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.

Evening intake of alpha-lactalbumin increases plasma tryptophan availability and improves morning alertness and brain measures of attention.

BACKGROUND: Brain serotonin function is thought to promote sleep regulation and cognitive processes, whereas sleep abnormalities and subsequent behavioral decline are often attributed to deficient brain serotonin activity. Brain uptake of the serotonin precursor tryptophan is dependent on nutrients that influence the availability of tryptophan via a change in the ratio of plasma tryptophan to the sum of the other large neutral amino acids (Trp:LNAA). OBJECTIVE: We tested whether evening consumption of alpha-lactalbumin protein with an enriched tryptophan content of 4.8 g/100 g increases plasma Trp:LNAA and improves alertness and performance on the morning after sleep, particularly in subjects with sleep complaints. DESIGN: Healthy subjects with (n = 14) or without (n = 14) mild sleep complaints participated in a double-blind, placebo-controlled study. The subjects slept at the laboratory for 2 separate nights so that morning performance could be evaluated after an evening diet containing either tryptophan-rich alpha-lactalbumin or tryptophan-low placebo protein. Evening dietary changes in plasma Trp:LNAA were measured. Behavioral (reaction time and errors) and brain measures of attention were recorded during a continuous performance task. RESULTS: Evening alpha-lactalbumin intake caused a 130% increase in Trp:LNAA before bedtime (P = 0.0001) and modestly but significantly reduced sleepiness (P = 0.013) and improved brain-sustained attention processes (P = 0.002) the following morning. Only in poor sleepers was this accompanied by improved behavioral performance (P = 0.05). CONCLUSION: Evening dietary increases in plasma tryptophan availability for uptake into the brain enhance sustained alertness early in the morning after an overnight sleep, most likely because of improved sleep.

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.

Neurobehavioural evaluation and kinetics of inhalation of constant or fluctuating toluene concentrations in human volunteers.

The health risks of inhalation exposure to volatile organic solvents may not only depend on the total external dose, but also on the pattern of exposure. It has been suggested that exposure to regularly occurring peak concentrations may have a stronger impact on the brain than constant exposure at the same average level. Recent animal experimental studies conducted in our laboratory using relatively high concentrations of toluene have shown different effects on discrimination performance and motor activity during and after exposure, depending on the exposure scenario. Relevance of these findings for man was evaluated in a volunteer study in which 11 healthy men (age 20-49 years) were exposed by inhalation for 4h to either a constant concentration of 40ppm toluene or to three 30-min exposure peaks at 110ppm during this 4h period. Selected tests from the Neurobehavioural Evaluation System (NES) were performed repeatedly during and after exposure. Blood concentrations of toluene as well as urinary o-cresol excretion were measured at relevant time points. The results show that toluene concentration in blood increased during constant exposure and fluctuated during occupationally relevant peak exposures. Presumably, brain concentrations showed similar qualitative patterns. No clear changes were observed on neurobehavioural measures of motor performance, attention, perceptual coding and memory, or on measures of mood and affect. The exposure conditions do not seem to induce significant acute changes in central nervous system function similar to those observed at much higher concentrations in animals, although a statistical correlation was found between one motor performance test (Finger Tapping Test with alternating hands) and blood toluene concentrations. Urinary o-cresol excretion appeared to be significantly higher during the first 2h after exposure.

Effects of Ginkgo biloba on corticosterone stress responses after inescapable shock exposure in the rat.

Extracts from the leaves of the Ginkgo biloba tree (GBE) are found to be clinically effective in neuroprotection, cerebral and cardiovascular function and cognitive processing. Recent animal findings suggest that GBE also may improve stress adaptation and prevent learned helplessness, as evidenced by its reduction of behavioral acquisition deficits of active avoidance after inescapable shock exposure. In the present report, the effects of two doses of GBE were studied on corticosterone stress responses and acquisition of active avoidance after inescapable shock exposure. Forty-eight rats were divided into three groups: either receiving a daily dose of 50 mg/kg or 150 mg/kg of GBE (containing 24% flavonoid and 6% terpenoid) or vehicle for 2 weeks. After 2 weeks of administration, animals were trained for active-avoidance acquisition following inescapable shock exposure (stress induction) or nonshock exposure (nonstress). Administration of 150 mg/kg but not of 50 mg/kg of GBE significantly prevented a corticosterone stress response after inescapable shock exposure (P<.0001) without any beneficial behavioral effect on active avoidance. Repeated administration of GBE particularly improves biological adaptation to noxious stimuli without beneficial behavioral consequences. Present findings do not support previous claims about the benefits of G. biloba on improving behavioral stress adaptation and acquisition of active avoidance and on reducing behavioral deficits indicative of "learned helplessness."

Application of physiologically based toxicokinetic modelling to study the impact of the exposure scenario on the toxicokinetics and the behavioural effects of toluene in rats.

The toxicity of inhalatory exposure to organic solvents may not only be related to the total external dose, but also to the pattern of exposure. In this study physiologically based toxicokinetic (PBTK) modelling has been used to study the impact of the exposure scenario on the toxicokinetics and the behavioural effects of the model solvent toluene in rats. After construction of the model with parameters from literature, toxicokinetic data were collected from rats exposed to either a constant concentration or fluctuating concentrations at total external dose levels of 20,000 and 10,000 ppm x h for model validation. At the same exposure conditions the effects on learned performance were evaluated in separate groups of rats using a visual discrimination task. In general, the PBTK model provided reliable predictions of the toxicokinetics of toluene at different exposure scenarios, but it also tended to underestimate the blood and brain concentrations in the descending parts of the tissue concentration-time curves. At these high dose levels the differences in toxicokinetics between the constant and the fluctuating exposure groups were relatively small. The visual discrimination experiments demonstrated a slowing of response speed and disinhibition of responding in all toluene-exposed groups. The results suggest that the brain concentration of toluene is one of the major determinants of its effect on disinhibition of responding.