Response to Doxorubicin of Exfoliated Human Buccal Epithelium Cells: Comparison of Three Methods of Cell Staining and Calcium Assessment.

Abstract

Doxorubicin is an anthracycline antibiotic which inhibits DNA and RNA synthesis intercalating DNA double helix and inducing free radicals. Doxorubicin is used in the treatment of cancer diseases. The influence of Doxorubicin on human buccal cell was used as a model for the assessment of toxic effects in vitro. We studied the possibility of using the process of heterochromatinization in cell nuclei in toxicological investigations of drugs in vitro. The exfoliated buccal epithelium cells of two donors (men) - Donor A (24 years) and Donor B (23 years) were used. Cells were subjected to Doxorubicin in concentration 0.2; 2; 20, and 200 µg/ml for 2 h. A significant increase of chromatin condensation in a dose-dependent way was shown. Doxorubicin induced chromatin condensation for cells of donors A and B if ≥ 0.2 µg/ml, and ≥ 2 µg/ml, correspondingly. Cell viability assessed by combined staining with Hoechst 33342 and ethidium bromide revealed a significant increase of damaged cells if ≥ 2 µg/ml. Indigo carmine staining also revealed a significant increase in permeability of cell membranes if ≥ 20 µg/ml. In cells of donor A the intranuclear calcium concentration increased if Doxorubicin concentration was ≥ 0.2 µg/ml. In cells of donor B cytoplasmic and intranuclear calcium concentration decreased if Doxorubicin concentration ≥ 0.2 µg/ml. The comparison revealed high sensitivity of the method of chromatin changes registration in human buccal epithelium cells as a method of assessment of drug toxicity in vitro, and this method may be recommended for toxicological investigations.