Daniel L. Villeneuve

Adverse outcome pathways: A conceptual framework to support ecotoxicology research and risk assessment

Ecological risk assessors face increasing demands to assess more chemicals, with greater speed and accuracy, and to do so using fewer resources and experimental animals. New approaches in biological and computational sciences may be able to generate mechanistic information that could help in meeting these challenges. However, to use mechanistic data to support chemical assessments, there is a need for effective translation of this information into endpoints meaningful to ecological risk-effects on survival, development, and reproduction in individual organisms and, by extension, impacts on populations. Here we discuss a framework designed for this purpose, the adverse outcome pathway (AOP). An AOP is a conceptual construct that portrays existing knowledge concerning the linkage between a direct molecular initiating event and an adverse outcome at a biological level of organization relevant to risk assessment. The practical utility of AOPs for ecological risk assessment of chemicals is illustrated using five case examples. The examples demonstrate how the AOP concept can focus toxicity testing in terms of species and endpoint selection, enhance across-chemical extrapolation, and support prediction of mixture effects. The examples also show how AOPs facilitate use of molecular or biochemical endpoints (sometimes referred to as biomarkers) for forecasting chemical impacts on individuals and populations. In the concluding sections of the paper, we discuss how AOPs can help to guide research that supports chemical risk assessments and advocate for the incorporation of this approach into a broader systems biology framework.

Endocrine disrupting chemicals in fish: Developing exposure indicators and predictive models of effects based on mechanism of action

Knowledge of possible toxic mechanisms (or modes) of action (MOA) of chemicals can provide valuable insights as to appropriate methods for assessing exposure and effects, thereby reducing uncertainties related to extrapolation across species, endpoints and chemical structure. However, MOA-based testing seldom has been used for assessing the ecological risk of chemicals. This is in part because past regulatory mandates have focused more on adverse effects of chemicals (reductions in survival, growth or reproduction) than the pathways through which these effects are elicited. A recent departure from this involves endocrine-disrupting chemicals (EDCs), where there is a need to understand both MOA and adverse outcomes. To achieve this understanding, advances in predictive approaches are required whereby mechanistic changes caused by chemicals at the molecular level can be translated into apical responses meaningful to ecological risk assessment. In this paper we provide an overview and illustrative results from a large, integrated project that assesses the effects of EDCs on two small fish models, the fathead minnow (Pimephales promelas) and zebrafish (Danio rerio). For this work a systems-based approach is being used to delineate toxicity pathways for 12 model EDCs with different known or hypothesized toxic MOA. The studies employ a combination of state-of-the-art genomic (transcriptomic, proteomic, metabolomic), bioinformatic and modeling approaches, in conjunction with whole animal testing, to develop response linkages across biological levels of organization. This understanding forms the basis for predictive approaches for species, endpoint and chemical extrapolation. Although our project is focused specifically on EDCs in fish, we believe that the basic conceptual approach has utility for systematically assessing exposure and effects of chemicals with other MOA across a variety of biological systems.

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.

Vision & Strategy: Predictive Ecotoxicology in the 21st Century

Abstract : In the 20th century, predicting ecological risk from the use of certain chemicals relied on testing programs that directly measured adverse outcomes (death, disease, reproductive failure, or developmental dysfunction) using in vivo toxicity tests. Extrapolation from these tests?from one species to another or from controlled laboratory tests to uncontrolled real-world environments - was based on largely conservative assumptions or arbitrary uncertainty factors. The result - Costly, time-consuming, unfocused, and contentious assessments that often failed to inspire public confidence in related regulatory and policy decisions.

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.

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.

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.

Gene expression responses in male fathead minnows exposed to binary mixtures of an estrogen and antiestrogen

BackgroundAquatic organisms are continuously exposed to complex mixtures of chemicals, many of which can interfere with their endocrine system, resulting in impaired reproduction, development or survival, among others. In order to analyze the effects and mechanisms of action of estrogen/anti-estrogen mixtures, we exposed male fathead minnows (Pimephales promelas) for 48 hours via the water to 2, 5, 10, and 50 ng 17α-ethinylestradiol (EE2)/L, 100 ng ZM 189,154/L (a potent antiestrogen known to block activity of estrogen receptors) or mixtures of 5 or 50 ng EE2/L with 100 ng ZM 189,154/L. We analyzed gene expression changes in the gonad, as well as hormone and vitellogenin plasma levels.ResultsSteroidogenesis was down-regulated by EE2 as reflected by the reduced plasma levels of testosterone in the exposed fish and down-regulation of genes in the steroidogenic pathway. Microarray analysis of testis of fathead minnows treated with 5 ng EE2/L or with the mixture of 5 ng EE2/L and 100 ng ZM 189,154/L indicated that some of the genes whose expression was changed by EE2 were blocked by ZM 189,154, while others were either not blocked or enhanced by the mixture, generating two distinct expression patterns. Gene ontology and pathway analysis programs were used to determine categories of genes for each expression pattern.ConclusionOur results suggest that response to estrogens occurs via multiple mechanisms, including canonical binding to soluble estrogen receptors, membrane estrogen receptors, and other mechanisms that are not blocked by pure antiestrogens.

Adverse outcome pathways during early fish development: a conceptual framework for identification of chemical screening and prioritization strategies.

The fish early life-stage (FELS) test guideline (OECD 210 or OCSPP 850.1400) is the most frequently used bioassay for predicting chronic fish toxicity and supporting aquatic ecological risk assessments around the world. For each chemical, the FELS test requires a minimum of 360 fish and 1 to 3 months from test initiation to termination. Although valuable for predicting fish full life-cycle toxicity, FELS tests are labor and resource intensive and, due to an emphasis on apical endpoints, provide little to no information about chemical mode of action. Therefore, the development and implementation of alternative testing strategies for screening and prioritizing chemicals has the potential to reduce the cost and number of animals required for estimating FELS toxicity and, at the same time, provides insights into mechanisms of toxicity. Using three reference chemicals with well-established yet distinct adverse outcome pathways (AOPs) in early life stages of fish, we proposed FELS-specific AOPs as conceptual frameworks for identifying useful chemical screening and prioritization strategies. The reference chemicals selected as case studies were a cardiotoxic aryl hydrocarbon receptor agonist (2,3,7,8-tetrachlorodibenzo-p-dioxin), neurotoxic acetylcholinesterase inhibitor (chlorpyrifos), and narcotic surfactant (linear alkylbenzene sulfonate). Using qualitative descriptions for each chemical during early fish development, we developed generalized AOPs and, based on these examples, proposed a three-tiered testing strategy for screening and prioritizing chemicals for FELS testing. Linked with biologically based concentration-response models, a tiered testing strategy may help reduce the reliance on long-term and costly FELS tests required for assessing the hazard of thousands of chemicals currently in commerce.