3R-Project 70-99

Sensory irritant screening using a stable cell line expressing the vanilloid receptor

Thomas Baumann
Dept. o Neurological Surgery, Oregon Health and Sciences University, Portland; OR 97239-3098, USA
, baumannt@ohsu.edu

Keywords: lung; sensory irritation; cell cultures: transgenic; reduction; replacement; toxicity testing: sensory irritants

Duration: 2 years Project Completion: 2003

Background and Aim
Sensory irritation (stinging or burning sensations) caused by chemical agents is commonly measured in mice using the reflexive decrease in respiratory rate when the animals inhale the irritant. Studies in mice indicate that for many chemicals the sensory irritation is a process that is mediated by a membrane receptor protein, the so-called "sensory irritant receptor." This “sensory irritant receptor” is thought to be activated by a number of different sensory irritants, including capsaicin. A capsaicin receptor, the vanilloid receptor 1 (VR1), has recently been cloned. In this project, VR1 will be incorporated into a self-propagating cell line. The aim is to develop the first animal-free assay to assess the sensory aspect of an exposure to an irritant chemical.

Method and Results
The VR1 receptor was stably transfected into a cell line that does not constitutively express VR1. The VR1 receptor is a non-selective cation channel. Therefore activation of the receptor/ion channel can be monitored by measuring the increase in intracellular Ca²⁺concentration. Changes in cytosolic calcium levels will be monitored with optical imaging (Fura-2). A number of sensory irritant chemicals will be tested using capsaicin as a positive control and capsazepine as a competitive antagonist. We will evaluate whether sensory irritation by known sensory irritants is entirely attributable to the activation of VR1 and whether the sensory irritants use the same binding sites as capsaicin. Finally, the sensitivity and specificity of the bioassay will be evaluated.

Conclusions and Relevance for 3R
Alternative methods already exist for the cytotoxic or tissue damaging activity of chemical irritants and for the ciliotoxic and ciliostatic potential of chemical additives. However, no in vitro method is yet available to detect the sensory irritant properties of chemicals. In the proposed cell culture model, the results will be scored with optical measurement systems amenable to a high level of automation. If the present project is successful, it may obviate the need to use animals for sensory irritant screening and contribute to the refinement of the existing mouse inhalation assay.

References
Nielsen G.D. (1991) Mechanisms of activation of the sensory irritant receptor by airborne chemicals, Critical Reviews in Toxicology 21: 183-208

Caterina M.J., Schumacher M.A., Tominaga M., Rosen T.A., Levine J.D. and Julius D. (1997) The capsaicin receptor: a heat-activated ion channel in the pain pathway, Nature 389: 816-824.