicity bioassays were cadmium
(Cd) and copper (Cu) prepared from analytical reagent grade
salts (99% purity, 3CdSO4d 8H2O and CuSO4d 5H2O,
respectively), paraquat (1,1V-dimethyl-4,4V-bipyridinium
dichloride hydrate, 95% purity), menadione (2-methyl-1,4-
naphthoquinone, vitamin K3, 95% purity) from Aldrich
Chemicals (Gillingham, UK) and endosulfan (alpha and
beta, 99.5% purity) from Labor Dr. Ehrenstorfer-Schafer
 C. Barata et al. / Comparative Biochemistry and Physiology, Part C 140 (2005) 175–186
GmbH (Augsburg, Germany). Enzymatic activities, lipid
peroxidation and protein assays were performed with
xanthine oxidase (EC 1.2.4.22), horse heart cytochrome c,
hypoxanthine, reduced glutathione (GSH), cumene hydro-
peroxide, glutathione reductase (EC 1.6.4.2), H2O2, h-
nicotinamide adenine dinucleotide phosphate (NADPH),
butylated hydroxytoluene (BHT), 1,1,3,3-tetramethoxypro-
pane (97% purity), quinine sulphate (99% purity) and serum
albumin from Sigma-Aldrich (St. Louis, MO, USA). All
other chemicals were analytical grade and were obtained
from Merck (Darmstadt, Germany).
2.2. Experimental animals and toxicity tests
To minimise variability in the response to the studied
chemicals, a single laboratory clone of D. magna Straus
cultured in the laboratory since 1995 was selected for this
study (Baird and Barata, 1998). Bulk cultures of 15 animals
each were maintained in ASTM hard synthetic water as
described by Barata et al. (2000). Animals were fed daily
with Scenedesmus subspicatus (corresponding to 2 mg C/L;
Boersma, 1995). The culture medium was changed every
other day and neonates were removed within 24 h. From
200 to 250 neonates were then transferred to 4-L tanks and
reared under the same conditions as their mothers until they
reached their fourth instar (4–5 days at 20 8C). At this stage,
groups of juveniles were used for enzymatic and toxicity
studies.
2.3. Toxicity bioassays
To determine toxicity, enzymatic activities and lipid
peroxidation in D. magna individuals, exposure to the
studied compounds was conducted by using acute toxicity
bioassays. Lethal, enzymatic and lipid peroxidation
responses were obtained after 48-h exposures of 30
juveniles in 300 mL ASTM hard water to 3–6 concen-
trations of the studied chemicals in the absence of food.
Each treatment was replicated from 3 to 6 times. Due to the
large sample size required for biochemical determinations,
enzymatic and lipid peroxidation analysis were performed in
two consecutive experimentally identical trials. Acetone
(HPLC grade b1 mL/L) was used as a carrier for menadione
and endosulfan treatments including controls. Preliminary
experiments showed that acetone at this concentration did
not affect survival neither biochemical responses. Stock
solutions of Cd and Cu were prepared by adding analytical
reagent grade salts (i.e. 3CdSO4d 8H2O and CuSO4d 5H2O,
respectively) to deionised water (Milli-Q; 18 MV cm!1
resistivity). Nominal test concentrations of each metal were
subsequently prepared by adding appropriate aliquot vol-
umes of each metal stock solution to the synthetic ASTM
test water. For paraquat, stock and test solutions were
prepared in ASTM hard water. Experiments were performed
following established OECD protocols (Barata et al., 2001).
At the end of experiments, animals were counted and
177
collected to determine enzymatic activities, lipid peroxida-
tion and lethal concentration effects.
2.4. Sample preparation
Preliminary studies were performed in order to optimize
the methodology involving the use of whole body D. magna
individuals. Groups of 20 juveniles for CAT and GST
analysis, and of 60 juveniles for SOD, GPX and lipid
peroxidation determination surviving chemical exposure
were pooled in a microcentrifuge tube and immediately
frozen at !80 8C until further biochemical analysis.
2.5. Antioxidant enzymes analyses
Juveniles (pooled wet mass 0.04–0.12 g) were homo-
genized at 4 8C in 1:4 wet wt./buffer volume ratio in 100
mM phosphate buffer, pH 7.4 containing 100 mM KCl and
1 mM EDTA. Homogenates were centrifuged at 10,000"g
for 10 min and the supernatants were immediately used as
enzyme sources.
Biochemical measurements were carried out using a
dual-beam spectrophotometer (Uvikon 941) at 25F0.5 8C.
Assays were run at least in duplicate. CAT activity was
measured by the decrease in absorbance at 240 nm due to
H2O2 consumption (e=40 M!1 cm!1) according to Aebi
(1974). The reaction volume was 1 mL and contained 50
mM phosphate buffer, pH 6.5, 50 mM H2O2 (Ni et al.,
1990). SOD activity was determined by an indirect method
involving the inhibition of cytochrome c reduction. The
S
reduction of cytochrome c by O2! is monitored by the
absorbance increase at 550 nm (Mc Cord and Fridovich,
1969). The reaction volume was 1 mL and contained 50
mM phosphate buffer, pH 7.8, 0.1 mM EDTA, 50 AM
hypoxanthine, 5.6 mU xanthine oxidase and 10 AM
cytochrome c (Livingstone et al., 1990). The results of
this enzymatic assay are given in units of SOD activity per
milligram of protein (U mg!1), where 1 U of SOD is
defined as the amount of sample causing 50% inhibition of
cytochrome c reduction. GPX activity was measured as
described by Livingstone et al. (1992). The reaction
mixture contained 100 mM phosphate buffer (pH 7.5), 2
mM GSH, 2 U glutathione reductase, 0.12 mM NADPH,
sodium azide (0.5 mM), 0.2 mM H2O2 and 3 mM cumene
hydroperoxide (CHP). GPX activity was monitored by
following the decrease in NADPH concentration (at 340
nm), which is consumed during the generation of GSH
from oxidized glutathione (e=6.2 cm!1 M!1), using H2O2
(Se-dependent activity) and cumene hydroperoxide (total-
GPX) as substrate. GST activity towards 1-chloro-2,4-
dinitrobenzene (CDNB) was measured as described by
Habig et al. (1974). The reaction mixture contained 100
mM phosphate buffer (pH 7.5), 1 mM CDNB and 1 mM
GSH. The formation of S-2,4-dinitrophenyl glutathione
conjugate was evaluated by monitoring the increase in
absorbance at 340 nm. Proteins were measured by the
178 C. Barata et al. / Comparative Biochemistry and Physiology, Part C 140 (2005) 175–186
method of Lowry et al. (1951) using serum albumin as
standard.
2.6. Lipid peroxidation analysis
Lipid peroxidation measured by the thiobarbituric
reactive species (TBARS) assay, which measures the
production of malondialdehyde (MDA) that reacts with
thiobarbituric acid (Ohkawa et al., 1979), and lipofucsin
levels (Sohal, 1981), were determined from total lipid
extracts to account for differences in total lipid content
across treatments (Oakes and Van Der Kraak, 2003). Both
biomarkers were determined in a Hitachi F-2500 fluo-
rescence spectrophotometer (Hitachi, Tokyo, Japan). Lipids
were extracted from lyophilised samples of whole body
daphnids (pool wet mass 0.1–0.2 g, n=3) by homogeni-
sation in 2 mL ice-cold chloroform/methanol (2:1 v/v) plus
0.01 (w/v) butylated hydroxytoluene (BHT) as an anti-
oxidant, following a modification of the method of Folch
et al. (1957). After homogenisation, 0.25 vol. of 0.88%
KCl were added to the homogenates and the solution
mixed. After phase separation, the chloroform layer was
removed, filtered and the solvent evaporated by flushing
with nitrogen. Total lipids were determined gravimetrically
(Mettler, 0.1 mg) and stored in chloroform/methanol (2:1
v/v) with 0.01% of BHT added as an antioxidant at a final
concentration of 10 Ag/AL. TBARS were measured
basically according to Oakes and Van Der Kraak (2003)
using total lipid extracts instead of fresh tissue. Subsam-
ples (100 Ag) of total lipid extracts were then treated with
32 AL of 8.1% sodium dodecyl sulfate, 280 AL of 20%
acetic acid (pH 3.5) and 280 AL of thiobarbituric acid. The
mixture was made up to 0.7 mL with distilled water and
then heated for 60 min in boiling water. After cooling, 175
mL of distilled water and 875 AL of the mixture of n-
butanol and pyridine (15:1 v/v) were mixed and shaken
vigorously before centrifugation at 4000"g for 10 min.
The supernatant (organic layer) was taken and its
fluorescence measured at an excitation/emission wave-
length of 530/550 nm, with a slit arrangement of 5 and
sensitivity of 700. TBARS concentrations were derived
from an external standard curve of 1,1,3,3-tetramethox-
ypropane and the values expressed in nanomoles per
milligram of total lipid (Oakes and Van Der Kraak, 2003).
Lipofucsin was measured according to Mourente and Dı́az-
Salvago (1999) and Sukhotin et al. (2002), using the same
total lipid extracts obtained from lipid analysis, since the
extraction procedures are almost identical. For measure-
ment of fluorescence pigments, aliquots of 100 Ag of total
lipid extract in chloroform/methanol (2:1 v/v) plus BHT
were diluted to 500 AL of the same solution and their
fluorescence measured. An emission spectrum between
350 and 550 nm was obtained at an excitation wavelength
of 350 nm. The luminescence of the sample was
determined at an emission maximum of 445 nm. Lip-
ofucsin levels were expressed as relative fluorescence
intensity according to Hill and Womersley (1991), using
0.1 quinine sulphate per 1 mL N H2SO4 as a standard per
mg of total lipid extracted. The linearity of the lumines-
cence response over the range of the fluorescence inten-
sities produced by D. magna extracts was confirmed by
using serial dilutions of the samples.
2.7. Statistical analysis
All measurements were performed in triplicate or
sixtuplicate and the results reported as meansFS.E.M.
Analyses of variance (one-way ANOVA) followed by
Tukey’s post-hoc multiple comparison tests were per-
formed to determine toxicant dependent effects on each
biochemical parameter studied. Bivariate Pearson correla-
tion analysis was also performed between exposure treat-
ments of the studied model compounds, enzyme activities
and end-products/lipid peroxidation to identifying patterns
of response of the studied oxidative stress indicators.
Significant differences were established at Pb0.05. Prior to
any analysis, data were tested for normality and variance
homoscedasticity and were log e transformed when
necessary (Zar, 1996). Lethal concentration values were
determined from probit analysis (Finney, 1971).
3. Results
Lethal concentration effects of the studied compounds on
D. magna juveniles expressed as nominal concentrations are
depicted in Table 1. Cd was the most toxic substance to
Daphnia juveniles followed by Cu, menadione, endosulfan
and paraquat. In relation to this, low and high sublethal
exposure levels were selected to study enzymatic and lipid
peroxidation responses. It is important to notice that,
although for paraquat, endosulfan and Cd some mortality
occurred at the highest exposure levels tested, only animals
alive were used for biochemical determinations.
Enzyme activity responses varied across chemicals.
SOD and CAT, which are considered the most important
antioxidant enzyme systems in invertebrate species,
showed different responses across chemicals (Fig. 1).
SOD activity was equally inhibited at all endosulfan
exposure levels, increased significantly under Cd exposure
and remained unchanged in Daphnia juveniles exposed to
Table 1
Lethal concentration effects (48-h LC 50) expressed as nominal concen-
tration of the five studied model compounds in D. magna juveniles
Compound LC 50 (Ag L!1)
Menadione 491.3 (350.2–709.3)
Paraquat 11320.1 (924.3–14560.4)
Endosulfan 950 (654.5–12154.0)
Cd 6.26 (5.6–8.12)
Cu 37.9 (20.5–61.6)
95% confidence intervals are shown in brackets.
 C. Barata et al. / Comparative Biochemistry and Physiology, Part C 140 (2005) 175–186 179

SOD CAT
(Units min-1 mg-1 protein) (µmol min-1 mg-1 protein)
25
600
20
15 400 c
a b
10
200
5
0 0
0 50 200 0 50 200
Menadione (µg L-1) Menadione (µg L-1)
25 d
600
b
20 b c
a a b
15 400
a
10
200
5
0 0
0 2 5 10 0 2 5 10
Paraquat (mg L-1) Paraquat (mg L ) -1

25
600
20
a 400
15 b b b b
10
200
5
0 0
0 200 400 600 800 0 200 400 600 800

Endosulfan (µg L-1) Endosulfan (µg L-1)

25
b b
20 400
a a a
15 a a
b
10 200

5
0 0
0 1 2 5 0 1 2 5

Cd (µg L-1) Cd (µg L-1)

25
20 400
ab b
15 ab a a
a
10 200
5
0 0
0 5 20 0 5 20

Cu (µg L-1) Cu (µg L-1)

Fig. 1. Activity of superoxide dismutase (SOD, left panel of graphs) and catalase (CAT, right panel of graphs) in D. magna juveniles exposed to menadione,
paraquat, endosulfan, Cd and Cu. Values are expressed as meanFS.E.M. (number of pools n=3–6). Significant differences ( Pb0.05) among exposure
treatments after Tukey’s post-hoc multiple comparison tests are followed by different letters.
180 C. Barata et al. / Comparative Biochemistry and Physiology, Part C 140 (2005) 175–186

Total GPx GST

(nmol min-1 mg-1 protein) (nmol min-1 mg-1 protein)
200
400
c
150
a b
100 a 200
b
50 c

0 0
0 50 200 0 50 200
Menadione (µg L-1) Menadione (µg L-1)
200
b b 400
b c
150
b
a a
100 200
a
50

0 0
0 2 5 10 0 2 5 10
Paraquat (mg L-1) Paraquat (mg L-1)

200
400 c d
150 b a b
a
a a ab
100 a 200
50

0 0
0 200 400 600 800 0 200 400 600 800

Endosulfan (µg L-1) Endosulfan (µg L-1)

200
400
150 b a a ab b

a a a
100 200
50

0 0
0 1 2 5 0 1 2 5
-1) L-1)
Cd (µg L Cd (µg
200
c 400
150 ab b
a
100
b
a 200
50

0 0
0 5 20 0 5 20

Cu (µg L-1) Cu (µg L-1)

Fig. 2. Activity of total glutathione peroxidase (total-GPX, left panel of graphs) and glutathione S-transferase (GST, right panel of graphs) in D. magna
juveniles exposed to menadione, paraquat, endosulfan, Cd and Cu. Values are expressed as meanFS.E.M. (number of pools n=3–6). Significant differences
( Pb0.05) among exposure treatments after Tukey’s post-hoc multiple comparison tests are followed by different letters.
 C. Barata et al. / Comparative Biochemistry and Physiology, Part C 140 (2005) 175–186
menadione. Daphnia juveniles exposed to paraquat had
greater SOD activities at the lowest and highest concen-
tration tested, whereas those exposed to Cu only showed a
marginal but not significant increase of SOD activity at 20
Ag L!1. CAT activity increased significantly under mena-
dione, paraquat and copper, remained unchanged in indi-
viduals exposed to endosulfan and decreased at high
exposure levels of Cd.
Glutathione-related antioxidant activities are depicted in
Fig. 2. Se-GPX activity, although detected (i.e. 3–7 nmol
min!1 mg!1 protein), was too close to detection limit of the
assay, to be considered with confidence as a responsive
marker. Total-GPX activity increased two- to three-fold
relatively to control animals under exposure to paraquat, Cd
and Cu, and to a lesser degree under endosulfan and Cd.
Alternatively, total-GPX activity decreased under mena-
dione exposure. GST activity increased under exposure to
the five studied chemicals with organic chemicals inducing
greater GST activity responses than metals.
Lipid peroxidation-related measurements are depicted in
Table 2 and Fig. 3. For paraquat and endosulfan, lipid
measurements were not conducted at the highest concen-
trations tested (10 mg/L and 800 Ag/L, respectively) since,
due to the existence of some mortality (10–20%), minimum
sample size was not achieved. For cadmium, an accidental
lost of the sample at 1 Ag/L prevented this treatment to be
included in the analysis. Lipid content in whole D. magna
juveniles changed little across treatments with mean values
(% total lipids of dry weight) varying from 9.6% to 13%
(Table 2). Moreover, juveniles exposed to Cd and paraquat
presented significant lower levels of lipids than those in
control treatments. Lipid peroxidation measured as TBARS
or/and lipofucsin levels tend to increase under exposure to
the five studied chemicals, although only under menadione,
Table 2
Lipid content expressed as % total lipid of dry weight of D. magna
juveniles exposed to the studied compounds
Compound Concentration % Lipid content (%)
Menadione (Ag/L) 0 11.9F0.2 a
50 11.2F0.9 a
200 10.8F0.6 a
Paraquat (mg/L) 0 12.7F0.6 a
2 10.0F0.1 b
5 10.1F0.1 b
Endosulfan (Ag/L) 0 11.7F0.2 a
200 12.3F1.0 a
400 11.9F1.4 a
600 10.5F0.3 a
Cd (Ag/L) 0 11.9F0.2 a
2 10.2F0.3 b
5 9.6F0.2 b
Cu (Ag/L) 0 12.1F0.1 a
5 13.0F1.0 a
20 11.2F0.9 a
Values are expressed as meanFS.E.M. (number of pools n=3). Significant
differences ( Pb0.05) among exposure treatments after Tukey’s post-hoc
multiple comparison tests are followed by different letters.
181
endosulfan and Cu exposure, D. magna juveniles showed
significant greater lipid peroxidation levels than control
animals.
The above-mentioned results were summarized in Table
3 where inter-individual correlations between exposure
treatments of the studied model compounds, enzyme
activities and end-products/lipid peroxidation denoted dif-
fering patterns of response of the studied oxidative stress
indicators across model toxicants.
4. Discussion
The enzymes included in the study differed in their
responses to prooxidative chemicals, with total-GPX and
GST being the most responsive enzymes followed closely
by CAT and SOD. In invertebrate species, SOD and CAT are
considered to play a greater antioxidant role than GPX,
whereas for vertebrate species the opposite is true (Simmons
and Jamall, 1988; Livingstone et al., 1992; Halliwell and
Gutteridge, 1999). Nevertheless, GPX recently has been
shown to occur within many invertebrate species (Table 2 in
Correia et al., 2003), playing an important antioxidant role
against exogenous ROS (Doyotte et al., 1997; Brouwner
and Brouwner, 1998; Geret et al., 2002; Geracitano et al.,
2002). Two different GPX have been identified in animal
tissues of vertebrate and invertebrate species (Aceto et al.,
1994, Arun et al., 1999), one reduces hydrogen peroxide
and organic hydroperoxides (Se-dependent activity),
whereas the other one reduces only organic hydroperoxides
(Se-independent activity). In our study, although Se-depend-
ent GPX activity was detected, it was too low to be
considered with confidence so only total-GPX including
both activities was reported. The results obtained for
glutathione peroxidase- and transferase-related activities
indicate that these enzymes may play a crucial and probably
an equally important role than CAT and SOD in the
Daphnia antioxidant defensive system. Indeed, except
total-GPX under menadione exposure, glutathione-related
enzymes increased in response to the studied chemicals,
with total-GPX activity increasing up to three fold under
paraquat and Cu, and GST activities being significantly
induced by all five compounds. In a previous study (Barata
et al., 2005), it was shown that D. magna antioxidant
enzyme activities including SOD, CAT and total-GPX were
in agreement with reported values in other invertebrate
species including crustaceans. Furthermore, the above
antioxidant enzyme activities were affected by endogenous
ROS during aging (Barata et al., 2005). In this study, it was
found that SOD, CAT, total-GPX and GST also responded
to exogenous chemical sources of ROS. In Daphnia species,
recent studies have reported antioxidant enzyme responses
including CAT, SOD, GST and oxidative tissue damage
(lipid peroxidation) to UV radiation, but information on
antioxidant defensive systems against prooxidant contami-
nants is absent (Vega and Pizarro, 2000; Borgeraas and
182 C. Barata et al. / Comparative Biochemistry and Physiology, Part C 140 (2005) 175–186

TBARS Lipofucsin content

(nmol mg-1 lipid) (RFI mg-1 lipid)
8 4
c
6
b
4 2 a

0 0
0 50 200 0 50 200
Menadione (µg L-1) Menadione (µg L-1)
8 4

4 2

0 0
0 2 5 0 2 5
-1 -1
Paraquat (mg L ) Paraquat (mg L )

8 4

6
ab b
2 a ab
4

0 0
0 200 400 600 0 200 400 600

Endosulfan (µg L-1) Endosulfan (µg L-1)

8 6

6
4
4
2
2

0 0
0 2 5 0 2 5
-1 -1
Cd (µg L ) Cd (µg L )
8 6
b
6
4
a a b
4
2
a a
2

0 0
0 5 20 0 5 20

Cu (µg L-1) Cu (µg L-1)

Fig. 3. Levels of lipid peroxide measured as TBARS (left panel of graph) and lipofucsin detected as relative fluorescence intensity (right panel of graphs) in
lipid extracts of D. magna juveniles exposed to menadione, paraquat, endosulfan, Cd and Cu. Values are expressed as meanFS.E.M. (number of pools n=3–6).
Significant differences ( Pb0.05) among exposure treatments after Tukey’s post-hoc multiple comparison tests are followed by different letters.
 C. Barata et al. / Comparative Biochemistry and Physiology, Part C 140 (2005) 175–186
Table 3
Inter-individual bivariate Pearson correlations between enzymatic activities, lipid peroxidation levels and exposure concentrations of the studied model
compounds
SOD CAT GST
Menadione !0.45 (9) 0.85 (9)T 0.82
Paraquat 0.56 (12)T 0.97 (12)T 0.89
Endosulfan !0.61 (25) 0.05 (25) 0.70
Cadmium 0.80 (12)T !0.40 (12) 0.62
Copper 0.33 (9) 0.51 (9) 0.75
Sample size is depicted between brackets. Variations in sample size are due to the use of different number of replicates and exposure treatments across model
compounds and biochemical parameters. For abbreviations, see text.
T Indicates significant correlations at Pb0.05.
Hessen, 2000, 2002a,b); thus, our results will be discussed
with existing information in other invertebrate and vertebrate
species.
Menadione, paraquat and Cu are potent redox cyclers (Di
Monte et al., 1986; Sandy et al., 1986; Livingstone et al.,
1990; Stohs and Bagghi, 1995), which are expected to
S
generate superoxide anion radical (O2!), hence enhancing
S!
the activities of SOD to dismutate O2 to H2O2, and of CAT
and GPX to further detoxify H2O2 and organic hydro-
peroxides (Halliwell and Gutteridge, 1999). For Cd and
endosulfan, however, little is known about the exact
mechanisms producing oxidative stress (Stohs and Bagghi,
1995; Hincal et al., 1995). In this study, SOD activity was
significantly induced by paraquat and Cd, marginally
increased by Cu, unaffected by menadione and inhibited
by endosulfan. Overall SOD activity was steady or varied
without a clear pattern across exposure levels (i.e. paraquat,
Cu), which may indicate that transient rather than main-
tained increases of SOD occurred. In agreement with the
previous argument, Wenning et al. (1988) found in mussels
that SOD responses to paraquat were maximal after 6–12 h
and declined to control levels by 24 h. Livingstone et al.
(1990), however, found in mussels that SOD responses to
menadione were only induced after 6 days.
CAT and total-GPX activity patterns, which are consid-
ered a second line of defense against ROS, varied across the
studied compounds. CAT activities and/or total-GPX
increased to some extent under exposure to most of the
studied compounds, thus denoting the ability of the studied
chemicals to enhance the production of hydrogen peroxide
or/and organic hydroperoxides. CAT and GPX have
complementary roles in hydrogen peroxide detoxication,
having different subcellular localisations (peroxisomal and
cytosolic, respectively) and target molecules (reduction of
H2O2 for CAT and H2O2 and toxic hydroperoxides for
GPX), with CAT and GPX showing the highest affinities for
high and low H2O2 levels, respectively (Halliwell and
Gutteridge, 1999; Orbea et al., 2000). Thus, CAT and GPX
activity patterns are expected to vary across redox cycling
compounds depending on the nature, amount and subcel-
lular localization of ROS produced.
Menadione increased CAT but inhibited Total-GPX
activities in a concentration-related manner. Alternatively,
endosulfan did not alter CAT, increasing total-GPX activity
183
Total-GPX TBARS LIPOFUCSIN
(9)T !0.87 (9)T 0.64 (9) 0.95 (9)T
(12)T 0.78 (12)T 0.21 (9) !0.43 (9)
(15)T 0.66 (15)T 0.16 (12) 0.64 (12)T
(12)T 0.74 (12)T 0.42 (9) 0.13 (9)
(9)T 0.95 (9)T 0.74 (9)T 0.75 (9)T
only at high concentrations. The distinct activity patterns of
CAT and total-GPX enzymes observed in Daphnia juveniles
exposed to menadione and endosulfan are in agreement with
previous studies. Livingstone et al. (1990) and Stephensen
et al. (2002) found that menadione induced CAT and
inhibited GPX in common mussel and rainbow trout after
two days of exposure. Padey et al. (2001) also reported the
inability of endosulfan to increase CAT activities and its
ability to induce GPX in the fish Channa punctatus.
Enhanced levels of total-GPX activity found in Daphnia
juveniles exposed to high endosulfan levels may also be
related to the enzymatic detoxication of hydroperoxides
produced by increased levels of hydroxyl radicals (Halliwell
and Gutteridge, 1999).
The observed high degree (up to two- and three-fold) of
inducible activity levels observed by total-GPX and CAT
under exposure to paraquat and Cu are in agreement with
reported studies in vertebrate and invertebrate species.
Paraquat up-regulates GPX in mammals (de Haan et al.,
1998) and may enhance CAT activity levels in mussels up to
two-fold (Wenning and Di Giulio, 1988; Wenning et al.,
1988; Livingstone et al., 1990). Cu is also known to
increase the activity of CAT and GPX enzymes in other
invertebrate species including crustacea and worms but not
molluscs (Doyotte et al., 1997; Brouwner and Brouwner,
1998; Geracitano et al., 2002; Geret et al., 2002). Although
Cd is known to produce oxidative damage (lipid perox-
idation) and alter antioxidant enzyme responses, there is no
consensus about its ability to produce ROS (Stohs and
Bagghi, 1995). In haemocytes, however, induction of ROS
by Cd has been reported to occur at moderate levels being
inhibited at high concentrations (Larson et al., 1989;
Gomez-Mendikute and Cajaraville, 2003). Therefore, induc-
tion of SOD and total-GPX activities of Daphnia juveniles
exposed to Cd could be related to ROS production.
All chemicals in our study caused moderate to two fold
increase of GST activity levels, with endosulfan and
menadione being the greater inducers, thus suggesting that,
in Daphnia, GST may be actively involved detoxifying
redox cycling chemicals. The relatively low induction levels
of GST by Cu and Cd relative to menadione and endosulfan
may be related to diminished levels of reduced glutathione
(GSH) susceptible of being conjugated. GSH may act either
as a nonenzymatic antioxidant (i.e. chelating metals) and as a
184 C. Barata et al. / Comparative Biochemistry and Physiology, Part C 140 (2005) 175–186
cofactor or substrate for GPX and GST enzymes, e.g. GSH is
oxidized to GSH disulfite (GSSG) in GPX catalyzed
reactions. To maintain the reduction potential of the cell,
GSSG is reduced back to GSH by glutathione reductase (GR,
EC 1.6.4.2) or exported from the cell, like some GSH-
conjugates, via multidrug resistance associated proteins
(Halliwell and Gutteridge, 1999). Another enzyme respon-
sible for maintaining the GSH pool is g-glutamylcysteine
synthetase (g-GCS, EC 6.3.2.2), the rate-limiting enzyme in
the novo synthesis of GSH. Indeed, it is known that redox
cycling chemicals can increase or decrease the intracellular
GSH pool by inducing conjugation, oxidation or by affecting
GR and g-GCS enzymes (Shi et al., 1994; Mulcahy et al.,
1997; Brouwner and Brouwner, 1998; Stephensen et al.,
2002). Therefore, if intracellular GSH became limiting,
competition for available GSH may modulate the activity of
GPX and GST enzymes (Brouwner and Brouwner, 1998;
Halliwell and Gutteridge, 1999). In relation to this, it is
interesting to note that those chemicals inducing greater
total-GPX activities (paraquat, Cu) and hence oxidizing
more GSH to GSSG also produced low induction levels of
GST, whereas the opposite was true for menadione and
endosulfan. Nevertheless, without measuring intracellular
GSSG and GSH levels, it is difficult to be conclusive.
Failure of antioxidant defenses to remove exogenous
ROS produced by redox cycling chemicals either by being
inhibited by those compounds or overwhelmed by an excess
ROS will disrupt the balance between antioxidant/proox-
idant system within the organisms leading to oxidative
damage (Livingstone, 2003). Although all the studied
chemicals are known to cause lipid peroxidation in many
species (Livingstone et al., 1990; Stohs and Bagghi, 1995;
Suntres, 2002; Dorval et al., 2003), disparity of results
suggest that the efficiency of antioxidant defensive systems
to remove ROS and prevent oxidative stress vary across
species. In this study, oxidative tissue damage was evaluated
determining lipid peroxidation measured as TBARS and
lipofucsin levels to account for the more transient responses
of MDA that tend to be excreted or incorporated into larger
lipofucsin molecules (Sohal, 1981; Sukhotin et al., 2002).
There was evidence for a significant increased lipid
peroxidation measured as TBARS and/or lipofucsin levels
only for menadione, endosulfan and Cu. The high induction
responses of SOD, CAT and total-GPX (depicted as positive
significant correlations) found in Daphnia individuals
exposed to paraquat may have prevented ROS to react with
membrane and other lipids, thus minimizing lipid perox-
idation. On the contrary, observed inhibitory responses
(negative correlations) of the antioxidant enzymes SOD and
total-GPX of individuals exposed to menadione and of SOD
and CAT when exposed to endosulfan may have resulted in
substantial lipid peroxidation. For Cu, low induction levels
of the antioxidant enzymes SOD and CAT (positive but no
significant correlations) may have resulted in an excess of
ROS and hence of lipid peroxidation levels. For Cd,
enhanced activity of SOD and of total-GPX but not of
CAT were also coupled with low or non existing lipid
peroxidation levels, thus suggesting that antioxidant
defenses were able to detoxify excess ROS. The relatively
great potential for Cu to cause oxidative tissue damage
relative to Cd may also be related to the redox cycling
properties of Cu (Stohs and Bagghi, 1995). Therefore, the
above mentioned results suggest that toxicants may induce
different antioxidant/prooxidant responses depending on
their ability to produce ROS and of antioxidant enzymes
to detoxify them (Livingstone, 2003).
Since many environmental contaminants exert toxic
effects related to oxidative stress, this phenomenon may
be an important feature for biomarker development. How-
ever, antioxidant enzymes are generally less responsive to
pollutants than other enzymatic endpoints (e.g. some P450
isoenzymes) and the relationships between response and
contaminant exposure are not well established. Their
function in detoxification processes, however, motivates
continued research on the potential use of antioxidant
enzymes in monitoring programs. Nonetheless, the results
reported in this and other studies indicate that antioxidant
enzyme responses are transient and variable for different
species, enzymes and chemicals (Livingstone, 2001).
Indeed, in field studies, higher, equal or lower activities of
various antioxidant enzymes have been observed in polluted
compared to cleaner areas (Narbonne et al., 1999; Ring-
wood et al., 1999; Porte et al., 2000). Thus, in order to
credibly use antioxidant enzyme activities as reliable
biomarkers of oxidative stress, controlled laboratory experi-
ments are still needed to elucidate enzymatic patterns of
response against different toxicants and species, and to
assess their physiological role detoxifying ROS and hence
preventing the disruption of the organism antioxidant/
prooxidant balance (Livingstone, 2003).
Acknowledgements
This study was funded by the MCyT Spanish projects
REN 2002-01709 and REN 2003-06917-CO2-O2. Carlos
Barata was supported by a Ramon y Cajal contract from
MCyT. The authors thank Miguel Angel Montolio for
helping when conducting lipid analysis.
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