Identification of one or more sets of in vitro models and assay endpoints that will provide an overall result predictive of in vivo neurotoxicity is a major hurdle in replacing the animal tests. Many batteries have been proposed (ECVAM, 2002; Prieto, 2005), but this continues to be a challenge.
A recent approach was the assessment by ECVAM researchers of an in vitro testing strategy composed of undifferentiated and differentiated PC12 cells and primary cerebellar granule cells for its ability to distinguish between cytotoxic and neurotoxic substances (Gartlon, et al., 2006). The different cell systems responded differently to the multiple endpoints evaluated. However, the conclusions were that "further work is required to determine suitable combinations of cell systems and endpoints capable of distinguishing neurotoxicants from cytotoxicants."
Another approach involved investigating the effect of metabolism on drug toxicity to cultured liver and neural cells. Four drugs were tested in metabolically competent mouse hepatocytes and human hepatoblastoma (HepG2) cells, and in neuroblastoma (SH-SY5Y) and astrocytoma (U-373 MG) cells (Mannerström, et al., 2006). The researchers reported "better estimations of neurotoxicity can be made by the combined use of metabolically competent hepatocytes and glial cells (e.g. U-373 MG) together with neuronal cells (e.g. SH-SY5Y)." The same experimental approach to evaluating post-metabolism cytotoxicity to other organ systems, including human neural cells, has been available for several years with the use of the integrated discrete multiple organ cell culture (IdMOC) system (Li, et al., 2004).
An ongoing problem in interpreting the results from in vitro experiments is the extrapolation of the results to the in vivo situation. Two recent experiments indicate the importance of determining intracellular concentrations of test substances in in vitro experiments. Mundy, et al. (2004) examined the intracellular concentration of the lipophilic polybrominated diphenyl ethers (PBDEs) using cultured neuronal and glial rat cells. Results showed a magnification of the applied doses - 100-fold for a 1 µM exposure of 60 minutes. Many experimental factors affected the intracellular concentrations of PBDE, including serum in the media, total volume of the media, and duration of exposure. Investigators concluded that media concentrations significantly underestimated cellular concentrations, and that tissue concentrations are the relevant parameter and should be determined for in vitro experiments.
Animal tests sometimes expose the test subjects to very high concentrations of chemicals; however, the concentration of neurotoxicants found in brain tissues is typically parts per million (ppm). Cells are exposed to concentrations that range from non-toxic to cytotoxic (causing cell death), usually in the micromolar (µM) range. Meacham, et al. (2005) addressed the question of whether the in vitro doses provided comparable intracellular concentrations to those found in vivo for certain lipophilic neurotoxicants. Intercellular accumulation of two compounds in three in vitro neuronal tissue models was tissue, time, and concentration-dependent and in the ppm range, leading to the conclusion that "tissue levels rather than exposure concentrations are a more appropriate metric for comparison of in vitro to in vivo effects."
A test of unique interest to CNS toxicology is blood-brain barrier (BBB) permeability. If a substance (or its metabolites) cannot penetrate the capillary bed separating the brain from the rest of the circulation (the BBB), then it cannot be toxic to the brain. Tähti, et al. (2003) reviewed the state of in vitro BBB models. Various species of capillary endothelial cells and endothelial cell lines have been cultured as monolayers on membranes to model the BBB. Astrocytes have been found essential for the brain capillaries to form a good barrier. Recent attempts to co-culture human microvascular endothelial cells and astrocytes as BBB models have been successful (Cucullo, et al., 2007; Siddharthan, et al., 2007).
Additional information on other emerging research and policies relevant to neurotoxicity testing will be provided on AltTox.org in the future; please check back again.
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