Understanding the NIH review process: a brief guide to writing grant proposals in neurotoxicology

During the past two years, the National Institutes of Health have made significant changes in the review process for investigator-initiated research grant applications in neurotoxicology. First, study sections that formerly dealt with toxicology and alcohol, respectively, have been merged. Neurotoxicology grant applications are now reviewed by ALTX-3, a study section in which the majority of members have expertise in the neuronal, biochemical or behavioral effects of alcohol, but usually not other neurotoxicants. Second, the NIH has instituted new review criteria, in which significance, approach, innovation, investigator expertise, and research environment must all be explicitly addressed by the reviews. In this article, past and present members of the ALTX-3 study section describe the NIH review process, with emphasis on how neurotoxicology applications are handled, and provide guidelines for preparing competitive applications.     

Arsenic and manganese exposure and children's intellectual function

Recently, epidemiologic studies of developmental neurotoxicology have been challenged to increase focus on co-exposure to multiple toxicants. Earlier reports, including our own work in Bangladesh, have demonstrated independent associations between neurobehavioral function and exposure to both arsenic (As) and manganese (Mn) in school-aged children. Our earlier studies, however, were not designed to examine possible interactive effects of exposure to both As and Mn. To allow investigation of possible synergistic impact of simultaneous exposures, we recruited a new sample of 299 8-11 year old children, stratified by design on As (above and below 10 μg/L) and Mn (above and below 500 μg/L) concentrations of household wells. When adjusted only for each other, both As and Mn in whole blood (BAs; BMn) were significantly negatively related to most WISC-IV subscale scores. With further adjustment for socio-demographic features and ferritin, BMn remained significantly associated with reduced Perceptual Reasoning and Working Memory scores; associations for BAs, and for other subscales, were expectably negative, significantly for Verbal Comprehension. Urinary As (per gram creatinine) was significantly negatively associated with Verbal Comprehension scores, even with adjustment for BMn and other contributors. Mn by As interactions were not significant in adjusted or unadjusted models (all p's>0.25). Findings are consistent with other reports documenting adverse impact of both As and Mn exposure on child developmental outcomes, although associations appear muted at these relatively low exposure levels.     

Neurotoxic exposures and effects: Gender and sex matter! Hänninen Lecture 2011

Although males and females differ both biologically and in their social and power relations throughout their life span, research in environmental and occupational neurotoxicology often ignore sex and/or gender as a characteristic that requires in-depth consideration. The neurotoxicology literature continues to confuse the terms sex (biological attributes) and gender (socially constructed roles and behavior) and the words are still used interchangeably. Throughout the lifespan, sex and gender are in interaction and both may play a role in influencing exposure and effect. Studies that have examined both males and females, provide evidence for sex differences in toxicokinetics and responses to neurotoxic assault as well as gender differences in exposure patterns, biomarkers of exposure, neurobehavioral performance and social consequences. Integrating sex and gender considerations into research in neurotoxicology would not only provide us with a better understanding of the mechanisms and pathways that lead to toxic assault, but also provide a means to improve preventive intervention strategies.     

Switching attention--additional aspects for the analysis

Problems related to attentional functions have often been reported within the neurotoxicology of long-term exposure to solvents, but knowledge about neuroanatomical sites involved in this degenerative process is still rare. However, some studies have emphasized the frontal cortex as a region of structural or functional changes in long-term exposed patients or accidental intoxications. Neurobehavioral tests using active switching tasks are widely used to detect frontal lobe dysfunction. Test batteries used in neurotoxicology provide such tasks but standard test interpretations often neglect these aspects. Rotogravure printing workers exclusively exposed to toluene were investigated in a longitudinal study with cross-sectional design. Data from two examination periods are presented. In the first sample 333 male workers were investigated. In the second examination period 278 workers could be retested. The workers differed with respect to level and duration of lifetime exposure to toluene. All subjects completed a neurobehavioral test battery including the task switching attention (EURONES). Different parameters were computed for consecutive trials with constant vs. shifted tasks and repetitions vs. changes of the response direction. In general the expected interaction between the two sequence factors could not be observed. In both examination periods the alternative strategy revealed no marked differences between task/response shifts. Considering the result of previous studies on task/response shifts, the results were highly unexpected. One explanation might be the strong deviations from equality for the relative frequencies of the four sequence conditions, resulting in biased expectancies. These may have superimposed on the basic effects of task shifts. However, in both examination periods weak effects of task shift could be revealed in the subgroup of the long-term exposed workers. In further studies it is necessary to balance conditions more carefully in order to exploit the sensitivity that the measurement of shift costs promises to provide. The exposure-related results of this study support this aspect.     

Cross species extrapolation in neurotoxicology: neurophysiological and neurobehavioral aspects.

The theory of phylogenetic continuity of animal species is the basis of any comparative or extrapolative endeavour (Calabrese, 1983). Cross species extrapolation is also a prerequisite for hazard identification in general and developmental neurotoxicology. Two steps must be distinguished: The first step is endpoint-based or qualitative, whereas the second is dose-based or quantitative. Comparison of different species, typically rodents, nonhuman primates and humans, in terms of endpoints is preferentially done within a framework of broad functional categories, such as sensory, motivational, cognitive, motor, and social variables. Within each category specific neurobehavioral as well as electrophysiological measures need to be considered; typically the degree of comparability is higher for electrophysiological than for most behavioral measures. For some frequently used behavioral endpoints in human neurotoxicology, such as psychometric IQ, there is no direct animal counterpart. Once the neural substrate of a particular neurotoxic effect has been identified, as is true for several chemicals such as the pyrethroid insecticides, the organophosphates, most nerve gases or MPTP, or if interspecies comparability in terms of endpoints has proven satisfactory, an effort towards quantitative, dose-based extrapolation is needed. Here species-specific differences in toxicokinetics and metabolism must be taken into consideration in order to arrive at valid translations of dose-response contingencies. If at all possible internal rather than external doses should serve as the frame of reference here. Neurotoxic chemicals of environmental concern for which an adequate data base is available for comparative purposes include alcohol, carbon monoxide, lead, methylmercury and polychlorinated biphenyls (PCB). Principles of cross species extrapolation in neurotoxicology will be illustrated by means of representative neurobehavioral and electrophysiological findings.     

Models for the in vitro assessment of neurotoxicity in the nervous system in relation to xenobiotic and neurotrophic factor-mediated events.

We have been investigating the use of three culture types for both screening and mechanistic neurotoxicology in vitro. These are the neuroblastoma cell lines (IMR32 - human; C-1300 - mouse), primary mixed monolayer cultures of the rat and chick embryonic midbrain ('micromass' systems) and organotypic whole rat brain reaggregate cultures. The performance of these models for neurotoxicity resting has been investigated with ethylcholine mustard aziridinium (ECMA), vincristine, aluminium, glutamate receptor antagonists, MPTP, and 'hypothyroidism'. From a 'screening' viewpoint, in vitro exposure through a tiered testing system (ranging from simple cytotoxicological parameters in the neural cell lines to neurotransmitter measurements in the organotypic cultures) may permit detection of CNS neurotoxicity and delineation of possible mechanisms. The type of developmental neurotoxicological information gained is highlighted in the cases of aluminum and the glutamate receptor antagonists. High concentrations of aluminum caused significant neural cell death in differentiated neuroblastoma cell lines after approximately two weeks exposure in vitro. In contrast, cell death was detected in the developing midbrain cultures as early as 24 - 48 hr. Studies in whole brain reaggregates suggest that cholinotoxicity may occur in a similar time-frame and is consistent with some of aluminium's effects in vivo. Preliminary experiments have shown that exposure of immature developing midbrain rat primary cultured neurones to the glutamate receptor antagonists, AP3 and MK-801 induces neural cell death which may relate to control of NGF by glutamate cells. Developing neural culture systems may prove useful for testing agents which cause neurotoxicity through disturbances of neurotrophic function.     

Overview of molecular, cellular, and genetic neurotoxicology

It has become increasingly evident that the field of neurotoxicology is not only rapidly growing but also rapidly evolving, especially over the last 20 years. As the number of drugs and environmental and bacterial/viral agents with potential neurotoxic properties has grown, the need for additional testing has increased. Only recently has the technology advanced to a level that neurotoxicologic studies can be performed without operating in a "black box." Examination of the effects of agents that are suspected of being toxic can occur on the molecular (protein-protein), cellular (biomarkers, neuronal function), and genetic (polymorphisms) level. Together, these areas help to elucidate the potential toxic profiles of unknown (and in some cases, known) agents. The area of proteomics is one of the fastest growing areas in science and particularly applicable to neurotoxicology. Lubec et al, provide a review of the potential and limitations of proteomics. Proteomics focuses on a more comprehensive view of cellular proteins and provides considerably more information about the effects of toxins on the CNS. Proteomics can be classified into three different focuses: post-translational modification, protein-expression profiling, and protein-network mapping. Together, these methods represent a more complete and powerful image of protein modifications following potential toxin exposure. Cellular neurotoxicology involves many cellular processes including alterations in cellular energy homeostasis, ion homeostasis, intracellular signaling function, and neurotransmitter release, uptake, and storage. The greatest hurdle in cellular neurotoxicology has been the discovery of appropriate biomarkers that are reliable, reproducible, and easy to obtain. There are biomarkers of exposure effect, and susceptibility. Finding the appropriate biomarker for a particular toxin is a daunting task. The appropriate biomarker for a particular toxin is a daunting task. The advantage to biomarker/toxin combinations is they can be detected and measured shortly following exposure and before overt neuroanatomic damage or lesions. Intervention at this point, shortly following exposure, may prevent or at least attenuate further damage to the individual. The use of peripheral biomarkers to assess toxin damage in the CNS has numerous advantages: time-course analysis may be performed, ethical concerns with the use of human subjects can partially be avoided, procedures to acquire samples are less invasive, and in general, peripheral studies are easier to perform. Genetic neurotoxicology comprises two focuses--toxin-induced alterations in genetic expression and genetic alterations that affect toxin metabolism, distribution, and clearance. These differences can be beneficial or toxic. Polymorphisms have been shown to result in altered metabolism of certain toxins (paraoxonase and paraoxon). Conversely, it is possible that some polymorphisms may be beneficial and help prevent the formation of a toxic by-product of an exogenous agent (resistance to ozone-induced lung inflammation). It has also become clear that interactions of potential toxins are not straightforward as interactions with DNA, causing mutations. There are numerous agents that cause epigenetic responses (cellular alterations that are not mutagenic or cytotoxic). This finding suggests that many agents that may originally have been thought of as nontoxic should be re-examined for potential "indirect" toxicity. With the advancement of the human genome project and the development of a human genome map, the effects of potential toxins on single or multiple genes can be identified. Although collectively, the field of neurotoxicology has recently come a long way, it still has a long way to go reach its full potential. As technology and methodology advances continue and cooperation with other disciplines such as neuroscience, biochemistry, neurophysiology, and molecular biology is improved, the mechanisms of toxin action will be further elucidated. With this increased understanding will come improved clinical interventions to prevent neuronal damage following exposure to a toxin.     

Hänninen lecture: problems and prevention: research in developing countries and immigrant populations from developing countries

The tradition of this excellent Triennial International Symposium, now in its Ninth gathering, has focused first on methods and later on effects in neurotoxicology. I respectfully suggest that it is time to add prevention to our agenda in order to focus not just on finding problems, but also on solving them. Otherwise our research runs the risk of making conditions worse for the participants because we inform them they have a problem but do not show them how to prevent that problem. Many barriers have been surmounted since the early days of this Symposium. Methods developed in industrialized nations have been modified and used in populations with limited education. Now, the primary barriers to effective collaborations are institutional. Perhaps the most challenging are companies that can bar a research study, or may welcome the opportunity to safeguard their workforce. A focus on prevention can increase the willingness of institutions, including companies, to accept and support research that examines neurotoxic effects of chemical exposures. This is because we are offering positive value, not simply uncovering a problem for them to fix. The cTRAIN software program is an illustration of a computer-based training method developed by neurotoxicologists that has been implemented in a variety of populations. This includes those from a developing country with limited education and those with college degrees. Training can provide prevention, can easily be appended to any neurotoxicology research study and is well within the skill set of the neurotoxicology community of scientists.     

Suboptimal performance on neuropsychological tests in patients with suspected chronic toxic encephalopathy

Suboptimal performance during neuropsychological testing can seriously complicate assessment in behavioral neurotoxicology. We present data on the prevalence of suboptimal performance in a group of Dutch patients with suspected chronic toxic encephalopathy (CTE) after long-term occupational exposure to solvents. One hundred and forty-five subjects referred to one of two Dutch national assessment centers for CTE were administered the Amsterdam Short-Term Memory Test (ASTM) and the Test of Memory Malingering (TOMM), two tests specifically developed for the detection of suboptimal performance. For both tests, very cautious cut-off scores were chosen with a specificity of 99%. Results indicated that suboptimal performance appears to be a substantial problem in this group of patients with suspected CTE after long-term exposure to organic solvents. Only 54% of our subjects obtained normal scores on both tests of malingering, i.e. at or above cut-off score. The two tests seemed to measure the same concept in that nearly all the subjects with low TOMM scores also had low ASTM scores. However, a higher proportion of subjects scored below the cut-off on the ASTM than on the TOMM.     

Microglia and macrophages in the developing CNS.

An understanding of microglial functions during normal CNS development is prerequisite for understanding developmental neurotoxicology. This review provides a brief summary of previous work regarding the origin of microglia and addresses differences and similarities between microglia and brain macrophages. Current concepts and ideas which implicate microglia in diverse developmental processes, such as apoptosis, axon growth, and vasculogenesis are discussed. The study of reactive microgliosis may prove useful in the histopathological analysis of neurotoxicant-induced brain damage during development.     

Prenatal stress in birds: pathways, effects, function and perspectives

Although most work on prenatal stress has been conducted on mammalian species, birds provide useful alternative models since avian embryos develop outside the mother's body in a concealed environment, the egg, which is produced during a short time window of 4-14 days. This facilitates measurement of maternal substances provided for and manipulation of the embryo without interfering with the mother's physiology. We critically review prenatal corticosterone mediated effects in birds by reviewing both studies were females had elevated levels of plasma corticosterone during egg formation and studies applying corticosterone injections directly into the egg. A selected review of the mammalian literature is used as background. The results suggest that besides prenatal exposure to corticosterone itself, maternal corticosterone affects offspring's behaviour and physiology via alteration of other egg components. However, results are inconsistent, perhaps due to the interaction with variation in the post-natal environment, sex, age, developmental mode and details of treatment. The potential role of adaptive maternal programming has not been tested adequately and suggestions for future research are discussed.     

Clinical neurological findings among metal degreasers exposed to chlorinated solvents

Among industrial solvents in present use trichloroethylene belongs to those which have been the concern of most neurological and occupational investigations since the beginning of this century. Reports on a broad spectrum of neurotoxicity are however mostly based on case-studies and accidental circumstances. The object of this study was to examine clinical neurological manifestations after long-term exposure to degreasing solvents, mainly trichloroethylene. The population was 99 metal degreasers, the design a historical cohort study. The most marked finding was a highly significant dose-response relation between solvent exposure and motor dyscoordination - a finding that was retained after multivariate control of relevant confounders. Vibration thresholds increased by solvent exposure at bivariate level, but the multivariate analysis showed that age was explaining most of the increase. No significant cranial nerve dysfunction was found.     

Delayed expression of neurotoxicity: the problem of silent damage.

A prolonged period of latency between toxic exposure and disease is a recognized phenomenon in oncology, but is a relatively new concept in neurotoxicology. Suggestions that exposure to neurotoxic compounds may be followed years or decades later by clinically evident neurological disease have received considerable anecdotal support but little empirical evidence. In recent years, evidence has emerged linking environmental factors to several neurodegenerative diseases, including amyotrophic lateral sclerosis and the Parkinsonism-dementia complex. While these diseases have not been definitively demonstrated to arise from exposure to toxins or toxicants, nevertheless the possibility exists that xenobiotic exposure could lead to neurological conditions possessing a period of clinical "silence" prior to expression. Detection of such damage using standard toxicology testing protocols poses a major challenge. Lessons learned from neurodegenerative diseases can be profitably applied to the study of the long-term effects of xenobiotics on the brain and elucidation of the role of aging on the expression of neurotoxicity.     

Environmental toxicology: Sensitive periods of development and neurodevelopmental disorders

Development of the mammalian central nervous system is a complex process whose disruption may have severe and long-lasting consequences upon brain structure and function, potentially resulting in a neurodevelopmental disorder (NDD). Many NDDs are known to be genetic in origin, with symptom onset and their underlying mechanisms now known to be regulated during time-dependent windows or ‘critical periods’ during normal brain development. However, it is increasingly evident that similar disturbances to the developing nervous system may be caused by exposure to non-genetic, environmental factors. Strikingly, at least 200 industrially applied or produced chemicals have been associated with neurotoxicity in humans and exposure to these modifying compounds, through consumer products or environmental pollution, therefore poses serious threats to public health. Through a combination of human epidemiological and animal experimental studies, we identified developmental periods for increased vulnerability to environmentally-modifying compounds and determined whether and how exposure during specific sensitive time-windows could increase the risk for the NDDs of autism, ADHD or schizophrenia in the developing organism. We report that many environmental toxicants have distinct sensitive time-windows during which exposure may disrupt critical developmental events, thereby increasing the risk of developing NDDs. The majority of these time-windows occur prenatally rather than postnatally. We propose four underlying mechanisms that mediate pathogenesis, namely oxidative stress, immune system dysregulation, altered neurotransmission and thyroid hormone disruption. Given the complexity of underlying mechanisms and their prenatal inception, treatment options are currently limited. Thus, we conclude that preventing early exposure to environmental toxicants, by increasing public awareness and improving government and industry guidelines, may ultimately lead to a significant reduction in the incidence of NDDs.     

Adolescent methylmercury exposure alters short-term remembering,but not sustained attention,in male Long-Evans rats

Methylmercury is an environmental neurotoxicant found in fish that produces behavioral deficits following early developmental exposure. The impact of adolescent exposure to this developmental neurotoxicant is only recently being explored in animal models. Here, short-term memory and sustained attention were examined using a rodent model of adolescent methylmercury exposure. Rats were exposed to 0, 0.5, or 5 ppm methylmercury throughout the adolescent period and tested on a two-choice visual signal detection task in adulthood. Methylmercury improved short-term remembering in this procedure but the dose-effect curve was nonmonotonic, as has been reported previously: effects on memory were observed in animals exposed to 0.5 ppm methylmercury, but not 5 ppm. Methylmercury did not significantly alter sustained attention, which is in contrast to effects following gestational exposure in human populations. The results may suggest that attention is not involved with previously reported effects of methylmercury during adolescence, but certain procedural issues remain unresolved.     

IN UTERO EXPOSURE TO ORGANIC SOLVENTS AND HUMAN NEURODEVELOPMENT

Recent reports of growth and mental retardation in infants whose mothers abused solvent-containing substances, and of an association between central nervous system malformations and solvent exposure, have suggested that in utero exposure to organic solvents may have a profound effect on the development of the human brain. The present investigation compared the neurobehavioral development of 41 children whose mothers worked with organic solvents during pregnancy with a group of matched, unexposed children. The children were compared on a variety of measures, including the McCarthy Scales of General Abilities, growth (weight, height and head circumference) and mother's report of developmental milestones, behavior and personality. Potential confounders were controlled for in multiple regression analyses. Despite adequate power, no differences could be found between the two groups on any of the measures of neurobehavioral development or growth. This study suggests that in utero exposure to relatively low levels of organic solvent is not associated with adverse neurodevelopmental outcome.     

Strategies for assessing learning and memory, 1978–1987: A comparison of behavioral toxicology, psychopharmacology, and neurobiology

Tests of learning and memory are currently not typically included in first-tier screening batteries even though there is ample evidence that chemical exposure can produce deficits in these cognitive processes. The approach taken in behavioral toxicology has been to restrict these cognitive tests to second tier or hazard characterization studies, yet there is little agreement on which tests are most appropriate. The present survey was designed to determine the current testing strategies in toxicology for detecting and characterizing the effects of chemical treatment on learning and memory, and to make comparisons to similar data from the fields of psychopharmacology and neurobiology. The survey data revealed a number of discipline-dependent effects on the selection of tests. A number of these effects were clearly related to the subject matter as well as the particular chemical/treatment being examined. Given the youth of the field, behavioral toxicology has the advantage of gaining valuable information from both of these disciplines. Behavioral testing in neurotoxicology should consider strategies which maximize unification of these closely related fields of neuroscience.     

Setting evidence-based occupational exposure limits for manganese

In 2004, a review by the Institute of Environment and Health (IEH) made recommendations on occupational exposure limits (OELs) for manganese and its inorganic compounds for inhalable and respirable fractions respectively. These OELs were based on a detailed comprehensive evaluation of all the scientific data available at that time. Since then, more published studies have become available and a number of occupational standard-setting committees (EU SCOEL, US ACGIH-TLV, and German MAK) have proposed OEL’s for manganese and its inorganic compounds that are somewhat lower that those proposed in the 2004 review.Based on current understanding, the key toxicological and human health issues that are likely to influence a health-based recommendation relate to: neurotoxicology; reproductive and developmental toxicology; and mutagenicity/carcinogenicity. Of these, it is generally considered that neurotoxicity presents the most sensitive endpoint. As such, many of the studies that have been reported since the IEH review have sought to use those neurofunctional tests that appear to be particularly sensitive at identifying the subtle neurological changes thought to associate with manganese toxicity. These recent studies have, however, continued to be limited to a significant extent by reliance on cross-sectional designs and also by use of unreliable exposure estimation methods. Consequently the strength of the potential association between manganese exposure and these subtle subclinical cognitive or neuromotor changes is still poorly characterised and the relevance of these minor differences in terms of either their clinical or quality of life consequences remains unknown.Based upon the overall evidence, it is concluded that the 8-h time weighted averages (TWA) for respirable (0.05 mg/m³ as Mn) and inhalable (0.2 mg/m³ as Mn) fractions as recommended by the SCOEL in 2011 are the most methodologically-sound, as they are based on the best available studies, most suited to the development of health-based OELs for both respirable and inhalable fractions. The dose-response characterisation informed by the examined studies used can be considered to establish a true human NOAEL for all the neurofunctional endpoints examined within the selected studies.