Leah C. Wehmas

Dynamic nature of alterations in the endocrine system of fathead minnows exposed to the fungicide prochloraz.

The vertebrate hypothalamic-pituitary-gonadal (HPG) axis is controlled through various feedback mechanisms that maintain a dynamic homeostasis in the face of changing environmental conditions, including exposure to chemicals. We assessed the effects of prochloraz on HPG axis function in adult fathead minnows (Pimephales promelas) at multiple sampling times during 8-day exposure and 8-day depuration/recovery phases. Consistent with one mechanism of action of prochloraz, inhibition of cytochrome P450 (CYP) 19 aromatase activity, the fungicide depressed ex vivo ovarian production and plasma concentrations of 17beta-estradiol (E2) in female fish. At a prochloraz water concentration of 30 microg/l, inhibitory effects on E2 production were transitory and did not persist during the 8-day exposure phase. At 300 microg/l prochloraz, inhibition of E2 production was evident throughout the 8-day exposure but steroid titers recovered within 1 day of cessation of exposure. Compensation or recovery of steroid production in prochloraz-exposed females was accompanied by upregulation of several ovarian genes associated with steroidogenesis, including cyp19a1a, cyp17 (hydroxylase/lyase), cyp11a (cholesterol side-chain cleavage), and follicle-stimulating hormone receptor. In male fathead minnows, the 8-day prochloraz exposure decreased testosterone (T) production, possibly through inhibition of CYP17. However, as for E2 in females, ex vivo testicular production and plasma concentrations of T recovered within 1 day of stopping exposure. Steroidogenic genes upregulated in testis included cyp17 and cyp11a. These studies demonstrate the adaptability of the HPG axis to chemical stress and highlight the need to consider the dynamic nature of the system when developing approaches to assess potential risks of endocrine-active chemicals.

Investigating Alternatives To The Fish Early-Life Stage Test: A Strategy For Discovering And Annotating Adverse Outcome Pathways For Early Fish Development

The fish early-life stage (FELS) test (Organisation for Economic Co-operation and Development [OECD] test guideline 210) is the primary test used internationally to estimate chronic fish toxicity in support of ecological risk assessments and chemical management programs. As part of an ongoing effort to develop efficient and cost-effective alternatives to the FELS test, there is a need to identify and describe potential adverse outcome pathways (AOPs) relevant to FELS toxicity. To support this endeavor, the authors outline and illustrate an overall strategy for the discovery and annotation of FELS AOPs. Key events represented by major developmental landmarks were organized into a preliminary conceptual model of fish development. Using swim bladder inflation as an example, a weight-of-evidence–based approach was used to support linkage of key molecular initiating events to adverse phenotypic outcomes and reduced young-of-year survival. Based on an iterative approach, the feasibility of using key events as the foundation for expanding a network of plausible linkages and AOP knowledge was explored and, in the process, important knowledge gaps were identified. Given the scope and scale of the task, prioritization of AOP development was recommended and key research objectives were defined relative to factors such as current animal-use restrictions in the European Union and increased demands for fish toxicity data in chemical management programs globally. The example and strategy described are intended to guide collective efforts to define FELS-related AOPs and develop resource-efficient predictive assays that address the toxicological domain of the OECD 210 test. Environ Toxicol Chem 2014;33:158–169.

Influence of ovarian stage on transcript profiles in fathead minnow (Pimephales promelas) ovary tissue

Interpretation of toxicogenomic experiments conducted with ovary tissue from asynchronous-spawning small fish species is complicated by background variation in the relative abundance and proportion of follicles at different stages within the ovary tissue sample. This study employed both real-time quantitative polymerase chain reaction and a 15,000 gene oligonucleotide microarray to examine variation in the fathead minnow (Pimephales promelas) ovarian transcriptional profile as a function of quantitative and qualitative differences in ovarian histology. The objectives were to provide data that could potentially aid interpretation of future toxicogenomics experiments, identify putative stage-related transcriptional markers, and generate insights into basic biological regulation of asynchronous oocyte development. Multiple lines of evidence from the present study indicate that variation in the transcriptional profile is primarily dependent on the relative abundance of previtellogenic versus vitellogenic follicles in the ovary. Due to the relatively small proportions of mature ovulated follicles or atretic follicles in the overall follicle population, few potential transcriptional markers of maturation, ovulation, or atresia could be identified. However, among the 460 differentially expressed genes identified in the present study, several targets, including HtrA serine peptidase 3 (htra3), tissue inhibitor of metalloproteinase 3 (timp3), aquaporin 8 (aqp8), transgelin 2 like (tagln2), Nedd4 family interacting protein 2 (ndfip2), chemokine ligand 12a (cxcl12a), midkine-related growth factor (mdka), and jagged 1b (jag 1b) exhibited responses and functional properties that support them as candidate molecular markers of significant shift in gross ovarian stage. Genes associated with a diversity of functions including cellular development, morphogenesis, coated vesicle transport, sexual reproduction, and neuron development, among others, were statistically enriched within the list of 460 genes differentially expressed among different ovarian classes. Overall, results of this study provide insights into background variation in ovary transcript profiles that should aid and enhance the interpretation of toxicogenomic data generated in experiments conducted with small, asynchronous-spawning fish species.

A time-course analysis of effects of the steroidogenesis inhibitor ketoconazole on components of the hypothalamic-pituitary-gonadal axis of fathead minnows

The objective of this study was to evaluate temporal effects of the model steroidogenesis inhibitor ketoconazole (KTC) on aspects of reproductive endocrine function controlled by the hypothalamic-pituitary-gonadal (HPG) axis in the fathead minnow (Pimephales promelas). Ketoconazole inhibits the activity of two cytochrome P450s (CYPs) key to sex steroid production in vertebrates, CYP11a (cholesterol side chain cleavage) and CYP17 (c17α-hydroxylase/17, 20-lyase). Sexually mature fish were exposed to water-borne KTC (30 or 300 μg/L) in a flow-through system for up to 8d, following which animals were allowed to recover in clean water. Fish were sampled after 1, 4 and 8d of exposure, and after 1, 8 and 16d of recovery. A shorter-term time-course experiment also was conducted in which females were sampled on seven occasions during a 12h KTC exposure. Ketoconazole consistently depressed ex vivo gonadal synthesis of testosterone (T) in both sexes, and 17β-estradiol (E2) in females during both exposure and recovery phases of the time-course studies. Effects on ex vivo steroidogenesis in females occurred within as little as 1h of exposure. Plasma concentrations of T in males and E2 in females also were depressed by exposure to KTC, but these decreases did not persist to the same degree as observed for the ex vivo effects. In females, after decreases within 12h, plasma E2 concentrations were similar to (or greater than) controls at 24h of exposure, while in males, plasma T returned to levels comparable to controls within 1d of cessation of KTC exposure. The discrepancy between the ex vivo and in vivo data at later stages in the test is consistent with some type of compensatory response to KTC in fish. However, we were unable to ascertain the mechanistic basis for such a response. For example, although a number of genes related to steroid synthesis (e.g., cyp11a, cyp17) were up-regulated in the gonads of both males and females during the exposure and early recovery phases of the experiment, this did not seem to account for the resurgence in plasma steroid concentrations in KTC-exposed fish. Further studies focused on metabolism and clearance of steroids might lend insights as to the effects of KTC on plasma steroid concentrations. Overall, our results demonstrate the complex, temporally dynamic nature of the vertebrate HPG system in response to chemical stressors.

Screening complex effluents for estrogenic activity with the T47D-KBluc cell bioassay: Assay optimization and comparison with in vivo responses in fish

Wastewater treatment plant (WWTP) effluents can contain estrogenic chemicals, which potentially disrupt fish reproduction and development. The current study focused on the use of an estrogen-responsive in vitro cell bioassay (T47D-KBluc), to quantify total estrogenicity of WWTP effluents. We tested a novel sample preparation method for the T47D-KBluc assay, using powdered media prepared with direct effluent. Results of the T47D-KBluc assay were compared with the induction of estrogen receptor-regulated gene transcription in male fathead minnows (Pimephales promelas) exposed to the same effluents. Effluent samples for the paired studies were collected over the course of three months. According to the T47D-KBluc assay, the effluent estrogenicity ranged from 1.13 to 2.00 ng 17β-estradiol (E2) equivalents/L. Corresponding in vivo studies exposing male fathead minnows to 0, 10, 50, and 100% effluent dilutions demonstrated that exposure to 100% effluent significantly increased hepatic vitellogenin (VTG) and estrogen receptor α subunit transcripts relative to controls. The induction was also significant in males exposed to 250 ng E2/L or 100 ng E2/L. The in vitro and in vivo results support the conclusion that the effluent contains significant estrogenic activity, but there was a discrepancy between in vitro- and in vivo-based E2 equivalent estimates. Our results suggest that the direct effluent preparation method for the T47D-KBluc assay is a reasonable approach to estimate the estrogenicity of wastewater effluent.

Developing Predictive Approaches to Characterize Adaptive Responses of the Reproductive Endocrine Axis to Aromatase Inhibition: I. Data Generation in a Small Fish Model

Adaptive or compensatory responses to chemical exposure can significantly influence in vivo concentration-duration-response relationships. This study provided data to support development of a computational dynamic model of the hypothalamic-pituitary-gonadal axis of a model vertebrate and its response to aromatase inhibitors as a class of endocrine active chemicals. Fathead minnows (Pimephales promelas) were either exposed to the aromatase inhibitor fadrozole (0.5 or 30 μg/l) continuously for 1, 8, 12, 16, 20, 24, or 28 days or exposed for 8 days and then held in control water (no fadrozole) for an additional 4, 8, 12, 16, or 20 days. The time course of effects on ovarian steroid production, circulating 17β-estradiol (E2) and vitellogenin (VTG) concentrations, and expression of steroidogenesis-related genes in the ovary was measured. Exposure to 30 μg fadrozole/l significantly reduced plasma E2 and VTG concentrations after just 1 day and those effects persisted throughout 28 days of exposure. In contrast, ex vivo E2 production was similar to that of controls on day 8-28 of exposure, whereas transcripts coding for aromatase and follicle-stimulating hormone receptor were elevated, suggesting a compensatory response. Following cessation of fadrozole exposure, ex vivo E2 and plasma E2 concentrations exceeded and then recovered to control levels, but plasma VTG concentrations did not, even after 20 days of depuration. Collectively these data provide several new insights into the nature and time course of adaptive responses to an aromatase inhibitor that support development of a computational model (see companion article).

Temporal evaluation of effects of a model 3β-hydroxysteroid dehydrogenase inhibitor on endocrine function in the fathead minnow

Inhibition of enzymes involved in the synthesis of sex steroids can substantially impact developmental and reproductive processes controlled by the hypothalmic-pituitary-gonadal (HPG) axis. A key steroidogenic enzyme that has received little attention from a toxicological perspective is 3β-hydroxysteroid dehydrogenase (3β-HSD). In these studies, we exposed reproductively-active fathead minnows (Pimephales promelas) to the model 3β-HSD inhibitor trilostane at two test concentrations (300 and 1,500 µg/L) over a 16-d period that included both 8-d exposure and 8-d recovery phases. Plasma concentrations of 17β-estradiol (E2) in females were depressed within hours of exposure to the drug and remained decreased at the highest trilostane concentration throughout the 8-d exposure. Reductions in E2 were accompanied by decreases in plasma concentrations of the estrogen-responsive protein vitellogenin (VTG). During the recovery phase of the test, plasma E2 and VTG concentrations returned to levels comparable to those of controls, in the case of E2 within 1 d. Up-regulation of ovarian expression of gene products for follicle-stimulating hormone receptor (fshr) and aromatase (cyp19a1a) suggested active compensation in trilostane-exposed animals. Effects of trilostane on HPG-related endpoints in exposed males were less pronounced, although, as in females, up-regulation of gonadal fshr was seen. Data from these time-course studies provide insights as to direct impacts, compensatory responses, and recovery from effects associated with perturbation of a comparatively poorly characterized enzyme/pathway critical to sex steroid synthesis. This information is important to the design and interpretation of approaches for assessing the occurrence and effects of HPG-active chemicals in both the laboratory and the field.

A Method for the Determination of Genetic Sex in the Fathead Minnow, Pimephales promelas, To Support Testing of Endocrine-Active Chemicals

Certain endocrine-active toxicants have been reported to completely sex reverse both male and female individuals in amphibian, avian, fish, invertebrate, and reptile species, resulting in a phenotype indistinguishable from unaffected individuals. Detection of low-level sex reversal often requires large numbers of organisms to achieve the necessary statistical power, especially in those species with predominantly genetic sex determination and cryptic/homomorphic sex chromosomes. Here we describe a method for determining the genetic sex in the commonly used ecotoxicological model, the fathead minnow (Pimephales promelas). Analysis of amplified fragment length polymorphisms (AFLP) in a spawn of minnows resulted in detection of 10 sex-linked AFLPs, which were isolated and sequenced. No recombination events were observed with any sex-linked AFLP in the animals examined (n=112). A polymerase chain reaction (PCR) method was then developed that determined the presence of one of these sex-linked polymorphisms for utilization in routine toxicological testing. Analyses of additional spawns from our in-house culture indicate that fathead minnows utilize a XY sex determination strategy and confirm that these markers can be used to genotype sex; however, this method is currently limited to use in laboratory studies in which breeders possess a defined genetic makeup. The genotyping method described herein can be incorporated into endocrine toxicity assays that examine the effects of chemicals on gonad differentiation.

Developing a Novel Embryo–Larval Zebrafish Xenograft Assay to Prioritize Human Glioblastoma Therapeutics

Glioblastoma is an aggressive brain cancer requiring improved treatments. Existing methods of drug discovery and development require years before new therapeutics become available to patients. Zebrafish xenograft models hold promise for prioritizing drug development. We have developed an embryo-larval zebrafish xenograft assay in which cancer cells are implanted in a brain microenvironment to discover and prioritize compounds that impact glioblastoma proliferation, migration, and invasion. We illustrate the utility of our assay by evaluating the well-studied, phosphatidylinositide 3-kinase inhibitor LY294002 and zinc oxide nanoparticles (ZnO NPs), which demonstrate selective cancer cytotoxicity in cell culture, but the in vivo effectiveness has not been established. Exposures of 3.125-6.25 μM LY294002 significantly decreased proliferation up to 34% with concentration-dependent trends. Exposure to 6.25 μM LY294002 significantly inhibited migration/invasion by ∼27% within the glioblastoma cell mass (0-80 μm) and by ∼32% in the next distance region (81-160 μm). Unexpectedly, ZnO enhanced glioblastoma proliferation by ∼19% and migration/invasion by ∼35% at the periphery of the cell mass (161+ μm); however, dissolution of these NPs make it difficult to discern whether this was a nano or ionic effect. These results demonstrate that we have a short, relevant, and sensitive zebrafish-based assay to aid glioblastoma therapeutic development.

Effects of the antimicrobial contaminant triclocarban, and co‐exposure with the androgen 17β‐trenbolone, on reproductive function and ovarian transcriptome of the fathead minnow (Pimephales promelas)

Triclocarban (TCC) is an antimicrobial agent routinely detected in surface waters that has been hypothesized to interact with the vertebrate endocrine system. The present study examined the effects of TCC alone and in combination with the model endocrine disruptor 17β-trenbolone (TRB) on fish reproductive function. Adult Pimephales promelas were continuously exposed to either 1 µg TCC/L or 5 µg TCC/L, to 0.5 µg TRB/L, or to a mixture (MIX) of 5 µg TCC/L and 0.5 µg TRB/L for 22 d, and a variety of reproductive and endocrine-related endpoints were examined. Cumulative fecundity was significantly reduced in fathead minnows exposed to TRB, MIX, or 5 µg TCC/L. Exposure to 1 µg TCC/L had no effect on reproduction. In general, both TRB and MIX treatments caused similar physiological effects, evoking significant reductions in female plasma vitellogenin, estradiol, and testosterone, and significant increases in male plasma estradiol. Based on analysis of the ovarian transcriptome, there were potential pathway impacts that were common to both TRB- and TCC-containing treatment groups. In most cases, however, those pathways were more plausibly linked to differences in reproductive status than to androgen-specific functions. Overall, TCC was reproductively toxic to fish at concentrations at or near those that have been measured in surface water. There was little evidence that TCC elicits reproductive toxicity through a specific mode of endocrine or reproductive action, nor that it could augment the androgenic effects of TRB. Nonetheless, the relatively small margin of safety between some measured environmental concentrations and effect concentrations suggests that concern is warranted. Environ Toxicol Chem 2017;36:231-242. Published 2016 Wiley Periodicals Inc. on behalf of SETAC. This article is a US government work and, as such, is in the public domain in the United States of America.