Perinatal exposure to the immune-suppressant di-n-octyltin dichloride affects brain development in rats.

Disruption of the immune system during embryonic brain development by environmental chemicals was proposed as a possible cause of neurodevelopmental disorders. We previously found adverse effects of di-n-octyltin dichloride (DOTC) on maternal and developing immune systems of rats in an extended one-generation reproductive toxicity study according to the OECD 443 test guideline. We hypothesize that the DOTC-induced changes in the immune system can affect neurodevelopment. Therefore, we used in-vivo MRI and PET imaging and genomics, in addition to behavioral testing and neuropathology as proposed in OECD test guideline 443, to investigate the effect of DOTC on structural and functional brain development. Male rats were exposed to DOTC (0, 3, 10, or 30 mg/kg of diet) from 2 weeks prior to mating of the F0-generation until sacrifice of F1-animals. The brains of rats, exposed to DOTC showed a transiently enlarged volume of specific brain regions (MRI), altered specific gravity, and transient hyper-metabolism ([18F]FDG PET). The alterations in brain development concurred with hyper-responsiveness in auditory startle response and slight hyperactivity in young adult animals. Genomics identified altered transcription of key regulators involved in neurodevelopment and neural function (e.g. Nrgrn, Shank3, Igf1r, Cck, Apba2, Foxp2); and regulators involved in cell size, cell proliferation, and organ development, especially immune system development and functioning (e.g. LOC679869, Itga11, Arhgap5, Cd47, Dlg1, Gas6, Cml5, Mef2c). The results suggest the involvement of immunotoxicity in the impairment of the nervous system by DOTC and support the hypothesis of a close connection between the immune and nervous systems in brain development.

Developmental immunotoxicity of ethanol in an extended one-generation reproductive toxicity study.

The susceptibility of developing immune system to chemical disruption warrants the assessment of immune parameters in reproductive and developmental testing protocols. In this study, a wide range of immune endpoints was included in an extended one-generation reproduction toxicity study (EOGRTS) design to determine the relative sensitivity of immune and developmental parameters to ethanol (EtOH), a well-known developmental toxicant with immunomodulatory properties. Adult Wistar rats were exposed to EtOH via drinking water (0, 1.5, 4, 6.5, 9, 11.5 and 14 % (w/v EtOH)) during premating, mating, gestation and lactation and continuation of exposure of the F(1) from weaning until killed. Immune assessments were performed at postnatal days (PNDs) 21, 42 and 70. Keyhole limpet hemocyanin (KLH)-specific immune responses were evaluated following subcutaneous immunizations on PNDs 21 and 35. EtOH exposure affected innate as well as adaptive immune responses. The most sensitive immune parameters included white blood cell subpopulations, ConA-stimulated splenocyte proliferation, LPS-induced NO and TNF-α production by adherent splenocytes and KLH-specific immune responses. Most parameters showed recovery after cessation of EtOH exposure after weaning in the 14 % exposure group. However, effects on LPS-induced NO and TNF-α production by adherent splenocytes and KLH-specific parameters persisted until PND 70. The results demonstrate the relative sensitivity to EtOH of especially functional immune parameters and confirm the added value of immune parameters in the EOGRTS. Furthermore, this study identified an expanded KLH-specific parameter set and LPS-induced NO and TNF-α production by adherent splenocytes as valuable parameters that can provide additional information on functional immune effects.

Potentially increased sensitivity of pregnant and lactating female rats to immunotoxic agents.

Characteristic susceptibility to environmental and pharmaceutical exposure may occur during periods in life of marked histophysiological changes of the immune system. Perinatal development is such a period; pregnancy followed by lactation is potentially another one. Here, we explored the influence of pregnancy and lactation on the model immunotoxic compound di-n-octyltin dichloride (DOTC) in rats using clinical and histopathological parameters. Female rats were exposed to 0, 3, 10, or 30 mg DOTC/kg feed during pregnancy and up to 20 (at weaning) or 56 days after delivery. Age-matched nonmated females were exposed during the same time periods. DOTC at the level of 10 and 30 mg/kg decreased thymus weight and affected thymus morphology in the lactating rats. In addition, DOTC decreased the numbers of neutrophils in the lactating rats. These effects were no longer apparent at day 56 despite continuous exposure to DOTC. This explorative study indicates that the innate and adaptive immune system may be especially sensitive to immunotoxicants during pregnancy and lactation.

Safety assessment of titanium dioxide (E171) as a food additive.

The present opinion deals with an updated safety assessment of the food additive titanium dioxide (E 171) based on new relevant scientific evidence considered by the Panel to be reliable, including data obtained with TiO2 nanoparticles (NPs) and data from an extended one-generation reproductive toxicity (EOGRT) study. Less than 50% of constituent particles by number in E 171 have a minimum external dimension < 100 nm. In addition, the Panel noted that constituent particles < 30 nm amounted to less than 1% of particles by number. The Panel therefore considered that studies with TiO2 NPs < 30 nm were of limited relevance to the safety assessment of E 171. The Panel concluded that although gastrointestinal absorption of TiO2 particles is low, they may accumulate in the body. Studies on general and organ toxicity did not indicate adverse effects with either E 171 up to a dose of 1,000 mg/kg body weight (bw) per day or with TiO2 NPs (> 30 nm) up to the highest dose tested of 100 mg/kg bw per day. No effects on reproductive and developmental toxicity were observed up to a dose of 1,000 mg E 171/kg bw per day, the highest dose tested in the EOGRT study. However, observations of potential immunotoxicity and inflammation with E 171 and potential neurotoxicity with TiO2 NPs, together with the potential induction of aberrant crypt foci with E 171, may indicate adverse effects. With respect to genotoxicity, the Panel concluded that TiO2 particles have the potential to induce DNA strand breaks and chromosomal damage, but not gene mutations. No clear correlation was observed between the physico-chemical properties of TiO2 particles and the outcome of either in vitro or in vivo genotoxicity assays. A concern for genotoxicity of TiO2 particles that may be present in E 171 could therefore not be ruled out. Several modes of action for the genotoxicity may operate in parallel and the relative contributions of different molecular mechanisms elicited by TiO2 particles are not known. There was uncertainty as to whether a threshold mode of action could be assumed. In addition, a cut-off value for TiO2 particle size with respect to genotoxicity could not be identified. No appropriately designed study was available to investigate the potential carcinogenic effects of TiO2 NPs. Based on all the evidence available, a concern for genotoxicity could not be ruled out, and given the many uncertainties, the Panel concluded that E 171 can no longer be considered as safe when used as a food additive.

2D and 3D assessment of neuropathology in rat brain after prenatal exposure to methylazoxymethanol, a model for developmental neurotoxicty.

To evaluate the ability of a tiered quantitative morphological approach to reveal developmental neurotoxicity, morphometric parameters were measured in the offspring of rats treated with methylazoxymethanol (MAM) during days 13-15 of pregnancy. Treatment was aimed at inhibiting the proliferation phase of hippocampal neurons while leaving cerebellar neurons unaffected. 2D and 3D assessment of brain morphology combined with straightforward measurement of brain size, weight and volume, and the usefulness of estimation of total neuron numbers were studied. Each tier indicated major effects of MAM, from macroscopic effects in the cerebrum (first tier) to a considerable loss of neurons in the hippocampal CA1 pyramidal layer (third tier). The cerebellum and the number of cerebellar granular neurons were not changed. Along with each step of the proposed tiered approach (brain size-->linear morphometry-->stereology), the discriminative strength of the endpoints, and thus the probability to pinpoint the extent and location of developmental brain lesions increased.

Behavioural effects and kinetics in brain in response to inhalation of constant or fluctuating toluene concentrations in the rat.

The toxicity of exposure by inhalation to organic solvents may not only be related to the total external dose, but also to the pattern of exposure. In this study the impact of the exposure scenario on the behavioural effects of the model solvent toluene in rats was investigated. Rats were exposed for 7.5h to either a constant concentration or fluctuating concentrations at total external dose levels of 20,000ppmh and 10,000ppmh. Different effects on measures of visual discrimination performance were observed in rats exposed to a constant or fluctuating concentrations, and when rats were tested immediately or sometime after the end of exposure to fluctuating concentrations. Motor activity was also differently affected by different exposure scenarios. Physiologically based toxicokinetic (PBTK) modelling was used to predict the toxicokinetics of toluene induced by these different exposure scenarios. The model was calibrated by measuring toluene concentrations in blood and brain during and after exposure. The results show that the acute effects of toluene on behaviour do not depend only on the concentration and duration of exposure, but primarily on the pattern of exposure.

Regulatory developmental neurotoxicity testing: a model study focussing on conventional neuropathology endpoints and other perspectives.

Our aim was to investigate a model of the morphologic approach proposed in guidelines for developmental neurotoxicity testing (DNT). Hereto, a limited DNT study [EPA Health Effects Test Guidelines OPPTS 870.6300, 1996a. Developmental Neurotoxicity Study "Public Draft", United States Environmental Protection Agency; Prevention, Pesticides and Toxic Substances (7101); EPA 712-C-96-239, June 1996. ] was carried out with different doses of methylazoxy methanol acetate (MAM), known to affect brain morphology and neuron numbers in the developing brain. After gross examination, the brains of F1-animals were further dissected along neuro-anatomical landmarks to ensure homology between tissues of different individuals. The (relative) weight of the brain (parts) was determined. One brain half (alternating left/right to avoid lateralization) was further used for microscopic slide reading and measurement of brain layer width (linear morphometry); the other was set aside for stereologic investigation in a later phase of the study. In the offspring, a clear reduction in brain size (gross macroscopy) and weight (MAM high- and top-dose groups) was observed on postnatal days (PN) 22 and 62, but this reduction was hard to pinpoint in the microscope as the changes primarily appeared quantitative in nature, rather than qualitative. Linear measurements of brain layer width appeared very sensitive and efficient. This first step of a project is presented and the perspectives of a further stereologic investigation are discussed.

Oral two-generation reproduction toxicity study with NM-200 synthetic amorphous silica in Wistar rats.

Synthetic amorphous silica (SAS) like NM-200 is used in a wide variety of technological applications and consumer products. Although SAS has been widely investigated the available reproductive toxicity studies are old and do not cover all requirements of current OECD Guidelines. As part of a CEFIC-LRI project, NM-200 was tested in a two-generation reproduction toxicity study according to OECD guideline 416. Male and female rats were treated by oral gavage with NM-200 at dose levels of 0, 100, 300 and 1000mg/kg bw/day for two generations. Body weight and food consumption were measured throughout the study. Reproductive and developmental parameters were measured and at sacrifice (reproductive) organs and tissues were sampled for histopathological analysis. Oral administration of NM-200 up to 1000mg/kg bw/day had no adverse effects on the reproductive performance of rats or on the growth and development of the offspring into adulthood for two consecutive generations. The NOAEL was 1000mg/kg body weight per day.

Zebrafish embryotoxicity test for developmental (neuro)toxicity: Demo case of an integrated screening approach system using anti-epileptic drugs.

To improve the predictability of the zebrafish embryotoxicity test (ZET) for developmental (neuro)toxicity screening, we used a multiple-endpoints strategy, including morphology, motor activity (MA), histopathology and kinetics. The model compounds used were antiepileptic drugs (AEDs): valproic acid (VPA), carbamazepine (CBZ), ethosuximide (ETH) and levetiracetam (LEV). For VPA, histopathology was the most sensitive parameter, showing effects already at 60µM. For CBZ, morphology and MA were the most sensitive parameters, showing effects at 180µM. For ETH, all endpoints showed similar sensitivity (6.6mM), whereas MA was the most sensitive parameter for LEV (40mM). Inclusion of kinetics did not alter the absolute ranking of the compounds, but the relative potency was changed considerably. Taking all together, this demo-case study showed that inclusion of multiple-endpoints in ZET may increase the sensitivity of the assay, contribute to the elucidation of the mode of toxic action and to a better definition of the applicability domain of ZET.

A category approach to predicting the developmental (neuro) toxicity of organotin compounds: the value of the zebrafish (Danio rerio) embryotoxicity test (ZET).

Zebrafish embryos were exposed to different organotin compounds during very early development (<100h post fertilization). Morphology, histopathology and swimming activity (in a motor activity test) were the endpoints analyzed. DBTC was, by far, the most embryotoxic compound at all time points and endpoints studied. In fact, we observed a clear concordance between the effects observed in our zebrafish embryo model, and those observed with these compounds in full rodent in vivo studies. All organotin compounds classified as developmental (neuro) toxicants in vivo, were correctly classified in the present assay. Together, our results support the ZET model as a valuable tool for providing biological verification for a grouping and a read-across approach to developmental (neuro) toxicity.

Delay and impairment in brain development and function in rat offspring after maternal exposure to methylmercury.

Maternal exposure to the neurotoxin methylmercury (MeHg) has been shown to have adverse effects on neural development of the offspring in man. Little is known about the underlying mechanisms by which MeHg affects the developing brain. To explore the neurodevelopmental defects and the underlying mechanism associated with MeHg exposure, the cerebellum and cerebrum of Wistar rat pups were analyzed by [(18)F]FDG PET functional imaging, field potential analysis, and microarray gene expression profiling. Female rat pups were exposed to MeHg via maternal diet during intrauterinal and lactational period (from gestational day 6 to postnatal day (PND)10), and their brain tissues were sampled for the analysis at weaning (PND18-21) and adulthood (PND61-70). The [(18)F]FDG PET imaging and field potential analysis suggested a delay in brain activity and impaired neural function by MeHg. Genome-wide transcriptome analysis substantiated these findings by showing (1) a delay in the onset of gene expression related to neural development, and (2) alterations in pathways related to both structural and functional aspects of nervous system development. The latter included changes in gene expression of developmental regulators, developmental phase-associated genes, small GTPase signaling molecules, and representatives of all processes required for synaptic transmission. These findings were observed at dose levels at which only marginal changes in conventional developmental toxicity endpoints were detected. Therefore, the approaches applied in this study are promising in terms of yielding increased sensitivity compared with classical developmental toxicity tests.

Developmental origins of adult diseases and neurotoxicity: epidemiological and experimental studies.

To date, only a small number of commercial chemicals have been tested and documented as developmental neurotoxicants. Moreover, an increasing number of epidemiological, clinical and experimental studies suggest an association between toxicant or drug exposure during the perinatal period and the development of metabolic-related diseases and neurotoxicity later in life. The four speakers at this symposium presented their research results on different neurotoxic chemicals relating to the developmental origins of health and adult disease (DOHaD). Philippe Grandjean presented epidemiological data on children exposed to inorganic mercury and methylmercury, and discussed the behavioral outcome measures as they relate to age and stage of brain development. Donald A. Fox presented data that low-dose human equivalent gestational lead exposure produces late-onset obesity only in male mice that is associated with neurodegeneration. Didima de Groot presented results on prenatal exposure of rats to methylazoxymethanol and discussed the results in light of the etiology of western Pacific amyotrophic lateral sclerosis and Parkinson-dementia complex. Merle G. Paule addressed the long-term changes in learning, motivation and short-term memory in aged Rhesus monkeys following acute 24 h exposure to ketamine during early development. Overall, these presentations addressed fundamental issues in the emerging areas of lifetime neurotoxicity testing, differential vulnerable periods of exposure, nonmonotonic dose-response effects and neurotoxic risk assessment. The results indicate that developmental neurotoxicity results in permanent changes, thus emphasizing the need to prevent such toxicity.

Zebrafish as potential model for developmental neurotoxicity testing: a mini review.

The zebrafish is a powerful toxicity model; biochemical assays can be combined with observations at a structural and functional level within one individual. This mini review summarises the potency of zebrafish as a model for developmental neurotoxicity screening, and its possibilities to investigate working mechanisms of toxicants. The use of zebrafish in toxicity research can ultimately lead to the refinement or reduction of animal use.

Locomotor activity assay in zebrafish larvae: influence of age, strain and ethanol.

Several characteristics warrant the zebrafish a refining animal model for toxicity testing in rodents, thereby contributing to the 3R principles (Replacement, Reduction, and Refinement) in animal testing, e.g. its small size, ease of obtaining a high number of progeny, external fertilization, transparency and rapid development of the embryo, and a basic understanding of its gene function and physiology. In this context we explored the motor activity pattern of zebrafish larvae, using a 96-well microtiter plate and a video-tracking system. Effects of induced light and darkness on locomotion of zebrafish larvae of different wild-type strains and ages (AB and TL, 5, 6 and 7 dpf; n=25/group) were studied. Locomotion was also measured in zebrafish larvae after exposure to different concentrations of ethanol (0; 0.5; 1; 2 and 4%) (AB and TL strain, 6 dpf; n=19/group). Zebrafish larvae showed a relatively high swimming activity in darkness when compared to the activity in light. Small differences were found between wild-type strains and/or age. Ethanol exposure resulted in hyperactivity (0.5-2%) and in hypo-activity (4%). In addition, the limitations and/or relevance of the parameters distance moved, duration of movements and velocity are exemplified and discussed. Together, the results support the suggestion that zebrafish may act as an animal refining alternative for toxicity testing in rodents provided internal and external environmental stimuli are controlled. As such, light, age and strain differences must be taken into account.

Developmental immunotoxicity of di-n-octyltin dichloride (DOTC) in an extended one-generation reproductive toxicity study.

Developmental immunotoxicity assessment is considered ready for inclusion in developmental toxicity studies. Further evaluation of proposed and additional assays is needed to determine their utility in assessing developmental immunotoxicity. In this study, a wide range of immunological parameters was included in an extended one-generation reproductive toxicity protocol. F(0) Wistar rats were exposed to DOTC via the feed (0, 3, 10, and 30mg/kg) during pre-mating, mating, gestation and lactation and subsequently F(1) were exposed from weaning until sacrifice. Immune assessments by several immune parameters were performed at PNDs 21, 42 and 70. The T cell-dependent antibody response to Keyhole Limpet hemocyanin (KLH) was assessed following subcutaneous immunizations with KLH on PNDs 21 and 35 and the delayed-type hypersensitivity response (DTH) against KLH was evaluated at PND 49. No effects were found on PND 21. While effects on lymphocyte subpopulations in the thymus were only observed in the 30mg/kg group on PND 42, effects on lymphocyte subpopulations in the spleen were found in the 30mg/kg group on both PNDs 42 and 70. The DTH response already showed an effect at 3mg/kg and was the overall critical endpoint. The results from this study support the inclusion of splenocyte subpopulation parameters in developmental toxicity studies and identified the DTH response as an important functional parameter.

Neural progenitor cells as models for high-throughput screens of developmental neurotoxicity: state of the science.

In vitro, high-throughput methods have been widely recommended as an approach to screen chemicals for the potential to cause developmental neurotoxicity and prioritize them for additional testing. The choice of cellular models for such an approach will have important ramifications for the accuracy, predictivity and sensitivity of the screening assays. In recent years neuroprogenitor cells from rodents and humans have become more widely available and may offer useful models having advantages over primary neuronal cultures and/or transformed cell lines. To date, these models have been utilized in only a limited number of toxicity studies. This review summarizes the state of the science regarding stem and neuroprogenitor models that could be used for screening assays, provides researchers in this field with examples of how these cells have been utilized to date, and discusses the advantages, limitations and knowledge gaps regarding these models. Data are available from both rodent and human stem and neuroprogenitor cell models that indicate that these models will be a valid and useful tool for developmental neurotoxicity testing. Full potential of these models will only be achieved following advances in neurobiology that elucidate differentiation pathways more clearly, and following further evaluation of larger sets of developmentally neurotoxic and non-toxic chemicals to define the sensitivity and predictivity of assays based on stem or progenitor cell models.

Developmental immunotoxicity of methylmercury: the relative sensitivity of developmental and immune parameters.

Current developmental and reproductive toxicity protocols include only a limited set of parameters for effects on the developing immune system. In this study, a wide range of immunological parameters were included in a pre- and postnatal developmental toxicity study. Dose-response data were compared to determine the relative sensitivity of different immune and developmental parameters. Mated female Wistar rats were dosed daily by gavage with methylmercury (0, 0.1, 0.4, 0.7, 1.0, 1.5, and 2.0 mg/kg BW/day) from gestational day 6 to postnatal day (PND) 10. In addition to general, reproductive, and developmental parameters, a wide range of immunological parameters were assessed in male offspring at PNDs 21, 42, and 70. The T cell-dependent antibody response to keyhole limpet hemocyanin (KLH) was assessed following sc immunizations on PNDs 21 and 35. Dose-response data were analyzed using the benchmark dose (BMD) approach by fitting dose-response models to the various endpoints. Methylmercury induced effects on developmental parameters, such as growth parameters and pup mortality. Effects on the immune system were found at doses without observed developmental toxicity. Immune effects differed at the three time points and consisted mainly of effects on functional parameters. The parameter with the lowest 5% lower confidence bound of the BMD (BMDL) was the primary KLH-specific IgG antibody response, which showed a dose-dependent decrease with a BMD of 0.039 mg/kg BW/day (CI 0.010-0.12). These data show the relatively high sensitivity of the developing immune system and thereby illustrate the relevance of testing immune parameters in reproductive and developmental toxicity testing protocols.