ETHANOL INDUCES TRANSIENT ARREST OF CELL-DIVISION (G(2)+M BLOCK) FOLLOWED BY G(0) G(1) BLOCK - DOSE EFFECTS OF SHORT-TERM AND LONGER-TERM ETHANOL EXPOSURE ON CELL-CYCLE AND CELL FUNCTIONS/

To study the cytophysiological effects of ethanol systematically, L929 cells, a fibroblastic cell line derived from mouse connective tissue, were exposed to various concentrations of ethanol(12.5, 50, 100 and 2 00 mM) for short (3 and 6 h) and longer (24 or 26 h) durations. Ethano l-induced cellular responses were analysed by a combination of the fol lowing assays: number of cells, amounts of DNA and protein, MTT [4,5-d imethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) assay and cell c ycle. Ethanol dose-dependently suppressed these cellular functions, ex cept that 12.5 mM exposures for both 6 and 26 h increased the amount o f protein in spite of almost no change in other cellular functions, co mpared to the control. The most marked dose-dependency was observed in a reduction of formazan product in an MTT assay after both 6 and 26 h exposures to ethanol, being independent of the number of cells and pr obably reflecting dose-dependent depression of mitochondrial respirati on. A G(2) + M block in the cell cycle, an inhibition of cell division , was induced after short-term exposures (3 and 6 h) to 100 and 200 mM ethanol, but the block was released before 24 h had passed. Alternati vely, prolonged exposures (24 h) to 50-200 mM ethanol induced a G(0)/G (1) block, resulting in a decrease in the amount of DNA below the cont rol value. Moreover, the percentage of the S phase was decreased gradu ally and dose-dependently throughout the 24 h exposure. Thus, high con centrations of ethanol (50, 100 and 200 mM) perturbed the cell cycle p rogression by causing both a transient G(2) + M block (an inhibition o f mitosis) and a continuous G(0)/G(1) block, though the latter was mas ked by the G(2) + M block during shortterm exposure. The cells seem fi nally to acquire some tolerance to ethanol so as to pass through mitos is, but much less tolerance to pass through the checkpoint from the G( 1) to the S phase, which results in a decline in proliferation.