Lindsey S. Blake

Ketoconazole in the fathead minnow (Pimephales promelas): Reproductive toxicity and biological compensation

Ketoconazole (KTC) is a model pharmaceutical representing imidazole and triazole pesticides, which inhibit fungal growth through blocking a cytochrome P450 (CYP)-mediated step in ergosterol biosynthesis. Several of these fungicides have been shown to be reversible inhibitors of CYPs in vertebrates (primarily mammals), including CYP isoforms involved in the pathway that converts cholesterol to active sex steroids. In these studies, we assessed the effects of KTC on aspects of steroidogenesis and reproductive function in the fathead minnow (Pimephales promelas). Exposure of spawning adults to the fungicide for 21 d significantly decreased egg production at a water concentration as low as 25 microg/L. Despite evidence of reduced ex vivo testosterone production by gonads from KTC-exposed fathead minnows, circulating plasma concentrations of sex steroids (testosterone, 17beta-estradiol) were not affected. Exposure to KTC caused an increase in the gonadosomatic index in both sexes and, in males, the fungicide caused a marked proliferation of interstitial (Leydig) cells. In addition, mRNA transcripts for two key steroidogenic enzymes, cytochrome P450 side-chain cleavage (CYP11A) and cytochrome P450 c17alpha hydroxylase/17,20 lyase (CYP17), were elevated by exposure to KTC. Both the changes in transcript levels and proliferation of gonad tissue represent potential adaptive or compensatory responses to impaired steroidogenic capacity. Overall our data indicate that, although KTC does adversely affect steroidogenesis and reproduction in the fathead minnow, the fish can compensate to some degree to mitigate effects of the fungicide. This has important implications for the interpretation of data from tests with endocrine-active chemicals.

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.

Hypoxia alters gene expression in the gonads of zebrafish (Danio rerio)

The objectives of this study were to characterize gene expression responses to hypoxia in gonads of mature zebrafish (Danio rerio), and to start characterizing modes of action by which hypoxia could potentially alter reproduction. Adult male and female zebrafish were maintained under normoxia (7mgO(2)/L), moderate hypoxia (3mgO(2)/L), and severe hypoxia (1mgO(2)/L) for 4 and 14 days and changes in gene expression in gonadal tissues (n=5 per sex per treatment) were evaluated using a commercial 21,000 gene zebrafish oligonucleotide microarray. Differentially expressed genes were determined using ANOVA (p<0.05), and enriched gene ontology (GO) categories (p<0.01) identified using GeneSpring GX software. Short-term (4d) exposure to hypoxia affected expression of genes associated with the initial adaptive responses such as: metabolism of carbohydrates and proteins, nucleotide metabolism, haemoglobin synthesis, reactive oxygen species metabolism, and locomotion. Prolonged (14d) hypoxia affected a suite of genes belonging to different GO categories: lipid metabolism, reproduction (e.g., steroid hormone synthesis), and immune responses. Results of the present study demonstrate that reproduction likely would be affected by hypoxia via multiple modes of action. These include previously hypothesized mechanisms such as modulation of expression of steroidogenic genes, and downregulation of serotonergic pathway. In addition, we propose that there are multiple other points of disruption of reproductive system function linked, for example, to reorganization of lipid transport and other mechanisms involved in responding to hypoxia (e.g., hydroxysteroid dehydrogenase alterations, downregulation of contractile elements, etc.).

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.

Characterization of the androgen‐sensitive MDA‐kb2 cell line for assessing complex environmental mixtures

Synthetic and natural steroidal androgens and estrogens and many other non-steroidal endocrine-active compounds commonly occur as complex mixtures in aquatic environments. It is important to understand the potential interactive effects of these mixtures to properly assess their risk. Estrogen receptor agonists exhibit additivity in mixtures when tested in vivo and in vitro. Little is known, however, concerning possible mixture interactions of androgen receptor agonists. In these studies we used the MDA-kb2 cell line, a human breast cancer cell line with endogenous androgen receptors and a stably transfected luciferase reporter gene construct to quantify the androgenic activity of seven natural and synthetic androgens: 17beta-trenbolone, dihydrotestosterone, methyltestosterone, testosterone, trendione, 17alpha-trenbolone, and androstenedione. We tested combinations of these androgens and compared the observed activity to expected androgenic activity based on a concentration addition model. Our analyses support the hypothesis that androgen receptor agonists cause additive responses in a mixture. Binary mixtures of 17beta-trenbolone with 17beta-estradiol or triclocarban (an anti-microbial found in the environment) were also tested. 17beta-Estradiol induced androgenic activity, but only at concentrations 600-fold greater than those found in the environment. Triclocarban enhanced the activity of 17beta-trenbolone. Additionally, three anti-androgens were each paired with three androgens of varying potencies. The relative potencies of the antagonists were a vinclozolin metabolite (M2) > procymidone > prochloraz regardless of the androgen used. The results of our studies demonstrate the potential utility of the androgen-responsive MDA-kb2 cell line for quantifying the activity of mixtures of endocrine-active chemicals in complex wastes such as municipal effluents and feedlot discharges.

I. Effects of a dopamine receptor antagonist on fathead minnow, Pimephales promelas, reproduction

Neurotransmitters such as dopamine play an important role in regulating fish reproduction. However, the potential for neuroendocrine active chemicals to disrupt fish reproduction has not been well studied, despite emerging evidence of their discharge into aquatic environments. This study is the first to apply the fathead minnow 21 d reproduction assay developed for the US Endocrine Disruptor Screening Program to evaluate the reproductive toxicity of a model neuroendocrine active chemical, the dopamine 2 receptor antagonist, haloperidol. Continuous exposure to up to 20 imcrog haloperidol/L had no significant effects on fathead minnow fecundity, secondary sex characteristics, gonad histology, or plasma steroid and vitellogenin concentrations. The only significant effect observed was an increase in gonadotropin-releasing hormone (cGnRH) transcripts in the male brain. Results suggest that non-lethal concentrations of haloperidol do not directly impair fish reproduction. Potential effects of haloperidol on reproductive behaviors and gene expression were examined in a companion study.

Effects of the insecticide fipronil on reproductive endocrinology in the fathead minnow.

Gamma-aminobutyric acid (GABA) and GABA receptors play an important role in neuroendocrine regulation in fish. Disruption of the GABAergic system by environmental contaminants could interfere with normal regulation of the hypothalamic-pituitary-gonadal axis, leading to impaired fish reproduction. The present study used a 21-d fathead minnow (Pimephales promelas) reproduction assay to investigate the reproductive toxicity of fipronil (FIP), a broad-spectrum phenylpyrazole insecticide that acts as a noncompetitive blocker of GABA receptor-gated chloride channels. Continuous exposure up to 5 µg FIP/L had no significant effect on most of the endpoints measured, including fecundity, secondary sexual characteristics, plasma steroid and vitellogenin concentrations, ex vivo steroid production, and targeted gene expression in gonads or brain. The gonad mass, gonadosomatic index, and histological stage of the gonad were all significantly different in females exposed to 0.5 µg FIP/L compared with those exposed to 5.0 µg FIP/L; however, there were no other significant effects on these measurements in the controls or any of the other treatments in either males and females. Overall, the results do not support a hypothesized adverse outcome pathway linking FIP antagonism of the GABA receptor(s) to reproductive impairment in fish.

II: Effects of a dopamine receptor antagonist on fathead minnow dominance behavior and ovarian gene expression in the fathead minnow and zebrafish.

Neurotransmitters such as dopamine play an important role in reproductive behaviors and signaling. Neuroendocrine-active chemicals in the environment have potential to interfere with and/or alter these processes. A companion study with the dopamine 2 receptor antagonist, haloperidol, found no evidence of a direct effect of the chemical on fish reproduction. This study considered haloperidols potential effects on behavior and ovarian gene expression. Male fathead minnows exposed to 50 microg haloperidol/L for 96 h were found to be significantly more dominant than control males. In terms of molecular signaling, investigated using oligonucleotide microarrays, there was little similarity in the identity and functions of genes differentially expressed in the ovaries of fathead minnows (Pimephales promelas) versus zebrafish (Danio rerio) exposed under the same conditions. Results suggest that non-lethal concentrations of haloperidol do not induce ovarian molecular responses that could serve as biomarkers of exposure to D2R antagonists, but may impact behavior.

Perturbation of gene expression and steroidogenesis with in vitro exposure of fathead minnow ovaries to ketoconazole.

Ketoconazole is a fungicidal drug that inhibits function of cytochrome P450s in the synthesis of steroids. To examine if inhibition of P450 function affects gene expression in a dynamic manner, we conducted in vitro exposures of ovary tissue from fathead minnows (Pimephales promelas) to 0.5 microM ketoconazole to investigate effects on steroid production and gene expression over time. Expression of four key steroidogenesis genes was examined at 1, 6, and 12h of exposure. 11 beta- and 20 beta-hydroxysteroid dehydrogenases were down regulated at 1h and Cytochrome P450 17 was down-regulated at 12h, consistent with the absence of steroid production. In contrast, cytochrome P450 19A was up-regulated at 6h, indicating feedback regulation. Microarray analysis of 12h exposures indicated enrichment of biological processes involved in neurotransmitter secretion, lymphocyte cell activation, sodium ion transport, and embryonic development. These data suggest that, with the exception of cytochrome P450 19A, these steroid metabolic genes are regulated in a feed forward manner and that the effects of ketoconazole may be broader than anticipated based on the mechanism of action alone.