John R. Bucher

A Framework for Human Relevance Analysis of Information on Carcinogenic Modes of Action

The human relevance framework (HRF) outlines a four-part process, beginning with data on the mode of action (MOA) in laboratory animals, for evaluating the human relevance of animal tumors. Drawing on U.S. EPA and IPCS proposals for animal MOA analysis, the HRF expands those analyses to include a systematic evaluation of comparability, or lack of comparability, between the postulated animal MOA and related information from human data sources. The HRF evolved through a series of case studies representing several different MOAs. HRF analyses produced divergent outcomes, some leading to complete risk assessment and others discontinuing the process, according to the data available from animal and human sources. Two case examples call for complete risk assessments. One is the default: When data are insufficient to confidently postulate a MOA for test animals, the animal tumor data are presumed to be relevant for risk assessment and a complete risk assessment is necessary. The other is the product of a data-based finding that the animal MOA is relevant to humans. For the specific MOA and endpoint combinations studied for this article, full risk assessments are necessary for potentially relevant MOAs involving cytotoxicity and cell proliferation in animals and humans (Case Study 6, chloroform) and formation of urinary-tract calculi (Case Study 7, melamine). In other circumstances, when data-based findings for the chemical and endpoint combination studied indicate that the tumor-related animal MOA is unlikely to have a human counterpart, there is little reason to continue the risk assessment for that combination. Similarly, when qualitative considerations identify MOAs specific to the test species or quantitative considerations indicate that the animal MOA is unlikely to occur in humans, such hazard findings are generally conclusive and further risk assessment is not necessary for the endpoint–MOA combination under study. Case examples include a tumor-related protein specific to test animals (Case Study 3, d-limonene), the tumor consequences of hormone suppression typical of laboratory animals but not humans (Case Study 4, atrazine), and chemical-related enhanced hormone clearance rates in animals relative to humans (Case Study 5, phenobarbital). The human relevance analysis is highly specific for the chemical–MOA–tissue–endpoint combination under analysis in any particular case: different tissues, different endpoints, or alternative MOAs for a given chemical may result in different human relevance findings. By providing a systematic approach to using MOA data, the HRF offers a new tool for the scientific communitys overall effort to enhance the predictive power, reliability and transparency of cancer risk assessment.

Improving the human hazard characterization of chemicals: a Tox21 update.

Background: In 2008, the National Institute of Environmental Health Sciences/National Toxicology Program, the U.S. Environmental Protection Agency’s National Center for Computational Toxicology, and the National Human Genome Research Institute/National Institutes of Health Chemical Genomics Center entered into an agreement on “high throughput screening, toxicity pathway profiling, and biological interpretation of findings.” In 2010, the U.S. Food and Drug Administration (FDA) joined the collaboration, known informally as Tox21. Objectives: The Tox21 partners agreed to develop a vision and devise an implementation strategy to shift the assessment of chemical hazards away from traditional experimental animal toxicology studies to one based on target-specific, mechanism-based, biological observations largely obtained using in vitro assays. Discussion: Here we outline the efforts of the Tox21 partners up to the time the FDA joined the collaboration, describe the approaches taken to develop the science and technologies that are currently being used, assess the current status, and identify problems that could impede further progress as well as suggest approaches to address those problems. Conclusion: Tox21 faces some very difficult issues. However, we are making progress in integrating data from diverse technologies and end points into what is effectively a systems-biology approach to toxicology. This can be accomplished only when comprehensive knowledge is obtained with broad coverage of chemical and biological/toxicological space. The efforts thus far reflect the initial stage of an exceedingly complicated program, one that will likely take decades to fully achieve its goals. However, even at this stage, the information obtained has attracted the attention of the international scientific community, and we believe these efforts foretell the future of toxicology.

Transforming Environmental Health Protection

We propose a shift from primarily in vivo animal studies to in vitro assays, in vivo assays with lower organisms, and computational modeling for toxicity assessments.

Role of Environmental Chemicals in Diabetes and Obesity: A National Toxicology Program Workshop Review

Background: There has been increasing interest in the concept that exposures to environmental chemicals may be contributing factors to the epidemics of diabetes and obesity. On 11–13 January 2011, the National Institute of Environmental Health Sciences (NIEHS) Division of the National Toxicology Program (NTP) organized a workshop to evaluate the current state of the science on these topics of increasing public health concern. Objective: The main objective of the workshop was to develop recommendations for a research agenda after completing a critical analysis of the literature for humans and experimental animals exposed to certain environmental chemicals. The environmental exposures considered at the workshop were arsenic, persistent organic pollutants, maternal smoking/nicotine, organotins, phthalates, bisphenol A, and pesticides. High-throughput screening data from Toxicology in the 21st Century (Tox21) were also considered as a way to evaluate potential cellular pathways and generate -hypotheses for testing which and how certain chemicals might perturb biological processes related to diabetes and obesity. Conclusions: Overall, the review of the existing literature identified linkages between several of the environmental exposures and type 2 diabetes. There was also support for the “developmental obesogen” hypothesis, which suggests that chemical exposures may increase the risk of obesity by altering the differentiation of adipocytes or the development of neural circuits that regulate feeding behavior. The effects may be most apparent when the developmental exposure is combined with consumption of a high-calorie, high-carbohydrate, or high-fat diet later in life. Research on environmental chemical exposures and type 1 diabetes was very limited. This lack of research was considered a critical data gap. In this workshop review, we outline the major themes that emerged from the workshop and discuss activities that NIEHS/NTP is undertaking to address research recommendations. This review also serves as an introduction to an upcoming series of articles that review the literature regarding specific exposures and outcomes in more detail.

The requirement for ferric in the initiation of lipid peroxidation by chelated ferrous iron.

When certain ferrous chelates are added to lipid, peroxidation of the lipid occurs following a short lag. This suggests that a product of ferrous autoxidation is required to initiate lipid peroxidation. This autoxidation product is apparently ferric iron, rather than the oxygen radicals which also result from ferrous autoxidation. Studies with oxy-radical scavengers and catalase suggest that O2-., H2O2, or the .OH are not involved in the initiation reactions, therefore, we propose that a ferrous-dioxygen-ferric chelate complex may be the initiating species.

Thiol-dependent lipid peroxidation

Abstract Initiation of lipid peroxidation in liposomes by cysteine, glutathione, or dithiothreitol required iron, and was not inhibited by superoxide dismutase. The absence of superoxide involvement in thiol autoxidation was confirmed by the inability of superoxide dismutase to inhibit thiol reduction of cytochrome c. Furthermore, the rate of cytochrome c reduction by thiols was not decreased under anaerobic conditions. We suggest that lipid peroxidation initiated by thiols and iron occurs via direct reduction of iron. Control of cellular thiol autoxidation, and reactions occurring as a consequence, such as lipid peroxidation, must therefore involve chelation of transition metals to control their redox reactions.

Systematic Review and Evidence Integration for Literature-Based Environmental Health Science Assessments

Background: Systematic-review methodologies provide objectivity and transparency to the process of collecting and synthesizing scientific evidence in reaching conclusions on specific research questions. There is increasing interest in applying these procedures to address environmental health questions. Objectives: The goal was to develop a systematic-review framework to address environmental health questions by extending approaches developed for clinical medicine to handle the breadth of data relevant to environmental health sciences (e.g., human, animal, and mechanistic studies). Methods: The Office of Health Assessment and Translation (OHAT) adapted guidance from authorities on systematic-review and sought advice during development of the OHAT Approach through consultation with technical experts in systematic review and human health assessments, as well as scientific advisory groups and the public. The method was refined by considering expert and public comments and through application to case studies. Results and Discussion: Here we present a seven-step framework for systematic review and evidence integration for reaching hazard identification conclusions: 1) problem formulation and protocol development, 2) search for and select studies for inclusion, 3) extract data from studies, 4) assess the quality or risk of bias of individual studies, 5) rate the confidence in the body of evidence, 6) translate the confidence ratings into levels of evidence, and 7) integrate the information from different evidence streams (human, animal, and “other relevant data” including mechanistic or in vitro studies) to develop hazard identification conclusions. Conclusion: The principles of systematic review can be successfully applied to environmental health questions to provide greater objectivity and transparency to the process of developing conclusions. Citation: Rooney AA, Boyles AL, Wolfe MS, Bucher JR, Thayer KA. 2014. Systematic review and evidence integration for literature-based environmental health science assessments. Environ Health Perspect 122:711–718; http://dx.doi.org/10.1289/ehp.1307972

The multiple effects of ethylenediaminetetraacetate in several model lipid peroxidation systems.

Abstract Experiments were performed which illustrate the various ways EDTA can influence lipid peroxidation. Either detergent-dispersed linoleate, or liposomes made from extracted microsomal phospholipids were utilized as substrates for peroxidation. Peroxidation was accomplished using Fe 2+ or Fe 3+ . In systems utilizing Fe 2+ , EDTA chelation facilitated Fe 2+ autoxidation which in turn caused peroxidation of detergent-dispersed linoleate. Peroxidation was not initiated during EDTA-Fe 2+ autoxidation when the substrate lipids were in a liposomal configuration. Systems utilizing Fe 3+ required an enzyme (either xanthine oxidase or NADPH-cytochrome P 450 reductase) to reduce the iron for peroxidative activity. EDTA chelation of Fe 3+ enhanced the xanthine oxidase and NADPH-cytochrome P 450 reductase-catalyzed peroxidation of detergent-dispersed linoleate, presumably by facilitating the reduction of Fe 3+ . Catalase and mannitol inhibited both EDTA-Fe 2+ - and EDTA-Fe 3+ -dependent lipid peroxidation. EDTA-Fe 3+ was not capable of initiating peroxidation of phospholipid liposomes following enzymatic reduction by either enzyme, but ADP-chelated iron effectively initiated liposomal peroxidation in similar systems. With xanthine oxidase-catalyzed peroxidation of liposomes with ADP-Fe 3+ , the inclusion of EDTA-Fe 3+ caused a modest enhancement of activity. EDTA-Fe 3+ greatly stimulated NADPH-cytochrome P 450 reductase-catalyzed peroxidation of liposomes with ADP-Fe 3+ . In contrast, the addition of EDTA, rather than EDTA-Fe 3+ inhibited the liposomal peroxidation catalyzed by either enzyme with ADP-Fe 3+ when the EDTA concentration exceeded the concentration of Fe 3+ .

Dose-Additive Carcinogenicity of a Defined Mixture of “Dioxin-like Compounds”

Use of the dioxin toxic equivalency factor (TEF) approach in human risk assessments assumes that the combined effects of dioxin-like compounds in a mixture can be predicted based on a potency-adjusted dose-additive combination of constituents of the mixture. In this study, we evaluated the TEF approach in experimental 2-year rodent cancer bioassays with female Harlan Sprague-Dawley rats receiving 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), 3,3′,4,4′,5-pentachlorobiphenyl (PCB-126), 2,3,4,7,8-pentachlorodibenzofuran (PeCDF), or a mixture of the three compounds. Statistically based dose–response modeling indicated that the shape of the dose–response curves for hepatic, lung, and oral mucosal neoplasms was the same in studies of the three individual chemicals and the mixture. In addition, the dose response for the mixture could be predicted from a combination of the potency-adjusted doses of the individual compounds. Finally, we showed that use of the current World Health Organization dioxin TEF values adequately predicted the increased incidence of liver tumors (hepatocellular adenoma and cholangiocarcinoma) induced by exposure to the mixture. These data support the use of the TEF approach for dioxin cancer risk assessments.

Key Characteristics of Carcinogens as a Basis for Organizing Data on Mechanisms of Carcinogenesis.

Background: A recent review by the International Agency for Research on Cancer (IARC) updated the assessments of the > 100 agents classified as Group 1, carcinogenic to humans (IARC Monographs Volume 100, parts A–F). This exercise was complicated by the absence of a broadly accepted, systematic method for evaluating mechanistic data to support conclusions regarding human hazard from exposure to carcinogens. Objectives and Methods: IARC therefore convened two workshops in which an international Working Group of experts identified 10 key characteristics, one or more of which are commonly exhibited by established human carcinogens. Discussion: These characteristics provide the basis for an objective approach to identifying and organizing results from pertinent mechanistic studies. The 10 characteristics are the abilities of an agent to 1) act as an electrophile either directly or after metabolic activation; 2) be genotoxic; 3) alter DNA repair or cause genomic instability; 4) induce epigenetic alterations; 5) induce oxidative stress; 6) induce chronic inflammation; 7) be immunosuppressive; 8) modulate receptor-mediated effects; 9) cause immortalization; and 10) alter cell proliferation, cell death, or nutrient supply. Conclusion: We describe the use of the 10 key characteristics to conduct a systematic literature search focused on relevant end points and construct a graphical representation of the identified mechanistic information. Next, we use benzene and polychlorinated biphenyls as examples to illustrate how this approach may work in practice. The approach described is similar in many respects to those currently being implemented by the U.S. EPA’s Integrated Risk Information System Program and the U.S. National Toxicology Program. Citation: Smith MT, Guyton KZ, Gibbons CF, Fritz JM, Portier CJ, Rusyn I, DeMarini DM, Caldwell JC, Kavlock RJ, Lambert P, Hecht SS, Bucher JR, Stewart BW, Baan R, Cogliano VJ, Straif K. 2016. Key characteristics of carcinogens as a basis for organizing data on mechanisms of carcinogenesis. Environ Health Perspect 124:713–721; http://dx.doi.org/10.1289/ehp.1509912