Lord Dowding Fund for humane research

 

National Antivisection Society

New approaches to neuro-toxicity testing

15 February 2008

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Two research projects by teams led by Dr Michael Coleman at Aston University with LDF support, have studied new approaches to testing for human neuro toxins.

Reactions in brain cells and their use in assessments of toxicity

This project has laid the groundwork for the development of a simple system, using human astrocytic cell lines for the high throughput screening of toxins.
The aim was to develop a co-culture model which would examine the relationship between human neuronal cells and astrocytic cells because the full role of astrocytes is not reflected in current neuro-toxicity models. Astrocytes have high levels of antioxidant systems compared to other brain cells, so they are important in fighting toxins in the central nervous system. These systems act to maintain normal levels of signalling molecules, detoxify chemicals and reduce the products of toxic exposure.

Co-cultures can show how two different types of cells behave, but individual cell types can only be observed if there are markers that are individual to each cell type, so the co-culture should be evaluated in terms of each cell type and collectively. There is a paucity of astrocytic-specific markers that give insight into how astrocytes respond to toxins etc. Astrocytes respond in a physically unique way, called astrogliosis, one component of which is an increase in the cellular levels of Glial Fibrillary Acidic Protein (GFAP) and cytokines IL-1 and IL-6.

Astrocytes can synthesise a number of chemical factors, the levels of which increase after the astrocytes are activated. These include growth factors, which have been shown to stimulate the recovery and regeneration of neurones, following trauma.

The project intended to extend the knowledge of astrogliosis and itís effect on astrocytes specific markers, so studies were designed to investigate the changes in GFAP and cytokines.

The main role of GFAP is thought to be in providing structural stability to glial cells by maintaining cytoskeleton integrity.

The use of GFAP as a marker for toxic effects in the CNS has been suggested since 1991 in vivo work. Various toxins have dose, time and region dependent effects on GFAP, so it can be used to indicate toxicity even in the absence of cytopathology. The increase in GFAP is not permanent and it was shown that astrocytes increase in volume rather than cell number.

By using human cell lines, the problem of extrapolation of the results is avoided. Prior to this study, the authors only knew of one previous study in this area using human astrocytes to monitor GFAP levels. Due to the lack of previous studies, there was no comparison of human cell lines Ė which can differ greatly in phenotype and genotypes & possibly in response to toxins.
The aims of the project were to select a suitable cell line that could then use toxins that have previously induced reactivity in the cells and hence an increase in GFAP. The relative cell toxicities will be determined and the sensitivities of the cells to them. The data can then be used as a reference for non-cytotoxic effects of the toxins. When a method for quantifying the GFAP is found, the effects of each toxin can be determined and related to existing assays. The effects of exposure time etc can be determined and also further investigation into the other markers of astrocytes reactivity.

Two potential cell lines were selected and it came to light that one of them had an extremely low level of GFAP. This is of interest to note, rather than implying that the particular line is not of any use as a marker of toxicity.
It was observed that GFAP was expressed at levels of toxin far lower than those which would be sufficient to cause cytotoxicity, so GFAP is an important indicator of the presence of a toxic stimuli rather than a crude indicator of serious damage. The ability to detect effects at such low concentrations, which mimic real-life, where exposure is a constant low-level and causes subtle effects on cellular function, is especially important.

With the cytokine response, one of the cell lines had a strong response to two of the chemicals at very low doses. This reaction supports the modelís further development as a human astrocytic model in itís own right, and as part of a co-culture system. There were certain limitations, in that one chemical, which was expected, was not detected, but this re-iterates the importance of a battery of cell models overcome any lack of response in the other models in certain areas.

There are now the foundations for a simple system, using human astrocytic cell lines for the high throughput screening of toxins. Using GFAP as a marker, the astrocytes response can be distinguished from other cell types.
The introduction of neurons would create a more accurate representation of the interaction between different cell types in the central nervous system (CNS) and how each cell type responds to toxic exposure

A 3-dimensional system to develop tests of neurotoxicity using human brain cells

Neurones and glial cells are fundamental components of the brain and are susceptible to damage due to various factors. Neurones in particular, have little regenerative capacity, which can lead to permanent impairment of the CNS.

Progress in human CNS research has been slow; there is a lack of clinical knowledge of even the basic pathology of many neurodegenerative diseases.
This LDF grant researched the use of human embryonal carcinoma cell line (NT2) which can be differentiated into neurons and astrocytes upon exposure to retinoic acid.

NT2 cells can be grown either in mono-layers or as cell aggregates. To differentiate in mono-layers takes 6 weeks, the aggregates take 24-28 days. Cell differentiation is a complex process, mediated by the exchange of ions and molecules through GAP junctions. GAP junctions allow electrical and metabolic communication. The major protein in GAP junctions is connexion, the different types of which are specific to different types of cells. To ensure that there was a mix of neurons and astrocytes in the cell aggregates, a number of connexins were looked at. This ensured different cell types were present and cell-cell communication could take place. The team have also been investigating how Gap junctions develop over time in the aggregates.
As well as looking at the connexins in the cells, the team will investigate changes within the cell.

The results so far show that the connexins being studied are present at all stages of differentiation, so astrocytes and neurons are present throughout the differentiation process. The lack of a specific marker has also highlighted that as soon as the retinoic acid is added the cells undergo differentiation and thus lose the pluripotency of the undifferen-tiated NT2 cells.

In the future the project will study the effect of toxins on the cell aggregates. They will also study any differences in the cells when they are differentiated in free-fall in the bio-reactor. The cells will then be exposed to a number of toxins.

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