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Sistema de Información Científica
Red de Revistas Científicas de América Latina y el Caribe, España y Portugal
OXIDATIVE STRESS RESPONSES AND HISTOLOGICAL HEPATIC ALTERATIONS IN
BARBEL,
Barbus bocagei
, FROM VIZELA RIVER, PORTUGAL
Francisco P. PEIXOTO
1,2
, João CARROLA
1,2
, Ana Maria COIMBRA
1,2
*, Conceição FERNANDES
3
,
Paulo TEIXEIRA
1,2
, Luís COELHO
1,4
, Ivo CONCEIÇÃO
1,4
,
Maria Manuel OLIVEIRA
1,4
and António FONTAÍNHAS-FERNANDES
1,2
1
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
2
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
3
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
4
Chemistry Research Center of Vila Real (Centro de Química de Vila Real-CQVR), UTAD
*Corresponding author: acoimbra@utad.pt
(Recibido agosto 2011, aceptado octubre 2012)
Key words: oxidative stress enzymes, lipid peroxidation, fsh, Freshwater, liver histopathology
ABSTRACT
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
S
-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
RESUMEN
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.
30
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.
INTRODUCTION
The hydrographical basin of the Ave River is
located in Minho region (northwest of Portugal) and
comprises an area of about 1390 km
2
, 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.
Soares
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,
Alves
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
(Alves
et al.
2009).
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-
nandes
et al.
2008a, Vieira
et al.
2011).
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,
contaminants.
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.
1997).
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
(Fernandes
et al.
2008b, Leonardi
et al.
2009).
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 (
Platichthys
fesus
) and Nile tilapia (
Oreochromis niloticus
) (Fer-
reira
et al.
2005, Figueiredo-Fernandes
et al.
2006).
Thus, oxidative stress biomarkers can be employed
HEPATIC ALTERATION IN
Barbus bocagei
FROM VIZELA RIVER
31
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
(Livingstone
et al.
1993, Ercal
et al.
2001), and have
a predictive importance as biomarkers of pollution
(Collen
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-
Fernandes
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.
MATERIALS AND METHODS
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×
g
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
c
. 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
2
O
2
consumed/
min/mg/protein.
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/
mg/protein.
The activity of glucose 6-phosphate dehydro-
genase (G6PD) was assayed by the method of Za-
heer
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
2
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.
32
Glutathione
S
-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.
2003).
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
formed/mg/protein.
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
consumed/mg/protein.
Peroxidative damage of lipids was determined
according to the method of Utley
et al.
(1967)
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
5
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 ×
magnifcation.
The hepatic lesions/alterations were identifed
according to general diagnostic categories (Kohler
et al.
2002, Lang
et al.
2006).
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.
(2008c).
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.
HEPATIC ALTERATION IN
Barbus bocagei
FROM VIZELA RIVER
33
RESULTS
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
S
-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
).
Histology
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).
DISCUSSION
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
TABLE
I
. ACTIVITY OF DIFFERENT ANTIOXIDANT ENZYMES, PROTEIN AND GSH CONTENT AND LIPID
PEROXIDATION IN
Barbus bocagei
cAPTURED FROM REFERENCE (CORGO RIVER) AND CON-
TAMINATED (VIZELA RIVER) SITES
Antioxidant enzymes
Reference
Vizela
Catalase (µmol O
2
/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**
Glutathione
S
-transferase (nmol CDNB /min/mg protein)
155.5
± 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 (
n=5-8
); *
P
<0.05, **
P
<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
A
e
e
Ma
La
BC
A
e
e
Ma
La
BC
F.P. Peixoto
et al.
34
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
(Baker
et al.
1997, 1998, Valavanidis
et al.
2006).
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.
2005,
Fernandes
et al.
2008c).
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
(Peixoto
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.
1997).
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.
1995,
Peixoto
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-
thesized
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)
7
AB
6
5
4
3
2
1
0
–1
7
6
5
4
3
2
1
0
Reference
Vizela
Reference
Vizela
–1
HEPATIC ALTERATION IN
Barbus bocagei
FROM VIZELA RIVER
35
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
2
. 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
et
al.
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.
2001,
Almroth
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
exposure.
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
et
al.
2005).
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.
2007).
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.
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