Neurotoxicity – The Way Forward
Published: Originally published: December 7, 2007 | Updated: July 27, 2011
Dr. Evelyn Tiffany-Castiglioni
Department of Veterinary Integrative Biosciences
Texas A&M University
College Station, TX 77843
The value of in vitro systems lies in their potential capacity to respond mechanistically to a toxicant in a manner similar to that occurring from in vivo exposure. However, the translation of in vitro results to in vivo applications has not been demonstrated in neurotoxicology, and the development of rapid, valid screening systems for unknown neurotoxicants remains largely theoretical. My purpose in this forum is to look at in vitro approaches to neurotoxicity testing, particularly mammalian cell and tissue culture, and consider how we can contribute more than we do as in vitro neurotoxicologists.
When to Use In Vitro Models
Although the mission of this forum is to explore strategies for moving toxicity testing towards a completely non-animal approach, that goal is still quite distant. In vitro systems are the best choice in two situations: 1) when technical limitations preclude in vivo observation, and 2) when cost, efficiency, or ethical considerations outweigh the value of in vivo testing. In vitro models excel at providing controlled environments for exposing known cell types to toxicants for short periods of time (minutes to weeks) and directly measuring their biological responses. However, the most commonly used in vitro models, which are primary cultures or cell lines, usually lack normal heterogeneous cell-cell interactions and do not allow the observation of toxic effects over a period of time comparable to mammalian developmental periods or life-spans.
Epidemiological and animal exposure studies with several neurotoxicants (lead, mercury and ethanol) show that the immature brain is usually more vulnerable than the mature brain, though in some cases the developing brain is known to be more resistant because of its plasticity. However, damage to the developing brain has life-long consequences to the individual and society in lost independence and productivity, and may also have delayed effects , such as neurodegenerative diseases late in life. These observations support the logic of using in vitro neurotoxicity testing for circumscribed, short-term studies of mechanisms of action in immature cells.
How to Use In Vitro Models
A reasonable expectation for in vitro models is that they facilitate the discovery of relevant mechanistic endpoints that help explain toxicity. Mechanism is a critical link for validation of in vitro results. Perhaps, most critically, there is a need to assess the insensitivity and the irrelevance of endpoints commonly tested in vitro, and progress has been rapid in recent years in this area. Neurotoxic endpoints can be classified according to the specificity of information they provide: 1) cell viability or death, 2) generic cell functions (respiration, ion transport, Ca2+ homeostasis, protein and DNA turnover, and oxidative stress responses), 3) differentiated cell functions (neurite extension, axonal transport, synapse function, myelination, cell-cell signaling, differentiated enzyme activities, and neurotransmitter uptake and metabolism), and 4) toxicant characteristics (uptake, accumulation, release, and metabolism). In general, the third and fourth types of endpoints should be most useful for in vivo validation. For example, if cytotoxicity (cell death) is not an important component of pathogenesis in vivo, then it is not a relevant endpoint for in vitro toxicity testing, beyond initial range-finding experiments to avoid cytotoxic exposure regimens.
Mechanism entails more than the molecular or cellular entities acted upon by the toxic substance; it is also the associated perturbations in physiology. Examples include the disruption of normal synaptic overproduction and pruning by exposure during development, impairment of plasticity and repair, and altered synaptic function. Each of these physiological processes has molecular components amenable to examination. In the case of synaptic function these include the molecular interactions involved in presynaptic neurotransmitter release, post-synaptic receptor function, and post-synaptic intracellular signaling. Examples specific to astrocytes include their roles in the distribution of water and ions related to tissue swelling, neurotransmitter uptake and inactivation, metal sequestration, protection of neurons from oxidative stress, and provision of metabolites to neurons. Each of these effects could be studied in a detailed fashion in vitro with validation in vivo.
Current work with in vitro models reflects the increased use of histotypic or hetereogeneous primary culture systems for certain types of studies. This trend counterbalances three decades of work on clonal cell lines that has dominated much of modern in vitro toxicology. Clonal cell lines have been the system of choice for many studies because they are well-characterized, easy to culture, and homogeneous in their responses to toxicants. Such cell lines still have considerable value for specific applications. However, many caveats must be considered in the interpretation of data from cell lines. For example, cell lines typically are more undifferentiated that primary cultures in committed cell lineages. Also wide morphological and functional heterogeneities exist in both neurons and glia in situ, so that toxic chemicals do not uniformly affect each member of a class of cells. Researchers are returning to the use of biologically more complex models, such as heterologous cell cultures, explants, and ex vivo tissue slices from toxicant-exposed animals. Their use is supported by improvements in analytical techniques that make single cells accessible to measurement, such as interactive laser cytometry, and thus facilitate the discovery of physiologically relevant mechanisms of toxic action.
The way forward: The following is a revised list of essential research objectives for in vitro neurotoxicology that I first outlined in 2004 (Tiffany-Castiglioni, 2004). This list is broadly applicable to environmental contaminants, such as heavy metals and pesticides, as well as ethanol, drugs, and endogenous neurotoxic proteins. Major areas are:
- Mechanistic integration of any known behavioral effects of the toxicant with its molecular and cellular substrates
- Molecular, physiologic, and morphologic effects of neurotoxicants on synaptogenesis, neuronal plasticity, and regeneration
- Stages of toxicant-induced carcinogenesis in glial cells
- Differences in sensitivity between immature and mature cells of all types (neurons, oligodendroglia, astroglia, and microglia) to neurotoxicants
- Interactions among neurotoxicants
- Influence of genetic polymorphisms on susceptibility to diseases induced by environmental contaminants.
Progress in these areas will be heavily dependent on progress in basic and applied neuroscience, and will be facilitated by close interdisciplinary collaborations. Addressing these and similar issues should provide significant advances in identifying, treating, and preventing diseases and functional impairments associated with neurotoxic exposures. As a platform for examining genetic susceptibility, in vitro neurotoxicology may become not only a central approach for risk assessment, but also for understanding commonalities between neurodegenerative diseases caused by chemicals in the environment and those caused by endogenous proteins.
©2011 Evelyn Tiffany-Castiglioni
Harry, G.J. & Tiffany-Castiglioni, E. (2005). Evaluation of neurotoxic potential by use of in vitro systems. Expert Opin. Drug Metab. Toxicol. 1(4), 1-13.
Tiffany-Castiglioni, E. (2004). In vitro neurotoxicology: introduction to concepts. In: In VitroToxicology: Principles and Challenges. E. Tiffany-Castiglioni, Ed., Humana Press, Totowa, New Jersey, pp. 1-28.
Tiffany-Castiglioni, E., Venkatraj, J.S., Qian, Y. & Wild, J.R. (2006). In vitro models for testing organophosphate-induced neurotoxicity and remediation. In: Toxicology of Organophosphate & Carbamate Pesticides, Ramesh C. Gupta, Ed., Elsevier, Amsterdam. pp. 315-337.