Effects of 17α-ethynylestradiol on EROD activity, spiggin and vitellogenin in three-spined stickleback (Gasterosteus aculeatus)

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Aquatic Toxicology 83 (2007) 33–42

Effects of 17�-ethynylestradiol on EROD activity, spiggin and vitellogeninin three-spined stickleback (Gasterosteus aculeatus)

Carin Andersson a,∗, Ioanna Katsiadaki b, Katrin Lundstedt-Enkel a, Jan Orberg a

a Department of Environmental Toxicology, Uppsala University, Norbyvagen 18A, SE-75236 Uppsala, Swedenb Centre for Environment, Fisheries and Aquaculture Science (CEFAS), Weymouth, Dorset, United Kingdom

Received 1 February 2007; received in revised form 12 March 2007; accepted 15 March 2007

bstract

The three-spined stickleback (Gasterosteus aculeatus) has quantifiable biomarkers of exposure to estrogens (vitellogenin), androgens (spiggin)nd aryl hydrocarbon receptor (AhR) agonists (EROD activity) and is therefore a promising test species for biomonitoring of reprotoxic chemicalsn aquatic environments. In this study we evaluated the effects of 17�-ethynylestradiol (EE2) on EROD activity, induction of vitellogenin andpiggin, hepatosomatic index (HSI), ovarian somatic index (OSI) and nephrosomatic index (NSI). Adult male and female three-spined sticklebacksere exposed to concentrations of 0–170 ng EE2/l (measured concentrations) in a flow-through system for 21 days. Exposure to 170 ng EE2/l

esulted in a significant 8- and 9-fold induction of gill EROD activity in males and females, respectively. In livers, EROD activity expressed inelation to microsomal protein content was suppressed due to a significant increase in microsomal protein content. Hepatic EROD activity per sexpressed as picomol/min was not affected by exposure to EE2. The lowest observed effect concentration for induction of vitellogenin in males was3.7 ng EE2/l. In females, vitellogenin levels were significantly higher in those exposed to170 ng EE2/l compared to controls. Spiggin production

as significantly inhibited and NSI lower in males exposed to 170 ng EE2/l. In both females and males LSI was significantly higher in fish exposed

o 170 ng EE2/l than in controls. In females exposed to 170 ng EE2/l, OSI was significantly lower and NSI higher than controls. The observedesults from this study show that a synthetic estrogen can affect the well-known biomarker of exposure for dioxin-like compounds, EROD activity,nd further that this response can differ between tissues. These findings are important for interpretation of biomonitoring data.

2007 Elsevier B.V. All rights reserved.

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eywords: EROD activity; Spiggin; Vitellogenin; Three-spined stickleback; 17

. Introduction

Several chemicals present in the environment can act asndocrine disrupters, i.e. they can affect organisms by alter-ng functions of the endocrine system. There is an increasingoncern that exposure to such chemicals can cause reproduc-ive disturbances in wildlife. Fish inhabiting areas that receiveffluent from sewage treatment plants have shown e.g. reducedonadal growth, high incidence of intersex (Jobling et al., 1998),elayed sexual maturation (Jobling et al., 2002) and reducedestosterone levels (Folmar et al., 1996). One group of chemicalshat causes reproductive disorders exerts its effects by inter-

cting with the estrogen receptors (ER) (Ankley et al., 1998;illesby and Zacharewski, 1998). Other compounds interactith the androgen receptors (AR) (Ankley et al., 1998) or bind to

∗ Corresponding author. Tel.: +46 18 471 26 18; fax: +46 18 51 88 43.E-mail address: [email protected] (C. Andersson).

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166-445X/$ – see front matter © 2007 Elsevier B.V. All rights reserved.oi:10.1016/j.aquatox.2007.03.008

ynylestradiol

he aryl hydrocarbon receptor (AhR)(reviewed by Denison andagy, 2003). Activation of all these receptors leads to inductionf certain proteins that can be measured for e.g. biomonitoringurposes.

The three-spined stickleback (Gasterosteus aculeatus) is aromising model species for monitoring of various contami-ants since they have quantifiable endpoints for estrogens andndrogens (Katsiadaki et al., 2002a, 2002b, 2006) and havelso been shown to be sensitive to AhR agonists (Anderssont al., 2006). Furthermore, the stickleback is euryhaline and cane used in field studies in different aquatic environments andlso in laboratory studies. In addition, sex-linked DNA-markersave been identified in three-spined sticklebacks (Griffiths etl., 2000; Peichel et al., 2004). This makes it possible to iden-ify genetic sex which is significant advantage in studies of

eprotoxic chemicals.

Vitellogenin (vtg) is an estrogen-inducible yolk precur-or protein normally produced only in mature female fish.owever, this protein can also be induced in juveniles and

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ales by estrogen receptor agonists. Presence of vitellogeninn male and juvenile fish is a well-known biomarker forstrogenic compounds (Sumpter and Jobling, 1995; Joblingt al., 1998) and sensitive assays have been developed foreveral fish species, e.g. zebrafish (Holbech et al., 2001;ilsen et al., 2004), fathead minnow (Mylchreest et al.,003; Nilsen et al., 2004; Tatarazako et al., 2004; Eidemt al., 2006), and three-spined stickleback (Katsiadaki et al.,002b; Hahlbeck et al., 2004). During breeding, male three-pined sticklebacks produce a glue-like secretion, which issed for nest-building (Jakobsson et al., 1999). This secre-ion contains a glycoprotein named spiggin (Jakobsson et al.,999), which is normally produced in the kidneys in sexu-lly mature males after stimulation by androgens (Jakobssont al., 1999). However, females and juveniles can also pro-uce spiggin after exposure to androgens (Katsiadaki et al.,002a). Hence, induction of spiggin in females or juvenilesan be used for identifying the presence of androgenic com-ounds. A sensitive ELISA for quantifying spiggin content inidneys has been developed and validated (Katsiadaki et al.,002a).

Cytochrome P4501A (CYP1A) is induced by a range ofhemicals, e.g. polyaromatic hydrocarbons (PAHs) and halo-enated aromatic hydrocarbons (HAHs) including coplanarolychlorinated biphenyls (PCBs) (reviewed by Denison andagy, 2003). These compounds bind to the AhR and sub-

equently induce the synthesis of CYP1A. The inductionf CYP1A is commonly measured as 7-ethoxyresorufin-O-eethylase (EROD) activity, which is used as a biomarker ofxposure to AhR ligands in aquatic environments (Goksoyr andorlin, 1992; Whyte et al., 2000). EROD activity in fish is tra-itionally determined in hepatic microsomes when monitoringaterborne AhR-agonists. However, when measuring EROD

ctivity in the liver there is a possibility that readily metabo-ized compounds that undergo first-pass metabolism in the gillsill remain undetected. Therefore, a sensitive method for mea-

uring EROD activity in intact gill-filament tips was developedy Jonsson et al. (2002).

This study is a part of a project investigating the poten-ial of the three-spined stickleback as a model species foriomonitoring of ER-, AR- and AhR-agonists in aquatic envi-onments and the present paper is focused on effects of the ERgonist 17�-ethynylestradiol (EE2). This substance, commonlysed in contraceptive pills, is a very potent estrogen that haseen detected in waterways receiving sewage treatment efflu-nt (Desbrow et al., 1998; Ternes et al., 1999). In rivers inhe UK, concentrations up to 15 ng EE2/l have been detectedAherne and Briggs, 1989) and in effluent water from a sewagereatment plant in Sweden a concentration of 4.5 ng EE2/l wasound (Larsson et al., 1999). Occasionally, much higher con-entrations of EE2 have been measured in the environment,.g. 125 ng EE2/l in surface water in Italy (Pojana et al., 2004)nd 62 ng EE2/l in sewage effluent in Germany (Stumpf et al.,

996).

The specific aim of this study was to determine effects ofE2 on selected biomarkers: vitellogenin, spiggin, liver and gillROD activity. In addition, we wanted to study eventual cor-

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xicology 83 (2007) 33–42

elations between the selected biomarkers and other variables,uch as coloration, ovarian somatic index (OSI), hepatosomaticndex (HSI) and nephrosomatic index (NSI).

. Materials and methods

.1. Animals and chemicals

Adult non-breeding (∼1 year old) three-spined sticklebacksGasterosteus aculeatus) were caught in Oresund on the Swedishouth-west coast in March 2004 and brought to Uppsala Uni-ersity. Females and males were kept separate in flow-throughreshwater holding tanks and maintained under a simulatedhort-day photoperiod (8 h light/16 h dark) and low tempera-ure (∼8 ◦C) to keep them in a reproductively quiescent state.he fish were kept under these conditions until the experimentstarted in November 2004.

All chemicals were obtained from Sigma–Aldrich (St. Louis,O, USA) unless otherwise stated.

.2. Experimental design

Male and female adult sticklebacks (1.7 ± 0.3 and.0 ± 0.2 g, respectively) were transferred from holding tanks to2 l glass aquaria (10 fish/aquarium) where they were exposedo either acetone (solvent control) or nominal EE2 concentra-ions of 5, 50 and 200 ng EE2/l for 21 days in a flow-throughystem. All treatments using females were performed in dupli-ates. Treatments of control males and males exposed to 50 ngE2/l were performed in triplicates, and those of males exposed

o 5 or 200 ng EE2/l in duplicates. To stimulate reproductiveaturation the light–dark period was 12 h light/12 h dark and

he water temperature was 15–17 ◦C. The fish were fed frozenrtemia once a day and faeces and debris were removed from

he aquaria every second day. Water (alkalinity: ∼280 mg/l;ater hardness: ∼16◦dH; calcium: ∼90 mg/l; pH ∼ 8) was sup-lied continuously into the aquaria at a rate of ∼120 ml/minnd the test solutions were added from stock solutions at aate of ∼150 �l/min by a peristaltic pump. Flow rates werehecked every second day and stock solutions were renewedvery fourth day. Maximum acetone concentration in the aquariaas less than 0.01%. Samples of water (1 l) from each aquar-

um were collected and stored in −20 ◦C for analysis of EE2ontent.

.3. Sampling

After three weeks the fish were anaesthetised with benzocainnd killed by cranial dislocation. The fish were weighed, pres-nce of breeding colour was noted using an arbitrary scale (1–4;= no breeding colour and 4 = full breeding colour), and gillsnd gonads were excised. Any overt liver or gonad abnormalitiesere noted. The fish were frozen in liquid nitrogen and, while

till frozen, the liver, heart and kidneys were excised and placedn pre-weighed eppendorf tubes and refrozen in −80 ◦C untilnalysis. The ovaries, kidneys and liver were weighed for calcu-ation of organosomatic indices (OSI, NSI and HSI) according

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o the formula organosomatic index = 100 × (organ weight/bodyeight).

.4. Gill EROD assay

The method to determine EROD activity in rainbow troutills, as described in Jonsson et al. (2002) was modified anddapted for use on three-spined sticklebacks. To optimize theill-EROD-assay mature female and male three-spined stickle-acks were exposed for 24 h to 1 �M �-naphtoflavone (�NF) oro solvent (acetone) in 1 l glass beakers. Parameters that wereested were pre-incubation times (10, 20 or 30 min) and incuba-ion times (10, 20, 30, 40, 50, 60 min) as well as two differenteaction buffers consisting of 1 �M or 2 �M 7-ethoxyresorufin.he effect of different concentrations of the DT-diaphorase

nhibitor dicumarol (0, 5, 10, 20, 50 and 100 �M) and the influ-nce of anaesthesia (benzocain and phenoxyethanol) on ERODctivity were also studied. Also, differences in EROD activityetween whole gill arches and gill filaments were evaluated.ased on the results from these tests the following protocol wassed for the analysis of gill-EROD activity: Three gill archesere excised and placed in ice-cold Hepes-Cortland (HC) buffer

0.38 g of KCl, 7.74 g of NaCl, 0.23 g of MgSO4·7H2O, 0.23 gf CaCl2·2H2O, 0.41 g of NaH2PO4·H2O, 1.43 g of HEPES, andg of glucose per 1 l of dH2O; pH 7.7). The gill filaments wereut off from the cartilage part of the gill arches and about 20laments in duplicates from each fish were placed in wells of12-well tissue culture plate (Falcon, Becton Dickinson USA).he filaments were pre-incubated under continuous shaking for0 min (at 17 ◦C) in 0.5 ml of a reaction buffer consisting of 7-thoxyresorufin (1 �M) and dicumarol (20 �M) in HC buffer.he reaction buffer was then replaced with 0.7 ml fresh buffernd after incubation for 40 and 60 min, 0.2 ml aliquots wereransferred to a 96-well plate (Nunc A/S Denmark). Resorufintandards (0–250 nM) were prepared by dilution of a stock solu-ion in methanol (100 �M) in the reaction buffer and 0.2 mlf each concentration in duplicates was transferred to the 96-ell plate. The fluorescence was determined in a multi-welllate reader (VICTOR3, Wallac Oy, Turku, Finland) at 590 nmfter excitation at 544 nm. A standard curve with resorufin wasetermined and EROD activity was calculated and expressed asicomol resorufin formed per filament and minute.

.5. Liver EROD assay

EROD activity in the livers was determined by a modi-ed method of (Eggens and Galgani, 1992). In short, liversere placed in glass-jars containing ice-cold homogenisationuffer (0.15 M KCl and 1 mM EDTA in 0.1 M sodium phosphateuffer, pH 7.4) cut into pieces and rinsed in homogenisationuffer. The livers were homogenised (at 1200 rpm, Potter-lvehjem homogeniser) in 1.5 ml homogenisation buffer and

hen centrifuged for 15 min at 10 000 × g (4 ◦C) (Rotana 460R,

ettich Zentrifugen; Tuttlingen, Germany). The resulting super-atant was centrifuged for 1 h at 105 000 × g (4 ◦C) in anltracentrifuge (L8–70; Beckman Instruments, Fullerton, CA,SA). The microsome pellet was resuspended in 1 ml of

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xicology 83 (2007) 33–42 35

epes-Cortland buffer pH 8.0 (HC 8). Resorufin standards0–250 nM) were prepared by dilution of a stock solution inethanol (100 �M) in HC 8. Aliquots of 50 �l of the standards

nd the microsome suspensions were transferred to a 96-welllate. A reaction buffer (160 �l) consisting of 7-ethoxyresorufin12.7 �M) and NADPH (2 mM) in HC-buffer was added to allells. The fluorescence was determined by repeated measure-ents during 10 min in a VICTOR3 multi-well plate reader at

90 nm after excitation at 544 nm. The protein concentrationn the microsome suspension was determined by adding fluo-escamine in acetonnitrile (300 �g/ml) to protein standards ando microsome suspensions in a 96-well plate. Protein standardsere prepared by dissolving bovine serum albumine (BSA) inC 8. The fluorescence was determined in a multi-well plate

eader at 460 nm after excitation at 355 nm. EROD activity wasxpressed as picomol resorufin formed per mg microsomal pro-ein and minute.

.6. Vitellogenin

The blood trapped in the heart of the fish provides an alterna-ive to plasma and can speed up the time consuming proceduref obtaining plasma from a small teleost such as the stick-eback. Plasma can only be obtained in this way when thesh have been frozen quickly after anaesthesia and the organissected out while the tissues are still frozen. The heartsere weighed and 100 �l of assay buffer (72 mM Na2HPO4,8 mM NaH2PO4, 140 mM NaCl, 27 mM KCl, Tween, 0.1%SA, 0.15 mM sodium azide, pH 7.8) were added to the tubesefore centrifugation at 13000 rpm for 5 min. The samples weretored at −20 ◦C until analysis. The vitellogenin assay waserformed using a 50 �l aliquot of the supernatant and theLISA described in Katsiadaki et al. (2002b) and Hahlbeck et al.

2004).

.7. Spiggin

The kidneys were weighed and placed in screw cap tubesnd 200 �l of denaturing buffer (100 mM Tris–HCl, 10 mMDTA, 8 M urea, 2% SDS, 200 mM �-mercaptoethanol) weredded. The samples were digested at 80 ◦C for 0.5–1 h and storedt −20 ◦C until analysis. The spiggin assay was performed asescribed in (Katsiadaki et al., 2002a).

.8. EE2 analyses

Water samples from each aquarium were collected duringhe last week of the experiments and analysed with liquidhromatography and mass spectrometry (LC/MS/MS) at anccredited analytical laboratory, Analycen (Lidkoping, Swe-en). The detection limit was 0.5 ng EE2/l.

.9. Statistics

For all basic statistics the software GraphPad Prism 4.03GraphPad Software Inc., San Diego, CA, USA) was used. Allata were tested for normality using the Kolmogorov-Smirnov

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est, and one-way analysis of variance (ANOVA) with Dunnett’sultiple Comparison Post-Test was performed on measured

ariables. To get a general picture of the trends and groupingsn the material, the data from all fish individuals were subjectedo multivariate data analyses, to principal component analysisPCA) using the software SIMCA-P+ 11.0 (Umetrics AB, Umea,weden). A significance level of 0.05 was used and data wereentred and scaled (to variance 1) prior to modelling (Wold etl., 1987).

. Results

.1. Analyses of EE2 content in water samples

In general, the measured EE2-concentrations in the wateramples were somewhat lower (82–86%) than the nominal con-entrations. At the nominal concentrations of 0, 5, 50, and 200 ngE2/l the measured EE2-concentrations were 0–1.1, 3.4–6.2,1–61, and 170 ng EE2/l, respectively.

.2. Growth parameters, condition and survival

Mortality rates were low (<10%) in all groups (Tables 1nd 2). There were no significant differences in body weightetween any of the groups either before or after the exposure. The

SI was significantly higher in both males and females exposed

o 170 ng EE2/l (measured mean concentration based on foureasurements) compared to control males and females, respec-

ively (Tables 1 and 2). However, the increase of HSI was morer(

able 1ortality, hepatosomatic index (HSI), ovarian somatic index (OSI), nephrosomatic

tickleback (Gasterosteus aculeatus) after exposure to 17�-ethynylestradiol (EE2) fo

ominal EE2 concentration (ng/l) Control

ortality 0/20SI (%) 4.1 ± 0.9SI (%) 6.6 ± 3.1SI (%) 1.2 ± 0.3TG (vtg units/mg heart) 2.2 ± 1.4piggin (spg units/mg bw) 0.05 ± 0.03

umber of observations varied between 17 and 20 per group. Data is presented as me* Significantly different from control, p < 0.05.

** Significantly different from control, p < 0.01, (one-way ANOVA followed by Dun

able 2ortality, hepatosomatic index (HSI), ovarian somatic index (OSI), nephrosomatic ind

Gasterosteus aculeatus) after exposure to 17�-ethynylestradiol (EE2) for 21 days

ominal EE2 concentration (ng/l) Control

ortality 0/30SI (%) 3.2 ± 0.7SI (%) 2.9 ± 0.7TG (vtg units/mg heart) 0.051 ± 0.13piggin (spg units/mg bw) 106.9 ± 75.7

umber of observations varied between 17 and 20 in the group exposed to nominaontrol group and in the group exposed to 50 ng EE2/l. Data is presented as mean ± S** Significantly different from control, p < 0.01, (one-way ANOVA followed by Dun

xicology 83 (2007) 33–42

ronounced in males than in females. Livers were paler in fishxposed to the highest concentrations of EE2 than livers from thether groups. In females, NSI was higher in individuals exposedo 170 ng EE2/l than in females in the other groups (Table 1). Inales, however, the NSI was lowest in individuals treated with

70 ng EE2/l (Table 2). NSI was higher in males than in femalesn all exposed groups except for the ones exposed to 170 ngE2/l (Tables 1 and 2). A reduction of OSI was seen in femalesxposed to the highest EE2 concentration (Table 1). Almost 60%f the fish (both males and females) exposed to 170 ng EE2/lnd 10% of the females exposed to 41 ng EE2/l (measured meanoncentration based on two measurements) had accumulation ofuid in the abdominal cavity. EE2 exposure affected the breed-

ng coloration of males; in ∼30% of males exposed to 170 ngE2/l the red ventral colouration was replaced by a bright bluisholouration.

.3. Vitellogenin

Plasma levels of vtg were significantly higher compared toontrols in males exposed to 53.7 (measured mean concentrationased on two measurements) or 170 ng EE2/l and in femalesxposed to 170 ng EE2/l (Tables 1 and 2).

In both males and females, exposure to 170 ng EE2/lesulted in significantly lower spiggin levels than in controlsTables 1 and 2).

index (NSI), vitellogenin (VTG) and spiggin levels in female three-spinedr 21 days

5 50 200

1/20 0/20 2/204.5 ± 0.6 4.3 ± 0.8 6.0 ± 1.1**

8.0 ± 4.0 4.9 ± 5.2 3.4 ± 2.5*

1.1 ± 0.3 1.3 ± 0.2 1.9 ± 0.9**

3.5 ± 2.5 6.5 ± 5.5 23.0 ± 12**

0.04 ± 0.03 0.05 ± 0.02 0.03 ± 0.02**

an ± S.D., except for mortality.

nett’s Multiple Comparison Post-Test).

ex (NSI), vitellogenin (VTG) and spiggin levels in male three-spined stickleback

5 50 200

0/20 0/30 2/203.0 ± 0.5 2.8 ± 0.5 5.5 ± 1.6**

2.7 ± 0.8 3.1 ± 0.8 2.0 ± 0.9**

0.042 ± 0.085 4.4 ± 6.1** 16.9 ± 5.3**

124.3 ± 96.1 146.1 ± 131.2 28.2 ± 5.4**

l concentration of 5 ng EE2/l and 200 ng EE2/l and between 27 and 30 in the.D., except for mortality.nett’s Multiple Comparison Post-Test).

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C. Andersson et al. / Aquatic Toxicology 83 (2007) 33–42 37

Table 3EROD activity and hepatic microsomal protein concentration in female three-spined sticklebacks (Gasterosteus aculeatus) after exposure to 17�-ethynylestradiol(EE2) for 21 days

Nominal EE2 concentration (ng/l) Control 5 50 200

Gill EROD activity (pmol resorufin/filament/min) 0.0002 ± 0.0001 0.0001 ± 0.00007 0.0003 ± 0.0002 0.002 ± 0.002**

Hepatic EROD activity (pmol resorufin/mg protein/min) 1.68 ± 1.0 1.42 ± 0.8 1.48 ± 0.9 0.81 ± 0.7**

Hepatic EROD activity (pmol resorufin/min) 0.068 ± 0.04 0.060 ± 0.04 0.057 ± 0.04 0.050 ± 0.05Microsomal protein concentration (mg/ml) 0.81 ± 0.3 0.90 ± 0.4 0.77 ± 0.3 1.3 ± 0.5**

N meanDun

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umber of observations varied between 17 and 20 per group. Data presented as** Significantly different from control, p < 0.01 (one-way ANOVA followed by

.5. EROD activity

EROD activities in gill filaments were significantly ele-ated in fish treated with the highest concentration of EE2Tables 3 and 4) with a 9- and 8-fold induction in femalesnd males, respectively. In all other groups the EROD activ-ties in gills were very low; around 0.0001–0.0004 pmolesorufin/filament/min (Tables 3 and 4). Conversely, hepaticROD activity normalised to microsomal protein contentas significantly lower in fish exposed to the highest con-

entration of EE2 (Tables 3 and 4). Microsomal proteinontent in livers of fish from that group was signif-cantly higher compared to fish from the other groupsTables 3 and 4). If hepatic EROD activity is expressedithout reference to protein content (pmol resorufin/min), no

ignificant decrease can be seen in either males or femalesTables 3 and 4).

.6. Multivariate data analysis

The resulting PCA model (R2X = 0.66; Q2 = 0.50; two com-onents) showed a clear trend with increasing EE2 concentrationnd that groupings could be seen (Fig. 1a). Females and malesormed two separate groups and most of the males in the70 ng EE2/l group (M3) showed similar response patterns ashe females in the 170 ng EE2/l group (F3). In the loading plotFig. 1b) it can be seen that several variables were correlated. Fornstance, with increasing EE2 concentration the variables vtg,W (hepatic weight), HSI, BW (bodyweight), and gill EROD

ctivities were increased. In addition, EROD activities in gillsnd livers were negatively correlated, meaning that an individ-al with high gill EROD activity had low liver EROD activityFig. 1b).

hltw

able 4ffects on EROD activity and hepatic microsomal protein concentration in malethynylestradiol (EE2) for 21 days

ominal EE2 concentration (ng/l) Control

ill EROD activity (pmol resorufin/filament/min) 0.0004 ± 0.0004epatic EROD activity (pmol resorufin/mg protein/min) 1.82 ± 1.2epatic EROD activity (pmol resorufin/min) 0.018 ± 0.016icrosomal protein concentration (mg/ml) 0.21 ± 0.14

umber of observations varied between 16 and 18 in the groups exposed to nominaontrol group and in the group exposed to 50 ng EE2/l. Data presented as mean ± S.D** Significantly different from control, p < 0.01, (one-way ANOVA followed by Dun

± S.D.nett’s Multiple Comparison Post-Test).

. Discussion

.1. EROD activity

The present study showed different responses in EROD activ-ty in gills and in livers. Exposure to170 ng/l EE2 resulted in aignificant induction of EROD activity in gills; 9-fold in femalesnd 8-fold in males. EE2 is not known as an AhR agonist and doesot share the “classical” structure of AhR agonists. Equilenin,owever, which belongs to equine estrogen family has structuralimilarities to typical AhR agonists and has recently been foundo induce hepatic EROD activity in mice (Jinno et al., 2006).uring the last decade many new AhR agonists have been iden-

ified whose structures are very different from the most studiedgonists e.g. PAHs and HAHs (Denison and Nagy, 2003).

Furthermore, all CYP1A inducers are not AhR agonists andherefore other pathways of CYP1A induction have been sug-ested (Delescluse et al., 2000; Nebert et al., 2000), e.g., it maynvolve tyrosine kinase activation or the retinoic acid receptorRAR) signalling pathway.

There are only a few studies on effects of EE2 exposure onROD activity. In one study EE2 treatment of chick embryoepatocytes resulted in a slight increase of EROD activitySundstrom et al., 1988) and in another study exposure to water-orne EE2 (51.38 �g/l) caused a 2-fold induction of ERODctivity in gill filaments of African sharptooth catfish (Clariasariepinus) (Mdegela et al., 2006). However, the induction inill filaments was only observed on day 3 in the 6-day study.urthermore, co-administration of EE2 and the well-known

igh affinity AhR agonist benzo[a]pyrene (BaP) resulted in aower EROD activity in gill filaments compared to administra-ion of BaP alone. No effect of EE2 on hepatic EROD activityas detected in that study. A similar result was obtained in

three-spined sticklebacks (Gasterosteus aculeatus) after exposure to 17�-

5 50 200

0.0003 ± 0.0003 0.0004 ± 0.0006 0.003 ± 0.002**

2.2 ± 1.3 1.95 ± 1 0.5 ± 0.6**

0.020 ± 0.016 0.016 ± 0.013 0.022 ± 0.0040.21 ± 0.15 0.18 ± 0.15 1.15 ± 0.54**

l concentration of 5 ng EE2/l and 200 ng EE2/l and between 28 and 30 in the.nett’s Multiple Comparison Post-Test).

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38 C. Andersson et al. / Aquatic Toxicology 83 (2007) 33–42

Fig. 1. Principal component analysis (R2X = 0.66; Q2 = 0.50; two components) of three-spined stickleback (n = 182 individuals) (a) observation plot with males (M)and females (F) exposed to varying concentrations of 17�-ethynylestradiol (EE2) for 21 days, groups; F0 and M0 (control), F1 and M1 (5 ng/l), F2 and M2 (50 ng/l),F3 and M3 (200 ng/l) and (b) the corresponding loading plot including the variables: BC-blue (blue body colour), BC-red (red body colour), BW (body weight),EyeC (blue eye colour), G-EROD (gill EROD activity), HSI (hepatosomatic index), HW (liver weight), NSI (nephrosomatic index), NW (kidney weight), L-EROD(hepatic EROD activity), SPG (spiggin), and VTG (vitellogenin).

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study on mosquitofish (Gambusia holbrooki). Exposure toaterborne EE2 (100 ng/l) did not affect either the basal or theNF-induced hepatic EROD activity (Aubry et al., 2005). Innother study with the same route of exposure, however, hep-tic EROD activity was lower in flounder (Platichthys flesus)xposed to EE2 (20 ng/l or 66 ng/l) in combination with an AhRgonist (dibenz(a,h)anthracene) than in individuals exposed tohe AhR agonist alone (Kirby et al., 2007). A suppression ofepatic EROD activity was observed also in carp (Cyprinus car-io) injected with EE2 (Sole et al., 2000). Maybe, the hepaticROD response to EE2 exposure is more dependent upon the

ested fish species than upon the route of administration.Several studies have presented data showing that 17�-

stradiol (E2) affects hepatic CYP1A activity in fish (Navas andegner, 2001; Elskus, 2004; Vaccaro et al., 2005). Exposure to2 caused a suppression of EROD activity in livers in male seaass (Dicentrarchus labrax), male rainbow trout (Oncorhynchusykiss), female winter flounder (Pseudopleuronectes ameri-

anus), grey mullet (Liza aurata) and female scup (Stenotomushrysops) (Gray Snowberger et al., 1991; Navas and Segner,001; Vaccaro et al., 2005; Cionna et al., 2006). In one study,sing rainbow trout hepatocytes, only the basal EROD activityas decreased by E2 exposure (Navas and Segner, 2001) but inther studies both basal and induced activities were decreasedElskus, 2004; Vaccaro et al., 2005). In addition, treatment withhe xenoestrogen 4-nonylphenol caused a suppression of basalepatic EROD activity in flounder, grey mullet and Atlanticalmon (Salmo salar) and of induced hepatic EROD activityn flounder and Atlantic salmon (Arukwe et al., 1997; Cionna etl., 2006; Kirby et al., 2007).

The mechanism behind the suppression of EROD activity bystrogenic compounds is not fully known, but observations thatoth CYP1A protein levels (Elskus et al., 1992) and CYP1ARNA (Williams et al., 1998; Navas and Segner, 2001) are

educed after E2 exposure indicates that E2 somehow interferest the gene level (Navas and Segner, 2001). Exposure to E2 inombination with tamoxifen (Tmx), an ER antagonist, abolishedhe inhibitory effect of E2 on EROD activity in rainbow trout hep-tocytes (Navas and Segner, 2001), which suggests that the ER isnvolved. In the present study hepatic EROD activity expressedn relation to microsomal protein content (pmol resorufin/mg

icrosomal protein/min) was suppressed in both females andales exposed to the highest concentration of EE2. However, this

ecrease in EROD activity is caused by an increase in microso-al protein content, probably vitellogenin, and not by a decrease

n the EROD activity per se (pmol resorufin/min). In this case annzyme activity expressed in relation to DNA-content instead ofrotein content had probably facilitated a correct evaluation ofE2-induced effects on hepatic EROD activity. To what extent

ncreased hepatic microsomal protein content also could explainhe estrogen (E2 and EE2) induced suppression of hepatic ERODctivity in the fish species mentioned above is not known.

.2. Vitellogenin

The sensitivity of vitellogenin induction in response to estro-enic compounds has been shown to differ between fish species.

cens

xicology 83 (2007) 33–42 39

n this study, the lowest observed effect concentration for induc-ion of vitellogenin in males was 53.7 ng EE2/l. Treatmentf juvenile sticklebacks with nominal concentration of 50 ngE2/l resulted in increased levels of vitellogenin (Hahlbeckt al., 2004). Rainbow trout (Oncorhynchus mykiss) has beenound to respond to low concentrations of EE2, and an induc-ion of vitellogenin was detected at a nominal concentrationf 2 ng/l (Jobling et al., 1996). In adult male zebrafish vitel-ogenin concentrations in plasma were elevated at nominal EE2oncentrations as low as 1.6 ng/l (Fenske et al., 2001) but inther studies the effective concentrations have been found to beomewhat higher in both adults (Rose et al., 2002) and juvenilesOrn et al., 2003). In another common test species in ecotoxi-ological studies, the fathead minnow, exposure to 1–5 ng EE2/lesulted in elevated vitellogenin concentrations (Panter et al.,002; Pawlowski et al., 2004). In Japanese medaka induction ofitellogenin seems to be relatively insensitive to EE2, with low-st effective concentrations ranging from 100 ng EE2/l to 500 ngE2/l (Tilton et al., 2005; Orn et al., 2006). In another study withapanese medaka a slight but significant elevation was observedfter exposure to 10 ng EE2/l (Scholz et al., 2004). Based onvailable data from the literature and on the results of this studyhe lowest concentration for EE2-induced vitellogenin synthesisn three-spined stickleback is between 5 and 50 ng/l.

.3. Spiggin

As expected, the levels of spiggin in all groups of femalesere very low and close to the detection limit of the assay. The

piggin level in control females was about 2000 times lowerhan that in control males. In females exposed to 170 ng EE2/l thepiggin level was significantly lower than that in control females.he reason for this is unknown but the biological significance of

his finding is probably negligible. Levels of spiggin were sig-ificantly lower in male fish exposed to 170 ng EE2/l comparedo male fish from the other groups. Similar results were reportedy Katsiadaki et al. (2006): high concentrations of estradiol hadn inhibitory effect on spiggin production. Decreased spigginroduction could be due to the inhibitory effects of estrogens onndrogen production (Bartke et al., 1977; Loomis and Thomas,000; Labadie and Budzinski, 2006). In addition, exposure toigh levels of estrogens has been shown to cause kidney lesionsWester et al., 1985; Herman and Kincaid, 1988; Folmar et al.,001; Zaroogian et al., 2001; Weber et al., 2003). Excessiveyaline material, most likely vitellogenin, accumulated in theidney glomeruli is one of the proposed causes for this kid-ey failure (Zaroogian et al., 2001; Van den Belt et al., 2002;eber et al., 2003). One sign of a possible kidney failure is

xtensive accumulation of fluid in the abdominal cavity. Such anccumulation of fluid was observed in Japanese medaka treatedith E2 (Hamazaki et al., 1987) and in zebrafish and in three-

pined sticklebacks exposed to EE2 (Van den Belt et al., 2002);atsiadaki, unpublished data). This fluid has been identified as

onsisting of vitellogenin and a chorion glycoprotein (Hamazakit al., 1987; Van den Belt et al., 2002). In the present studyo histological examination of the kidneys was carried out, butince several fish in this study exposed to high concentrations of

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E2 had excessive fluid in the abdominal cavity, an impairmentf kidney function is most likely responsible for the decreasedpiggin production.

.4. OSI

Ovarian somatic index is used in many studies to assess thempact of reprotoxic chemicals on the reproductive system in e.g.ebrafish and fathead minnow (Van den Belt et al., 2001, 2002;awlowski et al., 2004). In the present study, the OSI of femalesxposed to 170 ng EE2/l was significantly lower than the OSIf females in the other groups. An inhibition of ovarian growthfter exposure to EE2 has also been shown in other species e.g.n fathead minnows exposed to 100 ng EE2/l (Pawlowski et al.,004) and in zebrafish exposed to 10–100 ng EE2/l (Van denelt et al., 2002; Versonnen and Janssen, 2004; Hoffmann et al.,006). As suggested by Van den Belt et al. (2002) a decreasedituitary release of gonadotrophins through negative feed backf EE2 is a probable explanation to the observed effect (Van denelt et al., 2002).

.5. HSI

An increase of liver weight normally occurs during vitel-ogenin synthesis in female fish (van Bohemen et al., 1981), butn increase of HSI can also be due to enhanced detoxificationrocesses after exposure to toxic compounds. Oguro (Oguro,956) observed an increase in liver weight and morphologi-al changes of liver cells in male three-spined stickleback afterreatment with estradiol. Exposure to E2 resulted in high con-entrations of vitellogenin in plasma and an elevated HSI inuvenile rainbow trout (Thorpe et al., 2000) and in the flounderlatichthys flesus both HSI and vitellogenin synthesis were sig-ificantly increased after exposure to 10 ng EE2/l (Allen et al.,999). In the present study, HSI was elevated after exposure to70 ng EE2/l in both females and males and this increase in HSIs most likely caused by the increased production of vitellogeniny the liver.

.6. NSI

Estrogens have previously been shown either to inhibit nor-al kidney growth or to induce a reduction of kidney weight

n reproductive mature male sticklebacks resulting in subnor-al spiggin production (Oguro, 1957; Katsiadaki et al., 2006).hese effects were confirmed in the present study. The reason

or the observed increase in NSI in females after EE2 treatments unclear and needs further studying.

. Conclusion

At present EE2 is not known as an AhR agonist but in thistudy EE2 exposure caused a significant induction of gill EROD

ctivity. The mechanism behind this is not known. The EE2xposure also caused a marked increase in hepatic microsomerotein content, which resulted in suppressed hepatic ERODctivity when related to protein content (pmol resorufin/mg

D

xicology 83 (2007) 33–42

icrosomal protein/min). The hepatic EROD activity per sepmol resorufin/min), however, was not affected by the expo-ure and the observed suppression is probably due to “proteinilution”. In this case an enzyme activity expressed in relation toNA content instead of protein content had probably facilitatedcorrect evaluation of EE2 induced effects on hepatic EROD

ctivity. At fairly high exposure levels EE2 also induced the syn-hesis of vitellogenin in both sexes and inhibited the synthesisf spiggin in males. It appears that the three-spined sticklebacks somewhat less sensitive to estrogens than other fish speciesften used for monitoring of estrogenic substances, but sinceublished data for exposure to EE2 concentrations in the rangef 5–50 ng/l is lacking this needs to be further evaluated.

cknowledgements

We like to thank Bertil Borg at Stockholm University forssistance in collecting the fish, Asa Andersson at Stockholmniversity for assistance in the laboratory and Gunnar Steinholtz

t Uppsala University for help with the experimental set-up. Thisork was supported by the Swedish Environmental Protectiongency via the research program ReproSafe.

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