The present study was designed to investigate whether acute ethanol in
toxication increases the production of active oxidants, and subsequent
ly promotes apoptosis of hepatocytes, Hepatocytes were isolated from m
ale Wistar rats, and cultured in the presence or absence of ethanol, T
he fluorescence in situ nick end labeling method and an enzyme-linked
immunosorbent assay (ELISA) system to quantify fragmented DNA were use
d to estimate apoptotic change in hepatocytes, Nuclear morphological a
lterations and membrane barrier dysfunction of hepatocytes were assess
ed by staining with Hoechst 33342 and propidium iodide (PI). Intracell
ular glutathione level was determined as the fluorescence of monochlor
obimane (MCLB), which forms conjugate with glutathione to become fluor
escent. Ethanol (100 mmol/L) increased the amount of fragmented DNA an
d the number of apoptotic hepatocytes in vivo as well as in vitro, The
se ethanol-induced alterations in hepatocytes mere attenuated by simul
taneous incubation with either 4-methylpyrazole, an inhibitor of alcoh
ol dehydrogenase, or dimethylthiourea, an intracellular oxidant scaven
ger. Diethyl maleic acid (DMA), a glutathione depletor, enhanced the i
nduction of apoptotic change, and decreased membrane barrier function
in ethanol-treated hepatocytes, whereas ethanol per se did not increas
e the number of PI-positive hepatocytes. Furthermore, combination of e
thanol and DMA but not ethanol alone decreased the hepatocyte MCLB flu
orescence. Taken together, the present study suggests that active oxid
ants produced during ethanol metabolism mediate fragmentation of DNA i
n hepatocytes, and that intracellular antioxidants such as glutathione
play a critical role in the cytoprotective mechanisms of hepatocyte a
gainst lethal cell death, ie, apoptosis, induced by ethanol.