Systematic developmental neurotoxicity assessment of a representative PAH Superfund mixture using zebrafish.

Superfund sites often consist of complex mixtures of polycyclic aromatic hydrocarbons (PAHs). It is widely recognized that PAHs pose risks to human and environmental health, but the risks posed by exposure to PAH mixtures are unclear. We constructed an environmentally relevant PAH mixture with the top 10 most prevalent PAHs (SM10) from a Superfund site derived from environmental passive sampling data. Using the zebrafish model, we measured body burden at 48 hours post fertilization (hpf) and evaluated the developmental and neurotoxicity of SM10 and the 10 individual constituents at 24 hours post fertilization (hpf) and 5 days post fertilization (dpf). Zebrafish embryos were exposed from 6 to 120 hpf to (1) the SM10 mixture, (2) a variety of individual PAHs: pyrene, fluoranthene, retene, benzo[a]anthracene, chrysene, naphthalene, acenaphthene, phenanthrene, fluorene, and 2-methylnaphthalene. We demonstrated that SM10 and only 3 of the individual PAHs were developmentally toxic. Subsequently, we constructed and exposed developing zebrafish to two sub-mixtures: SM3 (comprised of 3 of the developmentally toxicity PAHs) and SM7 (7 non-developmentally toxic PAHs). We found that the SM3 toxicity profile was similar to SM10, and SM7 unexpectedly elicited developmental toxicity unlike that seen with its individual components. The results demonstrated that the overall developmental toxicity in the mixtures could be explained using the general concentration addition model. To determine if exposures activated the AHR pathway, spatial expression of CYP1A was evaluated in the 10 individual PAHs and the 3 mixtures at 5 dpf. Results showed activation of AHR in the liver and vasculature for the mixtures and some individual PAHs. Embryos exposed to SM10 during development and raised in chemical-free water into adulthood exhibited decreased learning and responses to startle stimulus indicating that developmental SM10 exposures affect neurobehavior. Collectively, these results exemplify the utility of zebrafish to investigate the developmental and neurotoxicity of complex mixtures.

Strategies to improve the regulatory assessment of developmental neurotoxicity (DNT) using in vitro methods.

Currently, the identification of chemicals that have the potential to induce developmental neurotoxicity (DNT) is based on animal testing. Since at the regulatory level, systematic testing of DNT is not a standard requirement within the EU or USA chemical legislation safety assessment, DNT testing is only performed in higher tiered testing triggered based on chemical structure activity relationships or evidence of neurotoxicity in systemic acute or repeated dose toxicity studies. However, these triggers are rarely used and, in addition, do not always serve as reliable indicators of DNT, as they are generally based on observations in adult rodents. Therefore, there is a pressing need for developing alternative methodologies that can reliably support identification of DNT triggers, and more rapidly and cost-effectively support the identification and characterization of chemicals with DNT potential. We propose to incorporate mechanistic knowledge and data derived from in vitro studies to support various regulatory applications including: (a) the identification of potential DNT triggers, (b) initial chemical screening and prioritization, (c) hazard identification and characterization, (d) chemical biological grouping, and (e) assessment of exposure to chemical mixtures. Ideally, currently available cellular neuronal/glial models derived from human induced pluripotent stem cells (hiPSCs) should be used as they allow evaluation of chemical impacts on key neurodevelopmental processes, by reproducing different windows of exposure during human brain development. A battery of DNT in vitro test methods derived from hiPSCs could generate valuable mechanistic data, speeding up the evaluation of thousands of compounds present in industrial, agricultural and consumer products that lack safety data on DNT potential.

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.

Neurodevelopmental fetal assessment using KANET scoring system in low and high risk pregnancies.

OBJECTIVE: To assess and compare fetal behavior and neurodevelopment (KANET) between low and high risk pregnancies. METHODS: Prospective, comparative, cohort study. One hundred and fifty-two consecutive pregnant women presenting for routine 2nd trimester and 3rd trimester scan had four dimensional ultrasound examinations (4D US) in order to assess fetal behavior and neurodevelopment. This was achieved by the study of: (1) isolated head movements, (2) isolated hand movements, (3) isolated leg movements, (4) cranial sutures, (5) hand to face/head movements, (6) finger movements, (7) yawning - mouthing, (8) facial expressions, (9) isolated eye blinking, (10) "Gestalt" perception. According to the maternal background risk, the population of the study was classified in low risk pregnancies (n = 78) and high risk pregnancies (n = 74) with IUGR fetuses (n = 12), diabetes mellitus (n = 24), and pre-eclampsia (n = 38). RESULTS: The neurodevelopmental score was statistically significant higher in the low risk group compared to the high risk group (p < 0.0004). The diabetes subgroup score was statistically significantly higher compared to the IUGR and the pre-eclampsia subgroup (p = 0.0001). CONCLUSIONS: The neurodevelopment fetal assessment by 4D ultrasound appears to be a feasible technique in the evaluation of high risk pregnancies. Further studies where any association between KANET score and neurological outcome of the childhood are warranted.

Four-dimensional sonographic assessment of fetal movement in the late first trimester.

OBJECTIVE: To evaluate, using four-dimensional (4D) sonography, the frequency of fetal movements during the late first trimester of normal singleton pregnancies. METHODS: Singleton pregnancies were studied-using transvaginal 4D sonography-for 10 minutes at 10-11 and 12-13 weeks of gestation. The frequencies of 5 fetal movements (isolated arm, isolated leg, short trunk, long trunk, and jumping movements) were evaluated. RESULTS: In the 17 pregnancies studied, the most frequent fetal movements were isolated arm movement at 10-11 weeks and jumping movement at 12-13 weeks. There was a significant difference in the frequency of jumping movement between 10-11 and 12-13 weeks (P<0.05). CONCLUSION: The difference in frequency of 5 fetal movements at 10-11 and 12-13 weeks of gestation may be caused by early neuromuscular development and differentiation of the neuromuscular system.

Workgroup report: incorporating in vitro alternative methods for developmental neurotoxicity into international hazard and risk assessment strategies.

This is the report of the first workshop on Incorporating In Vitro Alternative Methods for Developmental Neurotoxicity (DNT) Testing into International Hazard and Risk Assessment Strategies, held in Ispra, Italy, on 19-21 April 2005. The workshop was hosted by the European Centre for the Validation of Alternative Methods (ECVAM) and jointly organized by ECVAM, the European Chemical Industry Council, and the Johns Hopkins University Center for Alternatives to Animal Testing. The primary aim of the workshop was to identify and catalog potential methods that could be used to assess how data from in vitro alternative methods could help to predict and identify DNT hazards. Working groups focused on two different aspects: a) details on the science available in the field of DNT, including discussions on the models available to capture the critical DNT mechanisms and processes, and b) policy and strategy aspects to assess the integration of alternative methods in a regulatory framework. This report summarizes these discussions and details the recommendations and priorities for future work.

Assessment of fetal neurodevelopment via fetal magnetocardiography.

Fetal magnetocardiography (fMCG) offers unique capabilities for assessment of fetal heart rate (FHR) and fetal behavior, which are fundamental aspects of neurodevelopment. The most important attribute of fMCG for FHR monitoring is its high precision, which allows accurate assessment of beat-to-beat fetal heart rate variability (FHRV), including respiratory sinus arrhythmia. Using mathematical indices to assess FHRV, we find that short- and long-term FHRV both increase during gestation but not in the same manner. The largest increases in short-term FHRV occur during the last trimester, while the largest increases in long-term FHRV occur early on, with smaller changes occurring during the last trimester. The fMCG also allows assessment of fetal activity. This results from the high sensitivity of the signal to the position and orientation of the fetal heart. FMCG actograms are therefore specific for fetal trunk movement, which are thought to be more important than isolated extremity movements and other small fetal movements. The ability to assess FHR, FHRV, and fetal trunk movement simultaneously makes fMCG a valuable tool for neurodevelopment research.

Is neurological assessment of the fetus possible?

The possibility to assess the functional condition of the fetal nervous system is of great importance to the obstetrician, since a considerable part of early brain damage is of prenatal origin. Several attempts to develop such a technique are reviewed. In addition, a new method, the qualitative assessment of fetal general movements, is described as a successful tool to obtain reliable data on the fetal brain dysfunction. This new method is robust, non-intrusive and cost-effective. There is also the advantage that the same criteria for the diagnostic assessment can be used for the fetus as well as for the young infant.

Behavioural states: concomitants, clinical implications and the assessment of the condition of the nervous system.

Behavioural states do exist in the human fetus. These states have been called 1F to 4F and resemble states 1 to 4 in the neonate. States 1F and 2F are similar to 'non-REM sleep' or 'quiet sleep' and 'REM-sleep' or 'active sleep', respectively. The fetus spends most of the time in these two states. Each state can be characterized by a particular combination of 3 variables: presence or absence of fetal eye movements and body movements, and fetal heart rate patterns A, B, C and D. From about 36 wk these combinations can be recognized during longer periods without interruptions, and with clear state-transitions. At 32 wk a certain linkage between the three variables is already present but a step-wise increase can be seen between 36 and 38 wk. Fetal breathing, mouthing movements and fetal voiding are state concomitants. Breathing movements are much more regular during state 1F than during state 2F, while the incidence is increased in 2F. It is demonstrated that knowledge of fetal behavioural states is necessary for an adequate interpretation of the fetal heart rate patterns. The concept of behavioural states may be useful for the early detection of disturbances of the developing fetal nervous system. It is therefore concluded that further studies on fetal behaviour have to be 'standardized' for behavioural states.