Session E8: Organ-Specific Models III

Chairs: Eugene Elmore (USA) and June Bradlaw (USA)

E8: Using Normal Human Epithelial Cells to Predict Chemopreventive Agent Efficacy and Toxicity: Correlations with Clinical Trial Plasma Levels, Biomarkers, and Adverse Effects
E. Elmore¹, N. Desai¹, D. Stringer², V.E. Steele², E.W. Gerner², and J.L. Redpath¹. ¹Department of Radiation Oncology and Chao Family Comprehensive Cancer Center, University of California Irvine, Irvine, CA 92697, USA; ²Dept. of Radiation Oncology and Arizona Cancer Center, University of Arizona, Tucson, AZ, USA; ³Division of Cancer Prevention, National Cancer Institute, Bethesda, MD 20892, USA. eelmore@uci.edu.

The response data of several agents in the Human Epidermal Cell (HEC) Assay for chemo-preventive efficacy were compared to the clinical trial plasma levels to determine if concentrations that produce efficacy in vitro are achieved in vivo. The HEC Assay determines the efficacy of agents to inhibit carcinogen-induced changes in differentiation and growth of normal human epidermal cells. Aspirin, vitamin E acetate, perillyl alcohol, difluoromethylornithine (DFMO), and N-acetyl-l-cysteine had efficacious concentrations in vitro that were similar to plasma concentrations. The DFMO-induced changes in polyamine content were determined. The percent change in spermidine to spermine ratio and the depletion of putricine show excellent correlation with the same biomarkers from clinical trials and in vitro chemopreventive efficiacy. Dihydroepiandosterone (DHEA), and 9-cis-retinoic acid (RA) and were found in plasma at higher concentrations than were required for in vitro efficacy. In cytotoxicity studies with DHEA and RA using normal human epithelial cells from different target tissues show that agent concentrations found in plasma produce toxicity in vitro for oral mucosa and skin. These comparisons show that normal human cell assays for efficacy and toxicity are responsive at concentrations found in clinical trials.

E8: An Innervated Tissue Engineered Human Cornea for In Vitro Testing
May Griffith and Erik Suuronen. University of Ottawa Eye Inst., Ottawa Hospital–General Campus, 501 Smyth Road, Ottawa, Ontario, Canada K1H 8L6. mgriffith@ohri.ca.

We have previously fabricated a cell-based human corneal substitute that could serve as an alternative to animals in ocular irritancy testing. While these tissue-engineered (TE) human corneas reproduced key structural and physiological features of natural corneas and gave quantifiable changes in transparency corresponding to different degrees of cellular damage in response to different chemicals, they provided no indications of sensitivity and pain. The objective of this current work, therefore, was to innervate TE corneas to allow for a more complete range of testing capabilities, including neurotoxicity testing. Human corneal epithelial, stromal, and endothelial cell lines with extended lifespans were used to construct a cornea around and within a stabilized collagen-GAG scaffold. Dorsal root ganglia (DRG) dissected from early chick embryos served as the nerve source. The TE corneas show an innervation pattern that resembles that of natural corneas. The in-growing nerves possess the machinery to be excitable and propagate action potentials, and also show differential release of neurotransmitter, Substance P, in response to different neurotoxic substances. Differential wound healing rates in response to epithelial wounding were also observed. These innervated TE human corneas can therefore, in the future, be used as more complete alternatives to animals for ocular irritancy testing. We also show that modifications to the basic technology developed to date allows fabrication of other more complex tissues as in vitro alternatives to animals for testing applications.

E8: Validation of an Exposure Technique for Testing the Biological Activity of Gases and Complex Atmospheres In Vitro
M. Aufderheide, J.W. Knebel, and D. Ritter. Fraunhofer Institute of Toxicology and Aerosol Research, Nikolai-Fuchs-Str. 1, 30625 Hannover, Germany. aufderheide@ita.fhg.de.

In the field of inhalation toxicity, the use of in vitro methods is still limited, due to the difficulties in exposing cell cultures directly to gases and especially complex mixtures. To overcome these problems, a new experimental system, called CULTEX, was established and characterized. It is based on the membrane technique and allows direct exposure of cells at the air/liquid interface. For showing the efficiency of this technique to detect dose-dependent effects by airborne substances, the experimental setup was validated by using gases (ozone, nitrogen dioxide) as well as complex mixtures, like side-stream cigarette smoke and diesel exhaust, in comparison to clean air. In all cases, cellular reaction dependent on dose or dilution could be found in a reproducible manner, showing at the same time the stability of the system. The biological parameters estimated were cell number, metabolic activity, glutathione, and ATP content. Thus, the introduction of this exposure technique offers new test strategies for the toxicological evaluation of a broad range of inhalable substances in vitro.

E8: Epivaginal^TM, a Human Tissue Model for Vaginal Irritation Studies
M. Klausner¹, J. Kubilus¹, C. Cannon, J. Sheasgreen¹, and E. Lonardo². ¹MatTek Corporation, Ashland, MA 01721, USA; ²Johnson & Johnson, Personal Products Company, Skillman, NJ 08903, USA. mklausner@mattek.com.

Recently, a tissue culture-based model of the vaginal epithelium has developed. Normal, human ectocervico-vaginal (ECV) epithelial cells were induced to form a three-dimensional tissue, using specially formulated serum-free medium. The in vitro tissue reproduces many of the histological, ultra-structural, and protein expression properties of native tissue, including inter-digitation of cells, glycogen production, and cytokeratin expression. Initial experiments investigated the use of this tissue model and the MTT tissue viability assay for predicting ECV irritation. Vaginal anti-fungal products, contraceptives, and lubricants were exposed to the tissue model, and the exposure time, which causes a 50% reduction in tissue viability (ET-50), was determined. The ET-50s were compared to rabbit vaginal irritation scores for seven products that are currently marketed or in clinical trials. Using an ET-50 cutoff of 9.0 hours, the products could be successfully categorized into minimal or mild irritation classes. Based on these results and the problems associated with obtaining and the handling of human vaginal tissue, it is anticipated that the tissue model will be very useful in assisting product development scientists in developing safe, efficacious products.

E8: In Vitro Safety Evaluation of Anti-fungal Agents
E. Herreros, M.J. Almela, S. Lozano, and D. Gargallo-Viola. Glaxo Smithkline, Tres Cantos, 28760, Spain. eh13868@gsk.com.

Our laboratory has developed an in vitro screening system designed to determine which experimental drugs are most likely to be safe candidates for development and to decrease the number of animals used in the preclinical toxicity testing. Our screening panel consists of five cell lines from human and rodent origin and cultures of primary rat hepatocytes. Cell lines were exposed to two reference well-characterized antifungal agents (Fluconazole and Amphotericin B) and to six new in-house compounds during a 24-hour period. Cytotoxicity analysis was carried out, testing a battery of parameters for cell toxicity (protein synthesis, MTS, and Alamar Blue^TM). In general, no significant differences in basal cytotoxicity were observed between cell types derived from human and rodent origins. Among the end-point studies, protein synthesis was the most sensitive assay. MTS and AB assays produced similar dose-response curves. Next, the compounds were tested on cultures of rat hepatocytes by determining LDH, and glutathione content. The ranking of the drugs on the basis of the cell lines results showed the same order as for the rat hepatocytes model. We have found these combined in vitro tests to provide a good prediction of in vivo toxicity for several well-known antifungals, as well as for our drug candidates.