Adaptation of the adrenal medulla: sustained increase in choline acetyltransferase by psychosocial stimulation.

Sustained increases were produced in adrenal choline acetyltransferase of individually caged mice by placing them into groups for 10 or 15 minutes daily for 7 to 10 days. They were left undisturbed in their individual cages for the remainder of each day. As in previous experiments of similar design, adrenal catecholamines and adrenal weight were also increased, although body weight was not affected.

Vesicular and synaptoplasmic synthesis of acetylcholine.

The synthesis and distribution of acetylcholine has been studied in the isolated synaptosome. Binding or contamination of [(14)C]acetylcholine in the synaptic vesicle fraction represents 1.5 percent of the total amount found in the synaptoplasm (synaptosomal cytoplasm). However, when the [(14)C]acetylcholine is derived solely by synthesis from labeled choline, then the labeled acetylcholine in the synaptic vesicle is 15 percent of the amount of acetylcholine synthesized in the synaptoplasm. The results suggest that the [(14)C]acetylcholine found in the vesicle fraction of synaptosomes incubated with labeled choline is due to vesicular synthesis.

Subtle consequences of methylmercury exposure: behavioral deviations in offspring of treated mothers.

Overt neurological impairment is the endpoint currently used to document a case of methylmercury poisoning. No consideration is given to possible subtle consequences. Offspring from mice exposed to methylmercury on day 7 or 9 of pregnancy were apparently unaffected during postnatal development. However, subtle behavioral differences between treated and control offspring were found when the overtly normal animals were tested in an open field and evaluated in a swimming apparatus at 1 month of age. Brain weight, protein, choline acetyltransferase, and cholinesterase were not significantly altered.

Hyperactivity: a lead-induced behavior disorder.

Mice were exposed to lead from birth by substituting solutions of lead acetate for the drinking water of their mothers. The suckling mice were thus exposed to lead through their mother's milk and, at weaning, directly through the drinking water. Controls received equal concentrations of sodium acetate. No deaths of offspring or mothers occurred during the first 90 days of exposure. It has been suggested recently that lead exposure may account for some incidences of behavior disorders in children. Levels of motor activity of individual offspring were measured from weaning until 70 days of age in specially designed activity cages. Lead-treated mice were more than three times as active as age-matched or size-matched controls. Treated and control animals were administered drugs currently used in the treatment and diagnosis of hyperactivity in children. All control animals responded as expected to all drugs used in this study. However, lead-treated mice responded paradoxically to d- and l-amphetamine, methylphenidate, and phenobarbital. That is, the CNS stimulants suppressed their hyperactivity while phenobarbital exacerbated the lead-induced hyperactivity. These findings suggest that lead produces an animal model of hyperactivity which may have clinical relevance and which may explain some cases of hyperactivity in children.

Dermal toxicity: alternative methods for risk assessment.

Conceptually, irritant contact dermatitis (irritation) and allergic contact dermatitis (ACD) in man should provide the ideal platforms to launch in vitro toxicology into the pantheon of in vitro testing assays. In theory, irritant dermatitis has been considered by most a simple area of cutaneous biology, whereas ACD is a complex area of biology. However, both result in responses that are reasonably stereotypical and well characterized. The biology of the underlying mechanisms is becoming characterized and will thus allow development of mechanistically based in vitro assays that will be scientifically validated and thus acceptable to regulatory agencies.

Precursor dependence of acetylcholine release from rat brain in vitro.

These experiments were designed to test the extent to which the concentration of extracellular choline affects the synthesis and subsequent release of acetylcholine (ACh) by rat cortex in vitro. We found that the rate of potassium-depolarized ACh release from rat cortical minces was significantly accelerated when choline chloride was added to the incubation medium at concentrations of either 60 or 100 microM. The ACh content of the cortical minces was reduced by prolonged depolarization; this depletion was prevented by incubating minces with choline (100 microM). Raising the extracellular choline concentration of the incubation medium did not elevate the amount of ACh released spontaneously (4.7 mM K+) and had no effect on the accumulation of transmitter that occurs when cortical minces are incubated in physiologic buffer. A single dose of choline chloride administered orally to rats (20 mmol/kg) was without effect on the subsequent release of ACh and choline from cortical minces in vitro. The ACh and choline concentrations of rat cortex in vitro were similarly unaffected by in vivo choline administration. These results indicate that ACh release from rat cortex, in vitro, depends upon the direct availability of extracellular choline under conditions of prolonged neuronal depolarization.

Mechanisms of neurotoxicity as studied in tissue culture systems.

This presentation focused on describing the unique aspects of nervous tissue which make it susceptible to toxicological insult. It reviewed the published literature of compounds that have been studied in tissue culture of nerve tissue and compared the data in vivo toxicity studies with that obtained from the tissue culture system. Further, neuronal activity as a determinant of neurotoxicity was discussed as a basic mechanism to understand toxicological outcomes.

The use of spinal cord cell cultures in the study of neurotoxicological agents.

Primary cell cultures of mouse embryo spinal cords were used to study the biological effects of exposure to organophosphate esters and neurotoxic amino acids. The effects of exposure were correlated with markers of cholinergic function. The purpose of this study was to examine the mechanisms whereby xenobiotics produce neurotoxicologic damage.

Framework for validation and implementation of in vitro toxicity tests.

The development and application of in vitro alternatives designed to reduce or replace the use of animals, or to lessen the distress and discomfort of laboratory animals, is a rapidly developing trend in toxicology. However, at present there is no formal administrative process to organize, coordinate, or evaluate validation activities. A framework capable of fostering the validation of new methods is essential for the effective transfer of new technologic developments from the research laboratory into practical use. This committee has identified four essential validation resources: chemical bank(s), cell and tissue banks, a data bank, and reference laboratories. The creation of a Scientific Advisory Board composed of experts in the various aspects and endpoints of toxicity testing, and representing the academic, industrial, and regulatory communities, is recommended. Test validation acceptance is contingent on broad buy-in by disparate groups in the scientific community--academics, industry, and government. This is best achieved by early and frequent communication among parties and agreement on common goals. It is hoped that the creation of a validation infrastructure composed of the elements described in this report will facilitate scientific acceptance and utilization of alternative methodologies and speed implementation of replacement, reduction, and refinement alternatives in toxicity testing.

An approach to the development of in vitro toxicological methods.

Over the last few years there has been increased societal pressure on the one hand and self-generated scientific pressure on the other to develop new and better in vitro techniques for the evaluation of the safety of commercial products. In vitro methodology addresses four major areas within toxicology. It provides the basic science of the discipline with new information. In acute toxicity testing, it can provide alternatives to current approaches, e.g. the Draize irritation tests. In the area of chronic toxicity testing, it will allow us to define the mechanisms associated with organ specific insults. In the area of risk assessment, the combined knowledge obtained by superior methods will allow us to assess risk more accurately. The results of studies on alternatives to the Draize irritation tests being carried out at or supported by The Johns Hopkins Center for Alternatives to Animal Testing will be reviewed.

Development of alternative methods.

This presentation will raise specific issues and explore areas that need discussion and development of mechanistically based tests in toxicology. In addition, an approach to validation will be presented that is based on fundamental principles and modeled after actual practice in other areas of methodology acceptance. Further, it is recognized that the validation process is but only one aspect of incorporation of in vitro approaches into safety-evaluation and risk assessment.