Genotoxic effect of benzophenathridine alkaloids on human leukemia (HL-60) cells.
| Authors | |
|---|---|
| Year of publication | 2005 |
| Type | Article in Proceedings |
| Conference | Book of abstracts p.72, VI International Comet Assay Workshop, September 22-24 2005 |
| MU Faculty or unit | |
| Citation | |
| Field | Biochemistry |
| Keywords | benzo[c]phenanthridine alkaloids; genotoxicity; HL-60 cells; comet assay |
| Description | Sanguinarine and chelerythrine are the main alkaloids of benzo[c]phenanthridine group. Many significant biological effects of these compounds have been reported. Information about their cytotoxicity and genotoxicity concerning different cells, tissues and organisms is essential for their further investigation. The aim of the study was to assess a genotoxicity of two benzophenanthridine alkaloids, chelerythrine (CHEL) and sanguinarine (SANG), on the cultured leukemic cells line HL-60. Alkaline neutral modification of the comet assay method was employed. Cells were cultured for different time intervals (1-3 hours) with either CHEL or SANG in concentrations up to 1 mg per ml of culture medium. Hydrogen peroxide (30 uM) served as a positive control. At the end of incubation, cells were transferred into phosphate saline buffer, pH 7.4, and mixed with 0.8 % low melting point agarose. Aliquots of cell suspension were poured on microscopic slides covered with 1% normal agarose and overlaid with a cover glass. Lysis of the cells was carried out for 1 hour in a Tris-borate buffer (TBE), pH 8.4, containing 1mM EDTA and 2.5% SDS. After lysis, slides were placed into denaturating buffer for 20 min, neutralized in the TBE and laid side by side into electrophoresis chamber filled with cold TBE buffer. Electrophoresis has been performed for 15 min at constant 30 V (1 V/cm). Afterwards the slides were fixed and then silver stained. The microscope slides were evaluated under microscope Leitz Orthoplan (objective 16x, magnification 160x) attached to the TV camera or to the CCD camera. Images of the comets were saved in the bmp format and analyzed with suitable PC software. Both the alkaloids seem to have an effect on the cellular DNA in tested concentrations. CHEL at the concentration 10 ug/ml caused practically total damage of the HL-60 nucleoids, when comparing DNA amount in the comet head and tail (about 90 % of comet signal appeared in the tail). This damage is comparable with the damage of the positive control caused by cell culture incubation with 30 uM hydrogen peroxide. Comets in the presence of SANG at the same concentration show about 60% of the nuclear DNA in tails. Our experiments showed, that both studied substances, CHEL and SANG, cause damage to nuclear DNA of HL-60 cells and therefore might be considered to be genotoxic for cultured leukemic HL-60 cells. |
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