76 results on '"Levine WG"'
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2. Effect of sequence dispersity on morphology of tapered diblock copolymers from molecular dynamics simulations.
- Author
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Levine WG, Seo Y, Brown JR, and Hall LM
- Abstract
Tapered diblock copolymers are similar to typical AB diblock copolymers but have an added transition region between the two blocks which changes gradually in composition from pure A to pure B. This tapered region can be varied from 0% (true diblock) to 100% (gradient copolymer) of the polymer length, and this allows some control over the microphase separated domain spacing and other material properties. We perform molecular dynamics simulations of linearly tapered block copolymers with tapers of various lengths, initialized from fluids density functional theory predictions. To investigate the effect of sequence dispersity, we compare systems composed of identical polymers, whose taper has a fixed sequence that most closely approximates a linear gradient, with sequentially disperse polymers, whose sequences are created statistically to yield the appropriate ensemble average linear gradient. Especially at high segregation strength, we find clear differences in polymer conformations and microstructures between these systems. Importantly, the statistical polymers are able to find more favorable conformations given their sequence, for instance, a statistical polymer with a larger fraction of A than the median will tend towards the A lamellae. The conformations of the statistically different polymers can thus be less stretched, and these systems have higher overall density. Consequently, the lamellae formed by statistical polymers have smaller domain spacing with sharper interfaces.
- Published
- 2016
- Full Text
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3. Hepatic microsomal azoreductase activity. Reactivity of azo dye substrates is determined by their electron densities and redox potentials.
- Author
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Zbaida S, Brewer CF, and Levine WG
- Subjects
- Animals, Azo Compounds chemistry, Chemical Phenomena, Chemistry, Physical, Cytochrome P-450 Enzyme System metabolism, Electrons, Male, Nitroreductases, Oxidation-Reduction, Rats, Structure-Activity Relationship, Azo Compounds metabolism, Microsomes, Liver enzymology, NADH, NADPH Oxidoreductases metabolism
- Abstract
We have previously established that microsomal reduction of azo dyes requires polar electron-donating ring substituents [e.g. -OH, -NH2, -NHCH3, or -N(CH3)2]. Reduction of dyes substituted exclusively with such moieties on either phenyl ring is insensitive to both O2 and CO (I-substrates). In contrast, azoreduction of compounds containing additional electron-withdrawing substituents (e.g. -SO3H, -COOH, -COOCH3, and -AsO3H2) on the opposite (prime) phenyl ring is sensitive to both O2 and CO (S-substrates). We have recently shown that Hammett aromatic substituent constants and redox potentials of the dyes, determined by cyclic voltammetry (CV), distinguish between I- and S-substrates. Dyes that exhibit positive Hammett sigma substituent constants on 1 of the 2 rings are S-substrates, and undergo immediate quenching of their one electron-reduced intermediates upon exposure to air, as observed by CV. In contrast, dyes that have negative Hammett sigma values on one or both rings are I-substrates, and their one electron-reduced intermediates are relatively stable in air. In this study, we have investigated the susceptibility to microsomal azoreduction of monosubstituted dyes, with ring substituents possessing a wide range of Hammett sigma values. We have observed a direct correlation between the susceptibility of these compounds to microsomal azoreduction and the Hammett sigma constant of the aromatic ring substituent, and the presence of a positive potential in the dyes observed by CV. A Hammett sigma value of -0.37 or lower is required for substrate activity. Dyes with two substituents in the prime ring confirmed the previous correlation of negative and positive Hammett sigma values with I- and S-substrate activities, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)
- Published
- 1994
4. Substrates for microsomal azoreductase. Hammett substituent effects, NMR studies, and response to inhibitors.
- Author
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Zbaida S, Brewer CF, and Levine WG
- Subjects
- Animals, Magnetic Resonance Spectroscopy, Male, Oxidation-Reduction, Rats, Rats, Inbred F344, Structure-Activity Relationship, Substrate Specificity, Azo Compounds metabolism, Coloring Agents metabolism, Microsomes enzymology
- Abstract
In previous studies on azoreduction by microsomal cytochrome P-450, we identified two classes of substrates structurally related to 4-dimethylaminoazobenzene. Both require polar electron-donating groups for binding to enzyme and are differentiated by their structure, their redox potentials, their rates of chemical and enzymic reduction, and the influence on their metabolism of inducing agents, CO and O2. Azo compounds whose reductions are insensitive to CO and O2 (I-substrates) contain electron-donating substituents on either ring. Azo compounds whose reductions are O2- and CO-sensitive (S-substrates) also contain electron-withdrawing groups on the opposite (prime) ring. For all dyes, NMR studies revealed minor differences in the chemical shifts of the protons attached to the phenyl ring substituted with electron-donating substituents (ring A). This is consistent with the narrow range of pKa's (basicity) and KM values for all substrates. However, there are significant differences in the chemical shifts of the aromatic protons of the prime ring (ring B). The difference in chemical shifts is most pronounced for aromatic protons adjacent to the prime ring substituents, showing a clear distinction between I and S substrates. Furthermore, the Hammett sigma substituent constants on the prime ring clearly distinguish between the two classes of dyes. I- and S-substrates have negative and positive sigma Hammett values, respectively. This implies that the mechanism of microsomal azoreduction is critically dependent on the charge and redox potentials of the dyes and is exclusively determined by the nature of the substituents on the prime ring.(ABSTRACT TRUNCATED AT 250 WORDS)
- Published
- 1992
5. Multiple mechanisms in hepatic microsomal azoreduction.
- Author
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Levine WG, Stoddart A, and Zbaida S
- Subjects
- Animals, Cytochrome P-450 Enzyme System metabolism, Male, Microsomes, Liver enzymology, Oxidation-Reduction, Oxygen pharmacology, Rats, Rats, Wistar, Sensitivity and Specificity, p-Dimethylaminoazobenzene metabolism, Azo Compounds metabolism, Coloring Agents metabolism, Microsomes, Liver metabolism
- Abstract
1. Microsomal reduction of azo dyes related to dimethylaminoazobenzene (DAB) is catalysed by at least two types of cytochrome P-450. The first is selectively induced by clofibrate. The second is induced by phenobarbital, beta-naphthoflavone, isosafrole, and pregnenolone-16 alpha-carbonitrile, as well as clofibrate. 2. Azoreduction by the first type of P-450 is insensitive to both O2 and CO and involves dyes with only electron-donating substituents (I substrates). 3. Azoreduction by the second type of P-450 is inhibited by both O2 and CO and involves dyes with electron-withdrawing as well as donating substituents (S substrates). 4. All azo dye substrates exhibit two negative and one positive redox potential, as measured anaerobically by cyclic voltammetry. The negative potentials reflect one- and two-electron reductions while the positive potential permits electron transfer from microsomal P-450, the redox potential for which is reported to be negative (approximately 0.35 V). The positive potential is associated with a polar electron-donating group para to the azo linkage, which is an absolute requirement for microsomal reduction. Dyes without this functional group do not exhibit positive potentials and are not reduced. 5. The first negative potential of S substrates is quenched upon admitting air to the system, whereas this potential is unaffected in I substrates. The relative stability of the one-electron reduced state may be an explanation for the differential O2 sensitivity of I and S substrate reduction.
- Published
- 1992
- Full Text
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6. Azoreductase activity by purified rabbit liver aldehyde oxidase.
- Author
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Stoddart AM and Levine WG
- Subjects
- Aldehyde Oxidase, Aldehyde Oxidoreductases antagonists & inhibitors, Aldehyde Oxidoreductases isolation & purification, Animals, Benzenesulfonates metabolism, Hydrogen-Ion Concentration, Kinetics, Nitroreductases, Proadifen pharmacology, Rabbits, Vitamin K pharmacology, Aldehyde Oxidoreductases metabolism, Azo Compounds metabolism, Liver enzymology, NADH, NADPH Oxidoreductases metabolism
- Abstract
Our laboratory has investigated the azoreduction of dimethylaminoazobenzene (DAB) and its analogs by hepatic microsomal cytochrome P450. We have extended these studies to the cytosolic fraction of the mammalian liver using the molybdoflavoenzyme, aldehyde oxidase. Purified rabbit liver aldehyde oxidase readily reduced azo dyes which are mainly water soluble and contain charged groups. Lipophilic azo dyes, although readily reduced by microsomal cytochrome P450, were either poor substrates or not reduced at all. Kinetic measurements revealed no relationship between Vmax and Km for all dyes. More extensive studies were conducted on four azo dyes, o-methyl, red, 2'-pyridyl-DAB, sulfonazo III and Orange II, with characteristic functional groups. With each of these substrates, azoreductase activity was greatest when 2-hydropyrimidine (2-OHP) was the electron donor compared to N1-methylnicotinamide (N-MN), propionaldehyde and butyraldehyde. With 2-OHP as the electron donor, o-methyl red and 2'-pyridyl DAB exhibited maximal activity at pH 5.0 while sulfonazo III and Orange II showed maximal activity at pH 9.5 and 7.0, respectively. Km values for o-methyl red and 2'-pyridyl DAB were lower at their pH optima whereas that for sulfonazo III was higher at its pH optimum. There was also no correlation between maximal activity and Km; apparently Km is not a primary determinant for activity. The degree of ionization of function groups depends on pH. Since highest activity is seen at that pH in which maximal ionization of the substrate occurs, it can be concluded that rate of reduction is at least partially dependent on the charged state of the substrate. Azoreduction was inhibited by menadione and SKF 525-A. Sensitivity to inhibition by menadione was greatest at the pH where 2-OHP exhibited considerably higher activity than N-MN, but no differential was seen at the pH where activities with the two-electron donors were similar. On the other hand, sensitivity of azoreductase activity to inhibition by SKF 525-A was the same irrespective of electron donor, indicating that the mechanisms for these two inhibitors were different.
- Published
- 1992
- Full Text
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7. Role of electronic factors in binding and reduction of azo dyes by hepatic microsomes.
- Author
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Zbaida S and Levine WG
- Subjects
- Animals, Cytochrome P-450 Enzyme System metabolism, Electrons, Kinetics, Male, Mass Spectrometry, Microsomes, Liver enzymology, Oxidation-Reduction, Rats, Rats, Inbred F344, Sulfates metabolism, Azo Compounds metabolism, Coloring Agents metabolism, Microsomes, Liver metabolism
- Abstract
The factors which regulate the binding and reduction of azo dyes by rat liver microsomes have been investigated. Azo dyes having both electron-donating and -withdrawing substituents were reduced more readily both enzymically and chemically (dithionite) than those containing electron-donating substituents alone, which is consistent with less negative oxidation-reduction potentials of the former. A linear correlation between Vmax and Km was seen for substrates having only electron-donating substituents, suggesting a possible inverse relation between binding affinity and rate of reduction. This relationship was not apparent for substrates having both electron-donating and -withdrawing substituents. A lower Km was seen with substrates having a greater number of heteroatoms bearing nonbonding electrons in either electron-donating or -withdrawing groups. Furthermore, more basic dyes, which have a higher density of nonbonding electrons, showed an inverse correlation with both Vmax and Km. The requirement of nonbonding electrons was also observed with the binding of the fully reduced amine metabolites to microsomal cytochrome P-450. Type II binding spectra were observed for both aniline and 4-chloroaniline, but not with aniline derivatives bearing electron-withdrawing substituents such as methyl anthranilate and methyl-4-aminobenzoate. Electron-withdrawing substituents increase delocalization of nonbonding electrons on the amino residue; consequently, these are no longer available for binding to the enzyme. The binding constant of the reduced metabolite, aniline (36 microM), relative to the Km values of substrate azo dyes (range 0.31-1.73 microM) implies that the more weakly bound amine metabolites are readily released from the binding/catalytic site.(ABSTRACT TRUNCATED AT 250 WORDS)
- Published
- 1992
8. Protein binding, nuclear translocation and biliary secretion of metabolites of 3'-methyl-N,N-dimethyl-4-aminoazobenzene during hepatocarcinogenesis in rats.
- Author
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Srinivasan K, Levine WG, and Bhargava MM
- Subjects
- 2-Acetylaminofluorene toxicity, Animals, Body Weight drug effects, Chromatography, Gel, Cytosol chemistry, Cytosol metabolism, Electrophoresis, Polyacrylamide Gel, Liver chemistry, Liver metabolism, Liver Neoplasms, Experimental chemically induced, Male, Methyldimethylaminoazobenzene toxicity, Organ Size drug effects, Protein Binding, Proteins metabolism, Rats, Rats, Inbred Strains, Bile metabolism, Carcinogens toxicity, Cell Nucleus metabolism, Liver Neoplasms, Experimental metabolism, Methyldimethylaminoazobenzene metabolism
- Abstract
1. The hepatic content, biliary excretion, cytosolic protein binding and nuclear translocation of metabolites of i.v. administered 14C-3'-methyl-N,N-dimethyl-4-aminoazobenzene (3'-methyl-DAB) were investigated in rats at various stages of 2-acetamidofluorene (AAF)-induced hepatocarcinogenesis. 2. At nodular and post-nodular stages biliary excretion of radioactive metabolites was decreased, although hepatic content of radioactivity was similar to controls not dosed with AAF. The secretion in bile of a major azo dye binding protein was also decreased at these stages. 3. Binding of dye metabolites to cytosolic proteins was decreased by 40% at nodular and post-nodular stages compared to controls. 4. Translocation in vitro of dye metabolites from cytosol to nucleus at nodular and post-nodular stages was 40% less than that of controls. Since specific soluble proteins control translocation from cytosol into the nucleus (and bile), this decreased binding of metabolites may explain the diminished translocation of carcinogen metabolites into the nucleus.
- Published
- 1991
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9. Two classes of azo dye reductase activity associated with rat liver microsomal cytochrome P-450.
- Author
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Levine WG and Zbaida S
- Subjects
- Animals, Cytochrome P-450 Enzyme System classification, In Vitro Techniques, NADH, NADPH Oxidoreductases classification, NADPH-Ferrihemoprotein Reductase classification, NADPH-Ferrihemoprotein Reductase metabolism, Nitroreductases, Rats, Structure-Activity Relationship, Substrate Specificity, p-Dimethylaminoazobenzene chemistry, Cytochrome P-450 Enzyme System metabolism, Microsomes, Liver metabolism, NADH, NADPH Oxidoreductases metabolism, p-Dimethylaminoazobenzene metabolism
- Published
- 1991
- Full Text
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10. A novel application of cyclic voltammetry for direct investigation of metabolic intermediates in microsomal azo reduction.
- Author
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Zbaida S and Levine WG
- Subjects
- Animals, Azo Compounds chemistry, Electrochemistry methods, NADH, NADPH Oxidoreductases metabolism, Nitroreductases, Oxidation-Reduction, Rats, Substrate Specificity, p-Dimethylaminoazobenzene chemistry, p-Dimethylaminoazobenzene metabolism, Azo Compounds metabolism, Microsomes, Liver metabolism
- Abstract
We have established that reduction of azo dyes structurally related to 4-(dimethylamino)-azobenzene (DAB) by rat liver microsomal cytochrome P-450 requires a polar electron-donating substituent on one ring. Reduction of azo dyes containing only electron-donating substituents is insensitive to both oxygen and CO (I substrates). However, reduction of azo dyes containing electron-withdrawing substituents as well is sensitive to both oxygen and CO (S substrates). Positive, irreversible potentials were observed by cyclic voltammetry (CV) in anhydrous solutions for both I and S substrates but not for the nonreducible azo dyes. This positive potential permits electron transfer to dyes from NADPH-cytochrome P-450 reductase and from cytochrome P-450, both of which have negative potentials. Reduction products retaining electron-donating groups (amino, phenolic) also exhibited positive potentials, implying that these groups contribute the positive potential in the dye molecule. All substrates also exhibited two negative potentials with a clear distinction between I and S substrates. The latter exhibited, on average, potentials that were less negative than the former by about 0.6 V. This is consistent with the more rapid reduction of S substrates by liver microsomes [Zbaida and Levine (1990) Biochem. Pharmacol. 40, 2415-2423]. Admitting air to the system quenched the first potential for S but not for I substrates, which is consistent with the oxygen sensitivities of their reduction. Addition of water significantly shifted the second negative potential to a more positive value, ultimately leading to single negative potential. The water permits rapid protonation of the two-electron-reduced intermediate, facilitating further reduction.(ABSTRACT TRUNCATED AT 250 WORDS)
- Published
- 1991
- Full Text
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11. Metabolism of azo dyes: implication for detoxication and activation.
- Author
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Levine WG
- Subjects
- Animals, Azo Compounds chemistry, Azo Compounds toxicity, Bacteria metabolism, Biotransformation, Carcinogens metabolism, Coloring Agents chemistry, Coloring Agents toxicity, Humans, Inactivation, Metabolic, Mutagenesis, Oxidation-Reduction, Azo Compounds metabolism, Coloring Agents metabolism
- Abstract
Azo dyes are consumed and otherwise utilized in varying quantities in many parts of the world. Such widely used chemicals are of great concern with regard to their potential toxicity and carcinogenic properties. Their metabolism has been studied extensively and is significant for detoxication and metabolic activation. Both oxidative and reductive pathways are involved in these processes. The majority of azo dyes undergo reduction catalyzed by enzymes of the intestinal microorganisms and/or hepatic enzymes including microsomal and soluble enzymes. The selectivity of substrate and enzyme may to a large extent be determined by the oxygen sensitivity of reduction since a normal liver is mainly aerobic in all areas, whereas the microorganisms of the lower bowel exist in an anaerobic environment. However, it should be pointed out that the pO2 of centrilobular cells within the liver is only a fraction that of air, where pO2 = 150 torr. Therefore, an azo dye reduction experiment performed aerobically may not be an accurate predictor of reductive metabolism in all areas of the liver. Many of the azo dyes in common use today have highly charged substituents such as sulfonate. These resist enzymic attack and for the most part are poorly absorbed from the intestinal tract, providing poor access to the liver, the major site of the mixed-function oxidase system. Lipophilic dyes, such as DAB, which are often carcinogenic, readily access oxidative enzymes and are activated by both mixed-function oxidase and conjugating systems. Reduction of the carcinogenic dyes usually leads to loss of carcinogenic activity. By contrast, most of the highly charged water-soluble dyes become mutagenic only after reduction. Even then, most of the fully reduced amines required oxidative metabolic activation. An outstanding example is the potent human bladder carcinogen benzidine, which derives from the reduction of several azo dyes. Many problems regarding mutagenic and carcinogenic activation remain to be solved. At the present time, it is apparent that both oxidative and reductive pathways yield toxic products. Toxicologic assessment of azo dyes must consider all pathways and particularly the oxygen sensitivity of azoreduction. This is critical in the treatment of waste from chemical plants where there is a great need for soil bacteria which catalyze reduction aerobically. Consideration of secondary pathways are also of great concern. For example, azoreduction of carcinogenic dyes such as DAB removes carcinogenic activity although oxidative metabolism of the primary amines yield mutagenic products. Such apparent dilemmas must be dealt with when considering metabolism/toxicity relationships for azo dyes.
- Published
- 1991
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12. Characteristics of two classes of azo dye reductase activity associated with rat liver microsomal cytochrome P450.
- Author
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Zbaida S and Levine WG
- Subjects
- Animals, Carbon Monoxide pharmacology, Male, Oxidation-Reduction, Oxygen pharmacology, Rats, Rats, Inbred Strains, Structure-Activity Relationship, Azo Compounds metabolism, Coloring Agents metabolism, Cytochrome P-450 Enzyme System physiology, Microsomes, Liver enzymology, NADPH-Ferrihemoprotein Reductase physiology, p-Dimethylaminoazobenzene metabolism
- Abstract
Azo dyes are reduced to primary amines by the microsomal enzymes NADPH-cytochrome P450 reductase and cytochrome P450. Amaranth, a highly polar dye, is reduced almost exclusively by rat liver microsomal cytochrome P450 and the reaction is inhibited almost totally by oxygen or CO. Activity is induced by pretreatment with phenobarbital or 3-methylcholanthrene. In contrast, microsomal reduction of the hepatocarcinogen dimethylaminoazobenzene (DAB), a lipid soluble, weakly polar compound, is insensitive to both oxygen and CO. However, reconstitution of activity with purified NADPH-cytochrome P450 reductase and a partially purified cytochrome P450 preparation indicates that activity is catalyzed almost exclusively by cytochrome P450. Activity is induced by clofibrate but not phenobarbital, beta-naphthoflavone, 3-methylcholanthrene, isosafrol, or pregnenolone-16 alpha-carbonitrile. These observations suggest the existence of at least two classes of azoreductase activity catalyzed by cytochrome P450. To investigate this possibility, the reduction of a number of azo dyes was investigated using microsomal and partially purified systems and the characteristics of the reactions were observed. Microsomal reduction of azo dyes structurally related to DAB required a polar electron-donating substituent on one ring. Activity was insensitive to oxygen and CO if the substrates had no additional substituents on either ring or contained only electron-donating substituents. Introduction of an electron-withdrawing group into the prime ring conferred oxygen and CO sensitivity on the reaction. Substrates in the former group are referred to as insensitive and substrates in the latter group as sensitive. Inhibitors of cytochrome P450 activity depressed reduction of both insensitive and sensitive substrates. In a fully reconstituted system containing lipid, highly purified NADPH-cytochrome P450 reductase and a partially purified cytochrome P450 preparation, rates of reduction of various insensitive substrates varied several-fold, whereas rates of reduction of sensitive substrates varied by three orders of magnitude. Using purified enzymes, each of the insensitive substrates was shown to be reduced by reductase alone, but only at a fraction of the rate seen in the fully reconstituted system, implying that reducing electrons were transferred to the dyes mainly from cytochrome P450. Conversely, there was substantial, in some cases almost exclusive, reduction of sensitive substrates by purified reductase alone and almost no inhibition by CO. Their reduction, however, was inhibited by CO in microsomal systems.(ABSTRACT TRUNCATED AT 250 WORDS)
- Published
- 1990
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13. Microsomal reduction of dimethylaminoazobenzene (DAB). Selective effects of carbon dioxide asphyxiation versus decapitation of animals and variation with age, sex and species.
- Author
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Levine WG and Perez E
- Subjects
- Age Factors, Animals, Cricetinae, Death, Female, Fetus enzymology, Guinea Pigs, Kinetics, Male, Mesocricetus, Mice, Rabbits, Rats, Rats, Inbred Strains, Sex Factors, Species Specificity, Microsomes, Liver enzymology, NADH, NADPH Oxidoreductases metabolism
- Published
- 1990
- Full Text
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14. Azoreduction of dimethylaminoazobenzene (DAB) in primary cultures of rat hepatocytes. Effect of hypolipidemic agents.
- Author
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Stoddart AM and Levine WG
- Subjects
- Animals, Cells, Cultured, Clofibrate pharmacology, Cytochrome P-450 CYP4A, Cytochrome P-450 Enzyme System metabolism, Enzyme Induction drug effects, Liver drug effects, Male, Mixed Function Oxygenases antagonists & inhibitors, Mixed Function Oxygenases metabolism, NADH, NADPH Oxidoreductases antagonists & inhibitors, NADH, NADPH Oxidoreductases metabolism, Nafenopin pharmacology, Oxidation-Reduction, Rats, Rats, Inbred Strains, Azo Compounds metabolism, Hypolipidemic Agents pharmacology, Liver metabolism, p-Dimethylaminoazobenzene metabolism
- Abstract
This laboratory has investigated the azoreduction of the hepatocarcinogen, N,N-dimethyl-4-aminoazobenzene (DAB), by hepatic microsomal cytochrome P-450 (P-450) and its specific induction by the hypolipidemic drug, clofibrate. To extend these studies further, a primary hepatocyte culture system was developed as a model. Hepatocytes isolated from male Sprague-Dawley rats were incubated in a basal medium containing fetal calf serum, insulin, and hydrocortisone for up to 96 hr with varying concentrations of clofibrate or nafenopin, a related hypolipidemic agent. Both DAB azoreductase and laurate hydroxylase activities decreased rapidly in control cultures. However, there was gradual marked induction of both activities in medium supplemented with clofibrate: hydrocortisone was required for induction. Nafenopin stabilized and induced DAB azoreductase and laurate hydroxylase activities, respectively. The responses of both activities were dose dependent. DAB azoreductase and laurate hydroxylase activities in control hepatocytes retained their ability to respond to clofibrate for up to 96 hr, although the response gradually diminished after 24 hr. In all cases, maximal induction of both enzyme activities was observed 72 hr after addition of drug. Phenobarbital and beta-naphthoflavone did not induce DAB azoreduction, although the normal induction of other P-450-catalyzed pathways, 7-ethoxycoumarin O-deethylation and ethlymorphine N-demethylation, were seen. Suppression of DAB azoreductase activity by inhibitors of P-450 activity confirmed the involvement of this enzyme in DAB azoreduction. The results demonstrate that a primary culture of rat hepatocytes is a useful model for studying the regulation of DAB azoreductase activity.
- Published
- 1990
15. Transcriptional and translational control of nafenopin-induced ornithine decarboxylase activity in rat liver.
- Author
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Lam DC and Levine WG
- Subjects
- Animals, Cycloheximide pharmacology, Dactinomycin pharmacology, Enzyme Induction drug effects, Male, Ornithine Decarboxylase genetics, Phentolamine pharmacology, Rats, Carboxy-Lyases biosynthesis, Liver enzymology, Nafenopin pharmacology, Ornithine Decarboxylase biosynthesis, Propionates pharmacology, Protein Biosynthesis drug effects, Transcription, Genetic drug effects
- Abstract
The induction of liver ornithine decarboxylase activity by nafenopin was studied in rats. The alpha-adrenergic antagonist, phentolamine, injected 0.5 hr before nafenopin, greatly potentiated the response. Actinomycin D, injected before nafenopin, did not affect nafenopin-induced ornithine decarboxylase activity whereas the potentiating effect of phentolamine was inhibited. In contrast, administration of actinomycin D 5.0 hr after nafenopin markedly enhanced nafenopin-induced activity. The induction of ornithine decarboxylase activity by nafenopin, as well as the potentiating effect of phentolamine was inhibited by 1,3-diaminopropane and cycloheximide, implying a requirement for synthesis of new enzyme protein for both effects. These results suggest that nafenopin induced ODC through a posttranscriptional mechanism whereas the potentiating effect of phentolamine requires transcription of new messenger RNA.
- Published
- 1980
16. Uptake and hepatobiliary fate of two hepatocarcinogens, N,N-dimethyl-4-aminoazobenzene and 3'-methyl-N,N-dimethyl-4-aminoazobenzene, in the rat.
- Author
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Samuels AR, Bhargava MM, and Levine WG
- Subjects
- Animals, Chromatography, High Pressure Liquid, Half-Life, Male, Rats, Rats, Inbred Strains, Tissue Distribution, Bile metabolism, Liver metabolism, Methyldimethylaminoazobenzene metabolism, p-Dimethylaminoazobenzene analogs & derivatives, p-Dimethylaminoazobenzene metabolism
- Abstract
Two radiolabeled hepatocarcinogens, N,N-dimethyl-4-aminoazobenzene (DAB) and 3'-methyl-N,N-dimethyl-4-aminoazobenzene (3'-Me-DAB), were rapidly cleared from the blood of rats after i.v. administration, with half-lives of 40 and 70 sec, respectively. Rates of hepatic uptake and biliary secretion of [14C]-3'-Me-DAB were double that of [14C]DAB within 30 min of administration. Two hr after azo dye injection, the hepatic output into bile of [14C]-3'-Me-DAB-derived radioactivity was three times that of [14C]DAB. Fifty and 75% of the total 3'-Me-DAB-derived radioactivity was recovered in blood, liver, and bile 30 and 120 min after injection while only 30 to 40% of the administered [14C]DAB-derived radioactivity was recovered at these times. We postulate the existence of an extrahepatic azo dye accumulation site which may compete with the ability of the liver to clear azo dye from the circulation and which releases 3'-Me-DAB-derived radioactivity more readily than that of DAB. Azo dye metabolites were isolated from liver, bile, and blood. The chromatographic pattern of liver metabolites generated in vivo by rats which received either hepatocarcinogen was obtained and compared with that of biliary metabolites. With either azo dye, some metabolites were located exclusively in the liver, some were secreted immediately into bile, while others were present in both liver and bile, indicating selectivity in biliary excretion.
- Published
- 1983
17. Glutathione, lipid peroxidation and regulation of cytochrome P-450 activity.
- Author
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Levine WG
- Subjects
- Animals, In Vitro Techniques, Iron pharmacology, Liver metabolism, Male, Rats, Rats, Inbred Strains, p-Dimethylaminoazobenzene metabolism, Cytochrome P-450 Enzyme System analysis, Glutathione physiology, Lipid Peroxides metabolism
- Abstract
Depletion of hepatic glutathione leads to an increase in lipid peroxidation and depression of cytochrome P-450-catalyzed metabolism of the azo dye carcinogen, N,N-dimethyl-4-aminoazobenzene. This contributes to the marked decrease in biliary excretion of N-demethylated metabolites of the dye. Parallel time courses are seen for decreased hepatic glutathione, enhanced lipid peroxidation and depressed excretion of dye metabolites. In vitro metabolism of DAB by hepatic 10,000 g supernatant fractions is depressed by iron only after glutathione depletion. In view of the iron requirement for microsomal lipid peroxidation, it is proposed that glutathione depletion leads to an increase in the intracellular iron available for activation of lipid peroxidation. In this way, glutathione may contribute to the regulation of cytochrome P-450 activity.
- Published
- 1982
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18. Clofibrate selectively induces azoreduction of dimethylaminoazobenzene (DAB) by rat liver microsomes.
- Author
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Levine WG and Raza H
- Subjects
- Aerobiosis, Anaerobiosis, Animals, Biotransformation, Hydroxylation, Kinetics, Lauric Acids metabolism, Male, Microsomes, Liver drug effects, NADPH-Ferrihemoprotein Reductase metabolism, Oxidation-Reduction, Rats, Rats, Inbred Strains, Clofibrate pharmacology, Microsomes, Liver metabolism, p-Dimethylaminoazobenzene metabolism
- Published
- 1986
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19. Effect of dimethyl sulfoxide on the hepatic disposition of chemical carcinogens.
- Author
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Levine WG
- Subjects
- Animals, Bile metabolism, Cytosol metabolism, Female, Liver ultrastructure, Microsomes, Liver metabolism, Rats, Time Factors, Benzopyrenes metabolism, Carcinogens metabolism, Dimethyl Sulfoxide pharmacology, Liver metabolism, Methylcholanthrene metabolism
- Published
- 1975
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20. Effect of phenobarbital and beta-naphthoflavone on oxidative metabolism of N,N-dimethyl-4-aminoazobenzene by regenerating rat-liver microsomes and its response to sulphydryl compounds.
- Author
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Raza H and Levine WG
- Subjects
- Animals, Enzyme Induction drug effects, Hepatectomy, Kinetics, Male, Microsomes, Liver drug effects, Oxidation-Reduction, Rats, Rats, Inbred Strains, beta-Naphthoflavone, p-Aminoazobenzene metabolism, Benzoflavones pharmacology, Flavonoids pharmacology, Liver Regeneration, Microsomes, Liver metabolism, Phenobarbital pharmacology, Sulfhydryl Compounds pharmacology, p-Dimethylaminoazobenzene metabolism
- Abstract
The metabolism of the hepatocarcinogen, N,N-dimethyl-4-aminoazobenzene (DAB) is catalysed by selective forms of cytochrome P-450. DAB metabolism has been studied using microsomes from regenerating rat liver prepared 1, 2, 3, 7 and 10 d after partial hepatectomy. Greatly decreased N-demethylation of DAB was seen during liver regeneration, while virtually no effect on ring-hydroxylation was observed. Glutathione stimulated N-demethylation and ring-hydroxylation of DAB, while metabolism of the corresponding secondary amine N-methyl-4-aminoazobenzene (MAB) was not affected. During regeneration, response to the thiol was depressed in the early stages but later returned to normal. beta-Naphthoflavone (BNF) specifically induced N-demethylation of DAB. Induced activity was not depressed during liver regeneration. Phenobarbital (PB) induced total metabolism, which was depressed during regeneration. This indicates greater stability of BNF-induced cytochrome P-450 compared to control and PB-induced cytochrome P-450. The results indicate that during liver regeneration the metabolism of DAB associated with activation (N-demethylation) is depressed, whereas that associated with detoxication (ring-hydroxylation) is only slightly affected. This confirms the involvement of different forms of cytochrome P-450 in DAB metabolism.
- Published
- 1986
- Full Text
- View/download PDF
21. Biliary excretion of drugs and other xenobiotics.
- Author
-
Levine WG
- Subjects
- Aging, Animals, Bile Acids and Salts metabolism, Bile Ducts anatomy & histology, Cholestasis metabolism, Cyclic AMP metabolism, Liver enzymology, Liver metabolism, Liver Circulation, Molecular Weight, Protein Binding, Sex Factors, Subcellular Fractions metabolism, Bile metabolism, Pharmaceutical Preparations metabolism
- Published
- 1981
22. In vivo and in vitro effects of amrinone and milrinone on hepatic xenobiotic metabolism in rats.
- Author
-
Raza H and Levine WG
- Subjects
- Animals, Cytochrome P-450 Enzyme System biosynthesis, Enzyme Activation drug effects, Enzyme Induction drug effects, In Vitro Techniques, Liver drug effects, Male, Microsomes, Liver drug effects, Microsomes, Liver enzymology, Microsomes, Liver metabolism, Milrinone, Mixed Function Oxygenases metabolism, Rats, Rats, Inbred Strains, Amrinone pharmacology, Liver metabolism, Pharmaceutical Preparations metabolism, Pyridones pharmacology, Vasodilator Agents pharmacology
- Abstract
Studies were performed on the response of hepatic xenobiotic metabolizing enzymes to in vitro and in vivo exposure to amrinone and milrinone, two new inotropic compounds used in congestive heart failure. Both drugs exerted selective effects on various cytochrome P-450-dependent metabolic activities as well as conjugating pathways. Aminopyrine N-demethylation was selectively inhibited by in vitro addition of milrinone but not amrinone, and laurate hydroxylation was inhibited by both drugs. Cytosolic glutathione-S-transferase activity was profoundly inhibited by in vitro addition of both drugs. In vivo administration of either drug did not lead to significant inhibition of the pathways studied other than laurate hydroxylation which was depressed 20-30%. Irreversible binding of [14C]-amrinone-derived radioactivity to microsomal protein was partially NADPH-dependent. Inhibition by SKF 525-A, alpha-naphthoflavone and various antioxidants was observed. No binding of [14C]-milrinone-derived radioactivity was seen. It is suggested that amrinone may selectively inhibit certain hepatic drug-metabolizing enzymes through metabolic electrophilic intermediates.
- Published
- 1987
- Full Text
- View/download PDF
23. Biliary excretion of 3,4-benzpyrene in nafenopin-treated rats.
- Author
-
Levine WG and Bognacki J
- Subjects
- Animals, Female, Liver metabolism, Methylcholanthrene pharmacology, Rats, Time Factors, Nafenopin pharmacology, Propionates pharmacology
- Abstract
When rats treated for 2 days with nafenopin are injected i.v. with 3H-3,4-benzpyrene (BP), blood disappearance rates and liver levels of the carcinogen and the rate of biliary excretion of its metabolites are, in the main, similar to those of nontreated rats. This is in accord with the observation that nafenopin does not inhibit the metabolism of BP, which is the rate-limiting step in its biliary excretion. On the other hand, when 3H-BP metabolites are injected, nafenopin pretreatment slightly retards their rate of plasma disappearance and markedly inhibits their biliary excretion, as it does other organic anions. When rats are pretreated with 3-methylcholanthrene, the rate of metabolism of 3H-BP and consequently the biliary excretion of its metabolites is greatly stimulated. In this instance, metabolism may no longer be rate-limiting in the overall biliary excretion process and inhibition by nafenopin of liver-to-bile transport of metabolites can be observed. Since nafenopin pretreatment stimulates the synthesis of new liver tissue, it is presently a matter of conjecture as to whether or not the newly formed hepatocytes have the capacity to take up and excrete BP and its metabolites or whether nafenopin inhibits transport in all liver tissue.
- Published
- 1976
24. Effect of hypolipidemic drugs on the metabolism of lauric acid and dimethylaminoazobenzene by rat liver microsomes.
- Author
-
Raza H and Levine WG
- Subjects
- Animals, Hydroxylation, In Vitro Techniques, Male, Microsomes, Liver drug effects, Oxidation-Reduction, Rats, Rats, Inbred Strains, Hypolipidemic Agents pharmacology, Lauric Acids metabolism, Microsomes, Liver metabolism, p-Dimethylaminoazobenzene metabolism
- Published
- 1987
- Full Text
- View/download PDF
25. Effect of nafenopin (SU-13,437) on liver function: mechanism of choleretic effect.
- Author
-
Levine WG, Braunstein HR, and Mejier DK
- Subjects
- Animals, Bile analysis, Bile Acids and Salts analysis, Bile Ducts, Intrahepatic metabolism, Biliary Fistula, Body Water drug effects, Body Weight drug effects, Enterohepatic Circulation, Erythritol metabolism, Kinetics, Liver metabolism, Male, Nafenopin metabolism, Organ Size drug effects, Plasma Volume drug effects, Rats, Sodium analysis, Bile metabolism, Liver drug effects, Nafenopin pharmacology, Propionates pharmacology
- Abstract
Administration of nafenopin (SU-13-437) to male rats for two days leads to a doubling of bile production and a 50% increase in liver weight. These two effects have been shown not to be directly interrelated. A marked decrease in biliary salt concentration suggests that the bile salt independent flow is stimulated. The extra bile produced is probably of canalicular origin since bile to plasma concentration ratios of erythritol are unchanged. At least three polar metabolites of nafenopin have been observed in rat bile. Observations in rats with partial biliary fistulas indicate that the drug and its metabolites undergo extensive entero-hepatic circulation. Our studies support the view that much of the enhanced bile flow is associated with the presence of nafenopin and/or its metabolites within the hepatobiliary system. However, the response is too extensive to be explained merely by osmotic choleresis. Induced structural changes in the liver may also account forsome of this effect.
- Published
- 1975
- Full Text
- View/download PDF
26. Glutathione and hepatic mixed-function oxidase activity.
- Author
-
Levine WG
- Subjects
- Animals, Bile metabolism, Formaldehyde metabolism, Glutathione Peroxidase metabolism, Ketones pharmacology, Lipid Peroxides metabolism, Liver metabolism, Oxidation-Reduction, Rats, p-Dimethylaminoazobenzene metabolism, Glutathione metabolism, Liver enzymology, Mixed Function Oxygenases metabolism
- Abstract
It is apparent that hepatic GSH may function in drug metabolism not only as a substrate for conjugation but also in regulation of cytochrome P-450 activity. The remarkable aspect of the latter activity is its specificity. Loss of hepatic GSH depresses N-demethylation of DAB while ring hydroxylation is unaffected. On the other hand, the effect is to some degree nonspecific in that control as well as PB- or MC-induced N-demethylation is inhibited. Thus the response may not simply be specific to one isozyme of cytochrome P-450 but may be associated with one aspect of the enzymic activity of several cytochrome P-450 isozymes (i.e., N-demethylation). We have postulated that sensitivity of this activity to lipid peroxidation underlies the relationship to GSH since the tripeptide serves as a major protection against hepatic lipid peroxidation and its consequences. It is as yet not clear as to how or why this particular aspect of P-450 activity is more sensitive to lipid peroxidation than are other activities such as ring hydroxylation. Ongoing investigations include attempts to identify the cytochrome P-450 isozyme(s) which inhibit this response to GSH depletion. GSH-lipid peroxidation relationships have already been reported with isolated hepatocytes, and there may be a possible connection between this and the relative instability of cytochrome P-450 in cultured hepatocytes. Another factor which may be involved is heme oxygenase activity, which is markedly induced in the liver after GSH depletion, after cobalt administration (which also depresses cytochrome P-450 activity), and during incubation of isolated hepatocytes. This enzyme catalyzes the rate-limiting step in heme breakdown and may contribute to the loss of cytochrome P-450 activity associated with GSH depletion.
- Published
- 1983
- Full Text
- View/download PDF
27. Regulation of thiol environment of the N-demethylation and ring hydroxylation of N,N-dimethyl-4-aminoazobenzene (DAB) by rat liver microsomes.
- Author
-
Levine WG
- Subjects
- Animals, Cytochrome P-450 Enzyme System analysis, Dealkylation, Dithionitrobenzoic Acid pharmacology, Dithiothreitol pharmacology, Glutathione pharmacology, Hydroxylation, In Vitro Techniques, Male, NADPH-Ferrihemoprotein Reductase analysis, Phenobarbital pharmacology, Rats, Rats, Inbred Strains, p-Aminoazobenzene analogs & derivatives, p-Aminoazobenzene metabolism, Microsomes, Liver metabolism, Sulfhydryl Compounds pharmacology, p-Dimethylaminoazobenzene metabolism
- Abstract
A study was conducted on the regulation by thiol environment of microsomal metabolism of the azo dye hepatocarcinogen, N,N-dimethyl-4-aminoazobenzene (DAB). Physiological concentrations of glutathione (GSH) stimulated N-demethylation and ring hydroxylation of the dye in normal and phenobarbital (PB)-treated microsomes. However, little effect of GSH was seen with microsomes from beta-naphthoflavone (BNF)-treated rats. The synthetic thiol, dithiothreitol (DTT), stimulated ring-hydroxylation of DAB but inhibited N-demethylation at all concentrations in control nd PB-induced microsomes. A biphasic response to DTT was obtained with BNF microsomes; inhibition of N-demethylation was seen only at low concentrations (0.1 mM) and a return to control values occurred at higher concentrations. DTT inhibition was shown to be specific for the first N-demethylation step, whereas the second was slightly stimulated at concentrations greater than 3.0 mM. Agents which alkylate [N-ethylmaleimide (NEM), p-hydroxymercuribenzoate] or oxidize [5,5'-dithiobis(nitrobenzoic acid) or Ellman's reagent] protein SH groups inhibited DAB metabolism. Inhibition of microsomal NADPH-cytochrome c reductase activity by p-hydroxymercuribenzoate required an order of magnitude more inhibitor than was needed to block DAB metabolism. This suggests that DAB metabolism requires viable SH groups other than those involved in NADPH-cytochrome c reductase activity. NEM, in contrast, inhibited the N-demethylation of DAB and NADPH-cytochrome c reductase at approximately the same concentrations. Ring-hydroxylation was stimulated by high (greater than 1 mM) concentrations of NEM, implying a different enzymic mechanism for this pathway.(ABSTRACT TRUNCATED AT 250 WORDS)
- Published
- 1986
28. Effect of long-term administration of delta 9-tetrahydrocannabinol on hepatic mixed-function oxidase systems in the rat.
- Author
-
Morrill GA, O'Connell ME, Kostellow AB, and Levine WG
- Subjects
- Aminopyrine metabolism, Animals, Benzo(a)pyrene metabolism, Body Weight drug effects, Chemical Phenomena, Chemistry, Corticosterone blood, Dronabinol administration & dosage, Eating drug effects, Female, Oxidation-Reduction, Rats, Rats, Inbred Strains, Time Factors, Dronabinol pharmacology, Liver metabolism, Mixed Function Oxygenases metabolism
- Abstract
Chronic oral treatment of young female Fischer rats with 25 mg/kg delta 9-tetrahydrocannabinol (THC) per day inhibited aminopyrine demethylation and significantly increased benzo[a]pyrene oxidation by the liver. THC treatment also elevated serum corticosterone levels and produced a significant loss of body weight. The weight loss was not due to vehicle or food intake (pair-feeding). Pair-feeding did, however, produce a stimulation of both mixed function oxidase pathways as well as a marked elevation in serum corticosterone levels. The results indicate that THC has a differential effect on mixed function oxidase pathways in the liver that is not directly related to food intake or corticosterone levels.
- Published
- 1985
- Full Text
- View/download PDF
29. Effect of glutathione on the metabolism of N,N-dimethyl-4-aminoazobenzene by rat liver microsomes.
- Author
-
Levine WG and Lee S
- Subjects
- Animals, Cysteine pharmacology, Dithiothreitol pharmacology, Edetic Acid pharmacology, Male, Rats, Rats, Inbred Strains, Glutathione pharmacology, Microsomes, Liver metabolism, p-Dimethylaminoazobenzene metabolism
- Abstract
Rat liver microsomes catalyze both the N-demethylation and the 4'-hydroxylation of the azo dye carcinogen N,N-dimethyl-4-aminoazobenzene (DAB). It was found that addition of glutathione (GSH) to the microsomal system markedly stimulated both metabolic pathways. This occurred in the presence of either added NADPH or an NADPH-generating system. It was necessary that GSH be present when the reaction began; if added later, stimulation did not occur. This suggested a direct effect on microsomes rather than a chemical interaction with metabolic intermediates of DAB. Since stimulation occurred even in the presence of EDTA, the GSH effect cannot be satisfactorily explained in terms of suppression of lipid peroxidation which is totally inhibited by EDTA. Cysteine and cysteamine also stimulated both pathways but were less potent than was GSH; oxidized GSH was without significant effect. Dithiothreitol and beta-mercaptoethanol stimulated 4'-hydroxylation but inhibited N-demethylation, even in the presence of stimulatory concentrations of GSH. Apparently, the synthetic sulfhydryl compounds act through a mechanism different from that of GSH. Inhibition by dithiothreitol is consistent with formation of an N-oxide intermediate during N-demethylation. These observations also support previous findings that N-demethylation and 4' hydroxylation are, in the main, catalyzed by different isozymes of cytochrome P-450.
- Published
- 1983
30. Effects of adrenergic antigonists on prereplicative changes during nafenopin-induced liver growth in the rat.
- Author
-
Lam DC and Levine WG
- Subjects
- Amino Acids metabolism, Animals, DNA biosynthesis, Drug Synergism, Liver growth & development, Male, Ornithine Decarboxylase metabolism, Rats, Time Factors, Liver drug effects, Nafenopin pharmacology, Propionates pharmacology, Sympatholytics pharmacology
- Published
- 1979
- Full Text
- View/download PDF
31. Effect of nafenopin (SU-13,437) on liver function: influence on the hepatic transport of organic anions.
- Author
-
Meijer DK, Bognacki J, and Levine WG
- Subjects
- 9,10-Dimethyl-1,2-benzanthracene metabolism, Animals, Benzopyrenes metabolism, Bilirubin metabolism, Biological Transport drug effects, Half-Life, In Vitro Techniques, Indocyanine Green metabolism, Kinetics, Liver metabolism, Male, Perfusion, Rats, Sulfathiazoles metabolism, Sulfobromophthalein analogs & derivatives, Sulfobromophthalein metabolism, Taurocholic Acid metabolism, Time Factors, Anions, Bile metabolism, Liver drug effects, Nafenopin pharmacology, Propionates pharmacology
- Abstract
Rats treated with hypolipidemic agent, nafenopin (SU-13, 437) exhibit a higher plasma retention and a markedly reduced biliary excretion of organic anions, such as sulfobromophthalein (BSP) and its dibromo analog (DPSP), indocyaninegreen (ICG), succinylsulfathiazole (SST) and polar metabolites of bilirubin and the carcinogens 7, 12-dimethylbenzanthracene (DMBA) and 3,4 benzpyrene (BP), despite an increase in liver mass and a profound choleresis. However, taurocholate is not affected in this manner, which supports the idea of a transport mechanism for taurocholate that differs from that of other organic anions. A pharmacokinetic study was made for DBSP in vivo. After nafenopin treatment, primary hepatic uptake (k12) and transport from liver into bile (k23) are reduced in vivo. Infusion studies indicate that biliary transport maximum (Tm) for DBSP is also decreased although the calculated hepatic storage (S) is only moderately affected. In the isolated perfused liver, hepatic clearance and biliary excretion of BSP are reduced by two-thirds. The time course of anion transport inhibition and the hepato-biliary disposition of 14C-nafenopin suggest a direct effect of the drug. The extra liver mass induced by nafenopin appears to be hypo- or nonfunctional with respect to hepatic transport of organic anions.
- Published
- 1975
- Full Text
- View/download PDF
32. Hepatic uptake, metabolism, and biliary excretion of 7,12-dimethylbenzanthracene in the rat.
- Author
-
Levine WG
- Subjects
- Animals, Cytosol metabolism, Female, Liver cytology, Methylcholanthrene pharmacology, Metyrapone pharmacology, Microsomes, Liver metabolism, Phenobarbital pharmacology, Piperonyl Butoxide pharmacology, Proadifen pharmacology, Rats, Time Factors, Tritium, Benz(a)Anthracenes metabolism, Bile metabolism, Liver metabolism
- Published
- 1974
33. Azoreduction of N,N-dimethyl-4-aminoazobenzene (DAB) by rat hepatic microsomes. Selective induction by clofibrate.
- Author
-
Raza H and Levine WG
- Subjects
- Animals, Benzoflavones pharmacology, Cytochrome P-450 Enzyme System biosynthesis, Enzyme Induction, Hydroxylation, In Vitro Techniques, Male, Nafenopin pharmacology, Oxidation-Reduction, Proadifen pharmacology, Rats, Rats, Inbred Strains, beta-Naphthoflavone, Clofibrate pharmacology, Microsomes, Liver metabolism, p-Dimethylaminoazobenzene metabolism
- Abstract
Metabolism of the hepatocarcinogen, N,N-dimethyl-4-aminoazobenzene (DAB) by rat liver microsomes proceeds via N-demethylation, ring hydroxylation, and azoreduction. DAB azoreduction was induced in microsomes from rats treated with the hypolipidemic drug, clofibrate, whereas oxidative metabolism of the carcinogen was inhibited. In contrast, treatment with nafenopin, another hypolipidemic drug, inhibited microsomal azoreduction of DAB, whereas oxidative pathways were only slightly affected. No direct effect of either drug on azoreductase activity was observed. Both drugs markedly induced microsomal laurate hydroxylation. DAB azoreduction was increased slightly in microsomes from rats treated with beta-naphthoflavone while treatment with phenobarbital led to partial inhibition. Pretreatment with isosafrol or pregnenolone-16 alpha-carbonitrile did not significantly alter DAB reduction. Metyrapone, added in vitro, inhibited microsomal DAB azoreductase activity only in phenobarbital-treated microsomes, whereas alpha-napthoflavone and SKF 525-A inhibited activity in control and all induced microsomes. DAB azoreduction proceeds readily in air and is not sensitive to carbon monoxide. Neither clofibrate nor nafenopin affected NADPH-cytochrome c reductase activity. It is concluded that clofibrate-induced azoreductase activity is probably attributable to a specific isoform of cytochrome P-450 which can be distinguished from those which catalyze oxidative pathways of DAB or laurate hydroxylation.
- Published
- 1986
34. Effect of nafenopin (SU-13,437) on liver functions. Hepatic uptake and biliary excretion of ouabain in the rat.
- Author
-
Meijer DK, Bognacki J, and Levine WG
- Subjects
- Animals, Bile metabolism, Depression, Chemical, Liver drug effects, Male, Organ Size drug effects, Perfusion, Rats, Time Factors, Liver metabolism, Nafenopin pharmacology, Ouabain metabolism, Propionates pharmacology
- Abstract
Pretreatment of rats for 2 days with the hypolipidemic drug, nafenopin, 0.5 g/kg, results in an increase in liver weight and bile flow. Despite these changes, hepatic transport of ouabain is reduced. This was demonstrated in the isolated perfused liver as well as in vivo. Although net uptake into liver is diminished in treated rats, the initial rapid phase of the plasma disappearance curve is unaffected. This suggests that the primary uptake process is unaffected by nafenopin or is altered in a manner that is not reflected in this phase of plasma disappearance. Alternatively, nafenopin may increase ouabain efflux from liver to plasma. Biliary excretion of ouabain is markedly suppressed after nafenopin pretreatment and higher liver levels of ouabain were encountered after the first 20 min in treated animals. The inference is drawn that liver to bile transport of ouabain is also suppressed by nafenopin pretreatment.
- Published
- 1975
35. Brain disposition, liver metabolism and behavioural responses to barbiturates in inbred "sensitive" and "resistant" mice.
- Author
-
Halevy S, Frumin MJ, Rosenthal S, and Levine WG
- Subjects
- Animals, Carbon Radioisotopes, Cerebellar Cortex metabolism, Cerebellum metabolism, Drug Resistance, Female, Hexobarbital metabolism, Male, Medulla Oblongata metabolism, Mice, Mice, Inbred Strains, Microsomes, Liver metabolism, Organ Size, Sex Factors, Sleep drug effects, Spectrophotometry, Telencephalon metabolism, Time Factors, Behavior, Animal drug effects, Brain metabolism, Hexobarbital pharmacology, Liver metabolism
- Published
- 1974
- Full Text
- View/download PDF
36. Noncovalent binding of 3'-methyl-N,N-dimethyl-4-aminoazobenzene and its metabolites to liver cytosolic proteins and its role in their nuclear translocation.
- Author
-
Srinivasan K, Levine WG, and Bhargava MM
- Subjects
- Animals, Biological Transport, DNA metabolism, In Vitro Techniques, Male, Protein Binding, Rats, Rats, Inbred Strains, Cell Nucleus metabolism, Cytosol metabolism, Liver metabolism, Methyldimethylaminoazobenzene metabolism, p-Dimethylaminoazobenzene analogs & derivatives
- Abstract
When liver cytosol prepared from rats administered [14C]3'-methyl-N,N-dimethyl-4-aminoazobenzene was subjected to Sephadex gel chromatography, four peaks (I-IV) of radioactivity containing proteins and one peak (V) of radioactivity devoid of protein were obtained. Forty to fifty-five per cent of the radioactivity in the protein peaks was butanol-extractable. When the protein peaks were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis, over 90% of the radioactivity was separated from the proteins, indicating lack of covalent binding. Several differences in the metabolite patterns were seen when the butanol-extractable metabolites from the five chromatographic peaks were analyzed by TLC. When pooled fractions of the peaks were incubated with isolated rat liver nuclei, only radioactivity associated with peak II was translocated into the nucleus. Translocation was time- and temperature-dependent and was maximal at 40 min at 37 degrees C. Only 10 to 12% of the radioactivity associated with peak II could be translocated even in the presence of an excess of nuclei, indicating that specific protein metabolite adduct(s) present in this fraction is/are translocated. Five per cent of translocated radioactivity was irreversibly bound to DNA, 3% to RNA, 67% to non-histone proteins, and 7.5% to histones; the remaining was not associated with any of these macromolecules.
- Published
- 1987
37. Some biochemical changes associated with nafenopin-induced liver growth in the rat.
- Author
-
Levine WG, Ord MG, and Stocken LA
- Subjects
- Aminoisobutyric Acids metabolism, Animals, Body Weight drug effects, DNA biosynthesis, Eating drug effects, Liver drug effects, Liver metabolism, Liver Regeneration drug effects, Male, Organ Size drug effects, Ornithine Decarboxylase metabolism, Rats, Stimulation, Chemical, Time Factors, Liver growth & development, Nafenopin pharmacology, Propionates pharmacology
- Published
- 1977
- Full Text
- View/download PDF
38. Effect of nafenopin on the uptake of bilirubin and sulfobromophthalein by isolated perfused rat liver.
- Author
-
Gärtner U, Stockert RJ, Levine WG, and Wolkoff AW
- Subjects
- Animals, Indicator Dilution Techniques, Liver anatomy & histology, Liver drug effects, Male, Organ Size drug effects, Orosomucoid analogs & derivatives, Orosomucoid metabolism, Rats, Rats, Inbred Strains, Asialoglycoproteins, Bilirubin metabolism, Liver metabolism, Nafenopin pharmacology, Propionates pharmacology, Sulfobromophthalein metabolism
- Abstract
Hepatic uptake of bilirubin and sulfobromophthalein has kinetic characteristics suggesting facilitated diffusion. Because these compounds demonstrate mutual competition for uptake, a shared uptake mechanism has been presumed. Previous studies in isolated perfused regenerating liver revealed depressed uptake of bilirubin, sulfobromophthalein, and asialoorosomucoid, a desialylated glycoprotein which enters hepatocytes by receptor-mediated endocytosis. This study was designed to determine whether or not depressed transport seen in liver regeneration occurs in other states of hepatocellular proliferation. Rats were pretreated with nafenopin (200 mg/kg . day x 2), a drug that causes rapid hepatocellular proliferation similar to that seen in regeneration. Twenty-four hours after nafenopin treatment, liver weight increased by 40%. Influx, efflux, and sequestration rate constants in isolated perfused liver were quantitated by a computer fit to the model of Goresky. Results 1 day after nafenopin treatment revealed no change in transport parameters for bilirubin and asialoorosomucoid, but 55% and 49% reductions in influx of sulfobromophthalein and conjugated bilirubin, respectively. These studies suggest that hepatocellular proliferation alone is not responsible for the transport alterations seen during liver regeneration. Nafenopin effectively unmasks differences in uptake of bilirubin and other more water soluble organic anions such as sulfobromophthalein and conjugated bilirubin, suggesting that their uptake mechanisms are partially independent.
- Published
- 1982
39. Effect of hypolipidemic drugs, nafenopin and clofibrate, on the concentration of ligandin and Z protein in rat liver.
- Author
-
Fleischner G, Meijer DK, Levine WG, Gatmaitan Z, Gluck R, and Arias IM
- Subjects
- Animals, Immunodiffusion, Immunoelectrophoresis, Liver drug effects, Male, Organ Size, Proteins immunology, Rats, Carrier Proteins metabolism, Clofibrate pharmacology, Glutathione Transferase metabolism, Liver metabolism, Nafenopin pharmacology, Propionates pharmacology, Proteins metabolism
- Published
- 1975
- Full Text
- View/download PDF
40. Role of isozymes of cytochrome P-450 in the metabolism of N,N-dimethyl-4-aminoazobenzene in the rat.
- Author
-
Levine WG and Lu AY
- Subjects
- Animals, In Vitro Techniques, Male, Methylcholanthrene pharmacology, Metyrapone pharmacology, Microsomes, Liver enzymology, Microsomes, Liver metabolism, Phenobarbital pharmacology, Piperonyl Butoxide pharmacology, Pregnenolone Carbonitrile pharmacology, Proadifen pharmacology, Rats, Rats, Inbred Strains, Cytochrome P-450 Enzyme System metabolism, Isoenzymes metabolism, p-Dimethylaminoazobenzene metabolism
- Abstract
The metabolism of N,N-dimethyl-4-aminoazobenzene (DAB) was investigated in vitro by use of hepatic 10,000g supernatant fraction, microsomes, and purified cytochromes P-450 prepared from rats. Position-selective metabolism was studied in response to induction by 3-methylcholanthrene (MC), phenobarbital (PB), beta-naphthoflavone (BNF), and pregnenolone-16 alpha-carbonitrile (PCN) as well as inhibition by SKF 525-A, metyrapone, alpha-naphthoflavone, and piperonyl butoxide. The principal phase I pathways are demethylation of the tertiary (DAB) and secondary (MAB) amines and ring hydroxylation. When metabolism was measured with 10,000g supernatant fractions, each pathway responded differently and often independently to the inducers and inhibitors, suggesting that they are catalyzed preferentially by different isozymes of cytochrome P-450. Microsomes from PB-treated animals demethylated and hydroxylated DAB at the same rate as did control microsomes, based on cytochrome P-450 content, whereas microsomes from BNF- or MC-treated animals demethylated more rapidly and hydroxylated more slowly. Microsomes from PB-treated animals demethylated the secondary amine, MAB, more rapidly than the tertiary amine, DAB. Purified cytochrome P-448 from MC-treated animals catalyzed DAB demethylation very readily but hydroxylation very poorly. The turnover number was 10 times that seen in microsomes from MC-treated animals. Only one of the four cytochrome P-450 fractions isolated from PB-treated animals had significant activity with DAB and the turnover number of one of these (fraction B) was approximately that seen in microsomes. This study supports the concept of selectivity of various isozymes of cytochrome P-450 for the different steps in phase I metabolism of DAB. Furthermore, it is apparent that the association of certain inhibitors with specific isozymes of cytochrome P-450 is a generalization that requires qualification in terms of the substrates(s) involved.
- Published
- 1982
41. Effect of nafenopin (SU-13437) on liver function. Influence on the hepatic transport of phenolphthalein glucuronide and chlorothiazide.
- Author
-
Uesugi T and Levine WG
- Subjects
- Animals, Bile metabolism, Biological Transport, Active, Female, Kinetics, Liver drug effects, Male, Organ Size, Rats, Sex Factors, Chlorothiazide metabolism, Glucuronates metabolism, Liver metabolism, Nafenopin pharmacology, Phenolphthaleins metabolism, Propionates pharmacology
- Abstract
In rats treated with the hypolipidemic drug, nafenopin (NP), for 2 days the biliary excretion of phenolphthalein glucuronide (PPG) was markedly decreased, while in contrast that of chlorothiazide (CTZ) was enhanced. This suggests the existence of independent hepatic transport mechanisms for these two anions. For both PPG and CTZ blood disappearance curves showed an initial, rapid phase followed by a second, slow phase. The rapid phase for both compounds is affected only slightly by pretreatment with NP. Therefore, it is inferred that suppression of biliary excretion was attributable mainly to impairment of the liver-to-bile transport process. The increased bile flow induced by NP treatment was previously shown to be related to the biliary excretion of NP and its metabolites. Inhibition of NP choleresis by PPG may involve competition for biliary transport of these compounds. The marked hepatomegaly and choleresis seen after NP pretreatment was more evident in male rats than in females.
- Published
- 1976
42. Cytosolic factors that alter the metabolism of N,N-dimethyl-4-aminoazobenzene by rat liver microsomes.
- Author
-
Levine WG and Lee SB
- Subjects
- Animals, Dialysis, Edetic Acid pharmacology, Glutathione metabolism, In Vitro Techniques, Male, Metabolic Clearance Rate drug effects, NADP metabolism, Rats, Rats, Inbred Strains, Serum Albumin, Bovine pharmacology, Cytosol metabolism, Microsomes, Liver metabolism, p-Dimethylaminoazobenzene metabolism
- Abstract
N,N-Dimethyl-4-aminoazobenzene (DAB), an azo dye carcinogen, is N-demethylated and 4'-hydroxylated by rat liver microsomes. Addition of hepatic cytosol to the microsomal system stimulated both pathways. This occurred in the presence of added NADPH or an NADPH-generating system. Cytosol was effective only when present prior to addition of substrate; no stimulation was seen when added after the reaction had begun. This suggested a direct effect on the microsomes rather than a chemical interaction with one or more metabolic intermediates of DAB. The degree of stimulation was somewhat different when using microsomes from phenobarbital- or beta-naphthoflavone-treated animals, implying a selectivity of the cytosolic effect for various isozymes of cytochrome P-450. Some loss of stimulatory activity occurred with dialysis. Activity was restored by adding back glutathione (GSH) which can stimulate DAB metabolism even in the absence of cytosol. DAB metabolism is also stimulated by EDTA. Although both EDTA and cytosol inhibit lipid peroxidation, cytosol stimulated DAB metabolism even in the presence of EDTA. Therefore, suppression of lipid peroxidation does not explain satisfactorily the cytosolic effect. Separation of cytosolic proteins by gel filtration revealed a factor which inhibits N-demethylation but not 4'-hydroxylation of DAB. Heating at 100 degrees partially inactivated the stimulatory activity. However, inhibitory activity was less susceptible to heat inactivation than was stimulatory activity. These results indicate that, in the whole cell, microsomal metabolism of xenobiotics is regulated to an appreciable extent by macromolecular cytosolic substances.
- Published
- 1983
- Full Text
- View/download PDF
43. Studies on the mechanism of reduction of azo dye carcinogens by rat liver microsomal cytochrome P-450.
- Author
-
Zbaida S, Stoddart AM, and Levine WG
- Subjects
- Animals, Enzyme Induction, In Vitro Techniques, Kinetics, Male, Oxidation-Reduction, Rats, Spectrum Analysis, Azo Compounds metabolism, Cytochrome P-450 Enzyme System metabolism, Microsomes, Liver metabolism
- Abstract
This laboratory has described the azoreduction of p-dimethylaminoazobenzene (1c) by rat liver microsomal cytochrome P-450. To elucidate the mechanisms involved, the reduction of structurally related azobenzenes by hepatic microsomes was investigated. High substrate reactivity was observed for 1c, its corresponding secondary (1a) and primary (1b) amines and p-hydroxyazobenzene (1d). In contrast, only negligible rates were obtained for unsubstituted azobenzene (1g), hydrazobenzene (2g), p-isopropylazobenzene (1e) and 1f, the benzoylamide derivative of 1b. These results clearly indicate that electron-donating groups, such as hydroxyl or primary, secondary and tertiary amines, are essential for binding of azo dye carcinogens to liver microsomal cytochrome P-450 and, by implication, their enzymic reduction. No inhibition of azoreduction of 1c or 1d was obtained by addition of 1e, 1g, or 2g to the reaction mixture. In the presence of hepatic microsomes, a type I binding spectrum was obtained for 1d and type II binding spectra for 1a, 1b and 1c, the reactive azo dyes. In contrast, very weak binding was observed for the unreactive compounds 1e, 1f, 1g and 2g. Thus, there is good correlation between binding and substrate reactivity. The apparent lack of binding may explain the inability of the non-reactive compounds to inhibit azoreduction. The difference in the reduction rate observed for 1g vs. 1d suggested that hydroxylation would facilitate the reduction of an otherwise non-reactive azo dye. Support for such a mechanism was obtained in two experiments. In the first, marked facilitation of azoreduction of both the inactive compounds, 2g and 2f, was seen when they were incubated with microsomes under aerobic conditions where preliminary hydroxylation can occur. In the second, azobenzene was initially incubated aerobically with microsomes from phenobarbital- or beta-naphthoflavone-induced rats. The hydroxyazobenzene formed was then readily reduced anaerobically by microsomes from untreated rats.
- Published
- 1989
- Full Text
- View/download PDF
44. Metabolism and biliary excretion of NN-dimethylaminoazobenzene: a possible role for liver glutathione.
- Author
-
Levine WG
- Subjects
- Animals, Mixed Function Oxygenases metabolism, Oxidation-Reduction, Rats, Bile metabolism, Glutathione metabolism, Liver metabolism, p-Dimethylaminoazobenzene metabolism
- Published
- 1978
- Full Text
- View/download PDF
45. Effects of butylated hydroxyanisole (BHA) and butylated hydroxytoluene (BHT) on metabolism of N,N-dimethyl-4-aminoazobenzene (DAB) by rat liver microsomes.
- Author
-
Levine WG
- Subjects
- Animals, Cytochrome P-450 Enzyme System analysis, In Vitro Techniques, NADH, NADPH Oxidoreductases analysis, NADPH Oxidases, Rats, Anisoles pharmacology, Butylated Hydroxyanisole pharmacology, Butylated Hydroxytoluene pharmacology, Microsomes, Liver metabolism, p-Dimethylaminoazobenzene metabolism
- Abstract
The antioxidants, BHA and BHT, inhibited the N-demethylation and ring hydroxylation of N,N-dimethyl-4-aminoazobenzene (DAB) by liver microsomes from untreated and phenobarbital (PB)-treated rats. BHA was somewhat more potent in this regard than was BHT. Microsomal NADPH oxidase from PB-treated rats was stimulated by BHA but control microsomal activity was unaffected. Glutathione did not appreciably reverse the inhibitory effect of BHA on DAB metabolism and had no effect on NADPH oxidase activity. It is concluded that inhibition of DAB metabolism by BHA cannot be accounted for by interaction with NADPH oxidase, particularly in untreated microsomes. It more likely affects one or more species of cytochrome P-450.
- Published
- 1984
46. Biliary excretion of drugs in the rat during liver regeneration.
- Author
-
Uesugi T, Bognacki J, and Levine WG
- Subjects
- Animals, Azo Compounds metabolism, Benzenesulfonates metabolism, Bile Acids and Salts metabolism, Female, Glutathione metabolism, Male, Organ Size, Rats, Sex Factors, Sulfobromophthalein analogs & derivatives, Sulfobromophthalein metabolism, Time Factors, Bile metabolism, Liver Regeneration, Pharmaceutical Preparations metabolism
- Published
- 1976
- Full Text
- View/download PDF
47. Biliary excretion of N,N-dimethyl-4-aminoazobenzene (DAB) in the rat. Effects of pretreatment with inducers and inhibitors of the mixed-function oxidase system and with agents that deplete liver glutathione.
- Author
-
Levine WG and Finkelstein TT
- Subjects
- Animals, Bile drug effects, Enzyme Induction drug effects, In Vitro Techniques, Liver drug effects, Male, Mixed Function Oxygenases antagonists & inhibitors, Rats, Time Factors, Bile metabolism, Glutathione metabolism, Liver metabolism, Mixed Function Oxygenases metabolism, Oxidoreductases metabolism, p-Dimethylaminoazobenzene metabolism
- Published
- 1978
48. Studies on microsomal azoreduction. N,N-dimethyl-4-aminoazobenzene (DAB) and its derivatives.
- Author
-
Levine WG
- Subjects
- Aerobiosis, Animals, Carbon Radioisotopes, Male, Oxidation-Reduction, Rats, Rats, Inbred Strains, Structure-Activity Relationship, Microsomes, Liver metabolism, p-Dimethylaminoazobenzene analogs & derivatives, p-Dimethylaminoazobenzene metabolism
- Abstract
The azoreduction of N,N-dimethyl-4-aminoazobenzene (DAB) and N-methyl-4-amino-azobenzene (MAB) by rat liver microsomes was investigated. It was shown that measurement of azoreduction of DAB and structurally related azo dyes by the conventional method of substrate disappearance required an anaerobic environment since N-demethylated and ring-hydroxylated metabolites formed aerobically interfered with the assay system, producing quantitatively inaccurate results. Oxygen partially, but not totally, inhibited azoreduction of DAB. Glutathione (GSH) inhibited the azoreduction of DAB but stimulated the azoreduction of MAB. Dithiothreitol also stimulated azoreduction of MAB but had little effect on azoreduction of DAB. Para-hydroxymercuribenzoate (PHMB) and N-ethylmaleimide (NEM) blocked titratable microsomal thiol groups and inhibited azoreduction of MAB. However, the inhibitory action of NEM was weak with DAB azoreduction although PHMB was a potent inhibitor. These findings suggest that microsomal azoreduction of DAB and MAB may proceed via different mechanisms, possibly through different species of cytochrome P-450 which have selective dependence upon the sulfhydryl environment.
- Published
- 1985
- Full Text
- View/download PDF
49. The influence of dehydrocholate on hepatic uptake and biliary excretion of 3H-taurocholate and 3H-ouabain.
- Author
-
Meijer DK, Vonk RJ, Scholtens EJ, and Levine WG
- Subjects
- Animals, Bile drug effects, Liver drug effects, Ouabain blood, Rats, Taurocholic Acid blood, Bile metabolism, Dehydrocholic Acid pharmacology, Liver metabolism, Ouabain metabolism, Taurocholic Acid metabolism
- Abstract
The hepatic uptake and biliary excretion of 3H-taurocholate and 3H-ouabain was studied in the rat during saline (control) and dehydrocholate infusions. Dehydrocholate (140 mumol/hr) did not influence the plasma disappearance nor the biliary excretion of taurocholate after a single iv injection (37 mumol/kg). Bile production in the dehydrocholate experiment was increased 2- to 3-fold compared with controls. The biliary transport maximum for exogenously administered taurocholate was determined by constant infusion to be 135.0 +/- 3.0 mumol/hr (22 mumol/min/g of liver). Concomitant infusions of 140 mumol of dehydrocholate per hr did not alter the maximal taurocholate output. The effects of the two bile salts on bile flow were additive. Dehydrocholate (140 mumol/hr) reduced the biliary excretion of 3H-ouabain (0.8 mumol/kg) and elevated the secondary slow component of the plasma disappearance of the cardiac glycoside. The hepatic levels of ouabain were increased compared with controls. It is concluded that dehydrocholate interferes with ouabain transport at the canalicular level but not with primary hepatic uptake. Taurocholate (140 mumol/hr) failed to influence the total biliary output of ouabain. These differences and the lack of interaction between dehydrocholate and taurocholate suggest a hepatic transporting pathway for taurocholate which differs from that for taurocholate which differs from that for dehydrocholate and/or its metabolites.
- Published
- 1976
50. Biliary excretion of drugs and other xenobiotics.
- Author
-
Levine WG
- Subjects
- Animals, Drug Interactions, Drug-Related Side Effects and Adverse Reactions, Enterohepatic Circulation, Female, Humans, Male, Pharmaceutical Preparations urine, Sex Factors, Species Specificity, Structure-Activity Relationship, Bile metabolism, Pharmaceutical Preparations metabolism
- Abstract
A vast number and variety of xenobiotics appear in the bile. For some this is a final excretory process, for others it is merely one step in the active enterohepatic circulation. For still others it may be a vital step in a toxicologic or carcinogenic process. During the past ten years there has been a steady accumulation of observations in the literature bearing on biliary excretion mechanisms. Phenomena such as molecular weight thresholds and other aspects of species variation as well as response to inducing agents are described in many papers and much speculation is available as to their meaning. The clinical significance of this work is still somewhat dependent upon results obtained from lower animals, although studies occasionally appear on patients who have temporary bile drainage subsequent to surgery. It is important that efforts persist in obtaining data in humans since extrapolation from lower animals in the area of drug disposition is often precarious. The basic physiological and biochemical mechanism governing the biliary fate of drugs and other xenobiotics have yet to be elucidated fully. Perhaps the use of drugs and other pharmacological tools will hasten progress toward this goal.
- Published
- 1978
- Full Text
- View/download PDF
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