Elizabeth J. Durhan

LINKAGE OF BIOCHEMICAL RESPONSES TO POPULATION-LEVEL EFFECTS: A CASE STUDY WITH VITELLOGENIN IN THE FATHEAD MINNOW (PIMEPHALES PROMELAS)

A challenge in the field of ecotoxicology is the linkage of alterations at molecular and biochemical levels of organization to adverse outcomes in individuals and populations. In the present study, a predictive relationship between plasma vitellogenin (VTG) concentration and fecundity in female fathead minnows (Pimephales promelas) was derived from 21-d laboratory toxicity tests with five chemicals (17beta-trenbolone, 17alpha-trenbolone, prochloraz, fenarimol, and fadrozole) that inhibit VTG production through different mechanisms. Because VTG is key to egg production in female oviparous animals, changes in the lipoprotein could, theoretically, serve as an indicator of reproductive success. Regression of fecundity versus VTG concentration from the various studies yielded a highly significant linear model (fecundity = -0.042 + 0.95 x VTG, p < 0.01, r2 = 0.88). This relationship was integrated into a population model to translate changes in VTG concentrations of female fathead minnows to alterations in population growth. The model predicted relatively profound effects on population size of fish experiencing moderate decreases in vitellogenesis. For example, a fathead minnow population at a carrying capacity exposed to a chemical stressor that causes a 25% decrease in VTG concentration in females from baseline values would exhibit a 34.6% projected decrease in size after two years of exposure and reach an equilibrium population size that was only 30.2% of the preexposed population. Overall, the current study provides an example of how changes in a biomarker (VTG concentration) can be quantitatively translated into adverse effects at the individual and population levels.

Identification of metabolites of trenbolone acetate in androgenic runoff from a beef feedlot.

Little is known concerning the potential ecological effects of hormonally active substances associated with discharges from animal feeding operations. Trenbolone acetate is a synthetic anabolic steroid that is widely used in the United States to promote growth of beef cattle. Metabolites of trenbolone acetate include the stereoisomers 17α- and 17β-trenbolone, both of which are stable in animal wastes and are relatively potent androgens in fish and mammals. Our purpose in this study was to evaluate the occurrence of 17α- and 17β-trenbolone in a beef cattle feedlot discharge and in river water upstream and downstream from the discharge. In conjunction with that effort, we measured in vitro androgenic activity of the discharge using CV-1 cells that had been transiently cotransfected with human androgen receptor and reporter gene constructs. Samples were collected on nine different occasions during 2002 and 2003. Whole-water samples from the discharge caused a significant androgenic response in the CV-1 cells and contained detectable concentrations of 17α- and 17β-trenbolone. Further work is needed to ascertain the degree to which synthetic androgens such as trenbolone contribute to androgenic activity of feedlot discharges.

Direct effects, compensation, and recovery in female fathead minnows exposed to a model aromatase inhibitor.

Background Several chemicals in the environment have the potential to inhibit aromatase, an enzyme critical to estrogen synthesis. Objectives The objective of this study was to provide a detailed characterization of molecular and biochemical responses of female fathead minnows to a model aromatase inhibitor, fadrozole (FAD). Methods Fish were exposed via water to 0, 3, or 30 μg FAD/L for 8 days and then held in clean water for 8 days, with samples collected at four time points during each 8-day period. We quantified ex vivo steroid production, plasma steroids, and plasma vitellogenin (Vtg) concentrations and analyzed relative transcript abundance of 10 key regulatory genes in ovaries and 3 in pituitary tissue by real-time polymerase chain reaction. Results Ex vivo 17β-estradiol (E2) production and plasma E2 and Vtg concentrations were significantly reduced after a single day of exposure to 3 μg or 30 μg FAD/L. However, plasma E2 concentrations recovered by the eighth day of exposure in the 3-μg/L group and within 1 day of cessation of exposure in the 30-μg/L group, indicating concentration- and time-dependent physiologic compensation and recovery. Concentration-dependent increases in transcripts coding for aromatase (A isoform), cytochrome P450 side-chain cleavage, steroidogenic acute regulatory protein, and follicle-stimulating hormone receptor all coincided with increased E2 production and recovery of plasma E2 concentrations. Conclusions Results of this research highlight the need to consider compensation/adaptation and recovery when developing and interpreting short-term bioassays or biomarkers or when trying to predict the effects of chemical exposures based on mode of action.

Reproductive toxicity of vinclozolin in the fathead minnow: Confirming an anti‐androgenic mode of action

The objective of the present study was to characterize responses of the reproductive endocrine system of the fathead minnow (Pimephales promelas) to the fungicide vinclozolin (VZ), using a 21-d reproduction assay, and a shorter-term (approximately two weeks) test in which fish were cotreated with the VZ (a putative anti-androgen) and the androgen 17beta-trenbolone (TB). Effects on fecundity, gonadal histology, secondary sexual characteristics, reproductive hormones, and relative abundance of androgen receptor (AR) and 11beta-hydroxysteroid dehydrogenase (11betaHSD) mRNA transcripts were evaluated in one or both of these studies. Fecundity of VZ-exposed fish was decreased in a concentration-dependent manner in the 21-d test, culminating in complete reproductive failure at a concentration of 700 microg/L. Exposure to VZ decreased expression of male secondary sexual characteristics -- an effect typical of anti-androgens. The finding that exposure of females to TB-induced expression of prominent, male-like tubercles, which could be effectively blocked with VZ, provides powerful evidence of the anti-androgenic activity of VZ in vivo. In the two experiments VZ produced several responses possibly indicative of compensation or adaptation of the fish to the anti-androgen, including increases in gonad weight, AR and 11 betaHSD mRNA transcript abundance, and ex vivo gonadal production of testosterone and 11-ketotestosterone. Overall, our results demonstrate that the model anti-androgen VZ, which also is an environmental contaminant, impairs reproductive success of fathead minnows and elicits endocrine responses consistent with an anti-androgenic mode of action.

Ecotoxicogenomics to Support Ecological Risk Assessment: A Case Study with Bisphenol A in Fish

Effects of bisphenol A (BPA) on ovarian transcript profiles as well as targeted end points with endocrine/reproductive relevance were examined in two fish species, fathead minnow (Pimephales promelas) and zebrafish (Danio rerio), exposed in parallel using matched experimental designs. Four days of waterborne exposure to 10 μg BPA/L caused significant vitellogenin induction in both species. However, zebrafish were less sensitive to effects on hepatic gene expression and steroid production than fathead minnow and the magnitude of vitellogenin induction was more modest (i.e., 3-fold compared to 13,000-fold in fathead minnow). The concentration-response at the ovarian transcriptome level was nonmonotonic and violated assumptions that underlie proposed methods for estimating hazard thresholds from transcriptomic results. However, the nonmonotonic profile was consistent among species and there were nominal similarities in the functions associated with the differentially expressed genes, suggesting potential activation of common pathway perturbation motifs in both species. Overall, the results provide an effective case study for considering the potential application of ecotoxicogenomics to ecological risk assessments and provide novel comparative data regarding effects of BPA in fish.

Effects of a glucocorticoid receptor agonist, dexamethasone, on fathead minnow reproduction, growth, and development

Synthetic glucocorticoids are pharmaceutical compounds prescribed in human and veterinary medicine as anti-inflammatory agents and have the potential to contaminate natural watersheds via inputs from wastewater treatment facilities and confined animal-feeding operations. Despite this, few studies have examined the effects of this class of chemicals on aquatic vertebrates. To generate data to assess potential risk to the aquatic environment, we used fathead minnow 21-d reproduction and 29-d embryo-larvae assays to determine reproductive toxicity and early-life-stage effects of dexamethasone. Exposure to 500 µg dexamethasone/L in the 21-d test caused reductions in fathead minnow fecundity and female plasma estradiol concentrations and increased the occurrence of abnormally hatched fry. Female fish exposed to 500 µg dexamethasone/L also displayed a significant increase in plasma vitellogenin protein levels, possibly because of decreased spawning. A decrease in vitellogenin messenger ribonucleic acid (mRNA) expression in liver tissue from females exposed to the high dexamethasone concentration lends support to this hypothesis. Histological results indicate that a 29-d embryo-larval exposure to 500 µg dexamethasone/L caused a significant increase in deformed gill opercula. Fry exposed to 500 µg dexamethasone/L for 29 d also exhibited a significant reduction in weight and length compared with control fry. Taken together, these results indicate that nonlethal concentrations of a model glucocorticoid receptor agonist can impair fish reproduction, growth, and development.

Effects of a 3β-Hydroxysteroid Dehydrogenase Inhibitor, Trilostane, on the Fathead Minnow Reproductive Axis

A number of environmental contaminants and plant flavonoid compounds have been shown to inhibit the activity of 3beta-hydroxysteroid dehydrogenase/Delta(5)-Delta(4) isomerase (3beta-HSD). Because 3beta-HSD plays a critical role in steroid hormone synthesis, inhibition of 3beta-HSD represents a potentially important mode of endocrine disruption that may cause reproductive dysfunction in fish or other vertebrates. The objective of this study was to test the hypothesis that exposure to the model 3beta-HSD inhibitor, trilostane, would adversely affect reproductive success of the fathead minnow (Pimephales promelas). Results of in vitro experiments with fathead minnow ovary tissue demonstrated that trilostane inhibited 17beta-estradiol (E2) production in a concentration- and time-dependent manner, and that the effect was eliminated by providing a substrate (progesterone) that does not require 3beta-HSD activity for conversion to E2. Exposure of fish to trilostane caused a significant reduction in spawning frequency and reduced cumulative egg production over the course of the 21-day test. In females, exposure to 1500 mug trilostane/l reduced plasma vitellogenin concentrations, but did not cause significant histological alterations. In males, average trilostane concentrations as low as 50 mug/l significantly increased testis mass and gonadal somatic index. Trilostane exposure did not influence the abundance of mRNA transcripts coding for 3beta-HSD or other steroidogenesis-regulating proteins in males or females. As a whole, results of this study support the hypothesis that 3beta-HSD inhibition can cause reproductive dysfunction in fish, but did not yield a clear profile of responses at multiple levels of biological organization that could be used to diagnose this mode of action.

Effects of a short-term exposure to the fungicide prochloraz on endocrine function and gene expression in female fathead minnows (Pimephales promelas).

Prochloraz is a fungicide known to cause endocrine disruption through effects on the hypothalamic-pituitary-gonadal (HPG) axis. To determine the short-term impacts of prochloraz on gene expression and steroid production, adult female fathead minnows (Pimephales promelas) were exposed to the chemical (0 or 300 μg/L) for a time-course of 6, 12 and 24 h. Consistent with inhibition of cytochrome P450 17α-hydroxylase/17,20-lyase (CYP17) and aromatase (CYP19), known molecular targets of prochloraz, plasma 17β-estradiol (E2) was reduced within 6 h. Ex vivo E2 production was significantly reduced at all time-points, while ex vivo testosterone (T) production remained unchanged. Consistent with the decrease in E2 levels, plasma concentrations of the estrogen-responsive protein vitellogenin were significantly reduced at 24 h. Genes coding for CYP19, CYP17, and steroidogenic acute regulatory protein were up-regulated in a compensatory manner in ovaries of the prochloraz-treated fish. In addition to targeted quantitative real-time polymerase chain reaction analyses, a 15k feature fathead minnow microarray was used to determine gene expression profiles in ovaries. From time-point to time-point, the microarray results showed a relatively rapid change in the differentially expressed gene (DEG) profiles associated with the chemical exposure. Functional analysis of the DEGs indicated changes in expression of genes associated with cofactor and coenzyme binding (GO:0048037 and 0050662), fatty acid binding (GO:0005504) and organelle organization and biogenesis (GO:0006996). Overall, the results from this study are consistent with compensation of the fish HPG axis to inhibition of steroidogenesis by prochloraz, and provide further insights into relatively rapid, system-wide, effects of a model chemical stressor on fish.

Use of gene expression, biochemical and metabolite profiles to enhance exposure and effects assessment of the model androgen 17β-trenbolone in fish.

The impact of exposure by water to a model androgen, 17β-trenbolone (TRB), was assessed in fathead minnows using an integrated molecular approach. This included classical measures of endocrine exposure such as impacts on testosterone (T), 17β-estradiol (E2), and vitellogenin (VTG) concentrations in plasma, as well as determination of effects on the hepatic metabolome using proton nuclear magnetic resonance spectroscopy. In addition, the rates of production of T and E2 in ovary explants were measured, as were changes in a number of ovarian gene transcripts hypothesized to be relevant to androgen exposure. A temporally intensive 16-d test design was used to assess responses both during and after the TRB exposure (i.e., depuration/recovery). This strategy revealed time-dependent responses in females (little impact was seen in the males), in which changes in T and E2 production in the ovary, as well as levels in plasma, declined rapidly (within 1 d), followed shortly by a return to control levels. Gene expression measurements revealed dynamic control of transcript levels in the ovary and suggested potential mechanisms for compensation during the exposure phase of the test. Proton nuclear magnetic resonance spectroscopy revealed a number of hepatic metabolite changes that exhibited strong time and dose dependence. Furthermore, TRB appeared to induce the hepatic metabolome of females to become more like that of males at both high test concentrations of TRB (472 ng/L) and more environmentally relevant levels (33 ng/L).

Propiconazole Inhibits Steroidogenesis and Reproduction in the Fathead Minnow (Pimephales promelas)

Conazoles are designed to inhibit cytochrome P450 (CYP) 14α-demethylase, an enzyme key to fungal cell wall formation. In vertebrates, conazoles may inhibit other CYPs, potentially disrupting processes like sex steroid synthesis. Propiconazole is a current-use pesticide that is among the first chemicals being tested in the U.S. Environmental Protection Agency endocrine disruptor screening program. Fathead minnows (Pimephales promelas) were exposed to 0, 5, 50, 500, or 1000 µg propiconazole/l in a 21-day study that evaluated apical reproductive endpoints (fecundity, fertility, hatch); measures of endocrine function and steroid synthesis, such as cholesterol, vitellogenin (VTG), and sex steroid (testosterone [T], 17β-estradiol [E2]) concentrations in the plasma; and changes in gonadal expression of steroidogenic genes. Plasma E2 and VTG concentrations in females were reduced by exposure to propiconazole, and egg production was decreased in the 500 and 1000 µg/l treatment groups. These in vivo effects coincided with inhibition of E2 synthesis by ovary explants exposed to propiconazole in vitro. We also observed a compensatory response in females exposed to propiconazole, manifested as increased gonad weight and upregulation of genes coding for key steriodogenic proteins, including CYP19 (aromatase), CYP17 (hydroxylase/lyase), CYP11A (cholesterol side-chain-cleavage), and steroidogenic acute regulatory protein. Other than an increase in relative testis weight, effects on endocrine function in males were less pronounced than in females. This study provides important data relative to the potential endocrine activity of propiconazole in fish and, more generally, to the further delineation of pathways for the reproductive effects of steroid synthesis inhibitors in fish.