Cloning and Study of Vitellogenin Expression in Wild Cyprinid Petroleuciscus Esfahani As a 2 Biomarker of Endocrine Disruption Along the Zayandeh Roud River, Iran
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1 Cloning and study of vitellogenin expression in wild cyprinid Petroleuciscus esfahani as a 2 biomarker of endocrine disruption along the Zayandeh Roud River, Iran 3 Neda Gilannejada,b*, Salar Dorafshana, Fatemeh Paykan Heyratia, Nasrollah Mahboobi Soofiania, 4 Saeid Asadollaha, Juan Antonio Martos-Sitchab,c, Francisco Pratb, Manuel Yúferab, Gonzalo 5 Martínez-Rodríguezb 6 a Department of Natural Resources, Isfahan University of Technology, 84156-83111 Isfahan, Iran 7 b Instituto de Ciencias Marinas de Andalucía (ICMAN), CSIC, 11510 Puerto Real, Cadiz, Spain 8 c Department of Biology, Faculty of Marine Sciences, University of Cadiz, 11510 Puerto Real, Cadiz, Spain 9 10 * Corresponding author: [email protected] 11 12 Abstract 13 Aquatic environments are the ultimate sink for most of anthropogenic pollutants. The Zayandeh 14 Roud River is the most important river in the central Iranian Plateau, supplying water resources 15 to a large population. In order to determine the potential occurrence and in vivo effects of EDCs 16 with estrogenic properties we analyzed the wild populations of an extensively distributed 17 endemic fish species, Petroleuciscus esfahani. For this purpose, specimens were caught from 18 two sites upstream and two sites downstream of the expected major anthropogenic pollution 19 sources. P. esfahani full length cDNAs for vitellogenin (vtg), with 4,177 base pairs (bp) 20 encoding a 1,339 amino acids (aa), and for β-actin (actb), with 1,776 bp encoding a 375 aa, were 21 amplified and cloned. Hepatic vtg mRNA expression levels were measured by quantitative real- 22 time PCR. Condition factor, gonadosomatic index and sex ratio were calculated and compared 23 with vtg expression. Gonad histology was performed to study the possible presence of intersex 24 condition. Detection of vtg transcripts in male individuals from the two downstream sampling 25 sites supports the hypothesis of exposure to estrogenic compounds in these regions. Higher vtg 26 expression in male individuals, together with reduced gonad size and condition factor, in 27 specimens from the third site, located downstream of the major steel mill plant, suggest a major 28 endocrine disruption in this area. 29 Keywords: Endocrine disruption, Petroleuciscus esfahani, Vitellogenin expression, Zayandeh 30 Roud River 31 32 1. Introduction 33 There is a growing concern about a high variety of environmental pollutants affecting 34 human and wildlife health (Sun et al., 2011). Among them, there is a family of contaminants 35 known generically as endocrine disrupting chemicals (EDCs). One major class of EDCs is 36 formed by compounds with estrogenic properties, also known as xenoestrogens, which mainly 37 affect at the level of the hypothalamus-pituitary-gonad (HPG) axis (WHO/PCS, 2002; 38 WHO/UNEP, 2013). Many of the anthropogenic activities are potential sources of this group of 39 chemicals, which finally find their way to rivers and lakes and affect aquatic organisms (Jeffries 40 et al., 2010). 41 The Zayandeh Roud River is the only permanent and the most important river in the central 42 Iran, which plays a key role in providing Isfahan and the adjacent provinces with drinking water. 43 Land use along this river includes agricultural fields, cattle farms, municipal waste water 44 treatment plants and heavy industries such as steel mill companies, power plants and oil refinery 45 installations. Therefore, there is a high probability of contamination by EDCs. According to our 46 knowledge, the information about the presence and in vivo consequences of EDCs in water 47 ecosystems from Isfahan province or generally in Iran is very scarce. The limited data available 48 for the Zayandeh Roud River, which are dedicated to measuring some heavy metal and a series 49 of compounds belonging to polycyclic aromatic hydrocarbons, affirm their existence in water 50 and/or sediments (Nemati et al., 2009; Abdollahi, 2013). 51 Since chemical monitoring of the aquatic environment alone is restricted to identification 52 of some of the EDCs with estrogenic effects, and the detection methods are costly and complex, 53 the use of biomarkers in sentinel species has been considered an effective approach to assess the 54 health status of the aquatic ecosystems (Frenzilli et al., 2008; Blazer et al., 2012; Gilroy et al., 55 2012). Obviously, aquatic species are more exposed to EDCs than terrestrial animals 56 (WHO/PCS, 2002). Among different aquatic species used for EDCs screening, fishes are the 57 prominent model due to their particular features, the most important of which is that the basic 58 aspects of the function and structure of the HPG axis are conserved in all vertebrates. Therefore, 59 the results from their study can be extrapolated to other vertebrates including humans (Ankley et 60 al., 2009). Petroleuciscus esfahani is a small cyprinid fish endemic to the Zayandeh Roud basin 61 (Coad & Bogutskaya, 2010). This species is abundant along the river and is even available in 62 locations with constant input of contaminants. All these features, together with its limited 63 immigration capacity, make this fish a suitable candidate for sentinel species in this ecosystem. 64 Fish exposure to EDCs with estrogenic activity can lead to several consequences, including 65 i) vitellogenin induction in juveniles or males (Blazer et al., 2014; Duffy et al., 2014), ii) delay or 66 absence of secondary sexual characteristics and sexual behavior in males (Goksøyr et al., 2003), 67 iii) intersex condition (Puy-Azurmendi et al., 2013), iv) skewed sex ratio (Jobling et al, 1996), 68 and in extreme cases v) complete extinction of the population (Kidd et al., 2007). Vitellogenin 69 (Vtg) is a female-specific precursor protein for egg yolk, which is normally synthesized in 70 female hepatocytes in response to endogenous estrogens (Goksøyr et al., 2003). Males also 71 possess the vitellogenin genes (vtg), which are silenced but can be induced when exposed to 72 xenoestrogens (Hutchinson et al., 2005). Actually, vitellogenin induction in males is the 73 strongest biomarker for fish exposure to estrogenic compounds (Nadzialek et al., 2011). vtg 74 mRNA is induced rapidly (within a few hours of exposure) and therefore, are useful for short- 75 term screenings of the ecosystems, as well as it has been already field tested in many 76 ecotoxicological examinations (Frenzilli et al., 2008; Blanchet-Letrouvé et al. 2013; Bizarro et 77 al., 2014). 78 The aim of this study was to determine the possible exposure of xenoestrogens on P. 79 esfahani as a bioindicator in the Zayandeh Roud River ecosystem. For this purpose, full 80 sequence of the cDNA encoding vtg and actb was cloned for the first time in this species, and 81 hepatic vtg mRNA expression was quantified using real-time PCR. Moreover, condition factor, 82 gonadosomatic index and sex ratio were calculated and compared to vtg expression. Gonad 83 histology was also performed with the goal of detecting any state of intersex in the specimens. 84 2. Materials and methods 85 2.1. Studied area and sampling points 86 The Zayandeh Roud River, with a drainage area of 41,500 km2, is the largest river in the 87 central plateau of Iran. Sampling sites were selected throughout the river having in mind a 88 possible gradient of anthropogenic impacts. Cheshmeh Dimeh (S1) (32°31' N - 50°13' E), and 89 Khersoonak (S2) (32°31' N - 50°22' E), are located upstream of the Zayandeh Roud Dam and 90 were considered as uncontaminated sites. In these two sites, some extensive and scatter crop and 91 cattle production can be found. On the other hand, Chamgordan (S3) (32°23' N - 51°17' E) and 92 Safaieh Bridge (S4) (32°21' N - 51°26'E) are situated downstream of the Zayandeh Roud Dam 93 and were considered a priori as possible contaminated sites. S3 is a lagoon like reservoir which 94 is formed by entering the river into an enclosed area and it receives effluents from the nearby 95 major steel mill plant as well as agricultural fields. S4 is located downstream of the sewage 96 treatment plant discharges from the Zarin Shahr City (population in 2012: 95,326) (Figure 1). 97 During fish collections, temperature, dissolved oxygen, pH, and conductivity were measured on 98 site, and total dissolved solids (TDS), biochemical oxygen demand (BOD), and chemical oxygen 99 demand (COD) were determined in laboratory (Table 1). 100 Table 1. Water physico-chemical parameters in four sampling sites from the Zayandeh Roud River. Temperature Dissolved Oxygen Conductivity TDS BOD COD Site pH (°C) (mg/L) (µS/cm) (ppm) (ppm) (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 80 162 101 102 103 Figure 1. Sampling locations of P. esfahani from the Zayandeh Roud River. Cheshmeh Dimeh (S1), 104 Khersoonak (S2), Chamgordan (S3) and Safaieh Bridge (S4). 105 2.2. Fish sampling and analyzed parameters 106 Fish were captured during the non-reproductive period (January to February, 2012) by 107 seine net (8 m × 1 m, 5 mm mesh size) in S1, S2 and S4, and by boat electrofishing in S3 108 because of higher water depth in this site. After transporting the fish to the laboratory, 10 109 individuals were aquatically exposed to 17β-estradiol (E2) (2 µg/L) for 24 hours (Bowman et al., 110 2000) with the purpose of increasing the chance of cloning the vtg cDNA. At the end of this 111 period, male and female liver samples were preserved in RNAlater® (Invitrogen Life 112 Technologies) separately. On the other hand, fish from all the sampling sites were kept in aerated 113 tanks with water from the corresponding sampling site until processed (around 1 hour).