Dr. Arie Bruinink1 and Prof. Dr. Peter Maier2 were both head of a research group in Cellular Toxicology at the former Institute of Toxicology, ETH Zürich and University of Zürich, Switzerland.
Identification of neurotoxic chemicals in cell cultures
This 3R Research Foundation project is designed to develop co-cultures with primary cells. Such systems mimic more closely the situation in an organism. Accordingly they have the potential of replacing and reducing the use of animals during the initial step of drug development, during toxicity testing of chemicals and during final drug assessments.
Assessment of neurotoxicity
The identification of potential neurotoxic activity (damaging nervous tissue cells) of chemicals and drugs is an important task within the framework of the toxicity assessment of chemicals. Current guidelines recom-mend the use of animal tests, because of the possibility that a compound might be converted by xenobiotic metabolism to metabolites with neurotoxic activity. Current risk assessment guidelines for neurotoxic organophosphorus compounds (OP) are based on the in vivo hen model due to its high sensitivity.
Conversion of nontoxic chemicals to neurotoxic metabolites.
A chemical might exert its toxicity directly or indirectly, following metabolism in the liver. Stable metabolites can be released from liver cells into the blood stream, transported to the brain and there cross the blood brain-barrier. This pathway has been well established for some OPs, but cannot be reproduced in human or animal brain tumor (neuroblastoma) cell cultures. Accordingly such cell culture systems fail to identify indirectly acting neurotoxic compounds.
The 3R goals of the project
The goal of the project was to assess the possibility of identifying chemicals with a Iiver-mediated brain toxicity in a cell culture system[*]. Cocultures were established between primary rat hepatocytes (1) and chicken brain cells isolated from total brain of Tetra SL embryonic chicken at stage 29 (2). The hepatocytes (Fig 1a) represent the liver, the embryonic brain cells (Fig.1b) the ultimate target. Stable hepatic metabolites in the supernatant have a high chance of reaching brain tissue via the blood circulation. The test compound can be added either directly to the cocultures (Fig.2), or first to hepatocyte cultures, following which the supernatant is transferred to the brain cells (Fig.2). Comparison between the two results provides information on the stability of the hepatocyte-derived metabolites.
Brain cell toxicity was assessed by measuring the bioreductive capacity (conversion of the dye MTT), the lysososmal activity (neutral red uptake) and the brain specific acetycholinesterase (AchE) activity of the cells.
Hepatocytes release metabolites which are toxic to cocultured brain cells
Two chemicals were compared: Cylophosphamide (CP), known to be converted in liver to a metabolite which is cytotoxic for extrahepatic tissues but without a specific brain toxicity, and isofenphos (Bayer AG), a re-presentative organophosphate known to be neurotoxic in humans. Brain cells were exposed directly, in cocultures or with the supernatant collected from exposed hepatocytes (exposure time 6 h – 24 hours). Cultured chicken brain cells died only after CP had been in contact with hepatocytes (supernatant or cocultures) (Fig.3). This proves that the metabolic competence of the hepatocytes is preserved and comparable to the situation in the living organism.
Brain cell specific functions can be impaired without cell death
Isofenphos, in the dose range chosen, inhibited AchE activity without affecting cell viability, again only after contact with hepatocytes (Fig.4). This inhibition of an organ-specific function (in this case the cholinergic nerves) prior to an overall cytotoxicity contrasts with the behaviour of CP (3) and corresponds well with a tissue-specific neurotoxic activity.
Potential of cocultures
The addition of hepatocytes to cultures of chicken brain cells provides a metabolic activation system comparable to that of the liver. Testing the cytotoxicity of the supernatant from hepatocytes treated with the parent compounds provides a method to measure the stability and half-life of the reactive metabolites under investigation.