Title:Response to Doxorubicin of Exfoliated Human Buccal Epithelium Cells: Comparison of Three Methods of Cell Staining and Calcium Assessment
VOLUME: 15 ISSUE: 2
Author(s):Yuriy Shckorbatov*, Dmitriy Miroshnik and Igor Kovalenko
Affiliation:Department of Genetics, Institute of Biology, V.N. Karazin Kharkiv National University, Kharkiv, Department of Genetics, Institute of Biology, V.N. Karazin Kharkiv National University, Kharkiv, Institute for Problems of Cryobiology and Cryomedicine of the National Academy of Sciences of Ukraine, Kharkiv
Keywords:Cell nucleus, heterochromatin, cell membrane, ethidium bromide, indigo carmine, orcein,
fluo-3.
Abstract:Background: 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.
Objective: We studied the possibility of using the process of heterochromatinization
in cell nuclei in toxicological investigations of drugs in vitro.
Method: 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.
Results: 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.
Conclusion: 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.
