Prior studies in our laboratory have shown that exposure of cultured fetal rat hepatocytes to ethanol (E) blocks epidermal growth factor-dependent replication and that this is paralleled by cell membrane damage, mitochondrial dysfunction, membrane lipid peroxidation (LP), and enhanced generation of reactive oxygen species. These measures of E-mediated oxidative stress (OS) were mitigated by treatment with antioxidants, and cell replication could be normalized by maintaining cell glutathione (GSH) pools. We have now extended these studies to an in vivo model. Rats were administered E (4 g/kg, po) at 12-hr intervals on days 17 and 18 of gestation and killed on day 19, 1 hr following a final dose of E (a total of 5 doses). Fetal and maternal brain and liver were assayed for signs of OS. The 2-day in utero E exposure increased membrane LP in fetal brain as evidenced by increased malondialdehyde (MDA) levels from 1.76 +/- 0.12 SE (nMol/mg protein) to 2.00 +/- 0.08 (p < 0.05) and conjugated dienes from 0.230 +/- 0.006 SE (OD223/mg lipid) to 0.282 +/- 0.006 (p < 0.05). In fetal liver, MDA levels increased from 2.39 +/- 0.08 SE (nMol/mg protein) to 2.87 +/- 0.08 (p < 0.05), whereas dienes differed significantly only between ad libitum controls and the E and pair-fed control groups (p < 0.05). E decreased GSH levels in fetal brain by 19%, from 19.88 +/- 0.72 to 16.13 +/- 1.06 (nMol/mg protein) (p < 0.05). A 10% decrease in GSH was seen in fetal liver (p < 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)

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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.

Studies from our laboratory have shown that Short-term ethanol exposure inhibits epidermal growth factor-dependent replication of cultured fetal rat hepatocytes, along with a drop in ATP level, and that these effects could be caused, at least in part, by ethanolinduced oxidative stress. In these prior studies, mitochondrial morphology was abnormal and membrane lipid peroxidation products were increased, along with reduced transmembrane potential and enhanced permeability to sucrose. To define the effects of ethanol on mitochondrial function further, the present study examines the impact of ethanol exposure on mitochondrial electron transport chain components. A 24-hr exposure of cultured fetal fat hepatocytes to ethanol (2.5 mg/ml) reduced mitochondrial complex I activity by 16%(p e 0.05), complex IV by 28% (p e 0.05), and succinate dehydrogenase by 23% (p e 0.05). This reduction was paralleled by lower ADP translocase activity (24%, p < 0.05) and diminished mitochondrial glutathione (GSH) (20%, p < 0.05). Pretreatment with 0.1 mu Sadenosyl methionine, before ethanol exposure, normalized mitochondrial GSH along with activities of complex I, complex IV, and succinate dehydrogenase. A 3-hr exposure of isolated mitochondria (which do not metabolize ethanol) to ethanol (2.5 mg/ml), inhibited the activities of complex I (1970, p e 0.05), complex IV (24%, p < 0.05), and of ATP synthesis (20%, p e 0.05). It is concluded that: (1) short-term ethanol exposure modestly decreases ATP synthesis by inhibiting activities of fetal hepatic mitochondria1 electron transport chain components; (2) ethanol, in the absence of acetaldehyde (as in the isolated mitochondria), can directly impair mitochondrial respiratory chain components; and (3) protection from some of the adverse effects of ethanol by pretreatment with S-adenosyl methionine suggests that these effects are secondary to depletion of mitochondrial GSH.

The role of cocaine in cardiac ischemia and subsequent reversible and irreversible pathologic changes is well established. Nevertheless, the mechanisms leading to cardiac injury and irreversible cellular changes remain elusive. Reactive oxygen species (ROSs) are the critical mediators of cellular damage during ischemia-reperfusion. To explore the response of cardiac oxidative stress parameters to intravenous (i.v.) And intraperitoneal (i.p.) cocaine exposure, cardiac total glutathione (GSH, GSSG), malonaldialdehyde (MDA), Mn-superoxide dismutase (Mn-SOD), catalase (CAT), GSH-peroxidase (GSH-px), and GSH s-transferase (GST) were measured, along with biochemical and histologic markers indicative of cardiac injury. Repeated i.p. cocaine exposure produced significant impairment in cardiac integrity, demonstrated by increased circulating lactate (2.4-fold; p < 0.0001), creatine kinase (2.2-fold; p < 0.0001), and creatinine levels (1.7-fold; p < 0.0001). Infiltration of neutrophils into myocardial cavities also was evident. These changes paralleled increases in cardiac MDA (25%; p < 0.04), GSSG (55%; p < 0.001), protein carbonyls (23%; p < 0.05), and Mn-SOD (23%; p < 0.05) levels, indicative of oxidative stress, decreases in GSH (35%; p < 0.01), adenosine triphosphate (ATP; 26%; p < 0.04), GSH-px (28%; p < 0.03), CAT (32%; p < 0.01), and GST (50%; p < 0.001) levels. Intravenous cocaine administration also had similar effects on cardiac oxidative stress measures. In conclusion, our data indicate that cocaine administration compromised the heart's antioxidant defense system.