Karen H. Watanabe

Development and application of the adverse outcome pathway framework for understanding and predicting chronic toxicity: I. Challenges and research needs in ecotoxicology

To elucidate the effects of chemicals on populations of different species in the environment, efficient testing and modeling approaches are needed that consider multiple stressors and allow reliable extrapolation of responses across species. An adverse outcome pathway (AOP) is a concept that provides a framework for organizing knowledge about the progression of toxicity events across scales of biological organization that lead to adverse outcomes relevant for risk assessment. In this paper, we focus on exploring how the AOP concept can be used to guide research aimed at improving both our understanding of chronic toxicity, including delayed toxicity as well as epigenetic and transgenerational effects of chemicals, and our ability to predict adverse outcomes. A better understanding of the influence of subtle toxicity on individual and population fitness would support a broader integration of sublethal endpoints into risk assessment frameworks. Detailed mechanistic knowledge would facilitate the development of alternative testing methods as well as help prioritize higher tier toxicity testing. We argue that targeted development of AOPs supports both of these aspects by promoting the elucidation of molecular mechanisms and their contribution to relevant toxicity outcomes across biological scales. We further discuss information requirements and challenges in application of AOPs for chemical- and site-specific risk assessment and for extrapolation across species. We provide recommendations for potential extension of the AOP framework to incorporate information on exposure, toxicokinetics and situation-specific ecological contexts, and discuss common interfaces that can be employed to couple AOPs with computational modeling approaches and with evolutionary life history theory. The extended AOP framework can serve as a venue for integration of knowledge derived from various sources, including empirical data as well as molecular, quantitative and evolutionary-based models describing species responses to toxicants. This will allow a more efficient application of AOP knowledge for quantitative chemical- and site-specific risk assessment as well as for extrapolation across species in the future.

Fish tissue quality in the lower Mississippi River and health risks from fish consumption

Between 1990 and 1994, samples of three shellfish species (i.e. blue crab, Callinectes sapidus;crayfish, Procambarus acutis; and river shrimp, Macrobrachium ohionii) and 16 fish species and were collected at six sites along the lower Mississippi River by the Louisiana Department of Environmental Quality, Office of Water Resources in coordination with the US Environmental Protection Agency. The fish species included: bigmouth buffalo (Ictiobus cyanellus); blue catfish (Ictalurus furcatus); carp (Cyprinus carpio); channel catfish (Ictalurus punctatus); cobia (Rachycentron canadum); flathead catfish (Pylodictis olivaris); freshwater drum (Aplodinotus grunniens); largemouth bass (Micropterus salmoides); long nose gar (Lepisosteus osseus); red drum (Sciaenops ocellatus); red snapper (Lutjanus campechanus); smallmouth buffalo (Ictiobus bubalus); spotted gar (Lepisosteus oculatus); striped bass (Morone saxatilis); white bass (Morone chrysops); and white crappie (Pomoxis annularis). Organic compound and heavy metal concentrations were measured in 161 composite fish tissue samples where each composite included three to 10 individual fish. Nineteen chemicals, found at measurable levels in sample tissues, were used in calculations of lifetime excess cancer and non-cancer risks due to fish consumption. We calculated: 574 chemical-specific cancer risks; 41 total cancer risks; and 697 margins of exposure based on a consumption rate of one 8-ounce meal per week (0.032 kg/day), a body weight of 70 kg and reported cancer potency factors and reference doses. We identified nine species of concern (blue catfish, carp, channel catfish, cobia, crayfish, flathead catfish, red drum, spotted gar and striped bass) based on total cancer risk greater than 10(-4) or margin of exposure greater than 1, and whether or not samples collected in subsequent years resulted in lower risks. The compounds primarily responsible for the elevated risks were aldrin, dieldrin, alpha-benzene hexachloride, gamma-benzene hexachloride, heptachlor epoxide, arsenic and mercury.

Development and application of the adverse outcome pathway framework for understanding and predicting chronic toxicity: II. A focus on growth impairment in fish

Adverse outcome pathways (AOPs) organize knowledge on the progression of toxicity through levels of biological organization. By determining the linkages between toxicity events at different levels, AOPs lay the foundation for mechanism-based alternative testing approaches to hazard assessment. Here, we focus on growth impairment in fish to illustrate the initial stages in the process of AOP development for chronic toxicity outcomes. Growth is an apical endpoint commonly assessed in chronic toxicity tests for which a replacement is desirable. Based on several criteria, we identified reduction in food intake to be a suitable key event for initiation of middle-out AOP development. To start exploring the upstream and downstream links of this key event, we developed three AOP case studies, for pyrethroids, selective serotonin reuptake inhibitors (SSRIs) and cadmium. Our analysis showed that the effect of pyrethroids and SSRIs on food intake is strongly linked to growth impairment, while cadmium causes a reduction in growth due to increased metabolic demands rather than changes in food intake. Locomotion impairment by pyrethroids is strongly linked to their effects on food intake and growth, while for SSRIs their direct influence on appetite may play a more important role. We further discuss which alternative tests could be used to inform on the predictive key events identified in the case studies. In conclusion, our work demonstrates how the AOP concept can be used in practice to assess critically the knowledge available for specific chronic toxicity cases and to identify existing knowledge gaps and potential alternative tests.

Effects of estrogens and antiestrogens on gene expression of fathead minnow (Pimephales promelas) early life stages.

Endocrine disrupting chemicals (EDCs) are known to contaminate aquatic environments and alter the growth and reproduction of organisms. The objective of this study was to evaluate the sensitivity and utility of fathead minnow (Pimephales promelas) early life‐stages as a model to measure effects of estrogenic and antiestrogenic EDCs on physiological and gene expression endpoints relative to growth and reproduction. Embryos (<24‐h postfertilization, hpf) were exposed to a potent estrogen (17α‐ethinyl estradiol, EE2, 2, 10, and 50 ng L−1); a weak estrogen (mycotoxin zearalenone, ZEAR, same concentrations as above); an antiestrogen (ZM 189, 154; 40, 250, and 1000 ng L−1); and to mixtures of EE2 and ZM until swim‐up stage (∼170 hpf). Exposure to all concentrations of ZEAR and to the lowest concentration of ZM resulted in increased body sizes, whereas high concentrations of EE2 decreased body sizes. There was a significant increase in the frequency of abnormalities (mostly edema) in larvae exposed to all concentrations of EE2, and high ZEAR, and EE2 + ZM mixture groups. Expression of growth hormone was upregulated by most of the conditions tested. Exposure to 50 ng L−1 ZEAR caused an induction of insulin‐like growth factor 1, whereas exposure to 40 ng L−1 ZM caused a downregulation of this gene. Expression of steroidogenic acute regulatory protein gene was significantly upregulated after exposure to all concentrations of EE2 and luteinizing hormone expression increased significantly in response to all treatments tested. As expected, EE2 induced vitellogenin expression; however, ZEAR also induced expression of this gene to similar levels compared to EE2. Overall, exposure to EE2 + ZM mixture resulted in a different expression pattern compared to single exposures. The results of this study suggest that an early life stage 7‐day exposure is sufficient to recognize and evaluate effects of estrogenic compounds on gene expression in this fish model.

Calculated Cancer Risks for Conventional and “Potentially Reduced Exposure Product” Cigarettes

Toxicant deliveries (by machine smoking) are compiled and associated cancer risks are calculated for 13 carcinogens from 26 brands of conventional cigarettes categorized as “regular” (R), “light” (Lt), or “ultralight” (ULt), and for a reference cigarette. Eight “potentially reduced exposure product” (PREP) cigarettes are also considered. Because agency-to-agency differences exist in the cancer slope factor (CSF) values adopted for some carcinogens, two CSF sets were used in the calculations: set I [U.S. Environmental Protection Agency (EPA)–accepted values plus California EPA–accepted values as needed to fill data gaps] and set II (vice versa). The potential effects of human smoking patterns on cigarette deliveries are considered. Acetaldehyde, 1,3-butadiene, and acrylonitrile are associated with the largest calculated cancer risks for all 26 brands of conventional cigarettes. The calculated risks are proportional to the smoking dose z (pack-years). Using CSF set I and z = 1 pack-year (7,300 cigarettes), the calculated brand-average incremental lifetime cancer risk \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\overline{\mathrm{ILCR}}^{\mathrm{acetaldehyde}}_{1}\) \end{document} values are R, 6 × 10−5; Lt, 5 × 10−5; and ULt, 3 × 10−5 (cf. typical U.S. EPA risk benchmark of 10−6). These values are similar, especially given the tendency of smokers to “compensate” when smoking Lt and ULt cigarettes. \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\overline{\mathrm{ILCR}}^{\mathrm{sub{\Sigma}-lung}}_{1}\) \end{document}is the brand-average per pack-year subtotal risk for the measured human lung carcinogens. Using CSF set I, the \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\overline{\mathrm{ILCR}}^{\mathrm{sub{\Sigma}-lung}}_{1}\) \end{document} values for R, Lt, and ULt cigarettes account for ≤2% of epidemiologically observed values of the all-smoker population average per pack-year risk of lung cancer from conventional cigarettes. RPREP (%) is a science-based estimate of the possible reduction in lung cancer risk provided by a particular PREP as compared with conventional cigarettes. Using CSF set I, all RPREP values are <2%. The current inability to account for the observed health risks of smoking based on existing data indicates that current expressed/implied marketing promises of reduced harm from PREPs are unverified: there is little reason to be confident that total removal of the currently measured human lung carcinogens would reduce the incidence of lung cancer among smokers by any noticeable amount. (Cancer Epidemiol Biomarkers Prev 2007;16(3):584–92)

A computational model of the hypothalamic - pituitary - gonadal axis in female fathead minnows (Pimephales promelas) exposed to 17α-ethynylestradiol and 17β-trenbolone

BackgroundEndocrine disrupting chemicals (e.g., estrogens, androgens and their mimics) are known to affect reproduction in fish. 17α-ethynylestradiol is a synthetic estrogen used in birth control pills. 17β-trenbolone is a relatively stable metabolite of trenbolone acetate, a synthetic androgen used as a growth promoter in livestock. Both 17α-ethynylestradiol and 17β-trenbolone have been found in the aquatic environment and affect fish reproduction. In this study, we developed a physiologically-based computational model for female fathead minnows (FHM, Pimephales promelas), a small fish species used in ecotoxicology, to simulate how estrogens (i.e., 17α-ethynylestradiol) or androgens (i.e., 17β-trenbolone) affect reproductive endpoints such as plasma concentrations of steroid hormones (e.g., 17β-estradiol and testosterone) and vitellogenin (a precursor to egg yolk proteins).ResultsUsing Markov Chain Monte Carlo simulations, the model was calibrated with data from unexposed, 17α-ethynylestradiol-exposed, and 17β-trenbolone-exposed FHMs. Four Markov chains were simulated, and the chains for each calibrated model parameter (26 in total) converged within 20,000 iterations. With the converged parameter values, we evaluated the models predictive ability by simulating a variety of independent experimental data. The model predictions agreed with the experimental data well.ConclusionsThe physiologically-based computational model represents the hypothalamic-pituitary-gonadal axis in adult female FHM robustly. The model is useful to estimate how estrogens (e.g., 17α-ethynylestradiol) or androgens (e.g., 17β-trenbolone) affect plasma concentrations of 17β-estradiol, testosterone and vitellogenin, which are important determinants of fecundity in fish.

Quantitative Adverse Outcome Pathways and Their Application to Predictive Toxicology

A quantitative adverse outcome pathway (qAOP) consists of one or more biologically based, computational models describing key event relationships linking a molecular initiating event (MIE) to an adverse outcome. A qAOP provides quantitative, dose-response, and time-course predictions that can support regulatory decision-making. Herein we describe several facets of qAOPs, including (a) motivation for development, (b) technical considerations, (c) evaluation of confidence, and (d) potential applications. The qAOP used as an illustrative example for these points describes the linkage between inhibition of cytochrome P450 19A aromatase (the MIE) and population-level decreases in the fathead minnow (FHM; Pimephales promelas). The qAOP consists of three linked computational models for the following: (a) the hypothalamic-pitutitary-gonadal axis in female FHMs, where aromatase inhibition decreases the conversion of testosterone to 17β-estradiol (E2), thereby reducing E2-dependent vitellogenin (VTG; egg yolk protein precursor) synthesis, (b) VTG-dependent egg development and spawning (fecundity), and (c) fecundity-dependent population trajectory. While development of the example qAOP was based on experiments with FHMs exposed to the aromatase inhibitor fadrozole, we also show how a toxic equivalence (TEQ) calculation allows use of the qAOP to predict effects of another, untested aromatase inhibitor, iprodione. While qAOP development can be resource-intensive, the quantitative predictions obtained, and TEQ-based application to multiple chemicals, may be sufficient to justify the cost for some applications in regulatory decision-making.

Characterization of ontogenetic changes in gene expression in the fathead minnow (pimephales promelas)

Recently, researchers have begun looking at changes in gene expression in the fathead minnow (Pimephales promelas) after contaminant exposure as a way to develop biomarkers of exposure and effects. However, the bulk of this research has been conducted on adults, with few studies focusing on early life stages. Expression of selected genes important in growth, development, and reproduction in teleosts was quantified by quantitative polymerase chain reaction during different developmental time periods (from 0 to 28 d postfertilization [dpf]). Over the developmental period studied, there was a significant up-regulation of growth hormone mRNA and no significant changes in the expression of insulin-like growth factor 1. Thyroid hormone receptors A and B were detected in 4 dpf embryos and their expression stayed relatively constant. The variation in cytochrome P45019A mRNA expression was large during the first week of development, returning to 0 dpf expression levels thereafter. Estrogen receptor 2B was up-regulated during the first three weeks postfertilization, returning to prehatch values by 28 dpf. Expression of hydroxysteroid dehydrogenase 3B and steroidogenic acute regulatory protein increased after the third or fourth week postfertilization, respectively. Vitellogenin exhibited a large degree of variation within time points, especially after day 15, and a significant up-regulation for this gene was observed at 7 and 10 dpf. Knowledge of the normal changes in gene expression during embryo and larval development will allow for better experimental design and selection of suitable biomarkers when testing the potential toxicological effects of contaminants in this model fish species.

A bayesian approach to parameter estimation for a crayfish (Procambarus spp): Bioaccumulation model

Bioaccumulation models are used to describe chemical uptake and clearances by organisms. Averaged input parameter values are traditionally used and yield point estimates of model outputs. Hence, the uncertainty and variability of model predictions are ignored. Probabilistic modeling approaches, such as Monte Carlo simulation and the Bayesian method, have been recommended by the U.S. Environmental Protection Agency to provide a quantitative description of the degree of uncertainty and/or variability in risk estimates in ecological hazards and human health effects. In this study, a Bayesian analysis was conducted to account for the combined uncertainty and variability of model parameters in a crayfish bioaccumulation model. After a 5-d exposure in the LaBranche Wetlands (LA, USA), crayfish were analyzed for polycyclic aromatic hydrocarbon concentrations and lipid fractions. The posterior distribution of model parameters were derived from the joint posterior parameter distributions using a Markov chain Monte Carlo approach and the experimental data. The results were then used to predict the distribution of chrysene concentration versus time in the crayfish to compare the predicted ranges at the different study sites.

From primordial germ cells to primordial follicles: a review and visual representation of early ovarian development in mice

BackgroundNormal development of reproductive organs is crucial for successful reproduction. In mice the early ovarian developmental process occurs during the embryonic and postnatal period and is regulated through a series of molecular signaling events. Early ovarian development in mice is a seventeen-day process that begins with the rise of six primordial germ cells on embryonic day five (E5) and ends with the formation of primordial follicles on postnatal day two (P2).ResultsWe reviewed the current literature and created a visual representation of early ovarian development that depicts the important molecular events and associated phenotypic outcomes based on primary data. The visual representation shows the timeline of key signaling interactions and regulation of protein expression in different cells involved in ovarian development. The major developmental events were divided into five phases: 1) origin of germ cells and maintenance of pluripotency; 2) primordial germ cell migration; 3) sex differentiation; 4) formation of germ cell nests; and 5) germ cell nest breakdown and primordial follicle formation.ConclusionsThis review and visual representation provide a summary of the current scientific understanding of the key regulation and signaling during ovarian development and highlights areas needing further study. The visual representation can be used as an educational resource to link molecular events with phenotypic outcomes; serves as a tool to generate new hypotheses and predictions of adverse reproductive outcomes due to perturbations at the molecular and cellular levels; and provides a comprehendible foundation for computational model development and hypothesis testing.