The Society for Integrative and Comparative Biology
The Freshwater Mussel (Elliptio complanata) as a Sentinel Species: Vitellogenin and Steroid Receptors1
1 Department of Biology, Boston University, Boston, Massachusetts 02215 USA
2 Laboratory of Integrative Aquatic Biology, Field Science Center, Graduate School of Agricultural Science, Tohoku University, 15 Konorihama-mukai, Onegawa, Oshika 986-2242, Japan
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SYNOPSIS |
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Freshwater mussels, Elliptio complanata were collected from a reference and pollutant-impacted pond on Cape Cod, MA. Glutathione-S-transferase (GST) activity was measured in gill, hepatopancreas and foot. In addition, content of seven heavy metals were measured in whole bodies. GST activity was significantly elevated in hepatopancreas and foot, as was whole body cadmium level in animals from the contaminated site suggesting that these animals have been exposed to organic and inorganic contaminants. Sodium dodecyl acrylamide gel electrophoresis (SDS-PAGE) analysis showed putative vitellogenins with molecular weight 180 and 205 kDa bands only in the ovary. In non-denatured gel electrophoresis ovarian extracts revealed two higher molecular weight bands at 550 and 700 kDa, which were reproductive stage specific. Western blotting of SDS-PAGE and non-denatured gels using the anti-scallop yolk-protein antibody confirmed the presence of cross-reacting bands of the same molecular weights in the ovary but not other tissues. Although several experiments involving steroid hormone exposure were done, no significant changes in vitellogenin protein levels were observed. However, using an anti-human ERß antibody, ERß positive bands were observed both in female foot, and the ovary. No cross reactivity with the antibody was observed in hepatopancreas. Additional studies are required to resolve questions of vitellogenin regulation and the role of (xeno)estrogens in bivalve molluscs.
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INTRODUCTION |
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For a number of years we have studied the possible impact of xenobiotic contamination from a Superfund site, the Massachusetts Military Reservation (MMR), on local biota, and possibly humans. Contaminated ground water plumes which intersect with freshwater ponds, and probably impact the quality of residential well water, have been well described as to source, and flow of many contaminants (Air Force Center for Environmental Excellence AFCEE, 2003
). We have described reproductive impairment and evidence of xenobiotic exposure in a sub-population of freshwater turtles (Chrysemys picta; Rie et al., 2001; Kitana et al., 2003, 2004; Rie et al., 2005) in this area. Given the long-lived nature of these turtles, and low population number, we have developed other sentinel species for field and laboratory studies (see paper by Novillo et al., 2004; this symposium, for work on C. elegans). Here we report studies of Elliptio complanata, a species endemic to the fresh water ponds on Cape Cod in the vicinity of the MMR. This species is suitable as a model for both laboratory and field work because of its life history and exposure to the fresh water aquatic environment; as a filter feeder and bottom dweller it is likely to be exposed to all xenobiotics in the water and sediment.
Combining "sentinel species" with specific biomarkers provides important biological information on the potential impact of xenobiotics on the health of organisms and ecosystems (Van der Oost et al., 1997). Various biomarkers have been measured in mollusks and specifically in E. complanata, such as vitellin-like proteins, metallothionein-like proteins (MT), lipid peroxidation, protein-free DNA strands and glutathione S-transferase (GST) (Gagne et al., 2001a, 2004; Hoarau et al., 2004). In this study, we report on glutathione S-transferase (GST) activity and vitellogenin-like proteins of E. complanata as biomarkers of exposure to contaminant mixtures in the field, and our attempts to date to develop yolk protein synthesis as a bioindicator of xenoestrogen exposure in this species
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MATERIALS AND METHODS |
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Collection sites and maintenance of mussels
Freshwater mussels were collected from Johns Pond and Masphee Pond on Cape Cod, MA. The two ponds are located in the area surrounding the MMR and are used for human recreation. For our studies Masphee Pond is considered to be a potentially non-impacted site, and Johns Pond is considered to be a site impacted by intersecting contaminated ground water plumes. E. complanata gametes develop during winter and early spring (the vitellogenic period), and then are released between late spring and early summer. Zygotes develop within the mantle cavity and glochidia larvae are released in the early summer. Mussels from these ponds were collected by hand between early spring and late fall and returned to the laboratory. All mussels were maintained in an environmental chamber with an aeration system at 16°C and a 12 hr light/12 hr dark light cycle in the Department of Biology Aquarium Facility at Boston University. Mussels were kept in dechlorinated water and fed every other day with cultured green algae (Selenastrum capricornutum). All mussels were allowed to depurate at least for 24 hours in the aquarium before being used for experiments or assays.
Histology
The whole bodies of mussels at the vitellogenic stage were fixed in 10% neutral buffered formalin for 72 hours, and dehydrated through increasing concentrations of ethanol and embedded in paraffin. Serial sections (thickness, 5 µm) were cut, and stained with hematoxylin and eosin.
Preparation of tissue for metals analysis
After removal of the shell, whole unfixed mussels were used for metals analysis. Samples were stored in sterile polyethylene centrifuge tubes at –20°C until further analysis. Tissue samples (approximately 0.2 g, wet weight) were accurately weighed into acid cleaned Teflon vials and acid digested using 4 ml of concentrated nitric acid. Samples were digested in capped vials for 3 hours at 90°C. Vial caps were removed and tissues were dried at 90°C for 3 additional hours or until a residue formed. Residues were then diluted with 20 mls of 3% HNO3. The diluent was filtered using an acid-cleaned 60 cc syringe through a 0.45 µm filter into an acid cleaned 30 ml polyethylene storage bottle until further analysis. Blanks (1 per 20 digested tissue samples) consisting of nitric acid only were digested as described above and analyzed along with the digested tissue samples.
Tissue metals analysis
Tissue digests were analyzed for Cu, Cr, Mn, Ni and Zn by ICP-ES (inductively coupled plasma emission spectrometry) using a Jobin-Yvon JY170C combined simultaneous-sequential instrument. The following wavelengths were used: Cr (267.71 nm); Cu (324.75 nm); Mn (257.61 nm); Ni (231.60 nm); and Zn (213.85 nm). Cadmium and lead were analyzed using graphite furnace atomic absorption spectrophotometer (GFAA) (Perkin Elmer 5100), with Zeeman background correction. Samples were analyzed in duplicate and a mean value was calculated. A matrix modifier was used to stabilize sample matrix effects (0.6 mg/ ml Mg and 0.4 mg/ml Pb in 0.2% nitric acid). Blanks consisting of 3% HNO3 were analyzed every 10 samples to determine background and machine drift. A standard reference material (SRM, NIST #1974a, mussel) was digested as above and analyzed to verify the reliability of sample analysis. The standard curve range used was 0–11 µg/L for Cd and Pb and the limit of detection was 0.05 µg/L. Values were obtained as µg metal/L of sample, and were converted to µg/g based on the exact amount of tissue digested (g, wet weight).
Glutathione-S-transferase assay
Glutathione-S-transferase (GST) assay was performed on foot, gill and hepatopancreas from mussels collected from Masphee and Johns Pond by the method of Habig et al. (1974). The reaction mixture consisted of 1 mM 1,2 chlorodinitrobenzene (CNDB) dissolved in 100 mM Tris-HCl pH 7.4, 1 mM glutathione and 10 µl of the cytosolic fraction in 1 ml total volume. The activity of GST was determined at 340 nm at 25°C over 5 minutes. Data are expressed as µmol/ min/mg of cytosolic protein. Reduced glutathione was purchased from Sigma (St Louis, MO).
In vivo exposure of Elliptio complanata to vertebrate steroids
In the laboratory, mussels from Johns Pond were exposed to several steroid hormones at different concentrations (see Table 1): estrogen (17ß-estradiol, E), testosterone (17ß-hydroxy-4-androsten-3-one, T), progesterone (4-pregnene-3, 20-dione, P), 5-
-androstan-3ß, 17ß-diol (A), and coprostanol (5ß-cholestan-3ß-ol, C). E, T, and P were purchased from Fisher Biotech (Fair Lawn, NJ), and A and C from Sigma (St. Louis, MO). Mussels were exposed to steroid hormones by injection into either the foot or adductor muscle, or by addition to the water. Either ethanol or DMSO was used as hormone solvents depending on the exposure route. Control mussels were exposed to the proper volume of ethanol or dimethyl sulfoxide (DMSO) vehicle depending on the experiment. A total of four independent experiments were performed considering several variables such as: duration of treatment, method of exposure, physiological stage of mussels and steroid hormone tested (Table 1). When water was the exposure medium, water was changed at 2–3 day intervals. In Experiments 2 and 3, 25 µl of the different hormones per 40 g of total body plus shell weight were injected to each mussel. In Experiment 4, hormone doses were injected weekly for total exposure period of 30 days. At the end of the exposure period all mussels were sacrificed and sexed under a light microscope. Gonads, hepatopancreas, foot, gills were dissected and frozen in dry ice and stored at –70°C until needed.
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Protein extraction
For yolk protein extraction dissected gonads and hepatopancreas from mussels were homogenized in 0.05 M Tris-HCl buffer pH 7.5 with 0.1 M NaCl and protease inhibitors cocktail (containing ethylenediaminetetraacetic acid [EDTA], 4-[2-aminoethyl]benzenesulfonyl fluoride [AEBSF], bestatin, E-64, leupeptin, and aprotinin)(Sigma, St.Louis, MO), and then centrifuged at 4°C 12,000 x g for 30 min. The supernatant fraction was saved at –70°C until needed.
For extraction of estrogen receptor-like proteins, gonad, hepatopancreas and foot tissues were homogenized in extraction buffer of 50 mM Tris-HCl buffer, pH 7.4, containing 1 mM EDTA, 12 mM monothioglycerol and 30% (w/v) glycerol (buffer H), and a protease inhibitor cocktail (Sigma, St. Louis, MO). Tissue samples were weighed and protein extraction buffer was added in a ratio of 3:1 buffer/tissue. After homogenization, samples were centrifuged at 4°C for 10 minutes at 3,000 x g. The supernatant was centrifuged at 4°C at 100,000 x g for 1 hr to collect the final supernatant (cytosolic fraction). The samples were stored in aliquots at –70°C for further analysis.
Total protein concentration was determined by the Bradford method using a microplate reader (Bradford, 1976; Redinbaugh and Campbell, 1985), and bovine serum albumin was used as standard.
Electrophoresis and western blotting analysis
Yolk protein extracts were resolved by a 7.5% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE; Laemmli, 1970) and/or 4–15 or 4– 20% gradient gels (Bio-Rad, Hercules, CA), and 20 µg of gonad extract was loaded in each lane. Non-denatured polyacrylamide gel electrophoresis was carried out according to Davis (1964) with 4–20% gradient gels. To compare the molecular weights, 5 µg of 1 mg/ml thyroglobulin (MW 669,000), apoferritin (443,000), ß-amylase (200,000), and alcohol dehydrogenase (150,000) were used as standards in denatured gels. To detect estrogen receptor-like proteins 10% SDS-PAGE was prepared and 50 µg of total protein was loaded in each lane. Wide range markers (Sigma, St. Louis, MO) were used as standards. Proteins were transferred to nitrocellulose membrane (Towbin et al., 1979) using mini-transblot (Bio-Rad, Hercules, CA).
For immunodetection of yolk proteins, an anti-scallop yolk protein antibody and anti-rabbit IgG conjugated to alkaline phosphatase (Sigma, St. Louis, MO) were used. The membranes were developed using NBT-BCIP substrate system (Promega, Madison, WI). The intensity of yolk protein bands by Western blotting was measured using the ImageJ program, and values were normalized with respect to cytosolic proteins added in wells. For immunodetection of estrogen receptor-like protein, a polyclonal anti-human ERß antibody (H-150, Santa Cruz Biotechnology, Santa Cruz, CA) was used as the primary antibody and a horseradish peroxidase conjugated anti-rabbit IgG antibody (Sigma, St. Louis, MO) was the secondary antibody. For detection ECL reagents were used (Amersham Biotechnology, Rockville, MD). Cytosolic fractions of thymus (mice) and HeLa cells were used as positive and negative control, respectively (Courtesy of Dr. P. Gomez, Harvard Univ., MA).
Statistical analysis
Data was analyzed using 1-way ANOVAs and t-tests using Sigma Stat, Ver 2.03 (Jandel Scientific). Differences were accepted as significant at the level of P < 0.05.
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RESULTS |
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Histology of the gonad (Fig. 1A–E)
As is shown in the Figure 1A–E, the gonadal tissue from Elliptio complanata is composed of cysts or nests of developing oocytes or sperm surrounded by accessory/auxiliary cells. One hermaphroditic animal was found in fifteen animals used for histological studies. In this gonad an area of oocyte development was surrounded by testicular tissue with sperm.
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Glutathione-S-transferase (GST) activity (Fig. 2)
GST, a phase II conjugation enzyme, was examined in hepatopancreas, foot and gills of mussels (Fig. 2). Its activity was significantly elevated in both hepatopancreas and foot (P < 0.05) in animals from Johns Pond compared to Mashpee Pond. The mean GST activity in the gills of mussels from the two ponds studied was not different.
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Heavy metal levels in several tissues of Cape Cod fresh water mussels Elliptio complanata (Table 2)
The levels of seven heavy metals were measured in whole mussel tissue (Table 2). Of the heavy metals, only cadmium was significantly (P < 0.05) elevated in Johns Pond mussels compared to Mashpee Pond animals. Levels of other metals (Cu, Cr, Pb, Mn, Ni and Zn) were not different between mussels of two ponds studied.
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Identification of yolk proteins in Elliptio complanata (Figs. 3 and 4)
Males and females were sexed on the basis of fresh tissue examination of gonadal tissue under the microscope at autopsy, using the presence of oocytes or sperm as indicators. Protein extracts of both sexes, from the gonads and hepatopancreas were resolved by SDS-PAGE; two large molecular weight bands (180 and 205 kDa) were found only in the ovary (Fig. 3A), but not in testis or hepatopancreas, of either sex, in reproductively active animals. Further, non-denatured PAGE of ovarian extracts showed one or two higher molecular weight bands (550 and 700 kDa) depending upon the stage of the reproductive cycle (Fig. 4A). The 550 kDa band becomes stronger as maturation of the ovaries progresses. Western blotting of SDS-PAGE and non-denatured gels using the anti-scallop yolk-protein antibody confirmed the presence of cross reacting bands of the same molecular weights only in the ovary of reproductively active animals (Figs. 3B and 4B).
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Exposure of Elliptio complanata to steroids (Table 1 and Fig. 5)
In vivo exposure of mussels collected from Johns Pond, Cape Cod, MA to several vertebrate steroids did not significantly alter yolk protein expression as assessed by western-blot analysis with anti-scallop yolk protein antibody (Table 1 and Fig. 5). In four separate experiments performed as described in Materials and Method and summarized in Table 1, no significant changes in vitellogenin protein levels were observed, regardless of length of exposure, dosage level or injection protocol (data from a representative experiment is shown in Fig. 5).
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Detection of an estrogen receptor-like protein in Elliptio complanata tissues (Fig. 6)
Protein extracts from different mussel tissues (foot, hepatopancreas and gonads) were resolved by SDS-PAGE, and immunodetection of estrogen receptor-like proteins was performed by Western blot analysis using a polyclonal anti-human ERß antibody. Strong ERß positive bands were observed in both female foot and ovary (Fig. 6). No cross reactivity was observed in the hepatopancreas (Fig. 6). In order to confirm the specificity of the antibody, cytosolic extracts of mouse thymocytes and HeLa cells were used as positive and negative controls, respectively. The smallest band detected in the foot and thymocytes was at the approximate molecular weight for the ERß protein (55 kDa) of vertebrates. Additional larger bands were seen in the foot and ovary (75 and 165 kDa).
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DISCUSSION |
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The freshwater mussel, a bottom dweller and filter feeder, is sedentary, abundant, and long-lived. Although consistently exposed to the contaminants that accumulate from water or sediment, they are quite resistant to contamination (Halvik and Marking, 1987
). For these reasons, marine mussels have been used as biosensors in environmental monitoring studies for over 30 years. In 1978 "International Mussel Watch" using Mytilus edulis was established to monitor pollution levels in U.S. coastal waters. The eastern elliptio (Elliptio complanata), as a freshwater species, is also well placed and widely distributed to provide a freshwater counterpart of the blue mussel. This species has been used to monitor mercury contamination in the Sudbury River watershed, MA (Mierzykowsky and Carr, 2001) and to evaluate the effects of endocrine disrupting chemicals in municipal effluents at St. Lawrence River, Quebec, Canada (Gagne et al., 2001a; Gagne et al., 2004). In this study, we provide evidence for site-specific differences in the heavy metal load and in activity level of GST using Elliptio complanata from Cape Cod, MA. Mussels from the contaminated site accumulate significantly more cadmium than the reference site mussels, and GST level was also significantly higher in these mussels. In this study, there is no clear evidence for the correlation between high GST and high cadmium level in mussels. Other authors, however, have shown a positive correlation between metalothionein levels (MT) and GST activity in digestive glands of Elliptio complanata placed downstream from a municipal effluent. These data suggest that GST activity may be modulated by the presence of heavy metals (Gagne et al., 2004). Further, cadmium injection experiments in vertebrates increased GST level (Ognjanovic et al., 1995; Casalino et al., 2004). The high levels of GST activity, plus high levels of whole body cadmium in mussels indicate that exposure to different organic and inorganic chemicals occurs at the study site. The data are consistent with reports (AFCEE, 1998) that Johns Pond is influenced by contaminated ground water plumes from the MMR.
Beyond assessment of local Cape Cod mussel populations for classical indicators of toxic exposure as determined by heavy metal and GST, we and others (Gagne et al., 2001b, c) have attempted to use vitellogenin as a biomarker of exposure to xenoestrogens, as in vertebrates (Sumpter and Jobling, 1995). The studies described here have added to our basic understanding of the site of vitellogenin synthesis/secretion in bivalve molluscs, as well as identity and molecular size in E. complanata. Using SDS-PAGE and western-blot analysis we have identified two specific yolk protein bands (molecular weight 180 and 205 kDa) in the ovary of Elliptio complanata. In addition, the level of expression of the yolk proteins appears to be reproductive stage specific. An intense band at 205 kDa was found in gonadal extracts from females classed as preovulatory versus 180 kDa from females classed as postovulatory (based on the presence of advanced glochidia larvae in the gills). These data suggest the presence of processing/translational variants or several yolk protein genes which are active at different times. Other invertebrates, such as C. elegans express several vitellogenin genes (Blumenthal et al., 1984). No cross-reactivity with anti-scallop yolk protein antibody was found in the testis or hepatopancreas in either sex (data not shown). Native gels also demonstrated sex- and stage-specific cross reacting proteins at 700 kDa (preovulatory) and 550 kDa (postovulatory). It is possible, therefore, that vitellogenin exists normally as a polymer or is further glycosylated in the ovary.
Vitellogenins and yolk proteins of molluscs have been isolated and characterized in only a few species. The native molecular weight of vitellogenin in E. complanata is similar to that reported for other molluscs, such as Pacific Oyster, Crassostrea gigas (500 kDa; Suzuki et al., 1992) and Japanese Scallop, Patinopectin yessoensis (450 to 700 kDa; Osada et al., 1992). That we only identified yolk proteins in gonadal tissue agrees with evidence that bivalves synthesize vitellogenin within the gonad via a heterosynthetic mechanism. Using immunocytochemical methods Osada et al. (2003) showed in P. yessoensis that the auxiliary cells adjacent to the developing oocytes are the source of the yolk granules in the oocytes. In the same study, in situ hybridization experiments demonstrated the expression of vitellogenin mRNA in the auxiliary cells closely associated with growing oocytes. The authors concluded that synthesis of a major yolk protein occurs via a heterosynthetic pathway de novo in the ovary of the Japanese scallop (Osada et al., 2004).
Earlier studies, based on histological evidence, suggest that vitellogenesis in the marine mussel Mytilus edulis (Pipe, 1987) and Pacific oyster Crassostrea gigas (Suzuki et al., 1992) was homosynthetic, occurring within the oocytes. Further, Eckelbarger and Davis (1996) suggested that vitellogenesis in the Eastern Oyster, Crassostrea virginica, occurs in both the developing oocyte and the hemocoel. Our experiments support the idea that synthesis of vitellogenin occurs in the ovary of E. complanata, possibly first as a 700 kDa protein which is processed to 550 kDa during oocyte maturation. Molluscs thus appear to be different from other invertebrates, (C. elegans, arthropods; Wahli, 1988) in which vitellogenin is synthesized outside the ovary and transported via the hemocoel or blood, as in vertebrates (Byrne et al., 1989).
In order to develop vitellogenin as a biomarker for xenobiotic exposure and endocrine disruption in invertebrates, an understanding of the hormonal control mechanisms is necessary. Vitellogenin synthesis and its regulation in molluscs are not yet understood, but there is some evidence that estradiol-17ß and some xenobiotics may up-regulate an alkali labile phosphoprotein (Gagne et al., 2001b, c). In addition, exposure to estradiol has been reported to increase vitellogenin synthesis in the Japanese scallop (by injection; Matsumoto et al., 1997) and Pacific Oyster (by addition to sea water; Li et al., 1998). In earlier studies Mori et al. (1969) observed that estradiol injected into the gonad accelerates sexual maturation in female Pacific oysters. These studies indicate that estradiol may be a major regulatory factor in vitellogenin synthesis and reproduction in molluscs.
In our studies so far, in vivo exposure of Elliptio complanata to vertebrate steroids or compounds which may yield bioactive steroids via metabolism, such as coprostanol, did not significantly alter vitellogenin levels under several exposure paradigms using mussels at different reproductive stages. Similar studies performed in E. complanata by Gagne et al. (2001b), showed that after exposure to relatively low concentrations of estradiol (50 nM) for 72 hours, the level of alkali labile phosphoprotein increased (vitellin-like proteins). The discrepancy in results between the two studies can be explained by several factors: a) our exposure experiments used lower doses of estradiol (<30 nM) compared to Gagne et al. (2001b) who obtained an effect at 50 nM, b) other studies have used the relative non-specificity of the method used to measure vitellogenin, which was not specific alkali labile phosphoprotein as an indicator of vitellogenin c) It is also possible that the studies of Gagne et al. (2001b, c) may in fact represent changes in other estrogen-sensitive proteins not detected by this antibody.
Of importance to these studies, estradiol was found by HPLC and radioimmunoassay in the gonadal tissues of the marine mussel M. edulis (Zhu et al., 2003) and steroid (estrogen and progesterone) receptors have been identified in Octopus vulgaris gonadal tissues using classical methods (Di Cosmo et al., 2001). Cytosolic and membrane bound forms of ERß (55 kDa) have been identified in M. edulis (Stefano et al., 2003). Although we have not demonstrated estrogenic action in E. complanata, using Western blot, we demonstrate a protein that cross-reacts with anti human ERß antibody in the gonad and foot, but not hepatopancreas. Three distinct bands at 165 kDa, 75 kDa and 55 kDa were seen in the foot, whereas only two higher molecular weight bands (165 and 75 kDa) were seen in the gonad. Female hepatopancreas was negative. The 55-kDa-protein band may represent the ERß band in the control tissue (thymocytes); which also correlates with the major band found in M. edulis and vertebrate tissues (Stefano et al., 2003).
In conclusion, it should be emphasized that E. complanata is of value as a freshwater bioindicator of xenobiotic contamination. However, exposure to low doses of estradiol (from 6.81 pg/animal to 68.1 ng/ animal) for 72 hours and high doses (2.5 mg/animal) for 30 days was not sufficient to elicit significant changes in vitellogenin protein in the gonad. Results obtained with vertebrate ERß antibody must be interpreted with caution until steroid receptors and the enzyme aromatase are definitively identified using stringent molecular techniques. Nonetheless, the convergence of invertebrate endocrinology, endocrine disruption, toxicology, and environmental signaling promises exciting new findings.
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ACKNOWLEDGMENTS |
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This work was supported by EPA ES 07381 to Ian P. Callard. Apolonia Novillo was supported by a postdoctoral fellowship from Ministry of Science and Technology of Spain.
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FOOTNOTES |
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1 From the Symposium EcoPhysiology and Conservation: The Contribution of Endocrinology and Immunology presented at the Annual Meeting of the Society for Integrative and Comparative Biology, 5–9 January 2004, at New Orleans, Louisiana.
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References |
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SYNOPSISINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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Air Force Center for Environmental Excellence/Massachusetts Military Reservation., 1998. Final Ecological Quarterly Data Summary Report: Summer 1996. AFCEE/MMR Installation Restoration Program.
Air Force Center for Environmental Excellence/Massachusetts Military Reservation., 2003. Draft Final 2nd Five-Year Review, 1998–2002 Massachusetts Military Reservation (MMR) Superfund Site Otis Air National Guard Base, MA. 2003. AFCEE/ MMR Installation Restoration Program.
Blumenthal, T., M. Squire, S. Kirtland, J. Cane, M. Donegan, J. Spieth, and W. Sharrock. 1984. Cloning of a yolk protein gene family from Caenorhabditis elegans. J. Mol. Biol, 174:1-18.[CrossRef][Medline]
Bradford, M. M. 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem, 72:248-254.[CrossRef][ISI][Medline]
Byrne, B. M., M. Gruber, and G. Ab. 1989. The evolution of egg yolk proteins. Prog. Biophys. Molec. Biol, 53:33-69.[CrossRef][Medline]
Casalino, E., C. Sblano, V. Landriscina, G. Calzaretti, and C. Landriscina. 2004. Rat liver glutathione S-transferase activity simulation following acute cadmium or manganese intoxication. Toxicology, 200:29-38.[Medline]
Davis, B. J. 1964. Disc electrophoresis—II. Method and application to human serum proteins. Ann. N.Y. Acad. Sci, 121:404-427.[ISI][Medline]
Di Cosmo, A., C. Di Cristo, and M. Paolucci. 2001. Sex steroid hormone fluctuations and morphological changes of the reproductive system of the female of Octopus vulgaris throughout the annual cycle. J. Exp. Zool, 289:33-47.[CrossRef][ISI][Medline]
Eckelbarger, K. J., and C. V. Davis. 1996. Ultrastructure of the gonad and gametogenesis in the eastern oyster, Crassostrea virginica, I. Ovary and oogenesis. Mar. Biol, 127:79-87.[CrossRef]
Gagne, F., C. Blaise, M. Salazar, S. Salazar, and P. D. Hansen. 2001a. Evaluation of estrogenic effects of municipal effluents to the freshwater mussel Elliptio complanata. Comp. Biochem. Physiol. Pt.C, 128:213-225.[CrossRef]
Gagne, F., C. Blaise, B. Lachance, G. I. Sunahara, and H. Sabik. 2001b. Evidence of coprostanol estrogenicity to the freshwater mussel Elliptio complanata. Environ. Poll, 115:97-106.[Medline]
Gagne, F., D. J. Marcogliese, C. Blaise, and A. D. Gendron. 2001c. Occurrence of compounds estrogenic to freshwater mussels in surface waters in an urban area. Environ. Toxicol, 16:260-268.[Medline]
Gagne, F., C. Blaise, and J. Hellou. 2004. Endocrine disruption and health effects of caged mussels, Elliptio complanata, placed downstream from primary-treated municipal effluent plume for 1 year. Comp. Biochem. Physiol. Pt.C, 138:33-44.[CrossRef]
Habig, W. H., M. J. Pabst, and W. B. Jacoby. 1974. Glutathione-S-transferase, the first step enzymatic step in mercapturic acid formation. J. Biol. Chem, 249:7130-7139.
Halvik, M. E., and L. L. Marking. 1987. Effects of contaminants on naiad mollusks (Unionidae): a review, v. 164. U.S. Department of the Interior, Fish and Wildlife Service Resource Publication.
Hoarau, P., G. Garello, M. Gnassia-Barelli, M. Romeo, and J. P. Girard. 2004. Effect of three xenobiotic compounds on Glutathione S-Transferase in the clam Ruditapes decussates. Aquat. Toxicol, 68:87-94.[Medline]
Kitana, N., S. J. Won, and I. P. Callard. 2003. Potential endocrine and reproductive disruption by xenobiotic in freshwater turtles (Chrysemys picta) from Cape Cod. Massachusetts Superfund Basic Research Program annual meeting.
Kitana, N., S. J. Won, and I. P. Callard. 2004. Environmental and hormonal impacts on reproduction and development in the freshwater turtles. Society for Integrative and Comparative Biology 2004 annual meeting.
Laemmli, U. K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature, 227:680-685.[CrossRef][Medline]
Li, Q., M. Osada, T. Suzuki, and K. Mori. 1998. Changes in vitellin during oogenesis and effect of estradiol-17beta on vitellogenesis in the Pacific oyster, Crassostrea gigas. Invertebr. Repr. Dev, 33:87-93.
Matsumoto, T., M. Osada, Y. Osawa, and K. Mori. 1997. Gonadal estrogen profile and immunohistochemical localization of steroidogenic enzymes in the oyster and scallop during sexual maturation. Comp. Biochem. Physiol. Pt. B, 118:811-817.[CrossRef]
Mierzykowsky, S. E., and K. C. Carr. 2001. Total mercury and methyl mercury in freshwater mussel (Elliptio complanata) from the Sudbury River Watershed, Massachusetts. U.S. Fish and Wildlife Service Maine Field Office.
Mori, K. 1969. Effect of steroid on oyster—IV. Acceleration of sexual maturation in female Crassostrea gigas by estradiol-17ß. Bullet. Jap. Soc. Sci. Fish, 35:1077-1079.
Novillo, A., S. J. Won, C. Li, and I. P. Callard. 2004. Changes in nuclear receptor and vitellogenin gene expression in response to steroids and heavy metal in Caenorhabditis elegans. Integr. Comp. Biol45:61-71.
Ognjanovic, B., R. V. Zikic, A. Stajn, Z. S. Saicic, M. M. Kostic, and V. M. Petrovic. 1995. The effects of selenium on the antitoxidant defense system in the liver of rats exposed to cadmium. Physiol. Res, 44:293-300.[Medline]
Osada, M., T. Unuma, and K. Mori. 1992. Purification and characterization of a yolk protein from the scallop ovary. Nippon Suisan Gakkashi, 58:2283-2289.
Osada, M., T. Takamura, H. Sato, and K. Mori. 2003. Vitellogenin synthesis in the ovary of scallop, Patinopecten yessoensis: Control by estradiol-17ß and the central nervous system. J. Exp. Zool. Pt. A, 299:172-179.
Osada, M., H. Masahiko, M. Kishida, and A. Kihima. 2004. Molecular cloning and expression analysis of vitellogenin in scallop, Patinopecten yessoensis (Bivalva, Mollusca). Mol. Reprod. Dev, 67:273-281.[Medline]
Pipe, R. K. 1987. Oogenesis in the marine mussel Mytilus edulis: An ultrastructural study. Mar. Biol, 95:405-414.[CrossRef]
Redinbaugh, M. G., and W. H. Campbell. 1985. Adaptation of the dye-binding protein assay to microtiter plates. Anal. Biochem, 147:144-147.[CrossRef][Medline]
Rie, M. T., K. A. Lendas, and I. P. Callard. 2001. Cadmium: Tissue distribution and binding protein induction in the painted turtle, Chrysemys picta. Comp. Biochem. Physiol. Pt. C, 130:41-51.[CrossRef]
Rie, M. T., K. A. Lendas, S. J. Won, and I. P. Callard. 2005. Reproductive endocrinology disruption in a sentinel species (Chrysemys picta) on Cape Cod, Massachusetts. Arch. Environ. Contam. Toxicol. (In press).
Stefano, G. B., P. Cadet, K. Mantione, J. J. Cho, D. Jones, and W. Zhu. 2003. Estrogen signaling at the cell surface coupled to nitric oxide release in Mytilus edulis nervous system. Endocrinology, 144:1234-1240.
Sumpter, J. P., and S. Jobling. 1995. Vitellogenesis as a biomarker for estrogenic contamination of the aquatic environment. Environ. Health Perspect, 103:173-178.
Suzuki, T., A. Hara, K. Yamaguchi, and K. Mori. 1992. Purification and immunolocalization of a vitellin-like protein from the Pacific oyster Crassostrea gigas. Mar. Biol, 113:239-245.
Towbin, H., T. Staehelin, and J. Gordon. 1979. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: Procedure and some applications. Proc. Natl. Acad. Sci. U. S. A, 76:4350-4354.
Van der Oost, R., E. Vindimian, P. J. van der Brink, K. Satumalay, H. Heida, and N. P. E. Vermeulen. 1997. Biomonitoring aquatic pollution with feral eel (Anguilla anguilla). III. Statistical analyses of relationships between contaminant exposure and biomarkers. Aquat. Toxicol, 39:45-75.[CrossRef]
Wahli, W. 1988. Evolution and expression of vitellogenin genes. Trend. Genet, 4:227-232.[CrossRef][Medline]
Zhu, W., K. Mantione, D. Jones, E. Salamon, J. J. Cho, P. Cadet, and G. B. Stefano. 2003. The presence of 17-beta estradiol in Mytilus edulis gonadal tissues: Evidence for estradiol isoforms. Neuroendocrinol. Lett, 24:137-140.[Medline]
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