D. Roman, A. deBrugerolle-deFraissinette, B. Greiner, J. Marty, B. Vogel, M. Kolopp, A. Cordier, H. Van Cauteren and M. Bobadilla. Preclinical Safety, Basel, Switzerland.

Introduction
Cell proliferation index, apoptotic rate, or cell cycle analysis are parameters often included as surrogate markers for the assessment of drug toxicity and/or efficacy. The recently developed Laser Scanning Cytometer (CompuCyte Corporation, Cambridge, Mass.) has been adapted for evaluating these parameters on skin punch biopsies from a dermal toxicity study in minipigs. The microscope-based cytofluorometer quantifies the fluorescence of individual cells in cross sections or cytologic slides. It presents the advantages of rapidity and objectivity, in comparison with traditional immunohistopathological evaluation. The possibility of combining these measurements with the visual image of the corresponding cell represents an advantage since morphological structure can be preserved.

Experimental Procedure  1. Animal treatment:   Anti-mitotic compound was topically applied on minipig at different concentrations. Visual skin observations were done daily. Skin biopsies from the application site were taken when the skin irritation reached grade 3 to 4. 2. Cell isolation:   Single-epidermal cell suspensions were obtained by using the thermolysin-trypsin procedure1. The isolated cells were centrifuged on slides, fixed in ice-cold methanol and stored at -20°C until analysis. 3. Cell cycle studies:   The slides were washed with PBS and the cells were stained with a solution of propidium iodide, covered, and analysed with the LSC. 4. Apoptosis measurements:   Assessment of apoptosis induction was accomplished by detecting the expression of Bax and Bcl-2. The slides were counterstained with a solution of PI, covered, and analysed with the LSC. LSC Analysis The fluorescence emission of the stained cells was measured on the LSC. Slides were scanned using a 20X objective and an argon-ion laser operating at the 488-nm line. A minimum of 5,000 cells were examined. Green and red fluorescence were collected by separate photomultipliers. Contouring parameter: PI fluorescence. .....For each cellular contour, the values of green and red fluorescence intensities within that contour area were automatically processed by the software to generate a list of properties for that cell including the integrated intensity (sum of all pixels in the contour), value of maximal pixel, and position of the contour. Overlapping nuclei were automatically excluded from the counting by special statistical filters.

Cell Cycle Investigation

Figure 1. Bivariate distributions of DNA content (max pixel) versus area in minipig skin cells from non-treated and treated animals. As the mitotic cells progress from G2 to M, the scattergram shows an increase in the PI max Pixel and a decrease in size of nuclei as the chromatin condenses2.

Apoptosis Assessment

Figure 2. Detection of Bax translocation from cytosol to mitochondria by analysis of the increase of max pixel of Bax immunofluorescence3. Bivariate distributions of DNA content versus Bax expression in minipig skin cells from non-treated and treated animals. R1: cells not expressing Bax and R2: cells expressing Bax.

Scan of a Paraffin Section

Figure 3. Paraffin section of minipig skin biopsy stained for Bcl-2. WinCyte software enables you to color a region in a scattergram, find the corresponding cells on the position dot plot, and relocate the cells on the slide to confirm and document results.

Discussion / Conclusion
Measurement of DNA content of a cell provides information about the cell cycle position with respect to major phases (G1 vs. S vs. G2 vs. M). The present accumulation of cells in the M phase (Figure 1) may reflect division failure. The concomitant decrease of the percentage of cells in the G1 phase supports a cell cycle arrest.
.....Major regulators of the apoptotic pathways are members of the Bcl-2 family. Some family members (Bcl-2, Figure 3)) suppress apoptosis, while others (Bax, Figure 2) promote cell death. The decision as to whether apoptosis will be induced appears to depend on the Bax/Bcl-2 ratio. Quantitation of the apoptosis regulatory proteins resulted in an elevated Bax/Bcl-2 ratio providing conditions facilitating activation of the apoptotic execution machinery in epidermal cells.
.....The LSC has been shown to be a very powerful tool for the evaluation of cell-cycle and apoptosis-related parameters in skin biopsy from minipigs during early development of dermal products.

References
1. Glade CP, Seegers BAMPA, Meulen EFJ, van Hooijdonk CAEM, van Erp PEJ and van de Kerkhof PCM (1996). Arch Dermatol Res 288: 203-210.
2. Luther Ed and L. Kamentsky (1996). Resolution of mitotic cells using laser scanning cytometry. Cytometry 23:272-278.
3. Darzynkiewicz Z, Bedner E, Li X, Gorczyca W and Melamed MR (1999). Minireview. Laser scanning cytometry: a new instrumentation with many applications. Exp Cell Res 249, 1-12.

|Copyright © 1999. All rights reserved. CompuCyte Corporation