Chemosphere 144 (2016) 1342e1350

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Vitellogenin expression in wild cyprinid Petroleuciscus esfahani as a biomarker of endocrine disruption along the Zayandeh Roud River, Iran

* Neda Gilannejad a, b, , Salar Dorafshan a, Fatemeh Paykan Heyrati a, Nasrollah Mahboobi Soofiani a, Saeid Asadollah a, Juan Antonio Martos-Sitcha b, c, Francisco Prat b, Manuel Yúfera b, Gonzalo Martínez-Rodríguez b a Department of Natural Resources, Isfahan University of Technology, 84156-83111 Isfahan, Iran b Instituto de Ciencias Marinas de Andalucía (ICMAN), CSIC, 11519 Puerto Real, Cadiz, Spain c Department of Biology, Faculty of Marine Sciences, University of Cadiz, 11519 Puerto Real, Cadiz, Spain highlights

An endemic cyprinid was used as a bioindicator of estrogenic exposure in the Zayandeh Roud River. vtg mRNA was detected in males from sites located downstream of anthropogenic pollution sources. The site downstream of a steel mill plant had the max vtg expression and growth impairment. article info abstract

Article history: Aquatic environments are the ultimate sink for most of anthropogenic pollutants. The Zayandeh Roud Received 9 June 2015 River is the most important river in the central Iranian Plateau, supplying water to a large population. In Received in revised form order to determine the potential occurrence and in vivo effects of Endocrine Disrupting Chemicals (EDCs) 28 September 2015 with estrogenic or anti-androgenic properties we analyzed the wild populations of an extensively Accepted 29 September 2015 distributed endemic fish species, Petroleuciscus esfahani. For this purpose, specimens were caught from Available online xxx two sites upstream and two sites downstream of the expected major anthropogenic pollution sources. P. esfahani full-length cDNAs for vitellogenin (vtg), with 4177 base pairs (bp) encoding a 1339 amino acids Keywords: b fi Bioindicator (aa), and for -actin (actb), with 1776 bp encoding a 375 aa, were ampli ed and cloned. Hepatic vtg EDCs mRNA expression levels were measured by quantitative real-time PCR. Condition factor, gonadosomatic Quantitative PCR index and sex ratio were calculated and compared with vtg expression. Gonad histology was performed River ecosystem to study the possible presence of intersex condition. Detection of vtg transcripts in male individuals from the two downstream sampling sites supports the hypothesis of exposure to EDCs in these regions. Higher vtg expression in male individuals, together with reduced gonad size and condition factor, in specimens from the site located downstream of the major steel mill plant suggest a major endocrine disruption in this area. © 2015 Elsevier Ltd. All rights reserved.

1. Introduction 2011). Among them, there is a family of contaminants known generically as Endocrine Disrupting Chemicals (EDCs). EDCs with There is a growing concern about a high variety of environ- estrogenic and anti-androgenic properties affect at different levels mental pollutants affecting human and wildlife health (Sun et al., in the living organisms, such as steroid receptors, steroid synthesis, distribution and excretion, hypothalamus-pituitary-gonad (HPG) axis, as well as indirect mechanisms including thyroid and growth hormone disruption (Rempel and Schlenk, 2008). Many of the * Corresponding author. Department of Natural Resources, Isfahan University of anthropogenic activities are potential sources of this group of Technology, 84156-83111 Isfahan, Iran. fi fi E-mail address: [email protected] (N. Gilannejad). chemicals, which nally nd their way to rivers and lakes and affect http://dx.doi.org/10.1016/j.chemosphere.2015.09.106 0045-6535/© 2015 Elsevier Ltd. All rights reserved. N. Gilannejad et al. / Chemosphere 144 (2016) 1342e1350 1343 aquatic organisms (Jeffries et al., 2010). was quantified using real-time PCR. Moreover, condition factor, The Zayandeh Roud River is the only permanent and the most gonadosomatic index and sex ratio were calculated and related to important river in the central Iran, which plays a key role in vtg expression. Gonad histology was also performed with the goal providing Isfahan and the adjacent provinces with drinking water. of detecting intersex specimens. Land use along this river includes agricultural fields, cattle farms, municipal waste water treatment plants and heavy industries such 2. Materials and methods as steel mill companies, power plants and oil refinery installations. Therefore, there is a high probability of contamination by EDCs. 2.1. Studied area and sampling points According to our knowledge, the information about the presence and in vivo consequences of EDCs in water ecosystems from Isfahan The Zayandeh Roud River, with a drainage area of 41,500 km2, province or generally in Iran is very scarce. The limited data avail- is the largest river in the central plateau of Iran. Sampling sites able for the Zayandeh Roud River, which are dedicated to were selected throughout the river having in mind a possible measuring some heavy metal and a series of compounds belonging gradient of anthropogenic impacts. Cheshmeh Dimeh (S1) (32310 to polycyclic aromatic hydrocarbons, affirm their existence in water N e 50130 E), and Khersoonak (S2) (32310 N e 50220 E), are and/or sediments (Nemati et al., 2009; Abdollahi, 2013). located upstream of the Zayandeh Roud Dam and were considered Since chemical monitoring of the aquatic environment alone is as uncontaminated sites. In these two sites, some extensive and restricted to identification of some of the EDCs, and the detection scatter crop and cattle production can be found. On the other methods are costly and complex, the use of biomarkers in sentinel hand, Chamgordan (S3) (32230 N e 51170 E) and Safaieh Bridge species has been considered an effective approach to assess the (S4) (32210 N e 51260 E) are situated downstream of the health status of the aquatic ecosystems (Frenzilli et al., 2008; Blazer Zayandeh Roud Dam and were considered a priori as possible et al., 2012; Gilroy et al., 2012). Obviously, aquatic species are more contaminated sites. S3 is a lagoon like reservoir which is formed exposed to EDCs than terrestrial (Damstra et al., 2002). by entering the river into an enclosed area and it receives effluents Among different aquatic species used for EDCs screening, fishes are from the nearby major steel mill plant as well as agricultural fields. the prominent model due to their particular features, the most S4 is located downstream of the sewage treatment plant dis- important of which is that the basic aspects of the function and charges from the Zarin Shahr City (population in 2012: 95,326) structure of the HPG axis are conserved in all vertebrates. There- (Fig. 1). During fish collections, dissolved oxygen, temperature, pH, fore, the results from their study can be extrapolated to other conductivity, and total dissolved solids (TDS) were measured on vertebrates including humans (Ankley et al., 2009). Petroleuciscus site using WTW model OXI 196 Hand-Held Dissolved Oxygen esfahani is a small cyprinid fish endemic to the Zayandeh Roud Meter and HANNA model HI98129 pH/Conductivity/TDS Tester. basin (Coad and Bogutskaya, 2010). This species is abundant along Additionally, biochemical oxygen demand (BOD), and chemical the river and is even available in locations with constant input of oxygen demand (COD) were determined in laboratory according to contaminants. All these features, together with its limited migra- Radojevic and Bashkin (2006) (Table 1). tion capacity, make this fish a suitable candidate for sentinel spe- cies in this ecosystem. Fish exposure to EDCs with estrogenic and anti-androgenic ac- 2.2. Fish sampling and analyzed parameters tivity can lead to several consequences (Rempel and Schlenk, 2008), including i) vitellogenin induction in juveniles or males (Blazer Fish were captured during the non-reproductive period et al., 2014; Duffy et al., 2014), ii) delay or absence of secondary (January to February, 2012) by seine net (8 m 1 m, 5 mm mesh sexual characteristics and sexual behavior in males (Goksøyr et al., size) in S1, S2 and S4, and by boat electrofishing in S3 because of 2003), iii) intersex condition (Puy-Azurmendi et al., 2013), iv) higher water depth in this site. After transporting the fish to the skewed sex ratio (Jobling et al., 1996), and in extreme cases v) laboratory, on one side, 10 individuals were exposed to 17b- complete extinction of the population (Kidd et al., 2007). Vitello- estradiol (E2)(2mg/L) for 24 h (Bowman et al., 2000) with the genin (Vtg) is a female-specific precursor protein for egg yolk, purpose of increasing the chance of cloning the vtg cDNA. At the which is normally synthesized in female hepatocytes in response to end of this period, male and female liver samples were preserved ® endogenous estrogens (Goksøyr et al., 2003). Males also possess the in RNAlater (Invitrogen Life Technologies) separately. On the vitellogenin genes (vtg), which are silenced but can be induced other side, on fish arrival to the laboratory, the rest of the fish from when exposed to xenoestrogens (Hutchinson et al., 2005) and all the sampling sites were kept in aerated tanks with water from antiandrogens (Bhatia et al., 2014). Actually, vitellogenin induction the corresponding sampling site until processed (around 1 h). Fish in males is the strongest biomarker for fish exposure to estrogenic were anaesthetized by immersion in 100 ppm MS222. Length compounds (Nadzialek et al., 2011). Although male vtg induction is (standard length ± 1 mm) and body weight (body weight ± 0.01 g) usually considered as a consequence of xenoestrogen exposure, it were measured. Gonads were excised to assign sex (by visual in- should be taken into account that other types of chemicals may spection or with the help of a stereoscope) and weight (gonad elicit this response. For example, flutamide and cyproterone, which weight ± 0.01 g). After that, the condition factor (CF) and gona- are anti-androgen reagents, exerted feminizing effects such as up dosomatic index (GSI) were calculated by (body regulation of vitellogenin, b-estradiol and/or estrogen receptors in weight length 3 100) and (gonad weight body male fish (Kim et al., 2003; Jensen et al., 2004; Filby et al., 2007; weight 1 100) formulas, respectively. The middle section of Bhatia et al., 2014). vtg mRNA is induced rapidly (within a few gonads (and in small individuals the whole organ) were fixed in hours of exposure) and, therefore, is useful for short-term screen- 10% formalin, dehydrated in a graded ethanol series and ings of the ecosystems (Frenzilli et al., 2008; Blanchet-Letrouve embedded in paraffin blocks. 7e8 mm sections were mounted on et al., 2013; Bizarro et al., 2014). microscope slides stained with haematoxylin and eosin (Presnell The aim of this study was to determine the possible presence of and Schreibman, 1997). Slides were examined by light micro- EDCs in the Zayandeh Roud River ecosystem using P. esfahani as a scopy in order to detect any intersex condition. From each sam- bioindicator. For this purpose, full sequence of the cDNA encoding pling site, around 30 mg of liver from 20 specimens (around 10 vtg and b-actin (actb; to be used as an internal control) was cloned individuals from each sex) were selected randomly, preserved ® for the first time in this species, and hepatic vtg mRNA expression individually in RNAlater , and stored at 20 C till processed. 1344 N. Gilannejad et al. / Chemosphere 144 (2016) 1342e1350

Fig. 1. Sampling locations in the Zayandeh Roud River. Cheshmeh Dimeh (S1), Khersoonak (S2), Chamgordan (S3) and Safaieh Bridge (S4).

Table 1 Water physicoechemical parameters in four sampling sites from the Zayandeh Roud River.

Site Temperature (C) Dissolved oxygen (mg/L) pH Conductivity (mS/cm) TDS (ppm) BOD (ppm) COD (ppm)

S1 6 9.7 8.15 194 388 40 120 S2 4.8 9.6 7.88 215 431 30 110 S3 10.2 9 8.50 1140 570 70 151 S4 6.6 13.6 8.97 456 912 80 162 N. Gilannejad et al. / Chemosphere 144 (2016) 1342e1350 1345

2.3. Cloning vtg and actb in Petroleuciscus esfahani from 1000 replicates (Felsenstein,1985). Branches corresponding to less than 50% of the replicates were collapsed. The evolutionary The extended Material and Methods for the molecular biology distances were computed using the Poisson correction method procedures (parts 2.3, 2.4, and 2.5) is available as Supplementary (Zuckerkandl and Pauling, 1965) and are in the units of the number material. Total RNA extraction from P. esfahani preserved liver of amino acid (aa) substitutions per site. The tree is drawn to scale, samples (30 mg), and measurement of RNA quality and quantity with branch lengths in the same units as those of the evolutionary were carried out according to the procedures explained in distances of the tree. The analysis involved 57 aa sequences Baldisserotto et al. (2014). Total RNA was extracted on one side from retrieved from the NCBI protein database (www.ncbi.nlm.nih.gov/ the liver sample of a female captured from S1 and on the other side pubmed, accessed in January 2013). All positions containing gaps from the pooled liver samples of E2 treated males. In order to in- and missing data were eliminated. crease the chance of vtg cloning, the mentioned two total RNA samples were mixed prior to cDNA synthesis. This pooled total RNA 2.5. Real-time PCR (QPCR) sample was reverse-transcribed using the qScript™ cDNA synthesis kit (Quanta BioSciences). Total RNA isolation, quantification and the assessment of quality Degenerated primers for vtg were designed from conserved were performed as described above. Quantification was made in a ® regions along the full length cDNA sequences from Cyprinus carpio Mastercycler epgradient S Realplex2. Results were normalized to vtgb1 (GenBank: AB331884), Carassius auratus vtg (GenBank: actb as an internal control and a calibrator sample, a female DQ641252), Cirrhinus molitorella vtgb1 (GenBank: GU324313), Catla captured from S1, was measured on every QPCR plate to correct for catla vtgb1 (GenBank: EF190987), Gobiocypris rarus vtgao1 (Gen- inter-assay differences. Optimization of QPCR conditions was done Bank: EU623080), Pimephales promelas vtg (GenBank: AF130354), using different primer combinations (3 pairs for actb and 12 pairs Phoxinus oxycephalus vtg1 (GenBank: EF639845). For actb, primers for vtg), on a temperature gradient for annealing (50e60 C), available at the laboratory, used for the cloning of Rhamdia quelen primers concentrations (100 nM, 200 nM and 400 nM) and tem- actb (GenBank: KC195970) partial sequence (Baldisserotto et al., plate concentration (five 1:10 dilution series from 10 ng to 1 pg of 2014), were also used (see Supplementary material, Table 1). Af- input ARN). The resulting curves had amplification efficiencies and terwards, PCR reactions were performed with BIOTAQ™ DNA po- r2 of 0.86 and 0.999 for vtg and of 0.84 and 0.998 for actb, respec- lymerase (Bioline) and samples were cycled (95 C, 10 min; [95 C, tively. Amplicon sizes were 128 bp for vtg and 131 bp for actb. QPCR 30 s; 60.4 C, 30 s; 72 C, 30 s] 35 cycles; 72 C, 10 min). The PCR reactions (10 mL) were performed with 10 ng of cDNA (assumed products were run in agarose gel. Bands with expected size were from RNA input), sense and antisense primers (200 nM each; ® purified using the NucleoSpin Gel and PCR Clean-up Kit indicated in Supplementary material, Table 1) and PerfeCTa™ ® (MachereyeNagel). Cloning and sequencing were performed ac- SYBR Green FastMix™ (Quanta BioSciences). Relative gene quan- cording to Baldisserotto et al. (2014). The intermediate vtg and actb tification was performed using the DDCT method (Livak and products were 1278 bp and 334 bp long, respectively. Schmittgen, 2001). The PCR thermal profile was as follows: 95 C, Since the distance from the intermediate fragment of vtg cDNA 5 min; [95 C, 15 s; 60 C, 30 s] 40 cycles; melting curve to the 30 end was expected to be very long, an elongation strategy [60e95 C, 20 min]. PCR products were visualized in agarose gel, was followed. For that purpose, several PCR reactions were per- cloned and sequenced, and sequences matched the genes of in- formed using 3 nested forward specific oligonucleotides, designed terest. In total, vtg expression was quantified in 9, 6, 7, 13 females from the 30 end of the intermediate vtg fragment (overlapping a and in 9, 10, 9, 8 male specimens from S1, S2, S3, and S4, minimum of 470 bp), and 3 reverse degenerate primers (shown in respectively. Supplementary material, Table 1), designed from the conserved areas of other cyprinids. The rest of the procedure was as described 2.6. Statistics above. With this approach, vtg was extended 1724 additional base pairs toward the 30 end. Statistical analyses were performed using SPSS version 22 and Using total RNA as template, the 30 and 50 ends of vtg or actb GraphPad Prism version 6.00 for Windows. For length, weight, CF, ® mRNAs were amplified using the FirstChoice RLM-RACE Kit and GSI, normality and homogeneity of the data was confirmed by (Ambion, Life Technologies). To amplify the 30-ends, specific for- ShapiroeWilk test and Levene's test, respectively. Then, their dif- ward primers were designed in the 30-end of the previously ob- ferences between sites were analyzed using one-way analysis of tained fragments (see above) at 4 (for vtg) or 2 (for actb) different variance (ANOVA) followed by the post-hoc Duncan test. To positions and were used in combination with the 30-RACE Outer or compare the vtg expression levels between sites, since the data Inner primers supplied in the kit. For 50-RACE amplifications, spe- were not normally distributed, KruskaleWallis and Dunn's multiple cific reverse primers were designed in the 50-end of the previously comparison were used. All statistics were run separately for males cloned fragments at 2 different positions and were used in com- and females from each sampling site. Sex ratio differences from the bination with the 50-RACE Outer or Inner primers from the kit expected 1:1 ratio at each site were checked by chi-square tests. (Supplementary material, Table 1). The primers were designed to Data were considered statistically significant when p < 0.05. obtain a minimum overlapping of 164 bp for vtg and 45 bp for actb between the RACE clones and the previously achieved partial 3. Results cDNAs. The cloning and sequencing of PCR products were per- formed as described above. Finally, the obtained cDNA sequences 3.1. Assessed parameters in studied sites were assembled using the algorithm merger available in EMBOSS explorer (http://pro.genomics.purdue.edu/emboss/). For length and weight, the highest values were observed in S2 and S1, respectively. For CF in males, S2 and S3 showed maximum 2.4. vtg phylogenetic and evolutionary analysis and minimum values, respectively. However, no significant differ- ences were detected between S4 and S1 or S2. In females, S2 and S3 The evolutionary history was inferred using the neighbor- had the highest and lowest CF, respectively, whereas S1 did not joining method (Saitou and Nei, 1987) and was conducted in show any significant difference to S3 and S4 (Table 2). Histological MEGA5 (Tamura et al., 2011). The bootstrap consensus tree inferred assessment revealed no evidence of oocytes in testis tissue in the 1346 N. Gilannejad et al. / Chemosphere 144 (2016) 1342e1350

Table 2 Morphometric comparisons of P. esfahani collected from different sampling sites in the Zayandeh Roud River.

Site Individuals (N) Length (mm) Weight (g) Condition factor (CF)

Females Males Females Males Females Males Females Males

S1 82 21 108.39 ± 3.07b 101.33 ± 5.08b 18.98 ± 1.57b 14.75 ± 2.19b 1.33 ± 0.02bc 1.34 ± 0.05b S2 50 31 120.70 ± 3.03a 114.83 ± 2.64a 28.43 ± 2.03a 22.56 ± 1.45a 1.58 ± 0.06a 1.48 ± 0.05a S3 67 21 73.64 ± 5.74d 57.30 ± 1.74d 6.66 ± 1.87c 2.36 ± 0.29d 1.25 ± 0.05c 1.21 ± 0.05c S4 48 52 91.13 ± 2.31c 90.92 ± 1.61c 10.73 ± 0.88c 10.79 ± 0.65c 1.38 ± 0.03b 1.40 ± 0.02ab

Note: Data are reported as mean ± standard error of the mean (SEM). Values not sharing a common letter within a given parameter inside same sex are statistically different (p < 0.05). four investigated stations (data are not shown). In S1, S2 and S3, sex Platichthys flesus (GenBank: AAF63665). Both vtg and actb se- ratio was skewed from 1:1 in favor of females, forming 80%, 62% quences were sent to GenBank with acc. nos. KF766534 and and 76% of the total capture, respectively (Fig. 2). For GSI, there was KF766533, respectively. no significant difference between the four sites for females, while for males it was significantly lower in S3 (Fig. 3). 3.3. vtg phylogenetic and evolutionary analysis

3.2. Cloning vtg and actb full-length cDNAs in Petroleuciscus Phylogenetic analysis of vertebrates vitellogenin aa sequences esfahani demonstrates that Vtg of P. esfahani is clearly clustered within the family, and more closely related to Vtg1 from P. esfahani amplified and cloned full-length cDNA for vtg was P. oxycephalus (GenBank: ABR27689), and Vtg from P. promelas 4177 base pairs (bp) long, encoding for a protein of 1339 aa (GenBank: AAD23878) and Tanichthys albunoides (GenBank: (Supplementary material, Fig. 1). The cDNA sequence showed 96% ABN13867) (Fig. 4). identity to P. promelas vtg (GenBank: AF130354), 94% to P. oxycephalus vtg1 (GenBank: EF639845), 91% to C. carpio vtgb1 3.4. vtg mRNA quantification (GenBank: AB331884), and 90% to C. molitorella vtgb1 (GenBank: GU324313), but slightly lower to C. carpio vtgb2 (GenBank: Quantification of vtg mRNA showed that for females, there are AB106873) and Danio rerio vtg2 (NCBI: NM_001044913), 88% and not significant differences between the four sampling sites. In male 85%, respectively. Similarly, the deduced aa sequence showed a 93% specimens, no vtg mRNA transcription was detected in S1 and S2. identity to P. promelas Vtg (GenBank: AAD23878), 88% to While, in S3 and S4, vtg expression was detected in 5 out of 9 and 2 P. oxycephalus Vtg1 (GenBank: ABR27689), C. carpio Vtgb1 (Gen- out of 8 male samples, respectively. S3 had the highest vtg Bank: BAF73406), and C. molitorella Vtgb1 (GenBank: ADB77954), expression level; while, in S4, it was not significantly different from while the identity was 84% and 74% to C. carpio Vtgb2 (GenBank: the other 3 sites (Fig. 5). BAD51933) and D. rerio Vtg2 (NCBI: NP_001038378), respectively. P. esfahani full-length cDNA for actb was 1776 bp long, encoding for 4. Discussion a 375 aa protein (Supplementary material, Fig. 2). The nucleotide sequence for this gene showed 97% identity of to D. rerio actb2 Vitellogenin induction is being used extensively as a biomarker (NCBI: NM_181601) and Hemibarbus mylodon actb1 (GenBank: of estrogenic disruption in many research programs around the EF554924), and 96% identity to actb from Elopichthys bambusa world (Blanchet-Letrouve et al., 2013). Study of the gene tran- (GenBank: JN102135), C. molitorela (GenBank: DQ007446), and scriptional responses can be fundamental for understanding the Rhodeus uyekii (GenBank: KJ867513). The deduced aa sequence possible pathways in which chemicals can affect the organisms, showed 99% identity to b-actin in C. molitorela (GenBank: because they are the primary interaction points between chemicals AAY25518), E. bambusa (GenBank: AEK69350), Labeo calbasu available in the environment and the organism (Moens et al., 2007). (GenBank: AAL57317), Pargus major (GenBank: BAD88412), and In this study exposure to estrogenic and/or anti-androgenic chemicals in the Zayandeh Roud River was assessed by vtg expression in P. esfahani. Several vtg genes have been identified in 100 * ** teleosts, including cyprinids (Finn et al., 2009). The identity of the 21 cDNA cloned and the deduced aa sequences compared to other 21 cyprinids was higher for vtg1 than vtg2 form, suggesting that our 80 31 52 cDNA probably encodes vtg1. In this study, we did not find any other cDNA encoding other types of Vtg. To date, a single vtg form has 60 Males Females been also found in another cyprinid species, C. molitorella (Liang and Fang, 2012). Miracle et al. (2006) found that vtg1 induction 40 82

% Sex ratio % Sex 67 levels in male fathead (P. promelas) were 4 folds higher 50 a 48 than vtg3 when exposed to 17 -ethynylestradiol (EE2), which 20 suggests that vtg1 is an appropriate indicator for estrogenic exposure. 0 Absence of vtg transcripts in male individuals in the first two S1 S2 S3 S4 upstream sites confirms our hypothesis that in these regions EDCs Sites do not exist or the quantity is not enough to provoke vtg expression. On the other hand, quantifiable levels of vtg transcripts in S3 sug- Fig. 2. Analysis of sex ratio in P. esfahani collected from four different sites in the gest the fish biological response to EDCs. This finding is consistent Zayandeh Roud River. Asterisk (*) indicates statistically significant differences from the expected 1:1 ratio. The number inside each bar indicates the number of analyzed with the land use and topological properties of this region. Char- individuals. acteristics of effluents and hydrology of the area are important site- N. Gilannejad et al. / Chemosphere 144 (2016) 1342e1350 1347

Females Males

6 2.5 a a a

a a 2.0 a a 4 1.5

GSI (%) GSI (%) b 1.0 2

0.5

0 0.0 S1 S2 S3 S4 S1 S2 S3 S4

Sites Sites

Fig. 3. GSI for females and males of P. esfahani from four different sites in the Zayandeh Roud River. Data is shown as mean with SEM and different letters indicate statistically significant differences.

specific factors that should be considered in assessment of an size could be due to the negative effects of endocrine disruption ecosystem (Frenzilli et al., 2008). In this site, extremely poor water which has been confirmed by detection of vtg transcripts in male flow leads to accumulation of sediments, and therefore, an increase individuals. Since vtg does not have any biological role in males, it in bioavailability of these contaminants. According to studies on the imposes energetic expenses, which could have been used in body Zayandeh Roud River, concentrations of manganese (Mn), zinc (Zn), or testis growth (Hashimoto et al., 2000; Jeffries et al., 2008, nickel (Ni), lead (Pb) and cadmium (Cd) in sediments of this area Jerffries et al., 2010). Besides, there are evidences that EDCs can are significantly higher than in other parts of the river (Nemati affect other physiological systems such as hypothalamus-pituitary- et al., 2009; Tabatabaei, 2010). These metals could contribute or thyroid, and somatotropic axes and consequently cause deficiencies be responsible for the high levels of vtg found in this study. Labo- in growth and development (Jiao and Cheng, 2010; Reinecke, 2010; ratory experiments showed that some metals such as mercury (Hg), Arukwe et al., 2014; Li et al., 2014). Additionally, a high prevalence copper (Cu), Cd, Pb, and Zn have estrogenic activity and can up- of Ligula intestinalis in the population of P. esfahani in S3 has been regulate the vitellogenin gene or protein in fish species (Klaper reported (Shojaee et al., 2012). Various studies revealed that et al., 2006; Søfteland et al., 2010; Blickley et al., 2014; Huang chemicals such as polychlorinated biphenyls may produce immu- et al., 2014; Daiwile et al., 2015). Besides, in some studies re- nosuppressive effects which facilitate infection by parasites or ported from natural ecosystems, elevated vtg mRNA expression make them more prone to diseases (Sures, 2006; Schwacke et al., levels in male fish have been attributed to high levels of heavy 2012). This suggests that, in S3, the general welfare and health of metals (Canapa et al., 2007; Annabi et al., 2012). Many of them are the population is affected. endocrine disruptors and can affect at different levels including In the three first sampling sites, sex ratio was female biased. steroid receptor pathways and thyroid hormone. Therefore, the Although EDCs can influence fish sexual development and alter the metal estrogenic effect on vtg expression would be probably due to sex ratio from 1:1 (Cheek et al., 2001; Blazer et al., 2012), other interaction with the estrogen receptor alpha (Stoica et al., 2000; factor should be in mind when surveying wild populations. For Martin et al., 2003) rather than a direct effect. Surprisingly, in S4, species in which sex ratios do not deviate from 1:1 among families, vtg expression levels were not statistically different from the non it is very likely that the sex-determination system is chromosomal- contaminated sites. This can be explained by the fact that the water like. However, in other species, including some Cyprinids, in which flow is fast in this region and can dilute the EDCs. sex ratio differs from 1:1, sex determination is polyfactorial or Male GSI is typically reduced after exposure to estrogenic or controlled by environmental conditions (Devlin and Nagahama, anti-androgenic contaminants and there is a correspondence be- 2002; McKellar et al., 2009). Factors such as differences in tween the potency of these compounds and inhibition of testis behavior, habitat use or mortality rate between genders can cause growth. GSI decrease may happen due to suppression of testicular differences in possibility of capturing equal number of specimens development at puberty, inhibition of normal seasonal develop- from each sex (Konecna and Reichard, 2011). On the other hand, ment of the gonad, or atrophy (Hassanin et al., 2002; Milnes et al., considering that no vtg mRNA was detected in the males from S1 2006; Zhaobin and Jianying, 2008). Therefore, lower GSI in males and S2, existence of high concentrations of EDCs that can change from S3 could indicate the existence of compounds with estrogenic the sex determination pathways is implausible. Hence, we suggest or anti-androgenic activity in this area. However, water usage for that it is not possible to decide about the population sex ratio based cooling the steel mill plant installations in S3 causes a higher on a single sampling and more field study is needed. Moreover, in temperature and conductivity is this area. Water temperature can species in which mechanism of sex determination is not defined influence gonad development and hence GSI (Dorts et al., 2012). precisely, sex ratio should be used with caution as an index for EDC Interestingly, females in the same location, which are exposed to exposure. the same high temperatures, did not show any significant differ- No testis-ova or any other abnormal conditions were detected in ences for GSI in comparison to other sampling sites. Taking any of the histological samples even in males with vtg expression. together, these results suggest that the decrease of GSI in males can This result is in accordance with some other experimental assays be more likely due to the presence of EDCs than to the effect of high and field studies in which estrogenic or anti-androgenic contami- temperatures, although a temperature effect cannot be totally nants induced male vtg expression but did not cause intersexual excluded. gonads (Rey Vazquez et al., 2009; Allner et al., 2010; Bhatia et al., Growth suppression was clearly observed in the Zayandeh Roud 2014). Changing the gonad structure depends on the potency and River downstream sites, especially in S3. Decreased body and gonad concentration of the exposed compounds (Hassanin et al., 2002). 1348 N. Gilannejad et al. / Chemosphere 144 (2016) 1342e1350

96 Cyprinus carpio VtgB1 [BAF73406] 63 Carassius auratus Vtg [ABG22139] 58 Cyprinus carpio VtgB2 [BAD51933] Cirrhinus molitorella VtgB1 [ADB77954] 53 99 Catla catla Vtg [ABP04034] Tanichthys albonubes Vtg [ABN13867] Phoxinus oxycephalus Vtg1 [ABR27689] 8471 94 ♦Petroleuciscus esfahani Vtg 51 Pimephales promelas Vtg [AAD23878] Danio rerio Vtg7 [NP_001096141] Danio rerio Vtg1 [AAK58480] 100 100 95 Danio rerio Vtg6 [NP_001116082] Danio rerio Vtg4 [NP_001038759] 100 81 77 Danio rerio Vtg5 [NP_001020360] 99 Zacco platypus Vtg [AEE98103] Clarias macrocephalus Vtg [ABW96364] Siluriformes 63 Clupea harengus Vtg1 [ACJ65208] Clupeiformes Conger myriaster Vtg [BAD93275] Anguilliformes 100 Anguilla japonica Vtg2 [AAR82898] 60 100 Anguilla japonica Vtg1 [AAR82899] 100 Anguilla japonica Vtg [AAV48826] Oreochromis aureus Vtg [AAD01615] Cichliformes 100 Gambusia affinis Vtg [BAD93698] Cyprinodontiformes 100 Xiphophorus hellerii VtgB [AFH08753] 99 100 Xenotoca eiseni VtgB [ACI30218] Oryzias latipes Vtg [AAQ83616] Beloniformes 58 Melanogrammus aeglefinus VtgB [AAK15157] Gadiformes Mugil cephalus VtgB [BAF64836] Mugiliformes 88 Verasper moseri Vtg [BAD93696] Pleuronectiformes 98 99 Centrolabrus exoletus VtgAb1 [ACK36964] Labriformes 81 Labrus mixtus VtgAb2 [ACK36968] 28 Thunnus thynnus VtgB [ADD63987] Scombriformes Pagrus major Vtg [BAE43871] 49 Spariformes 64 Dicentrarchus labrax VtgAb [AFA26670] Moronidae 100 Morone saxatilis VtgAb [ADZ57173] 100 Morone americana VtgB [AAZ17416] 100 Acanthogobius flavimanus Vtg [BAC06190] Gobiiformes Acanthogobius hasta Vtg [AAV84912] 100 Xiphophorus hellerii VtgA [AFH08752] Cyprinodontiformes 99 Poecilia latipinna Vtg [ACV65040] 91 97 Xenotoca eiseni VtgA [ACI30217] 100 Kryptolebias marmoratus Vtg [AAQ16635] Oryzias latipes Vtg1 [BAB79696] Beloniformes 99 Colisa fasciata VtgA [ADE06081] Anabantiformes 100 Centrolabrus exoletus VtgAa [ACK36963] Labriformes 85 Labrus mixtus VtgAa [ACK36967] 55 Trematomus bernacchii Vtg [CBL95236] Perciformes Hippoglossus hippoglossus Vtg [ABQ58114] Pleuronectiformes 19 Sillago japonica Vtg [BAC20186] Sillaginidae 28 Mugil cephalus VtgA [BAF64835] 37 Mugiliformes 75 Dicentrarchus labrax VtgAa [AFA26669] Moronidae 100 Morone saxatilis VtgAa [ADZ57172] 97 Morone americana VtgA [AAZ17415] Scyliorhinus torazame Vtg [AEM05867] Carcharhiniformes 100 Acipenser baerii Vtg [ADM64616] Acipenseriformes 78 Larus argentatus VTG [AAL01527] 100 Gallus gallus VTG [AAA49139] AVES

0.1

Fig. 4. Phylogenetic tree of vitellogenin protein sequences from vertebrates. Numbers next to branch points are the percentage of replicate trees in which the associated taxa clustered together. For the sequences, GenBank accession numbers as well as the corresponding Order (or Family for those species with incertae sedis) are indicated in the figure. N. Gilannejad et al. / Chemosphere 144 (2016) 1342e1350 1349

Fig. 5. P. esfahani vtg mRNA expression in females and males, from four different sites in the Zayandeh Roud River. Data is shown as triangular points with median and range, and different letters indicate statistically significant differences.

Considering the presence of EDCs in S3, according to what our re- mechanism of action. Aquat. Toxicol. 92, 168e178. ~ sults suggest, their concentration and/or composition would not Annabi, A., Kessabi, K., Navarro, A., Saïd, K., Messaoudi, I., Pina, B., 2012. Assessment of reproductive stress in natural populations of the fish Aphanius fasciatus using have been sufficient to cross the threshold necessary for causing quantitative mRNA markers. Aquat. Biol. 17, 285e293. histological abnormalities. Arukwe, A., Olufsen, M., Cicero, N., Hansen, M.D., 2014. Effects on development, growth responses and thyroid-hormone systems in eyed-eggs and yolk-sac larvae of Atlantic salmon (Salmo salar) continuously exposed to 3,30,4,4'-tetra- 5. Conclusions chlorobiphenyl (PCB-77). J. Toxicol. Environ. Health A 77, 574e586. Baldisserotto, B., Martos-Sitcha, J.A., Menezes, C.C., Toni, C., Prati, R.L., Garcia, L.de O., In summary, the two upstream sites did not demonstrate signs Salbego, J., Mancera, J.M., Martínez-Rodríguez, G., 2014. The effects of ammonia and water hardness on the hormonal, osmoregulatory and metabolic responses of disruption by EDCs. However, detection of vtg transcripts in male of the freshwater silver catfish Rhamdia quelen. Aquat. Toxicol. 152, 341e352. individuals from the two downstream locations, especially in S3, Bhatia, H., Kumar, A., Ogino, Y., Du, J., Gregg, A., Chapman, J., McLaughlin, M.J., indicated the possible existence of these compounds. Highest vtg Iguchi, T., 2014. Effects of the commercial antiandrogen flutamide on the bio- markers of reproduction in male Murray rainbowfish (Melanotaenia fluviatilis). expression levels, along with other biological evidences such as Environ. Toxicol. Chem. 33, 1098e1107. growth impairment, reduced gonad size in males and high preva- Bizarro, C., Ros, O., Vallejo, A., Prieto, A., Etxebarria, N., Cajaraville, M.P., Ortiz- lence of parasites in fish from S3, suggest that in this area the fish Zarragoitia, M., 2014. Intersex condition and molecular markers of endocrine are affected by the presence of environmental contaminates disruption in relation with burdens of emerging pollutants in thicklip grey mullets (Chelon labrosus) from basque estuaries (South-East Bay of Biscay). Mar. released by the nearby steel mill plant. According to our findings, Environ. Res. 96, 19e28. P. esfahani could be considered a good sentinel species for water Blanchet-Letrouve, I., Lafont, A.G., Poirier, L., Baloche, S., Zalouk-Vergnoux, A., fi quality in the Zayandeh Roud River. Considering the important role Dufour, S., Mouneyrac, C., 2013. Vg mRNA induction in an endangered sh species (Anguilla anguilla) from the loire estuary (France). Ecotoxicol. Environ. of this river in different human needs, we recommend that future Saf. 97, 103e113. research involving characterization and measurement of different Blazer, V.S., Iwanowicz, L.R., Henderson, H., Mazik, P.M., Jenkins, J.A., Alvarez, D.A., groups of EDCs and determination of potential effects of these Young, J.A., 2012. Reproductive endocrine disruption in smallmouth bass (Micropterus dolomieu) in the Potomac river basin: spatial and temporal com- contaminants on human health should be addressed. parisons of biological effects. Environ. Monit. Assess. 184, 4309e4334. Blazer, V.S., Iwanowicz, D.D., Walsh, H.L., Sperry, A.J., Iwanowicz, L.R., Alvarez, D.A., Acknowledgments Brightbill, R.A., Smith, G., Foreman, W.T., Manning, R., 2014. Reproductive health indicators of fishes from Pennsylvania watersheds: association with chemicals of emerging concern. Environ. Monit. Assess. 186, 6471e6491. The present work has been partially financed by the Center for Blickley, T.M., Matson, C.W., Vreeland, W.N., Rittschof, D., Di Giulio, R.T., McClellan- International Scientific Studies & Collaboration (CISSC), Ministry of Green, P.D., 2014. Dietary CdSe/ZnS quantum dot exposure in estuarine fish: bioavailability, oxidative stress responses, reproduction, and maternal transfer. Science, Research and Technology of Iran on January 2011, with Aquat. Toxicol. 148, 27e39. project contract number 894/1. We acknowledge Ahmad Ghasemi Bowman, C.J., Kroll, K.J., Hemmer, M.J., Folmar, L.C., Denslow, N.D., 2000. Estrogen- for his valuable help and Ebrahim Motaghi, Neda Shojaee and Salar induced vitellogenin mRNA and protein in sheepshead minnow (Cyprinodon e Sohrabi for their assistance in field work and specimen collection. variegatus). Gen. Comp. Endocr. 120, 300 313. Canapa, A., Barucca, M., Gorbi, S., Benedetti, M., Zucchi, S., Biscotti, M.A., Olmo, E., Nigro, M., Regoli, F., 2007. 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