Maxine C. Croteau

New directions in toxicity testing.

In 2007, the U.S. National Research Council (NRC) published a groundbreaking report entitled Toxicity Testing in the 21st Century: A Vision and a Strategy. The purpose of this report was to develop a long-range strategic plan to update and advance the way environmental agents are tested for toxicity. The vision focused on the identification of critical perturbations of toxicity pathways that lead to adverse human health outcomes using modern scientific tools and technologies. This review describes how emerging scientific methods will move the NRC vision forward and improve the manner in which the potential health risks associated with exposure to environmental agents are assessed. The new paradigm for toxicity testing is compatible with the widely used four-stage risk assessment framework originally proposed by the NRC in 1983 in the so-called Red Book. The Nrf2 antioxidant pathway provides a detailed example of how relevant pathway perturbations will be identified within the context of the new NRC vision for the future of toxicity testing. The implications of the NRC vision for toxicity testing for regulatory risk assessment are also discussed.

A framework for the next generation of risk science.

Objectives: In 2011, the U.S. Environmental Protection Agency initiated the NexGen project to develop a new paradigm for the next generation of risk science. Methods: The NexGen framework was built on three cornerstones: the availability of new data on toxicity pathways made possible by fundamental advances in basic biology and toxicological science, the incorporation of a population health perspective that recognizes that most adverse health outcomes involve multiple determinants, and a renewed focus on new risk assessment methodologies designed to better inform risk management decision making. Results: The NexGen framework has three phases. Phase I (objectives) focuses on problem formulation and scoping, taking into account the risk context and the range of available risk management decision-making options. Phase II (risk assessment) seeks to identify critical toxicity pathway perturbations using new toxicity testing tools and technologies, and to better characterize risks and uncertainties using advanced risk assessment methodologies. Phase III (risk management) involves the development of evidence-based population health risk management strategies of a regulatory, economic, advisory, community-based, or technological nature, using sound principles of risk management decision making. Conclusions: Analysis of a series of case study prototypes indicated that many aspects of the NexGen framework are already beginning to be adopted in practice. Citation: Krewski D, Westphal M, Andersen ME, Paoli GM, Chiu WA, Al-Zoughool M, Croteau MC, Burgoon LD, Cote I. 2014. A framework for the next generation of risk science. Environ Health Perspect 122:796–805; http://dx.doi.org/10.1289/ehp.1307260

The Future of Toxicity Testing

In 2007, the U.S. National Research Council (NRC) released a report, “Toxicity Testing in the 21st Century: A Vision and a Strategy,” that proposes a paradigm shift for toxicity testing of environmental agents. The vision is based on the notion that exposure to environmental agents leads to adverse health outcomes through the perturbation of toxicity pathways that are operative in humans. Implementation of the NRC vision will involve a fundamental change in the assessment of toxicity of environmental agents, moving away from adverse health outcomes observed in experimental animals to the identification of critical perturbations of toxicity pathways. Pathway perturbations will be identified using in vitro assays and quantified for dose response using methods in computational toxicology and other recent scientific advances in basic biology. Implementation of the NRC vision will require a major research effort, not unlike that required to successfully map the human genome, extending over 10 to 20 years, involving the broad scientific community to map important toxicity pathways operative in humans. This article provides an overview of the scientific tools and technologies that will form the core of the NRC vision for toxicity testing. Of particular importance will be the development of rapidly performed in vitro screening assays using human cells and cell lines or human tissue surrogates to efficiently identify environmental agents producing critical pathway perturbations. In addition to the overview of the NRC vision, this study documents the reaction by a number of stakeholder groups since 2007, including the scientific, risk assessment, regulatory, and animal welfare communities.

Chronic Exposure of Rana pipiens Tadpoles to UVB Radiation and the Estrogenic Chemical 4-tert-octylphenol

While adverse effects of exposure to ultraviolet B radiation (UVBR) and environmental pollutants on amphibians have been documented, few studies examined the effects of interactions between environmental stressors on amphibian health. Here the impacts of chronic exposure to UVBR and the estrogenic chemical 4-tert-octylphenol (OP), alone and in combination, on the survival, development, growth, and metamorphosis of Rana pipiens (northern leopard frog) tadpoles are evaluated. Tadpoles were exposed to environmentally relevant levels of OP (0.01 or 10 nM), with and without exposure to UVBR (∼0.22 W/m2). After 8 mo of exposure, animals were transferred to a clean-water grow-out system for 3 additional mo. There was no effect of treatments on the weights of animals at wk 42 or on the age, snout–vent length (SVL), and weights of tadpoles at forelimb emergence (FLE). However, tadpoles exposed to UVBR had markedly delayed development and significantly fewer reached metamorphosis compared to control animals. Although exposure to UVBR and OP together produced the highest incidence of mortality and developmental anomalies, UVBR alone was sufficient to produce significant mortality of tadpoles, and exposure to UVBR or OP alone induced a significant increase in malformations and abnormalities. The malformations observed consisted mainly of spinal curvature. Our results show that chronic exposure to environmental levels of UVBR or OP alone exerts lethal and/or sublethal effects on R. pipiens tadpoles.

Assessment of thyroid system disruption in Rana pipiens tadpoles chronically exposed to UVB radiation and 4-tert-octylphenol

Many studies have considered recent increases in ultraviolet B radiation (UVBR) and endocrine disrupting chemicals polluting the environment as possible contributing factors to the reduction in amphibian populations. It has been demonstrated that exposure of amphibians to estrogenic chemicals or UVBR can affect the timing of larval development and metamorphosis. However, amphibians in the wild are exposed to multiple environmental stressors simultaneously. Therefore, our study examines the effects of UVBR and the estrogenic chemical 4-tert-octylphenol (OP), alone and in combination, on the thyroid system of Rana pipiens tadpoles, which is the main regulator of amphibian metamorphosis. Results demonstrate that thyroid gland histomorphology measurements in Gosner stage 31 tadpoles continuously exposed to UVBR (0.21W/m(2)) were not different than those measured in animals from the control group. In a separate experiment, tadpoles exposed to environmentally relevant levels of UVBR (0.22W/m(2)) and/or OP (0.01nM or 10nM) exhibited significantly delayed development starting from Gosner stage 29, given that fewer tadpoles developed past stage 29 in these groups. In addition, significantly fewer UVBR-treated tadpoles developed past stage 34 and metamorphosed. Samples were collected from stages 29 and 34 tadpoles for gene expression analysis in tail tissue and measurements of T3 (triiodothyronine) whole body levels (minus tail). UVBR and/or OP exposure did not affect T3 levels in stages 29 and 34 tadpoles. However, a decrease in deiodinase type 2 (D2) or increase in deiodinase type 3 (D3) mRNA levels was observed in groups of tadpoles with slowed developmental rates at those developmental stages. Given that D2 activates and D3 inactivates thyroid hormones (TH), UVBR/OP mediated disruptions in development are likely caused by dysfunctions in the localized metabolism of THs through alterations in the expression of these enzymes in peripheral tissues. This is the first study to our knowledge reporting a potential thyroid-based mechanism of action for the developmental delays in amphibians exposed to UVBR and/or OP.

Fate and Developmental Effects of Dietary Uptake of Methylmercury in Silurana tropicalis Tadpoles

Adverse effects of methylmercury (MeHg) exposure during amphibian metamorphosis remain to be fully characterized. Most previous investigations determined effects of short-term exposure to elevated dose rates, without information on mercury (Hg) depuration and degradation pathways. Since metamorphosis is primarily controlled by thyroid hormones (TH), alterations in this process suggest a disruption of the TH endocrine axis. The aim of this research was to (1) characterize patterns of MeHg accumulation and depuration in tadpoles and (2) examine effects of MeHg accumulation on metamorphosis and the TH axis. Silurana tropicalis tadpoles were exposed to environmental levels of dietary MeHg until metamorphic climax. Whole-body MeHg and total Hg (THg) levels were measured, as well as the number of metamorphs, rate of metamorphosis, body size, and whole-body triiodothyronine (T3) levels at metamorphosis. Tadpoles exposed to a higher level of MeHg exhibited increased mortality and size, and reduced metamorphosis. At lower levels of MeHg, body burdens increased rapidly and eventually reached a plateau, whereas no plateau was reached at a higher level of MeHg exposure. T3 levels were not affected. Data indicate that at low and medium levels of exposure, depuration of MeHg may prevent toxicity in tadpoles. However, depuration mechanisms may be insufficient at high doses, producing disruption of metamorphosis and death. Although there were no marked effects of MeHg on whole-body T3 levels, further investigation of other components of the TH axis is warranted.

Preexposure to ultraviolet B radiation and 4‐tert‐octylphenol affects the response of Rana pipiens tadpoles to 3,5,3′‐triiodothyronine

Exposure to multiple environmental stressors is negatively impacting the health of amphibians worldwide. Increased exposure to ultraviolet B radiation (UVBR) and chemical pollutants may affect amphibian populations by disrupting metamorphosis; however, the actual mechanisms by which these stressors affect development remain unknown. Because amphibian metamorphosis is controlled by thyroid hormones (TH), changes in developmental rates by environmental stress suggest a disruption of the thyroid system. Tadpoles were chronically exposed to environmental levels of UVBR (average of 0.15 W/m2) and 4-tert-octylphenol (OP; 10 nM), alone and combined, prior to being challenged to exogenous TH triiodothyronine (T3; 5 or 50 nM). This experimental approach was taken to determine whether exposure to these stressors affects the ability of T3 to elicit specific molecular and morphological responses. Exposure to OP increased mRNA levels of thyroid receptors (TRs) alpha and beta, deiodinase type 2 (D2), and corticotropin releasing hormone in the brain and of D2 in the tail of tadpoles. 4-tert-octylphenol also enhanced T3-induced expression of D2 in the brain. The combination of UVBR and OP affected the expression of TR alpha in the brain and the responses of TR alpha and beta genes to T3 in the tail, demonstrating the importance of considering the effects of multiple stressors on amphibians. Tadpoles exposed to UVBR were developmentally delayed and exhibited slowed tail resorption and accelerated hindlimb development following exposure to T3. Together, these findings indicate that UVBR alters the rate of development and TH-dependent morphological changes at metamorphosis, and that exposure to UVBR and/or OP disrupts the expression of genes important for development and the biological action of T3 in peripheral tissues. Our group is the first to demonstrate that environmental levels of UVBR and/or OP can affect the thyroid system of amphibians.

Using expert judgments to improve chronic wasting disease risk management in Canada

ABSTARCT Chronic wasting disease (CWD) is a neurodegenerative, protein misfolding disease affecting cervids in North America in epidemic proportions. While the existence of CWD has been known for more than 40 years, risk management efforts to date have not been able to curtail the spread of this condition. An expert elicitation exercise was carried out in May 2011 to obtain the views of international experts on both the etiology of CWD and possible CWD risk management strategies. This study presents the results of the following three components of the elicitation exercise: (1) expert views of the most likely scenarios for the evolution of the CWD among cervid populations in Canada, (2) ranking analyses of the importance of direct and indirect transmission routes, and (3) rating analyses of CWD control measures in farmed and wild cervids. The implications of these findings for the development of CWD risk management strategies are described in a Canadian context.

Expert elicitation on the uncertainties associated with chronic wasting disease

ABSTRACT A high degree of uncertainty exists for chronic wasting disease (CWD) transmission factors in farmed and wild cervids. Evaluating the factors is important as it helps to inform future risk management strategies. Expert opinion is often used to assist decision making in a number of health, science, and technology domains where data may be sparse or missing. Using the “Classical Model” of elicitation, a group of experts was asked to estimate the most likely values for several risk factors affecting CWD transmission. The formalized expert elicitation helped structure the issues and hence provide a rational basis for estimating some transmission risk factors for which evidence is lacking. Considered judgments regarding environmental transmission, latency of CWD transmission, management, and species barrier were provided by the experts. Uncertainties for many items were determined to be large, highlighting areas requiring more research. The elicited values may be used as surrogate values until research evidence becomes available.

Future directions in risk science

The NexGen framework published in Environmental Health Perspectives integrates three different views on the future of chemical risk assessment. The NexGen framework emulates a fundamental change towards in chemical testing for toxicity, as outlined 2007 NRC report, Toxicity Testing in the 21st Century: A Vision and a Strategy. This framework integrates population health approaches with chemical risk assessment methods, by integrating determinants of health into the risk assessment process. Additional perspective comes from the recommendations of the 2009 NRC report, Science and Decisions: Advancing Risk Assessment. The report also calls for changes within the risk assessment process, including the enhanced role of problem formulation, the unification of non-cancer and cancer methods for deriving dose-response relationships, and cumulative risk assessment. The integration of these three driving concepts is discussed in this review expanding the strengths of these three frameworks and what they brought to the NexGen framework for risk science.