Product Citations and Abstracts

Recent popular and scientific articles have reported the presence of estrogenic and other hormone mimicking chemicals in the environment and their potential for causing reproductive dysfunction in humans and wildlife. The purpose of this session was to present the best available, if not standard, analytical methods to assay for the effects of xenobiotic chemicals on a broad range of endocrine-mediated events, including reproduction, growth, development and stress responses in aquatic vertebrate and invertebrate animals.

Folmar, L.C., N.D. Denslow, K. Kroll, E.F. Orlando, J. Enblom, J. Marcino, C. Metcalfe and L.J. Guillette, Jr. 2001. Altered Serum Sex Steroids and Vitellogenin Induction in Walleye (Stizostedion vitreum) Collected near a Metropolitan Sewage Treatment Plant. Arch. Environ. Contam. Toxicol. 40(3):392-398. (ERL,GB 1044).

Several recent in situ studies have reported that domestic and mixed domestic/industrial sewage effluents contain one or more natural or anthropogenic estrogenic substances. Those studies examined caged or feral fish for the presence of the egg yolk precursor protein, vitellogenin (VTG), in the blood of male fish. We have previously reported that male, feral carp (Cyprinus carpio) obtained from the effluent channel of a major sewage treatment plant (STP) exhibited depressed serum testosterone (T) concentrations, as well as detectable levels of VTG. The present study examines male and female walleye (Stizostedion vitreum), a native species with a different life history and feeding habits, collected from the same Mississippi River locations below the St. Paul metropolitan STP. All male and female walleye collected from the effluent channel contained measurable levels of VTG in their blood. Males from that location also exhibited depressed serum T concentrations and elevated serum estradiol-17b (E2) concentrations compared with males from the Snake River reference site. Males obtained from Mississippi River Navigational Pool #2 (MRP-2), 3-20 miles downstream of the STP also exhibited reduced serum T concentrations, but showed no alterations in E2 concentrations or the presence of VTG in the serum. Females collected at the STP site had greatly elevated serum E2 concentrations, but serum T concentrations were not different from females collected in the Snake River. Our results demonstrate that the St. Paul metropolitan STP continues to relase an estrogenic effluent, capable of inducing VTG production and altering normal serum sex steroid concentrations in a commercially valuable, native fish, the walleye. Additional studies will be required to determine whether these observations portend long-term population level effects.

Folmar, Leroy C., George R. Gardner, Martin P. Schreibman, Lucia Magliulo-Cepriano, Lesley J. Mills, Gerald Zaroogian, Ruth E. Gutjahr-Gobell, Ramona Haebler, Doranne B. Horowitz and Nancy D. Denslow. 2001. Vitellogenin-Induced Pathology in Male Summer Flounder (Paralichthys dentatus). Aquat. Toxicol. 51(4):431-441. (ERL,GB 1084).

Male summer flounder (Paralichthys dentatus) were given two injections (initially and two weeks later) of 17 b-estradiol (E2) totaling 0.2 (2 x 0.1), 2.0 (2 x 1.0) or 20.0 (2 x 10.0) mg E2/kg body weight. Blood and tissue samples were collected 4, 6 and 8 weeks after the initial injection in the (2 x 0.1) mg/kg treatment, 4,6,8, and 15 weeks after the first injection in the (2 x 1.0) mg/kg treatment and at 4 weeks only in the (2 x 10.0) mg/kg treatment. Five of the 12 fish injected twice with 10.0 mg/kg were moribund before the first sampling period. Circulating levels of vitellogenin (VTG) in the blood of all E2-injected fish from all treatments were comparable with those concentrations found in the blood of wild male carp (Cyprinus carpio) and walleye (Stezostedion vitreum) previously collected near a sewage treatment plant (0.1 - 10.0 mg VTG/ml plasma). Excessive hyalin material accumulated in the livers, kidneys and testes of the treated fish. A portion of that material was identified as VTG by immunohistochemistry. The accumulation of VTG, and possibly other estrogen-inducible proteins, resulted in hepatocyte hypertrophy, disruption of spermatogenesis, and obstruction or rupture of renal glomeruli.

Folmar, Leroy C., Michael J. Hemmer, Nancy D. Denslow, Kevin Kroll, Jian Chen, Ann Cheek, Harold Richman, Hillary Meredith and E. Gordon Grau. 2002. Comparison of the Estrogenic Potencies of Estradiol, Ethynylestradiol, Diethylstilbestrol, Nonylphenol and Methoxychlor In Vivo and In Vitro. EPA/600/J-00/470. Aquat. Toxicol. 60(1-2):101-110. (ERL,GB 1123).

Five natural, pharmaceutical, or xenobiotic chemicals (17b-estradiol (E2), ethynylestradiol (EE2), diethystilbestrol (DES), methoxychlor (MXC), nonylphenol(NP)) were tested in two in vitro assays (yeast estrogen screen [YES], MCF-7 breast tumor cell proliferation (E-screen)], and compared with previously reported results from two in vivomale sheepshead minnow vitellogenin (VTG) production studies. The purpose of this investigation was to determine how accurately the two in vitro assays predicted responses observed in live animals. EC50 values for all five chemicals were approximately one order of magnitude less sensitive in the YES assay than in the MCF-7 assay. Based on the EC50 values, DES was 1.1 (YES) to 2.5 (MCF-7) times more potent in these receptor binding assays than was E2, while EE2 was slightly less potent than E2 in the YES assay (0.7) and nearly twice as potent (1.9) as E2 in the MCF-7 assay. EE2 and DES were of approximately equal potency in the 13-day sheepshead minnow VTG production bioassay. Both MXC and NP were 107 times less potent than E2 in the YES assay, MXC was 105 times less estrogenic than E2 in the MCF-7 assay, while both were approximately 100 times less potent than E2 in the live animal bioassay. The in vitro tests were substantially less sensitive (at least 1000 times) than the sheepshead minnow VTG assay for estimating estrogenic potency of the two xenobiotic chemicals, which suggests that in vitro-based, large-scale screening programs could potentially result in many false negative evaluations.

Folmar, Leroy C., Patrick Larkin, Michael J. Hemmer, Ariana J. Poston, H. Stephen Lee and Nancy D. Denslow. 2001. Use of DNA Macroarrays to Evaluate the Effects of Environmental Estrogens on Wildlife. In: Proceedings of the 2nd International Conference on Pharmaceuticals and Endocrine Disrupting Chemicals in Water, 9-11 October 2001, Minneapolis, MN. National Ground Water Association, Westerville, OH. 9 p. (ERL,GB 1143).

During the mid-1990s, several investigations in the United States and United Kingdom showed that wild fish of several species collected downstream of sewage treatment plants or industrial discharges presented expression of estrogen-responsive genes, or phenotypic sex reversal. Subsequently, numerous studies have shown up-regulation of vitellogenin and choriogenin proteins in male fish after exposure to natural estrogens, or estrogenic pharmaceutical, industrial and agricultural chemicals. In this study we have employed DNA macroarray technology to demonstrate a characteristic expression of several genes, including estrogen receptor a, vitellogenin a and b, choriogenins b and g, and transferrin in sheepshead minnows exposed to the natural estrogen, estradiol-17b.

Folmar, Leroy C. 1993. Effects of Chemical Contaminants on Blood Chemistry of Teleost Fish: A Bibliography and Synopsis of Selected Effects. EPA/600/J-93/062. Environ. Toxicol. Chem. 12(2):337-375. (ERL,GB 760). (Avail. from NTIS, Springfield, VA: PB93-168961)

The purpose of this article is to provide a bibliography of those publications which describe the effects of chemical contaminants (both organic and inorganic) on serum chemistry and hematology of teleost fish with a brief synopsis of those effects. Also included is a review of "normal" or "reference" values for various blood chemistry parameters and hormones measurable in a number of fish species.

Folmar, L.C., T. Moody, S. Bonomelli and J. Gibson. 1992. Annual Cycle of Blood Chemistry Parameters in Striped Mullet (Mugil cephalus L.) and Pinfish (Lagodon rhomboides L.) from the Gulf of Mexico. EPA/600/J-93/061. J. Fish Biol. 41(6):999-1011. (ERL,GB 771). (Avail. from NTIS, Springfield, VA: PB93-168953)

Annual cycle measurements were made on serum sodium (Na), potassium (K), chloride (Cl), iron (Fe), magnesium (Mg), calcium (Ca), carbon dioxide (CO2), total protein (TP), albumin (Albg), cholesterol (Chol), triglycerides (Trig), inorganic phosphorous (Pi), uric acid (Uric), blood urea nitrogen (BUN), creatinine (Crea), glucose (Glu), lactate dehydrogenase (LD-L), alkaline phosphatase (ALP), aspartate aminotransferase (AST), alanine aminotransferase (ALT) and creatine kinase (CK) in the striped mullet (Mugil cephalus L.) and the pinfish (Lagodon rhomboides L.). For each parameter, mean, standard deviation and coefficient of variation (C.V.) are reported. The lowest C.V.s were associated with the electrolytes and the greatest C.V.s with serum enzymes. The lowest variability for most parameters was observed in the vitellogenic and prespawning period.

Folmar, L.C., S. Bonomelli, T. Moody and J. Gibson. 1993. Effect of Short-Term Exposure to Three Chemicals on the Blood Chemistry of the Pinfish (Lagodon rhomboides). EPA/600/J-93/063. Arch. Environ. Contam. Toxicol. 24(1):83-86. (ERL,GB 784). (Avail. from NTIS, Springfield, VA: PB93-168979)

Injections of 3 ml/kg CCl4 caused significant elevations in the serum enzymes alanine aminotransferase (ALT), aspartate aminotransferase (AST), creatine kinase (CK) and lactate dehydrogenase (LD-L). Serum lipids and total protein were significantly lower, while serum glucose was significantly greater. Serum protein electrophoresis showed disassociation of albumin. Seawater species appear more tolerant of nitrite intoxication than freshwater species. Concentrations of fenthion as high as 30% of the 48-hr LC50 did not inhibit serum cholinesterase or alter serum chemistry.

Folmar, L.C., G.R. Gardner, J. Hickey, S. Bonomelli and T. Moody. 1993. Serum Chemistry and Histopathological Evaluations of Brown Bullheads (Ameiurus nebulosus) from the Buffalo and Niagara Rivers, New York. EPA/600/J-94/016. Arch. Environ. Contam. Toxicol. 25(3):298-303. (ERL,GB 830). (Avail. from NTIS, Springfield, VA: PB94-101599)

Cholangiomas and cholangiocarcinomas were observed in brown bullheads (Ameiurus nebulosus) collected from the Buffalo and Niagara Rivers (NY) and Old Woman Creek (OH). Significant increases in serum BUN, uric acid, triglycerides, inorganic phosphate, ALT, LDL, calcium and iron and a significant decrease in cholesterol were found in the fish from the Buffalo and Niagara Rivers compared to the Old Woman Creek reference area. When relationships between the pathology and serum chemistry measurements were tested by correlation and discriminant function analysis, no predictive relationships were apparent.

Folmar, L.C., J. Harshbarger, P.C. Baumann, G. Gardner and S. Bonomelli. 1995. Pathological and Serum Chemistry Profiles of Brown Bullheads (Ameiurus nebulosus) from the Black River and Old Woman Creek, Ohio. Bull. Environ. Contam. Toxicol. 54(1):50-59. (ERL,GB 842).

This study compares serum chemistry values for brown bullheads (Ameiurus nebulosus) from the industrially contaminated Black River, Ohio, with the rural and relatively uncontaminated Old Woman Creek, Ohio. Fifty-two percent (24 of 46) brown bullheads over 3-years-old had liver neoplasms: 30% (14) were of hepatocellular origin, not including 17% (8) that had foci of hepatocellular alteration and 48% (22) of cholangiocellular origin. Eleven fish had both types of liver tumors. In addition to liver tumors, four fish had squamous cell carcinomas, one had an epidermal papilloma and one had a mesothelioma. Only 1 of the 30 reference fish had a neoplasm of any type, a low grade cholangiocellular carcinoma. Of the 20 serum parameters measured, the mean values of nine were significantly different between the two populations. Seven of the nine were higher in the Black River fish (alanine and aspartate aminotransferase, lactate dehydrogenase, total protein, blood urea nitrogen, triglycerides and calcium). In contrast, manganese and inorganic phosphate were significantly lower. However, in individual fish, none of the serum parameters were significantly correlated with either type of liver neoplasm or either parasitosis.

Folmar, L.C., N.D. Denslow, R.A. Wallace, G. LaFleur, S. Bonomelli and C.V. Sullivan. 1995. Highly Conserved N-terminal Sequence for Teleost Vitellogenin with Potential Value to the Biochemistry, Molecular Biology and Pathology of Vitellogenesis. EPA/600/J-95/370. J. Fish Biol. 46(2):255-263. (ERL,GB 875).

N-terminal amino acid sequences for vitellogenin (Vtg) from six species of teleost fish (striped bass, mummichog, pinfish, brown bullhead, medaka, yellow perch and the sturgeon) are compared with published N-terminal Vtg sequences for the lamprey, clawed frog and domestic chicken. Striped bass and mummichog had 100% identical amino acids between positions 7 and 21, while pinfish, brown bullhead, sturgeon, lamprey, Xenopus and chicken had 87%, 93%, 60% 47%, 47-60% for four transcripts and had 40% identical, respectively, with striped bass for the same positions. Partial sequences obtained for medaka and yellow perch were 100% identical between positions 5 to 10. The potential utility of this conserved sequence for studies on the biochemistry, molecular biology and pathology of vitellogenesis is discussed.

Folmar, Leroy C., Nancy D. Denslow, Vijayasri Rao, Marjorie Chow, D. Andrew Crain, Jack Enblom, Joseph Marcino and Louis J. Guillette, Jr. 1996. Vitellogenin Induction and Reduced Serum Testosterone Concentrations in Feral Male Carp (Cyprinus carpio) Captured near a Major Metropolitan Sewage Treatment Plant. EPA/600/J-03/182. Environ. Health Perspect. 104(10):1096-1101. (ERL,GB 958).

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 compared to male carp collected from the St. Croix River, 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 which are biologically active as has been reported in Great Britain.

Folmar, Leroy C. 1996. Diagnostic Indicators for Aquatic Monitoring and Assessment Programs in the United States: Physiology and Biochemistry. In: Contaminant Effects on Fish: Symposium Proceedings: International Congress on the Biology of Fishes, San Francisco State University, July 14-18, 1996. Bruce Barton and Don MacKinlay, Editors. American Fisheries Society, Physiology Section, Bethesda, MD. Pp. 13-17. (ERL,GB 964).

During the past three decades, the scientific community and environmental regulatory agencies have focused attention on the long-term effects of xenobiotic chemicals on human and ecological health. Throughout the United States there have been numerous reports of chemically-contaminated lake, riverine and estuarine environments. The major sources of that contamination include: oil spills, point source dumping, sewage effluent, agricultural runoff and hazardous waste sites. In response to these concerns, several federal agencies established a variety of monitoring and assessment programs. Monitoring activities, current and past, have been dedicated to collecting information on chemical contaminants in sediment, water and edible flesh of fish or shellfish. In general, the emphasis of the early monitoring program was to estimate the risk of industrial and agricultural chemicals and effluents on human health with little consideration for the long-term effects of these chemicals on the ecosystems into which they were released. The more recent and current monitoring programs are more ecologically oriented.

Folmar, L.C., M. Hemmer, R. Hemmer, C. Bowman, K. Kroll and N.D. Denslow. 2000. Comparative Estrogenicity of Estradiol, Ethynyl Estradiol and Diethylstilbestrol in an In Vivo, Male Sheepshead Minnow (Cyprinodon variegatus), Vitellogenin Bioassay. Aquat. Toxicol. 49(1-2):77-88. (ERL,GB X963).

An in vivo bioassay for vitellogenin (VTG) synthesis was developed to screen individual chemicals or mixtures of chemicals for potentially estrogenic effects in a marine teleost model. An enzyme-linked immunosorbent assay (ELISA) was used to quantitate VTG synthesis in male sheepshead minnows (Cyprinodon variegatus) exposed to five concentrations of the natural estrogen (17b -estradiol), a synthetic, steroidal pharmaceutical estrogen (17a-ethynyl estradiol), or a synthetic, non-steroidal, pharmaceutical estrogen (diethystilbestrol) for 16 days. At an exposure concentration of 20 ng/l, only diethystilbestrol elicited a vitellogenic response. At all test concentrations greater than 100 ng/l, VTG appeared in the plasma in a dose-dependent manner for the three estrogen treatments. Liver VTG mRNA measurements were also made, exhibiting no clear correlations between quantities, nor temporal appearance of the message and mature protein were apparent. This assay is short-term, relatively inexpensive, shows a direct response, and easily quantitated.

A workshop titled "Using Sentinel Species Data to Address the Potential Human Health Effects of Chemicals in the Environmnet," sponsored by the U.S. Army Center for Environmental Health Research, the National Center for Environmental Assessment of the EPA, and the Agency for Toxic Substances and Disease Registry, was held to consider the use of sentinel and surrogate animal species data for evaluating the potential human health effects of chemicals in the environment. The workshop took a broad view of the sentinel species concept, and included mammalian and non-mammalian species, companion animals, food animals, fish, amphibians, and other wildlife. Sentinel species data included observations of wild animals in field situations as well as experimental animal data. Workshop participants identified potential applications for sentinel species data derived from monitoring programs or serendipitous observations and explored the potential use of such information in human health hazard and risk assessments and for evaluating causes or mechanisms of effect. Although it is unlikely that sentinel species data will be used as the sole determinative factor in evaluating human health concerns, such data can be useful as for additional weight of evidence in a risk assessment, for providing early warning of situations requiring further study, or for monitoring the course of remedial activities. Attention was given to the factors impeding the application of sentinel species approaches and their acceptance in the scientific and regulatory communities. Workshop participants identified a number of critical research needs and opportunities for interagency collaboration that could help advance the use of sentinel species approaches.

Hemmer, Michael J., Becky L. Hemmer, Chris J. Bowman, Kevin J. Kroll, Leroy C. Folmar, Dragoslav Marcovich, Marilynn D. Hoglund and Nancy D. Denslow. 2001. Effects of p-Nonylphenol, Methoxychlor and Endosulfan on Vitellogenin Induction and Expression in the Sheepshead Minnow, Cyprinodon variegatus. Environ. Toxicol. Chem. 20(2):336-343. (ERL,GB 1098).

Temporal and dose-response relationships of vitellogenin (VTG) mRNA induction and subsequent plasma VTG accumulation were established for sheepshead minnows (Cyprinodon variegatus) treated with p-nonylphenol (an alkylphenol) and the organochlorine pesticides methoxychlor and endosulfan. Thirty-two adult male fish per treatment were continuously exposed to measured concentrations of 0.64, 5.4, 11.8, 23.3 and 42.7 µg/L p-nonylphenol; 1.1, 2.5, 5.6, 12.1 and 18.4 µg/L methoxychlor; and in two separate tests, 15.9, 36.3, 68.8, 162, 277, 403, 590 and 788 ng/L endosulfan using an intermittent flow-through dosing apparatus. Separate triethylene glycol (50 µl/L) and 17b-estradiol (65.1 ng/L) treatments served as the negative and positive controls, respectively. Four fish were randomly sampled from each test concentration on days 2, 5, 13, 21, 25, and 42 of exposure and levels of hepatic VTG mRNA induction and serum VTG accumulation were determined for each individual. Overall, fish exposed to p-nonylphenol or methoxychlor demonstrated a rapid, dose-dependent synthesis of VTG mRNA up to day 5 of exposure, followed by a relatively constant dose-dependent expression through day 42. Both chemicals showed a dose-dependent increase in plasma VTG over the entire time course of exposure, with significantly elevated VTG levels by the fifth day of exposure to p- nonylphenol at concentrations of 5.4 µg/L or greater and to methoxychlor at concentrations of 2.5 µg/L or greater. Exposure to 0.64 µg/L p-nonylphenol resulted in highly variable plasma VTG levels of less than 6 mg/ml. Exposures with endosulfan failed to induce measurable levels of either hepatic VTG mRNA or serum VTG at the chemical concentrations tested. Our results demonstrate that the sheepshead minnow bioassay is a suitable estuarine/marine teleost model for in vivo screening of potentially estrogenic substances.

Bowman, Christopher J., Kevin J. Kroll, Michael J. Hemmer, Leroy C. Folmar and Nancy D. Denslow. 2000. Estrogen-Induced Vitellogenin mRNA and Protein in Sheepshead Minnow (Cyprinodon variegatus). Gen. Comp. Endocrinol. 120(3):300-313. (ERL,GB 1124).

Many environmentally persistent xenobiotic chemicals appear to disrupt normal endocrine function by acting as ligands for endogenous steroid receptors, including the estrogen receptor. Xenobiotics that bind to the estrogen receptor may elicit several effects, one of which is activating estrogen-responsive genes, such as vitellogenin (Vtg). Primers to vitellogenin mRNA have been used to amplify a portion of the coding sequence in sheepshead minnow (SHM) (Cyprinodon variegatus). Two Vtg cDNA fragments from SHM were isolated exhibiting 72% sequence homology and corresponding to the two Vtg genes identified in the mummichog, Fundulus heteroclitus. Using these Vtg cDNA fragments as sensitive genetic probes, we evaluated the initial estrogenic response of fish exposed to natural or anthopogenic chemicals. These probes were used to study in vivo gene induction in SHM exposed to 17b-estradiol (E2) and ethinylestradiol (EE2) under controlled laboratory conditions. Hepatic Vtg mRNA was upregulated and plasma Vtg synthesis in estrogen-induced SHM was assessed. Two in vivo time-course experiments were conducted; a single injection of E2 followed over 72 h and a double E2 injection examined for 12 days. These two protocols provided evidence for differential hepatic Vtg mRNA regulation resulting from a single or double injection. In a separate experiment using an aqueous flowthrough system, constant exposures to low doses of E2 (200 ng/L) and EE2 (100 ng/L) induced hepatic Vtg mRNA and plasma Vtg to levels comparable with the E2 injections. Larger aqueous exposure doses (2000 ng/L E2 or 1000 ng/L EE2) in the flowthrough experiment resulted in greater responses of hepatic Vtg mRNA and plasma Vtg at 7 days. Constant aqueous exposure to E2 (2000 ng/L) or EE2 (1000 ng/L) may thus be more effective than a single large-dose injection (5 mg/kg) to stimulate Vtg gene activation and synthesis.

Larkin, Patrick, Leroy C. Folmar, Michael J. Hemmer, Arianna J. Poston, H. Stephen Lee and Nancy D. Denslow. 2002. Array Technology as a Tool to Monitor Endocrine Disruption in Wild Fish Populations. Mar. Environ. Res. 54(3-5):395-399. (ERL,GB 1144). (PRIMO11)

A variety of anthropogenic chemicals are capable of binding to the estrogen receptor of vertebrate species. Binding of these compounds can interfere with homeostasis by disrupting normal gene expression patterns. The purpose of this study was to investigate the feasibility of applying array technology as a monitoring tool for detecting the presence and distribution of estrogenic compounds in coastal habitats using sheephead minnows as our model. cDNA clones that were isolated from differential display, including vitellogenin a and b, vitelline envelope protein (ZP2), and transferrin, among others, were spotted on the macroarray. The results of these experiments demonstrate a characteristic expression pattern of estrogen responsive genes in sheepshead minnows exposed to 17 b-estradiol (E2).

Larkin, Patrick, Leroy C. Folmar, Michael J. Hemmer, Arianna J. Poston and Nancy D. Denslow. 2003. Expression Profiling of Estrogenic Compounds Using a Sheepshead Minnow cDNA Macroarray. EPA/600/J-04/229. Environ. Health Perspect. 111(6):839-846. (ERL,GB 1171).

A variety of anthropogenic compounds are capable of binding to the estrogen receptor (ER) of vertebrate species. Binding of these chemicals to the ER can interfere with homeostasis by altering normal gene expression patterns. The purpose of this study was to characterize the expression of 30 genes using a sheepshead minnow (Cyprinodon variegatus) cDNA macroarray. Many of the genes on the array were previously identified by differential display reverse transcriptase-polymerase chain reaction to be upregulated or downregulated in sheepshead minnows treated through aqueous exposure to known or suspected estrogenic chemicals. The results of this study show that 17b-estradiol (E2), 17a-ethinyl estradiol (EE2), diethylstilbestrol (DES), and methoxychlor (MXC) have similar genetic signature for the 30 genes examined. The genetic signature of fish treated with p-nonlyphenol (pNP) was identical in pattern to fish treated with E2, EE2, DES, and MXC except for the additional upregulation of a cDNA clone that shares similarity to ubiquitin-conjugating enzyme 9. Endosulfan produced results that resembled the gene expression patterns of untreated control fish with exception of the upregulation of estrogen receptor a and the downregulation of a cDNA clone that shares similarity to 3-hydroxy-3-methylglutaryl-coenzyme A reductase. We show that our estrogen-responsive cDNA macroarray can detect dose-dependent changes in gene expression patterns in fish treated with EE2.

Mayer, Foster L., Donald J. Versteeg, Michael J. McKee, Leroy C. Folmar, Robert L. Graney, Delbert C. McCume and Barnett A. Rattner. 1992. Physiological and Nonspecific Biomarkers. In: Biomarkers: Biochemical, Physiological, and Histological Markers of Anthropogenic Stress. EPA/600/A-92/222. Robert J. Huggett et al., Editor. Lewis Publishers, Boca Raton, FL. Pp. 5-85. (ERL,GB 736). (Avail. from NTIS, Springfield, VA: PB93-119832)

This chapter summarizes the advantages and disadvantages of physiological and nonspecific biomarkers. It lists the criteria to be used in selecting biomarkers to address specific ecological questions. After a general discussion of this topic, several specific biomarkers of possible utility in environmental monitoring of exposure and effects are described individually. For each of these biomarkers, the chapter presents research needs for developing and validating ecologically meaningful biomarkers.

Denslow, Nancy D., Ming M. Chow and Leroy C. Folmar. 1994. Isoforms of Apolipoprotein A-I in the Serum of Brown Bullheads (Ameiurus nebulosus) with Liver Cancer. Can. J. Zool. 72(8):1522-1527. (ERL,GB 853).

Brown bullheads (Ameiurus nebulosus) express two different isoforms of apolipoprotein A-I in the serum, a predominant form characterized by a pI of 6.9 and a more acidic isoform with a pI of 6.65. The two proteins were identified by N-terminal amino acid sequencing, with 50 residues for the pI 6.9 isoform and 20 residues for the pI 6.65 isoform. Sequences for the two proteins were identical for the first 20 amino acids; however, differences in their primary sequences were deduced by analysis of peptide maps obtained through proteolytic digestion. The ratio of the isoforms changes from 75% pI 6.9/25% pI 6.65 (no tumors) to equal amounts of both isoforms in bullheads with hepato- or cholangio-cellular carcinomas. This is the first report of these serum isoforms in feral fish showing that the expression of these proteins is directly related to both cholangio- and hepato-cellular carcinomas. The potential use of these proteins as tumor biomarkers is discussed.

Heppell, Scott A., Nancy D. Denslow, Leroy C. Folmar and Craig V. Sullivan. 1995. Universal Assay of Vitellogenin as a Biomarker for Environmental Estrogens. EPA/600/J-96/079. Environ. Health Perspect. 103(Suppl 7):9-15. (ERL,GB 913).

Vitellogenin (VTG), the serum phospholipoglycoprotein precursor to egg-yolk, is potentially an ideal biomarker for environmental estrogens. This study was undertaken to develop antibodies against conserved regions on the vitellogenin molecule, antibodies that could form the basis for establishing bioassays to detect estrogen exposure in any oviparous vertebrate. We developed monoclonal antibodies (mAbs) generated against purified rainbow trout (Oncorhynchus mykiss) VTG, and selected for the property of specifically recognizing VTG purified from two phylogenetically distant vetebrates, trout and striped bass (Morone saxatilis). Results of enzyme-linked immunosorbant assay and Western blotting indicated that these mAbs specifically recognize purified VTG and VTG or other estrogen-inducible proteins in plasma or serum from representative species of 4 vertebrate classes (fish, amphibians, reptiles and birds). All of the mAbs generated were IgM class. A polyclonal antiserum was raised against a synthetic consensus peptide representing the conserved N-terminal amino acid sequence of VTG. The results of Western blotting indicate that this antiserum specifically recognizes VTG in plasma or serum from teleost fish of diverse families. It was used to detect VTG in Western blots of serum from brown bullhead (Ameiurus nebulosus) with cancer (hepatocellular and cholangio-carcinoma) collected from a contaminated industrial site outside of their normal vitellogenic season. Our results indicate that it is feasible to generate antibodies capable of recognizing VTG without regard to species and that development of a "universal" VTG assay is an achievable goal.

Ankley, Gerald T., Rodney D. Johnson, Naomi E. Detenbeck, Steven P. Bradbury, Gregory Toth and Leroy C. Folmar. 1997. Development of a Research Strategy for Assessing the Ecological Risk of Endocrine Disruptors. Rev. Toxicol. 1(5,6):71-106. (ERL,GB 963).

Correlational evidence suggests that specific populations of animals have been, or currently are being, adversely affected by exposure to environmental contaminants that manifest effects through different endocrine systems. However, there currently are insufficient data to resolve the ecological risk associated with endocrine-disrupting chemicals (EDCs). In recognition of this uncertainty, the Office of Research and Development (ORD) of the U.S. Environmental Protection Agency (EPA) sponsored a workshop that focused on a variety of issues central to the development of a research strategy for assessing ecological effects of endocrine disruptors. Specifically, the workshop was intended to address topics and concepts that would contribute to a strategy designed to establish: 1) a research framework focused upon the greatest uncertainties confronting risk assessment and risk management decisions concerning ECDs, and 2) proactive coordination and communication among Federal agencies whose research missions are especially relevant for the many different facets of this issue. During the first two days of the workshop, more than 60 international experts in the areas of risk assessment, comparative endocrinology and environmental toxicology were involved in presentations and discussions pertaining to the potential ecological risk of EDCs. On the final two days of the workshop, a group of Federal scientists representing EPA, the U.S. Fish and Wildlife Service, the National Biological Service and the National Oceanographic and Atmospheric Administration met to develop a research strategy based upon input from the larger meeting. These deliberations were structured in the context of the EPA ecological risk assessment framework, with special emphasis placed on evaluation of relevant measurement endpoints in the context of likely assessment endpoints, as well as exposure and effect characterization. The resultant research recommendations were developed conscious of the need to establish approaches to determine the relative ecological risk of EDCs to populations and communities, both from a prospective and retrospective standpoint, and to evaluate and potentially modify the current requirements for testing and evaluating chemicals and environmental samples to ensure that those exerting toxicity through specific endocrine axes will be adequately characterized.

Gardner, George R., Richard J. Pruell and Leroy C. Folmar. 1989. Comparison of Both Neoplastic and Non-Neoplastic Disorders in Winter Flounder (Pseudopleuronectes americanus) from Eight Areas in New England. Mar. Environ. Res. 28(1-4):393-397. (ERL,GB X757).

Distribution patterns of liver disease observed in winter flounder indigenous to the northeastern USA indicated that hepatocytic neoplasms were absent in populations from uncontaminated offshore areas and endemic in populations from moderately to highly contaminated inshore areas. Liver neoplasms in winter flounder collected from eight different locations ranged from 0% in animals collected offshore from Cape Cod to 32% in the nearshore area of New Bedford, MA. Similarly, an array of other hepatic lesions ranged from 9% in Martha's Vineyard to 79% in Boston Harbor. Proliferate lesions in endocrine, exocrine, respiratory, sensory, excretory and digestive organs and alteration of plasma protein were also characteristic of winter flounder populations residing in the nearshore environment. The concentrations of polycyclic aromatic hydrocarbons (PAHs), polychlorinated biphenyls (PCBs), other organic compounds and trace metals associated with marine sediment were elevated in urban embayments as compared with offshore locations. Degree of sediment chemical contamination and disease suggest a causal relationship.

Denslow, Nancy D., Majorie Chow, Ming M. Chow, Sherman Bonomelli, Leroy C. Folmar, Scott A. Heppell and Craig V. Sullivan. 1997. Development of Biomarkers for Environmental Contaminants Affecting Fish. In: Chemically Induced Alterations in Functional Development and Reproduction of Fishes. Rosalind M. Rolland, Michael Gilbertson, and Richard E. Peterson, Editors. SETAC Press, Pensacola, FL. Pp. 73-86. (ERL,GB X866).

A number of anthropogenic chemicals that have been introduced into the environment may compromise normal endocrine function and sexual development or lead to neoplastic lesions. It is therefore important to determine wildlife exposure to these chemicals and to begin to establish exposure-effect relationships. We used 2-dimensional polyacrylamide gel electrophoresis (2D-PAGE) to identify biomarkers associated with exposure of fish to contaminants. Two proteins, an acidic isoform of apolipoprotein A1 and vitellogenin (VTG), were more highly expressed in brown bullhead fish exposed to highly contaminated waters. We concentrated on VTG, the estrogen-inducible egg yolk precursor protein, as an ideal biomarker for estrogen-mimicking chemicals, and we developed a panel of high-affinity, vitellogenin-specific monoclonal antibodies (mAbs) (immunoglobulin G [IgG] type). In this report, we partially characterize 4 of the panels' most cross-reactive antibodies, which bind VTG from fish in a wide phylogenetic spectrum (including orders Perciformes, Atheriniformes, Batrachoidiformes, Salmoniformes, Cypriniformes, and Anguilliformes). While none of these antibodies cross-reacts with VTG in the order Siluriformes, another mAb 2D8 (immunoglobulin M [igM] type) does bind to VTG from fishes in this group as well as to VTGs from chickens and snakes, but with lower affinity. The panel of high-affinity IgG-type antibodies can be used in Western blots and enzyme-linked immunosorbent assays (ELISAs) to measure the expression of VTG in plasma from oviparous wildlife exposed to estrogen or estrogen-like contaminants.

Orlando, Edward F., Nancy D. Denslow, Leroy C. Folmar and Louis J. Guillette, Jr. 1999. Comparison of the Reproductive Physiology of Largemouth Bass, Micropterus salmoides, Collected from the Escambia and Blackwater Rivers in Florida. Environ. Health Perspect. 107(3):199-204. (ERL,GB X940).

Largemouth bass (LMB), Micropterus salmoides, were taken from the Escambia River (contaminated site) and the Blackwater River (reference site) near Pensacola, Florida. The Escambia River collection occurred downstream of the effluent from two identified point sources of pollution. These point sources included a coal-fired electric power plant and a chemical company. Conversely, the Blackwater River's headwaters and most of its length flow within a state park. Although there is some development on the lower part of Blackwater River, fish were collected in the more pristine upper regions. Fish were captured by electroshocking and were maintained in aerated coolers. Physical measurements were obtained, blood was taken, and liver and gonads were removed. LMB plasma was assayed for the concentration of 17 b-estradiol (E2) and testosterone using validated radioimmunoassays. The presence of vitellogenin was determined by gel electrophoresis (SDS-PAGE) and Western blotting using a monoclonal antibody validated for largemouth bass vitellogenin. No differences in plasma concentrations of E2 or testosterone were observed in females from the two sites. Similarly, males exhibited no difference in plasma E2. However, plasma testosterone was lower in the males from the contaminated site, as compared to the reference site. Vitellogenic males occurred only at the contaminated site. Additionally, liver mass was proportionately higher in males from the contaminated site, as compared to males from the reference site. These data suggest that reproductive steroid levels may have been altered by increased hepatic enzyme activity, and the presence of vitellogenic males indicates that an exogenous source of estrogen was present in the Escambia River.

Denslow, Nancy D., Christopher J. Bowman, Gillian Robinson, H. Stephen Lee, Ronald J. Ferguson, Michael J. Hemmer and Leroy C. Folmar. 1999. Biomarkers of Endocrine Disruption at the mRNA Level. In: Environmental Toxicology and Risk Assessment: Standardization of Biomarkers for Endocrine Disruption and Environmental Assessment. 8th Volume, ASTM STP 1364. D.S. Henshel, M.C. Black, and M.C. Harrass, Editors. American Society for Testing and Materials, West Conshohocken, PA. Pp. 24-35. (ERL,GB X978).

A large number of estrogen-mimicking, anthropogenic chemicals capable of disrupting normal reproductive function have been identified. The ubiquitous distribution of these compounds, many as components of complex industrial or municipal waste, has spurred an effort to develop methods to screen for chemicals which disrupt normal endocrine regulation of reproduction. We have developed assays that both allow exposure of animals in vivo and measure the response at the level of gene activation. We have developed a probe for measuring the induction of vitellogenin mRNA by Northern Blot in livers of sheepshead minnows treated with 17-b-estradiol. We have also developed a strategy for using Differential Display Polymerase Chain Reaction for determining gene induction profiles following exposure to estradiol. These methods should be adaptable to a variety of structurally diverse estrogen mimics.

Denslow, Nancy D., Christopher J. Bowman, Ronald J. Ferguson, H. Stephen Lee, Michael J. Hemmer and Leroy C. Folmar. 2001. Induction of Gene Expression in Sheepshead Minnows (Cyprinodon variegatus) Treated with 17B-Estradiol, Diethylstilbestrol, or Ethinylestradiol: The Use of mRNA Fingerprints as an Indicator of Gene Regulation. Gen. Comp. Endocrinol. 121(3):250-260. (ERL,GB X991).

The recent interest in hormonally active environmental contaminants has sparked a drive to find sensitive methods to measure their effects on wildlife. A molecular-based assay has been developed to measure the induction of gene expression in sheepshead minnows (Cyprinodon variegatus) exposed in vivo to the natural and pharmaceutical estrogens 17b-estradiol, ethinylestradiol, and diethylstilbestrol. This method used differential display reverse transcriptase polymerase chain reaction assays to compare the expression of individual mRNAs from control and estrogen-exposed fish. Forty-eight differentially expressed cDNAs were isolated by this method, including cDNAs for vitelline envelope proteins and vitellogenin. The mRNA expression patterns for fish injected with a pharmacological dose of estradiol (5 mg/kg) were identical to those obtained in fish receiving constant aqueous exposure to 212 ng estradiol/liter. Further, the cDNA "fingerprint" pattern observed in the estradiol-treated fish also matched that obtained in fish receiving continuous-flow aqueous exposures to 192 ng ethinyl estradiol/liter and a nominal concentration of 200 ng diethylstilbestrol/liter. The results demonstrate a characteristic expression pattern for genes upregulated by exposure to a variety of natural and anthropogenic estrogens and suggest this approach may be valuable to examine the potential effects of environmental contaminants on other endocrine-mediated pathways of reproduction, growth, and development.

Gooding, Meredith P., Vickie S. Wilson, Leroy C. Folmar, Dragoslav T. Marcovich and Gerald A. LeBlanc. 2003. Biocide Tributyltin Reduces the Accumulation of Testosterone as Fatty Acid Esters in the Mud Snail (Ilyanassa obsoleta). EPA/600/J-03/409. Environ. Health Perspect. 111(4):426-430. (ERL,GB X1033).

Imposex, the development of male sex characteristics by female gonochoristic snails, has been documented globally and is causally associated with exposure to the ubiquitous environmental contaminant tributyltin (TBT). Elevated testosterone levels in snails also are associated with TBT, and direct exposure to testosterone has been shown to cause imposex. We discovered previously that the mud snail (Ilyanassa obsoleta) biotransforms and retains excess testosterone primarily as fatty acid esters. The purpose of this study was to determine whether TBT interferes with the esterification of testosterone, resulting in the elevated free (unesterified) testosterone levels associated with imposex. Exposure of snails to environmentally relevant concentrations of TBT (greater than or equal to 1.0 ng/L as tin) significantly increased the incidence of imposex. Total (free + esterified) testosterone levels in snails were not altered by TBT; however, free testosterone levels increased with increasing exposure concentration of TBT. TBT-exposed snails were given [14 C]testosterone to measure the production of [14C]testosterone-fatty acid esters. The production of testosterone-fatty acid esters decreased with increasing exposure concentration of TBT. These results indicate that TBT elevates free testosterone levels in snails by decreasing the production of retention of testosterone-fatty acid esters. These findings were confirmed among field-sampled snails where individuals collected from a high-tin-affected site exhibited a greater incidence of imposex, higher free testosterone levels, and lower testosterone-fatty acid ester levels when compared with individuals sampled from a low-tin-affected site. Decreased testosterone-fatty acid esterification among TBT-treated snails was not caused by direct inhibition of the acyl coenzyme A:testosterone acyltransferase (ATAT) enzyme responsible for testosterone esterification, nor by suppressed ATAT protein expression. The target of TBT may be a co-contributor to the testosterone fatty esterification process or a factor in the enhanced hydrolysis of the testosterone-fatty acid pool.