COMPARISON OF VARIOUS METHODS OF ASSAYING THE CYTOTOXIC EFFECTS OF ETHANOL ON HUMAN HEPATOBLASTOMA CELLS (HUH-6 LINE)

The sensitivity of five kinds of cytotoxicity assays using ethanol on human hepatoblastoma cells (HUH-6 line), which were cultured as monola yers or spheroids, was compared. Ethanol was chosen as a test because it acts on cell membranes directly without being metabolized and exert s its cytotoxicity. The assay methods used were as follows: 3- (4, 5-d imethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT), lactate d ehydrogenase (LDH), colony formation, cell growth and DNA assays. The sensitivity of the assays was: LDH < DNA < cell growth < MTT < colony formation. LDH assay had the advantage that the same culture could be used for multiple assays, but when a small number of cells were assaye d, no significant increase in the release of LDH was detected in the a ssay cultures compared with the control cultures. Although the DNA and cell growth assays were more sensitive than the LDH assay, the extent of cell damage may be underestimated because the damaged cells and DN A present in the cultures are included in the assay samples. On the ot her hand, both MTT and colony formation assays showed a high sensitivi ty. The MTT assay was done within 24 h after ethanol was added to the cultures and was applicable to both monolayer and spheroid cultures, w hile the colony formation assay required 1-2 weeks and it was applicab le only to monolayer cultures. Taken together, the MTT assay was the m ost suitable method to evaluate the cytotoxic effects of ethanol on HU H-6 cells cultured as either monolayers or spheroids.