Andrew D. Kraft

Features of Microglia and Neuroinflammation Relevant to Environmental Exposure and Neurotoxicity

Microglia are resident cells of the brain involved in regulatory processes critical for development, maintenance of the neural environment, injury and repair. They belong to the monocytic-macrophage lineage and serve as brain immune cells to orchestrate innate immune responses; however, they are distinct from other tissue macrophages due to their relatively quiescent phenotype and tight regulation by the CNS microenvironment. Microglia actively survey the surrounding parenchyma and respond rapidly to changes such that any disruption to neural architecture or function can contribute to the loss in regulation of the microglia phenotype. In many models of neurodegeneration and neurotoxicity, early events of synaptic degeneration and neuronal loss are accompanied by an inflammatory response including activation of microglia, perivascular monocytes, and recruitment of leukocytes. In culture, microglia have been shown to be capable of releasing several potentially cytotoxic substances, such as reactive oxygen intermediates, nitric oxide, proteases, arachidonic acid derivatives, excitatory amino acids, and cytokines; however, they also produce various neurotrophic factors and quench damage from free radicals and excitotoxins. As the primary source for pro-inflammatory cytokines, microglia are implicated as pivotal mediators of neuroinflammation and can induce or modulate a broad spectrum of cellular responses. Neuroinflammation should be considered as a balanced network of processes whereby subtle modifications can shift the cells toward disparate outcomes. For any evaluation of neuroinflammation and microglial responses, within the framework of neurotoxicity or degeneration, one key question in determining the consequence of neuroinflammation is whether the response is an initiating event or the consequence of tissue damage. As examples of environmental exposure-related neuroinflammation in the literature, we provide an evaluation of data on manganese and diesel exhaust particles.

Microglia in the developing brain: a potential target with lifetime effects

Microglia are a heterogenous group of monocyte-derived cells serving multiple roles within the brain, many of which are associated with immune and macrophage like properties. These cells are known to serve a critical role during brain injury and to maintain homeostasis; yet, their defined roles during development have yet to be elucidated. Microglial actions appear to influence events associated with neuronal proliferation and differentiation during development, as well as, contribute to processes associated with the removal of dying neurons or cellular debris and management of synaptic connections. These long-lived cells display changes during injury and with aging that are critical to the maintenance of the neuronal environment over the lifespan of the organism. These processes may be altered by changes in the colonization of the brain or by inflammatory events during development. This review addresses the role of microglia during brain development, both structurally and functionally, as well as the inherent vulnerability of the developing nervous system. A framework is presented considering microglia as a critical nervous system-specific cell that can influence multiple aspects of brain development (e.g., vascularization, synaptogenesis, and myelination) and have a long term impact on the functional vulnerability of the nervous system to a subsequent insult, whether environmental, physical, age-related, or disease-related.

Developmental Neurotoxicity of Engineered Nanomaterials: Identifying Research Needs to Support Human Health Risk Assessment

Increasing use of engineered nanomaterials (ENM) in consumer products and commercial applications has helped drive a rise in research related to the environmental health and safety (EHS) of these materials. Within the cacophony of information on ENM EHS to date are data indicating that these materials may be neurotoxic in adult animals. Evidence of elevated inflammatory responses, increased oxidative stress levels, alterations in neuronal function, and changes in cell morphology in adult animals suggests that ENM exposure during development could elicit developmental neurotoxicity (DNT), especially considering the greater vulnerability of the developing brain to some toxic insults. In this review, we examine current findings related to developmental neurotoxic effects of ENM in the context of identifying research gaps for future risk assessments. The basic risk assessment paradigm is presented, with an emphasis on problem formulation and assessments of exposure, hazard, and dose response for DNT. Limited evidence suggests that in utero and postpartum exposures are possible, while fewer than 10 animal studies have evaluated DNT, with results indicating changes in synaptic plasticity, gene expression, and neurobehavior. Based on the available information, we use current testing guidelines to highlight research gaps that may inform ENM research efforts to develop data for higher throughput methods and future risk assessments for DNT. Although the available evidence is not strong enough to reach conclusions about DNT risk from ENM exposure, the data indicate that consideration of ENM developmental neurotoxic potential is warranted.

How credible are the study results? Evaluating and applying internal validity tools to literature-based assessments of environmental health hazards.

Environmental health hazard assessments are routinely relied upon for public health decision-making. The evidence base used in these assessments is typically developed from a collection of diverse sources of information of varying quality. It is critical that literature-based evaluations consider the credibility of individual studies used to reach conclusions through consistent, transparent and accepted methods. Systematic review procedures address study credibility by assessing internal validity or risk of bias - the assessment of whether the design and conduct of a study compromised the credibility of the link between exposure/intervention and outcome. This paper describes the commonalities and differences in risk-of-bias methods developed or used by five groups that conduct or provide methodological input for performing environmental health hazard assessments: the Grading of Recommendations Assessment, Development, and Evaluation (GRADE) Working Group, the Navigation Guide, the National Toxicology Programs (NTP) Office of Health Assessment and Translation (OHAT) and Office of the Report on Carcinogens (ORoC), and the Integrated Risk Information System of the U.S. Environmental Protection Agency (EPA-IRIS). Each of these groups have been developing and applying rigorous assessment methods for integrating across a heterogeneous collection of human and animal studies to inform conclusions on potential environmental health hazards. There is substantial consistency across the groups in the consideration of risk-of-bias issues or domains for assessing observational human studies. There is a similar overlap in terms of domains addressed for animal studies; however, the groups differ in the relative emphasis placed on different aspects of risk of bias. Future directions for the continued harmonization and improvement of these methods are also discussed.

Unmasking silent neurotoxicity following developmental exposure to environmental toxicants

Silent neurotoxicity, a term introduced approximately 25years ago, is defined as a persistent change to the nervous system that does not manifest as overt evidence of toxicity (i.e. it remains clinically unapparent) unless unmasked by experimental or natural processes. Silent neurotoxicants can be challenging for risk assessors, as the multifactorial experiments needed to reveal their effects are seldom conducted, and they are not addressed by current study design guidelines. This topic was the focus of a symposium addressing the interpretation and use of silent neurotoxicity data in human health risk assessments of environmental toxicants at the annual meeting of the Developmental Neurotoxicology Society (previously the Neurobehavioral Teratology Society) on June 30th, 2014. Several factors important to the design and interpretation of studies assessing the potential for silent neurotoxicity were discussed by the panelists and audience members. Silent neurotoxicity was demonstrated to be highly specific to the characteristics of the animals being examined, the unmasking agent tested, and the behavioral endpoint(s) evaluated. Overall, the experimental examples presented highlighted a need to consider common adverse outcomes and common biological targets for chemical and non-chemical stressors, particularly when the exposure and stressors are known to co-occur. Risk assessors could improve the evaluation of silent neurotoxicants in assessments through specific steps from researchers, including experiments to reveal the molecular targets and mechanisms that may result in specific types of silent neurotoxicity, and experiments with complex challenges reminiscent of the human situation.

The use of glial data in human health assessments of environmental contaminants.

Central nervous system (CNS) glia (i.e., astrocytes, microglia, and oligodendrocytes) are essential for maintaining neuronal homeostasis, and they orchestrate an organized cellular response to CNS injury. In addition to their beneficial roles, studies have demonstrated that disrupted glial function can have disastrous consequences on neuronal health. While effects on neuron-supportive glia are important to consider when evaluating neurotoxicity risk, interpreting glial changes is not always straightforward, particularly when attempting to discern pro-neurotoxic phenotypes from homeostatic processes or adaptive responses. To better understand how glia have been characterized and used in human health assessments of environmental contaminants (e.g., chemicals), an evaluation of all finalized assessments conducted by the U.S. Environmental Protection Agencys influential Integrated Risk Information System (IRIS) program between 1987 and 2013 was performed. Human health assessments to date have placed a clear emphasis on the neuronal cell response to potential toxicants, although more recent assessments increasingly include descriptions of glial changes. However, these descriptions are generally brief and non-specific, and they primarily consist of documenting gliosis following overt neuronal injury. As research interest in this topic continues to increase, methods for evaluating changes in glia continue to be expanded and refined, and assessors confidence in the reliability of these data is likely to rise. Thus, glial data are anticipated to have an increasingly influential impact on the interpretation of neurotoxicity risk and underlying mechanisms. As our understanding of the complex roles these cells play grows, this knowledge is expected to support the inclusion of more extensive and specific descriptions of glial changes, including informed interpretations of the potential impact on CNS health, in future human health assessments.

A systematic approach for identifying and presenting mechanistic evidence in human health assessments

Clear documentation of literature search and presentation methodologies can improve transparency in chemical hazard assessments. We sought to improve clarity for the scientific support for cancer mechanisms of action using a systematic approach to literature retrieval, selection, and presentation of studies. The general question was What are the mechanisms by which a chemical may cause carcinogenicity in the target tissue?. Di(2-ethylhexyl)phthalate was used as a case study chemical with a complex database of >3000 publications. Relevant mechanistic events were identified from published reviews. The PubMed search strategy included relevant synonyms and wildcards for DEHP and its metabolites, mechanistic events, and species of interest. Tiered exclusion/inclusion criteria for study pertinence were defined, and applied to the retrieved literature. Manual curation was conducted for mechanistic events with large literature databases. Literature trees documented identification and selection of the literature evidence. The selected studies were summarized in evidence tables accompanied by succinct narratives. Primary publications were deposited into the Health and Environmental Research Online (http://hero.epa.gov/) database and identified by pertinence criteria and key terms to permit organized retrieval. This approach contributes to human health assessment by effectively managing a large volume of literature, improving transparency, and facilitating subsequent synthesis of information across studies.

Study sensitivity: Evaluating the ability to detect effects in systematic reviews of chemical exposures

A critical step in systematic reviews of potential health hazards is the structured evaluation of the strengths and weaknesses of the included studies; risk of bias is a term often used to represent this process, specifically with respect to the evaluation of systematic errors that can lead to inaccurate (biased) results (i.e. focusing on internal validity). Systematic review methods developed in the clinical medicine arena have been adapted for use in evaluating environmental health hazards; this expansion raises questions about the scope of risk of bias tools and the extent to which they capture the elements that can affect the interpretation of results from environmental and occupational epidemiology studies and in vivo animal toxicology studies, (the studies typically available for assessment of risk of chemicals). One such element, described here as sensitivity, is a measure of the ability of a study to detect a true effect or hazard. This concept is similar to the concept of the sensitivity of an assay; an insensitive study may fail to show a difference that truly exists, leading to a false conclusion of no effect. Factors relating to study sensitivity should be evaluated in a systematic manner with the same rigor as the evaluation of other elements within a risk of bias framework. We discuss the importance of this component for the interpretation of individual studies, examine approaches proposed or in use to address it, and describe how it relates to other evaluation components. The evaluation domains contained within a risk of bias tool can include, or can be modified to include, some features relating to study sensitivity; the explicit inclusion of these sensitivity criteria with the same rigor and at the same stage of study evaluation as other bias-related criteria can improve the evaluation process. In some cases, these and other features may be better addressed through a separate sensitivity domain. The combined evaluation of risk of bias and sensitivity can be used to identify the most informative studies, to evaluate the confidence of the findings from individual studies and to identify those study elements that may help to explain heterogeneity across the body of literature.

Current Approaches to Risk Assessment for Developmental Neurotoxicity

Abstract Risk assessment for developmental neurotoxicity of chemicals follows a standardized paradigm that includes problem formulation and scoping, hazard identification, dose–response assessment, exposure assessment, and risk characterization. In each step, relevant data are systematically evaluated and integrated into a determination of potential risk to humans of neurological perturbation following exposure during periods of nervous system development. Human and animal data are supplemented by information from nonmammalian species, in vitro assays, toxicokinetic data, and other mechanistic studies when available. Developmental neurotoxicity risk assessment incorporates considerations of intrinsic susceptibilities, critical developmental windows of exposure and periods of assessment, the major manifestations of developmental toxicity (death, structural abnormalities, delayed growth, and functional effects), and an assessment of dose response. Methodological challenges are related to the outcomes assessed, the populations of concern, and the complexity and trajectory of nervous system development. Developmental neurotoxicity risk assessment is an indispensable tool to inform decisions for public health protection.

Quantitative meta-analytic approaches for the systematic synthesis of data and hazard identification: A case study of decreased pain sensitivity due to trimethylbenzene exposure.

Abstract Traditionally, human health risk assessments have relied on qualitative approaches for hazard identification, which involves weight of evidence determinations that integrate evidence across multiple studies. Recently, the National Research Council has recommended the development of quantitative approaches for evidence integration, including the application of meta‐analyses, to help summarize and evaluate the results of a systematic review. In the meta‐analytic approach, a pooled effect size is calculated after consideration of multiple potential confounding factors in order to determine whether the entire database under consideration indicates a chemical is a hazard. The following case‐study applies qualitative and quantitative approaches to determine whether trimethylbenzene (TMB) isomers represent a neurotoxic hazard, specifically focusing on pain sensitivity. Following a thorough literature search, the only pain sensitivity studies available for TMBs initially seem discordant in their results: effects on pain sensitivity are seen immediately after termination of exposure, appear to resolve 24 h after exposure, and then reappear 50 days later following foot‐shock. Qualitative consideration of toxicological and toxicokinetic characteristics of the TMB isomers suggests that the observed differences between studies are likely due to testing time and the application of external stressors. Meta‐analyses and –regressions support this conclusion: when all studies are included and possible confounders (isomer, testing time, laboratory, etc.) are accounted for, the pooled effect sizes are statistically significant, thus supporting that TMBs are a possible neurotoxic hazard to human health. Ultimately, this case study demonstrates how qualitative and quantitative methods can be combined to provide a robust hazard identification analysis by incorporating more of the available information. HighlightsApplication of meta‐analytical approaches for hazard identification are proposed.Quantitative methods compliment qualitative methods during evidence integration.Meta‐regression can identify modifiers that influence differences in study results.Systematic syntheses of evidence expand the utility of systematic review methods