Effect of chronic ethanol consumption on aldehyde dehydrogenase activity in the baboon

Baboon liver has detectable aldehyde dehydrogenase (A1DH) activity in the mitochondrial, microsomal and soluble fractions. Based on kinetic data, the mitochondrial and soluble fractions each appear to contain two forms of A1DH, one with a high, and another with a low, K_m for acetaldehyde. In the microsomes there was activity only with millimolar concentrations of acetaldehyde. In the baboon liver, about 75 per cent of total A1DH activity resides in the mitochondria and 20 per cent in the soluble faction. Chronic ethanol consumption decreased total and low K_m mitochondrial A1DH activity in baboon liver. In rats, ethanol consumption also resulted in decreased mitochondrial low K_m A1DH activity.     

Ethanol metabolism in vivo by the microsomal ethanol-oxidizing system in deermice lacking alcohol dehydrogenase (ADH)

To assess the importance of non-ADH ethanol metabolism, ADH-negative and ADH-positive deermice were fed liquid diets containing ethanol or isocaloric carbohydrate for 2-4 weeks. Blood ethanol disappearance rate increased significantly after chronic ethanol feeding in both strains. Although at low ethanol concentrations (between 5 and 10 mM) there was no significant difference between ethanol-fed and pair-fed control animals, at high ethanol concentrations (between 40 and 70 mM) blood ethanol elimination rates were increased significantly after chronic ethanol feeding in both ADH-positive and ADH-negative animals. There was no significant effect of the catalase inhibitor 3-amino-1,2,4-triazole on the ethanol elimination/rates in both strains. Whereas catalase and ADH activities were not altered after chronic ethanol treatment, the activity of the microsomal ethanol-oxidizing system (MEOS) was enhanced three to four times in both strains, and microsomal cytochrome P-450 content was also increased significantly. When MEOS activity was expressed per cytochrome P-450 content, it was higher in ADH-negative than in ADH-positive animals, and it increased after ethanol administration. When microsomal proteins were separated by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis, ethanol-fed animals had a distinct band which reflected the increase in microsomal cytochrome P-450 content and seemed to reflect a unique form of cytochrome P-450 induced by ethanol. Thus, despite the absence of the ADH pathway, a large amount of ethanol was metabolized by MEOS in ADH-negative deermice; this was associated with increased blood ethanol elimination rates, enhanced MEOS activity, and quantitative and qualitative changes of cytochrome P-450.     

Interaction of capsaicinoids with drug-metabolizing systems. Relationship to toxicity

The interaction of capsaicin with microsomal drug-metabolizing systems was assessed to determine the role that bioactivation of capsaicin may play in the induction of hepatotoxicity and neurotoxicity. Capsaicin produced a type I spectral change in rat hepatic microsomes in a high affinity (K_s = 8 μM) concentration-dependent manner and was approximately equipotent with SKF-525A in inhibiting ethylmorphine demethylation. Capsaicin (10 mg/kg, s.c.) inhibited biotransformation in vivo as measured by prolongation of pentobarbital sleep time. Reactive metabolites of capsaicin were studied using [³H]dihydrocapsaicin. [³H]Dihydrocapsaicin bound irreversiblyto hepatic microsomal protein after in vitro incubation or in vitro administration. No binding was observed in spinal cord or brain. Although the bioactivation and subsequent covalent binding of capsaicin equivalents may initiate events associated with the hepatotoxicity of capsaicin, it appears that capsaicin-induced neuropathy does not involve covalent interactions with neuroproteins in spinal cord or brain.     

Hepatic drug metabolism during ethanol ingestion in riboflavin deficient rats

Riboflavin deficiency was induced by feeding rats a riboflavin-deficient diet for 1 month. In order to find out if there are any combined effects of ethanol and riboflavin deficiency on drug metabolism, a group of riboflavin-deficient rats were also given ethanol in their drinking water. At the end of the fedding period, hepatic drug-metabolizing enzyme activities were determined. The hepatic phospholipid and protein contents were the same in rats receiving a standard diet and in those on a riboflavin-deficient diet. However, ethanol ingestion in both groups enhanced significantly the phospholipid content. Ethanol ingestion also markedly enhanced the hepatic cytochrome P-450 concentration in rats fed either a standard or riboflavin-deficient diet. Ethoxycoumarin O-deethylase activity was significantly lower in riboflavin-deficient rat livers than in those of the controls. In both groups ethanol ingestion nearly doubled the activities. Aryl hydrocarbon hydroxylase activity was also significantly decreased during riboflavin deficiency. However, ethanol administration did not change the activities of this enzyme. UDP glucuronosyltransferase activity was slightly lower in riboflavin-deficient rat livers than in those fed a standard diet. No significant decrease was found in the epoxide hydrase activity in the riboflavin-deficient rats. However, the riboflavin-deficient rats had enhanced activity after the ethanol ingestion.     

Differential effects of chronic ethanol feeding on cytochrome P-448- and P-450-mediated drug metabolism in the rat

The effects of chronic ethanol feeding on cytochrome P-448- and P-450-mediated drug metabolism have been studied both in vivo and in vitro in the rat, using caffeine, phenacetin, antipyrine and aminopyrine as test substrates. N-Demethylation of aminopyrine (P-450 mediated) was increased both in vivo and in vitro in rats after chronic ethanol feeding (P < 0.05) whereas in vivoN-demethylation of caffeine and O-dealkylation of phenacetin (P-448 mediated) were unchanged in the same animals. N-rmDemethylation of antipyrine was increased by both phenobarbital and 3-methylcholanthrene pretreatment and by chronic ethanol feeding (P < 0.05), possibly due to cytochrome P-450 induction. Furthermore, the Michaelis affinity constants. K_m, for hepatic microsomal aminopyrine N-demethylase and antipyrine N-demethylase were lower in chronic ethanol-fed animals (P < 0.05), suggesting a qualitative change in the enzymes resulting in greater substrate affinity. These findings suggest a differential effect of chronic ethanol feeding on the induction of cytochrome P-450- and cytochrome P-448-mediated drug metabolism, with a greater effect on the former microsomal system.     

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.     

Effectiveness of metyrapone in the treatment of acetaminophen toxicity in mice

Metyrapone tartrate, 400 mg/kg i.p. raised the LD₅₀ for acetaminophen from 340 mg/kg i.p. to 540 mg/kg i.p. in fasting male Swiss white mice. The minimum protective dose of metyrapone was 200 mg/kg. Metyrapone was effective in preventing death when given up to 2 h after acetaminophen administration. The LD₅₀ for metyrapone tartrate was 760 mg/kg i.p. Metyrapone decreased or prevented acetaminophen induced hepatic damage measured either by histology or plasma glutamate pyruvate transaminase activity. Metyrapone tartrate, 400 mg/kg i.p., inbibited the severe liver glutathione depletion seen with acetaminophen alone. It is proposed that metyrapone protects mice from acetaminophen induced liver toxicity and death by inhibiting the oxidation of acetaminophen to a toxic intermediate.     

Theobromine metabolism and pharmacokinetics in pregnant and nonpregnant Sprague-Dawley rats

The plasma kinetics of po administered theobromine (TBR) were determined in timed-pregnant (P) and nonpregnant (NP) Sprague-Dawley rats at doses of 5, 10, 50, and 100 mg/kg using TBR sodium acetate with 10 μCi [8-¹⁴C]TBR as a radioactive tracer. Since plasma radioactivity consisted of >99% TBR and <1% metabolites as shown by high-performance liquid chromatographic (HPLC) methods, liquid scintillation counting was used to quantify plasma TBR. No dose-dependent kinetics were observed in mean TBR plasma half-life, volume of distribution, systemic clearance, area under the curve-dose normalized, or time to reach maximum plasma concentration in either P or NP rats. The kinetic parameters of P rats were strikingly similar to NP rats at all TBR dosage levels employed. Analysis of urinary metabolites by HPLC and a radioactivity monitoring system after a single po TBR dose of 5 and 100 mg/kg with 10 μCi [8-¹⁴C]TBR revealed similar qualitative metabolic patterns in P and NP rats. Compounds identified in the urine were TBR (39 to 62%), 6-amino-5-[N-methylformylamino]-1-methyluracil (20 to 32%), 3-methylxanthine and 7-methylxanthine (8 to 15%), 3,7-dimethyluric acid (5 to 10%), and 7-methyluric acid (5 to 7%).     

Relationship of brain cyclic nucleotide levels and the interaction of ethanol with chlordiazepoxide

The effects of combined administration of ethanol (4 g/kg) and chlordiazepoxide (CDP, 12.5 mg/kg) on mouse brain c-AMP and c-GMP levels were investigated in order to test the hypothesis that the supra-additive effect of CDP on ethanol sleep time may be related to a supra-additive alteration in brain cyclic nucleotide levels induced by the combined drugs. Ethanol alone or CDP by itself did not cause any change in brain c-AMP levels, except for a transient decrease in the cerebral cortex and midbrain at 0.5 hr after ethanol injection, as well as a transient increase in the cerebellum at 0.5 hr after CDP injection. The combined drug treatment did not result in a supra-additive effect on c-AMP levels. On the other hand, c-GMP levels were depressed significantly for 4 hr after ethanol injection especially in the cerebellum. The mice regained the righting reflex when the c-GMP levels were still about 30 per cent of control values. Ethanol and CDP together induced a supra-additive decrease of c-GMP concentrations in the cerebellum at 2 and 4 hr. This resulted in a lengthened period (about 2.5 hr) during which the cerebellar c-GMP levels were below 30 per cent of control values, and this interval coincided with the increase in sleep time, suggesting a possible relationship between these two factors. Injection of ethanol and N-demethyl-chlordiazepoxide (NDCDP) simultaneously (the latter being a metabolite of CDP) also elicited a more than additive depression of cerebellar c-GMP levels at 4 hr. These data suggest that NDCDP or its metabolite could be responsible for the supra-additive effect of CDP on the ethanol-induced decrease in cerebellar c-GMP levels.     

Effect of heme on allylisopropylacetamide-induced changes in heme and drug metabolism in the rhesus monkey (macaca mulatta)

In rhesus monkeys, in which porphyria was induced by the administration of allylisopro-pylacetamide (AIA), hepatic δ-aminolevulinic acid synthase (ALA-S) was increased. Cytochrome P-450 and associated monooxygenase activities and microsomal heme oxygenase activity were decreased in these animals. Administration of heme for 4 days concurrently with AIA prevented the induction of hepatic ALA-S but produced further decreases in cytochrome P-450 and monooxygenase activities. The decrease in heme oxygenase activity elicited by AIA alone was partially reversed. Administration of heme alone caused an impairment of hepatic drug metabolism but had no significant effect on heme metabolism. The porphyric monkeys showed elevation of porphyrin levels in blood and urine. When heme was administered concurrently with AIA, blood porphyrin levels were further elevated, while the urinary excretion of porphyrins was lower than that following treatment of monkeys with AIA. Following the administration of heme alone, blood and urinary porphyrin levels were minimally affected. These results suggest that repeated heme administration in the primate may adversely affect drug metabolism by the liver.     

Role of red cell membrane lipid peroxidation in hemolysis due to phenylhydrazine

Although red cell membrane lipid peroxidation has been identified as a consequence of certain oxidizing hemolytic drugs, the relative contribution of lipid peroxidation to red cell damage leading to hemolysis is unclear. This has been evaluated by studying the response to phenylhydrazine of vitamin E-deficient rats as compared to vitamin E-supplemented rats. Following repetitive phenylhydrazinc injections, a lower hematocrit was observed in the vitamin E-deficient group which was associated with higher levels of lipid peroxidation, as indicated by the fluorescence of lipid-containing red cell extracts. However, no significant difference in the initial extent of hemolysis following phenyl-hydrazine injection was observed. Evidence was also obtained suggesting that malonaldehyde, a decomposition product of polyunsaturated fatty acids, is capable of cross-linking hemoglobin to the red cell membrane. These findings suggest that red cell membrane lipid peroxidation is of relatively minor consequence in the acute response to phenylhydrazine but may be of importance in chronic hemolysis due to this oxidizing drug.     

Effects of physiologic concentrations of lactate, pyruvate and ascorbate on glucose metabolism in unstressed and oxidatively stressed human red blood cells

Glucose metabolism was studied in human red blood cells incubated in the presence of physiologic concentrations of ascorbate (0.1 mM) and/or lactate (2 mM) plus pyruvate (0.1 mM). The total flux through glycolysis, as measured by 14C-labeling of glycolytic intermediates, was increased about 15% by ascorbate, 30% by lactate plus pyruvate, and 40% by ascorbate plus lactate plus pyruvate. We found, however, that physiologic concentrations of ascorbate and/or lactate plus pyruvate had no effect on flux of glucose or recycling of pentoses through the hexose monophosphate shunt. Increased formation of lactate accounted for most of the observed increase in glycolysis with little change in pyruvate formation, indicating that the increased flux of reducing equivalents from glucose was stored as lactate rather than being consumed by red cell metabolism. In all experiments, there was a net increase with time in the absolute amount of both lactate and pyruvate in red cell suspensions, indicating that lactate or pyruvate present at zero time did not function as a stoichiometric source or sink for reducing equivalents. There was little effect on steady-state levels of ATP or 2,3-diphosphoglycerate. Equilibration of ascorbate between red cells and the medium was complete before the addition of 14C-labeled glucose to the medium. Glucose metabolism prevented net oxidation of ascorbate in the incubation medium. Physiologic concentrations of ascorbate, lactate and pyruvate appear to increase flux through glycolysis by increasing the turnover of ATP and/or 2,3-diphosphoglycerate. Red cells were exposed to mild oxidative stress by incubation with 0.27 mM 6-hydroxydopamine, 0.27 mM 6-aminodopamine, 0.13 mM 1,4-naphthoquinone-2-sulfonic acid or 0.27 mM phenylhydrazine. The metabolic response to oxidative stress was determined by measuring the formation of methemoglobin, pyruvate, lactate and CO2 in the presence and absence of physiologic concentrations of lactate, pyruvate and ascorbate. Lactate, pyruvate and ascorbate had no effect on the net methemoglobin accumulation but rather on the distribution of the metabolic sources of reducing equivalents and on the flux of reducing equivalents to oxygen. Physiologic lactate and pyruvate allowed increased flow of reducing equivalents from glycolysis to methemoglobin and ultimately oxygen without the necessity of increased flux through glycolysis. This was accomplished by a decrease in the ratio of newly formed lactate to newly formed pyruvate with no increase in total lactate plus pyruvate.(ABSTRACT TRUNCATED AT 400 WORDS)     

Synergistic interaction between acivicin (AT-125) and 6-thioguanine in the murine leukemia L1210. Biochemical and cytokinetic considerations

The effect of the purine antagonist acivicin (AT-125) on the metabolism and cytotoxicity of 6-thioguanine was examined in the murine leukemia L1210. Cells exposed to 5 X 10(-6) M acivicin for 18 hr followed by 10(-5) M [14C]-6-thioguanine for 2 hr accumulated 2.90 +/- 0.17 nmoles 6-thioguanine/10(6) cells compared to 0.69 +/- 0.07 nmoles 6-thioguanine/10(6) cells in untreated controls. Intracellular accumulation of 6-thioguanine monophosphate, a lethal 6-thioguanine metabolite, increased from 0.27 +/- 0.05 to 1.08 +/- 0.13 nmoles 6-thioguanine monophosphate/10(6) cells following the same acivicin exposure. A similar increment was observed for the formation of 6-thioguanine triphosphate. These alterations in 6-thioguanine metabolism were associated with an increase in the intracellular level of 5-phosphoribosyl-1-pyrophosphate, an obligatory substrate in 6-thioguanine activation (57.9 +/- 7.6 vs 13.4 +/- 2.3 ng 5-phosphoribosyl-1-pyrophosphate/10(6) cells). In contrast, there was a 50% reduction in the amount of 6-thioguanine incorporated into RNA and DNA following acivicin pretreatment. Cytofluorometric analysis revealed that an 18-hr exposure to 5 X 10(6) M acivicin increased the population 5-phase cells, which are more sensitive to the actions of 6-thioguanine, by 50% relative to untreated controls. In both suspension culture growth and soft agar studies, the sequential administration of acivicin followed by 6-thioguanine resulted in substantial growth inhibitory activity; in contrast, the effects of the reverse sequence were subadditive. Pretreatment of L1210 cells with acivicin potentiates the action of subsequently administered 6-thioguanine, and the mechanism may involve both biochemical as well as cytokinetic factors. In vivo studies involving the sequential administration of these agents appear warranted.     

Studies on the toxicity and metabolism of cadmium-thionein

Cadmium, when bound to thionein from either rat or rabbit liver, was 7 to 8 times more toxic for the rat than was ionic Cd^{2 +}. Zinc-thionein was not only non-toxic at a dose level of 2.4 mg protein-bound-Zn^{2 +}/kg, but also protected the animals against a subsequent, normally lethal dose of cadmium-thionein. In contrast with the free cation, Cd^{2 +} administered intravenously as the metallothionein accumulated to its highest concentration in the kidney and, at a lethal dose, caused severe tubular damage. After administration of non-lethal doses of ¹⁰⁹Cd^{2 +} -labelled cadmium-thionein to rats thionein-bound-¹⁰⁹Cd^{2 +} accumulated in the kidneys, but in animals that were dosed with either the ³⁵S- or ³⁵H-labelled metalloprotein, little or none of the radioactive isotope was recovered in the renal metallothionein at 48 hr. The ³⁵S and ³H isotopes, however, were incorporated into high molecular weight proteins of the kidney soluble fraction and also were excreted in the urine, both as the metallothionein and as smaller, diffusible molecules. In vitro, ³H-labelled cadmium-thionein was degraded to acid-soluble products by homogenates of rabbit kidney cortex. It is suggested that parenterally administered cadmium-thionein is taken up by the renal tubules and catabolized, probably by the lysosomes of the tubular cells, with the liberation of Cd^{2 +} ions. These cations, if present in sufficiently high concentration, cause acute renal damage. Exposure of rats, with high hepatic concentrations of cadmium-thionein, to the hepatotoxins, carbon tetrachloride and retrorsine, did not cause the transfer of Cd^{2 +} from the liver to the kidney.     

Glucose metabolism of oxidatively stressed human red blood cells incubated in plasma or medium containing physiologic concentrations of lactate,pyruvate and ascorbate

Red cells suspended in either defined medium or buffered plasma were oxidatively stressed by incubation in the presence of 1, 4-naphthoquinone-2-sulfonate at concentrations which caused less than 50% methemoglobin accumulation, stimulation of the hexose monophosphate shunt to less than 15% of capacity, and about a 30% increase in flux through glycolysis. Normal plasma concentrations of lactate and pyruvate in either defined medium or buffered plasma allowed increased contribution of reducing equivalents from glycolysis in response to oxidative stress. Increased utilization of reducing equivalents by the red cell was observed as increased accumulation of pyruvate, whereas accumulation of lactate represented storage of reducing equivalents. Exogenous lactate or pyruvate did not serve as a net electron source or sink since the total content in red cell suspensions of both lactate and pyruvate was increased during exposure to oxidative stress. If exogenous lactate had been used as a net source of reducing equivalents, the lactate concentration would have decreased during incubation of red cell suspensions. Plasma ascorbate or other constituents did not alter the qualitative response of glycolysis to oxidative stress (decreased lactate accumulation, increased pyruvate accumulation, and increased total flux through glycolysis), but plasma constituents did raise significantly the dose of oxidant agent required to elicit a given quantitative response. At levels of oxidative stress likely to be encountered in vivo, glycolysis and the hexose monophosphate shunt may be equal in importance as aerobic/antioxidant pathways.     

Inhibition of the denaturation of human gamma globulin by a mixture of D-penicillamine disulfide and copper. A possible mechanism of action of D-penicillamine in rheumatoid arthritis.

Mixtures of d-penicillamine disulfide and copper(II) mimicked sulfhydryl-blocking reagents (viz. gold thiomalate, N-ethylmaeimide and p-chloromercuribenzoic acid) in inhibiting the formation of acetic acid-insoluble heat-denatured human gamma globulin and hyperviscous heat-denatured bovine serum albumin and diluted human serum. These studies suggest a mechanism by which d-penicillamine, after oxidation to d-penicillamine disulfide, could inhibit the denaturation of synovial fluid gamma globulin and in this way possibly suppress rheumatoid arthritis.