Sex-response differences of immunological and histopathological biomarkers in gill of Prochilodus...

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Contents lists avai

Fish & Shellfish Immunology

journal homepage: www.elsevier .com/locate / fs i

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Sex-response differences of immunological and histopathologicalbiomarkers in gill of Prochilodus argenteus from a polluted riverin southeast Brazil

Marcela Santos Procópio a, Heder José Ribeiro a,1, Luciano Almeida Pereira d,Gabriel Augusto Oliveira Lopes b, Antônio Carlos Santana Castro a, Elizete Rizzo a,Yoshimi Sato c, Remo de Castro Russo b, José Dias Corrêa Junior a,*aDepartamento Morfologia, Universidade Federal de Minas Gerais, Av. Antônio Carlos, 6627 Pampulha, Belo Horizonte, 31270 901 Minas Gerais, Brazilb Laboratório de Imunologia e Mecânica Pulmonar, Departamento de Fisiologia e Biofísica, Av. Antônio Carlos, 6627 Pampulha, Belo Horizonte,31270 901 Minas Gerais, BrazilcCompanhia 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, BrazildColégio Técnico, Universidade Federal de Minas Gerais, Belo Horizonte, Minas, Gerais, Brazil, Av. Antônio Carlos, 6627 Pampulha, Belo Horizonte 31270 901,Minas Gerais, Brazil

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Article history:Received 10 October 2013Received in revised form10 March 2014Accepted 14 April 2014Available online xxx

Keywords:BiomarkersHistopathologyMucous cellsRodlet cellsHyperplasiaInflammatory fociEdemaLamellar fusionMyeloperoxidaseEosinophil peroxidase

Abbreviation list: #1, first studied point; #2, seconindex; PEC, probable effect concentration; SQGV,values; TEC, threshold effect concentration.* Corresponding author. Departamento Morfolog

Minas, Gerais, Belo Horizonte, Minas Gerais, Av. AntBelo, Horizonte, MG 31270 901 Brazil. Tel.: þ55 31 34

E-mail addresses: [email protected], [email protected] Departamento Básico e Área de Saúde, Universid

Governador Valadares, Minas Gerais, Brazil.

http://dx.doi.org/10.1016/j.fsi.2014.04.0101050-4648/� 2014 Elsevier Ltd. All rights reserved.

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Please cite this article in press as: ProcópioProchilodus argenteus from a polluted river i

a b s t r a c t

The fish gill is in direct and standing contact with the immediate external environment and, therefore, ishighly vulnerable to aquatic pollutants. In this study, Prochilodus argenteus were caught at two differentpoints in São Francisco river. The first point is located near Três Marias dam, while the second is placeddownstream the Abaeté river. Chemical approaches showed the presence of metals contamination in thefirst point. Thus, the main goal of this study was to investigate the possible toxic effects of these con-taminants and the likely use of biomarkers on fish gills. Biometric data of length and weight of fish wereobtained in order to calculate the condition factor as an organismal biomarker. The histological changesin gills and alterations in mucous and rodlet cells occurrence were detected microscopically and eval-uated with quantitative analyses. Myeloperoxidase (MPO) and Eosinophil Peroxidase (EPO) were alsoassessed in fish gill. The analysis of the water and sediment samples revealed the presence of metals atthe two points. As and Cd were detected at higher concentrations at point 1. The presence of lamellar cellhyperplasia, 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 theenvironmental 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 achronical inflammation in gills due to the lowest activity of MPO and EPO, as well as low occurrence ofinflammatory foci and glycoprotein secretion by mucous cells, while female fish presented an oppositepattern 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.

� 2014 Elsevier Ltd. All rights reserved.

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d studied point; CI, conditionsediment quality guidelines

ia, Universidade Federal deônio Carlos, 6627 Pampulha,09 2792.frj.br (J.D. Corrêa Junior).ade Federal de Juiz de Fora,

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MS, et al., Sex-response diffen southeast Brazil, Fish & She

1. Introduction

Urban, industrial and agricultural activities have resulted in anincrease in the number of impacted environments due to therelease of contaminated wastewater in the freshwater systems. Inorder 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

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rences of immunological and histopathological biomarkers in gill ofllfish Immunology (2014), http://dx.doi.org/10.1016/j.fsi.2014.04.010

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the efficiency of the biomonitoring programs of the aquaticecosystem [1,2].

The gills represent the largest part of the surface area of the totalfish body which is in direct and standing contact with the envi-ronment [3e5]. Accordingly, the gill is considered a target organ ofwastewater pollutants [6]. Several studies, in laboratory and in thefields showed that histopathologies and innate immunologicalparameters are sensible tools to detect the toxic effects of chemicalcompounds in fish gill [7e13,23]. However, the largemajority of thestudies have based their conclusions on biomarkers that reach onlyone level of biological organization [14,15]. Thus, the association ofa variety of molecular, biochemical, physiological, histo-cytopathological and organismal biomarkers are recommended inorder to obtain an accurate evaluation of toxic compounds in thefreshwater ecosystem [16e18].

The condition index (CI), considered as an organismalbiomarker, is useful to monitor pollution as toxic compoundsdeplete energy reserves that were potentially destined for growth[19].

Nevertheless, fish exposure to metals contamination displayedthe presence of several histopathologies in fish gill, includinglamellar hyperplasia, lamellar edema, aneurysms, epithelial lifting,lamellar fusion and presence of inflammatory foci, which arehallmarks often observed in gill exposed to Cd, As, Zn and othermetals [15,20,21]. Immunological parameters in fish gills are alsoaffected by metal and others pollutants, as increased levels ofceruloplasmin and lysozym presented by fish exposed to Cd, Zn, Hg,Cr and Fe [13]. Immune modulation was also observed in liver andspleen of gilthead seabream (Sparus aurata) exposed to differentcontaminants [22e24], indicating a close relation between envi-ronmental stress response and immune system.

Rodlet cells which are characterized by a fibrous layer beneathplasma membrane and conspicuous cytoplasmic inclusions calledrodlets, have been observed in different marine and freshwatersfish species [25e27]. In the development of Cyprinus carpio, rodletcell in gills appear 14 days after fertilization, suggesting that thiscell is an innate constituent of the fish tissues [28]. Associated withthewide distribution of these cells among the epithelium of severalorgans and the increase of occurrence of these cells in exposure tostress factors, it is proposed that this cells act in immunologicalresponses in the organism [29e31]. However, the function devel-oped by this cell is still a matter of debate. Nevertheless, rodlet cellhas been used as biomarkers of exposure to metals, organochlo-rines, and parasitic infections [26,32,33].

Myeloperoxidase (MPO) in mammals, which is released fromcytoplasmic granules of neutrophils by a degranulation process,reacts with the H2O2 formed by the respiratory burst to form acomplex that can oxidize a large variety of substances [34]. Inzebrafish, (Danio rerio), MPO is present only in neutrophils [35e37]. In others teleost such as Labeo rohita and Carassius auratusMPO activity is also associated with neutrophils [38,39]. Eosino-phil in teleost, which has been thoroughly characterized recentlyin zebrafish, showed expression of GATA 2 transcription factorthat plays an essential role in eosinophil development in mam-mals. Moreover, degranulation and peroxidase release of purifiedgata2hi cells, analyzed using the OPD assay, was observed inresponse to Heligmosomoides polygyrus helminth extracts [37].Despite the widespread use and description of histopathological,cellular and immune biomarkers in environmental studies, fewapproaches consider the possible influence of fish sex on theresponse to stressors [19,40,41]. In addition to enhancing theinterpretation of the data, the discrimination of biological re-sponses according to the sex of animals could contribute to thesampling optimization and minimize the time-consuming anal-ysis procedure [42].

Please cite this article in press as: Procópio MS, et al., Sex-response diffeProchilodus argenteus from a polluted river in southeast Brazil, Fish & She

Prochilodus argenteus (Characiforme: Prochilodontidae) popu-larly known as curimba, is an endemic specie from São Franciscoriver with detritivore feeding habits [43]. This specie has economicimportance, representing 50% of all captured fish in the Três Mariasregion and configures itself as an important source of food for localpopulations [44,45]. Due to feeding habits P. argenteus is consideredideal specie to study metal contamination in freshwater ecosys-tems [46].

Several anthropogenic activities affect the São Francisco river inthe site between Três Marias dam and the Abaeté river. Metalcontamination of this site, due to the presence of a zinc producingindustry, 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 environmentalpollution in the São Francisco river; ii) discriminate the possibleinfluence of the fish sex in biomarkers responses; iii) assess theneed 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 contaminantexposure conditions, as described in Section 1. The first point islocated immediately downstream of the Três Marias dam reservoir(#1), while the second point, considered as reference, is locatedafter the São Francisco and Abaeté river confluence, at approxi-mately 34 km from the first point (#2) (Fig. 1). The study wasperformed during the dry season (2008 August) to avoid influencesof the fish reproductive period and of the thermal stratification ofthe Três Marias reservoir, since it is considered a warmmonomicticreservoir [51]. The reference point was defined based on previousstudies [52].

2.2. Environmental parameters and chemical analysis in water andsediment

Measures of water physicalechemical parameters and concen-tration of metals inwater and sediment were performed in the twostudied points. At each point, measurements were carried out onthe right and left river bank, as well as in the central channel. Theresults 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 a10% nitric acid solution for 48 h and kept refrigerated until analysis.Samples were filtered through a 0.45 mm membrane filter and theliquid portion was used in determining the concentration of cad-mium (Cd), manganese (Mn) and zinc (Zn). The sediment sampleswere collected using a Petersen dredge and 350 g were stored in500 mL plastic bottles previously decontaminated in a 10% nitricacid solution for 48 h and kept refrigerated until analysis. Next,sediment sampleswere dried at 60 �C for 24 h and passed through a65 mm sieve. Suspensions containing 10 mg of the sieved samplewere treated with HNO3 and HCl (PA) and used to analyze theconcentration of arsenium (As), cadmium (Cd), copper (Cu) andmanganese (Mn). The analyses were performed on the atomic ab-sorption spectrometry with graphite furnace HGA 900 (Perkin


Fig. 1. Sample points in São Francisco river. The point 1 is localized immediatelydownstream Três Marias dam and the point 1 in the confluence with the Abaeté river.

Table 1Values corresponding to the conductivity (mS cm�1), dissolved oxygen rate (mg L�1),temperature (

�C), turbidity (NTU), pH, and dissolved metals in water (mg L�1) and

sediment (mg kg�1) obtained at the two points. Q8

Sample points Point 1 Point 2

Physico-chemical water parametersConductivity 21.5 � 6.7* 5.6 � 0.17Dissolved oxygen 8.1 � 0.05* 8.7 � 0.04Temperature 22.9 � 0.06 23.3 � 0.25Turbidity 9.8 � 3.27 44.8 � 18.00pH 6.2 � 0.06* 6.6 � 0.08Metal concentration in waterCd 2.3 � 0.7* ndMn 213.6 � 25.9* 139.1 � 1.9Zn 101.1 � 2.1* 16.7 � 1.5Metal concentration in sedimentAs 50.4 � 3.4* 18.2 � 1.2Cd 5.2 � 1.6* 0.6 � 0.3Cu 11.6 � 1.7 5.6 � 0.3Mn 36.1 � 12.4 45.0 � 8.8

Values represented as mean � SEM. *p < 0.05; -n.d. not detectable. Q9

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Elmer, Norwalk, CT USA). The values were expressed in mg L�1 andmg Kg�1, respectively.

2.3. Fish sampling

For biological evaluations 42 specimens of P. argenteus totallength of 32.0 � 0.5 cm and body weight of 471 � 19.3 g) werecollected, while 20 (10 females and 10 males) were captured inpoint 1 and 22 (9 females and 13 males) in point 2. The fish capturewas authorized by the State Forestry Institute from Minas Geraisstate (IEF/MG), scientific fishing license NR 130-10, category D. Thespecimens were captured with the aid of cast nets and stored inplastic containers containing 500 L of water from the river andremained without food until euthanasia. For each animal, the bio-metric data obtained were the total length (TL), standard length(SL) and body weight (BW). Only animals that did not display anyapparent external morphological alterations were analyzed. Thecondition index (CI) was determined according to Nikolsky (1963)[46], using the following formula: (CI ¼ 100 � BW/SL3), where:BW ¼ Body weight (g) and TC ¼ total length (cm).

2.4. Tissue processing

After capture, the animals were sacrificed via cranial transectionwith surgical scissors. The operculum was removed and fragmentsof the first gill arch from the right side of each animal were fixed inBouin’s liquid and in modified Karnovsky’s solution (2.5% glutar-aldehyde, 2.0% paraformaldehyde in 0.2 M sodium cacodylate


buffer, pH 7.3). Some tissue samples were kept on dry ice andsubsequently stored in �80 �C freezer. The guideline was approvedby 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 placedin 70% ethanol (Merk KGaA, Darmstadt, HE), decalcified in Pereny’ssolution according to Bancroft and Gamble (2007) [54], dehydratedin a graded ethanol solutions up to pure ethanol and embedded inparaffin (Paraplast�). Next, 5-mm tissue sections were deparaffi-nized with xylene, hydrated in solutions of ethanol and stained byhematoxylin and eosin (H&E), PAS/AB 2.5 [54]. Gill epithelium andcell morphology were analyzed under an Olympus BX41 light mi-croscope. Digitalized images were obtained with an Olympus BX41light microscope coupled to a digital camera Q-Color 3 (OlympusCorporation, Shinjuku-Ku, TY).

2.6. Histomorphometry

Morphometrical analysis of gill histopathologies and rodlet cellsoccurrence were carried out in H&E stained-sections. For patho-logical morphometry, gill fragments from 10males and 8 females atpoint 1 and 11 males and 7 females at point 2 were assessed. Fromeach animal, 60 to 100 lamellae in three longitudinal filamentswere selected, totaling 2760 lamellae. Occurrence of lamellae withedema, hyperplasia, interlamellar fusion and inflammation werequantified using the criteria previously established [20]. The pres-ence of rodlet cells was considered only in longitudinal filament gillsections with interlamellar fused lamellae and only evident rodletcells with filamentous capsule and granules. Gill fragments from 9(4 males and 5 females) and 13 (8 males and 5 females) fish wereused in the first (point 1) and second (point 2) points, respectively.For each fish, 100 interlamellar spaces were analyzed, totaling 2200interlamellar spaces. Histopathologies and rodlet cells occurrencewere quantified using the Olympus BX 48 light microscope(Olympus Corporation, Shinjuku-Ku, TY) under 400�magnification.

2.7. Assessment of the distribution of types A, B and C mucous cellsand mucous in the interlamellar space

Types A, B and C mucous cells were quantified in the filamentsand lamellae of a total of 27 fish. In the first point were collected


Fig. 2. Graph containing the values of condition index (CI) of male and female fish atpoint 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 representsignificance among the groups, p < 0.0001.


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seven females and nine males were collected and in the secondpoint, three males and eight females. In PAS-AB 2.5 histochemistrytechnique, mucous cells of type A stains in purple, of type B in pinkand type C in magenta [55]. Two PAS-AB 2.5 stained-sections fromeach fish were selected and quantification was performed in 15fields of reticulated eyepiece, totaling 30 fields per fish (2430intersection points per fish), with an overall total of 780 fieldsanalyzed (65,610 points). The intersection points considered were:the types A, B and C mucosal cells present in the filaments and

Fig. 3. Histopathologies in the gill epithelium of P. argenteus. In A, arrowheads point to the(arrowheads). In C, inflammatory foci are seen between lamellae (arrowheads). In D, a(arrowhead) and edema (arrow). In F, observe the presence of parasitic cysts (arrowheads)


lamellae. Mucous types A, B, and C present in the interlamellarspaces follows the same criteria. All intersection points present inthe filaments, lamellae and spaces without tissue were alsomeasured. Only sections with longitudinal filaments and sym-metrical lamellae were included in the analysis. Mucous cells typeswere quantified using an Olympus BX 48 microscope with a 40�objective coupled to an Olympus� ax0069 reticulated eyepiecewith 81 intersections.

presence of lamellar fusion. In B, note the presence of lamellae with hyperplasia areasrrowhead shows detachment of filament epithelium. In E, lamellae with aneurysm. Bar A, B, C, D: 30 mm. Bar E: 60 mm. Bar F: 130 mm.

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Fig. 5. Graph displaying the occurrence of lamellae with hyperplasia areas in gill frommales and females sampled in impacted (Point 1) and reference (Point 2). Differentletters represent significance among the groups, p < 0.0001.


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2.8. Quantification of myeloperoxidase (MPO) and eosinophilperoxidase (EPO) activity in gill

For this purpose, 34 animals were used. Sixteen (7 males and 9females) were sampled in point 1 and eighteen (8 females and 10males) 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 MNa3PO4, 0.015 M Na2 EDTA, pH 4.7) and centrifuged at 4 �C for10 min at 10,000 rpm. The supernatant was then discarded and1.5 ml of 0.2% NaCl solution was added to the remaining precipitateand after 30 s, 1.5 ml of cold solutionwith 1.6% NaCl 5% glucose wasadded. The samples were homogenized and centrifuged at 4� C for10 min at 10,000 rpm. The supernatant was discarded followed bythe addition of 1.9 ml of sample buffer (0.05 M Na3PO4 and HETAB0.5%w/v) at pH 5.4, and homogenized. Three cycles of freezing withliquid nitrogen were performed. The samples were centrifuged at4 �C for 15 min at 10,000 rpm and the supernatant was collected forenzyme 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 with25 ml of the diluted sample. Next, 25 ml of TMB substrate (3, 30, 5, 50-tetramethylbenzidine) was added to each well and incubated at37 �C. After 5 min 100 ml of H2O2 (0.002%) was added and incubatedat 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 placedinto 96-well microplates in duplicate (white cavity: PBS); to eachwell, 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 theabsorbance was read at 492 nm in a spectrophotometer (Emax,

Fig. 6. Chart A showing the occurrence (%) of inflammatory foci between lamellae at sampflammatory foci at point 2 is seen; in B, note that females at point 2 show higher incidencreference. 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 GraphPad Prism, version 4.0. (San Diego, CA, USA). Results were consid-ered significant when p < 0.05. Data were assessed for normalityusing the method of Kolmogorov and Smirnov. Parametric data arepresented as mean � standard error of the mean (SEM) andnonparametric data presented as median. The data obtained by themeasurement of physicochemical parameters of water and for theevaluation of heavymetals inwater and sediment were analyzed byt test, followed by the F test for analysis of variation of standarddeviation. 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 exceptionof the foci of inflammatory infiltrate, data were analyzed bynonparametric Fisher test. For rodlet cell counts, a nonparametrictest (Chi-square) was used for comparing the points of the studywhile comparison of genders in the main study was performedwith Fisher test. T test was used for the analysis of the quantifica-tion of foci of inflammatory infiltrates, and measuring of theenzymatic activity of MPO and EPO when comparing only thepoints of the study. ANOVA (p < 0.05) followed by the Bonferroni’stest was used for comparison of the different genders in the studypoints. The correlation data of MPO and EPO was performed bySpearman’s test (p < 0.05). However, the data obtained bymorphometric mucosal cells were analyzed by t test (p < 0.05) andwhen necessary, followed by Welch’s correction. Furthermore, inorder to decrease variability, data was log transformed followed byT test.

3. Results

3.1. Environmental parameters and chemical analysis in water andsediment

The physic-chemical analysis of the water, such as conductivity(mS cm�1), dissolved oxygen (mg L�1) and pH showed differencesbetween 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 inwater showed higher values at the point immediately downstreamof the Three Marias (#1) when compared to the reference point (#2) (Table 1). In the sediment, metals such as As, Cd, Cu, Mn and Znwere detected in all samples collected in both study points.

le points and B according to gender and sample points. In A, higher incidence of in-e when compared to the other groups. F: female, M: male, Point 1: impacted, Point 2:

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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 theincidence 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.


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However, themean concentrations for As and Cd in the impacted (#1) point showed, respectively, values of 2.8 and 8.7 times higherthan those obtained in the reference point (# 2)(Table 1).

3.2. Fish biometrical data

There was no significant difference in the body weights and thecondition index (CI) (1.4� 0.16) betweenmales and females (Fig. 2).All animals did not display any apparent external morphologicalalterations.

3.3. Gill histopathologies

Histopathological changes in the gills of P.argenteus wereobserved 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) andparasitic cysts were also observed in the filaments (Fig. 3G). Themajority of the analyzed histopathologies were sex-influenced,with exception of the lamellar hyperplasia.

Male fish collected in the reference point (#2) showed moreoccurrences of fused lamellae and no difference was found whencomparing 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 withlamellar fusion than females (Fig. 4). In the impacted point (#1),males and females showed an increase of lamellaewith hyperplasiawhen compared to the reference site (#2). No difference betweensexes 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.

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occurrence of inflammation foci between neighboring lamella, fishfrom reference point (#2) almost duplicated its occurrence whencompared to impacted point (#1) (Fig. 6A). In this context, femalesfrom point (#2) showed the highest occurrence between thegroups, representing near six times higher from females in point(#1), while female fish from the impacted point (#1) showed thelowest occurrence of inflammation foci (Fig. 6B).

Differently, fish from the impacted point (#1) showed thehighest occurrence of rodlet cells compared with the referencepoint (#2) (Fig. 7A). In addition, males from point (#1) showed thehighest occurrence of rodlet cells, a two fold increase whencompared with males from the reference point (#2) (Fig. 7B).

With respect to lamellar edema, a sex and point dependentoccurrence were observed between the groups (Fig. 8).

3.4. Mucous cells

Fish collected in the impacted point (#1) showed a nearly twofold decrease in the volume of the filament occupied by type Amucosal cells and a threefold in the volume of the interlamellarspaces occupied by type A mucous (Table 2). This decrease in thevolume occupied by the filament-type mucous cells occurred in themale specimens (Table 3), whereas the decrease of the volumeoccupied by type A mucous was demonstrated in the interlamellarspaces of females (Table 4). However, in the impacted point (#1),males showed an increase in the volume of lamellae occupied bytype 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 toincrease 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 ofinterlamellar space occupied by type B mucous (Table 3).

Table 2Values in percentage of the volume occupied by the different types of mucus indifferent 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 � 0.1Lamella 1.6 � 0.3 1.7 � 0.3 3.3 � 0.6 2.4 � 0.6 0.4 � 0.1 0.3 � 0.1Interlamellar

space6.2 � 1.0a 19.3 � 5.0 6.2 � 1.0 11.2 � 2.0 0.4 � 0.2 0.7 � 0.3

Values represented by the mean � SEM.Point 1: impacted and Point 2: reference.

a significant values between the points (p < 0.05).

117118119120121122123124125126127128129130


Table 3Values 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

Filament 3.8 � 0.7a 8.0 � 1.8 4.0 � 0.5 5.0 � 0.6 0.5 � 0.2* 0.1 � 0.01Lamella 1.6 � 0.5 1.5 � 0.6 2.3 � 0.7 1.3 � 0.6 0.5 � 0.2a 0.1 � 0.01Interlamellar 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.


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3.5. MPO and EPO activity

The MPO and EPO activity in fishes from point (#2) showed adoubled increasewhen comparedwith fish from point (#1) (9A and9C). Male fishes from the impacted point (#1) showed lessMPO andEPO 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, resultshighlighted a sex-specific reaction of some biomarkers to theenvironmental conditions. Based on the metals contamination ofwater and sediment, point # 1 was the most affected while #2 wasconsidered the reference point in the São Francisco river.

The concentrations of Cd in the water in #1 were 2.3 timeshigher (1 mg/L) than the established values by the CONAMA Reso-lution 357/2005 [56]. Previous studies at #1 and in the same yearseason, showed no violation of standards for the concentration ofCd in water [48,49]. When comparing to the accepted values, Mnconcentration 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 probableeffect concentration (PEC) of 3.5 mg/kg, while the reference point(#2) had concentrations within the established limit for the 0.6mg/kg threshold effect concentration (TEC). Regarding As concentra-tion, the #1 showed concentrations nearly three times higher forthe established PEC (17 mg/kg), while in #2, the concentration waslower than the PEC. Silva (2007) at the same collection point (#1)found that the concentration of As in the sediment was below 5mg/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 atpoint #1, there is a high probability of incidence of adverse effectsto aquatic biota due to the concentrations of As and Cd [57].

In P.argenteus, no differences in CI were observed in fish fromthe 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

Table 4Values in percentage of the volume occupied by the mucous types in different regions o

Point Type A Typ

#1 #2 #1

Filament 5.0 � 2.0 5.5 � 0.7 4.3Lamella 1.6 � 0.4 2.4 � 0.7 4.3Interlamellar space 5.4 � 1.6a 19.2 � 6.7 8.8

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.


results showed no fish gender influence in CI. No gender variationwas also observed in the Gasterosteus aculeatus from a lowcontaminated stream [42]. Probably, the CI is not a sensible enoughbiomarker to be used in the São Francisco river points assessed.

Nevertheless, males from the impacted point (#1) showed feweroccurrences of MPO and EPO activity as well as inflammatory foci. Itwas associatedwith an increase in rodlet cells occurrence. In teleostfishes, the rodlet cells are suggested to be involved in inflammatoryresponses. However, the roles developed by these cells in the in-flammatory process are still unknown [28,29,32]. Rodlet cells inPsetta maxima showed the presence of S100 protein [31]. Inmammals, the subtype S100A9 is involved in the suppression ofpro-inflammatory macrophage activation contributing to the res-olution of inflammation [59]. Therefore, our results suggested thatthe lowest activity of MPO and EPO in males collected in theimpacted point (#1), associated with the highest occurrence ofrodlet cells may indicate that these cells could be involved in theresolution of the inflammation. MPO activity in mammals isenhanced by the influence of estrogens in females [60,61].

Previous studies carried out with P. argenteus at the samestudied points and season showed differences in the sex steroidsaccording to the points [62,63]. Higher levels of testosterone,17b�estradiol and 17a-hydroxiprogesterona were observed inmales and females of P. argenteus collected at point 2. Sincetestosterone 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 inP. argenteus.

However, we cannot fail to consider that the sex-differences ofMPO activity could be influenced by the feeding habits, since somestudies reported the modulation of non-specific immune re-sponses, including in MPO activity in different experimentallyinduced feeding habits [66,67]. In spite of some studies reporteddifferent sexual behavior in adaptation to stress conditions [68], toour knowledge, no studies related to the sexual behaving feedinghabits 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

f the gills of females collected at the two points.

e B Type C

#2 #1 #2

� 1.0 4.1 � 0.8 0.02 � 0.02 0.06 � 0.03� 0.7 2.8 � 0.7 0.4 � 0.2* 0.4 � 0.08� 2.0 12.7 � 3.0 0.1 � 0.1 0.6 � 0.3

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10

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 ahigher 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.


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probably a sexual hormone influence in MPO activity inP. argenteus.

The lamellar hyperplasia, characterized by the thickening of thegill epithelium, is considered a physiological barrier that impairedthe pollutant entrance into the blood circulation and consequentlyto a systemical spread [70,71]. Considering the studied sites, fishcollected in #1 presented more occurrence of hyperplasiacompared 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 fusionwere also observed in other fishspecies exposed to metal contamination [15,72,73]. Liza saliensfrom the Esmoriz-Paramos Coastal Lagoon in Portugal showed thesame histopathological alterations due to the contamination of Znand Cd [74].

Males from point #2 showed prevalence of fusion lamellae,probably due to a physiological adaptation to the higher oxygenconcentration 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 throughwhich the toxicant has to travel to reach the blood vessels, probablyacting as a physiological barrier to metal input [21]. It could alsocontribute to the fewer occurrences of inflammation foci andlamellar edema in male fish from the point #2. Our results alsoshow 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 glycoproteinsproduced by the mucous cells [75,78,80]. Hyperplasia of mucouscells 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 adecrease of area occupied by type A mucous in the filament and anincrease of type C mucous in lamellae. The same pattern was foundin Abramis brama exposed to oil spill in the Po river [83]. Howeverthe increase in tissue volume occupied by type C mucous did notovercome the decrease of type A mucous, suggesting a low secre-tion of mucous in males from point #1. In Solea senegalensis


chronically exposed to sediments contaminated with severalmetals, including Cu and Pd, mucous cells showed degeneration inthe 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. Mucouscoagulation on the gill surface also contributes to reduce pollutantsuptake [85,86].

In murine model of lung inflammation, several studies showedthat the increase of mucous secretion is positively associated withEPO activity [87,88]. Moreover, in inflammation, it has beenknown that eosinophils show numerous immune regulatoryfunctions, including production of a range of cytokines and che-mokines that leads to the exacerbation of inflammation andmucous secretion [89,90]. As occurs in mammals, our resultsshowed the association of the activities of EPO, with inflamma-tory foci and glycoprotein secretion by mucous cells. So, wesuggest that males collected in point (#1) showed chronicinflammation due to the lowest presence of inflammatory cells, aswell as, EPO and MPO activity and glycoprotein production. To ourknowledge, the present study is the first report to show the as-sociation of EPO activity, inflammatory foci occurrence andglycoprotein secretion in fish gill. Moreover, these data stronglysuggests sex-differences in fish adaptation to environmentalconditions.

Thus, our results show that the histopathological, cellular andbiochemical biomarkers assessed in gill of P. argenteus are reflectingthe environmental contamination in the São Francisco river.Moreover, most of the biomarkers studied showed a sex-specificresponse, 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 orderto comprehend the influence of the environment contaminants inthe biological mechanism.

Uncited reference

[53],[79] Q


Q3,4

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Acknowledgments

This work received financial support from Coordenação deAperfeiçoamento de Pessoal de Nível Superior, CNPq PRPq-UFMGand FAPEMIG (Brazilian Agencies for Science and Technology). Theauthors 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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References

[1] Schmitt CJ, Dethloff GM, editors. Biomonitoring of Environmental Status andTrends (BEST) Program: selected methods for monitoring chemical contami-nants and their effects in aquatic ecosystems. Columbia, (MO): U.S. GeologicalSurvey, Biological Resources Division; 2000. Information and TechnologyReport USGS/BRD-2000-0005.

[2] Au DW. The application of histo-cytopathological biomarkers in marinepollution monitoring: a review. Mar Pollut Bull 2004;48:817e34.

[3] Hughes CM. General anatomy of the gills. In: Hoar WS, Randall DJ, editors. Fishphysiology. New York: Academic Press; 1984. pp. 1e72.

[4] Arellano JM, Storch V, Sarasquete C. Ultrastructural and histochemical studyon gills and skin of the Senegal sole, Solea senegalensis. J Appl Ichthyol2003;20:452e60.

[5] Evans DH, Piermarini PM, Choe KP. The multifunctional fish gill: dominant siteof gas exchange, osmoregulation, acid-base regulation, and excretion ofnitrogenous waste. Physiol Rev 2005;85:97e177.

[6] Perry SF, Laurent P. Environmental effects on fish gill structure and function.In: Rankin JC, Jensen FB, editors. Fish ecophysiology. London: Chapman andHall; 1993. pp. 231e64.

[7] Jagoe CH, Haines TA. Changes in gill morphology of atlantic Salmon (SalmoSalar) smolts due to addition of acid and aluminum to stream water. EnvironPollut 1997;97(12):137e46.

[8] Erkmen B, Kolankaya D. Effects of water quality on epithelial morphology inthe gill of Capoeta tinca living in two tributaries of Kizilirmak River, Turkey.Bull Environ Contam Toxicol 2000;64:418e25.

[9] Ribeiro CAO, Belger L, Pelletier E, Rouleau C. Histopathological evidence ofinorganic mercury and methyl mercury toxicity in the arctic charr (Salvelinusalpinus). Environ Res 2002;90:217e25.

[10] Alberto A, Camargos AFM, Verani JR, Costa OFT, Fernandes MN. Health vari-ables and gill morphology in the tropical fish Astyanax fasciatus from asewage-contaminated river. Ecotoxicol Environ Saf 2005;61:247e55.

[11] Cengiz EI, Unlu E. Sublethal effects of commercial deltamethrin on thestructure of the gill, liver and gut tissues of mosquitofish, Gambusia afinis: amicroscopic study. Environ Toxicol Phar 2006;21:246e53.

[12] Capkin E, Altinok I, Karahan S. Water quality and fish size affect toxicity ofendosulfan, an organochlorine pesticide, to rainbow trout. Chemosphere2006;64:1793e800.

[13] Dautremepuits C, Marcogliese DJ, Gendron AD, Fournier M. Gill and headkidney antioxidant processes and innate immune system responses of yellowperch (Perca flavescens) exposed to different contaminants in the St. LawrenceRiver, Canada. Sci Total Environ 2009;407:1055e64.

[14] Thophon S, Kruatrachue M, Upatham ES, Pokethitiyook P, Sahaphong S,Jaritkhuan S. Histopathological alterations of white seabass, Lates calcarifer, inacute and subchronic cadmium exposure. Environ Pollut 2003;121:307e20.

[15] Abdel-Moneim AM, Al-Kahtani MA, Elmenshawy OM. Histopathological bio-markers in gills and liver of Oreochromis niloticus from polluted wetland en-vironments, Saudi Arabia. Chemosphere 2012;88:1028e35.

[16] McCarthy JF, Halbrook RS, Shugart LR. Conceptual strategy for design,implementation, and validation of a biomarker based biomonitoring capa-bility. Tennessee, USA: Environmental Science Division, Oak Ridge NationalLaboratory; 1991. Publication no. 3072, ORNL/TM-11783.

[17] Triebskorn R, Böhmer J, Braunbeck T, Honnen W, Köhler HR, Lehmann R. Theproject VALIMAR (validation of biomarkers for the assessment of small streampollution): objectives, experimental design, summary of results, and recom-mendations for the application of biomarkers in risk assessment. J Aquat EcosStress Rec 2001;8:161e78.

[18] Galloway TS, Brown RJ, Browne MA, Dissanayake A, Lowe D, Jones MB.A multibiomarker approach to environmental assessment. Environ Sci Tech-nol 2004;38:1723e31.

[19] Linde-Arias AR, Inácio AF, Novo LA, Albuquerque C, Moreira JC. Multi-biomarker approach in fish to assess the impact of pollution in a large Bra-zilian river, Paraiba do Sul. Environ Pollut 2008;156:974e9.

[20] Sanches JG, SpeareDJ, JohnsonGJ.Morphometric andhistochemical assessmentof the branchial tissue response of rainbow trout, Oncorhynchus mykiss (Wal-baum), associated with chloramine-T treatment. J Fish Dis 1997;20:375e81.

[21] Ahmed MK, Habibullah-Al-Mamun M, Parvin E, Akter MS, Khan MS. Arsenicinduced toxicity and histopathological changes in gill and liver tissue offreshwater fish, tilapia (Oreochromis mossambicus). Exp Toxicol Pathol2013;65:903e9.

[22] Guardiola FA, Gónzalez-Párraga P, Meseguer J, Cuesta A, Esteban MA. Modu-latory effects of deltamethrin-exposure on the immune status, metabolism


and oxidative stress in gilthead seabream (Sparus aurata L.). Fish ShellfishImmunol. http://dx.doi.org/10.1016/j.fsi.2013.10.020; 2013.

[23] Guardiola FA, Gónzalez-Párraga MP, Cuesta A, Meseguer J, Martínez S, Mar-tínez-Sánchez MJ, et al. Immunotoxicological effects of inorganic arsenic ongilthead seabream (Sparus aurata L). Aquat Toxicol 2013;134e135:112e9.

[24] Guardiola FA, Cuesta A, Meseguer J, Martínez S, Martínez-Sánchez MJ, Pérez-Sirvent C, et al. Accumulation, histopathology and immunotoxicological ef-fects of waterborne cadmium on gilthead seabream (Sparus aurata). FishShellfish Immunol 2013;35:792e800.

[25] Leino RL. Ultrastructure of immature, developing, and secretory rodlet cells infish. Cell Tissue Res 1974;155:367e78.

[26] Manera M, Dezfuli BS. Rodlet cells in teleosts: a new insight into their natureand functions. J Fish Biol 2004;65:597e619.

[27] Bielek E. Development of the endoplasmic reticulum in the rodlet cell of twoteleost species. Anat Rec A 2005;283:239e49.

[28] Mazon AF, Huising MO, Taverne-Thiele AJ, Bastiaans J, Verburg-vanKemenade BML. The first appearance of rodlet cells in carp (Cyprinus carpio L.)ontogeny and their possible roles during stress and parasite infection. FishShellfish Immunol 2007;22:27e37.

[29] Reite OB. The rodlet cells of teleostean fish: their potential role in host defencein relation to the role of mast cells/eosinophilic granule cells. Fish ShellfishImmunol 2005;19:253e7.

[30] Silphaduang U, Colorni A, Noga EJ. Evidence of widespread distribution ofpiscidin antimicrobial peptides in teleost fishes. Dis Aquat Organ 2006;72:241e52.

[31] Vigliano FA, Bermudez R, Nieto JM, Quiroga MI. Development of rodlet cells inthe gut of turbot (Psetta maxima L.): relationship between their morphologyand S100 protein immunoreactivity. Fish Shellfish Immunol 2009;26:146e53.

[32] Dezfuli BS, Giari L, Simoni E, Palazzi D, Manera M. Alteration of rodlet cellsin chub caused by the herbicide Stam�M-4 (Propanil). J Fish Biol 2003;63:232e9.

[33] Poltronieri C, Laurà R, Bertotto D, Negrato E, Simontacchi C, Guerrera MC, et al.Effects of exposure to overcrowding on rodlet cells of the teleost fish Dicen-trarchus labrax (L.). Vet Res Commun 2009;33:619e29.

[34] Liu G, Zhoua W, Wang LI, Park S, Miller DP, Xu LL, et al. MPO and SOD2polymorphisms, gender, and the risk of non-small cell lung carcinoma. CancerLett 2009;214:69e79.

[35] Bennett CM, Kanki JP, Rhodes J, Liu TX, Paw BH, Kieran MW, et al. Myelo-poiesis in the zebrafish, Danio rerio. Blood 2001;98(3):643e50.

[36] Lieschke GJ, Oates AC, Crowhurst MO, Ward AC, Layton JE. Morphologic andfunctional characterization of granulocytes and macrophages in embryonicand adult zebrafish. Blood 2001;98(10):3087e96.

[37] Balla KM, Lugo-Villarino G, Spitsbergen JM, Stachura DL, Hu Y, Bañuelos K,et al. Eosinophils in the zebrafish: prospective isolation, characterization,and eosinophilia induction by helminth determinants. Blood 2010;111(19):3944e54.

[38] Katzenback BA, Belosevic M. Isolation and functional characterization ofneutrophil-like cells, from goldfish (Carassius auratus L.) kidney. Dev CompImmunol 2009;33:601e11.

[39] Mohanty BR, Sahoo PK. Immune responses and expression profiles of someimmune-related genes in Indian major carp, Labeo rohita to Edwardsiella tardainfection. Fish Shellfish Immunol 2010;28:613e21.

[40] Nero V, Farwella A, Listerb A, Kraakb VGD, Leec LEJ, Meerd TV, et al. Gill andliver histopathological changes in yellow perch (Perca flavescens) and goldfish(Carassius auratus) exposed to oil sands process-affected water. EcotoxicolEnviron Saf 2006;63:365e77.

[41] Vega-López A, Galar-Martínez M, Jiménez-Orozco FA, García-Latorre E,Domínguez-López ML. Gender related differences in the oxidative stressresponse to PCB exposure in an endangered goodeid fish (Girardinichthysviviparus). Comp Biochem Physiol A 2007;146:672e8.

[42] Sanchez W, Piccini B, Maxence Ditche J, Porcher JM. Assessment of seasonalvariability of biomarkers in three-spined stickleback (Gasterosteus aculeatusL.) from a low contaminated stream: implication for environmental bio-monitoring. Environ Int 2008;34:791e8.

[43] Nakatani K, Agostinho AA, Baumgartner G, Bialetzki A, Sanches PV,Makrakis MC, et al. Ovos e larvas de peixes de água doce. Desenvolvimento emanual e identificação. 1a edição. Paraná: Editora EDUEM; 2001.

[44] Franco de Camargo SA, Petrere MJR. Social and financial aspects of the arti-sanal fisheries of Middle São Francisco River, Minas Gerais, Brazil. Fish ManagEcol 2001;8:163e71.

[45] Sato Y, Godinho HP. Migratory fishes of the São Francisco River. In:Carolsfelds J, Harvey B, Ross C, Baer A, editors. Migratory fishes of SouthAmerica: biology, fisheries and conservation status. Victoria: IDRC and WorldBank; 2003. pp. 195e232.

[46] Colombo JC, Bilos C, Remes-Lenicov M, Colautti D, Landoni P, Brochu C.Detritivorous fish contamination in the Río de la Plata estuary: a criticalaccumulation pathway in the cycle of anthropogenic compounds. Can J FishAquat Sci 2000;57:1139e50.

[47] Oliveira MR, Horn AF. Comparação da concentração de metais pesados naságuas do rio São Francisco em Três Marias, desde 1991 até hoje, relacionandoa atuação da CMM e Três Marias. Geonomos 2006;14(1, 2):55e63.

[48] Silva EFA. Pesquisa participativa no Rio São Francisco, região de Três Marias eMG: Contaminação ambiental e comunindade pesqueira. Dissertação demestrado. Depatamento de Química. Universidade Federal de São Carlos;2007. p. 94.



Q6

Q7


12345678910111213141516171819202122232425262728293031323334353637383940414243444546474849505152535455

5657585960616263646566676869707172737475767778798081828384858687888990919293949596979899

100101102103104105106

YFSIM2955_proof ■ 3 May 2014 ■ 10/10

[49] Ribeiro EV. Avaliação da qualidade da água do Rio São Francisco no segmentoentre Três Marias e Pirapora e MG: metais pesados e atividades antropo-gênicas. Dissertação de Mestrado. Departamento de Geografia. Instituto deGeociências. Universidade Federal de Minas Gerais; 2010. p. 196.

[50] Gomes MVT, Costa AS, Garcia CAB, Passos EA, Alves JPH. Concentrações eassociações geoquímicas de Pb e Zn em sedimentos do rio São Franciscoimpactados por rejeitos da produção industrial de zinco. Quim Nova2010;33(10):2088e92.

[51] Esteves FA, Amorim JC, Cardoso EL, Barbosa FAR. Caracterização limnológicapreliminar da represa de Três Marias (MG) com base em alguns parâmetrosambientais básicos. Ciência Cult 1985;37:608e17.

[52] Sato Y, Bazolli N, Rizzo E, Boschi MB, de Miranda MOT. Influence of the riverAbaeté in the reproductive success of the neotropical migratory teleost Pro-chilodus argenteus in the São Francisco River, downstream from the TrêsMarias dam, southeastern Brazil. River Res Applic 2005;8:939e50.

[53] Nikolsky GV. The ecology of fishes. 1th ed. Academic Press; 1963.[54] Bancroft JD, Gamble M. Theory and practice of histological techniques. 6th ed.

Churchill Livingstone; 2007.[55] Kumari U, Yashpal M, Mittal S, Mittal AK. Histochemical analysis of glyco-

proteins in the secretory cells in the gill epithelium of a catfish, Rita rita(Siluriformes, Bagridae). Tissue Cell 2009;41:271e80.

[56] CONAMA. Conselho Nacional de Meio Ambiente. Resolução 357 de Março de2005. Dispõe sobre e a classificação dos corpos de água e diretrizes ambientaispara o seu enquadramento, bem como estabelece as condições e padrões parao lançamento de efluentes, e dá outras providências. Disponível em: www.mma.gov.br/conama.

[57] Canadian Council of Ministers of the Environment. Summary tables of theCanadian sediment quality guidelines for the protection of aquatic life:updated in 2002. In: Canadian environmental quality guidelines, 1999. Win-nipeg: Canadian Council of Ministers of the Environment; 2002.

[58] de la Torre FR, Ferrari L, Salibia A. Biomarkers of a native fish species (Cnes-terodon decemmaculatus) application to the water toxicity assessment of aperi-urban polluted river of Argentina. Chemosphere 2005;59:577e83.

[59] De Lorenzo BHP, Godoy LC, Novaes e Brito RR, Pagano RL, Amorim-Dias MA,Grosso DM, et al. Macrophage suppression following phagocytosis ofapoptotic neutrophils is mediated by the S100A9 calcium-binding protein.Immunobiology 2010;215:341e7.

[60] Jansson G. Oestrogen-induced enhancement of myeloperoxidase activity inhuman polymorphonuclear leukocytes: a possible cause of oxidative stress ininflammatory cells. Free Radic Res Commun 1991;14:195e208.

[61] Liu G, Zhoua W, Wang LI, Park S, Miller DP, Xu LL, et al. MPO and SOD2polymorphisms, gender, and the risk of non-small cell lung carcinoma. CancerLett 2004;214:69e79.

[62] Arantes FP, Santos HB, Rizzo E, Sato Y, Bazzoli N. Profiles of sex steroids,fecundity, and spawning of the curimatã-pacu Prochilodus argenteus in the SãoFrancisco River, downstream from theTrês Marias Dam, Southeastern Brazil.Anim Reprod Sci 2010;118:330e6.

[63] Domingos FFT, Thomé RG, Arantes FP, Castro ACS, Sato Y, Bazzoli N, et al.Assessment of spermatogenesis and plasma sex steroids in a seasonalbreeding teleost: a comparative study in an area of influence of a tributary,downstream from a hydroelectric power dam, Brazil. Fish Physiol Biochem2012;38:1709e19.

[64] Thilagam H, Gopalakrishnan S, Bo J, Wang K. Effect of 17b-estradiol on theimmunocompetence of japanese Sea Bass (Lateolabrax Japonicus). EnvironToxicol Chem 2009;28(8):1722e31.

[65] Chaves-Pozo E, Cabas I, García-Ayala A. Sex steroids modulate fish immuneresponse. Sex Steroids. Scott M. Kahn editor. ISBN: 978-953-307-857-1:InTech. Available from: http://www.intechopen.com/books/sex-steroids/sex-steroids-modulate-fish-imnune-response.

[66] Alexander CP, Kirubakaran CJW, Michael RD. Water soluble fraction of Tino-spora cordifolia leaves enhanced the non-specific immune mechanisms anddisease resistance in Oreochromis mossambicus. Fish Shellfish Immunol2010;29:765e72.

[67] Zhang Y, Liu B, Ge X, Liu W, Xie J, et al. The influence of various feedingpatterns of emodin on growth, non-specific immune responses, and diseaseresistance to Aeromonas hydrophila in juvenile Wuchang bream (Megalobramaamblycephala). Fish Shellfish Immunol 2014;36(1):187e93.


[68] Øverli Ø, Sørensen C, Nilsson GE. Behavioral indicators of stress-coping stylein rainbow trout: do males and females react differently to novelty? PhysiolBehav 2006;87:506e12.

[69] Amutha C, Subramanian P. Cadmium alters the reproductive endocrinedisruption and enhancement of growth in the early and adult stages ofOreochromis mossambicus. Fish Physiol Biochem 2013;39:351e61.

[70] Richmonds C, Dutta HM. Histopathological changes induced by malathion inthe gills of bluegill Lepomis macrochirus. Bull Environ Contam Toxicol1989;43:123e30.

[71] Ortiz JB, Gonzalez de Canales ML, Sarasquete C. Histopathological changesinduced by lindane (g-HCH) in various organs of fish. Scientometrics Mar2003;67:53e61.

[72] Domingos FXV, Azevedo M, Silva MD, Randi MAF, Freire CA, Assis HAS, et al.Multibiomarker assessment of three Brazilian estuaries using oysters as bio-indicators. Environ Res 2007;105:350e63.

[73] Costa PM, Diniz MS, Caeiro S, Lobo J, Martins M, Ferreira AM, et al. Histologicalbiomarkers in liver and gills of juvenile Solea senegalensis exposed tocontaminated estuarine sediments: a weighted indices approach. AquatToxicol 2009;92:202e12.

[74] Fernandes C, Fontaínhas-Fernandes A, Monteiro SM, Salgado MA. Histopatho-logical gill changes in wild leaping grey mullet (Liza saliens) from the Esmoriz-Paramos Coastal Lagoon, Portugal. Environ Toxicol 2007;22(4):443e8.

[75] Bols NC, Brubacher JL, Ganassin RC, Lee LEJ. Ecotoxicology and innate im-munity in fish. Dev Comp Immunol 2001;25:853e73.

[76] Sollid J, Angelis PD, Gundersen K, Nilsson GE. Hypoxia induces adaptive andreversible gross morphological changes in crucian carp gills. J Exp Biol2003;206:3667e73.

[77] Nilsson GE. Gill remodeling in fish e a new fashion or an ancient secret? J ExpBiol 2007;210:2403e9.

[78] Schwaiger J, Ferling H, Mallow U, Wintermayr H, Negele RD. Toxic effects ofthe non-steroidal anti-inflammatory drug diclofenac. Part I: histopathologicalalterations and bioaccumulation in rainbow trout. Aquat Toxicol 2004;68:141e50.

[79] Shephard KL. Functions for fish mucus. Rev Fish Biol Fish 1994;4:401e29.[80] Mallatt J, Paulsen C. Gill ultrastructure of the pacific hagfish Eptatretus stouti.

Am J Anat 1986;177:243e69.[81] Robert SD, Powell MD. Comparative ionic flux and gill mucous cell histo-

chemistry: effects of salinity and disease status in Atlantic salmon (Salmo salarL.). Comp Biochem Physiol A 2003;134:525e37.

[82] Dezfuli BS, Squerzanti S, Fabbri S, Castaldelli G, Giari L. Cellular response insemi-intensively cultured sea bream gills to Ergasilus sieboldi (Copepoda) withemphasis on the distribution, histochemistry and fine structure of mucouscells. Vet Parasitol 2010;174:359e65.

[83] Giari L, Dezfuli BS, Lanzoni M, Castaldelli G. The impact of an oil spill onorgans of bream Abramis brama in the Po River. Ecotoxicol Environ Saf2012;77:18e27.

[84] Paulino MG, Souza NES, Fernandes MN. Subchronic exposure to atrazine in-duces biochemical and histopathological changes in the gills of a neotropicalfreshwater fish, Prochilodus lineatus. Ecotoxicol Environ Saf 2012;80:6e13.

[85] Grobler E, VanVuren JJJ, DuPreez HH. Routine oxygen consumption of Tilapiasparrmanii (Cichlidae) following acute exposure to atrazine. Comp BiochemPhysiol 1989;93:37e42.

[86] Iger Y, Lock RA, van der Meij JC, Wendelaar Bonga SE. Effects of water-bornecadmium on the skin of the common carp (Cyprinus carpio). Arch EnvironContam Toxicol 1994;26:342e50.

[87] Tomkinson A, Cieslewicz G, Duez C, Larson KA, Lee JJ, et al. Temporal associ-ation between airway hyperresponsiveness and airway eosinophilia inovalbumin-sensitized mice. Am J Respir Crit Care Med 2001;163:721e30.

[88] van der Ventel ML, Nieuwenhuizen NE, Kirsteina F, Hikuama C, Jeebhay MF,et al. Differential responses to natural and recombinant allergens in a murinemodel of fish allergy. Mol Immunol 2011;48:637e46.

[89] Trivedi DG, Lloyd CM. Biomedicine & Diseases: review eosinophils in thepathogenesis of allergic airways disease. Cell Mol Life Sci 2007;64(10):1269e89.

[90] Hogan SP, Rosenberg HF, Moqbel R, Phipps S, Foster PS, et al. Eosinophils:biological properties and role in health and disease. Clin Exp Allergy 2008;38:709e50.

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Sex-response differences of immunological and histopathological biomarkers in gill of Prochilodus...

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