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Sistema de Información Científica
Red de Revistas Científicas de América Latina y el Caribe, España y Portugal
Barbus bocagei
Francisco P. PEIXOTO
, Ana Maria COIMBRA
*, Conceição FERNANDES
Maria Manuel OLIVEIRA
School of Environment and Life Sciences (Escola de Ciências da Vida e do Ambiente-ECVA), University of
Trás-os-Montes e Alto Douro (UTAD), Apartado 1013, 5001-801 Vila Real, Portugal
Center for the Research and Technology of Agro-Environmental and Biological Sciences (Centro de Investi-
gação e de Tecnologias Agro-Ambientais e Biológicas-CITAB), UTAD
Agrarian Superior School (Escola Superior Agrária, Instituto Politécnico de Bragança, Centro de Investigação
de Montanha CIMO), Campus de Santa Apolónia, Apartado 1038, 5301-854 Bragança, Portugal
Chemistry Research Center of Vila Real (Centro de Química de Vila Real-CQVR), UTAD
*Corresponding author:
(Recibido agosto 2011, aceptado octubre 2012)
Key words: oxidative stress enzymes, lipid peroxidation, fsh, Freshwater, liver histopathology
Barbel (
Barbus bocagei
) a common species in Portuguese rivers was studied to as-
sess the impact of water contamination on hepatic oxidative stress response, lipid
peroxidation and histology. The Vizela River is a tributary of the Ave River, located
in one of the most industrialized areas of Portugal. The oxidative stress biomarkers
analyzed included superoxide dismutase, catalase, glutathione
-transferase, glutathione
reductase, glucose 6-phosphate dehydrogenase and xanthine oxidase activities. Levels
of reduced glutathione and lipid peroxidation were also evaluated. Except xanthine
oxidase activity, that did not show any alteration, all the other enzymatic activities
were increased in the liver of barbel captured in the Vizela River when compared with
reference barbel. While, no differences were observed for glutathione reductase content,
lipid peroxidation was higher in barbel from the Vizela River. Liver histological altera-
tions were determined and their severity scored. Though lymphocyte foci were only
observed in Vizela River barbel, macrophage aggregates were also present in reference
barbel, although the severity score was higher in Vizela fsh. The results oF this study
show that barbel liver oxidative stress responses, lipid peroxidation and histology are
sensitive to the contaminants present in Vizela River water and are valuable biomark-
ers for monitoring purposes.
Palabras clave: enzimas de estrés oxidativo, peroxidación lipídica, peces, agua dulce, histopatología hepática
Barbos (
Barbus bocagei
), una especie común en los ríos portugueses, se utilizó para
evaluar el impacto de la contaminación del agua en la respuesta hepática al estrés
oxidativo, en la peroxidación lipídica y en la histología del órgano. El río Vizela es un
Rev. Int. Contam. Ambie. 29 (1) 29-38, 2013
F.P. Peixoto
et al.
afuente del río Ave, situado en una de las regiones más industrializadas de Portugal.
Los biomarcadores de estrés oxidativo analizados fueron la actividad de las enzimas
superóxido dismutasa, catalasa, glutatión S-transferasa, glutation reductasa, glucosa
6 fosfato deshidrogenasa y de la xantina oxidasa. Los niveles de glutatión reducido
y de la peroxidación lipídica también fueron evaluados. Excepto la xantina oxidasa,
que no mostró ninguna alteración, todas las otras actividades enzimáticas han sufrido
incrementos en el hígado de los barbos capturados en el río Vizela, cuando se com-
paran con los barbos de referencia. No se observaron diferencias para el contenido
de glutatión reductasa, pero la peroxidación lipídica fue mayor en los barbos del río
Vizela. Las alteraciones en la histología hepática Fueron identi±cadas y clasi±cadas
de acuerdo con su gravedad. Mientras que los linfocitos de focos se observaron sólo
en barbos del Río Vizela, los agregados de macrófagos también estuvieron presentes
en barbos locales de referencia, aunque la gravedad de las alteraciones fue mayor en
los peces del río Vizela. Los resultados de este estudio muestran que las respuestas de
estrés oxidativo, la peroxidación lipídica y la histología hepática son sensibles a los
contaminantes presentes en el agua del Río Vizela, demonstrando ser biomarcadores
valiosos para propósitos de monitoreo.
The hydrographical basin of the Ave River is
located in Minho region (northwest of Portugal) and
comprises an area of about 1390 km
, being limited
up north by the Cavado River, east by the Douro
River and south by the basin of Leça River. The
Ave River has two main tributaries, the Este River
on the right edge and the Vizela River on left edge.
About 80 % of this basin is overpopulated. Textile
industry is one of the most important economic
activities in this region, with around 200 industrial
units, that include both manufacturing and tanning
units (Alves
et al.
2009). For many decades both
urban and industrial eFfuents were discharged di
rectly into the river basin and the presence of heavy
metals in sediments have already been reported.
et al.
(1999) observed high values of Cr
(0.36-0.67 g/kg volatile matter dry weight (DW)),
Cu (0.46-1.03 g/kg volatile matter DW) and Zn
(0.75-3.70 g/kg volatile matter DW). Ten years later,
et al.
(2009) found values of Cr (2.85-4.45),
Cu (0.62-0.69) and Zn (0.53-1.87). Recently, due to
the European legal requirements and the increasing
public awareness, Portugal has started to control the
quality of industrial discharges. As result, norms, like
ISO 14000 family, and legal contamination standards,
such as the EU Water Framework Directive, are be-
ing implemented in Portugal. Despite this, several
industrial eFfuents are still being discharged directly
into streams without any treatment, and several heavy
metals are present and can still be found in sediments
et al.
Heavy metals are non-degradable and may bio-
accumulate in organisms, possibly reaching toxic
levels. This can constitute a threat to public and/or
aquatic organisms’ health due to chronic exposure
to high concentrations (Fernandes
et al.
2007, Fer-
et al.
2008a, Vieira
et al.
Due to the high degree of pollution of the Ave
River basin, the biota in this aquatic ecosystem has
been progressively degraded. The Iberian barbel
Barbus bocagei
) is one oF the Few ±sh species still
widely distributed in this basin, being benthopelagic
and with a versatile diet (Magalhães 1992), and thus
susceptible to both, sediment and water column,
The evaluation of biochemical and histologi-
cal changes in ±sh liver has become an important
tool to monitor the environmental exposure oF ±sh
to contaminants (Hinton & Lauren 1990, Deviller
et al.
2005, Fernandes
et al.
2008b, Carrola
et al.
2009). Fish liver plays an important role in vital
functions and is the major organ for accumulation,
biotransformation, and excretion of contaminants in
±sh (Triebskorn
et al.
1994, Triebskorn
et al.
Histopathology oF ±sh liver is a monitoring tool,
which allows the assessment of the environmental
stressors eFFects, in ±sh. Indeed, it is one oF the most
reliable indicators of the health impairment induced
by anthropogenic stressors in aquatic organisms
et al.
2008b, Leonardi
et al.
Heavy metal exposure, in aquatic ecosystems, is
described as an enhancer of intracellular formation
of reactive oxygen species (ROS), which can give
rise to oxidative damage, as observed in fathead
grey mullet (
Mugil cephalus
), founder (
) and Nile tilapia (
Oreochromis niloticus
) (Fer-
et al.
2005, Figueiredo-Fernandes
et al.
Thus, oxidative stress biomarkers can be employed
Barbus bocagei
in environmental monitoring programs (McCarthy
and Shugart 1990, Fernandes
et al.
2008c). Lipid
peroxidation and protein oxidation are manifesta-
tions of oxidative damage induced by heavy metals
et al.
1993, Ercal
et al.
2001), and have
a predictive importance as biomarkers of pollution
et al.
2003, Bláha
et al.
2004, Almroth
et al.
2005). In addition, both antioxidant enzymes and
non-enzymatic antioxidants have been successfully
employed in aquatic monitoring studies (Figueiredo-
et al.
2006, Peixoto
et al.
2006). ROS
can be detoxifed by an enzymatic deFence system,
comprising superoxide dismutase (SOD), catalase
(CAT), and selenium-dependent glutathione per-
oxidase; or by a non-enzymatic system, with the
scavenging action of reduced glutathione. Moreover,
organic peroxides can be detoxifed by the activity
of glutathione S-transferase (GST) (Halliwell and
Gutteridge 1999).
The aim of this study was to assess biochemical
and histological biomarkers of exposure in the liver
of the Iberian barbel (
Barbus bocagei
) captured in
Vizela River. The values obtained will serve as base
for future surveys on barbel populations in the Vizela
River and evaluation of the impact of management
policies. Lipid peroxidation in liver and hepatic
activities of superoxide dismutase, catalase, gluta-
thione S-transferase, glutathione reductase, glucose
6-phosphate dehydrogenase, xantine oxidase, and the
amount of reduced glutathione were measured and
compared with the respective activities in reference
barbel. The biochemical evaluation was complement-
ed with hepatic histological analyses and possible
accumulation of metals in this organ was assessed.
Fish sampling
Twenty-four barbel (
Barbus bocagei
) were
captured in the Vizela River (41º22’16.85”N;
8º18’17.86”W) near the city of Caldas de Vizela.
Fish were captured in the autumn of 2009 using
pulsed DC backpack electrofishing equipment
with a DC-500 V generator. Reference barbel
were captured in Corgo River (41º17’14.62”N;
7º44’57.55”W), a low contaminated stream. Bar-
bels, From both locations, were sacrifced at the same
time, after being anaesthetized with 3-aminobenzoic
acid ethyl ester.
Liver sections were frozen in liquid nitrogen and
stored at –80 ºC For biochemical analysis, or fxed in
10 % buffered formalin during 24-48 h for histology.
In addition, a pool of 13 Vizela barbel was randomly
selected and the livers sub-sampled and stored in ep-
pendorfs at –20 ºC, for metals analysis.
Biochemical analysis
All chemicals used in the enzymatic activity were
of analytical purity from Sigma Chemical Co, except
when indicated.
One gram of liver tissue was homogenized in 5
ml of ice-cold sodium phosphate buffer (100 mM,
pH 7.4) and post-mitochondrial supernatant (PMS)
was obtained after centrifugation at 10 500×
for 20
min at 4 ºC.
Superoxide dismutase (SOD) activity was assayed
according to Paya
et al.
(1992) with minor modif
cations (Peixoto
et al.
2006). Nitrotetrazolium blue
chloride (NBT) was used as detection molecule instead
of cytochrome
. Assays were conducted in the pres-
ence of potassium phosphate buffer (100 mM, pH
7.0), hypoxanthine (10 mM), and NBT (10 mM).
The reaction was initiated by the addition of xan-
thine oxidase (0.023 U/mol) to enzymatic extract at
25 ºC. Activity was reported by its ability to inhibit
50 % reduction of NBT and the result is expressed
as U/min/mg/protein.
Catalase (CAT) activity was assayed by the
method Claiborne (1985). The reaction mixture
consisted in potassium phosphate buffer 50 mM, pH
7.4, hydrogen peroxide19 mM and PMS 10 %. The
reaction was carried out at 25 ºC and the change in
absorbance was recorded at 240 nm. CAT activity
was calculated in terms of µmol H
Glutathione reductase (GR) activity was as-
sayed by the method of Carlberg and Mannervik
(1975) as modifed by Mohandas
et al.
(1984). The
reaction system consisted of potassium phosphate
buffer (100 M, pH 7.4), EDTA 0.5 mM, oxidized
glutathione (GSSG) 1 mM, NADPH 0.1 mM and
PMS 10 %. Enzyme activity was quantifed at 25 ºC
by measuring the disappearance of NADPH at 340
nm and expressed as nmol NADPH oxidized/min/
The activity of glucose 6-phosphate dehydro-
genase (G6PD) was assayed by the method of Za-
et al.
(1965). The reaction mixture consisted
of Tris–HCl buffer 50 mM, pH 7.6, nicotinamide
adenine dinucleotide phosphate (NADP) 0.1 mM,
glucose 6-phosphate 0.8 mM, MgCl
8 mM (Merck,
Mumbai), PMS 10 % and 2.1 mL distilled water. The
change in absorbance at 25 ºC was recorded at 340
nm and the enzyme activity was expressed as nmol
NADP reduced/min/mg/protein.
F.P. Peixoto
et al.
-transferase (GST) activity was
measured according to Habig
et al.
(1974) with minor
modifcation. Reaction mixture contained 2 mL oF
potassium phosphate buffer 100 mM, triton X-100
10 %, 1-chloro-2, 4-dinitrobenzene (CDNB) 100 mM,
and GSH 100 mM. Reaction was started at 25 ºC by
adding the sample and the absorbance was monitored
at 340 nm. The GST activity was expressed in nmol
CDNB/min/mg/protein (Uguz
et al.
Xanthine oxidase (XOD) activity was assayed as
described by Stirpe and Dellacor (1969). The reac-
tion mixture containing 0.2 mL PMS diluted to 1 mL
with phosphate buffer and was incubated for 5 min at
25 ºC. The reaction was started by adding xanthine,
kept at 25 ºC for 20 min and stopped by the addition
of ice-cold perchloric acid (10 %). After 10 min, 2.5
mL distilled water was added to it and the mixture
was centrifuged at 4000 rpm for 10 min. The optical
density of the supernatant was read at 290 nm. The
activity of XOD was expressed as µmol uric acid
Reduced glutathione (GSH) was determined by us-
ing the method of Jollow
et al.
(1974). PMS 10 % was
precipitated with sulfosalicylic acid 4 % in 1:1 ratio.
The samples were kept at 4 ºC for 1 h and centrifuged
at 1500 rpm for 15 min at 4 ºC. The supernatant was
used for GSH estimation. The assay mixture contained
supernatant, phosphate buffer (100 mM, pH 7.4) and
5-5’-dithiobis-2-nitrobenzoic acid, DTNB (stocks
100 mM in 100 mM sodium phosphate buffer, pH
7.4) in total volume of 3 mL. GSH activity was deter-
mined spectrophotometrically by measuring reaction
product at 412 nm and expressed as nmol of GSH
Peroxidative damage of lipids was determined
according to the method of Utley
et al.
with some modifcations proposed by ±atima
et al.
(2000). The liver, 1 g, was homogenized in 5 mL of
chilled 100 mM potassium chloride solution. The
assay mixture contained 0.67 % thiobarbituric acid
(TBA), 10 % chilled trichloroacetic acid (TCA)
and liver homogenate (10 %). The rate of LPO is
expressed as nmol of thiobarbituric acid reactive
substance (TBARS) formed per gram of tissue using
a molar extinction coeFfcient oF 1.56=10
M/cm and
wavelength of 532 nm.
The protein content was determined according
to Lowry
et al.
(1951), with bovine serum albumin
as standard.
Liver histology
A liver sample/slice, 3 to 4 mm thick, from each
barbel was fxed in 10 % buFFered Formalin during
24-48 h, at room temperature and processed for par-
aFfn embedding. Then, 5 µm sections were cut in
a rotary microtome (Leica RM 2135), stained with
haematoxylin-eosin (H&E) and mounted for light
microscopy (LM) scrutiny. Sections sampled from
diverse blocks were studied. Microphotographies
were taken with a Nikon 4500 Coolpix digital cam-
era coupled to a Nikon Eclipse E 600 microscope.
±or each fsh 20 felds were evaluated using a 200 ×
The hepatic lesions/alterations were identifed
according to general diagnostic categories (Kohler
et al.
2002, Lang
et al.
The hepatic lesions (structures/cells that do not
appear in healthy tissues) and alterations (changes
in the number of structures/cells usually present in
the tissue) were scored according to a scale of 8
grades, based on Matos
et al.
(2007), as described
by Pinto
et al.
(2010). Brie²y, each lesion/alteration
score was assessed as a function of lesion/altera-
tion frequency and its severity (extension of the
lesion/alteration on each feld), From zero to seven.
Therefore, score zero is a tissue without any lesion/
alteration, score one represents a lesion/alteration
with very low frequency and a low severity, while
score seven represents extremely high frequency
and severity.
Liver metal content
Liver metal content was assayed using the
methodology described in Fernandes
et al.
Brie²y, For Al, Cr, Cu and Zn quantifcation, the
tissue was lyophilized and digested overnight with
nitric acid (supra pure grade) at 60 ºC. Samples were
analyzed in a graphite furnace atomic absorption
spectrophotometer (UNICAMP 939 AA - GF90).
Blank determinations were done using the same
procedure with Milli-Q50 water. Results were ex-
pressed in mg/kg dry weight (DW). The analytical
accuracy and precision were checked using certifed
reference materials, i.e DOLT-3 and DORM-2 (Na-
tional Research Council of Canada). The analyses
of the reference materials were always within the
certifed intervals.
Statistical analysis
All statistical analyses were performed with
SPSS statistical program. Quantitative differences
for histological lesion/alteration score and enzy-
matic activities, between barbel from Vizela River
and reference barbel, were tested by non-parametric
Mann-Whitney U-Test. A 5 % signifcance level was
employed throughout.
Barbus bocagei
Enzyme profle and stress indicators
Hepatic enzymes activities and lipid peroxida-
tion in fsh liver are presented in
Table I
. Generally
the higher activities were observed in fsh collected
From Vizela River, when compared to reFerence fsh
liver, with a 23 % increased of SOD, 31 % of CAT,
150 % of glutathione reductase activity and 47 %
of glutathione
-transferase. Glucose 6-phosphate
dehydrogenase showed an increase of 8 %. Xantine
oxidase activity was similar in both groups of barbel.
In the same way, GSH content did not differed be-
tween the two groups (
Table I
). As for lipid peroxida-
tion fsh captured in Vizela River exhibited an increase
of 41 % relative to the reference barbel (
Table I
Two hepatic lesions were observed and identi-
fed (
Fig. 1
): macrophage aggregates, which were
present on both groups (79 % of Vizela River bar-
bel and 40 % of reference barbel), and lymphocyte
foci that was only observed in Vizela River barbel
(100 %). Regarding the lesions scores (
Fig. 2
), the
macrophage aggregates and the lymphocytes foci
showed signifcant diFFerences between the reFer
ence and Vizela River barbels (p<0.05). In refer-
ence barbel, the median score for both macrophage
aggregates and lymphocyte foci, was zero, while
Vizela River barbel presented a score of three for
macrophage aggregates and oF fve For lymphocyte
foci (p<0.05).
Tissue metal content
Metals content in liver of barbel captured in the
Vizela River ranged between 9-15 mg/kg DW for Al
and 1.8-3.5 mg/kg DW for Cu and it was less than
the detection limits of 0.026 mg/kg DW for Zn and
0.006 mg/kg DW for Cr. The liver metals content
of the reference barbel were all below the detection
limits (0.011 mg/kg DW for Al and 0.003 mg/kg
DW for Cu).
In Portugal, there are frequent surveys on fresh-
water fsh populations dynamic, including the Iberian
barbel (Santos
et al.
2004), and on ecosystems health
and integrity (eg: Pinto
et al.
2010, Varandas and Cor-
tes 2010, Carvalho
et al.
2011). However, the studies
Barbus bocagei
Antioxidant enzymes
Catalase (µmol O
/min/mg protein)
4.61 ± 1.64
6.74 ± 1.26*
Superoxide dismutase (U/min/mg protein)
0.471 ± 0.084
0.618 ± 0.046**
-transferase (nmol CDNB /min/mg protein)
± 30.4
228.64 ± 48.4*
Glutathione reductase (nmol NADPH /min/mg protein)
14.93 ± 4.69
37.39 ± 5.14**
Glucose 6-phosphate dehydrogenase (nmol NADP /min/mg protein)
77.02 ± 0.05
83.23 ± 2.40*
Xanthine oxidase (µmol uric acid/min/mg of protein)
5.46 ± 2.41
4.22 ± 0.58
Protein (mg/g liver)
0.131 ± 0.019
0.165 ± 0.018**
TBARS (µM MDA/mg protein)
1.11 ± 0.67
1.88 ± 0.86*
Reduced glutathione (nmol GSH consumed/mg protein)
25.80 ± 3.20
25.40 ± 5.20
Values are expressed as mean ± SD (
); *
<0.05, **
<0.01 when compared with values oF reFerence fsh
Fig. 1.
Histological barbel liver sections stained with hematoxilin-eosin. Reference barbel (A);
Vizela River barbel (B-C). Ma-macrophage aggregate; La-Lymphocyte aggregate; e-
erythrocytes. Scale bar-100 µm
F.P. Peixoto
et al.
of exposure biomarkers, in freshwater species, are
inexistent being almost exclusive of estuarine species
(eg: Ferreira
et al.
2005, Cunha
et al.
2007, Gravato
et al.
2010). The present study aimed to evaluate
hepatic biochemical and histological biomarkers in
barbel captured in a polluted river, as a base for future
evaluation of the impact of management policies.
Many classes of environmental pollutants are
known to increase the intracellular formation of
ROS and several authors have already reported
physiological alterations induced by ROS, in fsh
et al.
1997, 1998, Valavanidis
et al.
Since induction of antioxidants represents a cel-
lular defense mechanism to counteract toxicity of
ROS, they have been extensively used in several
feld studies to assess the extent oF pollution in riv
ers, lakes and coastal waters (Ferreira
et al.
et al.
SODs are a group of metalloenzymes that play a
crucial antioxidant role and constitute the primary de-
fense mechanism against the toxic effect of oxygen,
in aerobic organisms. SOD catalyzes the dismutation
of the superoxide anion radical to water and hydrogen
peroxide, which aFterwards is detoxifed by CAT.
Therefore, a simultaneous activity induction of SOD
and CAT is usually an expected response. However,
this relation is not always observed (Peixoto
et al.
2006) and it is known to be species dependent (Ferreira
et al.
2005). In the present study, the liver of barbel
captured in the Vizela River presented high activity
values of both, SOD and CAT, suggesting a “coop-
erative” mechanism of the two enzymatic systems.
In addition to SOD and CAT, which are consid-
ered the major antioxidant enzymes, there are others
that may be useful biomarkers. These enzymes serve
as a backup function by replenishing GSH from
glutathione disulfde (GSSG) through the enzyme
GR and the reducing equivalent is provided by the
enzyme G6PD.
In the present study, higher values of GR and
G6PD were observed in the liver of barbel captured
in the contaminated location. Several authors re-
ported that fsh exposed to pollutants present higher
GR activity due to higher peroxidative components
et al.
2006, Sturve
et al.
2008). Equally, the
increase of G6PD activity should be related to the
increase of NADPH production, an important cofac-
tor necessary to recycle reduced glutathione through
glutathione reductase activity, in order to minimize
the oxidative stress condition. Thus, re±ecting an
adaptation to oxidative conditions to which fsh has
been exposed to (Lenartova
et al.
²urthermore, detoxifcation enzymes, and espe
cially GST, help to eliminate reactive compounds
by conjugation with glutathione and subsequent
elimination; thereby protecting cells against ROS
induced damage (Matos
et al.
2007). GST catalyzes
the conjugation of electrophilic compounds with the
tri-peptide glutathione and is a determinant enzyme
For herbicide detoxifcation (Villarini
et al.
et al.
2008). In Vizela barbel, GST activity
was double compared to the one in reference barbel,
this may indicate that in Vizela River fsh are exposed
to a higher load of compounds, present in textile
industry eF±uents.
GSH is an effective protector, capable of quench-
ing oxyradicals, and is an essential cofactor for GPx
and GST activity (Ross 1988). Despite the observed
increase in GST and GR activities in liver of barbel
captured in Vizela River, GSH content was not
increased. However, even if GSH was being syn-
de novo
in barbel captured in Vizela River,
since the oxidative stress condition were higher in
these fsh, GSH could be being used by GSH, GR
Fig. 2.
Box-whisker graphs showing score variation of hepatic lesions/alterations.
A-macrophage aggregates and B-lymphocyte Foci. Lowest box limit = per
centile-25, higher box limit = percentile-75; whiskers represent percentiles
10 and 90. Signifcant diFFerences (*p<0.05)
Barbus bocagei
and oxidized to GSSG, which would result in a de-
crease on GSH content. This situation could justify
the similar GST content observed between the two
barbel populations.
Xanthine oxidase (XO) catalyzes the conversion
of xanthine to uric acid. Uric acid, an excretory
product of purine catabolism, can act as a scavenger
of ROS such as OH
and O
. Therefore uric acid
can protect DNA and cellular membranes from
ROS-mediated damage (Stinefelt
et al.
2005). In the
present work XO activity was similar in the liver of
both groups of barbel.
When fsh are submitted to oxidative stress con
ditions, fatty acid peroxidation can occur. Indeed,
increased ROS production and subsequent oxidative
damage has been associated with pollutant-mediated
mechanisms oF toxicity in fsh liver (Livingstone
1993). Malondialdehyde (MDA) production is a
well-known oxidation product of polyunsaturated
Fatty acids, in±uencing cell membrane ±uidity as well
as the integrity of biomembranes (Ercal
et al.
et al.
2005), and can be used as an indica-
tor of lipid peroxidation. The present study revealed
high levels of lipid peroxidation, measured as MDA,
suggesting that antioxidant enzymes stimulation was
not capable of preventing hepatic lipid peroxidation,
probably induced by water contamination.
This study also put in evidence the histopatologi-
cal liver alterations observed in barbel from Vizela
River: macrophage aggregates and foci of lympho-
cytes. Liver lesions have been classifed and scored,
according to their relative importance, as indicators
of contaminant exposure. Macrophage aggregates are
related to storage of foreign material, such as parasitic
inFestations, and although it can be observed in fsh
living in low contaminated sites (Stentiford
et al.
2003), their prevalence and intensity can be used as a
potential biomarker to environmental contamination
(Couillard and Hodson 1996). In the present study,
prevalence of macrophage aggregates in Vizela River
barbel was higher than the one observed in reference
barbel. Furthermore, when looking to macrophage
aggregates median score in barbel, there is a clear
difference between the two groups of barbel, prob-
ably related to water contamination.
The hepatic foci of lymphocites, observed only in
fsh From Vizela River, could be the result oF chronic
in±ammatory conditions, by both, inFectious and
non-inFectious causes. This may re±ect a depleted
immunological status due to contaminated water
Several studies have established a causal rela-
tionship between metals concentrations and fsh
liver histopathological alterations (Au 2004). The
injuries are often dependent upon time of exposure
to metals (Yang and Chen 2003, Au 2004, Olojo
Metal accumulation in fsh organs re±ects bio
availability and exposure. In the present study, liver
of barbel captured in Vizela River presented low met-
als levels. This could be due to the low bioavailability
oF metals and/or occasional eF±uent discharges From
the textile Factories. ²urthermore, the textile eF±uents
carry other contaminants rather than metals and,
although Portugal is required to control industrial
discharges, several eF±uents, located in the study
area, are still discharging in the river bed without
any type of treatment.
Several studies showed a growing interest in the
use of bioindicators and biomarkers for assessment
and monitoring of the ecological systems (Braun-
beck and Völkl 1993, Vethaak and Wester 1996) in
addition to traditional biomonitoring studies, as a
way to understand the real bio-effects of pollution in
wildlife (Burger
et al.
2007), namely in fsh (Kirby
et al.
In conclusion, although being non specifc re
sponses, antioxidant enzymes activity and liver his-
topathology are useful tools to evaluate the impact
of industry wastewater. Therefore, these exposure
biomarkers can be used to assess the future impact
oF management policies on Vizela River fsh.
Almroth B.C., Sturve J., Berglund A. and Forlin L. (2005).
Oxidative damage in eelpout (
Zoarces viviparus
), mea-
sured as protein carbonyls and TBARS, as biomarkers.
Aquat. Toxicol. 73, 171-180.
Alves C.M., Boaventura R.R.A.R. and Soares H.M.V.M.
(2009). Evaluation of heavy metals pollution loadings
in the sediments of the Ave River Basin (Portugal). Soil
Sediment Contam. 18, 603-618.
Au D.W.T. (2004). The application of histo-cytopatho-
logical biomarkers in marine pollution monitoring: a
review. Mar. Pollut. Bull. 48, 817-834.
Baker R.T.M., Handy R.D., Davies S.J. and Snook J.C.
(1998). Chronic dietary exposure to copper affects
growth, tissue lipid peroxidation, and metal composi-
tion of the grey mullet,
Chelon labrosus
. Mar. Environ.
Res. 45, 357-365.
Baker R.T.M., Martin P. and Davies S.J. (1997). Ingestion
oF sub-lethal levels oF iron sulphate by AFrican catfsh
affects growth and tissue lipid peroxidation. Aquat.
Toxicol. 40, 51-61.
F.P. Peixoto
et al.
Bláha L., Kopp R., Šimková K. and Mareš J. (2004).
Oxidative stress biomarkers are modulated in silver
carp (
Hypophthalmichthys molitrix
Val.) exposed to
microcystin-producing cyanobacterial water bloom.
Acta Vet. Brno. 73, 477-482.
Braunbeck T. and Völkl A. (1993). Toxicant-induced
cytological alterations in fsh liver as biomarkers oF
environmental pollution? A case study on hepatocel-
lular effects of dinitro-o-cresol in golden ide (
ciscus idus melanotus
). In:
Fish - ecotoxicology and
(H.W. Braunbeck T., H. Segner Eds.).
VCH, Weinheim, pp. 55-80.
Burger J., Fossi C., McClellan-Green P. and Orlando E.F.
(2007). Methodologies, bioindicators, and biomarkers
for assessing gender-related differences in wildlife
exposed to environmental chemicals. Environ. Res.
104, 135-152.
Carlberg I. and Mannervik B. (1975). Purifcation and
characterization oF the ±avoenzyme glutathione re
ductase from rat liver. J. Biol. Chem. 250, 5475-5480.
Carrola J., Fontainhas-Fernandes A., Matos P. and Rocha
E. (2009). Liver histopathology in brown trout (
trutta f. fario
) from the Tinhela River, subjected to mine
drainage from the abandoned Jales Mine (Portugal).
Bull. Environ. Contam. Tox. 83, 35-41.
Carvalho L., Cortes R. and Bordalo A. (2011). Evaluation
of the ecological status of an impaired watershed by
using a multi-index approach. Environ. Monit. Assess.
174, 493-508.
Claiborne A. (1985). Catalase activity. In:
Handbook of
methods for oxygen radical research
(R. A. Greenwald,
Ed). Boca Raton, FL, pp. 283-284.
Collen J., Pinto E., Pedersen M. and Colepicolo P. (2003).
Induction of oxidative stress in the red macroalga
Gracilaria tenuistipitata
by pollutant metals. Arch.
Environ. Con. Tox. 45, 337-342.
Couillard C.M. and Hodson P.V. (1996). Pigmented mac-
rophage aggregates: A toxic response in fsh exposed to
bleached-kraFt mill eF±uent? Environ. Toxicol. Chem.
15, 1844-1854.
Cunha I., Neuparth T., Caeiro S., Costa M.H. and Guil-
hermino L. (2007). Toxicity ranking of estuarine
sediments on the basis of
Sparus aurata
Environ. Toxicol. Chem. 26, 444-453.
Deviller G., Palluel O., Aliaume C., Asanthi H., Sanchez
W., Nava M.A.F., Blancheton J.P. and Casellas C.
(2005). Impact assessment of various rearing systems
on fsh health using multibiomarker response and metal
accumulation. Ecotox. Environ. Safe. 61, 89-97.
Ercal N., Gurer-Orhan H. and Aykin-Burns N. (2001).
Toxic metals and oxidative stress Part I: Mechanisms
involved in metal-induced oxidative damage. Current
Topics in Medicinal Chemistry. 1, 529-539.
Fatima M., Ahmad I., Sayeed I., Athar M. and Raisuddin
S. (2000). Pollutant-induced over-activation of phago-
cytes is concomitantly associated with peroxidative
damage in fsh tissues. Aquat. Toxicol. 49, 243-250.
Fernandes C., Fontainhas-Fernandes A., Peixoto F. and
Salgado M.A. (2007). Bioaccumulation of heavy met-
als in
Liza saliens
from the Esmoriz-Paramos coastal
lagoon, Portugal. Ecotoxicol. Environ. Saf. 66, 426-431.
Fernandes C., Fontainhas-Fernandes A., Cabral D. and Sal-
gado M.A. (2008a). Heavy metals in water, sediment
and tissues of
Liza saliens
from Esmoriz-Paramos la-
goon, Portugal. Environ. Monit. Assess. 136, 267-275.
Fernandes C., Fontainhas-Fernandes A., Rocha E. and
Salgado M.A. (2008b). Monitoring pollution in
Esmoriz-Paramos lagoon, Portugal: Liver histologi-
cal and biochemical effects in
Liza saliens
. Environ.
Monit. Assess. 145, 315-322.
Fernandes C., Fontainhas-Fernandes A., Ferreira M. and
Salgado M.A. (2008c). Oxidative stress response in gill
and liver of
Liza saliens
, from the Esmoriz-Paramos
coastal lagoon, Portugal. Arch. Environ. Contam.
Toxicol. 55, 262-269.
Ferreira M., Moradas-Ferreira P. and Reis-Henriques
M.A. (2005). Oxidative stress biomarkers in two
resident species, mullet (
Mugil cephalus
) and ±ounder
Platichthys fesus
), from a polluted site in River Douro
Estuary, Portugal. Aquat. Toxicol. 71, 39-48.
Figueiredo-Fernandes A., Fontainhas-Fernandes A.,
Peixoto F., Rocha E. and Reis-Henriques M.A. (2006).
Effects of gender and temperature on oxidative stress
enzymes in Nile tilapia
Oreochromis niloticus
to paraquat. Pest. Biochem. Phys. 85, 97-103.
Gravato C., Guimarães L., Santos J., Faria M., Alves A.
and Guilhermino L. (2010). Comparative study about
the effects of pollution on glass and yellow eels (
guilla anguilla
) from the estuaries of Minho, Lima
and Douro Rivers (NW Portugal). Ecotox. Environ.
Safe. 73, 524-533.
Habig W.H., Pabst M.J. and Jakoby W.B. (1974). Glutathi-
one S-transferases - First enzymatic step in mercapturic
acid formation. J. Biol. Chem. 249, 7130-7139.
Halliwell B. and Gutteridge H. (1999). Free radicals in
biology and medicine. University Press, Oxford, 936 p.
Hinton D.E. and Lauren D.J. (1990). Liver structural altera-
tions accompanying chronic toxicity in fshes: Potential
biomarkers of exposure. In:
Biomarkers of environmen-
tal contamination
(J.F. McCarthy and L.R. Shugart,
Eds.). Lewis Publishers, Boca Raton FL, pp. 17-57.
Jollow D.J., Mitchell J.R., Zampaglione N. and Gillette
J.R. (1974). Bromobenzene-induced liver necro-
sis. Protective role of glutathione and evidence for
3,4-bromobenzene oxide as the hepatotoxic metabolite.
Pharmacol. 11, 151-169.
Barbus bocagei
Kirby M.F., Smith A.J., Rooke J., Neall P., Scott A.P. and
Katsiadaki I. (2007). Ethoxyresorufn-O-deethylase
(EROD) and vitellogenin (VTG) in Founder (
thys fesus
): System interaction, crosstalk and impli-
cations for monitoring. Aquat. Toxicol. 81, 233-244.
Kohler A., Wahl E. and Soffker K. (2002). Functional and
morphological changes of lysosomes as prognostic
biomarkers o± toxic liver injury in a marine Fatfsh
Platichthys fesus
(L.)). Environ. Toxicol. Chem. 21,
Lang T., Wosniok W., Barsiene J., Broeg K., Kopecka J.
and Parkkonen J. (2006). Liver histopathology in Baltic
Founder (
Platichthys fesus
) as indicator of biological
effects of contaminants. Mar. Pollut. Bull. 53, 488-496.
Lenartova V., Holovska K., Pedrajas J.R., Martinez Lara
E., Peinado J., Lopez Barea J., Rosival I. and Kosuth
P. (1997). Antioxidant and detoxi±ying fsh enzymes as
biomarkers of river pollution. Biomarkers 2, 247-252.
Leonardi M., Tarifeno E. and Vera J. (2009). Diseases of the
Chilean Founder,
Paralichthys adspersus
1867), as a biomarker of marine coastal pollution near
the Itata River (Chile): Part II. Histopathological lesions.
Arch. Environ. Contam. Toxicol. 56, 546-556.
Livingstone D.R., Lemaire P., Matthews A., Peters L.,
Bucke D. and Law R.J. (1993). Prooxidant, antioxidant
and 7-Ethoxyresorufn O-Deethylase (EROD) activity
responses in liver of Dab (
Limanda limanda
) exposed
to sediment contaminated with hydrocarbons and other
chemicals. Mar. Pollut. Bull. 26, 602-606.
Lowry O.H., Rosebrough N.J., Farr A.L. and Randall R.J.
(1951). Protein measurement with the folin phenol
reagent. J. Biol. Chem. 193, 265-275.
Magalhães M.F. (1992). Feeding ecology of the Iberian
Barbus bocagei
Steindachner, 1865 in a low-
land river. J. Fish Biol. 40, 123-133.
Matos P., Fontainhas-Fernandes A., Peixoto F., Carrola
J. and Rocha E. (2007). Biochemical and histological
hepatic changes of Nile tilapia
Oreochromis niloticus
exposed to carbaryl. Pest. Biochem. Phys. 89, 73-80.
McCarthy J.F. and Shugart L.R. (1990). Biological mark-
ers of environmental contamination. In:
of environmental contamination
(J.F. McCarthy and
L.R. Shugart, Eds.). Lewis Publishers, Boca Raton
FL, pp. 3-14.
Mohandas J., Marshall J.J., Duggin G.G., Horvath J.S.
and Tiller D.J. (1984). Differential distribution of
glutathione and glutathione-related enzymes in rabbit
kidney - possible implications in analgesic nephropa-
thy. Biochem. Pharmacol. 33, 1801-1807.
Olojo E.A.A., Olurin K.B., Mbaka G. and Oluwemimo
A.D. (2005). Histopathology of the gill and liver tis-
sues o± the A±rican catfsh
Clarias gariepinus
to lead. Afr. J. Biotechnol. 4, 117-122.
Paya M., Halliwell B. and Hoult J.R. (1992). Interac-
tions of a series of coumarins with reactive oxygen
species. Scavenging of superoxide, hypochlorous
acid and hydroxyl radicals. Biochem. Pharmacol.
44, 205-214.
Peixoto F., Alves-Fernandes D., Santos D. and Fon-
tainhas-Fernandes A. (2006). Toxicological effects
o± oxyFuor±en on oxidative stress enzymes in tilapia
Oreochromis niloticus
. Pest. Biochem. Phys. 85,
Peixoto F.P., Gomes-Laranjo J., Vicente J.A. y Madeira
V.M.C. (2008). Comparative effects of the herbicides
dicamba, 2,4-D and paraquat on non-green potato tuber
calli. J. Plant Physiol. 165, 1125-1133.
Pinto A., Varandas S., Coimbra A., Carrola J. and Fon-
taínhas-Fernandes A. (2010). Mullet and gudgeon liver
histopathology and macroinvertebrate indexes and
metrics upstream and downstream from a wastewater
treatment plant (Febros River-Portugal). Environ.
Monit. Assess. 169, 569-585.
Ross D. (1988). Glutathione, free-radicals and chemo-
therapeutic agents. Mechanisms of free-radical in-
duced toxicity and glutathione-dependent protection.
Pharmacol. Therapeut. 37, 231-249.
Santos J.M., Godinho F., Ferreira M.T. and Cortes R.
(2004). The organisation o± fsh assemblages in the
regulated Lima basin, Northern Portugal. Limnologica
34, 224-235.
Soares H.M.V.M., Boaventura R.A.R., Machado A.A.S.C.
and da Silva J.C.G.E. (1999). Sediments as monitors
of heavy metal contamination in the Ave river basin
(Portugal): multivariate analysis of data. Environ.
Pollut. 105, 311-323.
Stentiford G.D., Longshaw M., Lyons B.P., Jones G.,
Green M. and Feist S.W. (2003). Histopathological
biomarkers in estuarine fsh species ±or the assessment
of biological effects of contaminants. Mar. Environ.
Res. 55, 137-159.
Stinefelt B., Leonard S.S., Blemings K.P., Shi X.L. and
Klandorf H. (2005). Free radical scavenging, DNA
protection, and inhibition of lipid peroxidation medi-
ated by uric acid. Ann. Clin. Lab. Sci. 35, 37-45.
Stirpe F. and Dellacor E. (1969). The regulation of rat
liver xanthine oxidase. Conversion in vitro of enzyme
activity from dehydrogenase (Type D) to oxidase (Type
O). J. Biol. Chem. 244, 3855-3863.
Sturve J., Almroth B.C. and Forlin L. (2008). Oxidative
stress in rainbow trout (
Oncorhynchus mykiss
) exposed
to sewage treatment plant e±Fuent. Ecotox. Environ.
Safe. 70, 446-452.
Triebskorn R., Köhler H.-R., Honnen W., Schramm M.,
Adams S.M. and Müller E.F. (1997). Induction of heat
shock proteins, changes in liver ultrastructure, and al-
F.P. Peixoto
et al.
terations of Fsh behavior: are these biomarkers related
and are they useful to re±ect the state of pollution in the
Feld? J. Aquat. Ecosyst. Stress Rec. (²ormerly Journal
of Aquatic Ecosystem Health). 6, 57-73.
Triebskorn R., Kohler H.R., Flemming J., Braunbeck T.,
Negele R.D. and Rahmann H. (1994). Evaluation of
bis(tri-n-butyltin)oxide (Tbto) neurotoxicity in rain-
bow-trout (
Oncorhynchus mykiss
). 1. Behavior, weight
increase, and tin content. Aquat. Toxicol. 30, 189-197.
Uguz C., Iscan M., Erguven A., Isgor B. and Togan I.
(2003). The bioaccumulation of nonyphenol and its
adverse effect on the liver of rainbow trout (
rynchus mykiss
). Environ. Res. 92, 262-270.
Utley H.G., Bernheim F. and Hochstei. P (1967). Effect
of sulfhydryl reagents on peroxidation in microsomes.
Arch. Biochem. Biophys. 118, 29-32.
Valavanidis A., Vlahogianni T., Dassenakis M. and Scoul-
los M. (2006). Molecular biomarkers of oxidative
stress in aquatic organisms in relation to toxic environ-
mental pollutants. Ecotox. Environ. Safe. 64, 178-189.
Varandas S.G. and Cortes R.M. (2010). Evaluating mac-
roinvertebrate biological metrics for ecological assess-
ment of streams in northern Portugal. Environ. Monit.
Assess. 166, 201-221.
Vethaak A.D. and Wester P.W. (1996). Diseases of ±oun
Platichthys fesus
in Dutch coastal and estuarine
waters, with particular reference to environmental
stress factors. 2. Liver histopathology. Dis. Aquat.
Organ. 26, 99-116.
Vieira C., Morais S., Ramos S., Delerue-Matos C. and
Oliveira M.B. (2011). Mercury, cadmium, lead and
arsenic levels in three pelagic Fsh species from the
Atlantic Ocean: intra- and inter-speciFc variability
and human health risks for consumption. Food Chem.
Toxicol. 49, 923-932.
Villarini M., Moretti M., Scassellati-Sforzolini G., Monar-
ca S., Pasquini R., Crea M.G. and Leonardis C. (1995).
Studies on hepatic xenobiotic-metabolizing enzymes in
rats treated with insecticide deltamethrin. J. Environ.
Pathol. Toxicol. Oncol. 14, 45-52.
Yang J.L. and Chen H.C. (2003). Serum metabolic enzyme
activities and hepatocyte ultrastructure of common
carp after gallium exposure. Zool. Stud. 42, 455-461.
Zaheer N., Tewari K.K. and Krishnan P.S. (1965). Ex-
posure and solubilization of hepatic mitochondrial
shunt dehydrogenases. Arch. Biochem. Biophys. 109,