Modifications of drug metabolism by disulfiram and diethyldithiocarbamate. I. Mixed-function oxygenase.

Disulfiram and diethyldithiocarbamate were administered to rats for 4 days alone (300 mg/kg, daily, per os) or in combination with phenobarbital (80 mg/kg, daily, i.p.), in order to observe the effects of these compounds on the microsomal membrane components and on the mixed-function oxygenase system. Both disulfiram and diethyldithiocarbamate increased the liver to body weight ratio, and the total hepatic protein content. Disulfiram significantly increased also the microsomal protein and phospholipid contents. Diethyldithiocarbamate and disulfiram partially prevented the increase of microsomal protein and phospholipid contents caused by phenobarbital. Disulfiram and diethyldithiocarbamate decreased the amount of cytochrome P-450 and P-420, and the activity of p-nitroanisole O-demethylase. These changes were more pronounced after diethyldithiocarbamate than after disulfiram treatment. On the contrary, the activity of NADPH-cytochrome c reductase was enhanced only by disulfiram. The induction by phenobarbital of cytochrome P-450 and p-nitrosanisole O-demethylase was partially prevented on concomitant treatment with disulfiram and diethyldithiocarbamate. These compounds. however, had an additive effect with phenobarbital in enhancing the microsomal NADPH-cytochrome c reductase activity.     

Phenobarbital-induced increase of the hexose 6-phosphate dehydrogenase activity

The intracellular and intramicrosomal distributions of hexose 6-phosphate dehydrogenase were examined in the livers of normal and starved rats before and after phenobarbital treatment. The results have revealed that the administration of phenobarbital (75 mg/kg body weight, 4 days) causes a significant increase of the hexose 6-phosphate dehydrogenase activity only in smooth-surfaced, but not in rough-surfaced endoplasmic reticulum, with concomitant increases of the activities of NADPH-cytochrome c reductase and aminopyrine demethylase. Starvation prior to the phenobarbital treatment enhanced the effect of phenobarbital on the microsomal enzymes.     

Alpha-amidation of gastrin is impaired by diethyldithiocarbamate

The influence of gastrin alpha-amidation of the heavy-metal chelator diethyldithiocarbamate and disulfiram, its disulfide dimer, was studied in rat gastric antrum. Sensitive, sequence-specific immunoassays for glycine-extended and amidated gastrin were used to monitor extractions and chromatography. The results showed that intraperitoneal diethyldithiocarbamate administration (1000 mg/kg body weight) for two days caused a decrease in amidated gastrin from 2.6 +/- 0.4 to 1.4 +/- 0.3 nmol/g tissue (n = 11) with a simultaneous increase in glycine-extended gastrin from 0.84 +/- 0.15 to 2.4 +/- 0.3 nmol/g. Peroral administration of disulfiram (4 mg/kg body weight) for nine days did not change alpha-amidation significantly. The results of the present study demonstrate that the heavy-metal chelating agent diethyldithiocarbamate inhibits alpha-amidation of gastrin in vivo, in agreement with the inhibition of amidating activity observed in vitro. These results are in accordance with the previous observations that the presence of copper ions is necessary for the alpha-amidation to take place.     

Liver cytosolic aldehyde dehydrogenases in rats with different predisposition to induction by phenobarbital. Partial purification and characterization of the enzymes at basal state

Liver cytosolic aldehyde dehydrogenases were partially purified from rats with different genetic predisposition to induction of aldehyde dehydrogenase activity by phenobarbital. The enzymes were studied at basal state without any pretreatment with an inducer. The main aldehyde dehydrogenases from the non-, high- and intermediate reactor animals could not be differentiated by substrate specificity or thermostability. The enzyme from the non-reactor rats was more resistant to changes of pH and to the inhibitory effects of disulfiram than the enzymes from the high- or intermediate reactors. Immunochemical studies suggested a dissimilarity of these enzymes.     

Effect of styrene on testicular enzymes of growing rat.

Effect of styrene (100 or 200 mg/kg body wt/day) for 60 days was observed on testicular enzymes of postnatally maturing rats. A significant decrease in epididymal spermatozoa count was observed only at 200 mg/kg body weight dose. Activities of testicular sorbitol dehydrogenase and acid phosphatase decreased while activities of lactate dehydrogenase, beta-glucuronidase, glucose-6-phosphate dehydrogenase, and gamma-glutamyl transpeptidase significantly increased only in animals exposed to styrene at a dose of 200 mg/kg body weight. The results suggest that exposure to high dose of styrene during developmental period alters the activities of enzymes associated with specific cell type of testis.     

Dose-related effects of phenobarbital on hepatic microsomal enzymes

To study the relationship between the dose of phenobarbital (PB) and the magnitude of its effects on microsomal enzymes, cytochrome P-450, UDP-glucuronyl transferase (UDPGT), and glucose-6-phosphatase (G6P) activities were determined in liver homogenate and microsome preparations from control rats and rats treated for 6 days with PB at doses ranging from 1 to 125 mg/kg/day. Both P-450 and UDPGT activities were enhanced by PB in a dose-related fashion. However, while the lowest dose of the drug to produce significant induction of both enzymes was the same (3 mg/kg), maximal induction of P-450 (214%) and UDPGT (285%) was obtained with different doses of PB, namely 75 and 125 mg/kg, respectively. UDPGT induction could equally be demonstrated regardless of whether "native" enzyme or enzyme activated by UDP-N-acetyl glucosamine, digitonin or deoxycholate was employed. In contrast to these inducing effects of the drug on P-450 and UDPGT, PB treatment resulted in a dose-related inhibition of G6P activity. The inhibitory effect was observed with both "native" and deoxycholate-activated enzymes, and could be demonstrated whether the data were expressed as enzyme specific activity (nanomoles per minute per milligram microsomal protein) or as total G6P activity (micromoles per minute per 100 g body weight). These results indicate that: (I) enzyme induction by PB is dose-related; (ii) induction of both P-450 and UDPGT is obtained in the rat with doses of the drug similar to those given to man; and (iii) observed inhibition of G6P activity by PB does not solely reflect an enzymatic dilution secondary to the proliferated endoplasmic reticulum.     

Gossypol modulation of nucleotide metabolizing enzymes in the reproductive tract of male rats

The activities of several pivotal nucleotide metabolizing enzymes from the testis and vasal sperm of rats treated for 7 wk with 0, 20 or 30 mg X kg X day gossypol acetic acid were examined. Total testicular lactate dehydrogenase (LDH) activity increased 40% above control in the highest treatment group examined. However, the specific activity of the testis-specific isozyme of LDH, LDH-C4, decreased to 50 and 20% of control in the 20 and 30 mg X kg X day treatment groups, respectively. Basal soluble adenylate cyclase from a 100,000 X g supernatant of testis homogenate exhibited a 25% decrease in activity only in the 30-mg treatment group. Basal adenylate cyclase activity in the testicular membrane fraction increased 20 to 30% above control in response to gossypol administration. Testis membranes from the 20- and 30-mg treatment group exhibited a 2- and 4-fold greater activation of adenylate cyclase by guanine nucleotides. In vitro dose-response curves showed a half-maximal inhibitory concentration (IC50) for inhibition of soluble testicular adenylate cyclase by gossypol of 400 microM in each treatment group. Caudal epididymal sperm adenylate cyclase activity decreased to 25% of control levels in gossypol-treated animals, and the in vitro sensitivity of the enzyme to the inhibitory effects of gossypol increased 4-fold. IC50 values for gossypol inhibition of sperm adenylate cyclase decreased from 200 microM in control animals to 75 and 50 microM in the 20 and 30 mg X kg X day treatment groups, respectively. Cyclic adenosine 3':5' monophosphate phosphodiesterase activity in caudal sperm increased 6-fold in the 20- and 30-mg treatment groups. These results demonstrate that nucleotide metabolizing enzymes in sperm are major targets for the actions of gossypol and provide a possible mechanism for the inhibition of normal sperm function by this compound.     

Inhibition of peptide amidation by disulfiram and diethyldithiocarbamate

Peptidylglycine alpha-amidating monooxygenase is a copper- and ascorbate-dependent enzyme that converts peptides with COOH-terminal glycine residues into the corresponding alpha-amidated product peptides. The relatively selective copper chelator N,N-diethyldithiocarbamate (DDC) and its disulfide dimer, disulfiram (Antabuse), were used to determine whether the availability of copper affects the production of two alpha-amidated pro-ACTH/endorphin-derived peptides, alpha-melanotropin (alpha MSH) and joining peptide. When mouse pituitary corticotropic tumor cells (AtT-20) were grown in medium containing micromolar concentrations of disulfiram or DDC, alpha-amidation of newly synthesized joining peptide was specifically inhibited in a dose-dependent manner. In rats injected twice with disulfiram or DDC, the ability of the intermediate pituitary to alpha-amidate newly synthesized alpha MSH and joining peptide was inhibited in a dose-dependent manner; at disulfiram doses equivalent to those used in alcohol abuse therapy (4 mg/kg/day), only about 10% of the newly synthesized peptides were correctly alpha-amidated. Chronic treatment of rats with DDC or disulfiram produced a dose-dependent increase in the pituitary content of glycine-extended alpha MSH and joining peptide; the total amount of pro-ACTH/endorphin-related material was unaltered. After 11 days of treatment with 4 mg/kg/day disulfiram, about one-third of the pituitary alpha MSH and joining peptide were present in the glycine-extended rather than the alpha-amidated form; pituitary extracts normally contain almost entirely alpha-amidated peptides.     

Activity of cytoplasmic NADP-dependent dehydrogenase in rat liver during induction of cytochrome P-450 by phenobarbital

Activity of oxidation enzymes of the pentosephosphate way (glucose-6-phosphate dehydrogenase (EC 1.1.1.49) and 6-phosphogluconate dehydrogenase (EC 1.1.1.44), cytoplasmic malate dehydrogenase (decarboxylating oxaloacetate) (NADP+) (EC 1.1.1.40) and isocitrate dehydrogenase (NADP+) (EC 1.1.1.42) as well as the content of microsomal cytochromes b5 and P-450 in the rat liver have been studied 24 hours after 1, 2, 3, 4 and 5 intraperitoneal administrations of phenobarbital (4 mg per 100 g of the body weight). It is shown that the cytochrome P-450 content increases after a single administration of phenobarbital and then it gradually grows reaching its maximum after 4 administrations and falls after 5 administrations (though it remains high as compared to the control animals). The content of cytochrome b5 increases only after 4 administrations of phenobarbital and after 5th one it returns to the initial level. The content of microsomal gangliosides calculated per 1 mg of microsomal protein decreases after a single administration of phenobarbital and 5 days later it returns to the initial level. Activity of glucose-6-phosphate dehydrogenase increases after a single administration of phenobarbital, that of malate dehydrogenase--after 3 administrations, 6--phosphogluconate-dehydrogenase--after 4 administrations of the preparation. The 5 administrations of phenobarbital makes activity of all the mentioned dehydrogenases return to the initial level. Activity of isocitrate dehydrogenase under given conditions of the experiment does not change.     

Role of DL α-lipoic acid in gentamicin induced nephrotoxicity

The effect of DL -lipoic acid on the nephrotoxic potential of gentamicin was examined. Intraperitoneal injection of gentamicin (100 mg/kg/day) to rats resulted in decreased activity of the glycolytic enzymes-hexokinase, phosphoglucoisomerase, aldolase and lactate dehydrogenase. The two gluconeogenic enzymes—glucose-6-phosphatase and fructose-1, 6-diphosphatase, the transmembrane enzymes namely the Na⁺, K⁺-ATPase, Ca²⁺-ATPase, Mg²⁺-ATPase and the brushborder enzyme alkaline phosphatase, also showed decreased activities. This decrease in the activities of ATPases and alkaline phosphatase suggests basolateral and brush border membrane damage. Decreased activity of the TCA cycle enzymes isocitrate dehydrogenase (ICDH), succinate dehydrogenase (SDH) and malate dehydrogenase (MDH), suggests a loss in mitochondrial integrity. These biochemical disturbances were effectively counteracted by lipoic acid administration. Lipoic acid administration by gastric intubation at two different concentrations (10 mg and 25 mg/kg/day) brought about an increase in the activity of the glycolytic enzymes, ATPases and the TCA cycle enzymes. The gluconeogenic enzymes however showed a further decrease in their activities at both the concentrations of lipoic acid administered. These observations shed light on the nephroprotective action of lipoic acid against experimental aminoglycoside toxicity and the protection afforded at 25 mg/kg/day of lipoic acid was noted to be higher than that at 10 mg level.     

Determination of disulfiram and metabolites from biological fluids by high-performance liquid chromatography

A high-performance liquid chromatographic method is described for the determination of disulfiram, diethyldithiocarbamate, diethyldithiocarbamate methyl ester, carbon disulfide, and diethylamine from a single sample of plasma or urine. The analytical procedure is based on a quantitative stepwise extraction of disulfiram and diethyldithiocarbamate methyl ester, or the conversion of diethyldithiocarbamic acid, carbon disulfide and diethylamine to diethyldithiocarbamate methyl ester for chromatographical determination. The procedure is specific, precise and simple. The application of the analytical methods developed for the determination of disulfiram and the various metabolites in plasma from mice given disulfiram intraperitoneally or humans given Antabuse orally is illustrated.     

The effect of disulfiram on the aldehyde dehydrogenases of sheep liver.

1. The effect of disulfiram on the activity of the cytoplasmic and mitochondrial aldehyde dehydrogenases of sheep liver was studied. 2. Disulfiram causes an immediate inhibition of the enzyme reaction. The effect on the cytoplasmic enzyme is much greater than on the mitochondrial enzyme. 3. In both cases, the initial partial inhibition is followed by a gradual irreversible loss of activity. 4. The pH-rate profile of the inactivation of the mitochondrial enzyme by disulfiram and the pH-dependence of the maximum velocity of the enzyme-catalysed reaction are both consistent with the involvement of a thiol group. 5. Excess of 2-mercaptoethanol or GSH abolishes the effect of disulfiram. However, equimolar amounts of either of these reagents and disulfiram cause an effect greater than does disulfiram alone. It was shown that the mixed disulphide, Et2N-CS-SS-CH2-CH2OH, strongly inhibits aldehyde dehydrogenase. 6. The inhibitory effect of diethyldithiocarbamate in vitro is due mainly to contamination by disulfiram.     

Effect of triamcinolone administration on content of flavins in rabbit liver.

Triamcinoline acetonide (10 mg per kg of body weight a day) was administered to rabbit fed on a laboratory chow diet. The content of flavins in liver but not in kidney, muscle and brain started to decrease 24 h after a single dose. The activities of enzymes in the liver were determined: the activities of pyruvate dehydrogenase complex, lipoamide dehydrogenase (NADH:lipoamide oxidoreductase EC 1.6.4.3), NADH dehydrogenase (NADH : (acceptor) oxidoreductase EC 1.6.99.3) and D-amino acid oxidase (D-amino acid: oxygen oxidoreductase (deaminating) EC 1.4.3.3) were decreased but those of succinate dehydrogenase (succinate : (acceptor) oxidoreductase EC 1.3.99.1) and xanthine oxidase (xanthine : oxygen oxidoreductase EC 1.2.3.2) remained unchanged. The activities of enzymes in the kidney, however, remained unchanged except the decrease in the activity of pyruvate dehydrogenase complex.     

Enzymatic oxidation of vanillin, isovanillin and protocatechuic aldehyde with freshly prepared Guinea pig liver slices.

BACKGROUND/AIMS: The oxidation of xenobiotic-derived aromatic aldehydes with freshly prepared liver slices has not been previously reported. The present investigation compares the relative contribution of aldehyde oxidase, xanthine oxidase and aldehyde dehydrogenase activities in the oxidation of vanillin, isovanillin and protocatechuic aldehyde with freshly prepared liver slices. METHODS: Vanillin, isovanillin or protocatechuic aldehyde was incubated with liver slices in the presence/absence of specific inhibitors of each enzyme, followed by HPLC. RESULTS: Vanillin was rapidly converted to vanillic acid. Vanillic acid formation was completely inhibited by isovanillin (aldehyde oxidase inhibitor), whereas disulfiram (aldehyde dehydrogenase inhibitor) inhibited acid formation by 16% and allopurinol (xanthine oxidase inhibitor) had no effect. Isovanillin was rapidly converted to isovanillic acid. The formation of isovanillic acid was not altered by allopurinol, but considerably inhibited by disulfiram. Protocatechuic aldehyde was converted to protocatechuic acid at a lower rate than that of vanillin or isovanillin. Allopurinol only slightly inhibited protocatechuic aldehyde oxidation, isovanillin had little effect, whereas disulfiram inhibited protocatechuic acid formation by 50%. CONCLUSIONS: In freshly prepared liver slices, vanillin is rapidly oxidized by aldehyde oxidase with little contribution from xanthine oxidase or aldehyde dehydrogenase. Isovanillin is not a substrate for aldehyde oxidase and therefore it is metabolized to isovanillic acid predominantly by aldehyde dehydrogenase. All three enzymes contribute to the oxidation of protocatechuic aldehyde to its acid.     

Experimental analysis of gossypol and chloroquine interaction in serum and in liver of rat.

Interactive effects of gossypol and chloroquine as determined by activities of serum alanine transaminase (ALT), aspartate transaminase (AST) and liver lactate dehydrogenase (LDH), alkaline phosphatase (ALK-pase), glucose-6-phosphatase (G-6-pase) and cholesterol level were investigated in rats. Administration of gossypol for eight weeks, at a concentration of 20 mg per kg body wt. per day with or without chloroquine had no effect on the serum enzymes and glucose-6-phosphatase activities. When chloroquine at a concentration of 5 mg per kg body wt. thrice a week was administered alone, there was a marked decrease in total protein content and ALK-pose activities, while a significant increase in LDH activity was observed. Administration of either gossypol or chloroquine decreased the level of cholesterol. A greater decrease was recorded when both were given together. It is suggested that gossypol can be employed as a male contraceptive among malaria-infected populations.     

Effect of prenatal diazepam, phenobarbital, haloperidol and fluoxetine exposure on foot shock induced aggression in rats.

Different groups of pregnant rats were treated with diazepam (10 mg/kg), phenobarbital (10 mg/kg), haloperidol (0.1 mg/kg), fluoxetine (10 mg/kg) and vehicle (normal saline) intraperitoneally once a day during gestation days 13 to 21. After birth these pups were culled to 8 pups/dam and foster-nursed by lactating mothers for 3 weeks and were reared in colony cages thereafter. Sex and weight matched pairs of rat offsprings were subjected to foot shock induced aggression test at 8 weeks of age. Two parameters of aggressive behaviour were recorded namely, the latency to fight and total number of fighting bouts. The results indicate that prenatal exposure to diazepam, phenobarbital, haloperidol and fluoxetine caused significantly enhanced aggression in terms of number of fighting bouts.     

Protective effect of aspartate and glutamate on cardiac mitochondrial function during myocardial infarction in experimental rats

The present study investigates the effect of aspartate and glutamate on mitochondrial function during myocardial infarction (MI) in wistar rats. Male albino wistar rats were pretreated with aspartate [100 mg(kgbody weight)(-1) day(-1)] or glutamate [100 mg(kg body weight)(-1) day(-1)] intraperitoneally for a period of 7 days. Following amino acid treatment, MI was induced in rats by subcutaneous injection of isoproterenol [200 mg(kg body weight)(-1) day(-1)] for 2 days at an interval of 24 h. Isoproterenol (ISO) induction resulting in significant (P<0.05) increase in the levels of cardiac mitochondrial lipid peroxidation with a decrease in reduced glutathione level. The activities of glutathione peroxidase and glutathione reductase were significantly (P<0.05) decreased by ISO. ISO-induction also caused significant (P<0.05) decrease in the activities of mitochondrial tricarboxylic acid cycle enzymes (malate dehydrogenase, isocitrate dehydrogenase, succinate dehydrogenase, alpha-ketoglutarate dehydrogenase) and respiratory chain enzymes (NADH dehydrogenase and cytochrome-c-oxidase). ISO significantly (P<0.05) reduced the cytochrome contents, ATP production, ADP/O ratio and oxidation of succinate in state 3/state 4 whereas significantly (P<0.05) increased NADH oxidation. Pretreatment with aspartate or glutamate significantly (P<0.05) reduced the alterations induced by ISO and maintained normal mitochondrial function. The present findings reveal the protective effect of aspartate and glutamate on cardiac mitochondrial function in myocardial infarction-induced rats.     

Therapeutic Potential of Riboflavin,Niacin and Ascorbic Acid on Carbohydrate Metabolizing Enzymes in Secondary Endometrial Carcinoma Bearing Rats

Curative potential of riboflavin, niacin and ascorbic acid against tamoxifen mediated endometrial carcinoma was established by studies on carbohydrate metabolizing enzymes. The enzymes investigated were glycolytic enzymes namely, hexokinase; aldolase; phosphoglucoisomerase and the gluconeogenic enzymes namely, glucose-6-phosphatase and fructose-1, 6-biphosphatase in endometrial carcinoma bearing rats. A significant increase in glycolytic enzymes and a subsequent decrease in gluconeogenic enzymes were observed in plasma, liver and kidney of endometrial carcinoma animals. The administration of riboflavin (45 mg/kg bw/day), niacin (100 mg/kg bw/day) and ascorbic acid (200 mg/kg bw/day) along with tamoxifen (45 mg/kg bw/day) caused a significant decrease in the activity of glycolytic enzymes and a significant increase in the activities of gluconeogenic enzymes to near normal levels in experimental animals. Our results suggest that riboflavin, niacin and ascorbic acid have potential combination therapy against tamoxifen mediated secondary endometrial carcinoma in experimental rats. However, there were no deleterious side effects observed in combinants alone treated animals.     

Influence of various enzyme inductors and combinations of inductors on DDT residue elimination in rats]

A study was under-taken to investigate the influence of pretreatments with various hepatic microsomal enzyme inducers on the elimination of DDT residues in rats previously contaminated with p,p' -DDT at 5 mg/kg/day, during 20 days. The following inducers were used : phenobarbital (50 a mg/kg/day, i.p.), 3,4-benzopyrene (20 mg/kg/day, s.c.) and norethandrolone (20 mg/kg/day, s.c.), all given during 14 consecutive days. Each inducer was administered singly or in combination with the other two according to a 2 X 2 X 2 factorial experiment. The animals were then sacrificed for the measurement of p,p' -DDT, p,p' -DDD and p,p' -DDE residues in the blood, brain, abdominal fat, liver and kidneys. The results show that phenobarbital lowers markedly the concentration of total residues in the fat tissue and brain and that norethandrolone brings about a reduction of the residues in the blood, brain and kidneys, but not in the fat tissue. On the opposite, 3,4-benzopyrene produces an increase of the residues in the brain, liver and kidneys. Phenobarbital thus appears to be more efficacious than the other two inducers in facilitating the elimination of DDT residues from the fat tissue. In addition, it appears that under the experimental conditions used during this investigation, the elimination of DDT residues is not further accelerated by combining the inducers one with each other.     

Effect of triamcinolone administration on content of flavins in rabbit liver

Triamcinoline acetonide (10 mg per kg of body weight a day) was administered to rabbit fed on a laboratory chow diet. The content of flavins in liver but not in kidney, muscle and brain started to decrease 24 h after a single dose. The activities of enzymes in the liver were determined: the activities of pyruvate dehydrogenase complex, lipoamide dehydrogenase (NADH : lipoamide oxidoreductase EC 1.6.4.3), NADH dehydrogenase (NADH : (acceptor) oxidoreductace EC 1.6.99.3) and -amino acid oxidase ( -amino acid : oxygen oxidoreductase (deaminating) EC 1.4.3.3) were decreased but those of succinate dehydrogenase (succinate : (acceptor) oxidoreductase EC 1.3.99.1) and xanthine oxidase (xanthine : oxygen oxidoreductase EC 1.2.3.2) remained unchanged. The activities of enzymes in the kidney, however, remained unchanged except the decrease in the activity of pyruvate dehydrogenase complex.