Environmental Toxicology and Chemistry, Vol. 19, No. 4, pp. 972±981, 2000 Printed in the USA 0730-7268/00 $9.00 ϩ .00

FATHEAD MINNOW : COMPLEMENTARY DNA SEQUENCE AND MESSENGER RNA AND EXPRESSION AFTER 17␤-ESTRADIOL TREATMENT

JOSEPH J. KORTE,*² MICHAEL D. KAHL,² KATHLEEN M. JENSEN,² MUMTAZ S. PASHA,² LOUISE G. PARKS,³ GERALD A. LEBLANC,³ and GERALD T. A NKLEY² ²U.S. Environmental Protection Agency, Mid-Continent Ecology Division, 6201 Congdon Boulevard, Duluth, Minnesota 55804 ³North Carolina State University, Department of Toxicology, Box 7633, Raleigh, North Carolina 27695, USA

(Received 9 April 1999; Accepted 9 August 1999)

AbstractÐInduction of vitellogenin (VTG) in oviparous animals has been proposed as a sensitive indicator of environmental contaminants that activate the receptor. In the present study, a sensitive ribonuclease protection assay (RPA) for VTG messenger RNA (mRNA) was developed for the fathead minnow (Pimephales promelas), a species proposed for routine endocrine- disrupting chemical (EDC) screening. The utility of this method was compared with an enzyme-linked immunosorbent assay (ELISA) speci®c for fathead minnow VTG protein. Assessment of the two methods included kinetic characterization of the plasma VTG protein and hepatic VTG mRNA levels in male fathead minnows following intraperitoneal injections of 17␤-estradiol (E2) at two dose levels (0.5, 5.0 mg/kg). Initial plasma E2 concentrations were elevated in a dose-dependent manner but returned to normal levels within 2 d. VTG mRNA was detected within 4 h, reached a maximum around 48 h, and returned to normal levels in about 6 d. Plasma VTG protein was detectable within 16 h of treatment, reached maximum levels at about 72 h, and remained near these maximum levels for at least 18 d. While the RPA was about 1,000 times more sensitive than the ELISA, the ELISA appears superior for routine screening tests. The ELISA method is relatively simple to perform and, because males lack a clearance mechanism for VTG, the protein remains at relatively high concentrations in the plasma for an extended period of time. As part of the development of the RPA, the complementary DNA (cDNA) sequence for fathead minnow VTG was determined and the deduced amino acid sequence compared with VTG sequences for other ®sh species.

KeywordsÐVitellogenin Pimephales promelas 17␤-Estradiol mRNA cDNA

INTRODUCTION for screening EDCs is international in scope, with Organization Concern for possible adverse effects of endocrine-disrupt- for Economic Cooperation and Development member coun- ing chemicals (EDCs) on humans and wildlife prompted the tries working on methods development and test harmonization U.S. Environmental Protection Agency to identify research on with this species [8]. this topic as one of six high-priority issues [1]. Coincident There are a number of existing assay protocols with the with this, the U.S. Environmental Protection Agency received fathead minnow, including partial- and full-life cycle tests [9± a legislated mandate to develop and implement a screening 12], that would capture adverse effects on estrogen, androgen, program for detecting potential EDCs [2,3]. To meet this man- and/or thyroid function. However, the endpoints typically as- date, a number of expert workshops were initiated to help sessed in these tests (e.g., growth rate, developmental anom- identify potential screening methods [4±6], and a multistake- alies, reproductive success) are also re¯ective of potential im- holder advisory group was formed to provide recommenda- pacts associated with a variety of other toxicity mechanisms. tions to the U.S. Environmental Protection Agency for devel- Hence, in developing fathead minnow screening methods spe- oping and implementing an EDC screening program. This ci®c for EDCs, there has been an emphasis on assessment of group, the Screening and Testing Advi- biological endpoints that are directly related to mechanisms sory Committee, developed a list of screening assays suitable of concern. For example, the protocol recommended for tier for tier 1 (initial) testing for chemicals that could cause toxicity 1 screening incorporates assessment of plasma vitellogenin through disruption of reproductive and developmental pro- (VTG) concentrations in test animals [5,7]. Under normal con- cesses controlled by estrogen, androgen, and thyroid ditions, the production of VTG, a precursor to egg protein, [7]. The tier 1 battery recommended by the Endocrine Dis- is induced in the liver of oviparous females by the interaction ruptor Screening and Testing Advisory Committee includes of with the estrogen receptor (ER) [13,14]. Males three mammalian tests, one amphibian assay (with Xenopus also possess the gene for VTG, but plasma concentrations of laevis), and a test with the fathead minnow (Pimephales pro- the protein typically remain small, presumably due to low melas). Although the most immediate application is in the levels of endogenous estrogens [15,16]. However, exposure of United States, interest in using the fathead minnow as a model males to exogenous estrogens/estrogen mimics can result in a marked induction of plasma VTG, which tends to remain el- * To whom correspondence may be addressed evated because of lack of mechanisms to clear the protein[17]. ([email protected]). This has been successfully exploited as a biomarker of ex- This manuscript has been reviewed in accordance with of®cial posure of ®sh to ER agonists in the environment [e.g., 18± U.S. Environmental Protection Agency (U.S. EPA) procedures; how- ever, the content does not re¯ect U.S. EPA policy. Mention of trade 23]. Further, a number of studies have demonstrated a corre- names does not imply endorsement by the U.S. EPA or the U.S. lation between VTG induction and adverse reproductive effects government. both in male and female ®sh [24±28].

972 Expression of fathead minnow vitellogenin mRNA and protein Environ. Toxicol. Chem. 19, 2000 973

There are a number of techniques available for measuring compared with E2 standards. Extraction ef®ciency, as deter- VTG. An effective, but relatively nonspeci®c, measure of VTG mined by recovery of tritiated steroids extracted from plasma, can be made through assaying alkaline-labile protein-bound was 85 to 90% (data not shown). phosphorus in ®sh plasma [17,27,29]. More speci®c measure- ment techniques for VTG include radioimmunoassays and en- Plasma vitellogenin measurement zyme-linked immunosorbent assays (ELISA); these approach- Plasma VTG protein was measured using a competitive es have been used successfully for measuring fathead minnow ELISA with an antibody directed against fathead minnow VTG VTG [8,16,28,30]. It also is possible to assess events asso- [30]. Brie¯y, puri®ed fathead minnow VTG was serially di- ciated with VTG induction further upstream through mea- luted to obtain standard concentrations between 6 and 1,500 surement of VTG mRNA [31,32]; however, this approach has ng/ml. These standards and dilutions of plasma between 1:325 not been commonly utilized, in part, because of lack of a (controls and samples with little VTG expected) and 1:675,000 standard technique for measuring VTG mRNA. The purpose (treated samples with high VTG expected) were combined with of the present study, therefore, was to develop and validate a an equal volume of a 1:40,000 dilution of primary antibody ribonuclease protection assay (RPA) for measuring VTG and incubated for1hat37ЊC. Two hundred-microliter aliquots mRNA in the fathead minnow. As part of this study, we as- were removed in duplicate, added to 96-well microtiter plates sessed the kinetics of mRNA versus protein induction in male that had been coated with fathead minnow VTG, and incubated ®sh treated with 17␤-estradiol (E2). In conjunction with de- for1hat37ЊC. After washing the plates, a 1:40,000 or 1: velopment of the RPA, we also were able to completely se- 80,000 dilution of goat antirabbit peroxidase-conjugated sec- quence the fathead minnow VTG complementary DNA ondary antibody (Bio-Rad, Hercules, CA, USA) was added (cDNA) and compare the deduced amino acid sequence to and incubated for1hat37ЊC. After washing the plates again, published sequences for other ®sh species. the peroxidase substrate was added and allowed to react for 5 to 10 min. The reaction was stopped with 100 ␮lof1M MATERIALS AND METHODS phosphoric acid and the response measured using a 450-nm Treatment of ®sh ®lter in a plate reader (Model 3550, Bio-Rad). Some samples were reassayed at new dilutions because the values obtained To characterize induction of VTG mRNA and protein, adult were outside the reliable range of the standards. (seven±nine-month-old) male fathead minnows from on-site cultures were anesthetized via water exposure to tricaine meth- Preparation of probe and target RNA for the ribonuclease anesulfonate (100 mg/L) buffered with NaHCO3 (200 mg/L) protection assay and given intraperitoneal (ip) injections of E2 in 10% (v/v) Detailed presentation of the principles and procedures in- ethanol in corn oil. The ®sh were injected at 10 l/g of body ␮ volved in the development and use of the RPA for measure- weight to achieve E2 doses of 5.0 (high) and 0.5 mg/kg (low). ment of speci®c mRNA sequences can be found in the manual Control ®sh received injections of the ethanol-corn oil only. for the Ambion RPA IITM kit (Austin, TX, USA). The ®rst step After injection, the ®sh were held in 10-L tanks receiving 150 in development of the RPA was to obtain a probe that had the ml/min of sand-®ltered Lake Superior water at 25 C. The pho- Њ complementary sequence for the VTG mRNA. To achieve this, toperiod was 16:8 light:dark, and ®sh were fed frozen brine we isolated total RNA from a pool of from seven fathead shrimp (San Francisco Bay Brand, San Francisco, CA, USA) minnows 48 h after an ip injection of E2 at 5 mg/kg (see daily. At 2, 4, 8, 16, 24, 48, and 72 h and 6, 12, and 18 d, above) with Tri ReagentTM (Sigma, St. Louis, MO, USA). The ®ve ®sh from each treatment were anesthetized as above. RNA was suspended in buffer (10 mM Tris, 1 mM EDTA, pH Blood was removed from the caudal vein with heparinized 8.0) and the concentration determined by absorbance at 260 microhematocrit tubes and kept on ice until centrifuged. The nm. Then mRNA was isolated from 1.2 mg of total RNA using plasma ( 25 l) was transferred to microcentrifuge tubes con- ϳ ␮ the FastTrackTM 2.0 Kit (Invitrogen, Carlsbad, CA, USA). The taining heparin and aprotinin [30]. Livers were removed and concentration of mRNA was determined by absorbance at 260 frozen in liquid nitrogen. In addition to the animals sampled nm and used to make cDNA with the Universal Riboclone௡ above, ®ve additional untreated ®sh (no injection, but held cDNA Synthesis System (Promega, Madison, WI, USA). The under the same conditions) were sampled at the beginning and ®rst strand was synthesized using random hexamer primers end of the experiment. All samples were stored at 80 C until Ϫ Њ according to the instructions provided with the kit. Then 10 analyzed. ␮l of the second-strand buffer and 0.5 ␮l of RNAse H were added and allowed to react for 20 min at 37ЊC, followed by Plasma 17␤-estradiol measurement a 5-min incubation at 95ЊC. The cDNA was stored at Ϫ20ЊC Plasma E2 concentrations were measured by radioimmu- until needed. noassay [33] with E2 antiserum from Endocrine Sciences (Cal- An alignment of known VTG sequences from other species abasas, CA, USA) and tritiated E2 from Amersham Life Sci- was performed with the Clustal W 1.7 multiple sequence align- ences (Arlington Heights, IL, USA). Typically, 18 ␮l of plasma ment program [34] using the server at the Baylor College of was combined with 150 ␮l of buffer (0.01 M sodium phos- Medicine (http://dot.imgen.bcm.tmc.edu:9331/multi-align/

phate, pH 7.4, 0.15 M NaCl, and 0.1% NaN3) and extracted multi-align.html). The following VTG sequences and their ac- twice with 1.5 ml of ethyl ether. The ether fractions were cession numbers were obtained from GenBank (http:// combined, evaporated, and resuspended in 360 ␮l of buffer www.ncbi.nlm.nih.gov/PubMed/): rainbow trout (Oncorhyn- containing 1% BSA. A 100-␮l aliquot of the extracted sample, chus mykiss, 1296953), killi®sh (Fundulus heteroclitus, representing 5 ␮l of plasma, was then analyzed for E2. In 3123021), white sturgeon (Acipenser transmontanus, 3123009), some individuals, 18 ␮l of plasma was not available, but the African clawed frog (Xenopus laevis, 139636), and silver lam- ratio of 5 ␮l of plasma per 100 ␮l of resuspension buffer was prey (Ichthyomyzon unicuspus, 3123010). From the alignment, maintained. The amount of radioactivity in the samples was highly conserved regions were used to design consensus prim- 974 Environ. Toxicol. Chem. 19, 2000 J.J. Korte et al.

Table 1. Sequence of primers for polymerase chain reactions and sequencing; the location is based on the rainbow trout vitellogenin amino acid sequence [38]; single-letter abbreviations for amino acids and degenerate nucleotides are used for clarity; T7 refers to the promoter sequence for T7 RNA polymerase

Primer Location (orientation) Amino acid sequence Nucleotide sequence (5Ј to 3Ј)

1 195 upstream DFGLAYTE gaydtnggnytngcntayacnga 2 521 upstream ALRNIAK gcnytnmgnaayathgcnaa 3 545 upstream ALHPELRM gcnytncayccngarbtnmgnatg 4 521 downstream ALRNIAK ttngcdatrttncknarngc 5 547 downstream HPELRM catncknavytcnggrtg 6 854 downstream AVMGVNT gtrttnavncccatnacngc 7 Probe upstream AMVHDDAP catggtccatgatgatgctcc 8 Probe T7 downstream TASLAMHE(T7) ggatcctaatacgactcactatagggaggctcgtgcatagccaaactagctg 9 Target T7 upstream (T7)AMVHDDAP ggatcctaatacgactcactatagggaggcatggtccatgatgatgctcc 10 Target downstream TASLAMHE ctcgtgcatagccaaactagctg

ers (Table 1, primers 1±6), which were purchased from Inte- and were used to synthesize the speci®ed RNA sequences using grated DNA Technologies (Coralville, IA, USA). the MAXIscript kit. The reactions for the two RNAs were the Polymerase chain reactions (PCRs) using these primers same except that, for the probe, one half of the CTP was were performed in 0.5-ml tubes with the Hybaid TouchdownTM replaced by an equal amount of biotin-14-CTP (Gibco, Gai- thermal cycler (Teddington, Middlesex, UK). The reaction vol- thersburg, MD, USA). These RNA products were then puri®ed ume was 50 ␮l and contained 2.5 units of Taq DNA polymerase by cutting the full length product out of a polyacrylamide gel. (Promega), the buffer provided with the enzyme, 1.5 mM The RNAs then were eluted from the gel, quanti®ed by ab-

MgCl2, 40 pmol of each primer, 0.2 mM of each dNTP (dATP, sorbance at 260 nm, divided in 5-␮l aliquots, and stored at dCTP, dGTP, and dTTP; Promega), and 5 ␮l of cDNA that Ϫ80ЊC until needed. had been diluted ®vefold in water. The PCR was initiated by addition of the cDNA to the tube with the other reagents in Ribonuclease protection assay the thermal cycler at 95ЊC. The temperature was held at 95ЊC for 3 min. The thermal cycler was programmed for a modi®ed The ribonuclease protection assay (RPA) was performed touchdown PCR [35]: 10 cycles of 95ЊC for 1 min, 60ЊC (de- using the RPA II kit (Ambion). Total RNA was extracted creasing by 1ЊC per cycle to 51ЊC) for 1 min, and 72ЊC for 1 from the livers as described above, and the concentration was min; then 25 cycles of 95ЊC for 1 min, 50ЊC for 1 min, and determined by absorbance at 260 nm. Ten nanograms of sam- 72ЊC for 1 min; and, ®nally, a 72ЊC step for 7 min. ple RNA, 10 ␮g of yeast RNA from the kit, and 300 pg of The products of the PCR ampli®cations were analyzed by the fathead minnow VTG RNA probe were combined and electrophoresis in 1 to 3% (w/v) agarose gels in 40 mM Tris- precipitated. The sample was resuspended in a small volume acetate, 2 mM EDTA, pH 8.5. The size of the PCR products of hybridization buffer (from kit) and allowed to hybridize was determined by comparison to lambda DNA/EcoRI ϩ Hind overnight at 45ЊC. The samples were digested with a 500- III and ⌽174 DNA/Hae III molecular weight markers (Pro- fold dilution of a mixture of ribonuclease A and T1 for 30 mega) using ethidium bromide staining. Products of the correct min on ice. The protected RNA-probe hybrid was then pre- size were cut out of the agarose gels and the agarose digested cipitated, resolubilized in gel loading buffer, and electropho- using AgarACETM (Promega). The DNA was precipitated using resed in a 5% denaturing polyacrylamide gel. After electro- standard techniques [36] and was sequenced at the University phoresis, the samples were transferred from the gel to a pos- of Wisconsin Biotechnology Center (Madison, WI, USA). itively charged nylon membrane (Ambion) using the Similarity to other VTGs was con®rmed with the basic local PantherTM Semi-Dry Electroblotter (Owl Separation Systems, alignment search tool (BLAST) [37] at the National Center Portsmouth, NH, USA) at 400 mA for 30 to 45 min. The for Biotechnology Information (http://www.ncbi.nlm.nih.gov/ transferred RNA molecules were ®xed to the membrane in BLAST/). an 80ЊC oven for 15 to 20 min. For each set of unknown After obtaining the expected PCR product with primers 1 samples, a set of target RNA standards of known concentra- and 5 (Table 1), primers 7 to 10 were designed (as outlined tion (1.56, 3.12, 6.25, 12.5, 25.0, 50.0, and 100 pg), the in instructions for the MAXIscriptTM kit; Ambion) that were undigested probe, and a control with only yeast RNA were speci®c for fathead minnow VTG and that also incorporated also subjected to the procedure. the sequence of the T7 RNA polymerase promoter. These prim- The membrane was then analyzed for chemiluminescence ers were used, with the DNA product of the ®rst PCR as the using the BrightStarTM BioDetectTM kit (Ambion). The kit con- template, in another PCR to produce DNA templates contain- tains a streptavidin-alkaline phosphatase conjugate that binds ing the fathead minnow VTG sequence as well as the promoter to areas of the membrane containing the biotin-labeled probe. site necessary to synthesize RNA. The PCR conditions were Chemiluminescent images were obtained by exposing the similar to the ®rst PCR except that the touchdown steps were membrane to X-ray ®lm (high speed blue; Lake Superior X- not required, the annealing temperature was 60ЊC, and only Ray, Duluth, MN, USA) and the Fluor-STM MultiImager Sys- 30 cycles were performed. For the probe, the T7 promoter was tem (Bio-Rad). Quanti®cation of the amount of chemilumi- incorporated on the downstream side of the DNA product. For nescence produced by the samples was determined using Mul- synthesis of the target RNA, which is the same strand as the ti-Analyst௡ System software (Bio-Rad) and was compared mRNA, the T7 promoter was incorporated on the upstream with the response given by known amounts of target RNA. side of the DNA. These DNA products were isolated as above Simultaneous monitoring of a housekeeping gene to correct Expression of fathead minnow vitellogenin mRNA and protein Environ. Toxicol. Chem. 19, 2000 975

respectively. This was indicative of a very rapid assimilation into the bloodstream. The E2 also was cleared very rapidly from the plasma. After 48 h, E2 concentrations even in the ®sh given the highest dose were essentially the same as con- trols. Both untreated and sham-injected control animals had mean plasma E2 concentrations of 2 ng/ml or less.

Probe characteristics and measurement of vitellogenin mRNA Several combinations of consensus primers (Table 1) pro- duced PCR products of the correct size. The expected size was based on the location of the primer set in the multiple align- ment of ®sh VTGs. Primer 1 produced the products of the correct size when combined with either primer 4 or 5. Like- wise, expected products were obtained when primer 6 was combined with either primer 2 or 3. The PCR products from primers 1 and 5 and primers 2 and 6, both just over 1,000 bases, were isolated and sequenced. A BLAST search of the amino acid translation of these sequences revealed that both were very similar to VTG from other species. The PCR product of primers 1 and 5 was chosen as the template to continue the work of probe and target preparation for the RPA. Primers 7 and 8 were used to make the probe, and primers 9 and 10 were used to make the target RNA in separate PCR reactions. Both of these PCR products produced bands of the expected size, approximately 330 bases. These DNA templates were then eluted from the gel and used to synthesize RNA. The yields from RNA synthesis were about 5 ␮g for each. The actual protected part of these products would be 301 bases after ac- counting for the T7 promoter site. Using the probe derived for the RPA, VTG mRNA was detectable in the liver of E2-treated ®sh by 4 h (Fig. 1b). At both doses, mRNA initially appeared to increase at about the Fig. 1. Mean (Ϯ SE; n ϭ 5) plasma E2 (a), liver VTG mRNA (b), same rate; however, in ®sh receiving the high dose, the mRNA and plasma vitellogenin (VTG) protein (c) concentrations in adult continued to increase until around 48 h. After this point, VTG male fathead minnows after a single injection of E2 at 0.5 mg/kg or mRNA declined markedly in both groups, and by 6 d, the 5.0 mg/kg. The X-axis was split into segments of 0 to 25 h and 25 mRNA was not detected in the ®sh treated with the low dose to 500 h to clearly present all the results on the same plot. of E2 and had decreased substantially in ®sh given the higher dose. The VTG mRNA was not detectable in either group of control animals (untreated and sham injected) or in any of the for potential RNA losses was not done since the results were treated ®sh by 12 d after injection. replicated with ®ve ®sh per treatment. The RPA was extremely sensitive, giving a robust response with as little as 10 ng of total RNA (Fig. 2). In addition, the Sequencing the vitellogenin cDNA assay produced a linear response to at least 100 pg with a The MarathonTM cDNA Ampli®cation Kit (Clontech, correlation coef®cient of 0.99 (Fig. 3). Under the conditions Palo Alto, CA, USA) was used to obtain the sequence from used, a small background response was evident in controls and the remaining portions of the fathead minnow VTG cDNA. blank samples. This was easily subtracted using the Multi- In this method, adaptor segments of known DNA sequence AnalystTM System software. The background was apparently are ligated onto the ends of the cDNA. We used primer 7 due to residual DNA present from the synthesis of the probe. to generate a PCR product that extended to the 3Ј end of When lower amounts of probe were used, this background was the cDNA and primer 10 to give the corresponding 5Ј end. not detectable. For the analyses, the probe concentration was The AdvantageTM cDNA Polymerase Mix (Clontech) was kept relatively high to ensure at least a threefold molar excess used as the DNA polymerase enzyme in these long-range in the samples containing high levels of VTG mRNA. PCRs. We then sequenced these products and compared the results by multiple sequence alignment with known VTG Plasma vitellogenin sequences from other ®sh species. The ELISA method was very sensitive and required rel- RESULTS atively large dilutions of the plasma to obtain concentrations within the range of the standard curve. For example, dilu- Plasma 17␤-estradiol tions of 1:675,000 were required in samples containing the Measurement of the plasma E2 concentrations indicated largest amounts of VTG. Vitellogenin was not detected in that the peak level occurred at or before the initial 2-h sample most (5 out of 60 showed very low levels of Ͻ0.08 mg/ml) (Fig. 1a). The mean (Ϯ SE) concentrations for the high- and of the control ®sh when using a 325-fold dilution. In the low-dose ®sh at 2 h were 493 (139) and 190 (34) ng/ml, treated ®sh, plasma VTG was detectable by 16-h posttreat- 976 Environ. Toxicol. Chem. 19, 2000 J.J. Korte et al.

Fig. 3. Relative chemiluminescence produced from 1.56, 3.12, 6.25, 12.5, 25.0, 50.0, and 100 pg of target RNA. Target RNA has the complementary sequence to the probe and has the same sequence as the part of the fathead minnow vitellogenin (VTG) mRNA that hy- bridizes with the probe. The correlation coef®cient was 0.99. Fig. 2. Chemiluminescent image produced by the ribonuclease pro- tection assay using the Fluor-S MultiImager System. The image was inverted, light to dark, for clarity. Lane 1 (top left) was loaded with resis (data not shown). It is possible that this small discrepancy 12% of the sample from the undigested probe. All other samples is due to posttranslational modi®cations. contained the entire reaction. Lane 2 was a control with no sample RNA added. Lanes 3 to 9 contained 1.56, 3.12, 6.25, 12.5, 25.0, 50.0, DISCUSSION and 100 pg of target RNA, respectively. Lanes 10 to 15 were from samples that originally contained 10 ng of total RNA from control The kinetics of E2 clearance and induction of VTG in ®sh at 2, 4, 8, 16, 24, and 48 h, respectively. Lane 16 (bottom left) fathead minnows following exposure to E2 were similar to was the same as lane 5 and contained 6.25 pg of target RNA. Lanes those seen in other species. For example, plasma E2 was 17 to 20 were from samples that originally contained 10 ng of total observed at highest concentrations early after the ip dose and RNA from control ®sh at 72, 144, 288, and 432 hours, respectively. Lanes 21 to 30 were from samples that originally contained 10 ng of dropped rapidly, which is similar to observations in rainbow RNA from ®sh receiving the 5 mg/kg dose of E2 at 2, 4, 8, 16, 24, trout [39]. In terms of more speci®c comparisons, at 8 h, we 48, 72, 144, 288, and 432 hours, respectively. observed a mean E2 concentration of 77 ng/ml after a single injection of E2 at 5 mg/kg. This was similar to the 8-h value of 83 ng/ml determined in rainbow trout when the same dose ment and continued to rise until around 48 h (Fig. 1c). After was given [40]. The time course of VTG mRNA induction, 2 d, the VTG concentrations remained relatively constant peaking between 16 to 72 h and undetectable after 12 d, was through the end of the experiment. In the ®sh receiving the consistent with that observed in tilapia when treated with a high E2 dose, the VTG concentrations leveled at about 15 single ip injection of E2 [31]. Similarly, when exposed to to 20 mg/ml, whereas the plateau in ®sh receiving the low , an ER agonist, a substantial increase in VTG dose was about 8 mg/ml. mRNA expression, as indicated by reverse transcriptase PCR, occurred in rainbow trout, followed by a decrease in mRNA Fathead minnow vitellogenin sequence 48 to 72 h after placing the ®sh in clean water [32]. Also, The 5Ј and 3Ј ends of the cDNA were ampli®ed using long- in cultured rainbow trout hepatocytes, the mRNA response range PCR conditions. With primer 7, we obtained a product after a single treatment with E2 was detected within hours of greater than 3,000 bases, which theoretically should have and seemed to level off at 48 to 72 h [41]. These results all extended to the 3Ј end of the cDNA. Likewise, with primer differ somewhat from those reported in rainbow trout given 10, we were able to amplify an approximately 1,300 base a single injection of E2 where the peak VTG mRNA was product, which should have extended to the 5Ј end of the observed at 15 d posttreatment [39]. The cause of the dis- cDNA. These two fragments overlapped by about 300 bases crepancy in kinetics of VTG mRNA induction between that and, when assembled into a contiguous sequence that included study and others, including ours, is unclear. Signi®cantly, the codon for the methionine in the leader sequence and the even though the test conditions, species, and assay methods stop codon, resulted in a sequence that was 4,020 bases long. differed among studies, the peak concentrations of fathead The vast majority of the sequence was obtained by sequencing minnow VTG mRNA were approximately within an order of both strands of the DNA. Areas near the very ends of the magnitude of those found in both tilapia and rainbow trout templates could only be sequenced on one strand. The fathead treated with ip injections of E2. minnow VTG sequence information is available from Gen- In the fathead minnow, VTG protein concentrations re- Bank under accession number AF130354. mained elevated for at least 18 d after E2 treatment. This also Alignment of the deduced fathead minnow VTG amino acid was very similar to observations in tilapia [31] and is likely sequence with the rainbow trout VTG sequence reported by related to the lack of a mechanism for the removal of plasma Mouchel et al. [38] showed 56% identical residues (Fig. 4). VTG in male ®sh [17]. This slow clearance has critical im- A similar alignment with the killi®sh VTG I sequence showed plications for a screening assay since the effect of an ER 46% identical residues (data not shown). The deduced mass agonist would easily be measured long after initial exposure of the fathead minnow VTG protein monomer was 146,274 when the chemical may not be detectable in the ®sh. In fact, Daltons. This was slightly smaller than the 152,000 to 156,000 in the fathead minnow, it seems that the effects of the E2 Daltons determined by SDS polyacrylamide gel electropho- treatment on VTG likely could have been measured for days, Expression of fathead minnow vitellogenin mRNA and protein Environ. Toxicol. Chem. 19, 2000 977

if not weeks, after the last time point measured, 18 d, since Also, given the sensitivity of the RPA assay, it should be the concentration was not decreasing at this time. It has been feasible to use whole-body homogenates, as opposed to liver reported that the half-life of VTG in male amphibians is greater extracts, which would further simplify the assay. that 40 d, while in females the half-life is less than 2 d [42]. Of course, it is necessary to consider what the effects of Our data would suggest that the half-life for VTG clearance weak ER agonists would be in terms of sensitivity of the two in male fathead minnows may be at least this long. The actual assay methods. If one assumes that a proportional response VTG protein concentration stabilized at about 15 to 20 mg/ would be obtained, then low protein levels should be seen just ml in ®sh given the high dose of E2. Using the same detection as easily as low mRNA levels (see above for sensitivity versus method (including an antibody developed speci®cally for fat- abundance discussion). But it may be questionable whether head minnows), a value of 21 mg/ml was reported in male such sensitivity is needed since it may actually be dif®cult to fathead minnows receiving multiple injections of E2 [30]. In detect a true positive effect at low levels using either mRNA that same study, fathead minnows exposed to 1 nM E2 showed or protein. This stems from reports of a basal level of ex- plasma VTG concentrations well above 10 mg/ml after 21 d. pression that is already seen in untreated male ®sh when mea- Other work with male fathead minnows, using polyclonal an- suring either the protein [8,16,28] or the mRNA [31,44]. In tibodies developed for carp VTG, has found much lower VTG this study, we were able to discern measurable concentrations concentrations (Ͻ1 mg/ml) after similar exposures to E2 [28]. of VTG (0.02±0.08 mg/ml) in the plasma of 5 of 60 control Also, relatively low levels of VTG were measured with the male ®sh when diluting the plasma 325 times. However, the carp antibody in fathead minnows exposed to 100 ng/L E2 values were so low they were not meaningful in comparison during early life stages (24 h postfertilization to 30 d posthatch) with the treated ®sh. In the case of the RPA, we did not detect [8]. As previously suggested [30], it seems likely that these measurable VTG mRNA in control ®sh; however, all mea- values would have been higher if the homologous antibody surements were made on only small amounts of total RNA, and/or puri®ed fathead minnow VTG protein had been used and if more were used, there likely would have been positive in those studies. responses in the controls. Given these observations, the most In general, it appears that either the ELISA or RPA method sensitive assay may be unnecessary since it is possible to detect provides an extremely sensitive assay for evidence of exposure a response in some ostensibly untreated ®sh. of fathead minnows to ER agonists. Because of the tremendous Sequencing the fathead minnow VTG cDNA provided in- accumulation of VTG in male ®sh, the ELISA can be effec- sights regarding the relationship of this protein to VTG in other tively performed on less than 1 ␮l of serum, which is important ®sh species. For example, the positions of the , given the relatively small size of the ®sh and the need in some lipovitellin I domain, and phosvitin domain that have been instances to also measure plasma steroid concentrations [5]. identi®ed by Mouchel et al. [38] in rainbow trout appear to Likewise, the RPA can be performed using only a very small be very similar in the fathead minnow (Fig. 4). However, the amount of the total liver RNA present. This is due to the large serine-rich phosvitin domain is even smaller in the fathead amount of VTG mRNA synthesized and the great sensitivity minnow than in the trout, which previously had been reported of the RPA method. In an absolute comparison, the ELISA as the smallest known for . The most striking struc- can detect VTG at the nanogram level, while the RPA can tural difference, however, is that the lipovitellin II domain (C- detect VTG mRNA at picogram concentrations. In fact, on a terminal region of VTG) of the rainbow trout and other ®sh molar basis, the RPA is 1,000 times more sensitive than the species where the complete sequence is known is much larger ELISA. However, because the VTG protein accumulates to than in the fathead minnow. Alignment of the trout and fathead such high concentrations, the ability to detect expression of minnow VTG sequences shows that the two sequences are the gene is nearly the same between the two methods. Because similar on the N-terminal side of this domain, but the end of the ELISA is a much simpler method to perform, it would the fathead minnow VTG sequence is over 250 amino acids appear preferable to the RPA for routine screening of EDCs. before the end of the trout sequence. Mouchel et al. [38] sug- Not only does the ELISA enable a greater throughput of sam- gested that the 250 amino acid C-terminal region is important ples, but the time-frame during which the animals can be sam- for protein interactions. They also noted that much of this pled to detect induction is greatly expanded by measurement region has been shown to be absent in lipovitellin of the protein as opposed to mRNA. The window for the RPA complexes. The importance of the absence of this region in peak appeared from 1 to 3 d after E2 exposure; but in the the fathead minnow is not known but suggests some different ELISA, the measurement could have been made successfully characteristics for fathead minnow VTG compared with other anywhere from about 2 to 18 d and, perhaps, signi®cantly species. We should note that smaller VTG , similar in longer. In addition, considering cost effectiveness under the size to what we found, have been reported in several ®sh conditions used herein, the ELISA was preferable to the RPA species [17], although complete sequence information is not in terms of both supplies and labor. available for them. Additional support for the 3Ј end of the While it appears that the ELISA method may be more fathead minnow sequence is provided by a zebra®sh-expressed useful than the RPA for routine VTG screening, on some sequence tag (GenBank AA658675) that has the same four C- occasions, it would be advantageous to use the latter method. terminal amino acids preceding the same stop codon. Finally, For example, if there is interest in knowing if a response will the existence of more than one VTG gene is possible [45]; for occur just a few hours after exposure, than the RPA would example, two extremely similar VTG genes have been reported be superior to the ELISA. For routine screening, steps could in the rainbow trout, but most of the differences between the be taken to make the mRNA-based method easier to perform. two were in the introns [46]. This could certainly be the case For example, it is likely that, due to the highly speci®c in the fathead minnow as well, but under the conditions used of the probe, a slot-blot type of procedure [43] could be used, herein, we could not distinguish more than one gene. eliminating the electrophoresis, digestion, etc., steps used in The results of the fathead minnow cDNA sequence analysis this study. This would increase throughput and decrease cost. could be used in a multiple sequence alignment with other ®sh 978 Environ. Toxicol. Chem. 19, 2000 J.J. Korte et al.

Fig. 4. Alignment of the fathead minnow and rainbow trout vitellogenin amino acid sequences. The positions of the domains are shown based on the positions identi®ed in the rainbow trout by Mouchel et al. [38]. Expression of fathead minnow vitellogenin mRNA and protein Environ. Toxicol. Chem. 19, 2000 979

Fig. 4. Continued

VTGs to select a conserved sequence for use in designing ®sh In conclusion, a highly sensitive assay was developed for VTG monoclonal antibodies. This approach has been used measuring VTG mRNA in fathead minnows. However, given previously with some success by aligning the N-terminal se- the complexity of the RPA and the abundance of protein in quences of several ®sh VTGs [47,48]. The addition of another the plasma, coupled with a good ELISA method, the latter complete VTG sequence could be useful in the selection of a appears to be the more practical method to recommend for peptide sequence, other than the N-terminal region, that is routine EDC testing. However, as mentioned above, some of capable of producing effective antibodies in all ®sh. A mono- the complexities of the RPA could be avoided should a need clonal antibody that can be produced in large quantities from for a sensitive measurement for the mRNA arise. Currently, a known site within the VTG would be highly useful for EDC it would appear that an extremely sensitive method is not screening methods. It is, of course, possible that a conserved necessary since the existence of VTG can already be measured site capable of generating useful antibodies for all ®sh does in some ostensibly untreated (control) ®sh. not exist. If, however, this is the case, the sequence information could still be useful for design and/or analysis of fathead min- AcknowledgementÐHelpful comments on an earlier version of this now-speci®c VTG monoclonal antibodies. manuscript were supplied by Patricia Schmieder and Sigmund Degitz. 980 Environ. Toxicol. Chem. 19, 2000 J.J. Korte et al.

REFERENCES 1998. Identi®cation of estrogenic chemicals in STW ef¯uent. 1. 1. U.S. Environmental Protection Agency. 1996. Strategic plan for Chemical fractionation and in vitro biological screening. Environ the of®ce of research and development. EPA/600/R3-91-063. Sci Technol 32:1549±1558. Technical Report. Washington, DC. 24. Herman RL, Kincaid HL. 1988. Pathological effects of orally 2. U.S. Code. 1996. Food Quality Protection Act. PL 104±170. et administered estradiol to rainbow trout. Aquaculture 72:165±172. seq. 25. Jobling S, Sheahan D, Osborne JA, Matthiessen P, Sumpter JP. 3. U.S. Code. 1996. Safe Drinking Water Act. PL 104±182. et 1996. Inhibition of testicular growth in rainbow trout (Oncor- seq. hynchus mykiss) exposed to estrogenic alkylphenolic chemicals. 4. Gray LE, et al. 1997. Endocrine screening methods workshop Environ Toxicol Chem 15:194±202. report: Detection of estrogenic and androgenic hormonal and 26. Laenge R, Schweinfurth H, Croudace CP, Panter GH. 1997. antihormonal activity for chemicals that act via receptor and Growth and reproduction of fathead minnow (Pimephales pro- steroidogenic enzyme mechanisms. Reprod Toxicol 11:719± melas) exposed to the synthetic steroid ethynylestradiol 750. in a life-cycle test. Abstracts, 7th Annual Meeting, Society of 5. Ankley GT, et al. 1998. Overview of a workshop on screening Environmental Toxicology and ChemistryÐEurope, Amsterdam, methods for detecting potential (anti-) estrogenic/androgenic The Netherlands, April 7±10, p 43. chemicals in wildlife. Environ Toxicol Chem 17:67±87. 27. Kramer VJ, Miles-Richardson S, Pierens SL, Giesy JP. 1998. 6. Devito MJ, et al. 1999. Screening methods for chemicals that Reproductive impairment and induction of alkaline-labile phos- alter thyroid hormone action. Function and homeostasis work- phate, a biomarker of estrogen exposure, in fathead minnows shop. Environ Health Perspect 107:407±415. (Pimephales promelas) exposed to waterborne 17␤-estradiol. 7. U.S. Environmental Protection Agency. 1998. Endocrine disrup- Aquat Toxicol 40:335±360. tor screening and testing advisory committee (EDSTAC) report. 28. Panter GH, Thompson RS, Sumpter JP. 1998. Adverse repro- Of®ce of Prevention, Pesticides and Toxic Substances, Washing- ductive effects in male fathead minnows (Pimephales pro- ton, DC. melas) exposed to environmentally relevant concentrations of 8. Tyler CR, van Aerle R, Hutchinson TH, Maddix S, Trip H. 1999. the natural oestrogens, oestradiol and oestrone. Aquat Toxicol An in vivo testing system for endocrine disruptors in ®sh early 42:243±253. life stages using induction of vitellogenin. Environ Toxicol Chem 29. Craik JCA, Harvey SM. 1984. A biochemical method for distin- 18:337±347. guishing between the sexes of ®shes by the presence of yolk 9. U.S. Environmental Protection Agency. 1982. Users guide to con- protein in the blood. J Fish Biol 25:293±303. ducting life-cycle tests with fathead minnows (Pimephales pro- 30. Parks LG, Cheek AO, Denslow ND, Heppell SA, McLachlan JA, melas). EPA 600/8-81-011. Technical Report. Duluth, MN. LeBlanc GA, Sullivan CV. 1999. Fathead minnow (Pimephales 10. U.S. Environmental Protection Agency. 1989. Pesticide assess- promelas) vitellogenin: Puri®cation, characterization, and quan- ment guidelines. Subdivision E, hazard evaluation: Wildlife and titative immunoassay for the detection of estrogenic compounds. aquatic organisms. EPA 540/09-82-024. Technical Report. Wash- Comp Biochem Physiol C 123:113±125. ington, DC. 31. Lim EH, Ding JL, Lam TJ. 1991. Estradiol-induced vitellogenin 11. U.S. Environmental Protection Agency. 1994. Short-term meth- in a teleost ®sh, Oreochromis aureus. Gen Comp ods for estimating the chronic toxicity of ef¯uents and receiving Endrocrinol 82:206±214. waters to freshwater organisms, 3rd ed. EPA 600/4-91-002. Tech- 32. Lech JJ, Lewis SK, Ren L. 1996. In vivo estrogenic activity of nical Report. Cincinnati, OH. nonylphenol in rainbow trout. Fundam Appl Toxicol 30:229±232. 12. Organization for Economic Cooperation and Development. 1992. 33. Kagawa H, Takano K, Nagahama Y. 1981. Correlation of plasma Section 2. Guideline 204. Fish early-life stage toxicity test. Guide- estradiol-17␤ and progesterone levels with ultrastructure and his- lines for Testing Chemicals. Paris, France. tochemistry of ovarian follicles in the white spotted char, Sal- 13. Clemens MJ. 1978. The regulation of egg yolk protein synthesis velinus leucomaeni. Cell Tissue Res 218:315±329. by steroid hormones. Prog Biophys Mol Biol 28:71±108. 34. Thompson JD, Higgins DG, Gibson TJ. 1994. CLUSTAL W: 14. Ng TB, Idler DR. 1983. Yolk formation and differentiation in Improving the sensitivity of progressive multiple sequence align- teleost ®shes. In Hoar WS, Randall DJ, Donaldson EM, eds, Fish ment through sequence weighting, positions-speci®c gap penalties Physiology, Vol IXA. Academic, Orlando, FL, USA, pp 373± and weight matrix choice. Nucleic Acids Res 22:4673±4680. 404. 35. Don RH, Cox PT, Wainwright BJ, Baker K, Mattick JS. 1991. 15. Flouriot G, Vaillant C, Salbert G, Pelissero C, Guiroud JM, Val- ``Touchdown'' PCR to circumvent spurious priming during gene otaire Y. 1993. Monolayer and aggregate cultures of rainbow trout ampli®cation. Nucleic Acids Res 19:4008. hepatocytes: Long term and stable liver-speci®c expression in 36. Sambrook J, Fritsch EF, Maniatis T. 1989. Molecular Cloning. A aggregates. J Cell Sci 105:407±416. Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold 16. Tyler CR, van der Eerden B, Jobling S, Panter G, Sumpter JP. Spring Harbor, NY, USA. 1996. Measurement of vitellogenin, a biomarker for exposure to 37. Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ. 1990. oestrogenic chemicals, in a wide variety of cyprinid ®sh. J Comp Basic local alignment search tool. J Mol Biol 215:403±410. Physiol B 166:418±426. 38. Mouchel N, Trichet V, Betz A, Le Pennec J, Wolff J. 1996. Char- 17. Mommsen TP, Walsh PJ. 1988. and oocyte assem- bly. In Hoar WS, Randall VJ, eds, Fish Physiology, Vol XIA. acterization of vitellogenin from rainbow trout (Oncorhynchus Academic, San Diego, CA, USA, pp 347±406. mykiss). Gene 174:59±64. 18. Purdom CE, Hardiman PA, Bye VJ, Eno NC, Tyler CR, Sumpter 39. Pakdel F, Feon S, Le Gac F, Le Menn F, Valotaire Y. 1991. In JP. 1994. Estrogenic effects of ef¯uents from sewage treatment vivo estrogen induction of hepatic estrogen receptor mRNA and works. Chem Ecol 8:275±285. correlation with vitellogenin mRNA in rainbow trout. Mol Cell 19. Folmar LC, Denslow ND, Rao V, Chow M, Crain DA, Enblom Endocrinol 75:205±212. J, Marcino J, Guillette LJ Jr. 1996. Vitellogenin induction and 40. MacKay ME, Lazier CB. 1993. Estrogen responsiveness of vi- reduced serum testosterone concentrations in feral male carp (Cy- tellogenin gene expression in rainbow trout (Oncorhynchus my- prinus carpio) captured near a major metropolitan sewage treat- kiss) kept at different temperatures. Gen Comp Endocrinol 89: ment plant. Environ Health Perspect 10:1096±1101. 255±266. 20. Harries JE, et al. 1996. A survey of estrogenic activity in United 41. Flouriot G, Pakdel F, Valotaire Y. 1996. Transcriptional and Kingdom waters. Environ Toxicol Chem 14:1993±2002. post-transcriptional regulation of rainbow trout estrogen re- 21. Harries JE, Sheahan DE, Jobling S, Matthiessen P, Neall M, ceptor and vitellogenin gene expression. Mol Cell Endocrinol Sumpter JP, Taylor T, Zaman N. 1997. Estrogenic activity in ®ve 124:173±183. United Kingdom rivers detected by measurement of vitellogenesis 42. Tata JR. 1976. The expression of the vitellogenin gene. Cell 9: in caged male trout. Environ Toxicol Chem 16:534±542. 1±14. 22. Janssen PAH, Lambert JGD, Vethaak AD, Goos HJT. 1997. En- 43. Brown T, Mackey K. 1998. Analysis of RNA by northern and vironmental pollution caused elevated concentrations of oestra- slot blot hybridization. In Ausubel FM, Brent R, Kingston RE, diol and vitellogenin in the female ¯ounder, Platichthys ¯esus(L.). Moore DD, Seidman JG, Smith JA, Struhl K, eds, Current Pro- Aquat Toxicol 39:195±214. tocols in Molecular Biology, Vol1. John Wiley & Sons, New 23. Desbrow C, Routledge EJ, Brighty GC, Sumpter JP, Waldock M. York, NY, USA. Expression of fathead minnow vitellogenin mRNA and protein Environ. Toxicol. Chem. 19, 2000 981

44. Ren L, Lewis SK, Lech JJ. 1996. Effects of estrogen and non- 47. Folmar LC, Denslow ND, Wallace RA, LaFleur G, Gross TS, ylphenol on the post-transcriptional regulation of vitellogenin Bonomelli S, Sullivan CV. 1995. A highly conserved N-terminal gene expression. Chem Biol Interact 100:67±76. sequence for teleost vitellogenin with potential value to the bio- 45. Wahli W, Dawid IB, Ryffel GU, Weber R. 1981. Vitellogenesis chemistry, molecular biology and pathology of vitellogenesis. J and the vitellogenin gene family. Science 212:298±304. Fish Biol 46:255±263. 46. Mouchel N, Trichet V, Naimi BY, Le Pennec J, Wolff J. 1997. 48. Heppell SA, Denslow ND, Folmar LC, Sullivan CV. 1995. Uni- Structure of a ®sh (Oncorhynchus mykiss) vitellogenin gene and versal assay of vitellogenin as a biomarker for environmental its evolutionary implication. Gene 197:147±152. estrogens. Environ Health Perspect 103:9±15.