Ethanol oxidation by intestinal microsomes: increased activity after chronic ethanol administration.

In the rat, chronic ethanol ingestion increases microsomal protein of the mucosal cells of the proximal small intestine, enhances cytochrome P-450 content and NADPH cytochrome-C-reductase activity. Intestinal oxidation of ethanol by a microsomal system is demonstrated as well as its enhancement after chronic ethanol administration.     

Studies on turnover rates of rat gamma-glutamyltranspeptidase after chronic ethanol administration in vivo

Chronic ethanol administration to rats was shown to result in a significant increase of hepatic and serum GGT activities, contrasting to the decreased levels observed in pancreas, intestine, brain, and kidney by the new alcock regimen method. The kinetics of rat GGT synthesis and degradation in vivo among the different sources after chronic ethanol administration has been studied by use of acivicin, which irreversibly inactivates GGT. The comparison of kinetics of GGT return after acivicin injection showed that the kidney and serum GGT exhibits biphasic half-lives in contrast to liver, pancreatic, intestinal, and brain GGT half-lives in chronic ethanol-administered rats. The present studies on kinetics of GGT synthesis (Ks) and degradation (Kd) in vivo would seem to indicate the existence of three types of systems. That is, Ks rather than Kd may be preferential in liver and serum whereas Kd is apparently increased in kidney and intestine without noticeable change in Ks. The reverse phenomenon is also observed for pancreas and brain. These findings suggest that the contributions of alterations in the rates of GGT synthesis and degradation to changing levels of GGT have been evaluated as a mechanism for enzyme adaptation in animal tissues as a change from the control diet to the ethanol diet.     

Phospholipase C activation by ethanol in rat hepatocytes is unaffected by chronic ethanol feeding.

The activation of phosphoinositide-specific phospholipase C by ethanol was compared in hepatocytes isolated from ethanol-fed rats and from pair-fed control animals. Ethanol (100-300 mM) caused a dose-dependent transient increase in cytosolic free Ca2+ levels in indo-1-loaded hepatocytes from both groups of animals. The rate of Ca2+ increase was similar in hepatocytes from control and ethanol-fed rats, but the decay of the Ca2+ increase was somewhat slower in the latter preparation. The ethanol-induced Ca2+ increase caused activation of glycogen phosphorylase, with 50% response at 50 mM-ethanol and a maximal response at 150-200 mM-ethanol, not significantly different in hepatocytes from control and ethanol-fed animals. Ins(1,4,5)P3 formation in response to ethanol (300 mM) or vasopressin (2 nM or 40 nM) was also similar in the two preparations. It is concluded that long-term ethanol feeding does not lead to an adaptive response with respect to the ethanol-induced phospholipase C activation in rat hepatocytes. The ability of ethanol in vitro to decrease membrane molecular order in liver plasma membranes from ethanol-fed and control rats was measured by e.s.r. Membranes from ethanol-fed animals had a significantly lower baseline order parameter compared with control preparations (0.313 and 0.327 respectively), indicative of decreased membrane molecular order. Addition of 100 mM-ethanol significantly decreased the order parameter in control preparations by 2.1%, but had no effect on the order parameter of plasma membranes from ethanol-fed rats, indicating that the plasma membranes had developed tolerance to ethanol, similar to other membranes in the liver. Thus the membrane structural changes associated with this membrane tolerance do not modify the ethanol-induced activation of phospholipase C. The transient activation of phospholipase C by ethanol in hepatocytes may play a role in maintaining an adaptive phenotype in rat liver.     

Hepatic redox state: attenuation of the acute effects of ethanol induced by chronic ethanol consumption

The effects of an acute dose of a diet containing ethanol (3g/kg) on hepatic redox state was compared in rats fed ethanol for 25 days and in their littermates given isocaloric carbohydrate. In both groups, cytoplasmic and mitochondrial redox states of pyridine nucleotides shifted to a more reduced level, but the changes were much less extensive in rats chronically fed ethanol. This metabolic adaption may reflect the oxidation of ethanol by a pathway not involving alcohol dehydrogenase, such as the microsomal ethanol osidizing system, which increases in activity after chronic ethanol ingestion.     

Enhancement of rat brain tryptophan metabolism by chronic ethanol administration and possible involvement of decreased liver tryptophan pyrrolase activity.

1. Chronic ethanol administration enhances rat brain 5-hydroxytryptamine synthesis by increasing the availability of circulating tryptophan to the brain. This increased availability is not insulin-mediated or lipolysis-dependent. 2. Under these conditions, tryptophan accumulates in the liver and apo-(tryptophan pyrrolase) activity is completely abolished, but could be restored by administration of regenerators of liver NAD+ and/or NADP+. 3. All four regenerators used (fructose, Methylene Blue, phenazine methosulphate and sodium pyruvate) prevented the ethanol-induced increase in liver tryptophan concentration and the increased availability of tryptophan to the brain. 4. It is suggested that the enhancement of brain tryptophan metabolism by chronic ethanol administration is caused by the decreased hepatic tryptophan pyrrolase activity. The results are briefly discussed in relation to previous work with ethanol. 5. Fructose enhances the conversion of tryptophan into 5-hydroxyindol-3-ylacetic acid in brains of ethanol-treated rats, whereas Methylene Blue inhibits this conversion in both control and ethanol-treated animals.     

Long-lasting effects of chronic ethanol administration on the activity of antioxidant enzymes

The aim of this study was to investigate the long-lasting effects of prolonged administration of ethanol doses on oxidative processes of aged rats. We determined the activity of superox-ide dismutase (SOD), catalase (CAT), and gluta-thione peroxidase (GPx) in erythrocytes of rats 15, 20, and 24 months old treated with an average daily dose of 1.5 g/Kg of ethanol or saline administered intraperitoneally for 13 weeks and after a 2 month period of withdrawal from treatment. The activity of all three enzymes decreased significantly with aging in the controls, while no age-related changes were found among treated rats. These findings are important since they are the first to show a long-lasting toxic effect of low ethanol doses observed in association with the aging process.     

Effect of chronic ethanol administration on energy metabolism and phospholipase A2 activity in rat liver.

1. For a period of 31 days male rats were given a liquid diet containing 36% of its energy as ethanol. Liver mitochondria from these animals demonstrated lowered respiratory control with succinate as substrate, a diminished energy-linked anilinonaphthalene-sulphonic acid fluorescence response, and lowered endogenous ATP concentrations. The phospholipid/protein ratio in mitochondria from these animals was unchanged; only minor alterations in the phospholipid fatty acid composition were observed. 2. In experiments where mitochondria were incubated at 18 degrees C in iso-osmotic sucrose (aging experiments), the above energy-linked properties were lost at an earlier time in organelles from ethanol-fed animals. Phospholipase A2 acitivty was depressed in mitochondria from control animals until respiratory control was lost and ATP was depleted. In contrast, no lag in the expression of phospholipase activity was observed in mitochondria from ethanol-fed rats. This loss of control of the phospholipase resulted in an earlier degradation of membrane phospholipids under the conditions of the aging experiments. 3. The ATPase (adenosine triphosphatase) activities, measured in freshly prepared tightly coupled mitochondria and in organelles uncoupled with carbonyl cyanide p-trifluoromethoxyphenylhydrazone, were not significantly different in ethanol-fed and liquid-diet control animals. When the mitochondria were aged at 18 degrees C, the activity increased with time of incubation in organelles from both groups of animals. A lag was observed, however, as the ATPase activity increased in control preparations. This lag was not present as APTase activity increased in mitochondria from ethanol-fed animals. 4. The significantly lowered values observed for energy-linked functions with succinate as an energy source demonstrate that ethanol elicits an alteration in liver mitochondria that affects the site II-site III regions of the oxidative-phosphorylation system. The apparent lack of control of the phospholipase A2 and ATPase activities in mitochondria from ethanol-fed animals suggests that the membrane microenvironment of these enzymes has been altered such that they can exert their catabolic effects more readily under conditions of mild perturbation. The fatty acid analyses demonstrate that the observed alterations both in the energy-linked functions and in control of the phospholipase and ATPase are not mediated through changes in the acyl chain composition of bulk-phase phospholipids.     

Effect of chronic ethanol administration on hepatic eNOS activity and its association with caveolin-1 and calmodulin in female rats

Although chronic and excessive alcohol consumption is associated with liver disease, the mechanism of alcoholic liver injury is still not clear. Whether reduced hepatic production of nitric oxide, which is evident in models of liver injury, is associated with alcohol-induced liver injury has not been investigated. We measured nitric oxide synthase (NOS) activity in the liver of pair-fed rats receiving liquid diet with or without alcohol [3% (vol/vol)] for 12 wk. Compared with control rats, hepatic NOS activity was significantly reduced in alcohol-treated rats along with the evidence of liver injury. Interestingly, there was no difference in the hepatic expression of endothelial NOS (eNOS) between ethanol-fed and pair-fed rats. We then tested the hypothesis that an imbalance between the binding of eNOS with inhibitory and stimulatory proteins may underlie the reduced activity of eNOS because eNOS catalytic activity is regulated partly through dynamic interactions with the inhibitory protein caveolin-1 and the stimulatory protein calmodulin. We found that hepatic caveolin-1 was markedly increased in alcohol-treated rats compared with control rats, whereas calmodulin remained unaltered. The binding of caveolin-1 and calmodulin with eNOS was increased and decreased, respectively, in alcohol-treated rats. Our results suggest that chronic alcohol intake attenuates hepatic eNOS activity by increasing the expression of the inhibitory protein caveolin-1 and enhancing its binding with eNOS.     

Metabolic alterations produced in the liver by chronic ethanol administration. Comparison between the effects produced by ethanol and by thyroid hormones

1. Liver slices from rats treated with thyroxine show an increased rate of O₂ consumption. The extra consumption, but not the basal respiration, can be abolished by ouabain. 2. Dinitrophenol is not effective in increasing the rate of O₂ consumption of liver slices from thyroxine-treated animals but its effectiveness can be recovered in the presence of ouabain. 3. (Na⁺+K⁺)-stimulated adenosine triphosphatase activity of liver was increased by administration of thyroxine in vivo. No changes were found in total Mg²⁺-stimulated adenosine triphosphatase activity. 4. Mitochondrial α-glycerophosphate dehydrogenase and microsomal NADPH oxidase activity were increased by both thyroxine and chronic ethanol treatment. 5. Liver slices from animals chronically treated with ethanol synthesize urea at an increased rate. 6. Mitochondrial size (section area) is markedly increased in the liver of animals chronically treated with ethanol. 7. Acute administration of ethanol in doses of 4 and 6g/kg significantly increases the uptake of ¹³¹I-labelled thyroxine by the liver. 8. Work reported here, along with results from other investigators, indicates marked similarities between the effects produced in the liver by chronic administration of ethanol and by thyroid hormones.     

Metabolic alterations produced in the liver by chronic ethanol administration. Increased oxidative capacity

1. Administration of ethanol (14g/day per kg) for 21–26 days to rats increases the ability of the animals to metabolize ethanol, without concomitant changes in the activities of liver alcohol dehydrogenase or catalase. 2. Liver slices from rats chronically treated with ethanol showed a significant increase (40–60%) in the rate of O₂ consumption over that of slices from control animals. The effect of uncoupling agents such as dinitrophenol and arsenate was completely lost after chronic treatment with ethanol. 3. Isolated mitochondria prepared from animals chronically treated with ethanol showed no changes in state 3 or state 4 respiration, ADP/O ratio, respiratory control ratio or in the dinitrophenol effect when succinate was used as substrate. With β-hydroxybutyrate as substrate a small but statistically significant decrease was found in the ADP/O ratio but not in the other parameters or in the dinitrophenol effect. Further, no changes in mitochondrial Mg²⁺-activated adenosine triphosphatase, dinitrophenol-activated adenosine triphosphatase or in the dinitrophenol-activated adenosine triphosphatase/Mg²⁺-activated adenosine triphosphatase ratio were found as a result of the chronic ethanol treatment. 4. Liver microsomal NADPH oxidase activity, a H₂O₂-producing system, was increased by 80–100% by chronic ethanol treatment. Oxidation of formate to CO₂ in vivo was also increased in these animals. The increase in formate metabolism could theoretically be accounted for by an increased production of H₂O₂ by the NADPH oxidase system plus formate peroxidation by catalase. However, an increased production of H₂O₂ and oxidation of ethanol by the catalase system could not account for more than 10–20% of the increased ethanol metabolism in the animals chronically treated with ethanol. 5. Results presented indicate that chronic ethanol ingestion results in a faster mitochondrial O₂ consumption in situ suggesting a faster NADH reoxidation. Although only a minor change in mitochondrial coupling was observed with isolated mitochondria, the possibility of an uncoupling in the intact cell cannot be completely discarded. Regardless of the mechanism, these changes could lead to an increased metabolism of ethanol and of other endogenous substrates.     

Factors that modify the metabolism of ethanol in rat liver and adaptive changes produced by its chronic administration

1. 2,4-Dinitrophenol (0.1mm) increases by 100-160% the rate of ethanol metabolism by rat liver slices incubated in a medium saturated with a gas mixture containing O(2)+CO(2)+N(2) (18:5:77). Similar effects are produced by relatively low concentrations of arsenate (10mm). At higher concentrations (37.5 and 50mm) arsenate inhibits the rate of ethanol metabolism. 2. When liver slices are incubated under an atmosphere containing O(2)+CO(2) (95:5) the metabolism of ethanol increases by about 100% over that obtained with O(2)+CO(2)+N(2) (18:5:77). However, under these conditions the activating effect of dinitrophenol is no longer observed. 3. Chronic administration of ethanol to rats for 3-4 weeks, in doses from 3 to 8g/kg per day, increases by 70-90% the ability of the liver to metabolize ethanol. In the liver slices of these rats, although an O(2)+CO(2)+N(2) (18:5:77) mixture was used, dinitrophenol does not further increase the metabolism of ethanol. If the chronic administration of ethanol is discontinued for two weeks, the rate of ethanol metabolism is lowered to control values and the activating effect of dinitrophenol is recovered. 4. No change in alcohol dehydrogenase activity was found in the liver of the rats in which the metabolism of ethanol had been increased as a result of the chronic ethanol treatment; a 40% increase in the activity of succinate dehydrogenase was observed.     

Effect of chronic ethanol administration on testicular antioxidant system and steroidogenic enzyme activity in rats

In order to find out the effect of chronic ethanol administration on testicular antioxidant system and steroidogenic enzyme activity, male rats fed with ethanol 1.6g/kg body weight per day for four weeks were studied. Besides a drastic reduction in body and testis weight, there was decrease in ascorbic acid, reduced glutathione and activities of superoxide dismutase, catalase, glutathione reductase and glutathione peroxidase in the testicular tissue of the treated animals. Simultaneously, there was increase in lipid peroxidation and glutathione S-transferase activity. Activities of 3 beta-hydroxy steroid dehydrogenase and 17 beta-hydroxy steroid dehydrogenase were also found decreased in the treated animals. The results indicate that chronic ethanol administration resulted in increase in oxidative stress and decrease in the activities of steroidogenic enzymes in the rat testes.     

Modulation of alcohol dehydrogenase and ethanol metabolism by sex hormones in the spontaneously hypertensive rat. Effect of chronic ethanol administration

In young (4-week-old) male and female spontaneously hypertensive (SH) rats, ethanol metabolic rate in vivo and hepatic alcohol dehydrogenase activity in vitro are high and not different in the two sexes. In males, ethanol metabolic rate falls markedly between 4 and 10 weeks of age, which coincides with the time of development of sexual maturity in the rat. Alcohol dehydrogenase activity is also markedly diminished in the male SH rat and correlates well with the changes in ethanol metabolism. There is virtually no influence of age on ethanol metabolic rate and alcohol dehydrogenase activity in the female SH rat. Castration of male SH rats prevents the marked decrease in ethanol metabolic rate and alcohol dehydrogenase activity, whereas ovariectomy has no effect on these parameters in female SH rats. Chronic administration of testosterone to castrated male SH rats and to female SH rats decreases ethanol metabolic rate and alcohol dehydrogenase activity to values similar to those found in mature males. Chronic administration of oestradiol-17β to male SH rats results in marked stimulation of ethanol metabolic rate and alcohol dehydrogenase activity to values similar to those found in female SH rats. Chronic administration of ethanol to male SH rats from 4 to 11 weeks of age prevents the marked age-dependent decreases in ethanol metabolic rate and alcohol dehydrogenase activity, but has virtually no effect in castrated rats. In the intoxicated chronically ethanol-fed male SH rats, serum testosterone concentrations are significantly depressed. In vitro, testosterone has no effect on hepatic alcohol dehydrogenase activity of young male and female SH rats. In conclusion, in the male SH rat, ethanol metabolic rate appears to be limited by alcohol dehydrogenase activity and is modulated by testosterone. Testosterone has an inhibitory effect and oestradiol has a testosterone-dependent stimulatory effect on alcohol dehydrogenase activity and ethanol metabolic rate in these animals.     

Sensitivity of rat brain adenylate cyclase to activation by calcium and ethanol after chronic exposure to ethanol

Rats were fed ethanol (Lieber-DeCarli diet) for three weeks. Stimulation of cerebellar adenylate cyclase by calcium was measured in control (pair-fed), chronic-alcohol and alcohol-withdrawn animals. No differences in the sensitivity or maximal stimulation of this enzyme were observed among these groups. Ethanol $\text{in}$,$\text{vitro}$ (1%) stimulated brain adenylate cyclase approximately 50% in the presence or absence of calcium. Chronic alcohol exposure $\text{in}$,$\text{vivo}$ did not alter the sensitivity of adenylate cyclase to stimulation by alcohol $\text{in}$,$\text{vitro}$.     

Transducer for recording electrical and mechanical chronic intestinal activity.

An extraluminal displacement transducer has been developed for simultaneously recording the mechanical activity in two perpendicular directions and the electrical activity of the intestinal serosa. The length variations in two perpendicular directions were measured by means of strain gauges bounded on two pairs of lamellae embedded in a rigid stand. The electrical activity was recorded by means of four electrodes situated at the extremity of these lamellae. The electrical gauges of each pair of lamellae are connected to form a Wheatstone bridge. This device allows establishment of a correlation between the mechanical displacement of the intestinal wall serosa and electrical potentials by means of studies of long duration.     

Modulation of NK cell activity by moderate intensity endurance training and chronic ethanol consumption.

Chronic ethanol consumption can suppress natural killer (NK) cell activity. Exercise after ethanol administration may enhance blood ethanol clearance, which may benefit the immune response. This study examined the effects of moderate intensity endurance training and chronic ethanol consumption (20% wt/vol) on splenic NK cell activity. Mice were assigned to one of four groups: sedentary, water drinking (SED-H2O); sedentary, ethanol consuming (SED-EtOH); trained, water drinking (TR-H2O), and trained, ethanol consuming (TR-EtOH). TR groups ran 60 min/day, 5 days/wk, at 12 m/min for 10 wk. Mice were killed 48 h after exercise. Baseline NK cell activity was suppressed 30% in TR and EtOH groups compared with SED-H2O controls. Activation with recombinant human interleukin-2 increased cytolytic activity in all groups four- to fivefold. These results indicate that training did not abrogate the effects of chronic ethanol consumption on NK cell activity. Furthermore, moderate endurance training may contribute to suppressed nylon wool-enriched NK cell activity in murine splenocytes for as long as 48 h after exercise.     

Diethylnitrosamine-induced increase in gamma-glutamyltranspeptidase in rat liver: its association with thyroid hormone deficiency and its reversal by tri-iodothyronine.

1. The nodular phase of hepatic premalignancy was induced in male Fischer 344 rats by the administration of diethylnitrosamine, 200 mg/kg i.p., followed by promotion utilizing the Solt-Farber promoting regime. 2. Relative to the situation in normal non-treated control rats: the activity of gamma-glutamyltranspeptidase was found to be increased 9.42-fold in homogenate and 7.33-fold in plasma membrane fractions prepared from the livers of saline-injected control rats; and 81.37-fold in homogenates and 91.92-fold in plasma membranes prepared from the livers of diethylnitrosamine-injected rats; plasma levels of total T3 and total T4 were found to be decreased 42.06 and 47.45% in saline-injected control rats and 88.7 and 83.2% in diethylnitrosamine-injected rats, respectively. 3. An early pre-nodular phase of hepatic premalignancy was produced in young immature and mature adult male Fischer 344 rats by the administration of diethylnitrosamine, 75 mg/kg, without subsequent application of the promotion regime. 4. Relative to the situation in control rats: the activity of gamma-glutamyltranspeptidase was found to be increased in liver homogenates prepared from diethylnitrosamine-treated rats, 1.62-fold in young immature rats 1.20-fold in mature adult rats; plasma levels of total T3 were found to be reduced in diethylnitrosamine-treated rats, 28% in young immature rats 9% in mature adult rats. 5. Treatment of diethylnitrosamine-injected young immature male Fischer 344 rats at the prenodular phase of hepatic premalignancy with tri-iodothyronine at 0.005 micrograms/kg s.c. daily for 7 days reversed the diethylnitrosamine-induced increase in liver homogenate gamma-glutamyltranspeptidase activity and the decrease in plasma total T3, restoring these parameters to normal levels.(ABSTRACT TRUNCATED AT 250 WORDS)