Linking Environmental Exposure to Toxicity*

NOFFISAT OKIab, JEREMY LEONARDac, MARK NELMSa b, SHANNON BELLd, YU-MEI TANc, LYLE BURGOONe AND STEPHEN EDWARDS*b

aOak Ridge Institute for Science and Education, Oak Ridge, TN, 37831,

USA; bNational Health and Environmental Effects Research Laboratory, United States Environmental Protection Agency, 109 TW Alexander Dr Mail Code B305-01, Research Triangle Park, NC 27709, USA; cNational Exposure Research Laboratory, United States Environmental Protection Agency, Research Triangle Park, NC, 27709, USA; dNational Institute of Environmental Health Sciences, Research Triangle Park, NC, 27709, USA; Environmental Laboratory, US Army Engineer Research and Development Center, Vicksburg, MS, USA *E-mail: This email address is being protected from spam bots, you need Javascript enabled to view it

Introduction

Exposure to chemicals that have been released into the environment contribute to the overall disease burden along with genetic causes and lifestyle @he information in this document has been funded wholly (or in part) by the US Environmental Protection Agency. It has been subjected to review by the National Health and Environmental Effects Research Laboratory and approved for publication. Approval does not signify that the contents reflect the views of the Agency, nor does mention of trade names or commercial products constitute endorsement or recommendation for use.

Issues in Toxicology No. 31

Computational Systems Pharmacology and Toxicology Edited by Dale E. Johnson and Rudy J. Richardson © The Royal Society of Chemistry 2017 Published by the Royal Society of Chemistry, www.rsc.org decisions. Addressing the disease through pharmaceutical intervention can positively impact all sources of disease, but exposure to environmental stressors and lifestyle decisions represent areas where disease prevention can potentially avoid the need for medical treatment in many cases. while people are ultimately responsible for their lifestyle decisions when informed about how those decisions impact their health and well-being, governmental bodies around the world have assumed the responsibility for controlling exposure to environmental stressors (primarily, but not exclusively, chemicals) to ensure the safety of their citizens. This requires science to support those risk-based decisions as well as mechanisms for translating the scientific information into a form that can be readily used for this purpose. the field of toxicology has always been the scientific core of this process and continues in this role. However, an understanding of the processes leading to the exposure of humans and wildlife to the chemicals released in the environment is critical for determining the ultimate impact on health and well-being. Coupling this information with an understanding of the toxicology of these chemicals allows for the prevention of disease by eliminating the source of toxicity rather than treatment of the disease after the fact.

traditionally, the toxicity testing paradigm involved the use of large and expensive batteries of animal testing studies to determine the risk of adverse outcomes upon exposure to chemicals and other environmental toxicants. However, this traditional approach is quickly giving way to new approaches such as in vitro, in silico, and other alternative and more humane testing strategies for toxicity screening to support risk-based decision making. this paradigm shift has occurred, due in part, to the recognition that current in vivo testing methods are unable to keep pace with the ever-increasing number of chemicals in commerce and the environment, for which toxicity testing is needed. these issues have long been recognized, and toxicologists have operated under the principle of the three “R”s (reduction, replacement, and refinement of animal-based tests)1 in toxicity testing for decades. More recently, legislative mandates in the United States2 and Europe 3 have aimed at setting timelines for implementation of these principles into future toxicity testing strategies.

In 2007, the National Research Council (NRC) published a report outlining a new vision and strategy for toxicity testing in the 21st century.4 This report recommended a switch to in vitro based approaches, with the additional benefit of not only leading to the reduction of animals used, but also reduction in the time and cost of toxicity testing. The basis for these tests is the concept of a toxicity pathway, which is defined as a cellular/molecular pathway that when sufficiently perturbed, can lead to an adverse health outcome. The NRC also made suggestions for the type of technological tools and computational methods that might be used to achieve their vision and that would aid in identifying critical toxicity pathways.4 These tools and methods include high-throughput screening tests, microarrays, genomic data, the use of physiologically based pharmacokinetic (PBPK) models, and computational biology analysis methods. This new paradigm has led to an era of advances in toxicological testing methods driven by improved and increasingly cost-efficient analytical and high-throughput in vitro technologies that are capable of measuring cellular pathway responses.

In contrast to animal-based methods, in vitro high-throughput toxicity testing (HTT) technologies are able to achieve broader biological coverage at relatively lower cost, as these assays are capable of screening thousands of chemicals across several hundred toxicity pathway endpoints.56 in addition, these HTT technologies also aid in the prioritization of chemicals7 for more rigorous follow-up in vivo testing. Therefore, HTT technologies are expected to reduce the heavy footprints of traditional toxicity testing methods and are better suited to keep pace with the number of chemicals for which toxicity testing is needed. However, these new technologies and methods present challenges including the need to understand (1) the toxicity pathways and/ or mechanistic processes for which these in vitro technologies are acting as in vivo surrogates; (2) the other potential mechanistic processes required for manifestation of an adverse effect; and (3) the type and magnitude of exposure needed to generate the adverse reaction. with respect to challenge 3, animal-based tests have the advantage of including the ADME (absorption, distribution, metabolism, and excretion) processes that are needed to link external exposures to internal target tissue doses, albeit with no guarantee that the dosimetry will be consistent across different species. In vitro tests, in contrast, require greater understanding of the ADME processes in order to match the chemical concentrations that show effects in the in vitro assay with chemical concentrations to which humans and wildlife are exposed. Fortunately, advances in HTT have been occurring in parallel with advances in mechanistic toxicology and predictive exposure science such that these challenges are being addressed.

The mode of action (MOA) framework was initially described in 2001 67 and has been extensively refined over the past decade.68 The US Environmental Protection Agency (EPA) guidelines for carcinogen risk assessment (www. epa.gov/sites/production/files/2013-09/documents/cancer_guidelines_ final_3-25-05.pdf) defines an MOA as the sequence of key events that begin with the interaction of an agent (chemical or environmental toxicant) and result in cancer formation. In 2008, the World Health Organization (WHO) International Programme on Chemical Safety (IPCS) expanded this framework to include non-cancer adverse outcomes.8 Historically, MOA analyses, through the application of a weight-of-evidence approach, have been used to determine whether an MOA effect observed in an animal-based study will translate to the same effect in humans;9 and as a means to incorporate mechanistic data in human health risk assessment.

The adverse outcome pathway (AOP) framework emerged out of similar concepts that had been developed to support ecological risk assessment,10 and it provided a slightly more general framework because of the need to consider many species rather than a specific focus on humans. In a way, MOA could be thought of as a species-specific implementation of the AOP framework. More recently the AOP framework has been further generalized with an emphasis on integration of toxicological data including HTT to understand the common mechanisms of toxicity rather than the assembly of chemical-specific data for a single risk assessment.711 the aop framework maintains the concept of a key event as the MOA and separates key events into different levels of biological organization (molecular/cellular, tissue, organ, individual, and population), that connect events that are measurable in HTT with adverse outcomes of relevance to human or ecological risk assessment.

Being chemically agnostic7 allows the AOP to represent HTT in general and allow for increasing reliance on data from these assays for determining the potential for chemical toxicity as the confidence in the AOP increases. This is in contrast with traditional MOA analysis, where data used to build the MOA is typically derived exclusively from the chemical of interest. The chemically agnostic nature of AOPs also allows key events to be shared among AOPs, resulting in reusable components that allow parts of one AOP to be broadly applied to several adverse outcomes. For AOPs to be applicable to risk assessment, the chemical-specific data from an HTT can only inform potential for hazard. Any attempt at dose-response analysis requires that in vitro concentrations capable of perturbing a molecular target should be extrapolated to biologically effective doses, which can then be converted to external exposure levels through reverse dosimetry.12-14 Chemical-specific ADME behaviors, which are usually lacking in HTT assays, mediate the relationships between external concentrations, biologically effective doses, and resulting chemical toxicity. This chemical-specific information can then be integrated with the broad biological knowledge contained within the AOP framework to better inform risk assessment strategies.

Another important component of the NRC's vision for toxicity testing was the incorporation of exposure and population-based data in the testing strategy. The IPCS harmonization project document on risk assessment terminology (www.inchem.org/documents/harmproj/harmproj/harmproj1. pdf) defines exposure as the contact between an agent (e.g. chemical or environmental stressor) and a target (e.g. human or ecological receptor). Exposure science seeks to understand and characterize this interaction for the purpose of human and ecological health protection. Exposure assessments typically include measurements or estimates of the magnitude, frequency, route, pathway, and duration of exposure to the chemical agent, along with other population characteristics in the identification of public health hazards. Under the traditional risk assessment paradigm, exposure science is generally relegated to a supporting role, providing exposure estimates for comparison against hazard-based guidance values to determine whether unacceptable risks to public health exist. Also, historically, exposure data have been observational, which has limited them to serving as accompaniments to epidemiological studies for understanding impacts on individuals as well as populations. However, given the NRC's vision and the goal of more comprehensive assessments of thousands of chemicals/stressors for use in predictive toxicology, the need arose to complement toxicity testing with new strategies for exposure science in the 21st century.15

Major advances in analytical methods, biomonitoring, and computational tools have aided in the rapid transition of exposure science to a field that is more predictive, as well as data- and knowledge-driven. The need for an organizational and predictive framework for exposure science that furthers the application of systems-based approaches and fits the evolved exposure paradigm led to the conception of the aggregate exposure pathway (AEp) framework. the AEp is defined as a framework for organizing existing knowledge concerning biologically, chemically, and physically plausible, as well as empirically supported, links between the introduction of a chemical or stressor into the environment and its concentration at a site of action or target site of exposure.16 AEps allow for the organization of data and information emerging from an invigorated and expanding field of exposure science. the AEp framework is a layered structure that describes the elements of an exposure pathway, as well as the relationship between those elements.

Altogether, the aforementioned frameworks form a construct for understanding the processes that occur from the release of a stressor into the environment, subsequent exposure to that stressor, and the mechanism underlying any adverse outcome associated with the exposure. these frameworks also serve as a basis for informing risk assessment and decision making for endpoints of regulatory significance.

 
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