Impairment of hepatic microsomal drug metabolism in the rat during daily disulfiram administration.

Male rats were given 0.1 or 0.4 g/kg of disulfiram (DS) daily by gavage for up to 12 days, in order to study the effects of chronic DS administration on hepatic microsoma] drug metabolism. Administration of 0.4 g/kg of DS resulted in significant impairment of aniline (ANL) hydroxylase after 1 day. but 2 days of DS treatment were required for significant inhibition of ethylmorphine (EtM) metabolism and depression of cytochrome P-450 levels. At this time, maximum impairment of ANL and EtM metabolism and maximum reduction of cytochrome P-450 levels were seen. Continued administration of DS for 10 additional days produced no further change in these parameters. ANL hydroxylase was also significantly reduced in treated animals throughout a 12-day period during which 0.1 g/kg of DS was given. EtM N-demethylase activity and cytochrome P-450 levels were also reduced in animals so treated, but not until DS had been given for at least 5 days. However, by the end of the 12-day experimental period. EtM metabolism and cytochrome P-450 levels had returned almost to control levels and only ANL hydroxylase was significantly different from control activity. Daily DS administration (0.4 g/kg produced small but significant increases in microsomal cytochrome b₅ levels and in NADPH-cytochrome c reductase activity, whereas NADPH oxidase and NADPH-cytochrome P-450 reductase activities were significantly lower in treated rats. In addition to these effects in vivo, DS competitively inhibited EtM N-demethylase in vitro and bound to cytochrome P-450, producing a type I difference spectrum, thus providing additional mechanisms to account for impairment in vivo of drug metabolism by DS.     

Augmentation of sulfate ion absorption from the rat lung by heavy metals.

In rats injected ip with 500 mg of cyclotrimethylenetrinitramine (RDX)/kg, the respective mean times to first seizure and to death were 23.8 and 171.0 min, and the mean plasma concentrations of RDX at seizure and death were 5.2 and 13.8 μg/ml. Following 100 mg/kg po, the plasma concentration was 2.1 μg/ml at 4 hr and 3.0 μg/ml at 24 hr, while the urine concentration was 5.5 μg/ml at 4 hr and 6.9 μg/ml at 24 hr. In the 6 days following 50 mg/kg po, 0.7% was excreted as RDX in the feces and 2.4% in the urine. Irrespective of dosage or route of administration, the concentration of RDX was greatest in kidney, most variable in liver, and did not accumulate in the brain. Twenty-four hours after po dosing with 50 mg of [¹⁴C]RDX/kg, the liver and urine contained large amounts of RDX metabolites, and, after the first 4 days, 90% of the total radioactivity was recovered: 34% in the urine, 43% as ¹⁴CO₂, 3% in the feces, and 10% in the carcass. In miniature swine dosed with 100 mg/kg po, the plasma concentration was 1.6 μg/ml at 2 hr and 4.7 μg/ml at 24 hr, while the urinary concentration was 2.0 μg/ml at 2 hr and 3.6 μg/ml at 24 hr. At 24 hr, the concentrations of RDX in brain, heart, liver, kidney cortex, kidney medulla, and fat were between 4.4 and 9.1 μg/g. Convulsions in pigs occurred 12–24 hr after dosing with RDX.     

Iron-EDTA stimulated reduction of indicine N-oxide by the hepatic microsomal fraction, isolated hepatocytes, and the intact rat

Fe(III) complexes of EDTA and diethylenetriamine pentaacetic acid (DETAPAC) at low concentrations (between 1 and 100 microM) produced up to a 20-fold increase in anaerobic microsomal NADPH- and NADH-dependent reduction of indicine N-oxide. Under aerobic conditions microsomal indicine N-oxide reduction was stimulated to half the levels seen under anaerobic conditions. EDTA alone was much less effective at stimulating indicine N-oxide reduction, while FeCl3 alone had no effect on reduction. Other complexes of Fe(III) had little or no effect in stimulating microsomal indicine N-oxide reduction. Fe(III)-EDTA stimulated indicine N-oxide reduction by purified NADPH-cytochrome P-450 reductase and NADPH. It is probable that iron serves to transfer electrons between microsomal flavoprotein reductases and indicine N-oxide. The redox potential and the presence of an exchangeable ligand, such as water, in the inner ligand sphere of the iron complex are suggested to be important factors in determining which iron complexes will stimulate indicine N-oxide reduction. EDTA complexes of other transition metal ions do not stimulate indicine N-oxide reduction. Hydroxyl radicals, detected as the spin adduct of 5,5-dimethyl-1-pyroline-N-oxide, appear to be formed during Fe(II)-EDTA-dependent reduction of indicine N-oxide under anaerobic conditions. Fe(III)-EDTA at concentrations between 50 and 250 microM stimulated indicine N-oxide reduction by rat isolated hepatocytes up to 5-fold under anaerobic conditions and to half these values under aerobic conditions. By themselves, EDTA and FeCl3 at similar concentrations produced a small stimulation of indicine N-oxide reduction by hepatocytes under anaerobic conditions. Fe(III)-EDTA stimulated indicine N-oxide reduction by murine leukemia P-388 cells under aerobic conditions and by rat caecal flora under anaerobic but not aerobic conditions. Fe(III)-EDTA, EDTA or FeCl3 administered to rats produced a 3-fold increase in the 24-hr urinary excretion of indicine following an i.p. dose of indicine N-oxide.     

Impairment of hepatic microsomal and plasma esterases of the rat by disulfiram and diethyldithiocarbamate.

Twenty-four hr after oral administration (0.2 to 2.0 g/kg) of disulfiram (DS) to male rats. significant impairment of hepatic microsomal carboxylesterase and plasma carboxyl- and cholinesterase was observed. Plasma esterase activities returned to control values between 48 and 72 hr after a single oral dose of DS (2.0 g/kg), but microsomal carboxylesterase activity was still significantly lower in treated animals at both times. Daily administration of DS (0.1 or 0.4 g/kg) resulted in decreased microsomal carboxylesterase activity after 2 days. However, continued administration of DS for a total of 12 days did not produce further depression of microsomal esterase activity. Microsomal and plasma carboxylesterase activities were also decreased 24 hr after oral administration (1.0 to 2.0 g/kg) of sodium diethyldithiocarbamate (DDTC), the reduced metabolite of DS. Hepatic microsomal esterases that migrated rapidly toward the anode during polyacrylamide disc gel elcctrophoresis were the most sensitive to impairment by DS or DDTC. Esterase activity in the lung was also impaired after DS or DDTC administration, whereas esterases of the heart, kidney and testis were essentially unaffected. Incubation in vitro of liver microsomes with DS decreased microsomal carboxylesterase activity, while incubation with DDTC had little effect. Plasma carboxylesterase was inhibited in vitro to a greater extent than microsomal esterases by both DS and DDTC. Diethylamine and CS₂, the decomposition products of DDTC, were essentiallv inactive as esterase inhibitors in vitro.     

Effects of dietary pectin and cellulose on hepatic and intestinal mixed-function oxidations and hepatic 3-hydroxy-3-methylglutaryl-coenzyme a reductase in the rat

The aim of this study was to determine if feeding dietary fiber (cellulose or pectin) to male rats could influence hepatic and intestinal mixed-function oxidation. We simultaneously compared hepatic drug-oxidizing activity with the activity of 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase, the rate-controlling enzyme for cholesterol biosynthesis. Three groups of six animals were fed a purified diet containing by weight either 10.4% cellulose or 10.4% pectin, or a standard cereal-based diet containing 4.5% crude fiber; the caloric contributions by carbohydrate, protein and fat in the three diets were similar. In the cellulose-fed rats, the hepatic microsomal cytochrome P-450 content and the activities of ethylmorphine N-demethylase and aniline hydroxylase were significantly lower when compared with those of rats fed pectin or the cereal-based diet. The hepatic microsomal cytochrome P-450 content and the activities of ethylmorphine N-demethylase and aniline hydroxylase were similar in the pectin-fed and cereal diet-fed rats. Hepatic HMG-CoA reductase activity, hepatic microsomal cytochrome b₅ content, and intestinal benzo[a]pyrene hydroxylase activity were comparably lower in rats fed the purified diet with either dietary fiber when compared to those fed the cereal diet. It is concluded that dietary pectin and cellulose exert distinctly different influences on the hepatic microsomal mixed-function oxidase system for drug metabolism, but not on liver cholesterol synthesis or intestinal benzo[a]pyrene hydroxylation, suggesting that different physiological mechanisms control these enzyme systems.     

Receptor independent stimulatory effect of noradrenaline on Na,K-ATPase in rat brain homogenate. Role of lipid peroxidation

The effect of different adrenoceptor agonists on Na,K-ATPase activity and lipid peroxidation of rat brain homogenate was studied. Drugs which enhanced Na,K-ATPase activity--noradrenaline, adrenaline and oxymethazoline--were found to inhibit endogenous membrane lipid peroxidation. Other drugs--phenylephrine, xylazine and clonidine--which did not cause any change in the enzyme activity did not influence lipid peroxidation either. No increase of Na,K-ATPase activity by noradrenaline could be detected after preincubation of the homogenate for 5 min at 37 degrees. During this time endogenous lipid peroxidation of considerable extent could be observed. It is concluded that there is no correlation between the adrenoceptor agonist feature of noradrenaline and its stimulatory effect on Na,K-ATPase activity of rat brain homogenate. However, it seems likely that in rat brain homogenate the increase of Na,K-ATPase activity and inhibition of endogenous lipid peroxidation by noradrenaline are related.     

1-(4-Aminophenyl)isoquinoline derivatives. Potent inhibitors of calcium-independent and calcium-dependent phosphodiesterases from rat cerebral cortex

The effects of a series of 1-(4-aminophenyl)isoquinoline derivatives on the activity of calcium-independent and calcium-dependent phosphodiesterases purified from rat cerebral cortex were examined. Agents were approximately equipotent (IC50 values, 0.2 to 25 microM) in inhibiting the calcium-dependent hydrolysis of either cyclic AMP or cyclic GMP, while they were 6-35 times more effective as inhibitors of cyclic AMP hydrolysis when compared to cyclic GMP hydrolysis using the calcium-independent enzyme. The diastereomers of 3-(carbomethoxy)propenamido demonstrated a marked difference in specificity. The cis-isomer was very potent in inhibiting cyclic AMP or cyclic GMP hydrolysis by either enzyme (IC50 values, 0.2 to 8 microM) while the trans-isomer was only effective in inhibiting calcium-independent cyclic AMP hydrolysis (IC50 values, 2.5 microM). Kinetic analyses of the type of inhibition of the calcium-dependent enzyme revealed that the various agents were competitive inhibitors of cyclic GMP hydrolysis and noncompetitive inhibitors of cyclic AMP hydrolysis. A reverse pattern of inhibition by the isoquinoline derivatives was found using the calcium-independent phosphodiesterase, i.e. noncompetitive inhibition of cyclic GMP while competitive inhibition of cyclic AMP. Inhibition of phosphodiesterases by these agents was also manifest using intact brain slices prepared from rat cerebral cortex. Thus, the agents were found to potentiate forskolin-elicited accumulations of cyclic AMP by 100-700% and increased the half-time for the decline in cyclic AMP following forskolin stimulation from 3 to 6 min.     

Identification and quantification of 1,2,3,4-tetrahydro-β-carboline, 2-methyl-1,2,3,4-tetrahydro-β-carboline,and 6-methoxy-1,2,3,4-tetrahydro-β-carboline as in vivo constituents of rat brain and adrenal gland

The identification and quantification of three 1,2,3,4-tetrahydro-β-carbolines as normal constitutents of rat brain and adrenal gland, using combined gas chromatographic/mass spectrometric techniques, are reported. Qualitative analyses of these tissues led to the identification of 1,2,3,4-tetrahydro-β-carboline (THBC), 2-methyl-THBC (2-MTHBC) and 6-methoxy-THBC (6-MeOTHBC), as determined by observed peak retention times, mass fragments and ion mass ratios. Quantitative analyses, using deuterated internal standards, gave the following results: THBC (ng/g wet wt) in brain = 17.5 ± 4.86, adrenal = 500.3 ± 163. 6-MeOTHBC (ng/g wet wt) in brain = 35.6 ± 16.6, adrenal = 1113.7 ± 300. Mechanisms for the formation of these β-carbolines as well as their possible function in vivo are discussed.     

The relationship of metallothionein to the toxicity of cadmium after prolonged oral administration to rats

Male Sprague-Dawley rats were exposed to cadmium at concentrations of 10, 30, and 100 ppm in their drinking water for 24 weeks. The testicular function, blood pressure, heart rate, EKG, hematocrit, blood hemoglobin, plasma glucose, aniline hydroxylase, hexobarbital oxidase, cytochrome P-450, concentration of Cd in the tissues, concentration of metallothionein in the kidney and liver, organ weights, bone calcification, and histopathology of all the rats were recorded after 3, 6, 12, and 24 weeks. In addition, the weight gain, food and water intake, urine flow and protein excretion, and motor activity were measured weekly in the 24-week group. CNS function was assessed by measuring the motor activity. The hourly nocturnal and daily motor activities decreased with time for the 30- and 100-ppm rats when compared to the control rats. Renal injury was indicated by an increase in the concentration of protein in the urine with time for the 30- and 100-ppm rats when compared to the control rats. There was also slight and focal tubular necrosis in the 30- and 100-ppm rats by Week 24. The time- and dose-dependence of the concentration of the Cd in the intestine indicates that the suggested protective mechanism of intestinal metallothionein, where metallothionein sequesters dietary Cd in the mucosal cells and thus hinders the transfer of the metal to the systemic circulation, is quickly overloaded at concentrations of 10 ppm or less of Cd in the drinking water. The concentration of metallothionein and Cd in the kidney and liver increased with dose at all time intervals and increased with time at most doses. However, the rate of increase of the concentration of metallothionein and Cd in the liver and kidney was not the same. In the liver ratio of Cd to Cd-binding capacity of metallothionein reached a plateau with time, which may explain hepatic tolerance to Cd. On the other hand, in the kidney, were necrosis and dysfunction were observed, the ratio continued to increase with time.     

Mechanism of the drug-metabolizing enzymes' induction by 2-diethylaminoethyl-2-2-diphenylvalerate-HCl (SKF 525 A)

Repetitive administration of 2-diethylaminoethyl-2-2-diphenylvalerate-HCl (SKF 525 A) produces a biphasic effect on the pentobarbital sleeping time of rats. It causes a significant prolongation effect after one daily dose, but it significantly shortens it when more daily doses are given. Administration of three daily doses of SKF 525 A results in increased activity of ethylmorphine N-demethylase and cytochrome P-450 (P-450) content in liver microsomes while aniline hydroxylase and cytochrome c reductase activity in these preparations were not significantly increased. Repetitive administration of SKF 525 A increased [¹⁴]leucine incorporation and decreased ([¹⁴C]guanidino)arginine disappearance from microsomal proteins. Results suggest that effects on pentobarbital sleeping time and on drug metabolism resulting from repetitive SKF 525 A administration would result from increases in P-450 content which might derive from increased microsomal protein synthesis and from decreased microsomal protein degradation.     

Enzymatic and non-enzymatic reduction of N-acetyl-p-benzoquinone imine and some properties of the N-acetyl-p-benzosemiquinone imine radical

N-Acetyl-p-benzoquinone imine (NAPQI) is the postulated hepatotoxic intermediate in acetaminophen overdosage. NAPQI was rapidly metabolized by NADPH-cytochrome P-450 reductase, with an apparent Km of 1.8 to 4.0 microM and an apparent Vmax of 29.4 mumoles per min per mg, and exhibited substrate inhibition of metabolism at NAPQI concentrations above 10 microM. NADPH was oxidized by NAPQI at a slower rate in the absence of enzyme. NAPQI did not appear to undergo redox cycling at an appreciable rate to form superoxide, and it did not stimulate oxygen utilization or superoxide release by rat isolated hepatocytes. Electron spin resonance studies failed to show formation of a free radical by chemical or enzymatic reduction of NAPQI under anaerobic conditions in aqueous media.     

Cocaine-induced hepatotoxicity in mice

Cocaine, 75 mg/kg, ip, produced 53% mortality in male mice and 74% mortality in female mice. All deaths in both groups occurred during the first 3 hr following treatment. Phenobarbital (PB) pretreatment protected against this acute cocaine-induced mortality. However, either PB pretreatment or 3-methylcholanthrene pretreatment produced delayed toxicity characterized by hepatic dysfunction and necrosis. These lesions could not be induced in rats. PB plus cocaine treatment reduced hepatic glutathione concentrations by 40%. PB pretreatment did not alter the plasma concentrations of cocaine plus metabolites, but did increase the accumulation of cocaine plus metabolites in liver tissue. PB plus cocaine treatment produced a dose-related increase in serum glutamic-pyruvic transaminase activity and periportal necrosis. Enhancement of intracellular glutathione concentrations by pretreatment with cysteine protected the liver against cocaine-induced injury. Depletion of intracellular glutathione concentrations by pretreatment with diethylmaleate exaggerated the cocaine-induced injury. Cocaine covalently bound to hepatic protein. PB pretreatment increased by 85% the cocaine covalently bound to hepatic protein. Cocaine was N-dealkylated by hepatic microsomes, and PB pretreatment enhanced hepatic N-dealkylation of cocaine. Cocaine is biotransformed to a chemically reactive intermediate metabolite which produces hepatic dysfunction and periportal necrosis.     

Potentiation of duodenal ulcerogenic action of acrylonitrile by PCB or phenobarbital in the rat

Pretreatment of rats with the polychlorinated biphenyl (PCB) Aroclor 1254 or phenobarbital markedly increased the duodenal ulcerogenic action of acrylonitrile. The extent of forestomach and hepatic lesions in these rats, on the other hand, was not modified. The duodenal ulcers produced by Aroclor 1254 and acrylonitrile morphologically resembled the ulcers induced in other animal models of the human duodenal ulcer disease. The possible mechanisms of this potentiation of acrylonitrile action are discussed.     

Serotonin-sensitive aryl acylamidase activity of platelet acetylcholinesterase

Serotonin-sensitive aryl acylamidase (AAA, EC 3.5.1.13) was purified to apparent homogeneity from sheep platelets by affinity chromatography and it was shown to be associated with the platelet acetylcholinesterase (AChE, EC 3.1.1.7). The basis for the association of the two enzymes was the following. Both enzyme activities co-eluted from the affinity columns with constant ratios of specific activities and percentage recoveries. Both enzymes co-migrated on gel electrophoresis. Both enzymes co-eluted during Sepharose 6B gel filtration. Potent inhibitors of AChE such as bis(4-allyldimethyl ammoniumphenyl) pentan-3-one dibromide (BW 284C51), neostigmine and eserine also inhibited AAA potently. Both enzymes lost significant activity on treatment with deoxycholate or taurodeoxycholate and the loss could be partly restored by a mixture of phospholipids. The platelet AAA was specifically inhibited by serotonin and to a lesser extent by tryptamine but not by several other amines. It was also inhibited by acetylcholine and several of its analogues and homologues. It is suggested that in the platelets the two enzymes (AAA and AChE) are probably identical.     

Time-dependent inhibition of monoamine oxidase by β-phenethylamine

Several reports have suggested that monoamine oxidase activity towards β-phenethylamine is inhibited by high concentrations of that substrate. This inhibition is not found if initial velocities are measured, but there is a slower time-dependent inhibition at higher β-phenethylamine concentrations. Such time-dependent inhibition is not found with tyramine as substrate or upon incubation of the enzyme with the reversible inhibitor amphetamine. The inhibition is not due to the accumulation of phenacetaldehyde, phenylethanol or phenacetic acid, or to a reaction of any of these three products either with each other or with β-phenethylamine. Although the inhibition is time-dependent, the inactivated enzyme slowly regains activity upon removal of the β-phenethylamine. A model is proposed to explain the observed inhibition.     

Immunologic responses of cynomolgus monkeys after repeated inhalation exposures to enzymes and enzyme--detergent mixtures

Sera from monkeys repeatedly exposed by inhalation to bacterial enzymes, to a detergent, or to various enzyme-detergent mixtures were examined for the presence of enzyme-specific IgE and precipitating antibodies. In addition, lung sections were examined by immunofluorescence for deposits of immunoglobulins, complement, and fibrinogen. No clear-cut evidence was obtained for the presence of enzyme-specific IgE. However, precipitating antibodies were found in each group of monkeys exposed to either the enzyme alone or to the various enzyme-detergent mixtures. The precipitin responses were found to be dependent upon the dose of enzyme employed for inhalation exposure and to be potentiated by the presence of detergent in the inhalation mixtures. Results of immunofluorescent studies suggested that the presence of precipitating antibodies in the sera of the monkeys was not correlated with pulmonary pathological changes.     

Comparison between o-aminophenol glucuronidation in liver slices and homogenates from control and phenobarbital-treated Wistar and Gunn rats

Glucuronidation rates were studied in sliced and homogenised liver from control and phenobarbital-treated Gunn and Wistar rats of both sexes, aglycone concentration not being significantly rate-limiting. Kinetic parameters of o-aminophenol UDP-glucuronyltransferase (EC 2.4.1.17) were determined in fresh (“native”) homogenates and those activated by UDP-N-acetylglucosamine, digitonin and diethylnitrosamine added singly or together. Apparent K_{UDP-glucuronate}-values were increased by both UDP-N-acetylglucosamine and digitonin, but not by the specific activator diethylnitrosamine; values were similar for both strains and not significantly affected by phenobarbital pretreatment; sex differences were encountered. Apparent V_max-values were increased by 30- to 65-fold for Wistar and 400- to 1600-fold for Gunn rats in maximally-activated homogenates. Phénobarbital pretreatment approximately doubled V_max of maximally-activated enzyme in all animals, but had little effect on V_max of “native” or partially-activated preparations. Slices of Gunn rat liver glucuronidated o-aminophenol at rates 30–44 per cent those of Wistar rats; rates in both strains exhibited a consistent sex difference and increased after phenobarbital treatment. Comparison of results from slices and homogenates containing physiological concentrations of UDP-glucuronate suggested that in both sexes and strains UDP-glucuronyltransferase activity in vivo is higher than that observed in “native” unactivated homogenates, presumably because of endogenous activators; however, as glucuronidation in similar, but maximally-activated, homogenates was well above that in slices, the enzyme may still remain partially “latent” in vivo.