Vitellogenin induction and reduced serum testosterone concentrations in feral male carp (Cyprinus carpio) captured near a major metropolitan sewage treatment plant.

Endocrine disrupting chemicals can potentially alter the reproductive physiology of fishes. To test this hypothesis, serum was collected from common carp (Cyprinus carpio) at five riverine locations in Minnesota. Male fish collected from an effluent channel below the St. Paul metropolitan sewage treatment plant had significantly elevated serum egg protein (vitellogenin) concentrations and significantly decreased serum testosterone concentrations compared to male carp collected from the St. Croix River, classified as a National Wild and Scenic River. Carp collected from the Minnesota River, which receives significant agricultural runoff, also exhibited depressed serum testosterone concentrations, but no serum vitellogenin was apparent. These data suggest that North American rivers are receiving estrogenic chemicals that are biologically active, as has been reported in Great Britain. ImagesFigure 1.Figure 2. AFigure 2. B

1U.S. Environmental Protection Agency, Gulf Breeze, FL 32561 USA; 2Department of Biochemistry and Molecular Biology; and 3Department of Zoology, University of Florida, Gainesville, FL 32610 USA; 4Minnesota Department of Natural Resources, MN 55155 USA; 5Center for Bioenvironmental Research, Tulane University, New Orleans, LA 70112 USA.
Concern for estrogenic effects of environmental xenobiotic chemicals on human and wildlife health has existed for over 25 years (1,2). However, within the last 4 years, this interest has become focused and intensified (3)(4)(5). Among wildlife species, research has been focused on animals associated with wetland or aquatic habitats receiving sewage or industrial effluent and agricultural runoff. As with most studies in aquatic toxicology, fish have received the greatest attention. In fish, estrogenic responses have been associated with exposure to pesticides (6), surfactants (7), pulp mill effluent/phytoestrogens (8)(9)(10)(11), industrial waste (12), and sewage effluent (13,14). In these studies, one measure of estrogenic activity is the production of vitellogenin (VTG), an estrogen-inducible egg protein precursor. This protein, normally synthesized in the liver of female oviparous vertebrates, is estrogen dependent and increases markedly in the serum during oocyte development (15,16). In general, the VTG gene is present but not expressed in males [there are exceptions (17)(18)(19)(20)(21)]. However, administration of exogenous estrogen, estrogen mimics, or P450 aromatase inducers can elicit VTG synthesis in males (22)(23)(24). The primary purpose of this investigation was to determine whether male carp (Cyprinus carpio) collected in the Mississippi River below a major metropolitan sewage treatment plant (STP) exhibited elevated serum VTG concentrations and altered serum sex steroid concentrations when compared with a relatively pristine reference site, the St. Croix River. Either abnormality in male fish could indicate the presence of estrogenic chemicals in their riverine environment. To determine the extent of any observed effect, we collected carp from two additional Mississippi River locations below the STP and from a Mississippi River tributary receiving agricultural runoff, the Minnesota River.

Methods and Materials Fish Collection
Male carp were collected from five locations around the St. Paul, Minnesota, area ( Fig.  1): 1) a natural side channel of the Mississippi River receiving effluent from the St. Paul Metropolitan Sewage Treatment Works (STP, river mile 835, n = 10); 2) Mississippi River Navigational Pool #2 (MRP-2, between river miles 815 and 832, n = 10); 3) Mississippi River Pool #3 (MRP-3, between river miles 798 and 802, n = 10); 4) The Minnesota River, a Mississippi River tributary receiving extensive agricultural runoff (MNR, river mile 69, n = 10); and 5) The St. Croix River, our reference site classified as a National Wild and Scenic River (SCR, between river miles 63 and 125, n = 5). Additionally, female carp were collected from the effluent channel (n = 7) and from MRP-2 (n = 10). Fish were collected using a pulsed DC electrofishing boat. Fish, which were all in postspawning condition, were obtained from the effluent channel during the last week of August and from the other locations between 20 September and 16 October 1995. River flow rates at the time of collec-tion were > 200% of normal for this period. All fish were mature adults between ages III and V; mean length values are given in mm ± 1 standard error: STP, 472.2 ± 5.9 male (M) and 466.6 ± 9.3 female (F); MRP-2, 474.1 ± 10.7 (M) and 523.8 ± 14.5 (F); MRP-3, 500.2 ± 11.4; MRR, 496.2 ± 12.6; and SCR, 628.0 ± 13.3. Blood was drawn by cardiac puncture into unheparinized vacutainers and allowed to clot. The samples were centrifuged and the serum was pipetted into 1.5-ml microfuge tubes pretreated with aprotinen and frozen at -700 until analyzed.

Viltellogenin Analysis
The monoclonal antibody Mab HL 1147 (2D3-3A9) was raised against a mixture of fish vitellogenins injected into a Balb/C mouse in the Hybridoma Core Facility at the University of Florida. Briefly, a mouse was injected subcutaneously at three sites, first with purified striped bass VTG and then twice with a mixture of acrylamide gel pieces of purified brown bullhead and carp VTG in RIBI MPL and TDM adjuvant (RIBI Immuno Chemical, Inc., Hamilton, MT) at 2-to 3-week intervals. The mouse was then injected with a combination of 75 pg of purified carp VTG and gel pieces of carp and brown bullhead VTG, followed by a final intraperitoneal (ip) boost with 75 pg of purified carp VTG. Hybridomas were screened against carp, striped bass, and brown bullhead VTG by ELISA. We obtained hybridomas for carp and striped (Pierce Chemical Company, Rockford, IL) as the elution buffer. This purified antibody was used in the capture-ELISA assay. A polyclonal antisera (UF1 14) was raised in rabbits by injection of a mixture of polyacrylamide gel-purified VTGs from several species including carp, largemouth bass, striped bass, bluegill sunfish, and rainbow trout. The rabbit was boosted with three injections of the above VTG cocktail minus the rainbow trout VTG. Before use, antiserum UF1 14 was mixed with one-sixth volume of serum from control males to remove nonspecific reactivity. Precipitated material was removed by high speed centrifugation. The clear supernatant contained antisera that was specific and of high affinity for carp VTG. This antisera was used in the quantitative capture ELISA described below. For gel electrophoresis and Western blot analyses, serum samples were diluted 25-fold with Laemmli sample buffer (25), and 15 til of each sample was applied in duplicate to preformed wells in 7.5% Tris-tricine acrylamide gels (26). Immediately after electrophoresis, one set of gels was stained with Coomassie Brilliant Blue (R250) to visualize the proteins, and another set of gels was electroblotted overnight at 20 V and 40C onto Immobilon P membranes (Millipore Corporation, Bedford, MA) using 10 mM MES (morpholinoethane sulfonic acid), pH 6, 20% MeOH as the transfer buffer (27). For Western blot analyses, membranes were blocked with 5% non-fat dry milk (28) in TBST (10 mM Tris, pH 7; 150 mM NaCl; 0.5% Tween) for 2 hr at room temperature and then incubated directly with tissue culture supernatant containing Mab HL 1147 (2D3-3A9) for 16 hr at 40C. The membrane was then washed in TBST and probed with goat anti-mouse alkaline phosphatase-conjugat- ed secondary antibody (diluted 1 :1000) for 2 hr at room temperature. Blots were developed by incubating them with bromochloroindolyl phosphate/nitro blue tetrazolium substrate. Carp VTG was detected as a set of two high-molecular weight proteins of 180 kDa ( Fig. 2; thin band in blot) and 150 kDa (wide band). In addition, there were a number of degradation products of VTG detected by this sensitive assay in blots of samples with high levels of VTG. These bands make up less than 1% of the VTG protein and are not detected by Coomassie staining, thus illustrating the superior sensitivity of the Western blot assay.
Capture-ELISA Assay VTG was quantified using a capture-ELISA assay. Carp VTG, purified by chromatography on DEAE (diethylamino ethyl anion exchange medium; Perceptive Biosystems, Inc., Farmington, MA), was used as a standard. Protein concentration was determined by the Bradford assay (29). Purified Mab HL 1147 (2D3-3A9), diluted to a concentration of 10 pig/ml in phosphate buffered saline (PBS), was coated onto the surface of 96-well microtiter plates (50 pl/well) overnight at 4°C. The plates were washed with TBST and blocked with 360 id/well of 0.1% BSA in TBST for 2 hr at room temperature. Again, the plates were thoroughly washed with TBST. Fish plasma samples were diluted in the range of 1:500 to 1:5000 in 0.1% BSA in TBST. Fifty microliters of this dilution was added, in duplicate, to individual wells of the plate and allowed to incubate overnight. Standard curves were constructed by adding serial dilutions of purified carp VTG (0.1 to 2.0 pg/ml) to undiluted male serum diluted in the same range as the fish from the St. Croix River reference area (1:500 to 1:5000) and added  to the wells as indicated above. The next day, the plates were washed in TBST and then incubated with 50 pl/well rabbit anti-VTG polyclonal antisera (UF 114) diluted 1:500 to disclose the VTG captured by the monoclonal antibody in the first step; this incubation was done at room temperature for 2 hr. The polyclonal antiserum was in turn disclosed by a 1:1000 dilution of a third antibody, goat anti-rabbit IgG, linked to alkaline phosphatase. As above, this incubation was conducted for 2 hr at room temperature. After a final series of three washes with TBST, 100 pl of substrate solution (pnitrophenyl phosphate in carbonate buffer, pH 9.6) was added to each well and incubated for 30 min. The intensity of the yellow color that developed was quantified at 405 nm with an automated ELISA reader. VTG concentrations were calculated based on the standard curve after subtacting values for the male control serum.

Steroid Analysis
All samples and standards for radioimmunoassay analysis were prepared in duplicate and were analyzed in a single assay.

Statistics
To test for significant differences among collection sites, we analyzed the plasma steroid hormone concentrations using oneway ANOVA. If an overall significance was detected, Scheffe's S-tests were performed to determine differences between specific pairs. Prior to analysis, the raw data for serum T and E2 concentrations were log transformed to achieve homogeneity of variance. Steroid hormone data were also examined as an estrogen/androgen (E/A) ratio. These data were arc sine transformed prior to analysis. To determine any relationship between serum steroid and VTG concentrations, a Pearson's product moment correlation analysis was performed. All data are reported as mean ± 1 standard error (SE). Probability of significance was set at p < 0.05. All statistical analyses were conducted with Stat-View II software (Abacus Concepts, Berkeley, CA).

Serum Vitellogenin Concentrations
Gel electrophoresis and Western blot analysis is a very sensitive method to detect the presence of VTG in serum. Mab HL 1147 (2D3-3A9) reacts specifically with carp VTG present in vitellogenic females (or E2-induced males), but not with control males. In Figure 2B, the prominent VTG band in Lane 1 corresponds to 150 kDa. In some lanes, VTG is also expressed as a higher MW band of 180 kDa. The bands appearing below 150 kDa correspond to about 1% of the VTG protein and are breakdown products of VTG visualized by the high sensitivity of this assay. When male control serum is probed by Western blot, no bands appear, indicating that these bands are not the result of crossreactivity with normal serum proteins (data not shown). Plasma samples of all fish collected in this study were analyzed by gel electrophoresis and Western blot analysis. While most of the female fish collected at all sites had measurable amounts of VTG, both female and male fish collected in the sewage effluent canal (STP) had visible lev-els of VTG by Western blot analysis (Fig.  2B). The corresponding lanes in the Coomassie blue stained gels ( Fig. 2A) show that the equivalent amount of serum protein was loaded into each lane. As observed from the blot, 6 of the 7 females had high levels of VTG, while one had a lower level. Seven of the 10 males had visible levels, with 6 of those 7 in the range represented by the females. The lesser bands that are visible on the blot are degradation products of VTG and are most prominent in those samples with high VTG concentrations. The capture-ELISA assay was performed on the samples from the sewage effluent canal (STP) to quantitate the VTG concentrations in those fish ( = 0.022). As observed for serum T, a significant difference was observed among collection sites when the data were analyzed as E/A ratios (F = 4.73; df= 4,40; p = 0.003). Male fish collected in the effluent stream of the sewage treatment plant and from the Minnesota River exhibited significandy elevated E/A ratios compared to fish obtained from the reference site (St. Croix River). Reduced serum T, rather than increased serum estrogen, was responsible for that significant increase (Table 2). No statistically significant relationship was observed between either the serum E2 concentrations or the E/A ratios and serum VTG concentrations in male fish. However, the relationship between E/A ratio and serum VTG concentration did border on significant (r2 = 0.434; p = 0.054), suggesting that this relationship should be considered in future research.
Although this study focused primarily on males, adequate numbers of females were obtained from two sites, STP and MRP-2. Serum sex steroids in these females did not vary significantly between sites, but serum T concentrations were a quarter of that observed in males, whereas plasma E2 concentrations were 2-4 times greater than those detected in males ( Table 2). The E/A ratio (x 100) for females from STP was 32.8 (± 7.6) while females from MRP-2 averaged 60.9 (± 6.6). The disparity in female E/A ratio was due to variation in serum E2 concentrations, the opposite of that observed in the males. The E/A ratios for females were greatly elevated compared to those observed in males from any site.

Discussion
Our results show a pronounced estrogenic effect (VTG production in male carp) associated with exposure to municipal sewage effluent (Fig. 2). Serum VTG concentration was elevated without a coincident significant increase in the circulating concentration of the endogenous estrogen, E2 (Table 2). However, circulating T was reduced and the E/A ratio was elevated in male fish caught in the effluent channel.
Vitellogenin induction was not observed at any other sampling location, even in fish collected between 3 and 17 miles downstream of the effluent channel (MRP-2). No attempt was made to establish a gradient of VTG induction away from the effluent channel due to the diluting effect associated with an abnormally high flow rate of the Mississippi River in 1995 (160-300% of normal in May through October). The presence ofVTG in the serum of male carp suggests that there is an estrogenic component of the sewage discharge. Similar observations have been reported with caged fish in Great Britain rivers receiving domestic sewage (14,30). Purdom et al. (14) suggested that the most likely estrogenic substances in domestic sewage are ethynyl estradiol (EE2) and alkylphenol polyethoxylates (APEs); however, a wide range of environmental contaminants possessing estrogenic activity have been reported and may be associated with domestic sewage (31,32). Estrogens have been measured by radioimmunoassay in the effluent of a modern sewage treatment facility and shown to vary from 7 to 52 ng/l (33). Male sheepshead minnows can produce measurable quantities of VTG after a 3-day exposure to E2 at 10 ng/l (Folmar, unpublished observations). Purdom et al. (14) also showed that EE2 at 1-10 ng/l could induce a vitellogenic response in caged rainbow trout. Alkylphenol polyethoxylates are produced worldwide at >300,000 tons annually, and as much as 60% of that ends up in the aquatic environment (34). These chemicals have been shown to bind to the estrogen receptor of fish and mammals (7,35) and are capable of inducing VTG production in fish hepatocyte cell culture (7,13). Additionally, a wide array of chemicals, including pesticides and plasticizers commonly found in waste water, stimulate VTG production from fish hepatocytes in vitro (12). In general, there appears to be little similarity in structure among many of the documented estrogenic chemicals (36), but a para-substituted phenolic group (37) and structural rigidity (38,39) appear to enhance estrogenicity. Serum T concentrations were lowest in male fish collected from the effluent channel and the Minnesota River. No males from any location showed increased serum E2 concentrations, nor were those concentrations elevated when compared to females. Males from the effluent channel had detectable serum VTG concentrations, but male fish from the Minnesota River did not; this suggests that the elevated E/A ratios were not responsible for that increase. These findings indicate that vitellogenesis in the male fish from the sewage effluent channel was not induced by endogenous estrogens. Although we did not determine which chemical(s) was responsible for depressing serum T concentrations, phytoestrogens such as 3-sitosterol found in pulp mill effluent (10,40) are capable of dramatically reducing serum T concentrations by altering cholesterol availability (41). The E/A ratio appears to be a sensitive marker of abnormal serum sex steroid concentrations; however, when taken alone, the E/A ratio has little functional significance because normal ranges for this parameter have not been established for most wildlife species. Circulating sex steroid concentrations vary significantly in fish and other vertebrates throughout the annual reproductive cycle (42). Although the E/A ratio or serum sex steroid concentrations can be informative of contaminant exposure during embryonic development (43,44) and adulthood (10,41,42), samples must be collected in such a way as to minimize naturally occuring variation. Used in collaboration with serum VTG analysis, the serum sex steroid concentrations have provided important insight into the mechanism by which male fish are induced to express VTG. Although we have focused on induced VTG production in male fish exposed to chemical contaminants, earlier field studies reported either no change [winter flounder (45)] or decreases [English sole (46,47)] in serum VTG levels in female fish exposed to polynuclear aromatic hydrocarbons (PAHs) and polychlorinated biphenyls (PCBs). Similarly, several laboratory studies have shown decreases or no change in E2 and VTG synthesis in female fish exposed to acid, metals, cyanide, pesticides, PCBs, and PAHs (48)(49)(50)(51)(52)(53)(54). In females, elevated levels of various estrogen mimics could stimulate negative feedback in gonadotropin secretion, resulting in suppressed synthesis of endogenous estrogens, and thus, reduced VTG synthesis. Likewise, estrogen mimics that are weaker could occupy hepatic estrogen receptors but stimulate a lower level of VTG gene expression-an antiestrogenic effect. Our results show that the sewage Environmental Health Perspectives * Volume 104, Number 10, October 1996 effluent did not enhance endogenous estrogen synthesis, but it appears to act directly on the estrogen receptor (ER) or possibly through the disruption of other hormones such as the thyroid hormones, growth hormone, prolactin, or cortisol known to enhance hepatic yolk protein synthesis (55)(56)(57)(58). For example, a variety of pesticides, PCBs, and acidified water have been shown to alter circulating concentrations of triiodothyronine (T ) and thyroxine (T4) in fish (59)(60)(61)(62). Tsyroid hormones are known to potentiate estrogen induction of VTG mRNA and stimulate synthesis of estrogen receptors in frog hepatocytes (58). Likewise, contaminants at higher concentrations are capable of producing a generalized stress response with accompanying increases in circulating levels of cortisol. Cortisol has been shown to cause rapid but transient increases in male Oreochromis VTG mRNA (63), decreased E2 secretion in isolated rainbow trout ovarian follicles (64), increased serum binding capacity, decreased cytosolic and nuclear binding sites with no change in circulating E2 levels in immature female rainbow trout (65), and suppressed plasma VTG concentrations in female catfish (66). Future studies must begin to examine a wide range of hormones to determine the various mechanisms by which males are stimulated to express elevated serum concentrations of VTG. This study confirms similar observations from studies with caged fish in Great Britain (23,24) and indicates that similar problems exist in the United States, even with state-of-the-art sewage treatment plants. Future studies should focus on how such changes in serum sex steroid and VTG concentrations relate to reproductive dysfunction in individuals and consequential changes in populations.