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This article appeared in a journal published by Elsevier. The attachedcopy is furnished to the author for internal non-commercial researchand education use, including for instruction at the authors institution
and sharing with colleagues.
Other uses, including reproduction and distribution, or selling orlicensing copies, or posting to personal, institutional or third party
websites are prohibited.
In most cases authors are permitted to post their version of thearticle (e.g. in Word or Tex form) to their personal website orinstitutional repository. Authors requiring further information
regarding Elsevier’s archiving and manuscript policies areencouraged to visit:
Exposure to waterborne 4-tert-octylphenol induces vitellogenin synthesis anddisrupts testis morphology in the South American freshwater fishCichlasoma dimerus (Teleostei, Perciformes)
G. Rey Vázquez a,⁎, F.J. Meijide a,b, R.H. Da Cuña a,b, F.L. Lo Nostro a,b, Y.G. Piazza a,b, P.A. Babay c, V.L. Trudeau d,M.C. Maggese a,b, G.A. Guerrero a
a Lab. de Embriología Animal, Dpto. de Biodiversidad y Biología Experimental, FCEyN-UBA, Ciudad Universitaria, Pabellón II, 4° piso (C1428EHA), Buenos Aires, Argentinab CONICET, Rivadavia 1917 (C1033AAJ), Buenos Aires, Argentinac Gerencia de Química, Centro Atómico Constituyentes, CNEA. Av. Gral. Paz 1499 (1650), Pcia. de Buenos Aires, Argentinad Centre for Advanced Research in Environmental Genomics, Dept. of Biology, University of Ottawa, Ottawa, Ontario, Canada K1N 6N5
a b s t r a c ta r t i c l e i n f o
Article history:Received 29 January 2009Received in revised form 15 May 2009Accepted 19 May 2009Available online 27 May 2009
Keywords:Cichlasoma dimerusCichlidsLiver and testis pathologyOctylphenolSurface mucusTeleostsVitellogeninXenoestrogens
Exposure to environmental pollutants may disrupt endocrine functions and cause reproductive effects inhuman and wildlife populations. Various groups of chemicals have estrogen-like effects, includingdegradation products of alkylphenol polyethoxylates, such as 4-tert-octylphenol (OP). Laboratory studieshave shown that exposure of male fish to xenoestrogens results in induction of circulating vitellogenin (Vtg),inhibition of testicular growth, testis abnormalities and formation of intersex gonads. In this study, theimpact of the exposure to waterborne OP on reproductive aspects in the South American cichlid fish Ci-chlasoma dimerus was evaluated using qualitative changes in the levels of Vtg in plasma and surface mucusand histological alterations in the liver and gonads as endpoints. Adult males and females were exposed toOP via immersion during 60 days in aquaria under semi-static conditions, water changes being made every84 h. Treatment groups were: control (ethanol 0.005%), OP 30, 150 and 300 µg/L. Using Western and Dot blotanalysis, Vtg was detected in plasma and mucus of control and treated females and treated males, while noVtg was observed in samples from control males. Morphological changes in the hepatocytes due to theaccumulation of Vtg were observed in OP-exposed males. Impairment of testicular structure becameapparent in males treated with the highest OP concentrations. The most salient pathological change was thealteration of lobular organization with increased testicular fibrosis and progressive disruption ofspermatogenesis. No major changes were observed in ovarian architecture. Our results indicate thatdetection of Vtg in surface mucus may be a sensitive and non-invasive biomarker of the endocrine disruptingeffects of environmental estrogens, resulting in a useful method for field monitoring.
A wide range of chemicals introduced into the environment byhuman activities may be producing adverse health effects in humanand wildlife populations. The term endocrine disrupting chemicals(EDCs) is applied to awide range of compounds that interferewith thesynthesis, secretion, transport, binding, action or elimination ofnatural hormones in the body, that regulate homeostasis, reproduc-tion, development and behavior (Nishi et al., 2002). Among EDCs,various groups of chemicals have estrogen-like effects, being referredto as xenoestrogens. Alkylphenol polyethoxylates (APEs) are one ofthe classes of nonionic surfactants most widely used in themanufacturing of cleaning agents, plastics, pulp and paper, textiles,
agrochemicals, cosmetics and food products (Routledge and Sumpter,1997; Ying et al., 2002). Primary degradation of APEs in wastewatertreatment plants or in the environment generates more persistentshorter-chain APEs and alkylphenols (APs) such as 4-tert-octylphenol(OP), one of the most biologically active products. It has beensuggested that the levels of APE metabolites present in the aquaticenvironment may be well above the threshold necessary to induceendocrine disruption in wildlife. These findings have raised publicconcern over their environmental and human health effects (Yinget al., 2002). Laboratory and field studies have shown that exposure ofmale fish to alkylphenolic compounds results in induction ofcirculating vitellogenin (Vtg), inhibition of testicular growth, testisabnormalities and formation of intersex gonads, among other signs ofreproductive impairment (White et al., 1994; Sumpter and Jobling,1995; Jobling et al., 1996; Gray and Metcalfe, 1997; Christiansen et al.,1998; Gronen et al., 1999; Kinnberg et al., 2000; Folmar et al., 2001;Metcalfe et al., 2001; Van den Belt et al., 2001; Knörr and Braunbeck,
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⁎ Corresponding author. Tel.: +54 11 45763348; fax: +54 11 45763384.E-mail address: [email protected] (G. Rey Vázquez).
j ourna l homepage: www.e lsev ie r.com/ locate /cbpc
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2002; Kang et al., 2003; Rasmussen and Korsgaard, 2004; Rasmussenet al., 2005).
Vitellogenin is a complex phospholipoglycoprotein normallyproduced in the liver of mature female fish in response to increasingcirculating estrogen levels leading up to spawning (Arukwe et al.,2000). Circulating Vtg is taken up by developing oocytes and is aconstituent of yolk which serves as a source of nutrients for thedeveloping embryo. Adult male fish do not usually produce Vtg, butthey do possess the hepatic estrogen receptor and the Vtg gene, whichis normally silent. Therefore, Vtg synthesis in males can be induced bytreatment with xenoestrogens (Flouriot et al., 1997; Gronen et al.,1999; Ackerman et al., 2002; Park et al., 2003; Van den Belt et al.,2003; Knoebl et al., 2004;Meucci and Arukwe, 2005; Arukwe and Røe,2008). Various field investigations have reported Vtg detection inmale fish exposed to industrial and domestic effluents (Sumpter andJobling, 1995; Harries et al., 1997; Nichols et al., 1999; Wahli et al.,1998; Hashimoto et al., 2000; Folmar et al., 2001). These effluents areknown to carry awide range of structurally diverse chemicals with thecapability of exerting estrogenic actions.
Detection of Vtg in the surface mucus has been suggested as amean of determining sex in fish lacking sexual dimorphism or as ascreening method of maturity state (Kishida et al., 1992; Kishida andSpecker,1994, 2000; Takemura and Oka,1998). In addition, it has beenproposed that the possibility of detecting Vtg in the surface mucuscould result in a useful tool for monitoring the presence of estrogenicpollutants in the environment (Moncaut et al., 2003; Meucci andArukwe, 2005; Van Veld et al., 2005; Maltais and Roy, 2007; Arukweand Røe, 2008). On the other hand, the teleost liver is one of the mostsensitive organs to show alterations in biochemistry, physiology andstructure following exposure to various types of environmentalpollutants (Hinton and Couch, 1998; Andersson et al., 2007; PerezCarrera et al., 2007).
The South American cichlid fish Cichlasoma dimerus, a perciformteleost, is common in quiet shallow waters of the Paraguay River andmost of the Paraná River basins (Kullander, 1983). This species isrepresentative of teleosts in the La Plata River Basin and resultsrelevant to the Argentinean riverine ecosystems. In addition, C.dimerus adapts easily to captivity and shows notable reproductivefeatures such as a complex social and breeding behavior, whichincludes parental care and, most important, a high spawning rate(about every 25 days during 8 months) (Meijide and Guerrero, 2000),providing an amenable model for laboratory studies, includingecotoxicological testing.
Due to the lack of studies dealing with the effects of environmentalestrogens in South American perciform freshwater fish, the aim of thiswork was to evaluate the effects of waterborne exposure to OP atsublethal concentrations in C. dimerus using qualitative changes in thelevels of Vtg in plasma and surface mucus, and histological alterationsin the liver and gonads as endpoints.
2. Materials and methods
2.1. Fish
The adult specimens of C. dimerus used in this study were capturedfrom the natural environment in Esteros del Riachuelo, Corrientes,Argentina (27° 25′ S, 58° 15′ W). They were allowed to acclimate tocaptivity conditions for a month prior to the start of experimentation.A total of 32 fish (24 males and 8 females) were weighed and totallength (TL) measured (females: 27.14±8.95 g and 10.50±1.17 cm;males: 50.15±11.50 g and 12.78±0.98 cm) and kept in 100 L aquariaat 26.5±1 °C, pH 7.3, with 12:12 h photoperiod and an averagedensity of 6.4 g/L. Laboratory aquaria were well aerated and providedwith external filtration and a layer of gravel on the bottom. Fish werenormally fed once a day with pelleted commercial food (Tetra foodsticks).
2.2. Treatment and sampling
The test substance, 4-tert-octylphenol (OP) (N97% pure) wasobtained from Sigma-Aldrich (St. Louis, MO, USA). Concentrations ofOP for the exposures were selected from preliminary studies and a96 h acute toxicity test (LC 50–96 h=780 µg/L).
Previous to the onset of exposure, three males and one female fishwere transferred to each 50 L glass tank under the same physicalconditions and alimentary ratio, except that the layer of gravel on thebottom was removed. Animals were allowed to acclimate to the newexperimental conditions for a week before the experiment wasstarted. Fish were exposed to nominal concentrations of 0 (solventcontrol; ethanol 0.005%), 30, 150 and 300 µg/L OP in duplicateaquaria. Exposure to OP was performed during 60 days under semi-static conditions, water changes being made twice a week. Stocksolutions were prepared once aweek by dissolving OP in 100% ethanoland stored in darkness at 4 °C. During each water renewal, smallaliquots of the stock solutionwere added to filtered tap water in orderto obtain the desired concentrations.
At the end of the experiment, mucus and blood samples werecarefully and quickly collected after fish were softly narcotizedwith fish calmer (dose: 6 drops/2 L; active ingredients: acetone,dimethylketone alpha methyl quinoline; Jungle Hypno, USA). Mucuswas collected by scraping the body surface with a stainless steelspatula into microcentrifuge tubes containing the same volume of0.1 M phosphate-buffered saline, pH 7.4 with Tween 20 and 2.5 µL of1 mM PMSF (phenylmethylsulfonyl fluoride, protease inhibitor). Aftercentrifugation at 2500 g for 15 min at 4 °C, the supernatant wasseparated and stored at −20 °C. Peripheral blood was collected bypuncture of the caudal vein with a heparin-coated 25 gauge×1/2 in.needle, attached to a 1mL syringe. Samples with the addition of 2.5 µLof 1 mM PMSF were centrifuged at 2500 g for 15 min at 4 °C to obtainplasma which was stored at −20 °C. Immediately following, the fishwere sacrificed by decapitation under anesthesia and liver and gonadswere quickly removed. The samples were fixed for subsequenthistological and immunohistochemical examination.
2.3. Immunoblot
Plasma and surface mucus samples were analyzed by reducingsodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS/PAGE) using a 5% stacking gel and an 8% resolving gel followed byWestern blot. Samples were diluted in sample buffer containing0.3 M Tris/HCl, pH 6.8, 10% SDS, 12% glycerol, 11% β-mercaptoethanoland 0.2% bromophenol blue. Equal amounts of protein (20 µg), asmeasured by Lowry et al. (1951), were loaded into each lane.Molecular mass was estimated using pre-stained molecular massstandards (Invitrogen). Following separation by electrophoresis,proteins were transferred to a nitrocellulose membrane (Hybond,Amersham Pharmacia) for 90 min at 4 °C and 100 V in transfer buffer(25 mM Tris, 187 mM glycine, 20% (v/v) methanol). Non-specificbinding of membranes was blocked with 5% non-fat powdered milkin TBST (100 mM Tris–HCl, 0.9% NaCl, 0.1% Tween 20, pH 7.5)overnight at 4 °C. Membranes were then incubated with the primaryantiserum, rabbit anti-perch Vtg (donated by Dr. B. Allner, HessischesLandesamt für Umwelt und Geologie, Wiesbaden, Germany; seeHennies et al., 2003) 1:3000, for 90 min at room temperature. Thisantiserum has already proven to be effective for Vtg detection in C.dimerus (Moncaut et al., 2003). After three 5 min washes in TBST,membranes were incubated with a biotinylated anti-rabbit IgGantibody (Vector Lab., Burlingame, CA, USA) diluted 1:1000 for 1 hand washed again, followed by incubation with horseradishperoxidase-conjugated streptavidin (Dako, Carpenteria, CA, USA)diluted 1:3000 for 1 h in the dark. Immunoreactivity was developedwith 0.1% 3, 3′ diaminobenzidine in 0.1 M Tris buffer pH 7.6 (DAB)and 0.02% water peroxide (H2O2). Preadsorption with pure
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commercial carp Vtg (Biosense Lab A.S., Norway) before incubationand omission of the primary antiserumwere performed for Westernblot as specificity controls. For Dot blot, plasma and surface mucussamples (20–30 µg protein) were loaded into a nitrocellulosemembrane. Once dried, non-specific blocking, incubation anddeveloping were carried out as described before for Western blot. Aprimary antiserum omission control was performed.
2.4. Immunohistochemistry
Liver pieces were fixed in 0.1 M phosphate-buffered (pH 7.4) 10%formalin at room temperature, embedded in paraffin and sectioned at6 µm. Sections were deparaffinized, hydrated, rinsed for 10 min in PBSbuffer (0.1 mM phosphate-buffered saline, pH 7.6), and then treatedwith 3% hydrogen peroxide for 10 min to quench endogenousperoxidase activity. After buffer rinse, the slides were treated with aprotein blocker (5% of non-fat powderedmilk in PBS) for 40 min. Theywere subsequently incubated with the primary antiserum, anti-perchVtg diluted 1:500 for 1 h. Next, slides were incubated with thesecondary anti-rabbit biotinylated antibody (Sigma Chemical Co.,USA) diluted 1:300 for 1 h. Slides were then incubated with avidin–biotin complex (ABC, Vector Lab.) for 1 h. All the incubations wereperformed at room temperature. Immunostaining was visualizedusing 0.1% DAB in 0.1 M Tris buffer pH 7.6 and 0.02% H2O2. Sectionswere lightly counterstained with Carazzi hematoxylin, dehydratedand mounted with DPX. Micrographs were taken with a Nikon-Microphot FX microscope. Control sections were treated with PBSinstead of primary antiserum.
2.5. Light microscopy
For standard histological techniques, small pieces of ovaries, testesand liver were fixed in Bouin's fluid for 12 h at 4 °C and then preservedin 70% ethanol. Samples were embedded in paraffin or glycolmethacrylate (Leica Historesin) and sectioned at 6 µm and 4 µm,
respectively. Sections were stained with Hematoxylin–Eosin (H&E).Tissue pieces were also fixed in 3% glutaraldehyde in 0.1 M phosphatebuffer (pH 7.4), post-fixed in 1% osmium tetroxide in 0.1 M phosphatebuffer (pH 7.4) for 2 h, and embedded in Spurr resin. Semithinsections at 2 µm were stained with toluidine blue. Photomicrographswere taken with a Nikon-Microphot FX microscope.
2.6. Measurement of actual octylphenol concentrations
As a common situation in laboratory experiments is the frequentlylarge discrepancy between measured and nominal concentrations oftest chemicals, actual concentrations of OPweremeasured by reverse-phase HPLC coupled to fluorescence detection, following the approachby Ahel and Giger (1985) and Ahel et al. (1985). The column employedwas a PRP-1 of 250×4.1 mm, 10 µm particle size and 100 Å porediameter (Hamilton, USA), and elution was performed with a 80:20methanol/water mixture, at a flow rate of 0.8 mL/min. Detector wasset at excitation and emission wavelengths of 230 and 300 nm,respectively. Water samples from the 150 µg/L OP and control aquariawere taken every 24 h during the last week of the experiment andtreated by solid phase extraction (SPE) on C18 with methanol elutionbefore injection in the HPLC. Data were acquired and analyzed withthe Konikrom 5.2 software (Konik Instruments, Spain). For quantifica-tions, calibration curves were constructed for peak areas, frominjection of standard solutions daily prepared by adding knownamounts of OP to control water and processed in the same manner asthe samples. For each set of replicate samples, mean and standarddeviations were calculated after interpolation of OP chromatographicpeak area in the calibration curve (R2=0.99).
2.7. Statistical analysis
A repeated measures analysis of variance (ANOVA) was used todetermine differences in weight and length of animals betweentreatments, before and after the exposure.
Fig. 1. Immunodetection of vitellogenin (Vtg) in plasma and mucus samples of adult C. dimerus exposed to waterborne OP at 0 (Ctrl), 30, 150 and 300 µg/L during 60 days. A, B:Western blot analysis in plasma (A) and mucus (B), respectively. Each lane represents the plasma or surface mucus of one representative fish. Arrowheads indicate Vtg from controlvitellogenic females. Replacement of the primary antiserum by TTBS resulted in the suppression of Vtg immunoreactivity (not shown). Std: Broad range pre-stained molecular massstandards. Pread: Preadsorption control. Suppression of immunoreactivity was evidenced when plasma from control females was preadsorbed with Vtg. C, D: Dot blot analysisrevealing the presence of Vtg in plasma (C) and surface mucus (D) of control females (f ctrl) and treated males (m) and females (f). Ctrl−: Negative control. Omission of the primaryantiserum resulted in the absence of immunoreactivity. The difference in the intensity of immunoreactivity observed in mucus samples frommales and females exposed to 300 µg/LOP may be explained by males lacking a depositional site (ovary) for Vtg and the skin serving as an excretory route.
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3. Results
3.1. Survival and growth
All of the control and OP-exposed fish survived to the end of theexperiment. Statistical analysis indicated that theweight and length ofthe fish assigned to the different treatments did not vary significantlyduring the course of the experiment (pN0.05; data not shown).
3.2. Vitellogenin detection
A qualitative identification of Vtg was performed using Westernand Dot blot techniques. Vtg was detected in vitellogenic females andall the OP-treated fish. In control males, no corresponding proteinbands were observed, as expected (Fig. 1). In plasma from controlvitellogenic females, Vtg appeared as a major band of 115 kDa andthree additional bands of 158, 99 and 73 kDa (Fig. 1A). However, onlythe bands corresponding to 115 and 73 kDa occurred in the mucussample (Fig. 1B). Vtg was detected both in plasma and surface mucusof male fish exposed to waterborne OP, where in addition to the fouraforementioned Vtg bands, other immunoreactive (ir)-Vtg bandswere present (Fig. 1A, B).
Dot blot analysis proved to be a useful and easy method to detectthe presence of Vtg, since in both plasma and mucus samples frommale fish exposed to OP a positive immunostain for Vtg was evidenced(Fig. 1C, D).
No Vtg immunoreaction was observed in liver sections fromcontrol males (inset of Fig. 2A). Liver sections from females and OP-
treated males showed substantial amounts of accumulated ir-Vtg. In30 µg/L OP-exposed males, ir-Vtg was only present in the hepaticvascular system (inset of Fig. 2B) while males treated with 150 or300 µg/L OP also presented large amounts of ir-Vtg materialoccupying the cytoplasm of most hepatocytes (insets of Fig. 2C, D).
3.3. Hepatic histology
C. dimerus possesses a hepatopancreas, a common feature amongcertain teleost families. Under light microscopy, the liver parenchymaof control males showed longitudinal arrays of hepatocytes thatappeared as cord-like, arranged around sinusoids. The hepatic cellshad a polyhedric shape with a basal nucleus and usually onenucleolus. The nuclear membrane was weakly basophilic, while thecytoplasm primarily contained eosinophilic material. A fairly largeamount of lipid was evidenced in the cytoplasm of hepatocytes. Forthis reason, the usual H&E method caused the appearance of manyvacuolar structures within these cells (Fig. 2A). The pancreatic tissueappeared around the major blood vessels interspersed with thehepatic tissue. Gross inspection of the hepatopancreas from OP-treatedmales showed them to be softer than normal, more friable andclearer than those from control males. Histological examination of theliver from OP-treated males reflected that their physiological func-tional state appeared similar to the active period of vitellogenesis infemales. At lower OP concentrations, the liver parenchyma did notdiffer from that of control animals (Fig. 2A, B). At higher OPconcentrations, the hepatocyte nuclei were slightly enlarged. Thecells appeared swollen and their cytoplasm showed increased
Fig. 2. Liver cross-sections of control (A) and 30, 150 and 300 µg/L OP-treated males (B–D). H&E. Within the liver parenchyma, hepatocytes showed a longitudinal array aroundsinusoids. An altered hepatocyte morphology and an increase of cytoplasm basophilia were observed at progressively higher OP concentrations. Insets show immunolocalization ofVtg accumulated immunoreactive material (arrows). N: hepatocyte nucleus, S: hepatic sinusiod, P: pancreatic cells. Scale bar=15 µm (A–D), 16 µm (insets).
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Fig. 3. Testis cross-sections of control (A, B) and OP-treated males (C–H). Testes from control males (A, B) and 30 µg/L OP-exposed males (C, D) showed a lobular architecture withcysts containing all stages of spermatogenesis. Interstitial fibrosis became apparent in the testes of 150 µg/L OP-treated males (E, F), while a disarrangement of the lobularorganization and an abnormal spermatogenesis with absence of cysts were evidenced in males from the 300 µg/L OP treatment (G, H). C: cyst, I: interstitial tissue, L: lobule, LL:lobular lumen, M: cell under division, S: Sertoli cell, SG: spermatogonium, SC: spermatocyte, SP: sperm, V: blood vessel. Scale bar=60 µm (A, C, E, G); 15 µm (B, D, H, F). Stainings:H&E (A–H). Inset shows granulocytes stained with toluidine blue; scale bar=8 µm. E: erythrocyte; G: granulocyte.
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basophilia while the cell outline became blurry, displaying irregularshapes (Fig. 2C, D).
3.4. Gonadal histology
Histological examination of the testes revealed progressiveadverse effects on testicular structure following exposure to increas-ing OP concentrations. Similar morphological characteristics wereobserved in samples from replicates of a same treatment. In testesfrom control fish, all stages of spermatogenesis were present (Fig. 3A).Testicular lobules contained spermatogonia as well as numerous cystswith early (spermatocytes) and late (spermatids) stages of sperma-togenesis. A preponderance of sperm was usually observed in thelobular lumen, with spermatogonia and cysts of spermatocytesremaining in the lobule walls (Fig. 3A, B). At the light microscopiclevel, testes of males exposed to 30 µg/L appeared similar to those ofcontrol males (Fig. 3C, D). In 150 µg/L OP-exposed males, the testespreserved the lobular structure but an increased interstitial fibrosiswas observed. Within the germinal epithelium, cysts became moredifficult to identify. However, spermatogonia as well as spermatocytesand spermatozoa were observed. In addition, eosinophil granulocyteswere identified (Fig. 3E, F). Gonadal tissue of males treated with300 µg/L OP revealed a disarranged lobular structure as well asabsence of cysts. The majority of testes appeared as sacks of fibrousinterstitial tissue filled with sperm. Moreover, abnormality ofspermatogenesis was evidenced by the progressive degeneration ofthe germinal epithelium. Isolated Sertoli cells appeared squamous andfew spermatogonia could be found scattered throughout the remnantsof the germinal epithelium (Fig. 3G, H).
Ovaries of OP-treated females did not differ histologically fromovaries of control females (not shown).
3.5. Concentration of octylphenol in the water
The measured concentrations of OP in replicate water samplestaken every 24 h from the 150 µg/L treatment group is indicated inTable 1. The initial nominal and actual OP levels were in goodagreement. Over the 72 h test period, measured levels of OP declinedto approximately 20% of the initial concentrations. OP was notdetected in samples from the control treatment.
4. Discussion
In the present study, exposure to waterborne OP resulted in thepresence of vitellogenin both in the plasma and surfacemucus of maleC. dimerus. In addition, testicular damage was evidenced aftertreatment with the highest OP concentrations. Our results provideconfirmation that OP mimics the effects of 17β-estradiol in thisspecies (Moncaut et al., 2003), to stimulate Vtg synthesis andtherefore acts as a xenoestrogen. Estrogenic effects upon treatmentwith OP were reported in species from several orders of teleosts:
Cyprinodontiformes, guppy (Poecilia reticulata) (Bayley et al., 1999);Beloniformes, Japanese medaka (Oryzias latipes) (Gronen et al., 1999;Knörr and Braunbeck, 2002); Siluriformes, brown bullhead catfish(Ameiurus nebulosus) (Holland Toomey et al., 1999); Cypriniformes,zebrafish (Danio rerio) (Van den Belt et al., 2001, 2003); Salmoni-formes, rainbow trout (Oncorhynchus mykiss) (Van den Belt et al.,2003); Pleuronectiformes, flounder (Platichthys flesus) (Madsen et al.,2006). In Perciformes only three species were studied: sand goby(Pomatoschistus minutus) (Robinson et al., 2004), eelpout (Zoarcesviviparus) (Rasmussen and Korsgaard, 2004; Andreassen et al., 2005;Rasmussen et al., 2005) and Japanese sillago (Sillago japonica)(Yoon et al., 2008), all of them marine. To our knowledge, this isthe first report of the actions of OP in a South American fresh-water perciform fish. In this study, Vtg of C. dimerus was detectedusing a heterologous anti-perch antiserum generated againstPerca fluviatilis Vtg (Hennies et al., 2003). Validity of the cross-species reactions is based on the fact that both C. dimerus and P.fluviatilis are Perciformes. Preadsorption of the antiserum with Vtgresulted in a marked decrease in immunoreactivity for all theimmunoreactive bands in vitellogenic females, confirming the anti-serum's specificity to Vtg. In addition, the antiserum proved to beeffective for Vtg detection in other non-perciform species (Hennieset al., 2003; Allner, pers. com.). Immunoreactivity patterns showed aspecific reaction only in samples of vitellogenic females and OP-exposed males, while samples from control males and non-vitello-genic females did not show immunoreactivity.
In control vitellogenic females, a main band of 115 kDa and threeadditional bands of 158, 99 and 73 kDa were detected in plasmasamples, whereas only the bands of 115 and 73 kDa were present inmucus samples. Vtg expression seemed to be highly dependent on themoment of the reproductive cycle duringwhich the samplewas taken,since plasma and mucus samples from non-vitellogenic females didnot reveal the presence of Vtg. Given that ovarian cycles in C. dimerusare relatively short and the spawning interval may be as brief as25 days, females were expected to be at different stages of vitellogen-esis by the end of the 60-day exposure period. Themain Vtg protein infemales of C. dimerus has a slightly lower molecular mass (115 kDa)than that of other teleost species like Arctic charr (Salvelinus alpinus)(Johnsen et al., 1999), fathead minnow (Pimephales promelas) (Parkset al., 1999), platyfish (Xiphophorus maculatus) (Kinnberg et al., 2000),zebrafish (D. rerio) (Segner et al., 2003), Chinese minnow (Phoxinusoxycephalus) (Park et al., 2003), Atlantic salmon (Salmo salar) (Meucciand Arukwe, 2005) and copper redhorse (Moxostoma hubbsi) (Maltaisand Roy, 2007), in which Vtg ranges from 134 to 200 kDa. However, inmales exposed to high concentrations of OP, in addition to the 115 kDaprotein, the ir-Vtg band of 158 kDa also became conspicuous. Thisprotein could correspond to not fully processed Vtg precursors ofhigher molecular mass being released to the circulation upontreatment with high concentrations of OP, due to the intensity of thestimulus which would overwhelm synthetic pathways. The presenceof additional ir-Vtg bands of lower molecular mass is frequentlyobserved, and often ignored, when WB analysis is performed. Theseproteins could be breakdown products due to degradation of Vtg.Stability of this high molecular mass phospholipoglycoprotein is veryvariable between species; in some it is highly stable, showing only asingle immunoreactive band in Western blot techniques, whereas inother species similar additional bands can be detected, suggesting ahigher lability of Vtg (Silversand et al., 1993; Parks et al., 1999; Brionet al., 2000; Hennies et al., 2003). Some of the additional bands mightalso represent Vtg variants, since at least two Vtg genes have beendescribed in teleosts (Utarabhand and Bunlipatanon, 1996; Matsubaraet al., 1999).
The presence of Vtg in mucus samples from treated animals couldbe due to the fact that the skin may be serving as an excretorypathway, since this protein lacks a depositional site (ovary) in malesand large concentrations would exhaust the normal pathways of
Table 1Actual concentrations of OP in the aquarium water during the last week of theexperiment.
Measurements were made 0, 24, 48 and 72 h after addition of OP. Values are means±standard deviation of the concentrations recorded in two samples from the 150 µg/Ltreatment group. % are values for the measured OP concentration expressed aspercentage of the nominal concentration.
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elimination. The accumulation of such high quantities of Vtg in thebody of male and female fish may have deleterious effects, since highVtg concentrations have been associated with kidney failure andincreased mortality rates (Herman and Kincaid, 1988). Interestingly,Arukwe and Røe (2008) found that treatment with 4-nonylphenol(NP) in Atlantic salmon not only caused the presence of Vtg in skin,but also expression of the Vtg and estrogen receptor mRNA inepidermal cells. This suggests that the skin, in addition to its role as anexcretory pathway of surplus Vtg, is also capable of responding toxenoestrogens by Vtg synthesis. Larvae of some species of cichlidfishes are known to feed on the epidermal mucus of both male andfemale parents (Noakes, 1979). In these species, Vtg is present in thesurface mucus of parents (Kishida and Specker, 1994), along withother non-nutrients such as prolactin, growth hormone and thyroxine(Schütz and Barlow, 1997). Though this nipping behavior of larvae islimited to a few teleost species, the presence of Vtg in surface mucuscould be evolutionarily significant and related to nutrition.
In male fish, production of the yolk-precursor protein Vtg isconsidered to be a sensitive biomarker of estrogenic exposure(Sumpter and Jobling, 1995). The presence of Vtg is usually assessedin blood plasma samples. This study extends previous findings of ourresearch group (Moncaut et al., 2003), which demonstrated that anintraperitoneal injection of 17β-estradiol induced detectable levels ofVtg in the surface mucus of C. dimerus. Based on this property, assaysfor evaluation of vitellogenin levels in mucus were later developed inother teleosts (Van Veld et al., 2005; Maltais and Roy, 2007). MucusVtg induction was also recorded in juvenile Atlantic salmon afterexposure to waterborne NP (Meucci and Arukwe, 2005). Thedetection of Vtg in the surface mucus of xenoestrogen-exposed fishrepresents a sensitive and non-invasive method to monitor thepresence of known endocrine disruptors in the aquatic environment.This easy and fast technique then constitutes a useful tool for fieldmonitoring. Moreover, the non-invasive approach benefits protectedor endangered species, which cannot be submitted to some of thecommonly used destructive methods of risk assessment (Fossi et al.,1999; Maltais and Roy, 2007).
Numerous ecotoxicological studies indicate that the vertebrateliver is one of themost extensively studied organs, as it is themain siteof xenobiotics detoxification, glycogen storage and excretion. Undernatural conditions, the cytoplasm of hepatic cells of male fish containsa large number of glycogen granules, while in spawning females therough endoplasmic reticulum occupies the majority of the cell'scytoplasm, indicative of active protein synthesis (Yamamoto andEgami, 1974). In C. dimerus, hepatic cells in OP-treated male fish werehistologically similar to those of spawning females. Morphologicalchanges in hepatocytes associated with the accumulation of Vtgincluded an intense cytoplasmic basophilia, which reflects a significantdevelopment of the rough endoplasmic reticulum and the Golgiapparatus, and an enlargement of the hepatocytes nuclei, likely a directresult of intense Vtg synthesis. Immunohistochemical evaluation usinga heterologous antiserum against Vtg allowed us to identify the Vtgproducing cells in the hepatic parenchyma of vitellogenic females andOP-exposed males. This confirmed the identity of the accumulatedproteins within the hepatocyte cytoplasm as Vtg, and showed thatthese active cells were distributed diffusely over the whole liver.
Histopathology is employed as a sublethal test for evaluating toxiceffects of xenobiotic compounds. The histological examination of thetestes of OP-exposed male fish revealed dose-related testiculardamage. Exposure to 30 µg/L OP did not produce apparent effectsassessed at the light microscope level. However, treatment with150 µg/L OP resulted in an increased interstitial fibrosis whileexposure to 300 µg/L OP caused a disarrangement of the lobularorganization and an absence of Sertoli cells and cysts. In fish treatedwith the highest OP concentration, lobules resembled sacks of fibrousinterstitial tissue filled with spermatozoa. These structural changesare indicative of impairment of spermatogenesis and probably of testis
functionality. Similar to the effects we observed in C. dimerus,exposure to comparable concentrations of NP resulted in a reductionof the number of cysts containing all different spermatogenic stages inmale platyfish X. maculatus (Kinnberg et al., 2000). The mechanismswhereby OP causes the effects observed on the testes of C. dimerus arenot clear and need further investigation. Estrogenic chemicals may actindirectly via the hypothalamus–pituitary–gonad axis, altering thesynthesis and/or secretion of gonadotropin-releasing hormone and/or gonadotropins (Trudeau et al., 1993; Christiansen et al., 1998), orthey may act directly on the testes. Direct effects can be eithercytotoxic, when the disruption is caused by damage to the testis cells,or endocrine, inwhich the function of specific cells (e.g. Sertoli cells) isdisrupted due to an endocrine malfunction (Kime, 1999). The failureof Sertoli cell function is believed to be especially critical duringspermatogenesis, when the germ cells are under development. Asformation of cysts and regulation of spermatogenesis are two of manyimportant roles of Sertoli cells, it is possible that the absence of cystsobserved in C. dimerus is a result of the altered function of these cells.Exposure to estrogens was reported to inhibit spermatogenesis in seabream (Sparus aurata) and zebrafish by impeding cell division ofspermatogonia, and thus the development of germ cells beyond thisstage (Condeça and Canario, 1999; Ortiz-Zarragoitia and Cajaraville,2005). In C. dimerus, OP may act in a similar manner, which wouldexplain the absence of cysts containing spermatocytes or spermatids,since these germ cell stages would have finished meiosis and no newspermatogonia would have initiated meiotic division. Estrogenicchemicals may also exert their effects directly on the testis viainhibition of androgen synthesis (Trudeau et al., 1993). Estrogenreceptors have been identified in the testis of some teleosts andelasmobranchs (Miura et al., 1999; Betka and Callard, 1998), and showaffinity for xenoestrogens (Loomis and Thomas, 1999). Rasmussenet al. (2005) demonstrated that an anti-estrogen can abolish some ofthe OP-induced alterations in eelpout (Z. viviparus) testes, providingevidence that at least some of these effects may be mediated by theestrogen receptor. Acidophilic granulocytes, a type of phagocytic cell,are recruited to the testis in response to a physiological need underexperimental conditions. Therefore, it was not surprising to find largenumbers of these cells in testes of fish exposed to high OPconcentrations, since they would be associated with the involutiontaking place in this organ.
Whenever a static renewal system is employed during experimentsof exposure to chemical compounds, the difference between measuredandnominal concentrations inexposure chambers tends tobeespeciallynoticeable. In this study, the actual OP concentration decreased to lessthan 50% of the nominal concentration over a 24 h period and resultedonly a 20% towards the moment of water renewal. This tendency of OPdecline was determined frommeasurements done only in the 150 µg/Ltreatment; however comparable results are expectedwith the other twoconcentrations employed. In a similar experiment, Kinnberg et al.(2000) recorded a comparable drop of the actual concentration of NP toan average 15–23% of the nominal concentrations over a 3 day period.Likewise,Metcalfe et al. (2001) found that themeasured concentrationsof estradiol and ethinyl-estradiol in water over a 48 h period were, onaverage, 43% and 29% of the nominal concentrations, respectively, whileBalch and Metcalfe (2006) registered a decrease of NP level to a 29% ofnominal concentration in the same time period. The mechanisms forloss of estrogenic compounds in the aquariumwatermay include uptakeby thefish, adsorption to the glass,microbial degradation andphotolysis(Ekelund et al., 1993; Ahel et al., 1994; Lewis and Lech, 1996). Althoughthese studies reveal that actual concentrations of chemicals decreasenotoriously in the test chambers, exposure to the contaminants byregular pulses such as the ones used in our study results ecologicallyrelevant, since fish are not usually exposed to constant waterconcentrations of toxicants in the environment. Moreover, it indicatesthat the effects registered under these conditions are being produced byexposure to concentrations considerably lower than the nominal values.
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As two of the main breakdown products of alkylphenol poly-ethoxylates, both OP and NP are ubiquitous in the aquatic environ-ment. NP is the preponderant alkylphenol, constituting 80% of APsfound in surface waters and sediments (Ying et al., 2002). Incomparison, OP accounts for about 15% of the commercial alkylphenolinput (Bennett and Metcalfe, 1998). Although the estrogenic potencyof OP is higher than that of NP (White et al., 1994), it has been oftenoverlooked in ecotoxicological studies due to its lower usage. Theconcentrations of OP used in the present study span the range ofconcentrations recorded in North America and UK. In rivers and lakesof developed countries, concentrations of APs rarely exceed 20 µg/L(Blackburn and Waldock, 1995; Bennie et al., 1997), although in riversreceiving significant amounts of industrial and/or domestic effluents,point source contaminant levels may reach 600 to 1000 µg/L(Warhurst, 1995; Ying et al., 2002). In effluents from wastewatertreatment plants, concentrations of these compounds are generally inthe range of 10–300 µg/L (Bennie, 1999). APs are lipophilic (Ahel andGiger, 1993) and so tend to become associated with organic matter insediments, reaching higher concentrations than in surface waters(Ying et al., 2002). In South America, there is a scarcity of informationabout levels of APs in the aquatic environment. However, sinceindustrial activities are not well regulated in South Americandeveloping nations, environmental levels of these xenobiotics arelikely to be high. In nature, other factors must be taken into accountwhen assessing the estrogenic exposure of fish. These includeduration of exposure, bioaccumulation and the presence of otherchemicals. The adverse effects on the testes reported here occurred athigh concentrations of OP but the fish were exposed only during twomonths. Possibly, similar effects may be observed after long termexposure to lower concentrations. Because of their lipophilic proper-ties, APs tend to accumulate in aquatic organisms. Bioconcentrationfactors of up to 1300 have been reported for fish (Ekelund et al., 1990;Ahel et al., 1993). Other estrogenic chemicals usually present in theenvironment may have additive effects with APs. Then, it is possiblethat APs may have adverse effects on the reproductive system of malefish either by themselves or through their contribution to the pool ofenvironmental estrogens (Kinnberg et al., 2000).
In conclusion, the present study reveals an estrogen-like action ofOP in male C. dimerus. This is based on the induction of Vtg synthesis,an effect classically observed with 17β-estradiol. Moreover, theimpairment of testis morphology indicates that OP could be capableof decreasingmale fertility in this species. Such evidences suggest thatfurther assessment of OP as an endocrine disruptor in South Americanteleost fishes is required.
Acknowledgements
This study was performed with financial support from theUniversity of Buenos Aires, contract grant number EX 157, andCONICET, contract grant number PIP 5877, and from the NaturalSciences and Engineering Research Council of Canada.
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