Vitellogenin mRNA regulation and plasma clearance in male sheepshead minnows, (Cyprinodon variegatus) after cessation of exposure to 17β-estradiol and p-nonylphenol

Aquatic Toxicology 58 (2002) 99 – 112

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Vitellogenin mRNA regulation and plasma clearance in male sheepshead minnows, (Cyprinodon 6ariegatus) after cessation of exposure to 17b-estradiol and p-nonylphenol Michael J. Hemmer a,*, Christopher J. Bowman b, Becky L. Hemmer a, Stephanie D. Friedman a, Dragoslav Marcovich a, Kevin J. Kroll b, Nancy D. Denslow b a

US En6ironmental Protection Agency, Gulf Ecology Di6ision, 1 Sabine Island Dri6e, Gulf Breeze, FL 32561, USA b Department of Biochemistry and Molecular Biology, Uni6ersity of Florida, Gaines6ille, FL 32611, USA Received 16 May 2001; received in revised form 19 July 2001; accepted 3 August 2001

Abstract Research was conducted to determine the kinetics of hepatic vitellogenin (VTG) mRNA regulation and plasma VTG accumulation and clearance in male sheepshead minnows (Cyprinodon 6ariegatus) during and after cessation of exposure to either 17b-estradiol (E2) or para-nonylphenol (NP). Adult fish were continuously exposed to aqueous measured concentrations of 0.089 and 0.71 mg E2 per l, and 5.6 and 59.6 mg NP per l for 16 days using an intermittent flow-through dosing apparatus. Fish were sampled on days 8 and 16 of exposure followed by sampling at discrete intervals for up to 96 days post-exposure. At each interval five fish were randomly sampled from each concentration and hepatic VTG mRNA and serum VTG levels for individual fish determined by slot blot and direct enzyme-linked immunosorbent assay (ELISA), respectively. Exposure to E2 and NP resulted in a dose dependent increase in hepatic VTG mRNA and plasma VTG over the course of the 16-day exposure period. Mean plasma VTG levels at day 16 were \100 mg/ml for both high doses of E2 and NP, and \20 mg/ml for the low exposure treatments. Within 8 days post-exposure, hepatic VTG mRNA levels returned to baseline in both high and low E2 treatments but remained elevated 2–4 fold in the NP treatments. Due to a shortened sampling period, a clearance rate for plasma VTG in the 5.6 mg NP per l treatment could not determined. In the 0.089, 0.71 mg E2 per l, and 59.6 mg NP per l treatments, VTG levels began decreasing within 4 days after exposure cessation and exhibited an exponential rate of elimination from plasma. Clearance rates for 0.71 mg E2 per l and 59.6 mg NP per l were not significantly different (P= 0.47), however, both demonstrated significantly higher rates of clearance (P B0.02) than observed in the 0.089 mg E2 per l treatment. Our results indicate that hepatic VTG mRNA rapidly diminishes after cessation of estrogenic exposure in sheepshead minnows, but plasma VTG clearance is concentration and time dependent and may be detected at measurable levels for months after initial exposure to an estrogenic compound. © 2002 Elsevier Science B.V. All rights reserved. Keywords: Fish; Vitellogenin; mRNA; Nonylphenol; 17b-Estradiol; Endocrine disruption

* Corresponding author. Tel.: + 1-850-934-9243; fax: + 1-850-934-9201. E-mail address: [email protected] (M.J. Hemmer). 0166-445X/02/$ - see front matter © 2002 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 6 - 4 4 5 X ( 0 1 ) 0 0 2 3 8 - 7

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1. Introduction Concern over the possible relationship between environmental contaminants and alteration of endocrine functions in wildlife has prompted investigations to identify chemicals with endocrine moderating potential. Much of the current information available on the adverse effects of endocrine disrupting chemicals (EDC’s) is implied from a few isolated field observations (Fox, 1992; Guillette et al., 1994; Folmar et al., 1996, 2001; Harries et al., 1997; Allen et al., 1999) or extrapolated from chemicals that have been tested in mammalian tissue systems (Shelby et al., 1996) and in vitro cell proliferation, estrogen receptor binding and gene expression assays (Thomas and Smith, 1993; Jobling et al., 1995; Soto et al., 1995; Coldham et al., 1997; Petit et al., 1997; Andersen et al., 1999). Therefore, it is important to determine if the adverse effects observed in wildlife species at various locations are confined to isolated areas of contamination, or if they are representative of more widespread conditions which may impact population and community structure. This requires development of diagnostic procedures specifically targeted to investigate impairment of endocrine processes in whole organisms at multiple cellular and physiological levels which can be integrated into predictive exposure and effect models in support of risk assessment and management activities. Circulating levels of vitellogenin (VTG) measured in male fish have been used extensively as a marker of exposure to estrogenic chemicals (Purdom et al., 1994; Donohoe and Curtis, 1996; Arukwe et al., 1998; Christiansen et al., 1998; Panter et al., 1998; Kime et al., 1999; Folmar et al., 2000; Hemmer et al., 2001) and as an indicator of contaminated habitats (Harries et al., 1996; Allen et al., 1999). Vitellogenin is the egg yolk precursor protein synthesized in the liver of oviparous female fish in response to circulating levels of estrogen during the reproductive cycle (Mommsen and Walsh, 1988). Induction of VTG does not normally occur in male fish, but males are capable of VTG expression if exposed to an exogenous estrogenic substance. However, little is known concerning the stability of hepatic VTG

mRNA or persistence of VTG in the plasma after cessation of exposure to an estrogenic compound. This is especially important in relation to male fish, which possess no natural target tissue for the sequestration of VTG. A thorough understanding of the kinetic profile encompassing hepatic VTG mRNA regulation and plasma VTG protein accumulation and elimination is required for the effective field application of VTG endpoints as biomarkers of estrogenic exposure. In two previous studies, an in vivo aqueous exposure system and procedures for measuring hepatic VTG mRNA synthesis and serum VTG levels in male sheepshead minnows were described (Folmar et al., 2000; Hemmer et al., 2001). In those studies we established the relative potency of the native ligand, 17b-estradiol, two pharmaceutical estrogens (ethynyl estradiol and diethylstilbestrol), and three xenobiotic compounds (p-nonylphenol, methoxychlor and endosulfan) which have demonstrated varying degrees of estrogenic potency in assorted in vitro assays (Soto et al., 1995; Coldham et al., 1997). Using these validated procedures, this study examines the time-course of hepatic VTG mRNA regulation and VTG plasma accumulation and clearance in sheepshead minnows associated with the cessation of exposure to two known estrogenic substances; the native estrogen ligand, 17b-estradiol, and pnonylphenol, an alkylphenol ethoxylate. 2. Materials and methods

2.1. Chemicals and fish Test chemicals were obtained from the following sources: 17b-estradiol (98.0% purity, Sigma, St. Louis, MO); p-nonylphenol (96.4% purity, Schenectady International, Schenectady, NY). Chemical stock solutions at the appropriate concentrations were prepared using reagent grade triethylene glycol (TEG) (Spectrum, Gardena, CA) as the solvent. Radioimmunoassays were performed using estradiol antisera purchased from Endocrine Sciences (Calabassas, CA), tritiated estradiol from Amersham, International (Buckinghamshire, England) and Scintiverse BD from Fisher Scientific (Pittsburgh, PA).

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Adult male sheepshead minnows (Cyprinodon 6ariegatus) collected by bag seine from Big Sabine Point, Santa Rosa Sound, FL, were acclimated in 60 l tanks receiving a continuous flow of aerated, 209 2 parts per thousand (ppt), filtered seawater at 259 2 °C for a minimum of 2 weeks before being used in exposures. Fish were maintained under a 16-h light:8-h dark photoperiod and fed Tetramin® flakes to satiation at least twice daily. Sizes of experimental fish were as follows (mean9 standard deviation (S.D.)): TEG control (5.979 0.34 cm, 4.269 0.86 g), 17b-estradiol (5.9190.31 cm, 4.299 0.81 g), p-nonylphenol (5.889 0.34 cm, 4.2590.83 g).

2.2. Exposure conditions Concurrent flow-through exposures of sheepshead minnows were performed with 17bestradiol and p-nonylphenol at nominal exposure concentrations of 0.1 and 1.0 mg estradiol per l, and 10 and 100 mg p-nonylphenol per l. Future references to exposure concentrations refer to the average concentration measured in water samples from the aquaria. Lower exposure concentrations were based on the results of previous dose-response studies (Folmar et al., 2000; Hemmer et al., 2001) and selected to encompass the approximate threshold concentration for hepatic induction and plasma expression of VTG. Upper concentrations were chosen at one order of magnitude greater than threshold values to investigate possible dose-dependent downregulation of VTG mRNA and clearance of plasma VTG. Exposures were conducted in 135 l glass aquaria using two-six chamber dosing apparatus supplying 1 l of test solution per cycle to duplicates of each test concentration at a rate of 20 cycles per h. Chemical stock solutions were infused at 50 ml per cycle into the apparatus mixing chambers using six dual channel Hamilton Microlab 500B dispensers fitted with 100 ml Hamilton syringes (Hamilton Instruments, Reno, NV). Duplicate TEG treatments at 50 ml/l were used as negative (carrier) controls. For each chemical examined, 350 adult males were randomly divided among the treatments

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with each duplicate chemical exposure aquaria and TEG control aquaria receiving 35 fish. The fish were continuously exposed for a period of 16 days at which time chemical dosing was terminated. For the remainder of the experimental period the fish were kept under flow-through conditions using clean 209 2 ‰ seawater in a temperature controlled water bath (259 2 °C) with a constant photoperiod of 16-h light:8-h dark.

2.3. Sample collection The sampling intervals for this study were based on the results of a short duration preliminary experiment of 35 days (data not shown). Five fish were randomly sampled from the control, estradiol and high p-nonylphenol exposure concentrations on days 8 and 16 of exposure followed by sampling 2, 4, 8, 13, 17, 21, 26, 54 and 96 days post-exposure, or until all fish in that group were sacrificed. Since results of the preliminary experiment indicated an accelerated plasma VTG clearance rate in fish exposed at the lower p-nonylphenol concentration, a shorter sampling interval was initiated. Ten fish in the lower pnonylphenol concentration were sampled on days 8 and 16 of exposure followed by sampling 2, 4, 6, 8, 13 and 17 days post-exposure. Total length and wet weight (blotted) were recorded for each fish sampled and for mortalities occurring during the exposures. Blood was sampled from each fish by severing the tail at the caudal peduncle with a razor blade and collecting the blood in a heparinized capillary tube. The tubes were centrifuged for 3 min at 13 700× g, the plasma aspirated and transferred to a 1.5 ml Eppendorf microfuge tube, then quickly frozen at − 70 °C until analyzed for VTG. After decapitation, each fish was split ventrally, the liver removed and flash frozen in liquid nitrogen, then stored at −70 °C until assayed for levels of VTG mRNA. All fish were analyzed for plasma VTG levels. However, only fish samples from days 8 and 16 of exposure and days 2, 4, and 8 post-exposure were selected for analysis of hepatic VTG mRNA.

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2.4. Chemical analysis All chemical stock solutions were analyzed prior to the start of exposures. Water samples were collected from the exposure tanks 96 h after the start of chemical flow, 24 h after introduction of the fish and on days 7, 12 and 16 of the test to determine the exposure concentrations. After termination of chemical dosing on day 16, two subsequent water samples were collected on days 17 and 18 of the experiment. Half liter water samples were siphoned from mid-water column of each duplicate exposure aquaria and one TEG control aquaria. Water samples collected from the TEG aquaria were spiked with 25 ml of the chemical stock solutions diluted to equal the exposure concentrations and served as analytical controls.

2.4.1. 17i-estradiol Water samples were analyzed for estradiol as described by Hemmer et al. (2001). In brief, seawater samples were passed under vacuum through E-18 solid phase extraction tubes (Supelco, Bellefonte, PA) and eluted from the column with 5 ml methanol (MeOH). Sample volume was reduced under nitrogen and evaporated to dryness using a Centri-Vap (Savant Instruments, Farmingdale, NY), then stored at − 20 °C until analyzed by radioimmunoassay. Samples and standards were prepared in triplicate and analyzed in duplicate assays at the end of the experiment. Standard curves were prepared using radioinert estradiol in assay buffer (60 mM H3BO3, 1% bovine serum albumin (BSA), pH 8.0) at concentrations of 6.25, 12.5, 25, 50, 100 and 200 pg per tube. Samples were reconstituted using 1 ml of borate buffer (65 mM H3BO3/1% MeOH, pH 8.0). Assay tubes containing 200 ml estradiol antisera (1:22 000), 100 ml tritiated estradiol at 12 000 cpm per tube, 100 ml borate buffer, 100 ml assay buffer, and 100 ml of reconstituted sample or estradiol standard were vortexed and incubated for 24 h at 4 °C. Separation of bound and free estradiol was achieved by adding 500 ml of 5% charcoal/0.5% dextran followed immediately by centrifuging for 25 min at 1500× g. Five hundred ml of the supernatant was placed in 15 ml of Scintiverse BD and counted in a Packard 2500 TR liquid scintillation counter

(Packard Instruments, Meriden, CT). Estradiol concentrations in seawater samples were calculated from standard curves generated with each run using SAS statistical software (SAS, 1990) The mean percent recovery and standard deviation (S.D.) of three TEG control samples spiked at 0.1 and 1.0 mg estradiol per l was 919 12.8%.

2.4.2. p-Nonylphenol Seawater samples containing p-nonylphenol were acidified to pH  3 with 3 ml of 1.8 N H2SO4 and extracted with 25 ml of hexane on a magnetic stir plate for 45 min. Depending on the exposure concentration, 5 ml (100 mg/l concentration) or 15 ml (10 mg/l) of the hexane extract was concentrated to 1.0 ml under a gentle stream of nitrogen. Analyses were performed using a Hewlett– Packard model 5890 series II gas chromatograph equipped with a fused-silica non-polar capillary column and flame-ionization detector. Hewlett–Packard Chemstation software (Palo Alto, CA) was used to control gas chromatograph parameters and to collect and process the data. The mean percent recovery and S.D. of five TEG control samples spiked at 19.0 mg p-nonyphenol per l was 92 9 8.8%. 2.5. Analysis of hepatic VTG mRNA 2.5.1. Total RNA isolation and in 6itro synthesis of standards Analysis of VTG mRNA was accomplished using the methods of Bowman and Denslow (1999). Individual liver tissues were processed for RNA using RNeasy mini-spin columns (Qiagen, Valencia, CA) following manufacturer’s recommendations, then measured spectrophotometrically and electrophoretically to estimate the amount and quality of total RNA recovered from the isolation. Complementary VTG RNA (cRNA) standards were synthesized from the identical sheepshead minnow clone used to make the VTG cDNA probe. Transcription reactions were performed with the cloned VTG fragment as the template, T7 RNA Polymerase and Ambion’s Megascript kit (Austin, TX) according to the manufacturer’s directions. The VTG cRNA was analyzed for purity by electrophoresis and quantitated by spectrophotometry.

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2.5.2. cDNA probes The cDNA probes for VTG and b-actin were made from plasmids (pSHMVTGa2) and (pSHMbactA1), respectively (Bowman et al., 2000). The fragment of sheepshead minnow VTG or b-actin was cut using EcoRI from the plasmid vector pGEM-T Easy (Promega, Madison, WI) and purified by gel electrophoresis. The [a-32P]-labeled cDNA probes were synthesized using Ambion’s StripEZ kit and purified using TE-Midi Select-D, G50 columns (5 Prime-3 Prime, Boulder, CO). 2.5.3. Slot blot hybridization The synthesized cRNA standards and 12 mg of sample RNA were denatured (50% formamide, 7% formaldehyde, 0.15 M NaCl and 0.015 M Na3C6H5O7, pH 7.0) for 15 min at 68 °C and slot blotted onto Biodyne B nylon membranes (Life Technologies, Rockville, MD). Sheepshead minnow VTG cRNA standards ranging from 0.01 to 100 ng were applied to all blots. The RNA was UV-crosslinked using a Stratalinker 1800 (Stratagene, La Jolla, CA), then stained with methylene blue to ensure even loading of the nucleic acid (Bowman and Denslow, 1999). The membranes were pre-hybridized in ExpressHyb buffer using a Techne Hybridiser oven (Clontech, Palo Alto, CA) for 30 min at 68 °C followed by incubation at 68 °C for 1 h in fresh hybridization buffer containing 3× 105 dpm VTG probe per ml. The nylon membranes were washed twice with 2× standard saline citrate (SSC), 0.1% SDS for 20 min at 25 °C, then twice with 0.1× SSC, 0.1% SDS for 30 min at 50 °C. After washing, the nylon membranes were wrapped in plastic wrap and exposed to BioMax MR X-ray film (Eastman Kodak, Rochester, NY) and the results quantified on a PhosphorImager (Molecular Dynamics, Sunnvale, CA). The VTG cDNA data were used to generate a standard curve for each blot to determine the amount of VTG mRNA in ng per mg total mRNA per sample. The nylon membranes were then stripped using Ambion’s Strip EZ kit and re-probed using 3× 105 dpm b-actin probe per ml hybridization buffer under the same conditions described above. The VTG mRNA levels for each individual sample were normalized using the derived b-actin levels. An autoradiogram of a

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typical slot blot membrane used to quantitate VTG mRNA is presented in Fig. 1.

2.6. Analysis of plasma 6itellogenin Sheepshead minnow VTG was purified from serum of estradiol-injected males by anion exchange chromatography using the methods described by Kroll and Doroshov (1991). Purified VTG was adjusted to pH 7.0 and stabilized with aprotinin (10 KIU/ml) and sodium azide (0.02%), mixed with an equal volume of glycerol and stored at − 80 °C. Samples were thawed, used once as standards in an assay, then discarded. Plasma levels of VTG were quantitated by direct ELISA following the methods of Denslow et al. (1999). In brief, samples, blanks and standards (0, 0.01, 0.1, 0.2, 0.4, 0.6, 0.8, 1.0 mg/ml) were diluted with phosphate buffered saline (PBSZ, 10 mM NaH2PO4, 150 mM NaCl, 0.02% NaN3, pH 7.2 containing 10 KIU/ml aprotinin), loaded into a 96 well microtiter plate (50 ml per well) and incubated overnight at 4 °C. Samples were diluted in the range of 1:200 to 1:1 000 000 in PBSZ with

Fig. 1. Autoradiogram of a typical slot blot membrane used in determination of sheepshead minnow VTG mRNA level from the 17b-estradiol treatments. Column A contains in vitro synthesized vitellogenin cRNA fragments ranging from 0.01 to 100 ng used to generate the standard curve. Columns B – F contain replicate samples for each time point: column B = day 8, column C = day16, column D =day 18, column E = day 20, column F =day 24. Rows 1 – 3 contain replicate samples from the 0.089 mg/l treatments, rows 4 – 6 contain replicate samples from the 0.71 mg/l treatments. Row 7, columns B – D contain the TEG control samples.

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Table 1 Nominal and average measured concentrations ( 9 S.D.) of 17b-estradiol and p-nonylphenol, from five water samples taken during the period of flow-through exposure ending on day 16 of the experiment 17b-Estradiol

p-Nonylphenol

Nominal concentration (mg/l)

Measured water concentration (mg/l)

Nominal concentration (mg/l)

Measured water concentration (mg/l)

0.1 1.0

0.089 (0.040) 0.71 (0.11)

10 100

5.60 (1.14) 59.64 (13.00)

aprotinin for the values to fall into the range of the standard curve. The plates were washed four times with tris buffered saline with Tween (TBST, 10 mM Tris, 150 mM NaCl, 0.05% Tween) and blocked with 1.0% BSA in TBST for 2 h at room temperature. The plates were rewashed with PBST (PBSZ, plus 0.05% Tween), then incubated overnight at 4 °C with 0.1 mg/ml of primary antibody HL 1330 (5C9-4A8) raised against sheepshead minnow VTG. Colorimetric determination of VTG concentration was accomplished using a Pierce Immunopure ABC mouse IgG kit (Rockford, IL) with alkaline phosphatase following the manufacturer’s instructions. The optical density of the standards and samples were read at 405 nm, a standard curve generated, and sample VTG concentrations determined using a Molecular Devices Model 190 microplate reader and accompanying Softpro software. All samples and standards were run in triplicate. The coefficients of variation and correlation coefficients for this assay were 5 10% and ]0.95, respectively.

the Bonferroni procedure. Pairwise comparisons of the slopes were performed at an individual significance level of h= 0.0016 with an overall Bonferroni family significance level of h= 0.05 (Neter et al. 1990). All statistical analysis were conducted using the SAS General Linear Models procedure (SAS, 1990).

3. Results

3.1. TEG sol6ent controls Male fish in the TEG control exposures demonstrated a mortality rate of 16% of which 6% was attributable to fish jumping from the exposure aquaria. The TEG control fish demonstrated a mean basal hepatic level of 11.56 pg VTG RNA per mg total RNA (S.D.= 2.12). No VTG was detected in the plasma of any control fish during the course of the experiment.

3.2. 17i-Estradiol

2.7. Statistical analyses For each chemical, mean concentrations of hepatic VTG mRNA and plasma VTG were graphed to compare induction, accumulation and elimination over time at each of the exposure concentrations tested. Using a single compartment PK model, a first-order elimination rate constant and half life of plasma VTG was determined for fish from each treatment (Gibaldi, 1991). Analyses for differential rates of plasma VTG clearance among exposure concentrations were conducted by comparing the log transformed slopes of the VTG elimination curves using regression analysis and

Measured exposure concentrations of estradiol in the test aquaria were an average of 89 and 71% of the expected nominal 0.1 and 1.0 mg/l concentrations, respectively (Table 1). Mortality rates associated with the estradiol treatments were 5 11% over the exposure and depuration portions of the study. Fish exposed to estradiol demonstrated rapid dose-dependent synthesis of hepatic VTG mRNA (Fig. 2A). Through the 16-day exposure period, VTG mRNA induction increased 140 and 490fold in the 0.089 and 0.71 mg/l treatments, respectively. After cessation of exposure, hepatic VTG

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mRNA levels decreased rapidly in both treatments exhibiting a mean reduction in expression of ]98% within 4 days, and returning to basal levels within 8 days post-exposure (Fig. 2A). VTG mRNA half-life (t1/2) values of 1.06 days (26.5 h) and 0.91 days (21.9 h) were calculated for the 0.089 and 0.71 mg/l treatments, respectively. Concurrent with the elevation of hepatic VTG mRNA, dose-dependent increases in plasma VTG were observed throughout the exposure period (Fig. 3A). Plasma VTG accumulated at an estimated rate of 3.8 and 8.1 mg/ml per day and peaked approximately 2 and 4 days after cessation of exposure in the 0.089 and 0.71 mg/l estradiol treatments, respectively. Vitellogenin followed a dose-related exponential rate of elimination from

Fig. 2. Dose response and time course regulation of hepatic VTG mRNA levels (mean 9S.D.; n =3) in male sheepshead minnows during and following a 16 day exposure to (A) 0.089 and 0.71 mg/l 17b-estradiol; (B) 5.6 and 59.6 mg/l p-nonylphenol.

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the plasma at both the 0.089 and 0.71 mg/l treatments with calculated t1/2 values of 14.1 and 13.8 days, respectively (Fig. 4A and B). Comparisons of the regression slopes indicated that plasma VTG clearance rates were not significantly different for fish exposed at the two estradiol treatment levels (P= 0.02).

3.3. p-Nonylphenol Measured exposure concentrations of pnonylphenol were stable through the exposure period at approximately 56–60% of the expected nominal concentrations (Table 1). Mortalities in the p-nonylphenol treatments were 5 10% through the course of the experiment. A differential induction pattern of hepatic VTG mRNA was clearly discernable between the two exposure concentrations as illustrated in Fig. 2B. During the initial 16 days of exposure, VTG mRNA synthesis increased an average of 47-fold in fish exposed to 5.6 mg/l while fish in the 59.6 mg/l treatment demonstrated a 700 fold message induction. After cessation of chemical dosing, hepatic VTG mRNA levels showed a concentration-dependent decline with message levels decreasing 94–99% from peak induction within 8 days post-exposure. The calculated half-life value of 1.95 days (46.8 h) for the 5.6 mg/l treatment was 2-fold higher than the 1.12 days (26.8 h) determined for the 59.6 mg/l treatment. Messenger RNA levels failed to reach basal levels of expression within the given sample period (i.e. 8 days post-exposure) remaining 2 and 3.7 fold over baseline in fish from the 5.6 and 59.6 mg/l treatments, respectively (Fig. 2B). Para-nonylphenol precipitated a dose dependent increase in plasma levels of VTG over the 16-day exposure period of the experiment (Fig. 3B). Plasma VTG accumulated at an estimated rate of 2.5 and 8.0 mg/ml per day and peaked approximately 2 and 4 days after cessation of chemical exposure in the 5.6 and 59.6 mg/l treatments, respectively. During the depuration period, fish from the 5.6 mg/l treatment demonstrated a large degree of variability in the measured amount of plasma VTG with coefficients of variation between sample periods ranging from 45 to 96%.

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Fig. 3. Dose response and time course of plasma VTG accumulation and elimination (mean 9 S.D.; n = 5) in male sheepshead minnows during and following a 16 day exposure to (A) 0.089 and 0.71 mg/l 17b-estradiol; (B) 5.6 and 59.6 mg/l p-nonylphenol.

Due to the shorter sampling interval applied to the 5.6 mg/l treatment, no clearly discernable pattern of plasma VTG clearance was evident and therefore an elimination profile could not be determined (Fig. 5A). In contrast, plasma VTG followed a dose-dependent exponential rate of elimination at the 59.6 mg/l exposure level with a calculated t1/2 value of 13.3 days (Fig. 5B). The plasma clearance rate for VTG at this exposure level was not statistically different from the rate

determined for the upper 0.71 mg/l estradiol exposure (P= 0.49), but was significantly higher than the clearance rate observed in the lower 0.089 mg/l estradiol treatment (P = 0.0003).

4. Discussion The lower exposure concentrations of E2 and NP were chosen based on previously determined

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threshold concentrations for hepatic VTG mRNA induction in sheepshead minnows (Folmar et al., 2000; Hemmer et al., 2001). Selection of the upper exposure concentrations at one order of magnitude higher than threshold encompass the majority of the reported measured environmental concentrations for these com-

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pounds (Tyler et al., 1998; Blackburn and Waldock, 1995). Additionally, using a large span between the upper and lower exposure concentrations allowed examination of possible concentration-dependent differences in VTG mRNA reduction and plasma VTG clearance after cessation of exposure.

Fig. 4. Time course of plasma VTG elimination and exponential clearance rate function (black line) for male sheepshead minnows following cessation of exposure to (A) 0.089 mg estradiol per l; (B) 0.71 mg estradiol per l.

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Fig. 5. Time course of plasma VTG elimination and exponential clearance rate function (black line) for male sheepshead minnows following cessation of exposure to (A) 5.6 mg p-nonylphenol per l; (B) 59.5 mg p-nonylphenol per l.

In the present study, untreated control fish displayed a mean basal VTG mRNA level of 12 pg/mg total RNA which is consistent with previous studies with sheepshead minnows (Folmar et al., 2000; Bowman et al., 2000; Hemmer et al., 2001), and with reported levels of approximately 50 pg VTG mRNA per mg total RNA measured in untreated male tilapia, Oreochromis aureus

(Lim et al., 1991). Measurable levels of plasma VTG have also been observed in a small percentage of untreated male fathead minnow, (Pimephales promelas) controls although VTG message induction was not discernable (Korte et al., 2000). The authors attributed inability to measure induction in these fish to the small amount of total RNA used in their assay. The positive results

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stated above contrast reported findings from in vitro studies with rainbow trout, Oncorhynchus mykiss, hepatocytes (Flouriot et al., 1996; Islinger et al., 1999), and in vivo exposures with male rainbow trout (Lech et al. 1996), and flounder, Platichthys flesus (Jansen et al., 1997) in which VTG transcripts were not detected in untreated controls. The conflicting results from these studies suggest that male fish of only certain species have the capacity to constitutively express low levels of VTG mRNA, possibly due to interspecific differences in the plasma concentration of 17b-estradiol or other endogenous estrogens. However, these inconsistencies may be an artifact of assay variability caused by such factors as differences in the total RNA sample volume used, probe specificity, blotting technique employed, or other analytical conditions which result in decreased assay sensitivity. A definitive answer to the question of constitutive VTG message expression in males must await further studies using a greater number of fish species. Increasing hepatic VTG mRNA levels in E2and NP-treated sheepshead minnows clearly followed a dose-dependent induction pattern through the 16-day exposure period (Fig. 2A and B). Similar dose-dependent induction patterns have been observed in primary cultures of male rainbow trout hepatocytes treated with E2 (Vaillant et al., 1988; Flouriot et al., 1996), and E2 or NP (Islinger et al., 1999). In vivo studies with E2 and NP using injected or aqueous routes of exposure have also described similar concentration-dependent VTG transcriptional activity in a variety of teleost models including rainbow trout (Le Guellec et al., 1988; Lech et al., 1996), fathead minnow (Korte et al., 2000), Atlantic salmon, Salmo salar (Yadetie et al., 1999), and sheepshead minnow (Folmar et al., 2000; Hemmer et al., 2001). After termination of E2 and NP exposure on day 16, sheepshead minnows displayed a rapid reduction in hepatic VTG mRNA levels in all treatments. Comparison of VTG mRNA half-life values for both treatment levels of E2 and the upper 56 mg/l NP treatment indicate similar halflives ranging from 0.98 to 1.1 days suggesting VTG transcript stability may not be a concentra-

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tion-dependent function. Interestingly, a mRNA half-life of 24 h was determined for primary cultured rainbow trout hepatocytes treated with 10 − 6 M E2 for 48 h which agrees closely with our findings (Flouriot et al., 1996). Sheepshead minnows treated with E2 demonstrated a 94% decrease in VTG mRNA within 4 days post-exposure and returned to basal levels within 8 days (Fig. 2A). A similar pattern of VTG mRNA elimination was observed in adult fathead minnows receiving single injections of either 0.5 or 5.0 mg E2 per kg where VTG mRNA reached undetectable levels within 12 days after treatment (Korte et al., 2000). Since E2 is known to be directly responsible for induction of VTG, the sharp decrease in hepatic VTG mRNA observed after termination of aqueous E2 exposure can be reasonably attributed to the rapid systemic reduction in available E2 by normal catabolic processes (Pankhurst et al., 1986; Kime, 1987). In our study with sheepshead minnows, plasma E2 levels were not measured. However, Pankhurst et al. (1986) reported plasma E2 t1/2 values of 1.27 and 1.37 days for female goldfish administered saline injections of E2 at 2 and 20 mg/g body weight, respectively. Although differing exposure procedures were used, a strong correlation was apparent between their calculated plasma E2 t1/2 values and the VTG mRNA half-lives of 1.06 and 0.91 days we derived for sheepshead exposed to 0.89 and 0.71 mg E2 per l, respectively. Additional information is available concerning VTG mRNA up-regulation and stability in fish after single and/or multiple injections of E2 or NP, however, comparisons between continuous aqueous exposures and other routes of exposure are difficult to compare without knowledge of the measured tissue concentrations of these compounds over the timecourse of exposure and depuration. In the case of the lower 5.6 mg NP per l treatment, VTG transcript half-life was determined to be approximately 2-fold longer (1.95 days) than observed for the higher NP treatment and both E2 treatments. The reason for this departure is unclear but may reflect the continued availability of NP through tissue depuration which is masked in the 56 mg/l NP treatment and both high and low E2 treatments by the overall

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high amplitudes of induction observed. Further, tissue bioaccumulation and subsequent depuration may also be a factor in the failure of VTG mRNA levels to reach baseline within 8 days post-exposure in both NP treatments. NP has been shown to evenly distribute in liver, kidney, gills, skin, abdominal fat and brain of Atlantic salmon exposed to tritiated NP under static waterborne conditions for 72 h (Arukwe et al., 2000). NP bioconcentration factors (BCF’s) of 24 and 98 in carcass and viscera, respectively, have been reported for rainbow trout with depuration half-lives in muscle and fat of 18.5 and 20 h, respectively (Lewis and Lech, 1996). A rapid dose-dependent accumulation of plasma VTG was observed in male sheepshead exposed to either E2 or NP (Fig. 3). Plasma accumulation of VTG has been well documented in a variety of fish species treated by injection or waterborne routes with E2 (Emmersen et al., 1979; Le Guellec et al., 1988; Panter et al., 1998; Korte et al., 2000; Folmar et al., 2000) and NP (Christiansen et al., 1998; Giesy et al., 2000; Hemmer et al., 2001). Although increases in plasma VTG levels paralleled the hepatic VTG mRNA induction patterns for both upper and lower treatment levels of E2 and NP, an approximate 2– 4 day lag was apparent between the cessation of exposure on day 16 and peak plasma levels of VTG protein measured on sample days 18–20 (Figs. 3 and 4). The continued increase in VTG expression after the end of the exposure period suggest accumulated E2 and NP residues remaining in the liver, plasma and possibly other tissues continue to stimulate transcriptional activity for a short time beyond the period of external exposure. Only limited information is available on the clearance kinetics of plasma VTG in male fish induced through estrogenic exposure. Allen et al. (1999) reported a calculated VTG half-life in plasma of 13.5 days for flounder exposed for 3 weeks to 10 ng ethynylestradiol per l (EE2) under flow-through conditions. Their results are consistent with the calculated half lives of 14.1, 13.8 and 13.3 days for sheepshead minnows exposed to 0.089, 0.71 mg E2 per l, and 59.6 mg NP per l, respectively. In contrast, Schultz et al. (2001) determined VTG half-lives ranging from 42 to 145

h for rainbow trout given single intra-arterial bolus injections of EE2 ranging from 0.001 to 10 mg/kg body weight. The disparity in VTG half-lives between trout and other fish may reflect species differences in VTG metabolism. In conclusion, although the rates of VTG clearance appear to be dose related, in male fish the time-course of its elimination is dependent on the total accumulated amount of VTG in the plasma. Therefore, exposure to high levels of an estrogenic chemical, or to lower levels for an extended period of time can result in large accumulations of VTG in the blood stream which can take months to reach undetectable levels. This makes the measure of plasma VTG alone as a biomarker of estrogenic exposure problematic because the temporal aspect of exposure is unknown. However, because of the rapid disappearance of VTG transcripts after removal of an estrogenic stimulus, measurement for the presence or absence of hepatic VTG mRNA serves as an excellent temporal indicator of exposure. Using plasma VTG expression in male fish as a primary screen followed by examination of VTG mRNA levels in those fish demonstrating positive plasma values can indicate the relative current exposure status for an individual. Therefore, combining both VTG induction and expression profiles should provide a rapid and cost effective technique capable of differentiating between past exposure and that of continuing estrogenic exposure and activity in populations of male fish sampled from contaminated environments. Acknowledgements The mention of commercial trade names does not constitute endorsement by the US Government. This study was partially supported by EPA Cooperative Agreement CR826357-01 to Nancy D. Denslow.

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