Electronic Journal of Ichthyology July, 2005 1:10-20

METABOLIC CHANGES ASSOCIATED WITH 17ALPHA-ETHINYLESTRADIOL- EXPOSURE IN THE PREGNANT TELEOST VIVIPARUS (L)

Bodil Korsgaard Institute of Biology, University of Southern Denmark, Odense, Denmark e-mail: [email protected]

Abstract: Females of the Zoarces viviparus were exposed in the ambient seawater to the synthetic estrogen 17α-ethinylestradiol at different doses, 5, 10, 25, 50, 500 ng/L during early pregnancy and the effect on the maternal metabolism was studied. A significant dose- related increase in the level of calcium and the yolkprecursor-protein vitellogenin was ob- served in plasma of the exposed motherfish. The increased synthesis of vitellogenin could also be observed in the liver of the exposed motherfish by PCR and indirectly by the increase in the hepato-somatic index. No significant effects were observed in the concentration of glu- cose in plasma; the concentrations of amino acids in plasma, however, showed a significant decrease in the groups exposed to the higher doses of EE2. A general decrease in plasma os- molarity and chloride-concentrations were found in the exposed groups compared to controls. A dose-related decrease was observed in the activity of glutamate pyruvate-transaminase (GPT). The EE2- induced changes in the hepatic activity of the glycolytic enzyme pyruvate kinase (PK) and the gluconeogenetic enzyme PEPCK as well as of glucose-6-phosphate de- hydrogenase (G6PDH), an indicator for oxidative stress response, were less consistent. The activity of these 3 enzymes was observed to decrease significantly when expressed per unit liver wet weight but to increase in the high dose groups when activity was expressed normal- ized to the hepatosomatic index.

Key words: xenoestrogens, exposure, metabolism, eelpout, viviparous teleost

Introduction eventually lead to changes in growth, de- The synthetic estrogen 17α-ethinyl- velopment and reproductive performance. estradiol (EE2), used in contraceptive pills, Very few investigations have been is known to enter the aquatic environment made on metabolic processing in relation via seawage effluents, thereby posing a to xenoestrogenic exposure. Estrogen threat to aquatic wildlife. (Tyler et al., mimics may affect metabolism the same 1998). EE2 has been measured in the ef- way as the natural endogenous estradiol in fluents at concentrations at the low ng per relation to the increased demand of me- L range. EE2 has been shown to induce the tabolites associated with the intensive syn- synthesis of the yolk-precursor protein thesis of vitellogenin (Mommsen and vitellogenin in male rainbow trout at a Walsh, 1988), but may also exert dose- concentration as low as 0.1 ng/L (Purdom dependent stress-like effects on the general et al. 1994). The natural steroid 17ß- metabolic performance of the exposed estradiol has been observed at concentra- . Steroid hormones including estra- tions up to 80 ng/l in British rivers (Tyler diol have been shown to inhibit xenobiotic et al, 1998), a concentration at which vitel- biotransformation by downregulating the logenin synthesis may be induced in in induction of CYP450 (Winzer et al, their ambient environment and which may 2001b). Previous experiments have shown that estradiol-treatment also significantly

10 Korsgaard, B. 2005 Metabolic changes in Zoarces viviparus alters metabolic flux concurrent with the liver is the major synthesizing organ for hepatic induction of vitellogenin synthesis. vitellogenin it was relevant to study how During prolonged estradiol-treatment of the xenobiotic exposure affects metabo- rainbow trouts, carbohydrate metabolism lism and osmoregulation of the pregnant is generally affected by marked reductions female fish in relation to hepatic synthesis in liver glycogen with concomitant of vitellogenin or as a stress inducer. In changes of enzymes involved in the gly- addition to vitellogenin, enzymes indica- colytic and gluconeogenic metabolism of tive of glycolysis and gluconeogenesis the liver (Washburn et al.,1993; Korsgaard were measured to investigate effects of and Mommsen, 1993). Such changes in xenobiotic exposure on metabolic per- carbohydrate metabolism have also been formance. The enzyme glucose-6- observed during normal endogenous vitel- phosphate dehydrogenase was included in logenesis in turbot females (Scophtalmus the investigation, as this particular enzyme maximus) (Soengas et al, 1995) and during has been shown to be very sensitive to in- normal vitellogenesis and pregnancy in a activation by chronic exposure to polluted viviparous teleost, the eelpout Zoarces vi- marine habitats (Van Noorden et al, 1997). viparus (Korsgaard and Petersen, 1979). The enzyme is important as the major pro- Of particular concern are the effects vider of NADPH, required for detoxifica- which environmental estrogens may im- tion pathways (Winzer et al., 2002) and pose on embryonic development. In the may thus be regarded as a stress indicator. eelpout the maternal-fetal trophic relation- ship has been observed to be affected by dose-related concentrations of octylphenol Material and Methods or EE2 (Rasmussen et al., 2002; Korsgaard Zoarces viviparus (L) females were et al., 2002). Thus different compounds in caught during early pregnancy (October) ovarian fluid, the ambient medium of the by fyke nets at Dalby Bugt, Funen, Den- developing embryos, such as calcium, glu- mark and transferred to the Marine Biol- cose and amino acids, believed to be of ogy Research Center in Kerteminde to nutritional importance for normal growth large indoor tanks with aerated running of the embryos during their intraovarian seawater pumped from the Great Belt of development, were found to be affected Denmark. The fish were acclimated for by the EE2 exposure (Korsgaard et al. about one week before experiment and 2002). held at a 12L:12D cycle. The fish were Previous work in our laboratory has exposed under flow through conditions (20 shown that not only vitellogenesis but also ‰SW) in 50 L aquaria to 17α-ethinyl- other physiological processes may be af- estradiol (0, 5, 10, 25, 50, 500 ng/l) using fected by xenoestrogens. Treatment with estradiol-17ß (500 ng/l) as positive con- estradiol or 4-nonylphenol was shown to trol. The compounds were dissolved in significantly affect the osmoregulatory 100% isopropanol and applied directly to performance in the atlantic salmon (Salmo the experimental aquaria at a rate of 72 ml salar) during smoltification (Madsen et al,. per day. Each aquarium was fitted with a 1997). Cortisol is an important hormone in circulation pump to assure uniform mix- osmoregulation and may antagonize the ing. The continuous flow-through system negative effect of estradiol on the osmo- used in the present study was identical to regulatory response of the fish (Madsen the system used and validated in previous and Korsgaard, 1991). experiments (Korsgaard et al. 2002), in The present work was initiated to elu- which the actual water concentration of cidate how the pregnant female fish re- EE2 was kept constant throughout the ex- sponds to exposure to different doses of periment at approx. 80% of the nominal EE2 under flow-through conditions. As the concentrations. The experiment lasted 32

11 Korsgaard, B. 2005 Metabolic changes in Zoarces viviparus

days and the fish were not fed during the tion of reverse-transcribed Vtg mRNA by experimental period to avoid erratic feed- use of the primers described by Andreas- ing due to the xenobiotic exposure. By sen et al., (2002). ß-actin was amplified as sampling the fish were anaesthetized in the reference gene. Values are expressed 0.2‰ phenoxyethanol. Fish were weighed as means ± SEM. Statistics were per- and blood was collected from the caudal formed as one-way analysis of variance vein into heparinized eppendorf tubes. (ANOVA) followed by Tukey’s multiple Plasma was collected after centrifugation. comparison tests and significance level Fish were killed by decapitation and the was chosen at the 0.05 level. livers were carefully removed, weighed and frozen in liquid N2 and stored at – o 80 C until use. Results Vitellogenin in plasma was measured Dose-response effects of waterborne by ELISA as described by Korsgaard and Pedersen, (1998). 17α-ethinylestradiol on the maternal me- Plasma total calcium was measured by tabolism of the eelpout Zoarces viviparus atomic absorption spectrophotometry, were investigated during early pregnancy. plasma glucose by the glucose oxidase Estradiol-17ß was used as a positive con- method using a commercial kit (Boe- trol for estrogenic effects of the xenobiotic hringer Mannheim) and total amino acids compound. During the 32 day period of by the Ninhydrin method (Moore and exposure to EE2 the yolk-precursor protein Stein, 1948). Total osmotic concentration vitellogenin (Vtg) and total plasma cal- of plasma was measured on a Knauer cium increased in a dose dependent man- automatic osmometer using the freezing ner, as did the hepatosomatic index (Figure point depression method and the plasma 1). chloride concentration by a Radiometer Vtg mRNA was induced in the liver chloride automatic titrator. samples from EE2- and E2 (not shown) The hepatic enzymes, GPT (glutamat exposed pregnant female fish (Figure 2A). pyruvate transaminase, PEPCK (phos- Induction of Vtg mRNA was also ob- phoenolpyruvate carboxykinase) and PK served in the embryos during their in- (pyruvate kinase) were measured accord- traovarian development (Figure 2B). How- ing to methods described by Mommsen et ever, Vtg cDNA was only amplified in the al. (1980), using a 50 mM imidazole buffer embryos of the EE2-group at the high dose and following the appearance or disap- (500 ng/l). Glucose levels in plasma re- pearance of NADH at 340 nm. For de- mained fairly stable in the different temination of G-6-PDH (glucose-6- groups, whereas the level of plasma amino phosphate dehydrogenase) activity liver acids (NPS) decreased significantly in the was homogenized in 0.66 mM EDTA groups exposed to the higher concentra- buffer (pH 7.5) and assayed in a medium tions of EE2. (Figure 3). Interestingly the containing 50 mM triethanolaminbuffer, total osmotic concentration and the con- 30 mM NADP+ and 40 mM glucose 6- centration of chloride in plasma appeared phosphate. The total volume of the reac- to decrease significantly in the groups ex- tion mixture was 1 ml. The reaction was posed to the higher doses of EE2 (Figure started by substrate addition and the in- 4), thus indicating an effect of the estro- crease in absorbance monitored at 340nm. genic compound on the osmoregulatory PCR was carried out as described by performance of the pregnant fish. Cortisol Andreassen et al., (2002). Briefly, RNA levels in plasma (not shown) were not sig- was extracted from the liver by TRIzol and nificantly different in the exposed groups 1 µg of total RNA was reverse-transcribed when compared to controls. in 20µl reactions followed by amplifica-

12 Korsgaard, B. 2005 Metabolic changes in Zoarces viviparus

The activity of enzymes related to crease in activity of PK and PEPCK was amino acid and glucose metabolism was observed in the high dose group (500ng) measured and evaluated as activity per unit when expressed in total hepatic units. liver WW, or expressed normalized to the As the major provider of NADPH act- hepatosomatic index (HSI) of the maternal ing as a reductant in various detoxification liver (Figure. 5A-H). When evaluating ef- pathways, G6PDH is a biomarker enzyme fects of xenobiotics on hepatic metabolism for oxidative stress (Winzer et al. 2002). in general it should be taken into consid- The results show that the xenoestrogenic eration that a decrease in enzyme ac- tivity 80

70

60 mg Vtg/ml 50

40

Figure 1: The effect of a 32 day ex- 30 posure to 17α-ethinylestradiol on the concentration of vitellogenin 20 and calcium in plasma and the 10 hepatosomatic index in pregnant 0 female eelpout. 300 indicates significant difference٭ (P<0.05) from control values. N=6 250

in each group. 200

150 µg Ca/ml µg

100

50 0

3

2 HSI 1

0 Control 5ng EE2 10ng 25ng 50ng 500ng 500ngE2 expressed per unit liver wet weight EE2 EE2 EE2 EE2 may be compensated for by an induced compound EE2 reduced the activity of this overall increase in the total hepatic wet enzyme in a dose dependent way ex- weight. A significant dose-related decrease pressed per unit liver wet weight with sig- was observed in GTP activity by the two nificant decreases in activities in the different expressions. The activity of the groups exposed to the higher doses of the glycolytic enzyme pyruvate kinase (PK) EE2. and the gluconeogenic enzyme phosphoe- nolpyruvate carboxykinase (PEPCK) ex- pressed per unit liver weight decreased significantly. However a nonsignificant in-

13 Korsgaard, B. 2005 Metabolic changes in Zoarces viviparus

A

Vtg

Control EE2

B

Vtg

Control EE2 (500)

Figure 2: The effect of 32 days exposure to17α-ethinylestradiol on the VtgmRNA in pregnant females (A) and her embryos (B) measured by PCR. Vtg cDNA was only amplified in the embryos at the high dose of EE2.

45 40 35 30 25 20 15

mg Glu/100 ml Glu/100 mg 10 5 0

10 9 8 7 6 5 4 3 2 mg NPS/100 ml NPS/100 mg 1 0 control 5ng EE2 10ng EE2 25ng EE2 50ng EE2 500ng EE" 500ng E2

Figure 3: The effect of a 32 day exposure to 17α-ethinylestradiol on the concentra- tion of glucose and amino acids (NPS) in plasma. indicates significant difference (P<0.05) from control values. N=6 in each ٭ group.

14 Bodil Korsgaard, 2005 Metabolic changes in Zoarces viviparus

180

170

160

mmol Chlorid/L mmol 150

140

425

400

375 mosmol/L

350

325 Control 5ng 10ng 25ng 50ng 500ng 500ng EE2 EE2 EE2 EE2 EE2 E2

Figure 4: The osmotic concentration and the concentration of chloride in plasma of eelpout females after a 32 day exposure to 17α-ethinylestradiol. *indicates significant difference (P<0.05) from control values. N=6 in each group.

15 Bodil Korsgaard, 2005 Metabolic changes in Zoarces viviparus

GPT GPT 70 70 60 60 50 50 40 40

30 30 20 20 10 10 0 0

15 10 PEPCK PEPCK 8 10 -1 6 HSI

4 5 -1 2 min 0 0 -1 25 20 PK PK 20 15 15

10 µmol min

µmol g 10 5 5

0 0

40 G-6-PDH 40 G-6-PDH 35 35 30 30 25 25 20 20 15 15 10 10 5 5 0 0 control 5ng 10ng 25ng 50ng 500ng 500ng control 5ng 10ng 25ng 50ng 500ng 500ng EE2 EE2 EE2 EE2 EE2 E2 EE2 EE2 EE2 EE2 EE2 E2

Figure 5: Activities expressed in µmol g-1 min-1(A) or relative to the hepatosomatic in- dex, HSI (B) of enzymes in glycolysis, gluconeogenesis or oxidative stress response (G6PDH) in livers of pregnant female eelpout after 32 days of exposure to EE2. *indicates significant difference (P<0.05) from control values. N=6 in each group.

16 Bodil Korsgaard, 2005 Metabolic changes in Zoarces viviparus

Discussion nificant effect on plasma cortisol or glu- The results show that, overall, the ma- cose. The E2 treatment of tilapia, how- ternal metabolism is affected in a dose- ever, was shown to decrease the metabolic dependent manner by exposure to the syn- capacity of the liver, lowering the activity thetic estrogen 17α-ethinylestradiol. The of key enzymes in the liver. concentration of vitellogenin is the com- A general dose-related decrease was mon used end point biomarker for estro- observed in the present study in the activi- genic exposure and the marked dose- ties per unit liver WW of all four enzymes related response in the present indicated (GTP, PEPCK , PK and G6PDH) in the that the female fish also responds to the groups exposed to EE2 as well as to E2. estrogenic compound during pregnancy, These overall changes in the glycolytic when Vtg is no longer synthesized in the and gluconeogenic key enzymes in the ex- liver due to a marked decrease in estrogen posed groups indicate that major changes levels during pregnancy (Korsgaard occur in carbohydrate and protein metabo- 1994). The Vtg response was also verified lism. Such changes have been observed in by the PCR-analysis showing that Vtg previous experiments in which fish have mRNA was induced in all liver samples been treated with estradiol-17ß (Washburn from the exposed groups but not in the et al. (1993); Korsgaard & Mommsen controls. This effect of EE2 was also ob- (1993). Washburn et al (1993) observed served in the embryos in ovario, respond- that in male rainbow trout, implanted with ing to the high dose of EE2 by VtgmRNA estradiol-17ß, the process of gluconeo- induction. VtgmRNA induction is re- genesis by isolated hepatocytes was sig- garded a very sensitive indicator for estro- nificantly depressed resulting in lower genic exposure as observed in several fish glucose concentration in plasma. Similar species (Jobling et al. 1996; Lech et al. observations were made in immature 1996, Rasmussen et al. 2002). Exogenous rainbow trout after intraperitoneal injec- induction of vitellogenin synthesis in male tion of estradiol-17ß one or two weeks af- and immature females may cause serious ter the injection. After 6 weeks however, metabolic stress due to the drain on energy de novo glucose synthesis was signifi- reserves diverting amino acids, lipids, cantly higher in the estradiol-treated group glucose and calcium from their respective compared to controls when expressed target tissues. Increases in cortisol and normalized to the hepatosomatic index glucose concentrations in plasma in re- (Korsgaard and Mommsen, 1993). In the sponse to stress factors have been reported present experiments the fish were not fed by Mommsen et al. (1999). In the present, and as the experiment lasted 32 days the plasma glucose remained fairly stable in circulating glucose would be expected to the EE2-exposed groups compared with derive from glucose synthesis from non- the control and cortisol levels in plasma carbohydrate precursors rather than from of the exposed groups were not signifi- the process of glycogenolysis. Previous cantly different from that of controls. experiments have shown that estrogen The present results indicate that the treatment first mobilizes liver glycogen osmoregulatory performance of the ex- then leads to enhanced gluconeogenesis posed pregnant fish is affected by the es- (Korsgaard and Mommsen, 1993; Whiting trogenic compounds as expressed by a de- and Wiggs, 1978.). Accordingly, the liver crease in plasma osmolarity. Vijayan et al. depots are expected to be gradually ex- (2001) observed that estradiol treatment of hausted in relation to the steady increase in the euryhaline tilapia, Oreochromis mos- vitellogenin synthesis by the prolonged sambicus, prevented recovery of plasma exposure to xenobiotic compounds, osmolality in 50% seawater with no sig- thereby increasing the need for extra-

17 Bodil Korsgaard, 2005 Metabolic changes in Zoarces viviparus hepatic precursors such as amino acids. experiment a dose-related decrease was Amino acids enter the liver to be incorpo- observed in the activity of this enzyme in rated into vitellogenin or to be shunted into the groups exposed to the higher doses of the gluconeogenic pathways by the respec- EE2. A reduction in G6PDH activity in tive transaminases. The transaminase GTP the liver was also observed in bullhead decreased in the groups exposed to the (Cottus gobio) exposed to paper mill efflu- higher doses of EE2, but this decrease did ents (Bucher et al. 1993). Thus the ob- not seem to have an effect on the circulat- served decrease in hepatic activity of ing glucose, which was found to be at G6PDH in the present may reflect meta- similar (nonsignificant) levels in the vari- bolic imbalance after xenoestrogenic stress ous groups. The circulating amino acids in the pregnant fish and may indicate that (NPS), however, showed significant de- susceptibility for xenobiotic toxicity may creases in the groups exposed to the higher increase by long term exposure or by in- doses of EE2. This decrease in amino ac- creasing concentrations of xenoestrogens ids in plasma may well be due to the dose- in the environment. related increase in the synthesis of protein In conclusion the present experiments (vitellogenin), but may also reflect the en- show that long-term exposure to EE2 has a hanced shunting into the gluconeogenic marked dose-related effect on the overall process. However, the observed activity metabolic and osmoregulatory perform- of the gluconeogenic key enzyme PEPCK ance of the pregnant female eelpout with did not provide any clear indication for the PCR-results indicating that the xenoes- this possibility, as it shows a dose-related trogenic exposure may also affect the em- decrease in activity in the liver. This de- bryos during their development in ovario crease, however, could have been compen- sated for by an increase in liver mass, as observed in the high-dose group or by an Acknowledgements increased gluconeogenic activity of the The author wishes to thank laboratory muscular mass. Estradiol-treatment of fish technician, Jette Porsgaard, for excellent has been reported to induce increases in work in the Laboratory and the Danish liver wet weight by hypertrophic or hyper- Natural Science Research Council for fi- plastic growth (Korsgaard and Emmersen, nancial support of the study (SNF 21-02- 1976). Expressed as total metabolic poten- 0520) tial of the liver (multiplied by the hepato- somatic index) activities of GTP were shown to decrease significantly in the high References: dose groups, while PEPCK and PK activi- Andreassen, T.K., Skjoedt, K., Anglade, I., ties were observed at an increased level in Kah, O. & Korsgaard, B. (2003). Mo- the same group. lecular cloning, characterization and tis- A decrease in activity of the enzyme sue distribution of oestrogen receptor G6PHD of the flounder (Platichthys fle- alpha in Eelpout (Zoarces viviparus). sus) has been shown to be one of the short- Gen. Comp. Endocrinol. 132, 356-368 term responses of the liver to oxidative Bucher, F., Hofer, R., Krumschnabel, G., stressors (Winzer et al, 2002). The authors & Doblander, C. (1993). Disturbances suggest that the observed inhibition of in the prooxidant-antioxidant balances G6PDH may reflect early cellular imbal- in the liver of bullhead (Cottus gobio) ances due to xenobiotic stress in direct re- exposed to treated paper mill effluents. lation to the limited availability of Chemosphere 27, 1329-1338. NADPH. They also observed this inhibi- Jobling, S., Sheahan, D., Osborne, J.A. tion of G6PDH to occur in a more robust Matthiessen, P. & Sumpter, J. (1996). way in the female flounder. In the present Inhibition of testicular growth in rain-

18 Bodil Korsgaard, 2005 Metabolic changes in Zoarces viviparus

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cinogenesis in livers of flat- notransferase activity of brook trout fish. Biochem. Biophys. Acta 1342: Salvenius fontinalis. Mitchell. Comp. 141-148. Biochem. Physiol. 60: 463-465. Vijayan, MM., Takemura, A. & Momm- Winzer, K., Becker, W., Van Norden, sen, T.P. (2001). Estradiol impairs hy- C.J.F. & Kohler, A. (2001b). Sex- poosmoregulatory capacity in the eury- specific biotransformation and detoxifi- haline tilapia, Oreochromic mossambi- cation after xenobiotic exposure of pri- cus. Amer. J. Physiol. 281(4):1161- mary cultured hepatocytes of european 1168. flounder (Platichthys flesus). Aquatic Washburn, B.S., Krantz, J.S., Avery, E.H. Toxicol. 52: 143-155. & Freedland, R.A. (1993). Effects of es- Winzer, K., Van Norden, C.J.F. & Kohler, trogen on gluconeogenesis and related A. (2002). Glucose-6-phosphate dehy- parameters in male rainbow trout. Am. drogenase: the Key to sex-related xeno- J. Phys. 264:720-725. biotic toxicity in hepatocytes of euro- Whiting, S.J. & Wiggs, A.J. (1978.). Ef- pean flounder (Platichthys flesus). fects of sexual maturation and estradiol- Aquatic Toxicol. 56: 275-288. 17ß on liver glycogen and tyrosine ami-

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