delta-Aminolevulinate synthetases in the liver cytosol fraction and mitochondria of mice treated with allylisopropylacetamide and 3,5-dicarbethoxyl-1,4-dihydrocollidine.
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Hepatic delta-aminolevulinate (ALA) synthetase was induced in mice by the administration of allylisopropylacetamide (AIA) and 3,5-dicarbethoxy-1,4-dihydrocollidine (DDC). In both cases, a significant amount of ALA synthetase accumulated in the liver cytosol fraction as well as in the mitochondria. The apparent molecular weight of the cytosol ALA synthetase was estimated to be 320,000 by gel filtration, but when the cytosol ALA synthetase was subjected to sucrose density gradient centrifugation, it showed a molecular weight of 110,000. In the mitochondria, there were two different sizes of ALA synthetase with molecular weights of 150,000 and 110,000, respectively; the larger enzyme was predominant in DDC-treated mice, whereas in AIA-treated mice and normal mice the enzyme existed mostly in the smaller form. When hemin was injected into mice pretreated with DDC, the molecular size of the mitochondrial ALA synthetase changed from 150,000 to 110,000. The half-life of ALA synthetase in the liver cytosol fraction was about 30 min in both the AIA-treated and DDC-treated mice. The half-life of the mitochondrial ALA synthetase in AIA-treated mice and normal mice was about 60 min, but in DDC-treated mice the half-life was as long as 150 min. The data suggest that the cytosol ALA synthetase of mouse liver is a protein complex with properties very similar to those of the cytosol ALA synthetase of rat liver, which has been shown to be composed of the enzyme active protein and two catalytically inactive binding proteins, and that ALA synthetase may be transferred from the liver cytosol fraction to the mitochondria with a size of about 150,000 daltons, followed by its conversion to enzyme with a molecular weight of 110,000 within the mitochondria. The process of intramitochondrial enzyme degradation seems to be affected in DDC-treated animals. (+info)
Cytochrome CYP sources of N-alkylprotoporphyrin IX after administration of porphyrinogenic xenobiotics to rats.
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Cytochrome P-450 (CYP) 3A2 and CYP2C11 are sources of 70 and 30%, respectively, of N-vinylprotoporphyrin IX (N-vinylPP) formation after administration of 3-[(arylthio)ethyl]sydnone (TTMS) to rats. Female rats receiving TTMS were pretreated with dexamethasone, which induces CYP3A1 preferentially to CYP3A2. The resulting 12-fold increase in N-vinylPP formation showed that CYP3A1 was also a source of N-vinylPP. Phenobarbital (PB) pretreatment, which induces CYP2B1/2 and 3A1/2 in male rats, increased N-vinylPP formation after TTMS administration. Troleandomycin, a selective CYP3A inhibitor, was unable to decrease TTMS-mediated N-vinylPP formation in PB-treated male rats, indicating that CYP2B1/2 were sources of N-vinylPP. This conclusion was supported by demonstrating a 15-fold increase in TTMSinduced N-vinylPP formation in female rats after CYP2B1/2 induction with PB pretreatment. Allylispropylacetamide (AIA) inactivates rat CYP2B1/2, 2C6, 2C7, 2C11, and 3A1/2. Troleandomycin was unable to decrease N-AIA protoporphyrin IX adduct (N-AIAPP) formation, showing that CYP3A1/2 were not susceptible to AIA-mediated N-alkylation. N-AIAPP formation in females was approximately 30% of that in males, and thus we attribute 30% of N-AIAPP formation in males to the non-gender-specific isozymes (CYP2C6, 2C7, and/or 2B1/2), whereas approximately 70% originates from CYP2C11. PB treatment in female rats resulted in a 5-fold increase in N-AIAPP formation, showing that CYP2B1/2 were also susceptible to N-alkylation mediated by AIA. 1-Aminobenzotriazole elicited formation of equivalent amounts of N'N-aryl bridged protoporphyrin IX in male and female rat liver, demonstrating that nonselective mechanism-based inactivation is accompanied by nonselective conversion of the CYP heme moieties to N'N-aryl bridged protoporphyrin IX. (+info)
On the sequence of reactions leading to cytochrome P-450 synthesis-effect of drugs.
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The effect of phenobarbital on the rates of the synthesis of the protein and heme moieties of cytochromeP-450 has been studied. For this purpose, cytochrome P-450 has been partially purified as its P-420 derivative and the labeled amino acid incorporation into the protein has been studied after subjecting a partially purified preparation to sodium dodecyl sulfate gel electrophoresis. The incorporation studies into the protein species after sodium dodecyl sulfate gel electrophoresis reveal that the drug primarily accelerates the rate of apoptotein synthesis followed by an increase in the rate of heme synthesis. The messenger for apocytochrome P-450 appears to be fairly stable. (+info)
Cobalt stimulation of heme degradation in the liver. Dissociation of microsomal oxidation of heme from cytochrome P-450.
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The administration of cobalt to rats caused a marked increase in the oxidative degradation of heme (hematin, iron protoporphyrin-IX) BY HEPATIC MICROSOMAL ENZYMES. The onset of this enzyme stimulation was very rapid, beginning within 2 hours after injection of the metal and reaching its maximum in 16 to 24 hours. During the rapid phase of stimulation, i.e. the first 2 to 4 hours, when heme oxidation was 450% above control values, there was a significant decrease in microsomal oxidative N-demethylation activity and in microsomal oxidative Ndemethylation activity and in microsomal content of heme with an insignificant decrease in cytochrome P-450 content. Within 24 hours the oxidative activity of the microsomal electron transport chain for drugs was decreased to about 30% of the control. However, during the same period the oxidation of heme approached levels 800% above control. During this period there was a further decrease in the microsomal content of heme with a significant decrease in cytochrome P-450 content and an increase in the activity of delta-aminolevulinate synthetase. The activity of delta-aminolevulinate synthetase reached its maximum within 8 hours after cobalt treatment. Repeated injections (at 24-hour intervals) of cobalt were necessary to maintain these changes in microsomal enzyme activities since, after single injections of the metal, these parameters returned to normal within 72 hours. The inducing effect of cobalt on the oxidation of heme could be inhibited by the administration of actinomycin D and puromycin. Furthermore, this stimulatory effect could not be elicited by in vitro treatment of microsomes with cobalt nor could the effect be attributed to any soluble components of the cytoplasm. Cobalt protoporphyrin-IX was less effective than cobalt chloride in stimulating heme oxidation. 3-Amino-1, 2, 4-triazole did not enhance hepatic heme oxidation activity, while allylisopropylacetamide decreased this activity. The oxidative degradation of heme was found not to be cytochrome P-450 dependent since the highly increased levels of heme oxidation in microsomes from cobalt-treated animals could be retained despite the fact that the cytochrome P-450 content of such microsomes was decreased to spectrally undetectable amounts and drug oxidation was eliminated by treatment of the microsomes with 4 M urea. These findings exclude an obligatory role for cytochrome P-450 in the oxidation of heme compounds, although the possibility that this process is a heme-dependent oxidation is not ruled out. (+info)
Effect of allylisopropylacetamide on Nuclear Ribonucleic Acid synthesis in rat liver.
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The porphyrogenic drug allylisopropylacetamide, a potent inducer of delta-aminolaevulinate synthetase, specifically increases nucleoplasmic RNA synthesis in rat liver. The drug-mediated increase in nucleoplasmic RNA synthesis is blocked by cycloheximide and haemin, which also inhibit the enzyme induction. (+info)
Degradation of cytochrome P-450 haem by carbon tetrachloride and 2-allyl-2-isopropylacetamide in rat liver in vivo and in vitro. Involvement of non-carbon monoxide-forming mechanisms.
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Degradation of intrinsic hepatic [(14)C]haem was analysed as (14)CO formation in living rats and in hepatic microsomal fractions prepared from these animals 16h after pulse-labelling with 5-amino[5-(14)C]laevulinic acid, a precursor that labels bridge carbons of haem in non-erythroid tissues. NADPH-catalysed peroxidation of microsomal lipids in vitro (measured as malondialdehyde) was accompanied by loss of cytochrome P-450 and microsome-associated [(14)C]haem (largely cytochrome P-450 haem), but little (14)CO formation. No additional (14)CO was formed when carbon tetrachloride and 2-allyl-2-isopropylacetamide were added to stimulate lipid peroxidation and increase loss of cytochrome P-450 [(14)C]haem. Because the latter effect persisted despite inhibition of lipid peroxidation with MnCl(2) or phenyl-t-butylnitrone(a spin-trapping agent for free radicals), it was concluded that carbon tetrachloride, as reported for 2-allyl-2-isopropylacetamide, may promote loss of cytochrome P-450 haem through a non-CO-forming mechanism independent of lipid peroxidation. By comparison with breakdown of intrinsic haem, catabolism of [(14)C]methaemalbumin by microsomal haem oxygenase in vitro produced equimolar quantities of (14)CO and bilirubin, although these catabolites reflected only 18% of the degraded [(14)C]haem. This value was increased to 100% by addition of MnCl(2), which suggests that lipid peroxidation may be involved in degradation of exogenous haem to products other than CO. Phenyl-t-butylnitrone completely blocked haem oxygenase activity, which suggests that hydroxy free radicals may represent a species of active oxygen used by this enzyme system. After administration of carbon tetrachloride or 2-allyl-2-isopropylacetamide to labelled rats, hepatic [(14)C]haem was decreased and haem oxygenase activity was unchanged; however, (14)CO excretion was either unchanged (carbon tetrachloride) or decreased (2-allyl-2-isopropylacetamide). These changes were unaffected by cycloheximide pretreatment. From the lack of parallel losses of cytochrome P-450 [(14)C]haem and (14)CO excretion, one may infer that an important fraction of hepatic [(14)C]haem in normal rats is degraded by endogenous pathways not involving CO. We conclude that carbon tetrachloride and 2-allyl-2-isopropylacetamide accelerate catabolism of cytochrome P-450 haem through mechanisms that do not yield CO as an end product, and that are insensitive to cycloheximide and independent of haem oxygenase activity. (+info)
Characterization of the anticonvulsant profile and enantioselective pharmacokinetics of the chiral valproylamide propylisopropyl acetamide in rodents.
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1. Propylisopropyl acetamide (PID) is a new chiral amide derivative of valproic acid. The purpose of this study was to evaluate the anticonvulsant activity of PID in rodent models of partial, secondarily generalized and sound-induced generalized seizures which focus on different methods of seizure induction, both acute stimuli, and following short-term plastic changes as a result of kindling, and to assess enantioselectivity and enantiomer-enantiomer interactions in the pharmacokinetics and pharmacodynamics of racemic PID and its pure enantiomers in rodents. 2. Anticonvulsant activity of (S)-PID, (R)-PID and racemic PID was evaluated in the 6 Hz psychomotor seizure model in mice, in the hippocampal kindled rat, and in the Frings audiogenic seizure susceptible mouse. The pharmacokinetics of (S)-PID and (R)-PID was studied in mice and rats. 3. In mice (S)-PID, (R)-PID and racemic PID were effective in preventing the 6 Hz seizures with (R)-PID being significantly (P < 0.05) more potent (ED(50) values 11 mg kg(-1), 46 mg kg(-1) and 57 mg kg(-1) at stimulation intensities of 22, 32 and 44 mA, respectively) than (S)-PID (ED(50) values 20 mg kg(-1), 73 mg kg(-1) and 81 mg kg(-1) at stimulation intensities of 22, 32 and 44 mA, respectively). (S)-PID, (R)-PID and racemic PID also blocked generalized seizures in the Frings mice (ED(50) values 16 mg kg(-1), 20 mg kg(-1) and 19 mg kg(-1) respectively). 4. In the hippocampal kindled rat a dose of 40 mg kg(-1) of (R)- and (S)-PID prevented the secondarily generalized seizure, whereas racemic PID also blocked the expression of partial seizures following an i.p. dose of 40 mg kg(-1). Racemic PID also significantly increased the seizure threshold in this model. 5. Mechanistic studies showed that PID did not affect voltage-sensitive sodium channels or kainate-, GABA- or NMDA- evoked currents. 6. The pharmacokinetics of PID was enantioselective following i.p. administration of individual enantiomers to mice, with (R)-PID having lower clearance and longer half-life than (S)-PID. In rats and mice, no enantioselectivity in the pharmacokinetics of PID was observed following administration of the racemate, which may be due to enantiomer-enantiomer interaction. 7. This study demonstrated that PID has both enantioselective pharmacokinetics and pharmacodynamics. The better anticonvulsant potency of (R)-PID in comparison to (S)-PID may be due to its more favorable pharmacokinetic profile. The enhanced efficacy of the racemate over the individual enantiomers in the kindled rat may be explained by a pharmacokinetic enantiomer-enantiomer interaction in rats. This study also showed the importance of studying the pharmacokinetics and pharmacodynamics of chiral drugs following administration of the individual enantiomers as well as the racemic mixture. (+info)
Hepatic heme metabolism and its control.
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This review summarizes heme metabolism and focuses especially upon the control of hepatic heme biosynthesis. Activity of delta-aminolevulinic acid synthetase, the first enzyme of heme biosynthesis, is of primary importance in controlling the overall activity of this biosynthetic pathway. Delta-aminolevulinic acid synthetase is subject to inhibition and repression by heme, and numerous basic and clinical studies support the concept that there exists within hepatocytes a "regulatory" heme pool which controls activity of delta-aminolevulinic acid synthetase. In addition, activity of this enzyme is repressed by feeding, especially by ingestion of carbohydrates (the so-called "glucose effect"). Studies pertaining to the mechanisms underlying this effect are also reviewed. The "glucose effect" appears to be mediated by glucose or perhaps by glucose-6-phosphate or uridine diphosphate glucose, rather than by metabolites further removed from glucose itself. Unlike the situation in E. coli, the "glucose effect" in liver of higher organisms is not mediated by alterations in intracellular concentrations of cyclic AMP. Effects of heavy metals, especially iron, on hepatic heme metabolism are also considered. Iron has been found to inhibit formation and utilization of uroporphyrinogen III and to lead to decreased concentrations of microsomal heme and cytochrome P-450. Administration of large amounts of iron is also associated with an increase in activity of heme oxygenase, a property shared by several other metal ions, most notably cobalt. This effect of iron or cobalt administration is similar to the effect of heme administration in increasing heme oxygenase activity; however, we believe it is unlikely that iron, rather than heme itself, is a physiologic regulator of hepatic heme metabolism, although this hypothesis has lately been proposed. (+info)