The Center for Alternatives to Animal Testing is an academic center affiliated with the Division of Toxicological Sciences in the Department of Environmental Health Sciences of the Johns Hopkins University Bloomberg School of Public Health.

 

Johns Hopkins School of Public Health

Research Grants 2001-2002

Sensory Irritant Screening Using a Stable Cell Line Expressing the Vanilloid Receptor

Thomas K. Baumann, PhD
Oregon Health Sciences, Portland, Oregon

The recently cloned vanilloid receptor (VR1) has allowed us to proceed with the development of a useful in vitro assay for chemical sensory irritation. This assay will help minimize the use of animals in screening for sensory irritation by chemical agents.

We established a Chinese hamster ovary (CHO) cell line that stably expresses the VR1 vanilloid receptor/ion channel and then tested the effects of various chemicals on the cell line. The VR1-expressing cell line showed membrane currents in response to the sensory irritant piperine (10 µM) and the inflammatory mediator 12-(S)- hydroperoxy-eicosatetraenoic acid (10 µM). Some sensory irritants (e.g. nicotine) were ineffective.

Since the VR1 receptor gates a non-selective cation channel that is highly permeable to calcium, we also measured changes in cytosolic levels of calcium resulting from sensory irritant stimulation. Capsaicin (100 and 311 nM) caused huge increases in the intracellular calcium, as indicated by the ratio of emitted fluorescence intensities for the 340 and 380 nm excitation wavelengths. We also demonstrated that the stable cell line responds reliably with a [Ca2+]i increase in the presence of eugenol (1 mM). Responses to menthol (1 mM) were more variable.

None of the currently available alternative tests is able to assess the sensory aspects of exposure to a chemical. The initial results of this study demonstrate that an in vitro assay can provide a means for assessing the presence of sensory irritants that may excite chemonociceptive trigeminal ganglion neurons and thus give rise to sensory irritation (stinging or burning sensations). The initial success of this approach is encouraging. However, lack of response of the VR1 cell line to some known sensory irritants suggests that a battery of cell lines expressing different irritant receptors may be needed for sensory irritant screening in vitro.