On the Disulfiram-Like Effect of Coprine,the Pharmacologically Active Principle of Coprinus atramentarius

Abstract Coprine or disulfiram was given to rats in various doses at various time intervals before the administration of 2 g/kg ethanol. The ratio acetaldehyde/ethanol in the alveolar air was measured by gas chromatography and was taken as an index of the aldehyde dehydrogenase (ALDH) activity. The activity of dopamine β-hydroxylase (DBH) was estimated in the same animals by measuring the amount of ¹⁴C-octopamine formed from ¹⁴C-tyramine in the heart. Coprine and disulfiram both caused an increase in the acetylaldehyde/ethanol ratio, coprine being more potent than disulfiram. Disulfiram, but not coprine, reduced the net yield of ¹⁴C-octopamine. In rats pretreated with either coprine or disulfiram, blood-pressure and heart-rate were recorded before and after intraperitoneal injections of 0.4 g/kg ethanol. In both cases ethanol caused a marked and rapid fall in blood-pressure. However, this effect was accompanied by tachycardia only in animals treated with coprine. It is concluded that coprine like disulfiram inhibits ALDH, but only disulfiram causes an additional inhibition of DBH. This difference may account for differences in the cardiovascular response to ethanol     

Effects of Phenobarbital and Ethanol on Rat Liver Aldehyde Dehydrogenases

The aldehyde dehydrogenases and the drug-metabolizing system (the mixed function oxidase) were studied in Wistar rats treated with ethanol and phenobarbital. Phenobarbital treatment had no effect on the microsomal aldehyde dehydrogenase activity, whereas the mitochondrial aldehyde dehydro-genase activity was slightly decreased and the activities of ethylmorphine demethylase, NADPH-cytochrome c reductase and the concentration of cytochrome P-450 were increased two to threefold. Ethanol treatment caused no changes in the activities of the aldehyde dehydrogenases, and the drug metabolizing system was only slightly affected. Very little aldehyde dehydrogenase activity was found in the cytosol from livers of either control, ethanol-treated or phenobarbital-treated rats. The results are discussed with particular reference to the oxidation of acetaldehyde and other aldehydes during ethanol metabolism.     

Disulfiram as a Tool in the Studies on the Metabolism of Acetaldehyde in Rats

Abstract The liver acetaldehyde dehydrogenases and the acetaldehyde level in the blood during ethanol metabolism were studied in rats 24 hrs after the administration of disulfiram. High doses of disulfiram (150–600 mg/kg) caused a threefold decrease in the activity of the mitochondrial low-K_m enzyme, whereas no significant effects were found on the activity of the high-K_m enzymes present in the mitochondrial, the microsomal and the cytosolic fractions. The concentration of acetaldehyde was threefold higher in the hepatic venous blood and fivefold higher in the peripheral blood in rats given disulfiram compared to rats given ethanol only. Low doses of disulfiram (25–50 mg/kg) decreased the activity of the low-K_m enzyme by 26 %, and caused a significant increase in the liver output of acetaldehyde. The rate of ethanol elimination decreased by 35 % at a high dose of disulfiram, whereas the alcohol dehydrogenase activity was not influenced. It is suggested that the mitochondrial low-K_m enzyme has a primary role in the regulation of the hepatic output of acetaldehyde, and the results will be discussed with special reference to the site and kinetics of acetaldehyde oxidation during ethanol metabolism in rat liver.     

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.     

Inhibition of the Acetaldehyde Dehydrogenases in Rat Liver by a Cyanamide Derivative present in a Commercial Standard Diet for Small Animals

Abstract The inhibition of the acetaldehyde dehydrogenase activity in rat liver by an unknown dietary factor, present in a commercial standard diet for rats, was studied. The inhibitory factor was located in the calcinated bonemeal fraction of the diet and was identified by infrared spectrophotometry and thin–layer and paper chromatography as cyanamide or a cyanamide derivative. The occurrence and formation of cyanamide in calcinated bonemeal, its effects on the acetaldehyde dehydrogenases and the metabolism of acetaldehyde in rat liver, are discussed.     

A Comparative Study on the Effects of Disulfiram,Cyanamide and 1-Aminocyclopropanol on the Acetaldehyde Metabolism in Rats

Abstract 1-aminocyclopropanol (ACP) is a potent inhibitor of aldehyde dehydrogenase (ALDH) in vivo and in vitro. Like cyanamide it has a rapid onset of action in vivo with the highest inhibition occurring after 2-24 hrs. and a long duration of action like disulfiram with measurable inhibition after 144 hrs. All the three inhibitors decreased the activity of the mitochondrial low-K_m ALDH strongly in vivo, however, in markedly different doses. Cyanamide inhibited the high-K_m ALDH only in vivo, whereas in vitro, the high-K_m ALDH was unaffected by cyanamide but significantly inhibited by disulfiram and ACP. The inhibition produced by the inhibitors appeared to be irreversible. Acetaldehyde protected the low-K_m enzyme at different extents depending on the inhibitor used. The inhibition of ALDH in intoxicated and control rats and its relation to acetaldehyde oxidation and the disulfiram-ethanol reaction are discussed.     

Placental and Foetal Metabolism of Acetaldehyde in Rat I. Contents of Ethanol and Acetaldehyde in Placenta and Foetus of the Pregnant Rat during Ethanol Oxidation

Abstract: Following the administration of ethanol to pregnant rats, the ethanol content of the maternal aortic blood was compared with that of the intact placenta and whole foetuses. The results support previous observations that ethanol is present in the placenta and foetus in about the same amount as in the maternal circulation. In contrast, only 25 % of the acetaldehyde content present in the maternal aortic blood could be found in the intact placenta and no acetaldehyde was found in the intact foetal tissue. These findings suggest that during maternal ethanol oxidation there is no accumulation of acetaldehyde in the foetal tissue.     

Genetic Variation of Drug–Metabolizing Enzymes in the Wistar Rat

Abstract Rats of the Wistar/Af/Han/Mol/(Han 67) strain were preselected for the inducibility of the hepatic soluble aldehyde dehydrogenase, after treatment with phenobarbital (80 mg/kg daily, intraperitoneally, for 4 days) and open liver biopsy. Animals with highly induced aldehyde dehydrogenase also had a 50 and 40 % difference in the induction of D–glucuronolactone dehydrogenase and NADPH–cytochrome c reductase respectively. Other enzymes of the hepatic drug hydrox–ylation and glucuronidation did not show significant variation in the induction pattern between the two genetic groups. After inbreeding, the second filial generation was used in order to determine enzyme activities without any pretreatment. The basal activities of aldehyde and D–glucuronolactone dehydrogenase, but not of NADPH–cytochrome c reductase, were also different in the untreated animals. Urinary excretion of D–glucaric acid was the same in the two groups, both in the basal and the induced state.     

Inducible Aldehyde Dehydrogenases in the Hepatic Cytosol of the Rat

Abstract Rats of the Wistar/Af/Han/Mol/(Han 67) strain have previously been shown to respond in a variable way to phenobarbital treatment, as far as the induction of aldehyde dehydrogenase activity is concerned ( Marselos 1976 ). This biochemical property is genetically determined and concerns the high-Km aldehyde dehydrogenase of the hepatic cytosol. In this study, administration of phenobarbital (1 mg/ml of drinking water, for 1 week) produces a uniform induction of aldehyde dehydrogenase in all rats, when measured with micromolar substrate concentration. The inducible low-Km enzyme of the cytosol is not genetically determined like the high-Km enzyme, and shows a wide specificity for aliphatic as well as for aromatic aldehydes. Despite the inducibility of the cytosolic enzymes, no alterations are found in the mitochondrial aldehyde dehydrogenase activities after phenobarbital treatment. The oxidation of D-glucuronolactone takes place only in the cytosol, and seems to be dependent on the low-Km aldehyde dehydrogenase. This is consistent with NMR studies, which showed that a very minimal amount of D-glucuronolactone is in aldehyde form under the measurement conditions usually applied. Further, the oxidation of D-glucuronolactone is also enhanced by phenobarbital in all rats without a genetic predisposition, and its dose-response curve is very similar to that of the low-Km aldehyde dehydrogenase.     

In vitro Metabolism of Propoxyphene in Rat Liver: Reaction of a Carbinol Metabolite with Acetaldehyde

Abstract: The metabolism of the analgesic drug propoxyphene (α-d-propoxyphene) has been investigated in the rat liver 9,000Xg supernatant fraction. The incubations were analyzed by HPLC. The major metabolite was norpropoxyphene carbinol, obtained through demethylation and ester hydrolysis. The demethylated metabolite of propoxyphene, norpropoxyphene, was also detected. Addition of acetaldehyde to the incubation mixture decreased the metabolism of propoxyphene. Reactions between norpropoxyphene carbinol and acetaldehyde resulted in a fast disappearence of the carbinol and the formation of a reaction product, the significance of which is discussed.     

On the Aromatic Hydroxylation of Amphetamine in Rat Liver Microsomes and Perfused Liver Preparations: Effects of Long-term Administration

Abstract The liver microsomal p-hydroxylation of amphetamine to parahydr-oxyamphetamine (pOHA) was dependent on NADP and inhibited by carbon monoxide indicating the involvement of cytochrome P-450. SKF 525-A, fenfluramine and desmethylimipramine were the most effective inhibitors of this pathway of amphetamine metabolism. Repeated administration of phenobarbital resulted in reduced p-hydroxylation of amphetamine in vitro. Chronic administration of amphetamine reduced the microsomal p-hydroxylation of amphetamine without apparent changes in the cytochrome P-450 levels or in the activity of NADPH-cytochrome c reductase. The aromatic hydroxylation of aniline and the demethylation of ethylmorphine was not affected by this treatment. However, the 455 nm complex formed during the microsomal metabolism of N-hydroxy-amphetamine was increased by the long-term administration of amphetamine. These results indicate some pecularities of the in vitro hydroxylation of amphetamine by rat liver microsomes. Amphetamine disappeared from the perfusate of the perfused liver at the same rate in rats given a single dose of amphetamine and in rats given amphetamine orally for four weeks. The excretion of pOHA and its conjugate increased at 60 and 90 min. and 30, 60 and 90 min. respectively in the perfusate of the same experiment as compared to the controls. The total excretion of radioactive amphetamine metabolites at the end of the perfusion was increased in the perfusate and reduced in the bile compared to the control experiment.     

Electrical stimulation and lesions of the medial forebrain bundle of the rat: Changes in voluntary ethanol consumption and brain aldehyde dehydrogenase activity

Repeated sessions of electrical stimulation or lesions in the ventral aspect of the medial forebrain bundle (VMFB) region of the brain in rats resulted in a significant increase and a decrease in voluntary ethanol (10% v/v) intake, respectively. Whole brain and midbrain-diencephalon (MB-DE) aldehyde dehydrogenase (ALDH) were measured in different groups of experimental and control animals before, immediately after, and 30 days after the termination of the stimulation regimen, or 8 days and 30 days after the induction of the lesions. By the end of the stimulation regimen, the levels of MB-DE ALDH of the experimental (ethanol-drinking: stimulated) animals were significantly higher than those of control animals (ethanol-drinking: nonstimulated, water-drinking: stimulated, and water-drinking: nonstimulated). A marked decrease in MB-DE ALDH activity was noted in lesioned animals but not in cyanamide-treated or in implanted control animals. Neither the stimulation nor the lesions had any demonstrable effect on whole brain ALDH activity. Cyanamide administration caused a pronounced decrease in ethanol intake and in levels of liver ALDH activity. The increase in MB-DE ALDH activity in the ethanol-drinking, stimulated animals was attributed to the interaction between the VMFB activation and the ethanol drinking, while the reduction in ALDH activity was attributed to the degeneration of biogenic-amine-containing nerve fibers.     

Age–Dependent Effects of Ethanol on Central Monoamine Synthesis in the Male Rat

Abstract: In the present study, the effect of ethanol on central monoamine synthesis in developing and adult male rats was studied by measuring the accumulation of DOPA and 5–hydrotryptophan after inhibition of aromatic amino acid decarboxylase. Before adolescence, ethanol caused a decrease of DOPA accumulation in the whole rat brain, while after adolescence ethanol increased the DOPA synthesis. Ethanol had no effect on the serotonin synthesis at any of the ages studied. It is suggested that hormonal events occuring during puberty may be of importance for the stimulatory properties of ethanol observed at adult age     

Development of Alcohol Dehydrogenase and Aldehyde Dehydrogenases in the Offspring of Female Rats Chronically Treated with Ethanol

Abstract The influence of maternal ethanol drinking on the development of alcohol dehydrogenase and aldehyde dehydrogenases in the liver of the offspring was studied in Wistar rats. No statistically significant differences were found between the ethanol group and the control group.     

Aldehyde toxicity and metabolism: the role of aldehyde dehydrogenases in detoxification,drug resistance and carcinogenesis

Aldehydes are carbonyl compounds found ubiquitously in the environment, derived from both natural and anthropogenic sources. As the aldehydes are reactive species, therefore, they are generally toxic to the body. To reduce the toxicity and pathogenesis related to aldehydes, the human body contains several aldehyde metabolizing enzyme systems including aldehyde oxidases, cytochrome P450 enzymes, aldo-ketoreductases, alcohol dehydrogenases, short-chain dehydrogenases/reductases and aldehyde dehydrogenases (ALDHs). These enzyme systems maintain a low level of aldehydes in the body by catalytically converting them into less-harmful and easily excreted products. The human ALDH (hALDH) superfamily consists of 20 functional ALDH genes identified so far at distinct chromosomal locations, expressing 20 ALDH proteins, which belong to 11 different ALDH families. They are involved in the NAD(P)⁺-dependent oxidation of a wide range of exogenous and endogenous aldehydes to their corresponding carboxylic acids. The hALDHs are present in all sub-cellular locations and have a wide tissue distribution. This review gives an account of aldehydes; their source, toxicity and metabolism, different aldehyde metabolizing enzymes with special emphasis on ALDHs including their biochemical, physiological and pathophysiological roles in the body.     

Placental and Foetal Metabolism of Acetaldehyde in Rat II. Studies on Metabolism of Acetaldehyde in the Isolated Placenta and Foetus

Abstract: The aldehyde-oxidizing capacity of placenta and foetal liver from the rat was studied using acetaldehyde as substrate. In the placenta the activity was approximately 8 % and in foetal liver 49 % of that found in the liver of adult animal. The placenta seems to prevent most of the acetaldehyde found in maternal blood during ethanol oxidation from entering the foetal circulation.     

In Vitro and in Vivo Metabolism of the Anti-Cancer Agent CI-1040, a MEK Inhibitor, in Rat, Monkey, and Human

PURPOSE: The use of in vitro and in vivo models using both rodent and non-rodent species plays an important role with regard to metabolism during the drug development process. In this study, we compared the metabolism of a MEK inhibitor (CI-1040) using in vitro and in vivo models with that observed in a cancer patient. METHODS: Radiolabeled CI-1040 was assessed for metabolism using rat and monkey liver microsomes and hepatocytes, as well as in Wistar rats and cynomolgus monkeys via oral administration. Human bile and plasma samples were obtained immediately after administration of CI-1040 to a patient with advanced colon cancer. A combination of HPLC-radiochromatography (HPLC-RAM), LC/MS and LC/MS/MS experiments were used to analyze all resulting metabolites. Unlabeled CI-1040 was administered (100 mg/day, QD) for 15 days to a patient suffering from colon cancer. Bile was collected by the insertion of a T-tube directly into the bile duct over a 14-h period. Metabolites were also monitored in the patient's plasma. RESULTS: Analysis of the metabolites in all species using in vitro and animal models demonstrated that CI-1040 undergoes extensive oxidative metabolism (14 metabolites identified) with subsequent glucuronidation of the hydroxylated metabolites. Metabolites were predominantly excreted through the bile in the animal models. CONCLUSIONS: Overall, the in vitro and animal models in combination provided comprehensive coverage for all metabolites observed in human bile and plasma. In conclusion, the results obtained in this study demonstrate the utility of conducting investigations across species in order to gain complete coverage for successfully predicting human metabolites of new compounds in development.     

二甲双胍对糖尿病大鼠肝脏葡萄糖-6-磷酸酶表达的影响

目的：探讨二甲双胍抑制肝糖输出，减轻肝脏胰岛素抵抗的分子学机制。方法：建立糖尿病大鼠模型并将之分为正常对照组、糖尿病组、低剂量二甲双胍干预组、高剂量二甲双胍干预组，饲养2周后取出肝脏，以半定量RT-PCR方法测定肝脏葡萄糖-6-磷酸酶（G6Pase）mRNA的表达，蒽酮比色法测定肝脏糖原含量。结果：（1）糖尿病组肝脏G6Pase催化亚基（P36）及转运亚基（P46）mRNA的灰度高于正常对照组（P〈0．05），而肝脏糖原含量低于正常对照组（P〈0．05）。（2）二甲双胍可以明显减少肝脏G6Pase mRNA表达并增加肝糖原含量（P〈0．05），且呈剂量依赖性。结论：二甲双胍能够抑制糖尿病状态下肝脏G6Pase催化亚基及转运亚基mRNA表达，使得6-磷酸葡萄糖向葡萄糖的转化减少，从而减少肝糖输出，达到减轻胰岛素抵抗的作用。     

Uptake of Nanoparticles by Rat Glomerular Mesangial Cells in Vivo and in Vitro

Glomerular mesangial cells play a major role in the structure of capillary loops, generation of mediators of inflammation, and uptake of macromolecules. We demonstrate here that isobutylcyanoacrylate nanoparticles loaded with actinomycin D (ADNP) concentrate in rat mesangial cells in vitro and in vivo, as compared to the free drug (AD). In normal rats injected with 20 µg of ³H-ADNP or ³H-AD, the uptake ratios ³H-ADNP/³H-AD measured in whole kidneys at 30 and 120 min were 2.2 ± 1.0 and 2.3 ± 0.9, respectively. The same ratios calculated for isolated rat glomeruli and tubules, were 4.1 ± 0.5 and 0.8 ± 0.2 at 30 min, and 2.6 ± 0.5 and 0.6 ± 0.3 at 120 min, respectively. In the glomeruli, the absolute uptake of ³H-ADNP corresponded to 7.5 (30 min) and 1.8 (120 min) % I.D./100 mg of protein. In rats with experimental glomerulonephritis, the uptakes of ³H-ADNP and ³H-AD by the glomeruli were 6.9 and 4.0 times higher than in normal rats, respectively. In vitro experiments demonstrated up to 5 times higher uptake by glomerular mesangial cells than by epithelial cells. Uptake was maximum after 60 min, higher at 37°C than at 4°C, dependent on the presence of fresh serum and inhibited by cytochalasin-B. Drug targeting by nanoparticles is thus possible to renal cells involved in inflammatory processes, especially mesangial cells and macrophages. Nanoparticles could also be useful for lowering drug concentration in tubular cells, to reduce any tubular toxicity. Targetting of the mesangial cells is of particular interest for drugs such as corticosteroids capable of reducing glomerular inflammation in various pathological conditions.     

Kinetic Studies of Dose-Dependent Metabolism of Alprenolol: In Vitro and In Vivo Studies in Different Species

Abstract Kinetic studies of the metabolism of alprenolol were performed with isolated microsomes from the rat, guinea-pig, dog and man at an initial substrate concentration of 0.17-150 μM. In all species the rate of aromatic hydroxylation reached a plateu above 50 μM of alprenolol in contrast to the rate of desisopropylation, where consistent saturation level was not obtained. The Km-values for the aromatic hydroxylation in the guinea-pig and man, 2 ,7 μM and 1.3 μM respectively, showed no concentration dependency in contrast to the rat (Km₁ = 0.20 μM, Km₂ = 26 μM) and the dog (Km₁ = 0.78 μM, Km₂ = 66 μM). The apparent Km-value of 0.20 μM for aromatic hydroxylation in the rat seemed to be of the same order of magnitude as reported spectral dissociation constant (Ks = 0.34 μM). In vivo experiments in the rat by oral administration of 7-700 μmol/kg demonstrated a dose-dependent presystemic elimination of alprenolol. The urinary excretion of hydroxy-alprenolol was significantly lower after the highest dose. It is proposed, that the saturation of the aromatic hydroxylation, catalyzed by a high affinity site or subspecies of cytochrome P-450 with a low capacity. contributes to the dose-dependent kinetics in vivo.