Effects of castration and testosterone administration on rat liver alcohol dehydrogenase activity

The effects of castration and testosterone administration on the activity of liver alcohol dehydrogenase and on the rate of ethanol elimination were determined in male Sprague-Dawley rats. Castration increased liver alcohol dehydrogenase activity. The total liver activity in castrated animals was 2.37 ± 0.229 (S.E.) $\text{mmoles}\text{hr}$ as compared with a value of 1.39 ± 0.125 $\text{mmoles}\text{hr}$ in sham-operated controls (P < 0.01). Testosterone administration partially suppressed the enhanced activity of liver alcohol dehydrogenase produced by castration. By contrast, in control animals testosterone administration resulted in a small paradoxical increase in liver alcohol dehydrogenase. The increase in the enzyme activity in castrated animals was associated with a parallel increase in the rate of ethanol elimination. Castrated and control animals showed decreases in free cytosolic and mitochondrial NAD⁺/NADH ratios after ethanol administration. These observations suggest that testosterone (and probably other as yet unknown factors modified by castration) affects liver alcohol dehydrogenase activity, and that the total enzyme activity can be a principal limiting factor in ethanol elimination.     

Influence of epinephrine on alcohol dehydrogenase activity in rat hepatocyte culture

The effects of epinephrine on alcohol dehydrogenase activity and on rates of ethanol elimination were determined in rat hepatocyte culture. Continuous exposure of the hepatocytes to epinephrine (10 microM) in combination with dexamethasone (0.1 microM) enhanced alcohol dehydrogenase activity on days 4-7 of culture, whereas neither hormone alone had an effect. The increased alcohol dehydrogenase activity was associated with an increased rate of ethanol elimination. Acute addition of 10 microM epinephrine to hepatocytes maintained in culture with 0.1 microM dexamethasone did not change alcohol dehydrogenase activity, but resulted in an immediate marked, but transitory, increase in ethanol elimination within the first 5 min after the addition of the hormone. Prazosin, an alpha 1-adrenergic blocker, and antimycin, an inhibitor of mitochondrial respiration, were powerful inhibitors of the transient increase in ethanol elimination, whereas 4-methylpyrazole was only partially inhibitory. These observations indicate that epinephrine has a chronic effect in increasing alcohol dehydrogenase activity and ethanol elimination and, also, an acute transient effect of increasing ethanol elimination which is not limited by alcohol dehydrogenase activity.     

Effect of testosterone on ethanol oxidation during chronic alcoholism in castrated rats

It has been shown in non-alcoholized male rats that castration significantly and appreciably raises the level of endogenous ethanol. In chronic alcoholization of castrated and non-castrated rats, the rate of ethanol elimination (REE) is noticeably increased, with testosterone producing no essential effect on the REE. In the liver, alcohol dehydrogenase activity rises insignificantly under the effect of testosterone, whereas aldehyde dehydrogenase activity declines 30--100%.     

Effect of ethanol concentration on rates of ethanol elimination in normal and alcohol-treated rats in vivo.

Ethanol was infused intravenously to yield in the blood concentrations between 30 and 40 mM (low dose) or 80 and 90 mM (high dose). Duplicate blood samples were taken every 30 min for gas Chromatographie determination of ethanol. Elimination curves for both low and high does of ethanol were linear in normal rats until ethanol concentrations reached values of less than 5 mM. At the low and high doses, average rates of ethanol elimination were 179 ± l and 266 ± 13 μmoles/g/hr respectively. The stimulation of ethanol metabolism due to the high dose did not diminish as the concentration declined. At both doses in both normal and ethanol—pretreated rats, elimination rates were diminished over 80 per cent by prior treatment with 4-methylpyrazole. Pretreatment with aminotriazole produced a 20–25 per cent decrease in the rate at the high dose in normal rats and at both doses in ethanol-pretreated rats, but had no effect at the low dose in normal rats. From these data we conclude that a concentration effect of ethanol on rates of ethanol elimination, which has both an alcohol dehydrogenase—and a catalase-H₂O₂-dependent component, exists in vivo. Moreover, the adaptive increase in ethanol elimination due to chronic pretreatment with ethanol also involves both components. Pyruvate and ethanol pretreatment stimulated ethanol elimination at the low but not at the high dose of ethanol. It is further concluded that NADH reoxidation is rate-limiting for ethanol utilization at the low dose whereas the activity of alcohol dehydrogenase becomes limiting at the high dose and after pretreatment with ethanol in the fed state in vivo.     

Role of the pituitary and neonatal androgenic imprinting in the hormonal regulation of liver alcohol dehydrogenase activity

Liver alcohol dehydrogenase activity is increased by thyroidectomy, orchidectomy, or hypophysectomy. We investigated the mechanisms of these hormonal effects by examining the effects of testosterone, dexamethasone and thyroid hormone on liver alcohol dehydrogenase activity in hypophysectomized rats and in cultured hepatocytes, and the effect of administration of androgens to neonatal female rats. Testosterone did not lower alcohol dehydrogenase activity in hypophysectomized rats, whereas dexamethasone and thyroxine produced moderate decreases in activity. Triiodothyronine reduced alcohol dehydrogenase activity of cultured hepatocytes from male and hypothyroid female rats in a dose-dependent fashion, confirming that thyroid hormone had pituitary-independent effects on the enzyme activity. Dexamethasone was required for the expression of alcohol dehydrogenase activity in cultured cells, and it increased the enzyme activity when present at supraphysiologic concentrations. Treatment of neonatal female rats with testosterone reduced the activity of the enzyme in adulthood. The difference in alcohol dehydrogenase activity in adult male and female rats appears to be determined in part by neonatal imprinting by androgens and in part by an effect of testosterone that is either mediated by or dependent upon the pituitary. Thyroid hormone reduces alcohol dehydrogenase activity by a direct effect on the liver.     

Effect of triiodothyronine on alcohol dehydrogenase and aldehyde dehydrogenase activities in rat liver. Implications for the control of ethanol metabolism

Treatment of rats with 20 micrograms of 3,3',5-triiodo-L-thyronine (T3) per 100 g body wt for a period of 6 days led to a 45% decrease in total liver alcohol dehydrogenase and a 36% decrease in total liver aldehyde dehydrogenase. Most of the latter decrease was directly attributable to a 57% fall in the level of the physiologically-important low Km mitochondrial isoenzyme. The high Km isoenzyme of the postmitochondrial and soluble fractions was much less affected by T3-treatment. T3, at concentrations up to 0.1 mM, did not inhibit the activity of aldehyde dehydrogenase in vitro. Despite these large losses of the two enzymes most intimately involved in ethanol metabolism, the rate of ethanol elimination in vivo was the same in T3-treated and control animals. Moreover, there was no difference between the two groups in the susceptibility of ethanol elimination to inhibition by 4-methylpyrazole, making it unlikely that an alternative route of ethanol metabolism had been significantly induced by treatment with T3. As it had been suggested that T3 might create a "hypermetabolic state" in which constraints normally imposed on alcohol dehydrogenase and aldehyde dehydrogenase are removed thereby compensating for any loss in total enzymic activity, 2,4-dinitrophenol (0.1 mmoles/kg body wt) was administered to rats in order to raise the general metabolic rate. However, the uncoupler proved to be lethal to T3-treated animals and did not stimulate ethanol elimination in controls. The results do not support the notion that ethanol elimination in vivo is normally governed either by the level of alcohol dehydrogenase or by that of hepatic aldehyde dehydrogenase. However, the mode of control remains unclear.     

Effect of a low-protein diet on acetaldehyde metabolism in rats

The effect of dietary changes on liver alcohol and aldehyde dehydrogenase activities as related to effects on ethanol and acetaldehyde metabolism was investigated. Feeding rats for 8 weeks on diets rich in carbohydrate or fat, but with normal protein content, induced minor changes relative to giving a balanced diet. A low-protein, high-carbohydrate diet (5 per cent and 80 per cent of calory content, respectively) caused a significant reduction of both alcohol and aldehyde dehydrogenase activities in the liver. The activity of the high-Km aldehyde dehydrogenase in the microsomal and soluble fractions appeared to be more reduced than that of the low-Km enzyme in the mitochondrial fraction. The tail blood acetaldehyde was significantly higher in rats on the protein deficient diet in spite of their reduced ethanol elimination rates. The results suggest that protein deficiency deranges acetaldehyde metabolism and may thus increase the possible contribution of acetaldehyde to the effects caused by ethanol metabolism.     

Castration and tolerance induces changes in the levels of the activity of acetylcholinesterase in the isolated vas deferens of the rat

Changes in the activity of acetylcholinesterase (AChE) of the isolated vas deferens from normal, castrated, morphine and ethanol-tolerant rats were studied. Three days after the termination of treatment with morphine and on the last day of treatment with ethanol, a significant inhibition of the activity of AChE was detected. This reduction in the enzymatic activity persisted in morphine-tolerant rats for 15 days, but not for 30 days, at which time the levels of AChE were determined to be normal. However, in ethanol-tolerant rats, there were no significant changes found at days 15 or 30. The activity of AChE was decreased significantly in castrated rats, but this effect was reversed by treatment with testosterone. During withdrawal from morphine or ethanol, the levels of AChE were significantly increased. The results indicate that morphine and ethanol may be inducing changes in the feedback mechanism which regulates the levels of AChE at post-synaptic sites, and these changes could play an important role in the development of tolerance to morphine and to ethanol.     

Effect of epinephrine on ethanol metabolism by isolated rat hepatocytes

The effect of epinephrine on ethanol metabolism was determined in isolated rat hepatocytes. Epinephrine (10 microM) enhanced an initial rapid rate of ethanol elimination observed in the first 5 min. Thereafter, between 5 and 90 min, the rate of ethanol elimination was slower and not affected by epinephrine. Epinephrine resulted in higher acetaldehyde concentrations at 2 min, but not thereafter. Acetaldehyde production in the presence and absence of epinephrine was inhibited by 4-methylpyrazole, by a low free extracellular calcium concentration, and by the alpha 1-adrenergic blocker prazosin. Ethanol alone and epinephrine alone increased oxygen consumption, but the effects were not additive. The ethanol-induced decreases in the cytosolic NAD-/NADH and NADP++NADPH ratios and in the mitochondrial NAD+/NADH ratio were delayed by the presence of epinephrine. An accelerated initial alcohol dehydrogenase activity sufficient to account for the rapid initial rate of ethanol elimination shown with epinephrine was demonstrated by coupling ethanol oxidation with lactaldehyde reduction, a system which increases the rate of dissociation of NADH from the enzyme and its oxidation back to NAD+. The findings in this study indicate that an increased reoxidation of NADH during ethanol oxidation by alcohol dehydrogenase is the basis for the rapid transient increase in ethanol elimination produced by epinephrine.     

Influence of the Diet on the Metabolism of Acetaldehyde in Rats

Abstract The acetaldehyde dehydrogenases of rat liver, the hepatic output of acetaldehyde and the rate of ethanol elimination were studied in two groups of female Sprague-Dawley rats fed on two different commercial standard diets (diet 1 and diet 2). The activity of the mitochondrial low-K_m enzyme was 2–3 times lower in rats fed on diet 1 as compared to rats fed on diet 2, whereas only slight differences were found in the activities of the high-K_m enzymes in the mitochondrial, the microsomal and the cytosol fractions. No differences were observed between the two groups in the activities of alcohol dehydrogenase, glutamate dehydrogenase, glucose-6-phosphatase or malic enzyme. The rate of ethanol elimination was 15 % lower, and the acetaldehyde level in the blood was 2–4 times higher in rats fed on diet 1 as compared to rats fed on diet 2. When the diets were interchanged between the two groups, the activity of the low-K_m enzyme increased or decreased twofold within 12 hrs. Starvation increased the activity of the low-K_m enzyme in rats fed on diet 1, but had no effect on the activity in rats fed on diet 2. The results suggest that diet 1 contains an inhibitor of the low-K_m enzyme, and that dietary factors may be of importance in the regulation of the hepatic output of acetaldehyde during ethanol metabolism.     

Rate-determining factors for ethanol metabolism in fasted and castrated male rats

The effects of castration and fasting upon the alcohol elimination rate, liver alcohol dehydrogenase (LADH) maximum activity (V_max), and hepatic concentrations of ethanol, acetaldehyde, and free NADH during ethanol oxidation were examined in male Wistar rats. Castration increased the V_max of LADH and, to a lesser extent, the alcohol elimination rate in vivo. On the other hand, fasting reduced the V_max of LADH and the alcohol elimination rate in sham-operated and castrated rats but it did not nullify the effect of castration. Castration produced small but significant changes in the hepatic concentrations of ethanol, acetyldehyde and free NADH in fed rats during ethanol oxidation. Fasting also caused significant increases in the concentration of free NADH during alcohol oxidation in both the sham-operated and castrated groups. The ratio of the steady-state velocities of LADH in situ to the maximum velocities of LADH ($\text{ν}\text{V}{}_{\mathrm{<mtext>max</mtext>}}$) under the different experimental conditions was calculated by using the steady-state rate equation for the enzyme mechanism of rat LADH and its kinetic constants. The calculated $\text{ν}\text{V}{}_{\mathrm{<mtext>max</mtext>}}$ ratios were 50–62%, indicating that LADH activity was limited to about the same extent by its substrates and products under these conditions and that the changes in alcohol elimination rates produced by fasting and castration mainly reflected changes in the V_max of LADH. The calculated steady-state velocities in situ (ν) were 14–28% lower than the measured rates of alcohol elimination in vivo. The extent of agreement is probably acceptable in view of the assumptions needed to determine the free NADH concentration in liver and the existence of non-LADH-related processes for alcohol elimination in vivo.     

Acute interaction of halothane and enflurane with the metabolism of ethanol in isolated hepatocytes and liver cytosol preparations from the rat

The effects of halothane and enflurane on ethanol (40 mM) oxidation were studied in isolated rat hepatocytes. Anaesthetic (halothane, enflurane and diethyl ether) effect on the activity of alcohol dehydrogenase (ADH) was studied in incubations of cytosol preparations from rat liver. Mean rates of ethanol metabolism ranged from 0.44 to 0.49 mumol ethanol metabolized/mg cell protein/hour in control hepatocytes from fasted and fed animals. These rates were enhanced by 2- and 3-fold in hepatocytes from fed and fasted animals, respectively, when pyruvate (5 mM) was added. Halothane and enflurane both caused dose dependent inhibition of ethanol metabolism (15-40%) in all hepatocytes without exogenous addition of pyruvate. The inhibitory effect was present also after pyruvate stimulation in hepatocytes from fasted animals, but disappeared in hepatocytes from fed animals when pyruvate was added. The rate of ethanol oxidation by cells from fed rats was enhanced by approximately 40% when the concentration of ethanol was increased from 20 mM to 80 mM. The anaesthetic inhibition of ethanol metabolism was about 20% more pronounced at the higher ethanol concentration compared to the lower concentration when no pyruvate was added. In the presence of pyruvate the effect of anaesthetics was again reversed regardless of ethanol concentration. Halothane (2 mM) and enflurane (2 mM) both caused about 25% inhibition of the ADH-activity in cytosol preparations while ether (30 mM) caused more than 50% inhibition. No inhibition of hepatocyte uptake of ethanol was caused by any of the three anaesthetics.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of long-term ethanol treatment on aldehyde and alcohol dehydrogenase activities in rat liver.

Administration of intoxicating doses of ethanol by gavage for 3 weeks caused weight loss and reduced hepatic aldehyde dehydrogenase activity in the soluble, mitochondrial and microsomal fractions. Rats receiving equivalent amounts of ethanol as a constituent of a liquid diet for 5 weeks gained weight and showed no changes in aldehyde dehydrogenase activity. Alcohol dehydrogenase activity was decreased in the rats treated by gavage and unchanged in those given ethanol in the diet, but in spite of this the rate of ethanol elimination was accelerated in both groups. In the livers of two strains of rats genetically selected for their difference in voluntary alcohol consumption, the mitochondrial and microsomal aldehyde dehydrogenase activities had previously been shown to be significantly higher in the alcohol-consuming (AA) than in the alcohol-avoiding (ANA) rats. Similar differences were now found after long-term intragastric ethanol administration, although in both strains the absolute levels of aldehyde dehydrogenase were reduced. Profound reduction of mitochondrial low-K_m aldehyde dehydrogenase activity and high blood acetaldehyde were observed, especially in the ANA rats. This suggests a possible connection between the low activity of this enzyme and the increased acetaldehyde level.     

Disulfiram enhances ethanol pharmacological activity and impairs its elimination

Disulfiram or diethyldithiocarbamate (DDC) significantly prolonged ethanol-induced loss of righting reflex in mice. The disappearance of ethanol from blood, and brain was significantly delayed in disulfiram-treated animals, suggesting an impairment in the activity of alcohol dehydrogenase in these animals. DDC, an active metabolite of disulfiram, inhibited mouse liver alcohol dehydrogenase (LADH) in vitro. Pyrazole, a known inhibitor of alcohol dehydrogenase, affected ethanol elimination and ethanol-induced loss of righting reflex in mice in a manner similar to that seen with disulfiram.     

Effect of castration on the turnover of rat liver alcohol dehydrogenase

Castration increased liver alcohol dehydrogenase activity and enzyme protein in male rats. The turnover of alcohol dehydrogenase determined from the decline in radioactivity present in immunoprecipitated enzyme after injection of NaH¹⁴CO₃ was decreased after castration. The fractional rate of degradation (K_d) for the enzyme was 0.11 · day⁻¹ in the castrated as compared with 0.13 · day⁻¹ in the control animals (P < 0.05). The fractional rate of synthesis (K_s) of the enzyme was not affected by castration, while the absolute rate of synthesis was increased slightly. This study shows that a decrease in the rate of degradation is the principal cause for the increase in liver alcohol dehydrogenase following castration.     

Quantitative correlation of ethanol elimination rates in vivo with liver alcohol dehydrogenase activities in fed, fasted and food-restricted rats.

The effects of nutritional states upon liver alcohol dehydrogenase (ADH) activity and ethanol elimination rate in vivo have been examined in the rat. Male Sprague-Dawley rats, 250–280 g, were studied in the fed state, after fasting for 24, 48 and 72 hr, and after 9 days of food restriction (5g food/day). Total ADH activity per liver or per animal (2.20 m-moles/hr in fed rats) decreased after a 24-hr fast and was 1.32 and 0.94 m-moles/hr after a 48-hr fast and food restriction respectively. Cytosolic protein and liver wet weight decreased in parallel with total ADH activity, but DNA content exhibited only a 10% decrease with fasting and a 20% decrease with food restriction. Ethanol elimination rate in vivo per animal after intraperitoneal injection of 2g ethanol/kg was 1.92, 1.14 and 0.84 m-moles/hr in the fed, 48 hr-fasted and food-restricted rats, respectively. These data indicate that the decrease in the ethanol elimination rate with fasting and food restriction may be caused by decreasing ADH activity, since the cytosolic free NAD⁺/ NADH in liver after acute administration of alcohol in vivo has been reported to be nearly identical in the fed and 48 hr-fasted rats. The close agreement between liver ADH activity and ethanol elimination rate in vivo suggests that the total enzymatic activity of liver ADH is an important rate-limiting factor in ethanol metabolism under the nutritional conditions examined.     

Cholinesterase activity in the plasma and acid-secretory gastric mucosa of normal, castrated, and castrated oestrogen-treated male rats

Cholinesterase activity in plasma and fundic mucosa of male, normal, castrated, and castrated and oestrogen-treated rats has been measured. Castration significantly elevated cholinesterase activity in both plasma (0.02<P<0.05) and fundic mucosa (P<0.001). Castrated rats given 17β-oestradiol (120 μg, s.c. daily for 10 days) had enzyme activity in plasma and fundus not significantly different from that in normal rats.     

吲哚-3-原醇对乙醇损伤性大鼠肝切片的保护作用

目的　采用精密肝切片技术 ,研究十字花科类蔬菜提取物吲哚 3 原醇 (I3C)对乙醇肝损伤的作用及机制。方法　制作大鼠乙醇损伤肝切片模型 ,观察不同剂量I3C对培养液中肝损伤标志酶及肝细胞浆苯胺羟化酶 (ANH)、乙醇脱氢酶 (ADH)活性的影响 ,并进行组织学检查。结果　乙醇5 0mmol·L-1作用肝切片 4h时 ,培养液谷丙转氨酶、谷草转氨酶、乳酸脱氢酶和谷胱甘肽S 转移酶活性明显升高 ,同时肝细胞浆ANH活性升高、ADH活性降低 ;加入 10 0～ 4 0 0μmol·L-1的I3C后 ,培养液中各酶活性降低的同时 ,肝细胞ANH和ADH活性恢复正常。肝切片病理学检查也证实I3C的保护作用。结论　I3C能有效拮抗乙醇所致的肝损伤 ,其机制与改变乙醇代谢途径有关。     

Ethanol oxidation by isolated hepatocytes from fed and starved rats and from rats exposed to ethanol,phenobarbitone or 3-amino-triazole: No evidence for a physiological role of a microsomal ethanol oxidation system

In isolated hepatocytes from fed and starved rats, basal rates of ethanol oxidation were 1.15 and 0.71 μmoles/g wet wt, respectively, and were unchanged over the ethanol concentration range 8–96 mM. The addition of 4-methyl pyrazole (4 mM), a competitive inhibitor of alcohol dehydrogenase, largely abolished ethanol oxidation from 8mM ethanol, while at an ethanol concentration of 96 mM, the oxidation rate was inhibited by 87 per cent. Pyrazole was a less effective inhibitor of alcohol oxidation than 4-methyl pyrazole. In hepatocytes isolated from rats treated with ethanol, phenoharbitone or 3-amino-triazole, basal rates of ethanol oxidation were the same at ethanol concentrations of 8–96 mM and the rates were similar to, and never exceeded, the rate found in hepatocytes from normal fed rats. 4-Methyl pyrazole inhibited ethanol oxidation to the same extent in all liver cell preparations. regardless of the treatment the donor animal had received. Pyruvate stimulated cellular ethanol oxidation irrespective of the prior treatment of the donor animal. This stimulation, together with the ethanol-induced accumulation of lactate, was abolished by 4-methyl pyrazole. This suggests that the capacity for alcohol oxidation in isolated liver cells is generally limited by the lack of suitable acceptors for the hydrogen generated in the cytoplasm by the alcohol dehydrogenase-catalysed oxidation of ethanol to acetaldehyde. Methylene blue, phenazine methosulphate and menadione stimulated both ethanol oxidation and respiration, irrespective of the prior treatment of the donor animal. This enhancement of ethanol oxidation and respiration was prevented by 4-methyl pyrazole. These artificial electron acceptors appear to act by circumventing normal pathways for the oxidation of cytoplasmic NADH generated in the conversion of ethanol to acetaldehyde. In cells from each treatment group, antimycin was more effective than rotenone as an inhibitor of ethanol oxidation; inhibition of ATP formation by oligomycin had least effect on alcohol oxidation. Ethanol oxidation by cells from alcohol-treated rats was most affected by these inhibitors of mitochondrial respiration. These results indicate that under a wide variety of experimental conditions the contribution of the postulated microsomal ethanol oxidizing system to ethanol oxidation in isolated, intact liver cells appears minimal. Thus they cast doubt on a physiological role for this system in vivo.     

Action of metadoxine on isolated human and rat alcohol and aldehyde dehydrogenases. Effect on enzymes in chronic ethanol-fed rats

Metadoxine (pyridoxine-pyrrolidone carboxylate) has been reported to accelerate ethanol metabolism. In the present work we have investigated the effect of metadoxine on the activities of isolated alcohol and aldehyde dehydrogenases from rat and man, and on the activity of these enzymes in chronic ethanol-fed rats. Our results indicate that in vitro metadoxine does not activate any of the enzymatic forms of alcohol dehydrogenase (classes I and II) or aldehyde dehydrogenase (low-Km and high-Km, cytosolic and mitochondrial). At concentrations higher than 0.1 mM, metadoxine inhibits rat class II alcohol dehydrogenase, although this would probably not affect the physiological ethanol metabolism. Chronic ethanol intake for 5 weeks results in a 25% decrease of rat hepatic alcohol dehydrogenase (class I) activity as compared with the pair-fed controls. The simultaneous treatment with metadoxine prevents activity loss, suggesting that the positive effect of metadoxine on ethanol metabolism can be explained by the maintenance of normal levels of alcohol dehydrogenase during chronic ethanol intake. No specific effect of chronic exposure to ethanol or to metadoxine was detected on rat aldehyde dehydrogenase activity.