Development of an Evidence-Based Risk Assessment Framework.

Assessment of potential human health risks associated with environmental and other agents requires careful evaluation of all available and relevant evidence for the agent of interest, including both data-rich and data-poor agents. With the advent of new approach methodologies in toxicological risk assessment, guidance on integrating evidence from mul-tiple evidence streams is needed to ensure that all available data is given due consideration in both qualitative and quantitative risk assessment. The present report summarizes the discussions among academic, government, and private sector participants from North America and Europe in an international workshop convened to explore the development of an evidence-based risk assessment framework, taking into account all available evidence in an appropriate manner in order to arrive at the best possible characterization of potential human health risks and associated uncertainty. Although consensus among workshop participants was not a specific goal, there was general agreement on the key consider-ations involved in evidence-based risk assessment incorporating 21st century science into human health risk assessment. These considerations have been embodied into an overarching prototype framework for evidence integration that will be explored in more depth in a follow-up meeting.

Integrated Risk Information System (IRIS) response to "Assessing risk of bias in human environmental epidemiology studies using three tools: different conclusions from different tools".

"Assessing risk of bias in human environmental epidemiology studies using three tools: different conclusions from different tools," a recent publication in this journal, applied the study evaluation approach developed by the U.S. Environmental Protection Agency's Integrated Risk Information System (IRIS), as well as other approaches, to a set of studies examining polybrominated diphenyl ethers (PBDEs) and neurodevelopment. They concluded that use of the IRIS approach resulted in exclusion of studies, which would lead to hazard conclusions based on an incomplete body of evidence. As scientists in the IRIS program, we support the comparison of approaches to improve systematic review methods for environmental exposures; however, we believe the IRIS approach was misrepresented. In this letter, we demonstrate that the ratings attributed to the IRIS approach were not consistent with our own application of the tool. We also clarify the use of studies rated as "low confidence" and the use of an overall study confidence rating in our systematic reviews. In conclusion, the IRIS study evaluation approach is a transparent method to inform certainty in our evidence synthesis decisions and ensures consistency in the development of IRIS health assessments.

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.

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.

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 Program's (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.

Association Between Microglia, Inflammatory Factors, and Complement with Loss of Hippocampal Mossy Fiber Synapses Induced by Trimethyltin.

Complement-associated factors are implicated in pathogen presentation, neurodegeneration, and microglia resolution of tissue injury. To characterize complement activation with microglial clearance of degenerating mossy fiber boutons, hippocampal dentate granule neurons were ablated in CD-1 mice with trimethyltin (TMT; 2.2 mg/kg, i.p.). Neuronal apoptosis was accompanied by amoeboid microglia and elevations in tumor necrosis factor [Tnfa], interleukin 1ß [Il1b], and Il6 mRNA and C1q protein. Inos mRNA levels were unaltered. Silver degeneration and synaptophysin staining indicated loss of synaptic innervation to CA3 pyramidal neurons. Reactive microglia with thickened bushy morphology showed co-localization of synaptophysin+ fragments. The initial response at 2 days post-TMT included transient elevations in Tnfa, Il1b, Il6, and Inos mRNA levels. A concurrent increase at 2 days was observed in arginase-1 [Arg1], Il10, transforming growth factor ß1 [Tgfb1], and chitinase 3 like-3 [Ym1] mRNA levels. At 2 days, C1q protein was evident in the CA3 with elevated C1qa, C1qb, C3, Cr3a, and Cr3b mRNA levels. mRNA levels remained elevated at 5 days, returning to control by 14 days, corresponding to silver degeneration. mRNA levels for pentraxin3 (Ptx3) were elevated on day 2 and Ptx1 was not altered. Our data suggest an association between microglia reactivity, the induction of anti-inflammatory genes concurrent with pro-inflammatory genes and the expression of complement-associated factors with the degeneration of synapses following apoptotic neuronal loss.

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 Agency's 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.

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.

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.

Activated microglia proliferate at neurites of mutant huntingtin-expressing neurons.

In Huntington's disease (HD), mutated huntingtin (mhtt) causes striatal neurodegeneration which is paralleled by elevated microglia cell numbers. In vitro corticostriatal slice and primary neuronal culture models, in which neuronal expression of mhtt fragments drives HD-like neurotoxicity, were employed to examine wild type microglia during both the initiation and progression of neuronal pathology. As neuronal pathology progressed, microglia initially localized in the vicinity of neurons expressing mhtt fragments increased in number, demonstrated morphological evidence of activation, and expressed the proliferation marker, Ki67. These microglia were positioned along irregular neurites, but did not localize with mhtt inclusions nor exacerbate mhtt fragment-induced neurotoxicity. Prior to neuronal pathology, microglia upregulated ionized calcium binding adaptor molecule 1 (Iba1), signaling a functional shift. With neurodegeneration, interleukin-6 and complement component 1q were increased. The results suggest a stimulatory, proliferative signal for microglia present at the onset of mhtt fragment-induced neurodegeneration. Thus, microglia effect a localized inflammatory response to neuronal mhtt expression that may serve to direct microglial removal of dysfunctional neurites or aberrant synapses, as is required for reparative actions in vivo.

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.

Voluntary exercise protects hippocampal neurons from trimethyltin injury: possible role of interleukin-6 to modulate tumor necrosis factor receptor-mediated neurotoxicity.

In the periphery, exercise induces interleukin (IL)-6 to downregulate tumor necrosis factor (TNF), elevate interleukin-1 receptor antagonist (IL-1RA), decreasing inflammation. Exercise also offers neuroprotection and facilitates brain repair. IL-6 production in the hippocampus following exercise suggests the potential of a similar protective role as in the periphery to down-regulate TNFα and inflammation. Using a chemical-induced model of hippocampal dentate granule cell death (trimethyltin, TMT 2.4 mg/kg, ip) dependent upon TNF receptor signaling, we demonstrate neuroprotection in mice with 2 weeks access to running wheel. Exercise attenuated neuronal death and diminished elevations in TNFα, TNF receptor 1, myeloid differentiation primary response gene (MyD) 88, transforming growth factor ß, chemokine (C-C motif) ligand 2 (CCL2), and CCL3. Elevated mRNA levels for IL-1α, IL-1RA, occurred with injury and protection. mRNA and protein levels of IL-6 and neuronal expression of IL-6 receptor α, were elevated with injury and protection. Microarray pathway analysis supported an up-regulation of TNFα cell death signaling pathways with TMT and inhibition by exercise. IL-6 pathway recruitment occurred in both conditions. IL-6 downstream signal events differed in the level of STAT3 activation. Exercise did not increase mRNA levels of brain derived neurotrophic factor, nerve growth factor, or glial derived neurotrophic factor. In IL-6 deficient mice, exercise did not attenuate TMT-induced tremor and a diminished level of neuroprotection was observed. These data suggest a contributory role for IL-6 induced by exercise for neuroprotection in the CNS similar to that seen in the periphery.

Injury-induced neurogenesis: consideration of resident microglia as supportive of neural progenitor cells.

The induction of neurogenesis in the adult subgranular zone (SGZ) by injury is often accompanied by changes in the extracellular environment that can have significant impacts on neural progenitor cells (NPCs). We examined the induction of neurogenesis in the SGZ at 72 h following an injection of the hippocampal toxicant, trimethyltin (TMT; 2 mg/kg, ip) inducing apoptosis in dentate granule neurons. BrdU+ incorporation during the active period of neuronal death indicated NPC proliferation and migration of newly generated cells into the granule cell layer (GCL). BrdU+ cells were transiently in contact with process bearing microglia within the inner SGZ layer. Contact with GFAP+ astrocyte processes occurred once cells were within the GCL. A small percentage of the BrdU+ cells within the SGZ region showed immunoreactivity for tumor necrosis factor (TNF) p75 receptor (TNFp75R). In mice deficient for TNFp75R, TMT injection produced an equivalent level of dentate granule cell death however; BrdU+ cells were localized at the SGZ as compared to the presence of cells within the GCL in the WT mice dosed with TMT. These data suggest that cells generated by NPCs in the SGZ induced with a focal lesion to the dentate granule neurons of adolescent mice maintain the capacity to utilize the neuroinflammation and microglia responses within their environment for migration into the GCL.

Imaging upregulated brain arachidonic acid metabolism in HIV-1 transgenic rats.

Human immunodeficiency virus (HIV)-associated infection involves the entry of virus-bearing monocytes into the brain, followed by microglial activation, neuroinflammation, and upregulated arachidonic acid (AA) metabolism. The HIV-1 transgenic (Tg) rat, a noninfectious HIV-1 model, shows neurologic and behavioral abnormalities after 5 months of age. We hypothesized that brain AA metabolism would be elevated in older HIV-1 Tg rats in vivo. Arachidonic acid incorporation from the plasma into the brain of unanesthetized 7-to-9-month-old rats was imaged using quantitative autoradiography, after [1-(14)C]AA infusion. Brain phospholipase (PLA(2)) activities and eicosanoid concentrations were measured, and enzymes were localized by immunostaining. AA incorporation coefficients k* and rates J(in), measures of AA metabolism, were significantly higher in 69 of 81 brain regions in HIV-1 Tg than in control rats, as were activities of cytosolic (c)PLA(2)-IV, secretory (s)PLA(2), and calcium independent (i)PLA(2)-VI, as well as prostaglandin E(2) and leukotriene B(4) concentrations. Immunostaining of somatosensory cortex showed elevated cPLA(2)-IV, sPLA(2)-IIA, and cyclooxygenase-2 in neurons. Brain AA incorporation and other markers of AA metabolism are upregulated in HIV-1 Tg rats, in which neurologic changes and neuroinflammation have been reported. Positron emission tomography with [1-(11)C]AA could be used to test whether brain AA metabolism is upregulated in HIV-1-infected patients, in relation to cognitive and behavioral disturbances.

Heterogeneity of microglia and TNF signaling as determinants for neuronal death or survival.

Microglia do not constitute a single, uniform cell population, but rather comprise cells with varied phenotypes, some which are beneficial and others that may require active regulatory control. Thus, gaining a better understanding of the heterogeneity of resident microglia responses will contribute to any interpretation regarding the impact of any such response in the brain. Microglia are the primary source of the pro-inflammatory cytokine, tumor necrosis factor (TNF) that can initiate various effects through the activation of membrane receptors. The TNF p55 receptor contains a death domain and activation normally leads to cellular apoptosis; however, under specific conditions, receptor activation can also lead to the activation of NF-kappaB and contribute to cell survival. These divergent outcomes have been linked to receptor localization with receptor internalization leading to cell death and membrane localization supporting cell survival. A second TNF receptor, TNF p75 receptor, is normally linked to cell growth and survival, however, it can cooperate with the p55 receptor and contribute to cell death. Thus, while an elevation in TNFalpha in the brain is often considered an indicator of microglia activation and neuroinflammation, a number of factors come into play to determine the final outcome. Data are reviewed demonstrating that heterogeneity in morphological response of microglia and the expression of TNFalpha and TNF receptors are critical in identifying and characterizing neurotoxic events as they relate to neuroinflammation, neuronal damage and in stimulating neuroprotection.

The Nrf2/ARE pathway as a potential therapeutic target in neurodegenerative disease.

Nuclear factor E2-related factor 2 (Nrf2) is a transcription factor known to induce expression of a variety of cytoprotective and detoxification genes. Several of the genes commonly regulated by Nrf2 have been implicated in protection from neurodegenerative conditions. Work from several laboratories has uncovered the potential for Nrf2-mediated transcription to protect from neurodegeneration resulting from mechanisms involving oxidative stress. For this reason, Nrf2 may be considered a therapeutic target for conditions that are known to involve free radical damage. Because common mechanisms of neurodegeneration, such as mitochondrial dysfunction and build-up of reactive oxygen species, are currently being uncovered, targeting Nrf2 may be valuable in combating conditions with variable causes and etiologies. Most effectively to target this protein in neurodegenerative conditions, a description of the involvement of Nrf2 and potential for neuroprotection must come from laboratory models. Herein, we review the current literature that suggests that Nrf2 may be a valuable therapeutic target for neurodegenerative disease, as well as experiments that illustrate potential mechanisms of protection.

The Nrf2-ARE pathway: an indicator and modulator of oxidative stress in neurodegeneration.

Transcriptional activation of protective genes is mediated by a cis-acting element called the antioxidant responsive element (ARE). The transcription factor Nrf2 (NF-E2-related factor 2) binds to the ARE. Activation of this pathway protects cells from oxidative stress-induced cell death. Increased oxidative stress is associated with neuronal cell death during the pathogenesis of multiple chronic neurodegenerative diseases, including Alzheimer's disease, Parkinson's disease, Huntington's disease, and amyotrophic lateral sclerosis. We hypothesize that Nrf2-ARE activation is a novel neuroprotective pathway that confers resistance to a variety of oxidative, stress-related, neurodegenerative insults. In recent studies, primary neuronal cultures treated with chemical activators of the Nrf2-ARE pathway displayed significantly greater resistance to oxidative stress-induced neurotoxicity. Similar cultures generated from ARE-hPAP reporter mice demonstrated selective activation of the Nrf2-ARE pathway in astrocytes, suggesting that Nrf2 activation in astrocytes somehow confers resistance to naive neurons. Further, in chemical models of neurodegeneration, Nrf2 knockout mice are significantly more sensitive to mitochondrial complex I and II inhibitors. Combining these observations with the results implying that the astrocyte is central to Nrf2-ARE-mediated neuroprotection, we transplanted Nrf2-overexpressing astrocytes into the mouse striatum prior to lesioning with malonate. This procedure led to dramatic protection against malonate-induced neurotoxicity. Translating this to other chemical and genetic models of neurodegeneration will be discussed.

Neuroinflammation and microglia: considerations and approaches for neurotoxicity assessment.

BACKGROUND: The impact of an inflammatory response, as well as interactions between the immune and nervous systems, are rapidly assuming major roles in neurodegenerative disease and injury. However, it is now appreciated that the exact nature of such responses can differ with each type of insult and interaction. More recently, neuroinflammation and the associated cellular response of microglia are being considered for their contribution to neurotoxicity of environmental agents; yet, so far, the inclusion of inflammatory end points into neurotoxicity assessment have relied primarily on relatively limited measures or driven by in vitro models of neurotoxicity. OBJECTIVE: To present background information on relevant biological considerations of neuroinflammation and the microglia response demonstrating the complex integrative nature of these biological processes and raising concern with regards to translation of effects demonstrated in vitro to the in vivo situation. Specific points are addressed that would influence the design and interpretation of neuroinflammation with regards to neurotoxicology assessment. CONCLUSION: There is a complex and dynamic response in the brain to regulate inflammatory processes and maintain a normal homeostatic level. The classification of such responses as beneficial or detrimental is an oversimplification. Neuroinflammation should be considered as a balanced network of processes in which subtle modifications can shift the cells toward disparate outcomes. The tendency to overinterpret data obtained in an isolated culture system should be discouraged. Rather, the use of cross-disciplinary approaches to evaluate several end points should be incorporated into the assessment of inflammatory contributions to the neurotoxicity of environmental exposures.

Activation of the Nrf2-ARE pathway in muscle and spinal cord during ALS-like pathology in mice expressing mutant SOD1.

Oxidative stress plays a key role in the neuronal loss exhibited in amyotrophic lateral sclerosis (ALS), an event precipitating irreversible muscle atrophy. By crossing ALS mouse models (SOD(G93A) and SOD(H46RH48Q)) with an antioxidant response element (ARE) reporter mouse, we identified activation characteristics of the ARE system throughout the timecourse of motor neuron disease. Surprisingly, the earliest and most significant activation of this genetic sensor of oxidative stress occurred in the distal muscles of mutant SOD mice. The resultant data supports existing hypotheses that the muscle is somehow implicated during the initial pathology of these mice. Subsequently, Nrf2-ARE activation appears to progress in a retrograde fashion along the motor pathway. These data provide timely information concerning the contributions of the Nrf2-ARE pathway in ALS disease progression.