Fish & Shellfish Immunology 39 (2014) 108e117

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Fish & Shellfish Immunology

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Full length article

Sex-response differences of immunological and histopathological

biomarkers in gill of Prochilodus argenteus from a polluted river
in southeast Brazil
Marcela Santos Procópio a, Heder José Ribeiro a, Luciano Almeida Pereira d,
Gabriel Augusto Oliveira Lopes b, Antônio Carlos Santana Castro a, Elizete Rizzo a,
Yoshimi Sato c, Remo Castro Russo b, José Dias Corrêa Junior a, *
a
Departamento Morfologia, Universidade Federal de Minas Gerais, Av. Antônio Carlos, 6627 Pampulha, Belo Horizonte, 31270 901 Minas Gerais, Brazil
b
Laboratório de Imunologia e Mecânica Pulmonar, Departamento de Fisiologia e Biofísica, Av. Antônio Carlos, 6627 Pampulha, Belo Horizonte,
31270 901 Minas Gerais, Brazil
c
Companhia de Desenvolvimento dos Vales do São Francisco e Parnaíba, CODEVASF, Estação de Piscicultura e Hidrobiologia de Três Marias,
Caixa Postal n
11, 39.205-000 Três Marias, Minas Gerais, Brazil
d
Colégio Técnico, Universidade Federal de Minas Gerais, Belo Horizonte, Minas, Gerais, Brazil, Av. Antônio Carlos, 6627 Pampulha, Belo Horizonte 31270 901,
Minas Gerais, Brazil

a r t i c l e i n f o a b s t r a c t

Article history: The fish gill is in direct and standing contact with the immediate external environment and, therefore, is
Received 10 October 2013 highly vulnerable to aquatic pollutants. In this study, Prochilodus argenteus were caught at two different
Received in revised form points in São Francisco river. The first point is located near Três Marias dam, while the second is placed
10 March 2014
downstream the Abaeté river. Chemical approaches showed the presence of metals contamination in the
Accepted 14 April 2014
first point. Thus, the main goal of this study was to investigate the possible toxic effects of these con-
Available online 2 May 2014
taminants and the likely use of biomarkers on fish gills. Biometric data of length and weight of fish were
obtained in order to calculate the condition factor as an organismal biomarker. The histological changes
Keywords:
Biomarkers
in gills and alterations in mucous and rodlet cells occurrence were detected microscopically and eval-
Histopathology uated with quantitative analyses. Myeloperoxidase (MPO) and Eosinophil Peroxidase (EPO) were also
Mucous cells assessed in fish gill. The analysis of the water and sediment samples revealed the presence of metals at
Rodlet cells the two points. As and Cd were detected at higher concentrations at point 1. The presence of lamellar cell
Inflammation hyperplasia, lamellar fusion, lamellar edema and inflammatory foci varied according to the point.
Additionally, mucous and rodlet cells and MPO and EPO activities showed variability according to the
environmental conditions. Furthermore, with exception of lamellar hyperplasia and eosinophil peroxi-
dase activity, all others parameters showed sex-variation responses. At the first point, male fish showed a
chronical inflammation in gills due to the lowest activity of MPO and EPO, as well as low occurrence of
inflammatory foci and glycoprotein secretion by mucous cells, while female fish presented an opposite
pattern of response to the same environmental conditions. Therefore, we suggest the use of such bio-
markers in future monitoring of aquatic systems, taking into account the sex-variation responses.
Ó 2014 Elsevier Ltd. All rights reserved.

1. Introduction

Abbreviation list: #1, first studied point; #2, second studied point; CI, condition
index; PEC, probable effect concentration; SQGV, sediment quality guidelines
values; TEC, threshold effect concentration.
* Corresponding author. Departamento Morfologia, Universidade Federal de
Minas, Gerais, Belo Horizonte, Minas Gerais, Av. Antônio Carlos, 6627 Pampulha,
Belo, Horizonte, MG 31270 901 Brazil. Tel.: þ55 31 3409 2792.
E-mail address: correajr@ufmg.br (J.D. Corrêa Junior).
Urban, industrial and agricultural activities have resulted in an
increase in the number of impacted environments due to the
release of contaminated wastewater in the freshwater systems. In
order to assess the adverse effects of combination of various con-
taminants in the aquatic organisms, biomarkers have been associ-
ated with chemical and physical water analysis aiming to improve

http://dx.doi.org/10.1016/j.fsi.2014.04.010
1050-4648/Ó 2014 Elsevier Ltd. All rights reserved.
 M.S. Procópio et al. / Fish & Shellfish Immunology 39 (2014) 108e117
the efficiency of the biomonitoring programs of the aquatic
ecosystem [1,2].
The gills represent the largest part of the surface area of the total
fish body which is in direct and standing contact with the envi-
ronment [3e5]. Accordingly, the gill is considered a target organ of
wastewater pollutants [6]. Several studies, in laboratory and in the
fields showed that histopathologies and innate immunological
parameters are sensible tools to detect the toxic effects of chemical
compounds in fish gill [7e13,23]. However, the large majority of the
studies have based their conclusions on biomarkers that reach only
one level of biological organization [14,15]. Thus, the association of
a variety of molecular, biochemical, physiological, histo-
cytopathological and organismal biomarkers are recommended in
order to obtain an accurate evaluation of toxic compounds in the
freshwater ecosystem [16e18].
The condition index (CI), considered as an organismal
biomarker, is useful to monitor pollution as toxic compounds
deplete energy reserves that were potentially destined for growth
[19].
Nevertheless, fish exposure to metals contamination displayed
the presence of several histopathologies in fish gill, including
lamellar hyperplasia, lamellar edema, aneurysms, epithelial lifting,
lamellar fusion and presence of inflammatory foci, which are
hallmarks often observed in gill exposed to Cd, As, Zn and other
metals [15,20,21]. Immunological parameters in fish gills are also
affected by metal and others pollutants, as increased levels of
ceruloplasmin and lysozym presented by fish exposed to Cd, Zn, Hg,
Cr and Fe [13]. Immune modulation was also observed in liver and
spleen of gilthead seabream (Sparus aurata) exposed to different
contaminants [22e24], indicating a close relation between envi-
ronmental stress response and immune system.
Rodlet cells which are characterized by a fibrous layer beneath
plasma membrane and conspicuous cytoplasmic inclusions called
rodlets, have been observed in different marine and freshwaters
fish species [25e27]. In the development of Cyprinus carpio, rodlet
cell in gills appear 14 days after fertilization, suggesting that this
cell is an innate constituent of the fish tissues [28]. Associated with
the wide distribution of these cells among the epithelium of several
organs and the increase of occurrence of these cells in exposure to
stress factors, it is proposed that this cells act in immunological
responses in the organism [29e31]. However, the function devel-
oped by this cell is still a matter of debate. Nevertheless, rodlet cell
has been used as biomarkers of exposure to metals, organochlo-
rines, and parasitic infections [26,32,33].
Myeloperoxidase (MPO) in mammals, which is released from
cytoplasmic granules of neutrophils by a degranulation process,
reacts with the H2O2 formed by the respiratory burst to form a
complex that can oxidize a large variety of substances [34]. In
zebrafish, (Danio rerio), MPO is present only in neutrophils [35e
37]. In others teleost such as Labeo rohita and Carassius auratus
MPO activity is also associated with neutrophils [38,39]. Eosino-
phil in teleost, which has been thoroughly characterized recently
in zebrafish, showed expression of GATA 2 transcription factor
that plays an essential role in eosinophil development in mam-
mals. Moreover, degranulation and peroxidase release of purified
gata2hi cells, analyzed using the OPD assay, was observed in
response to Heligmosomoides polygyrus helminth extracts [37].
Despite the widespread use and description of histopathological,
cellular and immune biomarkers in environmental studies, few
approaches consider the possible influence of fish sex on the
response to stressors [19,40,41]. In addition to enhancing the
interpretation of the data, the discrimination of biological re-
sponses according to the sex of animals could contribute to the
sampling optimization and minimize the time-consuming anal-
ysis procedure [42].
109
Prochilodus argenteus (Characiforme: Prochilodontidae) popu-
larly known as curimba, is an endemic specie from São Francisco
river with detritivore feeding habits [43]. This specie has economic
importance, representing 50% of all captured fish in the Três Marias
region and configures itself as an important source of food for local
populations [44,45]. Due to feeding habits P. argenteus is considered
ideal specie to study metal contamination in freshwater ecosys-
tems [46].
Several anthropogenic activities affect the São Francisco river in
the site between Três Marias dam and the Abaeté river. Metal
contamination of this site, due to the presence of a zinc producing
industry, has already been reported in chemical approaches.
However, investigation of the possible toxic effects of these con-
taminants on fish gill has not been conducted [47e50]. Therefore,
the main goals of the present work were to: i) establish gill bio-
markers in P. argenteus in order to assess the environmental
pollution in the São Francisco river; ii) discriminate the possible
influence of the fish sex in biomarkers responses; iii) assess the
need for using biomarkers of various biological levels.
2. Material and methods
2.1. Study area
Two different points in the São Francisco River, localized be-
tween Três Marias and São Gonçalo do Abaeté cities (Minas Gerais,
southern Brazil), were chosen based on the different contaminant
exposure conditions, as described in Section 1. The first point is
located immediately downstream of the Três Marias dam reservoir
(#1), while the second point, considered as reference, is located
after the São Francisco and Abaeté river confluence, at approxi-
mately 34 km from the first point (#2) (Fig. 1). The study was
performed during the dry season (2008 August) to avoid influences
of the fish reproductive period and of the thermal stratification of
the Três Marias reservoir, since it is considered a warm monomictic
reservoir [51]. The reference point was defined based on previous
studies [52].
2.2. Environmental parameters and chemical analysis in water and
sediment
Measures of water physicalechemical parameters and concen-
tration of metals in water and sediment were performed in the two
studied points. At each point, measurements were carried out on
the right and left river bank, as well as in the central channel. The
results were obtained by the average of three measurements per-
formed on different segments of the river. The values of the con-
ductivity of water (mS/cm), dissolved oxygen (mg/L), temperature


( C), turbidity (NTU) and pH were obtained by using a water quality
multiprobe (Horiba U10, IrvineeUSA).
Water samples, for determination of metal concentrations, were
collected in 500 mL plastic bottles previously decontaminated in a
10% nitric acid solution for 48 h and kept refrigerated until analysis.
Samples were filtered through a 0.45 mm membrane filter and the
liquid portion was used in determining the concentration of cad-
mium (Cd), manganese (Mn) and zinc (Zn). The sediment samples
were collected using a Petersen dredge and 350 g were stored in
500 mL plastic bottles previously decontaminated in a 10% nitric
acid solution for 48 h and kept refrigerated until analysis. Next,
sediment samples were dried at 60
C for 24 h and passed through a
65 mm sieve. Suspensions containing 10 mg of the sieved sample
were treated with HNO3 and HCl (PA) and used to analyze the
concentration of arsenium (As), cadmium (Cd), copper (Cu) and
manganese (Mn). The analyses were performed on the atomic ab-
sorption spectrometry with graphite furnace HGA 900 (Perkin
110 M.S. Procópio et al. / Fish & Shellfish Immunology 39 (2014) 108e117

buffer, pH 7.3). Some tissue samples were kept on dry ice and
subsequently stored in 80
C freezer. The guideline was approved
by the Ethics Committee on Animal Experimentation (CETEA -
UFMG) under protocol number 175/11.

2.5. Histology

After 24 h fixation in Bouin’s solution, the samples were placed

in 70% ethanol (Merk KGaA, Darmstadt, HE), decalcified in Pereny’s
solution according to Bancroft and Gamble (2007) [54], dehydrated
in a graded ethanol solutions up to pure ethanol and embedded in
paraffin (ParaplastÒ). Next, 5-mm tissue sections were deparaffi-
nized with xylene, hydrated in solutions of ethanol and stained by
hematoxylin and eosin (H&E), PAS/AB 2.5 [54]. Gill epithelium and
cell morphology were analyzed under an Olympus BX41 light mi-
croscope. Digitalized images were obtained with an Olympus BX41
light microscope coupled to a digital camera Q-Color 3 (Olympus
Corporation, Shinjuku-Ku, TY).

2.6. Histomorphometry

Morphometrical analysis of gill histopathologies and rodlet cells

Fig. 1. Sample points in São Francisco river. The point 1 is localized immediately
downstream Três Marias dam and the point 1 in the confluence with the Abaeté river.
Elmer, Norwalk, CT USA). The values were expressed in mg L1 and
mg Kg1, respectively.
occurrence were carried out in H&E stained-sections. For patho-
logical morphometry, gill fragments from 10 males and 8 females at
point 1 and 11 males and 7 females at point 2 were assessed. From
each animal, 60 to 100 lamellae in three longitudinal filaments
were selected, totaling 2760 lamellae. Occurrence of lamellae with
edema, hyperplasia, interlamellar fusion and inflammation were
quantified using the criteria previously established [20]. The pres-
ence of rodlet cells was considered only in longitudinal filament gill
sections with interlamellar fused lamellae and only evident rodlet
cells with filamentous capsule and granules. Gill fragments from 9
(4 males and 5 females) and 13 (8 males and 5 females) fish were
used in the first (point 1) and second (point 2) points, respectively.
For each fish, 100 interlamellar spaces were analyzed, totaling 2200
interlamellar spaces. Histopathologies and rodlet cells occurrence
were quantified using the Olympus BX 48 light microscope
(Olympus Corporation, Shinjuku-Ku, TY) under 400
magnification.

2.3. Fish sampling 2.7. Assessment of the distribution of types A, B and C mucous cells
and mucous in the interlamellar space
For biological evaluations 42 specimens of P. argenteus total
length of 32.0  0.5 cm and body weight of 471  19.3 g) were Types A, B and C mucous cells were quantified in the filaments
collected, while 20 (10 females and 10 males) were captured in and lamellae of a total of 27 fish. In the first point were collected
point 1 and 22 (9 females and 13 males) in point 2. The fish capture
was authorized by the State Forestry Institute from Minas Gerais
Table 1
state (IEF/MG), scientific fishing license NR 130-10, category D. The Values corresponding to the conductivity (mS$cm1), dissolved oxygen rate


specimens were captured with the aid of cast nets and stored in (mg$L1), temperature ( C), turbidity (NTU), pH, and dissolved metals in water
plastic containers containing 500 L of water from the river and (mg$L1) and sediment (mg$kg1) obtained at the two points.
remained without food until euthanasia. For each animal, the bio- Sample points Point 1 Point 2
metric data obtained were the total length (TL), standard length
Physico-chemical water parameters
(SL) and body weight (BW). Only animals that did not display any Conductivity 21.5  6.7* 5.6  0.17
apparent external morphological alterations were analyzed. The Dissolved oxygen 8.1  0.05* 8.7  0.04
condition index (CI) was determined according to Nikolsky (1963) Temperature 22.9  0.06 23.3  0.25
[46,53], using the following formula: (CI ¼ 100  BW/SL3), where: Turbidity 9.8  3.27 44.8  18.00
pH 6.2  0.06* 6.6  0.08
BW ¼ Body weight (g) and TC ¼ total length (cm).
Metal concentration in water
Cd 2.3  0.7* nd
2.4. Tissue processing Mn 213.6  25.9* 139.1  1.9
Zn 101.1  2.1* 16.7  1.5
Metal concentration in sediment
After capture, the animals were sacrificed via cranial transection As 50.4  3.4* 18.2  1.2
with surgical scissors. The operculum was removed and fragments Cd 5.2  1.6* 0.6  0.3
of the first gill arch from the right side of each animal were fixed in Cu 11.6  1.7 5.6  0.3
Bouin’s liquid and in modified Karnovsky’s solution (2.5% glutar- Mn 36.1  12.4 45.0  8.8

aldehyde, 2.0% paraformaldehyde in 0.2 M sodium cacodylate Values represented as mean  SEM. *p < 0.05; -n.d. not detectable.
 M.S. Procópio et al. / Fish & Shellfish Immunology 39 (2014) 108e117 111

Fig. 2. Graph containing the values of condition index (CI) of male and female fish at
point 1 (impacted) and point 2 (reference). Fig. 4. Graph showing the occurrence of fused lamellae in gill from males and females
sampled in impacted (Point 1) and reference (Point 2). Different letters represent
significance among the groups, p < 0.0001.

seven females and nine males were collected and in the second
point, three males and eight females. In PAS-AB 2.5 histochemistry
technique, mucous cells of type A stains in purple, of type B in pink
and type C in magenta [55]. Two PAS-AB 2.5 stained-sections from
each fish were selected and quantification was performed in 15
fields of reticulated eyepiece, totaling 30 fields per fish (2430
intersection points per fish), with an overall total of 780 fields
analyzed (65,610 points). The intersection points considered were:
the types A, B and C mucosal cells present in the filaments and
lamellae. Mucous types A, B, and C present in the interlamellar
spaces follows the same criteria. All intersection points present in
the filaments, lamellae and spaces without tissue were also
measured. Only sections with longitudinal filaments and sym-
metrical lamellae were included in the analysis. Mucous cells types
were quantified using an Olympus BX 48 microscope with a 40
objective coupled to an OlympusÒ ax0069 reticulated eyepiece
with 81 intersections.

Fig. 3. Histopathologies in the gill epithelium of P. argenteus. In A, arrowheads point to the presence of lamellar fusion. In B, note the presence of lamellae with hyperplasia areas
(arrowheads). In C, inflammatory foci are seen between lamellae (arrowheads). In D, arrowhead shows detachment of filament epithelium. In E, lamellae with aneurysm
(arrowhead) and edema (arrow). In F, observe the presence of parasitic cysts (arrowheads). Bar A, B, C, D: 30 mm. Bar E: 60 mm. Bar F: 130 mm.
112 M.S. Procópio et al. / Fish & Shellfish Immunology 39 (2014) 108e117
Fig. 5. Graph displaying the occurrence of lamellae with hyperplasia areas in gill from
males and females sampled in impacted (Point 1) and reference (Point 2). Different
letters represent significance among the groups, p < 0.0001.
2.8. Quantification of myeloperoxidase (MPO) and eosinophil
peroxidase (EPO) activity in gill
For this purpose, 34 animals were used. Sixteen (7 males and 9
females) were sampled in point 1 and eighteen (8 females and 10
males) at the reference point (point 2). Samples of gills (100 mg)
were homogenized with 1.9 ml of buffer (0.1 M NaCl, 0.02 M
Na3PO4, 0.015 M Na2 EDTA, pH 4.7) and centrifuged at 4
C for
10 min at 10,000 rpm. The supernatant was then discarded and
1.5 ml of 0.2% NaCl solution was added to the remaining precipitate
and after 30 s, 1.5 ml of cold solution with 1.6% NaCl 5% glucose was
added. The samples were homogenized and centrifuged at 4
C for
10 min at 10,000 rpm. The supernatant was discarded followed by
the addition of 1.9 ml of sample buffer (0.05 M Na3PO4 and HETAB
0.5% w/v) at pH 5.4, and homogenized. Three cycles of freezing with
liquid nitrogen were performed. The samples were centrifuged at
4
C for 15 min at 10,000 rpm and the supernatant was collected for
enzyme assay.
For the MPO enzyme assay, 96-well microplates in duplicate
(white cavity: 0.05 M Na3PO4 and HETAB 0.5% w/v) were filled with
25 ml of the diluted sample. Next, 25 ml of TMB substrate (3, 30 , 5, 50 -
tetramethylbenzidine) was added to each well and incubated at
37
C. After 5 min 100 ml of H2O2 (0.002%) was added and incubated
at 37
C for 5 min. For the blocking reaction, 100 ml of H2SO4 (1 M)
was added and the absorbance was read at 450 nm in a spectro-
photometer (Emax, Molecular Devices).
For the EPO enzyme assay, 75 ml of diluted sample was placed
into 96-well microplates in duplicate (white cavity: PBS); to each
well, 75 ml substrate (OPD 1.5 mM, Tris-0.075 mM HCl, pH 8.0,
6.6 mM H202) were added After 30 min of incubation in the dark,
the reaction was blocked by adding 50 ml of H2SO4 (1 M) and the
absorbance was read at 492 nm in a spectrophotometer (Emax,
Fig. 6. Chart A showing the occurrence (%) of inflammatory foci between lamellae at sample points and B according to gender and sample points. In A, higher incidence of in-
flammatory foci at point 2 is seen; in B, note that females at point 2 show higher incidence when compared to the other groups. F: female, M: male, Point 1: impacted, Point 2:
reference. Values represented as mean  SEM. *p < 0.05 and **p < 0.0001.
Molecular Devices). Both results were expressed as mean absor-
bance on 100 mg of wet tissue.
2.9. Statistical analysis
Statistical analyzes were performed using the software Graph
Pad Prism, version 4.0. (San Diego, CA, USA). Results were consid-
ered significant when p < 0.05. Data were assessed for normality
using the method of Kolmogorov and Smirnov. Parametric data are
presented as mean  standard error of the mean (SEM) and
nonparametric data presented as median. The data obtained by the
measurement of physicochemical parameters of water and for the
evaluation of heavy metals in water and sediment were analyzed by
t test, followed by the F test for analysis of variation of standard
deviation. The parameters that showed variability in standard de-
viations were analyzed by t test with Welch’s correction (p < 0.05).
The factor of Fulton’s condition was analyzed by ANOVA (p < 0.05).
As for the quantification of gill histopathologies, with the exception
of the foci of inflammatory infiltrate, data were analyzed by
nonparametric Fisher test. For rodlet cell counts, a nonparametric
test (Chi-square) was used for comparing the points of the study
while comparison of genders in the main study was performed
with Fisher test. T test was used for the analysis of the quantifica-
tion of foci of inflammatory infiltrates, and measuring of the
enzymatic activity of MPO and EPO when comparing only the
points of the study. ANOVA (p < 0.05) followed by the Bonferroni’s
test was used for comparison of the different genders in the study
points. The correlation data of MPO and EPO was performed by
Spearman’s test (p < 0.05). However, the data obtained by
morphometric mucosal cells were analyzed by t test (p < 0.05) and
when necessary, followed by Welch’s correction. Furthermore, in
order to decrease variability, data was log transformed followed by
T test.
3. Results
3.1. Environmental parameters and chemical analysis in water and
sediment
The physic-chemical analysis of the water, such as conductivity
(mS cm1), dissolved oxygen (mg L1) and pH showed differences
between the points of the study (#1 and # 2), while the tempera-


ture ( C) turbidity (NTU) did not differ between the two points
(Table 1). The mean concentrations of Cd, Mn and Zn dissolved in
water showed higher values at the point immediately downstream
of the Three Marias (#1) when compared to the reference point (#
2) (Table 1). In the sediment, metals such as As, Cd, Cu, Mn and Zn
were detected in all samples collected in both study points.
 M.S. Procópio et al. / Fish & Shellfish Immunology 39 (2014) 108e117
Fig. 7. Graph showing percentage of interlamellar spaces containing the presence of rodlet cells. In A, note the higher incidence of rodlets cells in fish at point 1; B shows the
incidence between genders and at sample points. At point 1, males have a higher incidence than females; also, males at point 1 show higher incidence than males at point 2. F:
female, M: male, Point 1: impacted, Point 2: reference. Values represented by the median. *p < 0.0001.
However, the mean concentrations for As and Cd in the impacted (#
1) point showed, respectively, values of 2.8 and 8.7 times higher
than those obtained in the reference point (# 2)(Table 1).
3.2. Fish biometrical data
There was no significant difference in the body weights and the
condition index (CI) (1.4  0.16) between males and females (Fig. 2).
All animals did not display any apparent external morphological
alterations.
3.3. Gill histopathologies
Histopathological changes in the gills of P.argenteus were
observed in fish from both sex and studied points. The most rele-
vant gill changes were: lamellar fusion (Fig. 3A), lamellar hyper-
plasia (Fig. 3B), lamellar edema (Fig. 3C) and inflammatory foci
(Fig. 3D). Aneurysms (Fig. 3E) and epithelial lifting (Fig. 3F) and
parasitic cysts were also observed in the filaments (Fig. 3G). The
majority of the analyzed histopathologies were sex-influenced,
with exception of the lamellar hyperplasia.
Male fish collected in the reference point (#2) showed more
occurrences of fused lamellae and no difference was found when
comparing females from different points. In the impacted point
(#1) no difference between males and females were observed,
while in point 2, males had higher occurrence of lamellae with
lamellar fusion than females (Fig. 4). In the impacted point (#1),
males and females showed an increase of lamellae with hyperplasia
when compared to the reference site (#2). No difference between
sexes was observed in the points (Fig. 5). Concerning the
Fig. 8. Graphs displaying the incidence of lamellar edema in gill from males and fe-
males sampled in impacted (Point 1) and reference (Point 2). Different letters repre-
sent significance among the groups, p < 0.01.
113
occurrence of inflammation foci between neighboring lamella, fish
from reference point (#2) almost duplicated its occurrence when
compared to impacted point (#1) (Fig. 6A). In this context, females
from point (#2) showed the highest occurrence between the
groups, representing near six times higher from females in point
(#1), while female fish from the impacted point (#1) showed the
lowest occurrence of inflammation foci (Fig. 6B).
Differently, fish from the impacted point (#1) showed the
highest occurrence of rodlet cells compared with the reference
point (#2) (Fig. 7A). In addition, males from point (#1) showed the
highest occurrence of rodlet cells, a two fold increase when
compared with males from the reference point (#2) (Fig. 7B).
With respect to lamellar edema, a sex and point dependent
occurrence were observed between the groups (Fig. 8).
3.4. Mucous cells
Fish collected in the impacted point (#1) showed a nearly two
fold decrease in the volume of the filament occupied by type A
mucosal cells and a threefold in the volume of the interlamellar
spaces occupied by type A mucous (Table 2). This decrease in the
volume occupied by the filament-type mucous cells occurred in the
male specimens (Table 3), whereas the decrease of the volume
occupied by type A mucous was demonstrated in the interlamellar
spaces of females (Table 4). However, in the impacted point (#1),
males showed an increase in the volume of lamellae occupied by
type C mucous cells (Table 3). A similar trend was observed in fe-
males of the same point (#1) (Table 4).
Males from the impacted point (#1) also showed a tendency to
increase the volume occupied by the filament-type C cells (Table 3),
not observed in females of the same point (#1) (Table 4). In addi-
tion, males from #1 also showed an increase in the volume of
interlamellar space occupied by type B mucous (Table 3).
Table 2
Values in percentage of the volume occupied by the different types of mucus in
different regions of the gill between the points.
Point Type A Type B Type C
#1 #2 #1 #2 #1 #2
Filament 3.7  0.5a 6.2  0.7 4.0  0.5 4.0  0.6 0.4  0.2 0.1  0.1
Lamella 1.6  0.3 1.7  0.3 3.3  0.6 2.4  0.6 0.4  0.1 0.3  0.1
Interlamellar 6.2  1.0a 19.3  5.0 6.2  1.0 11.2  2.0 0.4  0.2 0.7  0.3
space
Values represented by the mean  SEM.
Point 1: impacted and Point 2: reference.
a
significant values between the points (p < 0.05).
114 M.S. Procópio et al. / Fish & Shellfish Immunology 39 (2014) 108e117

Table 3
Values in percentage of the volume occupied by the different types of mucous in different regions of the gills of males collected at the two points.

Point Type A Type B Type C

#1 #2 #1 #2 #1 #2
a
Filament 3.8  0.7 8.0  1.8 4.0  0.5 5.0  0.6 0.5  0.2* 0.1  0.01
Lamella 1.6  0.5 1.5  0.6 2.3  0.7 1.3  0.6 0.5  0.2a 0.1  0.01
Interlamellar space 9.5  3.2 19.6  9.4 11.5  1.5a 4.2  0.4 0.5  0.3 0.9  0.9

Values represented by the mean  SEM.

*p ¼ 0.07.
Point 1: impacted and Point 2: reference. e log (y) transformed, and t-test (p < 0.05).
a
significant values between the two points.

3.5. MPO and EPO activity
The MPO and EPO activity in fishes from point (#2) showed a
doubled increase when compared with fish from point (#1) (9A and
9C). Male fishes from the impacted point (#1) showed less MPO and
EPO activity compared to the other groups (Fig. 9BeD).
4. Discussion
The results of the present study provide evidence that P.
argenteus gill biomarkers differ between the studied points ac-
cording to the water and sediment quality. Furthermore, results
highlighted a sex-specific reaction of some biomarkers to the
environmental conditions. Based on the metals contamination of
water and sediment, point # 1 was the most affected while #2 was
considered the reference point in the São Francisco river.
The concentrations of Cd in the water in #1 were 2.3 times
higher (1 mg/L) than the established values by the CONAMA Reso-
lution 357/2005 [56]. Previous studies at #1 and in the same year
season, showed no violation of standards for the concentration of
Cd in water [48,49]. When comparing to the accepted values, Mn
concentration at the #1 was 2-fold higher the accepted while at #2,
it was about 1.3 times (100 mg/L). Concerning metals in the sedi-
ment, the # 1 showed Cd values above the considered probable
effect concentration (PEC) of 3.5 mg/kg, while the reference point
(#2) had concentrations within the established limit for the 0.6 mg/
kg threshold effect concentration (TEC). Regarding As concentra-
tion, the #1 showed concentrations nearly three times higher for
the established PEC (17 mg/kg), while in #2, the concentration was
lower than the PEC. Silva (2007) at the same collection point (#1)
found that the concentration of As in the sediment was below 5 mg/
kg, which is almost 10 times lower than that obtained in the pre-
sent study, Thus, based on the toxicity proposed by SQGV (Sedi-
ment quality guidelines values), it is possible to assume that at
point #1, there is a high probability of incidence of adverse effects
to aquatic biota due to the concentrations of As and Cd [57].
In P.argenteus, no differences in CI were observed in fish from
the points #1 and #2. Similarly, specimens of Cnesterodon decem-
maculatus from a polluted river in Argentina did not show differ-
ences in CI even in the presence of Cd, Cr and Zn [58]. Also, our
results showed no fish gender influence in CI. No gender variation
was also observed in the Gasterosteus aculeatus from a low
contaminated stream [42]. Probably, the CI is not a sensible enough
biomarker to be used in the São Francisco river points assessed.
Nevertheless, males from the impacted point (#1) showed fewer
occurrences of MPO and EPO activity as well as inflammatory foci. It
was associated with an increase in rodlet cells occurrence. In teleost
fishes, the rodlet cells are suggested to be involved in inflammatory
responses. However, the roles developed by these cells in the in-
flammatory process are still unknown [28,29,32]. Rodlet cells in
Psetta maxima showed the presence of S100 protein [31]. In
mammals, the subtype S100A9 is involved in the suppression of
pro-inflammatory macrophage activation contributing to the res-
olution of inflammation [59]. Therefore, our results suggested that
the lowest activity of MPO and EPO in males collected in the
impacted point (#1), associated with the highest occurrence of
rodlet cells may indicate that these cells could be involved in the
resolution of the inflammation. MPO activity in mammals is
enhanced by the influence of estrogens in females [60,61].
Previous studies carried out with P. argenteus at the same
studied points and season showed differences in the sex steroids
according to the points [62,63]. Higher levels of testosterone,
17bestradiol and 17a-hydroxiprogesterona were observed in
males and females of P. argenteus collected at point 2. Since
testosterone and 17b-estradiol increase innate immunological re-
sponses in fish, including myeloperoxidase (MPO) activity [64,65],
we suggest a possible hormonal influence in MPO activity in
P. argenteus.
However, we cannot fail to consider that the sex-differences of
MPO activity could be influenced by the feeding habits, since some
studies reported the modulation of non-specific immune re-
sponses, including in MPO activity in different experimentally
induced feeding habits [66,67]. In spite of some studies reported
different sexual behavior in adaptation to stress conditions [68], to
our knowledge, no studies related to the sexual behaving feeding
habits were carried out in the field.
Moreover, metals such as Cd are considered an endocrine dis-
rupted chemical in fish, altering the synthesis of testosterone,
progesterone, estradiol and other reproduction-related hormones
[69]. Thus, our results showed a sex-variation in MPO activity, and

Table 4
Values in percentage of the volume occupied by the mucous types in different regions of the gills of females collected at the two points.

Point Type A Type B Type C

#1 #2 #1 #2 #1 #2

Filament 5.0  2.0 5.5  0.7 4.3  1.0 4.1  0.8 0.02  0.02 0.06  0.03
Lamella 1.6  0.4 2.4  0.7 4.3  0.7 2.8  0.7 0.4  0.2* 0.4  0.08
Interlamellar space 5.4  1.6a 19.2  6.7 8.8  2.0 12.7  3.0 0.1  0.1 0.6  0.3

Values represented by the mean  SEM.

*p ¼ 0.058.
Point 1: impacted and Point 2: reference. e log (y) transformed, t test (p < 0.05).
a
significant values between points.
 M.S. Procópio et al. / Fish & Shellfish Immunology 39 (2014) 108e117
Fig. 9. Charts displaying MPO and EPO absorbance rate in male and female fish gills at the two points. A and B refers to MPO and C and D to EPO. In A, note the higher rate at point 2,
and in B in males at point 2. C shows the highest EPO absorbance rate at point 2; in D, males at point 2 show a higher rate than males at point 1 whereas females at point 2 present a
higher rate than males at point 1. F: female, M: male, Point 1: impacted, Point 2: reference. Values represented as mean  SEM. *p < 0.01 and **p < 0.0001.
probably a sexual hormone influence in MPO activity in
P. argenteus.
The lamellar hyperplasia, characterized by the thickening of the
gill epithelium, is considered a physiological barrier that impaired
the pollutant entrance into the blood circulation and consequently
to a systemical spread [70,71]. Considering the studied sites, fish
collected in #1 presented more occurrence of hyperplasia
compared with fish from #2 probably due to the high contamina-
tion of Cd, As, Mn and Zn in this point. Lamellar hyperplasia,
lamellar edema and lamellar fusion were also observed in other fish
species exposed to metal contamination [15,72,73]. Liza saliens
from the Esmoriz-Paramos Coastal Lagoon in Portugal showed the
same histopathological alterations due to the contamination of Zn
and Cd [74].
Males from point #2 showed prevalence of fusion lamellae,
probably due to a physiological adaptation to the higher oxygen
concentration in the water in this point when compared to point
(#1) [76,77]. No reports in the literature suggest differential re-
sponses to oxygen concentration related to fish sex. Furthermore,
lamellar fusion also contributes to increase the distance through
which the toxicant has to travel to reach the blood vessels, probably
acting as a physiological barrier to metal input [21]. It could also
contribute to the fewer occurrences of inflammation foci and
lamellar edema in male fish from the point #2. Our results also
show the presence of epithelial lifting and aneurysms as a conse-
quence of severe edema in some fish [78].
Another barrier to pollutants input in gill is the glycoproteins
produced by the mucous cells [75,78e80]. Hyperplasia of mucous
cells is observed in several fish species exposed to salinity varia-
tions, parasite infections, metal contamination, oil spills and her-
bicides [81e84]. Our results showed that males from point #1 had a
decrease of area occupied by type A mucous in the filament and an
increase of type C mucous in lamellae. The same pattern was found
in Abramis brama exposed to oil spill in the Po river [83]. However
the increase in tissue volume occupied by type C mucous did not
overcome the decrease of type A mucous, suggesting a low secre-
tion of mucous in males from point #1. In Solea senegalensis
115
chronically exposed to sediments contaminated with several
metals, including Cu and Pd, mucous cells showed degeneration in
the gill epithelium and decrease in mucous secretion [73]. More-
over, type C mucous is composed of acid sulphated and carboxi-
lated glycoproteins that present high-viscosity. Mucous
coagulation on the gill surface also contributes to reduce pollutants
uptake [85,86].
In murine model of lung inflammation, several studies showed
that the increase of mucous secretion is positively associated with
EPO activity [87,88]. Moreover, in inflammation, it has been known
that eosinophils show numerous immune regulatory functions,
including production of a range of cytokines and chemokines that
leads to the exacerbation of inflammation and mucous secretion
[89,90]. As occurs in mammals, our results showed the association
of the activities of EPO, with inflammatory foci and glycoprotein
secretion by mucous cells. So, we suggest that males collected in
point (#1) showed chronic inflammation due to the lowest pres-
ence of inflammatory cells, as well as, EPO and MPO activity and
glycoprotein production. To our knowledge, the present study is the
first report to show the association of EPO activity, inflammatory
foci occurrence and glycoprotein secretion in fish gill. Moreover,
these data strongly suggests sex-differences in fish adaptation to
environmental conditions.
Thus, our results show that the histopathological, cellular and
biochemical biomarkers assessed in gill of P. argenteus are reflecting
the environmental contamination in the São Francisco river.
Moreover, most of the biomarkers studied showed a sex-specific
response, with exception of lamellar hyperplasia and EPO activity.
Therefore, our results highlight the necessity of fish sex discrimi-
nation and the importance in the use of several biomarkers in order
to comprehend the influence of the environment contaminants in
the biological mechanism.
Acknowledgments
This work received financial support from CAPES, CNPq, PRPq-
UFMG and FAPEMIG (Brazilian Agencies for Science and
116 M.S. Procópio et al. / Fish & Shellfish Immunology 39 (2014) 108e117
Technology). The authors are also grateful to the following Brazilian
institutions: Hydrobiology and Hatchery Station of Três Marias and
CODEVASF/CEMIG (for the help with fish sampling).
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