Effect of ethanol on rat fetal hepatocytes: Studies on cell replication, lipid peroxidation and glutathione

Abstract

Studies have shown that ethanol at moderate concentrations inhibits epidermal growth factor-dependent replication of fetal rat hepatocytes in culture. This may account for the growth/development impairment associated with fetal alcohol syndrome and decreased liver regeneration in alcoholic liver disease. In this study, we further define the mechanism(s) of the negative impact of ethanol on fetal rat hepatocytes and provide evidence that ethanol-induced injury to these cells is associated with membrane damage caused by lipid peroxidation, altered cell glutathione homeostasis and deranged mitochondrial structure and function. Exposure of fetal rat hepatocyte replication to ethanol (2 mg/ml) promptly resulted in blockade of replication, as indicated by a 40% reduction in DNA synthesis (p < 0.05). Assessment of cell injury on the basis of lactate dehydrogenase and ALT leakage indicated a statistically significant but not appreciable effect, whereas 51Cr leakage was more substantially increased (p < 0.05). Within 6 hr of ethanol exposure, superoxide radical levels increased more than twofold (p < 0.05). We noted a 56% increase in levels of diene conjugates, a 131% increase in malonaldehyde concentration and a 66% increase in fluorescent products of lipid peroxidation (all p < 0.05). Glutathione levels were decreased to 47% below control values (p < 0.05). Electron microscopic studies illustrated a slight disruption of mitochondrial structure (enlargement of mitochondria and dilation of cristae). This disruption was accompanied by mitochondrial swelling (increased permeability), altered mitochondrial membrane potential (a 16% decrease in rhodamine uptake), a 28% decrease in succinate dehydrogenase activity and a 30% decrease in cellular ATP level (p < 0.05). Pretreatment of fetal rat hepatocytes with 0.1 mmol/L N-acetylcysteine or S-adenosylmethionine for 24 hr prevented the ethanol-induced reduction of ATP and glutathione levels, essentially restored cell replication, ameliorated 51Cr leakage and decreased malonaldehyde and diene conjugate levels to 41% to 65% and 25% above control values, respectively. Pretreatment with 0.1 mmol/L vitamin E fully normalized malonaldehyde and diene conjugate levels and 51Cr leakage but failed to improve ATP levels or to increase significantly cell replication and glutathione levels. Concomitant administration of glutathione precursors with ethanol, rather than pretreatment, did not alter the impaired cell replication. Thus our data underscore the importance of cellular glutathione and ATP in preventing ethanol-induced decreases in fetal cell replication and suggest that alleviation of cellular lipid peroxidation alone is not sufficient to prevent this abnormality in fetal rat hepatocyte function.

Overexpression of Manganese Superoxide Dismutase Prevents Alcohol-induced Liver Injury in the Rat

“…Ethanol (200 mM) caused 78 Ϯ 12% depletion of mitochondrial GSH in Ad.lacZ-infected animals compared with hepatocytes incubated with vehicle alone (*, p Ͻ 0.05, four individual experiments). These findings are consistent with previously published work in which a high concentration of ethanol (Ͻ100 mM) caused a similar loss of mitochondrial GSH (38,39). In Ad.SOD2-infected rats, mitochondrial GSH levels were depleted to a similar extent by ethanol.…”

Effects of Oxidants and Glutamate Receptor Activation on Mitochondrial Membrane Potential in Rat Forebrain Neurons

“…Rats with a congenitally enhanced capacity to free radical generation have a reduced rate of respiration and oxidative phosphorylation and a decreased z~W (Salganik et al, 1994). Also, lipid peroxidation of the mitochondrial inner membrane bilayer could increase permeability of the membrane and mitochondrial swelling and result in uncoupling and depolarization of the inner membrane (Devi et al, 1993;Kowaltowski et al, 1996a;Gadeiha et al, 1997;Brookes et al, 1998).…”

Effect of short-term ethanol on the proliferative response of Swiss 3T3 cells to mitogenic growth factors

“…Previously, experiments with acetaldehyde and with the alcohol dehydrogenase inhibitor 4-methylpyrazole suggested that the inhibitory effect was due to ethanol itself and not to its metabolite acetaldehyde (Guizzetti and Costa, 1996). Alleviation of cellular radical production alone was not sufficient to prevent the abnormality in fetal rat hepatocyte function (Devi et al, 1993). When ethanol was pretreated for 1 hour and removed by PBS-washing, there were no changes in the growth factor-induced signaling pathways.…”

“…Tissue injury by either acute or chronic exposure to ethanol is due to several factors including accumulation of acetaldehyde and modified proteins, alterations in cellular redox state, microsomal activation of toxins, deranged mitochondrial function, and the enhancement of lymphocyte cytotoxicity (Devi et al, 1993;Lieber, 1994;Arnon et al, 1995). In addition, ethanol also interferes with the proliferation of several diverse cell lines in vitro, including astrocytes, human T cells, and primary rat hepatocytes.…”