National Antivisection Society
2004-05 Brain Research - Design of neurotoxicity assays using human tissues
The study of substances which are poisonous to human nerves presents a challenge because of the complexity of the central nervous system (CNS), consisting of the brain and spinal cord. The distinct areas of the brain communicate with each other through a network of nerve tissues. These tissues have a limited ability to regenerate and are more vulnerable to irreversible damage when exposed to a toxin than other tissues. Therefore it is essential to evaluate the potential toxicity of therapeutic drugs and their breakdown products in relevant models of the human brain.
Current evaluation of the toxicity of a substance to the nervous system involves administration to an animal and observing behavioural changes – meaning animal suffering and results that poorly predict the human situation. With LDF support, Dr Mike Coleman at the School of Pharmacy, Aston University, is developing a system for testing potential neurotoxins using co-cultures of human nerve cells.
It is estimated that over 2 million mice are used annually in nerve toxicity studies worldwide. Mercury, for example, continues to be tested on animals despite its devastating effects on the nerves having been well documented in humans since the 1960s. To test the neurotoxicity of mercury, monkeys are exposed to it whilst still in the womb. The long-term effects of mercury on the eyesight and hearing are also measured in primates. Many primates are still used to test neurotoxic agents, such as the street drug ecstasy. In the UK in 2003 there were a total of 3,500 experimental procedures involving toxicity evaluation in primates. Worldwide, approximately 10,000 primates are used specifically in neurotoxicity studies, up to half of these in the USA.
Animal brain tissues are used widely in vitro (in culture) to study neurotoxicity. However, the effects of a specific toxin can differ widely between animal and human tissues. For example, although much research has been directed at the development of primate models of human Parkinsonism using MPTP, the drug-induced condition still does not adequately mimic the human disease. Recent studies of human brain biochemistry indicate that many brain disorders and the effectiveness of drugs to treat them depend upon biochemical systems unique to humans. There is a need for a dependable human-based model for human neurotoxicity.
The design of culture tests which reflect the unique sophistication of human brain tissue is in its infancy, due to the difficulty in obtaining human nerve tissue, its deterioration after death, inconsistency, high costs and risk of infection. Isolation of nerve cells from human embryonic tissues raises ethical concerns and data produced from waste tissues is not reproducible. As a first step in the replacement of animals, Dr Coleman’s test system has been designed using human cell lines to reliably detect potential neurotoxicity. The project uses cells from a human teratocarcinoma (tumour type) cell line, chemically treated to form nerve cells, and human astrocytic cells – brain cells responsible for regulation of immunity and inflammation. Nerve cells are more resistant to toxicity when in contact with astrocytic cells (large neuroglia cells of nervous tissue). The astrocytic cells will be grown on cellulose inserts placed on top of the other cell lines.
It is essential to determine whether toxic breakdown products can be produced from an apparently harmless substance. Therefore, to break down substances, enzymes normally present in the human brain are placed in a compartment above that containing the cells, separated by a cellulose membrane barrier. The system will reflect the complexity of the human brain and be maintained at body temperature in a water bath.
The toxicity of a substance to the nerve cells will be detected by studying damage to mitochondria within the cells with the use of a fluorescent dye. Mitochondria are the principal energy store or ‘powerhouse’ of the cell and regulate nerve cell viability. Also, as a number of compounds are capable of inducing apoptosis (selective cell death) flow cytometry (a method of counting cells and measuring their viability while they are in suspension) will be used to quantify the final stages of apoptosis in cell populations as well as cell viability.
Dr Coleman says this work intends to “help reinforce the UK’s position at the forefront of worldwide endeavour to design and implement novel in vitro neurotoxicity tests, sufficiently predictive of the human situation to meet society’s needs for safe and effective toxicity testing without animal suffering”.
“If a novel human tissue neurotoxicity test were able to model successfully even a narrow aspect of human neural damage sufficiently well to gain worldwide regulatory acceptance, several thousand animal neurotoxicity experiments would become obsolete.”
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