Product Citations and Abstracts

Mammalian P-glycoprotein is a highly conserved integral membrane protein functioning as an energy-dependent efflux pump which decreases the concentration of certain lipophilic aromatic compounds entering the cell by diffusion. Expression of such a transporter in teleost species could play a significant role in conferring resistance to fish populations exposed to xenobiotic stressors and may serve as a potential indicator of species at risk to certain environmental contaminants. In previous studies we demonstrated that a strong correlation existed between corresponding mammalian and teleost tissues showing immunoreactivity to specific mammalian P-glycoprotein antibodies. In the present study, comparisons of staining pattern, intensity, and tissue specificity between tissues treated in Bouin's Dietrich's and Lillie's histological fixatives were determined in the sheepshead minnow, Cyprinodon variegatus,using monoclonal antibodies C219, C494, JSB-1 and polyclonal antiserum MDR(Ab-1). Immunoreactivity of these antibodies was found to be fixative-dependent. Results are presented illustrating the differential staining patterns and tissue specificity observed for each tissue type, fixative, and antibody combination. Our data indicate tissue fixation has a significant impact on P-glycoprotein antibody immunoreactivity in teleost tissues and must be considered in the comparison and interpretation of results.

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.

Hemmer, Michael J., Christopher J. Bowman, Becky L. Hemmer, Stephanie D. Friedman, Dragoslav Marcovich, Kevin J. Kroll and Nancy D. Denslow. 2002. Vitellogenin mRNA Regulation and Plasma Clearance in Male Sheepshead Minnows, (Cyprinodon variegatus) After Cessation of Exposure to 17B-Estradiol and p-Nonylphenol. EPA/600/J-01/429. Aquat. Toxicol. 58(1-2):99-112. (ERL,GB 1137).

Research was conducted to determine the kinetics of hepatic vitellogenin (VTG) mRNA regulation and plasma VTG accumulation and clearance in male sheepshead minnows (Cyprinodon variegatus) during and after cessation of exposure to either 17b-estradiol (E2) or para-nonylphenol (NP). Adult fish were continuously exposed to aqueous measured concentrations of 0.089 and 0.71 µg E2 per l, and 5.6 and 59.6 µg NP per l for 16 days using an intermittent flow-through dosing apparatus. Fish were sampled on days 8 and 16 of exposure followed by sampling at discrete intervals for up to 96 days post-exposure. At each interval five fish were randomly sampled from each concentration and hepatic VTG mRNA and serum VTG levels for individual fish determined by slot blot and direct enzyme-linked immunosorbent assay (ELISA), respectively. Exposure to E2 and NP resulted in a dose dependent increase in hepatic VTG mRNA and plasma VTG over the course of the 16 day exposure period. Mean plasma VTG levels at day 16 were >100 mg/ml for both high doses of E2 and NP, and >20 mg/ml for the low exposure treatments. Within 8 days post-exposure, hepatic VTG mRNA levels returned to baseline in both high and low E2 treatments but remained elevated 2-4 fold in the NP treatments. Due to a shortened sampling period, a clearance rate for plasma VTG in the 5.6 µg NP per l treatment could not be determined. In the 0.089 µg E2/L, 0.71 µg E2 per l, and 59.6 µg NP per l treatments, VTG levels began decreasing within 4 days after exposure cessation and exhibited an exponential rate of elimination from plasma. Clearance rates for 0.71 µg E2 per l and 59.6 µg NP per l were not significantly different (P = 0.47), however, both demonstrated significantly higher rates of clearance (P < 0.02) than observed in the 0.089 µg E2 per l treatment. Our results indicated that hepatic VTG mRNA rapidly diminishes after cessation of estrogenic exposure in sheepshead minnows, but plasma VTG clearance is concentration and time dependent and may be detected at measurable levels for months after initial exposure to an estrogenic compound.

Hemmer, Michael J., Geraldine M. Cripe, Becky L. Hemmer, Larry R. Goodman, Kimberly A. Salinas, John W. Fournie and Calvin C. Walker. 2008. Comparison of Estrogen-Responsive Plasma Protein Biomarkers and Reproductive Endpoints in Sheepshead Minnows Exposed to 17B-Trenbolone. Aquat. Toxicol. 88(2):128-136. (ERL,GB 1321).

Protein profiling can be used for detection of biomarkers that can be applied diagnostically to screen chemicals for endocrine modifying activity. In previous studies, mass spectral analysis revealed four peptides (2950.5, 2972.5, 3003.4, 3025.5 m/z) in the plasma of estrogen agonist-treated male and gravid female sheepshead minnows (Cyprinodon variegatus, SHM), which served as distinct estrogenic biomarkers. In this study, a 21 day reproductive assay with adult SHM was conducted to investigate possible dose-related effects of the synthetic androgen, 17$-trenbolone, on expression of these four estrogen-responsive peptides. In addition, the response of the peptide biomarkers were compared to traditional reproductive endpoints of fecundity, histopathology, secondary sex characteristics, length, weight, hepatosomatic index, female gonadosomatic index and plasma vitellogenin (VTG) levels. Fish were continuously exposed to 0.005, 0.05, and 5.0 :g/l, a solvent control (triethylene glycol, TEG), and a seawater control (SW) using an intermittent flow-through dosing system. Plasma was analyzed for the presence of the four peptide biomarkers by MALDI-TOF MS and VTG protein by quantitative ELISA. Male fish from the trenbolone treatments and controls showed no expression of the four peptide biomarkers or measurable levels of VTG. The estrogen-responsive biomarkers and plasma VTG were constitutively expressed in females from the SW, TEG, 0.005 and 0.05 :g/l exposures. All four peptide biomarkers were significantly reduced (p<0.0002 to p<0.005) at the 5.0 :g/l treatment level which correlated with significant reductions in fecundity and changes in ovarian morphology. A distinct but non-significant reduction in VTG was also observed in female fish from the 5.0 :g/l treatment. Results of this study suggest application of these estrogen-responsive protein biomarkers may be a cost effective alternative to fecundity measures which are labor intensive and expensive to conduct.

Hemmer, Michael J., Kimberly A. Salinas and Peggy S. Harris. 2011. Application of Protein Expression Profiling to Screen Chemicals for Androgenic Activity. Aquatic. Toxicol. 103(1-2):71-78. (ERL,GB 1386).

Protein expression changes can be used for detection of biomarkers that can be applied diagnostically to screen chemicals for endocrine modifying activity. In this study, Surface Enhanced Laser Desorption/Ionization Time-of-Flight Mass Spectrometry (SELDI-TOF-MS) coupled with a short term fish assay was used to investigate changes in plasma protein expression as a means to screen chemicals for androgenic activity. Adult gravid female sheepshead minnows (Cyprinodon variegatus) were placed into separate aquaria for seawater control, ethanol solvent control, and the following androgen agonist treatments at 5.0 µg/L: dihydrotestosterone (DHT), methyldihydrotestosterone (MDHT), testosterone (T), methyltestosterone (MT) and trenbolone (TB). Treatments of 0.6 µg/L endosulfan and 40 µg/L chlorpyrifos (CP) served as non-androgenic negative stressor controls. Test concentrations were maintained using an intermittent flow-through dosing apparatus supplying exposure water at 20 L/hour. Fish were sampled at 7 days, the plasma diluted, processed on weak cation exchange CM10 Proteinchip arrays and analyzed. Spectral processing resulted in 249 individual m/z peak clusters for the androgen exposed fish. Partial least squares-discriminant analysis was used to develop an androgen-responsive model using sample spectra from exposures with DHT and unexposed solvent control fish as the training set. The androgen classification model performed with 79% specificity (% true negative) and 70% sensitivity (% true positive) for non-aromatizable androgens. The aromatizable androgens T and MT were classified as androgenic with specificities of 42 and 79%, respectively. The reduction in sensitivity observed with T is thought to be caused by its metabolic conversion to an estrogen by aromatase. The results of these studies show diagnostic plasma protein expression models can correctly classify chemicals by their androgenic activity using a combination of high throughput mass spectrometry and multivariate approaches

Hemmer, Michael J., Mace G. Barron and Richard M. Greene. 2010. Comparative Toxicity of Eight Oil Dispersant Products on Two Gulf of Mexico Aquatic Test Species. U.S. Environmental Protection Agency. Office of Research and Development. Washington, DC http://www.epa.gov/emergency/bpspill/reports/ComparativeToxTest.Final.6.30.10.pdf. Pp. 11. (ERL,GB 1395). (06/30/2010)

This report is the first of a round of toxicity testing data for eight oil dispersants that have been authorized for use on the National Contingency Plan (NCP) Product Schedule, which is a list of authorized dispersants and other chemicals that may be used to respond to oil discharges. This report was initated as part of the EPA response to the explosion of the Deepwater Horizon oil exploration platform on April 20, 2010 and the period following the BP Oil spill in the Gulf of Mexico. The dispersants were tested on two aquatic toxicity test species; the mysid shrimp, Americamysis bahia and the inland silverside, Menida beryllina

Hemmer, Michael J., Mace G. Barron and Richard M. Greene. 2010. Comparative Toxicity of Louisiana Sweet Crude Oil (LSC) and Chemically Dispersed LSC to Two Gulf of Mexico Aquatic Test Species. http://www.epa.gov/bpspill/reports/phase2dispersant-toxtest.pdf. 13p. (ERL,GB 1400).

Environmental Protection Agency released peer reviewed results from the second phase of its independent toxicity testing on mixtures of eight oil dispersants with Louisiana Sweet Crude Oil. EPA conducted the tests as part of an effort to ensure that EPA decisions remain grounded in the best available science and data. EPA’s results indicate that the eight dispersants tested have similar toxicities to one another when mixed with Louisiana Sweet Crude Oil. These results confirm that the dispersant used in response to the oil spill in the gulf, Corexit 9500A, when mixed with oil, is generally no more or less toxic than mixtures with the other available alternatives. The results also indicate that dispersant-oil mixtures are generally no more toxic to the aquatic test species than oil alone.

Hemmer, Michael J., Mace G. Barron and Richard M. Greene. 2011. Comparative Toxicity of Eight Oil Dispersants, Louisiana Sweet Crude Oil (LSC) and Chemically Dispersed LSC to Two Aquatic Test Species. Environ. Toxicol. Chem. 30(10):2244-2252. (ERL,GB 1412).

This study describes the acute toxicity of eight commercial oil dispersants, Louisiana sweet crude oil (LSC), and chemically dispersed LSC. The approach utilized consistent test methodologies within a single laboratory in assessing the relative acute toxicity of the eight dispersants, including Corexit 9500A, the predominant dispersant applied during the DeepWater Horizon spill in the Gulf of Mexico. Static acute toxicity tests were performed using two Gulf of Mexico estuarine test species, the mysid shrimp (Americamysis bahia) and the inland silversides (Menidia beryllina). Dispersant-only test solutions were prepared with high energy mixing, while water accommodated fractions of LSC and chemically dispersed LSC were prepared with moderate energy followed by settling and testing of the aqueous phase. LC50 values for the dispersant-only tests were calculated using nominal concentrations whereas tests conducted with LSC alone and dispersed LSC were based on measured total petroleum hydrocarbon concentrations (mg TPH/L). For all eight dispersants in both test species, the dispersants alone were less toxic (LC50s: 3 to >5600 ppm) than the dispersant-LSC mixtures (0.4 to 13 ppm; mg TPH/L). LSC alone had generally similar toxicity to mysids (LC50: 2.7 ppm) and Menidia (LC50: 3.5 ppm) as the dispersant-LSC mixtures. The results of this study indicate that Corexit 9500A had generally similar toxicity as other available dispersants when tested alone and as a mixture with LSC

Hemmer, Michael J. 1980. Pressurized Proportional Diluter for Aquatic Toxicological Studies. EPA-600/J-80-002. Water Res. 14(3):243-246. (ERL,GB 381). (Avail. from NTIS, Springfield, VA: PB80-184328)

A half-liter proportional diluter using positive pressure was designed and tested. Its primary siphons are activated by a pulse of pressure instead of by a partial vacuum as in conventional diluters. This method eliminates the need for venturi tubes, vacuum manifolds, and individual water blocks. Pressure can be supplied by air pumps, air cylinders, or air compressor lines. The diluter's advantages are ease of construction, calibration, compact size, amd rapid cleaning. Unit cost is approximately $250.00.

Hemmer, Michael J., Douglas P. Middaugh and James C. Moore. 1990. Effects of Temperature and Salinity on Menidia beryllina Embryos Exposed to Terbufos. EPA/600/J-90/375. Dis. Aquat. Org. 8:127-136. (ERL,GB 667). (Avail. from NTIS, Springfield, VA: PB91-163881)

Embryos of the inland silverside, Menidia beryllina, were exposed to the organophosphorus pesticide terbufos at nine combinations of temperature (20°, 25° and 30° C) and salinity (5, 12.5 and 20 o/oo). Nominal exposure concentrations were 12.5, 25, 50 and 100 µg terbufos l-1 with an acetone and seawater control for each temperature/salinity combination. Test durations were temperature dependent and ranged from 5 to 14 days. Endpoints were embryo survival, hatching and percentage of larvae with normal vertebrae. Embryo survival was significantly (a= 0.05) lower in tests conducted at 20° C for all salinities. Salinity affected survival only at combinations of 20 o/oo and 100 µg terbufos l-1. Both temperature and salinity affected the percentage hatch, with the lowest hatching occuring in 20° C tests, and in tests conducted at 20 o/oo. The percentage of larvae with normal vertebrae was significantly (a=0.05) reduced from controls at terbufos concentrations of 25 (7 to 32 %), 50 (44 to 62 %) and 100 µg l-1 (58 to 73 %) for the 3 temperatures tested, whereas salinity showed no significant effect. Anomalies in the development occurred across all temperature and salinity combinations, and were observed at concentrations as low as 12.5 µg terbufos l-1.

Hemmer, Michael J., Douglas P. Middaugh and Valerie Comparetta. 1992. Comparative Acute Sensitivity of Larval Topsmelt, Atherinops affinis, and Inland Silversides, Menidia beryllina to 11 Chemicals. EPA/600/J-92/209. Environ. Toxicol. Chem. 11(3):401-408. (ERL,GB 718). (Also available from NTIS, Springfield, VA: PB92-195668)

Larval topsmelt (Atherinops affinis) and inland silversides (Menidia beryllina) were exposed in 96-hr static acute toxicity tests to eleven chemicals to determine the relative sensitivity to the two atherinid species. High to low LC50 ratios for endosulfan, methoxychlor, carbophenothion, chlorpyrifos, terbufos, fenvalerate, permethrin, 4-nitrophenol, and sodium lauryl sulfate were within a factor of < 2 for the two species. A. affinis was more sensitive to both azinphos-methyl and 2,4-dinitrophenol by factors of 6.7 and 4.4, respectively. Comparison of the relative sensitivity of A. affinis with three freshwater fish species (Lepomis macrochirus, Oncorhynchus mykiss, Pimephales promelas) and one estuarine fish species (Cyprinodon variegatus) are also presented. Sensitivities were similar between A. affinis and the two most sensitive freshwater species, L. macrochirus and O. mykiss. A. affinis is easily transported, cultured and maintained in the laboratory, and readily adaptable for use in toxicological studies.

Hemmer, Michael J., Lee A. Courtney and Lisa S. Ortego. 1995. Immunohistochemical Detection of P-glycoprotein in Teleost Tissues Using Mammalian Polyclonal and Monoclonal Antibodies. EPA/600/J-95/372. J. Exp. Zool. 272(1):69-77. (ERL,GB 899).

Mammalian P-glycoprotein is a highly conserved 170-kD integral plasma membrane protein functioning as an energy dependent efflux pump of exogenous and endogenous lipophilic aromatic compounds entering the cell by diffusion. In this study, the tissue specificity of one polyclonal (pAb) and three monoclonal (mAbs) antibodies to mammalian P-glycoprotein were identified in paraffin-embedded, parasagittal whole body sections of the guppy, Poecilia reticulata. Pab mdr (Ab-1) and mAbs C219, C494 and JSB-1 demonstrated differential staining patterns in the following tissues: bile canaliculi in the liver, exocrine pancreas, lumenal surface of the intestinal epithelium, renal tubules, interrenal tissue, branchial blood vessels, gas gland, pseudobranch, and the gill transverse septa. Positive P-glycoprotein expression in P. reticulata correlates well with published results for homogogous mammalian tissues of secretory and execretory function. These data indicate that one or more highly conserved members of the P-glycoprotein transporter family exist in a teleost species which can be detected using commercially available mammalian antibodies.

Previously it was demonstrated that biliary excretion of a single dose of [14 C]dieldrin or [3H]7,12-dimethylbenz[a]anthracene (DMBA) was stimulated up to 700% and 300%, respectively, in rainbow trout fed 0.3-0.4 mg dieldrin/kg/d for 9-12 wk. This was not explained by increased activities of hepatic microsomal xenobiotic-metabolizing enzymes or increased amounts of any of six cytochrome P-450 isozymes quantitated by Western blots. It was hypothesized that stimulated excretion was explained by induction of (1) cytosolic binding proteins that facilitated intracellular trafficking of DMBA to sites of metabolism, or (2) ATP-dependent proteins that transport xenobiotic metabolites from liver to bile. Binding of 15 and 60 nmol [3H]DMBA/mg protein increased about 200% in hepatic cytosol from dieldrin-fed fish. A 50-fold molar excess of unlabelled DMBA reduced binding of 15 nmol [3H]DMBA/mg protein (non-specific binding) by the same amount in cytosol from control and dieldrin-fed fish, indicating that dieldrin induced specific binding. Liver sections from control and dieldrin-fed fish were treated with multidrup resistance (MDR) protein monoclonal antibodies C494, C219 and JSB-1, and polyclonal antibody MDR Ab-1. There were no marked differences in optical densities of immunohistochemical staining near bile canaliculi of control and dieldrin-fed fish. Induction of xenobiotic binding capacity in cytosol of dieldrin-fed rainbow trout at least partially explained altered DMBA disposition in fish pretreated with this cyclodiene insecticide.

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.

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.

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.

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.

Walker, Calvin C., Kimberly A. Salinas, Peggy S. Harris, Sherry S. Wilkinson, James D. Watts and Michael J. Hemmer. 2007. A Proteomic (SELDI-TOF-MS) Approach to Estrogenic Activity Screening. Toxicol. Sci. 95(1):74-81. (ERL,GB 1266).

*A small fish model and surface enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI) were used to investigate plasma protein expression as a means to screen chemicals for estrogenic activity. Adult male sheepshead minnows (Cyprinodon variegatus) were placed into aquaria for seawater control, solvent control, and treatments of 1 71B-estradiol (E2), methoxychior, bisphenol-A, 4-tert-pentylphenol, endosulfan and chlorpyriphos. Fish plasma was applied to weak cation exchange (CM1 0) ProteinChip® arrays , processed and analyzed. The array produced approximately 42 peaks for E2 plasma and 30 peaks for solvent control plasma. Estrogen responsive mass spectral biomarker peaks were identified by comparison of E2 treated and control plasma spectra. Thirteen potential protein biomarkers with a range from 1-13 kDa were up- or down regulated in E2 treated fish and their performance as estrogenic effects markers was evaluated by comparing spectra from control, estrogen agonist and non-agonist stressor treated males and normal female fish plasma. One of the biomarkers, m/z 3025.5, was identified by MS/MS as C. variegatus zona radiata protein, fragment 2. The weak environmental estrogens methoxychlor, bisphenol-A and 4-tort-pentylphenol elicited protein expression profiles consistent with the estrogen expression model. Estrogen responsive peaks were not detected in plasma from fish in the seawater, vehicle, endosulfan or chlorpyriphos treatments. No difference was found between plasma protein expression of seawater control and solvent control fish. We show that water exposure of fish to estrogen agonists produces distinct plasma protein biomarkers that can be reproducibly detected at low levels using protein chips and mass spectrometry.

Salinas, Kimberly A., Michael J. Hemmer, Peggy S. Harris and Calvin C. Walker. 2008. A Simple and Rapid Matrix-Assisted Laser Desorption/Ionization Time of Flight Mass Spectrometry Method to Screen Fish Plasma Samples for Estrogen-Responsive Biomarkers. Environ. Toxicol. Chem. 27(5):1175-1183. (ERL,GB 1295).

In this study, we describe and evaluate the performance of a simple and rapid mass spectral method for screening fish plasma for estrogen-responsive biomarkers using matrix assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF-MS) coupled with a short term fish assay. Adult male sheepshead minnows (Cyprinodon variegatus) were placed into aquaria consisting of duplicate tanks for vehicle control and the following estrogen agonist treatments: 0.00713, 0.0163, 0.04, 0.05, 0.1305 0.222, 0.399 and 0.906u g 17B-estradiol/l; 100 ug 4-tert-pentylphenol /1; 6 and 12 ug methoxycor/l; and 120.5 and 1079.5 ug bisphenol-Al. Treatments with 80 ug chlorpyris/l and 0.6 ug endosulfan/l served as non-estrogenic negative controls. Test concentrations were maintained using an intermittent flow-through dosing apparatus, and fish were sampled at seven or 10 days. Plasma was obtained from individuals, diluted and applied to an inert gold array and analyzed. Multiple protein peaks, ranging from 2.9 to 12.9 KDa, were identified as markers of estrogeniC effects when comparing estrogen-treated and control fish using interpercentile reference values. A binary classification tree model was constructed from plasma protein profiles of the vehicle control and 0.222 ug/l 17B-estradioI treatments and used to evaluate all samples. Treatments with estrogen agonists 17B-estradiol, 4-tert-pentylphenol, methoxychlor and bisphenol-A generated reproducible diagnostic biomarkers based on the presence of specific estrogen-responsive plasma proteins. The controls and non-estrogenic compounds chlorpyrifos and endosulfan did not produce this estrogen responsive protein profile. An average measured NOEC for 17B-estradiol at 0.04 ug/l was estimated from concentration response exposures. MALDI-TOF-MS is a sensitive and specific tool for protein expression profiling and can be used to screen chemicals for mode of action in ecotoxicological studies.

Cripe, Geraldine M., Becky L. Hemmer, Larry R. Goodman, John W. Fournie, Sandy Raimondo, Joann C. Vennari, Rodney L. Danner, Kent Smith, Blaze R. Manfredonia, Dannielle H. Kulaw and Michael J. Hemmer. 2009. Multigenerational Exposure of the Estuarine Sheepshead Minnow (Cyprinodon variegatus) to 17-estradiol. I. Organism-Level Effects Over Three Generations. Environ. Toxicol. Chem. 28(11):2397-2408. (ERL,GB 1343). (Cyprinodon variegatus)

This study reports the effects of 17-estradiol (E2) on reproductive processes through two complete generations of the sheepshead minnow, Cyprinodon variegatus, and determined the need for multiple generation exposure testing for assessing the risks of endocrine disrupting chemicals (EDCs). Adult F0 fish were continuously exposed to nominal concentrations of 0.01, 0.03, 0.08, 0.2 and 0.5 g E2/L for 21 days, as was the F1 generation; the F2 generation was exposed to  0.2 g E2/L during the 40-week test. A cumulative 21-day production of normal embryos was significantly reduced in the F0 generation at 0.5µg E2/L and in the F1 and F2 generations at > 0.08µg E2/L. However, analyses of the last 7 days of the 21-day egg production indicated that it was significantly reduced in all three generations at  0.08 g E2/L. The proportion of infertile eggs increased significantly in F1 fish (0.03 g E2/L) and F2 fish ( 0.08 g E2/L). Changes in liver, kidney and gonadal tissues were seen in the F0 and F1 generation exposed to > 0.2µg E2/L. E2 affected the female gonadosomatic index at 0.5µg/L in the F0 and F1 generations, but not the hepatosomatic index in any treatment. All F1 fish in 0.5µg E2/L were phenotypically female. There was no significant treatment x generation effect on any non-lethal endpoint. However, exposure of fish to E2 throughout their life cycle (F1 and F2 generations) significantly decreased embryo production at concentrations lower than those affecting the F0 generation. Our results emphasize the importance of evaluating the impact of an estrogenic chemical on reproductive performance through a minimum of two sequential generations

Middaugh, Douglas P. and Michael J. Hemmer. 1984. Spawning of the Tidewater Silverside, Menidia peninsulae (Goode and Bean), in Response to Tidal and Lighting Schedules in the Laboratory. Estuaries. 7(2):139-148. (ERL,GB 441).

Tidewater silverside, Menidia peninsulae were maintained in 1.3 m diameter holding tanks in identical laboratory recirculating systems. During two weeks under constant conditions (a current velocity of 8 cm s-1 and continous illumination, 24 L:O D) there was a low relative frequency of arrhythmic spawning. In the subsequent two-week period, fish in one pair of tanks were maintained under the singular influence of twice daily decreases in current velocity from 8 to 0 cm s-1 at 0600-0700 and 1800-1900, under continous illumination. The relative frequency of spawning remained low and there was no evidence of a daily spawning rhythm. However, the mean number of eggs per spawn increased substantially. Fish in the second holding system were subjected to diel light cycle of 13 L:11 D with a constant current velocity of 8 cm s-1 for two weeks. The relative frequency of spawning remained low and there was no indication of spawning rhythmicity; moreover, there was only a slight increase in the mean number of eggs per spawn. During the third two-week period, fish in the first pair of tanks were provided a 13 L:11 D diel light cycle, in conjunction with preexisting twice daily decreases in current velocity; those in the second pair of tanks were provided twice daily decreases in current velocity in conjunction with the preexisting 13 L:11 D light cycle. Under the combined influence of decreases in current velocity and a diel light cycle, there was a marked increase in the relative frequency of spawning in both pairs of tanks. Fish manifested a discernible spawning periodicity, spawns typically occurred between 1800 and 2400; the mean number of eggs per spawn also increased. When fish were returned to constant conditions, current velocity 8 cm s-1 and 24 L:0 D for two weeks, the frequency of spawning decreased and there was no indication of a spawning periodicity. Results of another experiment (decreased current velocities at 1200-1300 and 2400-0100 with 13 L:11 D light cycle) indicated gradual expression of a tidal spawning rhythm during nighttime, 2000-0359. Our laboratory results indicate that M. peninsulae is predominantly a nocturnal spawner and that spawning coincides with decreased current velocities.

Middaugh, Douglas P., Michael J. Hemmer and Yara Lamadrid-Rose. 1986. Laboratory Spawning Cues in Menidia beryllina and M. peninsulae (Pisces, Atherinidae) with Notes on Survival and Growth of Larvae at Different Salinities. EPA/600/J-86/086. Environ. Biol. Fishes. 15(2):107-117. (ERL,GB 508). (Avail. from NTIS, Springfield, VA: PB86-208543)

Spawning patterns of inland silversides, Menidia beryllina, and tidewater silversides, Menidia peninsulae, were examined in the laboratory under several combinations of 'tidal' and diel light cycle cues. M. beryllina showed a high frequency of spawning throughout the day when held under constant conditions (24L: OD, current velocity 8 cm sec-1) and when 'tidal' and diel light cycles were presented singly or in combination. In contrast, M. peninsulae demonstrated a high frequency of spawning only when presented a combination of 'tidal' and diel light cycle cues and spawned predominantly at night. Menidia beryllina embryos were euryhaline. Hatching ranged from 73 to 78% at salinities of 5, 15 and 30 o/oo. M. peninsulae embryos showed an inverse relationship between the percentage hatch and the incubation salinity, 90% at 5 o/oo and only 65% at 30 o/oo. Survival and growth of larval M. beryllina from the day of hatching through 16 days old was optimal at 15 o/oo. Although survival of M. peninsulae larvae was optimal at 30 o/oo, no trend was apparent in growth of larvae held for 16 days at 5, 15, or 30 o/oo salinity.

Middaugh, Douglas P., Michael J. Hemmer and Daniel E. Penttila. 1987. Embryo Ecology of the Pacific Surf Smelt, Hypomesus pretiosus (Pisces: Osmeridae). EPA/600/J-87/468. Pac. Sci. 41(1-4):44-53. (ERL,GB 557). (Avail. from NTIS, Springfield, VA: PB89-208623)

A study of the ecology of developing embryos of the Pacific surf smelt, Hypomesus pretiosus, was conducted. Embryos were maintained in the laboratory at 7.6, 12.1 and 17.6°C and the time to specific embryonic stages determined. Embryos held at 7.6° C developed to stage 24, 18 days after collection; those held at 12.1°C hatched after 13 days; at 17.6°C hatching occurred 8.5 days after collection. Embryos maintained at 15°C and salinities of 20, 25 and 30 o/oo averaged 84% survival. There was no significant difference in survival among groups (ANOVA, P=0.53). Field observations indicated that embryos are spawned in patches in the upper intertidal zone near the time of high tide. They are attached to gravel substrates by the zona radiata membrane which ruptures and quickly turns inside out at the time embryos are fertilized. After several days of development, stage 18 to 22 embryos detach from the original spawning substrates and are washed seaward and down into the gravel substrate in the intertidal zone. However, there was no significant difference (ANOVA, P >=0.09) in the number of eggs found at each of 4 depth strata in the upper, middle and lower intertidal zones.

Middaugh, Douglas P. and Michael J. Hemmer. 1987. Reproductive Ecology of the Tidewater Silverside, Menidia peninsulae (Pisces: Atherinidae) from Santa Rosa Island, Florida. Copeia. 1987(3):727-732. (ERL,GB 561). (Avail. from NTIS, Springfield, VA: PB88-171061)

The reproductive ecology of the tidewater silverside, Menidia peninsulae, was studied during 1982-1983 along the shoreline of Santa Rosa Island, Florida. Adult Menidia were observed at low tide spawning on a red alga, Ceramium byssoideum. Pinfish, Lagodon rhomboides, were noted preying upon newly spawned Menidia eggs. The annual reproductive cycle of M. peninsulae extended from Feb.-July with the highest spawning activity during March-June at water temperatures of 16.7 to 30.8° C. Several statistical analyses of tidal stages and gonadal indices failed to reveal significant differences between spring and neap tides and reproductive activity. However, on four occasions peaks in the percentage occurrence of mature eggs within ovaries coincided with the time that the moon was positioned over the equator. Moreover, analysis of young-of-the-year Menidia (6-28 mm SL) revealed distinct length classes, suggesting that spawning and subsequent hatching of larvae may have occurred in periodic pulses throughout the spring and early summer.

Middaugh, Douglas P. and Michael J. Hemmer. 1994. Fish Model as an Indicator for Teratogenic Substances. In: Biological Monitoring of the Environment: A Manual of Methods. EPA/600/A-94/202. J. Salanki, D. Jeffrey, and G.M. Hughes, Editors. CAB International, Wallingford, England. Pp. 116-120. (ERL,GB 621). (Avail. from NTIS, Springfield, VA: PB95-122966)

A fish model, suitable for use as an indicator for teratogenic substances, is described. Individual blastula stage embryos of the inland silverside, Menidia beryllina, are exposed to teratogens in sealed tissue culture tubes containing 6 ml of saline test media, 5 o/oo salinity and 25 +- 1°C. Individual embryos are examined daily and terata enumerated using a system that ranks craniofacial (CR), cardiovascular (CV) and skeletal (SK) responses. Procedures for statistical analysis of data are described.

Middaugh, Douglas P., Michael J. Hemmer, Jonathan M. Shenker and Toru Takita. 1990. Laboratory Culture of Jacksmelt, Atherinopsis californiensis, and Topsmelt, Atherinops affinis (Pisces: Atherinidae), with a Description of Larvae. Calif. Fish Game. 76(1):4-13. (ERL,GB 646).

Embryonic and larval jacksmelt, Atherinopsis californiensis, and topsmelt, Atherinops affinis, were cultured in the laboratory. Larval A. californiensis were grown for 24 days at 10, 20 and 30 o/oo salinity. Survival, 80-91%, was highest at 10 o/oo salinity. Increases in standard length (SL) and wet weight were greatest for larvae cultured at 10 or 20 o/oo. Survival of larval A. affinis cultured at 10, 20 and 30 o/oo for 24 days ranged from 99-100%. Increases in SL and wet weight were greatest for larvae cultured at 20 or 30 o /oo salinity. Illustrations of day of hatch, 8-, and 24-day-old larvae are presented with morphometric descriptions for each species. Unique melanophore patterns provide a useful character for identification of these two closely related atherinid fishes which occur sympatrically in California bays and estuaries.

Middaugh, Douglas P., John W. Fournie and Michael J. Hemmer. 1990. Vertebral Abnormalities in Juvenile Inland Silversides Menidia beryllina Exposed to Terbufos During Embryogenesis. EPA/600/J-90/382. Dis. Aquat. Org. 9(2):109-116. (ERL,GB 695). (Avail. from NTIS, Springfield, VA: PB91-163956)

Embryos of the inland silverside, Menidia beryllina, were exposed to a nominal concentration of 50 µg terbufos 1-1 during the first five days of embryogenesis. Silversides were maintained in clean dilute seawater until 37 days after hatching. Radiographs revealed compressed and fused vertebrae and dorsal-ventral misalignment of pre- and post-zygapophyseal processes. Histopathological examination of individuals exposed to terbufos during hyperostoses to almost complete fusion of some vertebrae.

Goodman, Larry R., Michael J. Hemmer, Douglas P. Middaugh and James C. Moore. 1992. Effects of Fenvalerate on the Early Life Stages of Topsmelt (Atherinops affinis). EPA/600/J-92/217. Environ. Toxicol. Chem. 11(3):409-414. (ERL,GB 719). (Also available from NTIS, Springfield, VA: PB92-195742)

Flow-through acute and early life-stage (ELS) toxicity tests were conducted with topsmelt (Atherinops affinis), a Pacific Coast saltwater fish, and fenvalerate, a synthetic pyrethroid insecticide. The 96-h LC50 for juvenile fish was 0.66 µg/L. In the 30-d ELS test with laboratory-spawned embryos, average measured fenvalerate concentrations were nondetectable (< 0.075 µg/L) in two control treatments, 0.14, 0.34, 0.82, 1.5, and 3.2 µg/L. Survival of embryos to hatching ranged from 94% to 100%, with no statistically significant difference among treatments. No fry survived exposure to fenvalerate concentrations > 0.82 µg/L; overall survival in lower concentrations and control treatments ranged from 86% to 97%. There were no consistent concentration-dependent differences in weight between fish in the carrier-control treatment and fish exposed to fenvalerate. Mean wet weights of surviving fish ranged from 16.9 mg in 0.34 µg/L to 20.3 mg in 0.14 µg/L. The average bioconcentration factor for fish exposed to 0.14 and 0.34 µg fenvalerate/L was 315.

Middaugh, Douglas P. and Michael J. Hemmer. 1992. Reproductive Ecology of the Inland Silverside, Menidia beryllina (Pisces: Atherinidae) from Blackwater Bay, Florida. EPA/600/J-92/220. Copeia. 1992(1):53-61. (ERL,GB 724). (Also available from NTIS, Springfield, VA: PB92-195775)

The reproductive ecology of the inland silverside, Menidia beryllina, was studied during Feb. 1988--March 1989 at Robinson Point, Blackwater Bay, Florida. Environmental variables including pH, rainfall, salinity, water temperature, and dissolved oxygen were measured weekly or biweekly. Fish were sampled weekly with a seine designed to catch adult, juvenile, and young-of-the year (YOY) individuals. Most reproductive activity occurred during Feb.--April 1988. The maximum mean weekly female gonadosomatic index (GSI) of 12.5 occurred in April. Fecundity ranged from 63 to 419 hydrated eggs/female. The maximum mean weekly male GSI of 6.1 occurred in early March. Catches of YOY individuals 7.6-37.5 mm SL were greatest in May. Some of these YOY individuals matured in July-Sept. and spawned. This reproductive activity resulted in recruitment of a second group of YOY fish into the population during Aug.-Oct. Growth rates of YOY in May-July, calculated by regression methods from weekly frequency distributions of standard length, was 0.34 mm/day for females and 0.31 mm/day for males. The reproductive pattern of M. beryllina from Blackwater Bay, Florida indicates that qualitatively it is an r-strategist with rapid growth of YOY, sexual maturation at an early age, relatively high fecundity, and multiple spawning within the first reproductive period for YOY fish in July-Sept. and again as 1- to 1-plus-year-old individuals.

Middaugh, Douglas P., Michael J. Hemmer and Larry R. Goodman. 1987. Methods for Spawning, Culturing and Conducting Toxicity Tests with Early Life Stages of Atherinid Fishes. EPA/600/8-87/004. U.S. Environmental Protection Agency, Environmental Research Laboratory, Gulf Breeze, FL. 56 p. (Avail. from NTIS, Springfield, VA: PB87-174934)

Procedures are presented for spawning, culturing and conducting acute and chronic toxicity tests with four atherinid fishes: the inland silverside, Menidia beryllina, Atlantic silverside, M. menidia, tidewater silverside, M. peninsulae, and California grunion, Leuresthes tenuis. Guidelines also are provided for growing of food organisms (Chlorella sp., Brachionus plicatilis, and Artemia sp.) that are required for successful culture and testing of the atherinid fishes.

Scott, Geoffrey I., Tommy I. Sammons, Douglas P. Middaugh and Michael J. Hemmer. 1982. Impacts of Water Chlorination and Coliform Bacteria on the American Oyster, Crassostrea virginica (Gmelin). In: Physiological Mechanisms of Marine Pollutant Toxicity. W.B. Vernberg, A. Calabrese, and F.P. Thurberg, Editors. Academic Press, Inc., New York, NY. Pp. 505-529. (ERL,GB X232).

In estuaries such as Murrells Inlet, South Carolina, public health officials have chlorinated nonpoint source run-off so that bacterial water quality can be maintained and oyster resources made harvestable. There is a trade-off as the more immediate risk of bacterial pollution is reduced to an acceptable level, but in its place chlorination by-products such as bromoform, a potent carcinogen, are introduced into estuarine waters where they may be bioconcentrated by oysters and may ultimately affect human consumers. Throughout this attempt to control bacterial pollution, the primary concern has been focused on protecting human health without giving consideration to the potential physiological effects of the disinfection process on oysters. The purpose of this paper was to review and contrast the potential physiological effects of coliform bacteria and chlorine on the American oyster, Crassostrea virginica (Gmelin) and to evaluate the potential risks and benefits of each pollutant type in an effort to gain insight into the proper management of shellfish resources.

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.

Knoebl, Iris, Michael J. Hemmer and Nancy D. Denslow. 2004. Induction of Zona Radiata Proteins and Vitellogenins in Estradiol and Nonylphenol Exposed Male Sheepshead Minnows (Cyprinodon variegatus). Mar. Environ. Res. 58(2-5):547-551. (ERL,GB X1059).

Several genes normally induced by estradiol (E2) in female fish, those for vitellogenins (VTGs) and zona radiata proteins (ZRPs), are also inducible in males exposed to estrogenic chemicals. Male sheepshead minnows (SHM) were exposed to both E2 and para-nonylphenol (NP), at several doses and times to determine a dose-response. Quantitative real time PCR was used to measure mRNA for VTG1, VTG2, ZRP2 and ZRP3. Both E2 and NP elicited a dose-response increase in all of the mRNAs tested. Exposure to both chemicals resulted in VTG2 expression at about a 10-fold lower level than VTG1, and ZRP2 expression at a lower level than ZRP3.

Knoebl, Iris, Jason L. Blum, Michael J. Hemmer and Nancy D. Denslow. 2006. Temporal Gene Induction Patterns in Sheepshead Minnows Exposed to 17B-Estradiol. J. Exp. Zool. 305A(9):707-719. (ERL,GB X1086).

Gene arrays provide a powerful method to examine changes in gene expression in fish due to chemical exposures in the environment. In this study, we expanded an existing gene array for sheepshead minnows (Cyprinodon variegatus) (SHM) and used it to examine temporal changes in gene expression for male SHM exposed to 100 ng 17-estradiol (E2)/L for five time points between 0 and 48 hr. We found that in addition to the induction of genes involved in oocyte development (vitellogenin [VTG], zona radiata [ZRP]), other genes involved in metabolism and the inflammatory response are also affected. We identified five patterns of temporal induction in genes whose expression was modified due to E2 exposure. We validated the gene array data for the expression of VTG 1, VTG 2, ZRP 2 and ZRP 3 and found that with low levels of exogenous E2 (100 ng E2/L) exposure, ZRP expression precedes VTG expression. However, at higher concentrations of E2 (500 ng E2/L), the difference in temporal expression appears to be lost. Exposure to high levels of environmental contaminants may affect the normal ordered expression of genes required for reproduction. Gene expression profiling using arrays promises to be a valuable tool in the field of environmental toxicology. As more genes are identified for species used in toxicological testing, researchers will be better able to predict adverse effects to chemical exposures and to understand the relationships between changes in gene expression and changes in phenotype.