RÉSUMÉ
The tobacco-specific nitrosamine, 4-(methylnitrosoamino)-1-(3-pyridyl)-1-butanone, is activated to lung DNA methylating and pyridyloxobutylating intermediates. It is likely that both pathways play a role in lung tumor initiation by this nitrosamine. Previous studies indicated that O(6)-methylguanine (O(6)-mG) persistence is critical for lung tumor formation in A/J mice. The model pyridyloxobutylating agent, 4-(acetoxymethylnitrosamino)-1-(3-pyridyl)-1-butanone (NNKOAc), enhanced the tumorigenic activity of a model methylating agent, acetoxymethylmethylnitrosamine (AMMN), presumably by increasing O(6)-mG persistence in lung DNA. We have been testing the hypothesis that the pyridyloxobutylation pathway increases the mutagenic activity of the DNA methylation pathway by preventing the repair of O(6)-mG by O(6)-alkylguanine-DNA alkyltransferase (AGT). In this study, we report that NNKOAc depletes AGT in lungs but not livers of A/J mice. The consequences of AGT depletion by NNKOAc were then compared with those observed with a known AGT inhibitor, O(6)-benzylguanine (O(6)-bG). NNKOAc and O(6)-bG had similar effects on the levels of AMMN-derived O(6)-mG at 4 and 96 h postinjection. This increase in O(6)-mG levels correlated to increased lung tumor multiplicity in animals simultaneously treated with AMMN (0.75 or 1 micromol) and NNKOAc or O(6)-bG. Only NNKOAc significantly increased lung tumor multiplicity at doses of 0.25 or 0.5 micromol AMMN. The results from these studies indicate that the pyridyloxobutylating agent, NNKOAc, can influence the tumorigenic activity of methylating agents in two ways. At low AMMN doses, the increase in tumor multiplicity is dominated by the additive tumorigenic properties of AMMN and NNKOAc. At higher AMMN doses, NNKOAc appears to enhance the tumorigenic activity of AMMN through enhanced depletion of the repair protein, AGT, leading to increased O(6)-mG persistence. It is likely that similar interactions are important for the organospecific effects of 4-(methylnitrosoamino)-1-(3-pyridyl)-1-butanone.
Sujet(s)
Agents alcoylants/pharmacologie , Cancérogènes/pharmacologie , N-Méthyl-N-nitroso-méthanamine/analogues et dérivés , N-Méthyl-N-nitroso-méthanamine/pharmacologie , Guanine/pharmacologie , Tumeurs du poumon/induit chimiquement , Nitrosamines/pharmacologie , O(6)-methylguanine-DNA methyltransferase/antagonistes et inhibiteurs , Pyridines/pharmacologie , Animaux , Méthylation de l'ADN/effets des médicaments et des substances chimiques , Réparation de l'ADN , Synergie des médicaments , Femelle , Guanine/analogues et dérivés , Guanine/métabolisme , Foie/effets des médicaments et des substances chimiques , Foie/enzymologie , Poumon/effets des médicaments et des substances chimiques , Poumon/enzymologie , Poumon/métabolisme , Tumeurs du poumon/enzymologie , Tumeurs du poumon/métabolisme , Souris , Souris de lignée A , O(6)-methylguanine-DNA methyltransferase/métabolismeRÉSUMÉ
The quinazoline antifolate N-(5-[N-(3,4-dihydro-2-methyl-4-oxoquinazolin-6-ylmethyl)-N- methylamino]-2-thenoyl)-L-glutamic acid (ZD1694; Tomudex) is a potent inhibitor of thymidylate synthase and causes cell death through disruption of DNA synthesis and repair by blocking the obligatory thymidine nucleotide synthesis. B43(anti-CD19)-PAP immunotoxin is a potent inhibitor of protein synthesis in CD19+ B-lineage acute lymphoblastic leukemia (ALL) cells and causes apoptosis. In this model, 100% of SCID mice challenged with 1 x 10(6) human NALM-6 B-lineage ALL cells develop overt and invariably fatal leukemia. All of the 22 control SCID mice treated with phosphate-buffered saline died of disseminated human leukemia between 31 and 61 days with a median survival of 41.2 days. Treatment with ZD 1694 resulted in improved leukemia-free survival with a median survival of 69.2 days (P < 0.001, log-rank test). B43-PAP treatment was more effective than ZD1694 (P=0.026) and resulted in 51.0% long-term leukemia-free survival with a median survival of 187.5 days (P < 0.0001. log-rank test). The combination of ZD1694 and B43-PAP was more effective than either agent alone and resulted in 100% long-term leukemia-free survival. To our knowledge, this preclinical study is the first to demonstrate the feasibility and therapeutic advantage of combining an anti-leukemia immunotoxin with a thymidylate synthase inhibitor.
Sujet(s)
Antimétabolites antinéoplasiques/administration et posologie , Antinéoplasiques d'origine végétale/administration et posologie , Antienzymes/administration et posologie , Immunotoxines/administration et posologie , N-Glycosyl hydrolases , Protéines végétales/administration et posologie , Leucémie-lymphome lymphoblastique à précurseurs B et T/thérapie , Quinazolines/administration et posologie , Thiophènes/administration et posologie , Animaux , Antigènes CD19/immunologie , Lymphocytes B/anatomopathologie , Lignage cellulaire , Association thérapeutique , Modèles animaux de maladie humaine , Humains , Souris , Souris SCID , Leucémie-lymphome lymphoblastique à précurseurs B et T/immunologie , Leucémie-lymphome lymphoblastique à précurseurs B et T/anatomopathologie , Leucémie-lymphome lymphoblastique à précurseurs B et T/physiopathologie , Protéines inactivant les ribosomes de type 1 , Analyse de survie , Thymidylate synthase/antagonistes et inhibiteursRÉSUMÉ
Garlic organosulfur compounds exert chemopreventive effects at several organ sites in rodents after administration of chemical carcinogens, possibly by inhibiting carcinogen activation via cytochrome P-450-mediated oxidative metabolism. It has been suggested that the variability in potency of tumor inhibition by garlic sulfur compounds is due to structural differences, such as the number of allyl and sulfur groups. In this study, diallyl sulfide (DAS), diallyl disulfide (DADS), and allyl methyl sulfide (AMS) were administered to acetone-treated adult male Sprague-Dawley rats by gastric gavage at a dose of 1.75 mmol/kg in cottonseed oil. After 15 hours, hepatic microsomal cytochrome P-450 activity and content were examined. The activity of p-nitrophenol (pNP) hydroxylase (E.C. 1.14.13.29) was significantly decreased by all garlic compounds, whereas benzphetamine N-demethylase and ethoxyresorufin O-deethylase activities were not changed. The activity of pNP hydroxylase was decreased to 31%, 54%, and 65% of control activity, and immunodetectable CYP2E1 protein levels were decreased in a similar manner by DAS, DADS, and AMS, respectively. Additional acetone-treated rats were given 4-methyl pyrazole, a ligand specific for CYP2E1, intraperitoneally five hours after garlic compound administration. Ten hours later, pNP hydroxylase activity was decreased to 73%, 78%, and 67% of control levels by DAS, DADS, and AMS, respectively. Further studies are needed to determine whether the variable potency of inhibition of CYP2E1 enzyme activity is related to chemopreventive efficacy of garlic sulfur compounds.
Sujet(s)
Composés allyliques/pharmacologie , Anticarcinogènes/pharmacologie , Cytochrome P-450 enzyme system/métabolisme , Disulfures/pharmacologie , Foie/enzymologie , Sulfures/pharmacologie , Composés allyliques/administration et posologie , Animaux , Inhibiteurs du cytochrome P-450 CYP2E1 , Disulfures/administration et posologie , Antienzymes/pharmacologie , Ail , Lavage gastrique , Mâle , Plantes médicinales , Rats , Rat Sprague-Dawley , Sulfures/administration et posologieRÉSUMÉ
n-Butyraldoxime (n-BO) is known to cause a disulfiram/ethanol-like reaction in humans, a manifestation of the inhibition of hepatic aldehyde dehydrogenase (AIDH). As with a number of other in vivo inhibitors of AIDH, n-BO does not inhibit purified AIDH in vitro, suggesting that a metabolite of n-BO is the actual inhibitor of this enzyme. In re-examination of the effect of n-BO on blood acetaldehyde levels following ethanol in the Sprague-Dawley rat, we found that pretreatment with substrates and/or inhibitors of cytochrome P450 blocked the n-BO-induced rise in blood acetaldehyde in the following order of decreasing potency: 1-benzylimidazole (0.1 mmol/kg) > 3-amino-1,2,4-triazole (1.0 g/kg) > ethanol (3.0 g/kg) > phenobarbital (0.1% in the drinking water, 7 days) > SKF-525A (40 mg/kg). Rat liver microsomes were shown to catalyze the conversion of n-BO to an active metabolite that inhibited yeast AIDH. This reaction was dependent on NADPH and molecular oxygen and was inhibited by CO and 1-benzylimidazole. Hydroxylamine, postulated by others to be a metabolite of n-BO, inhibited AIDH via a catalase-mediated reaction and not through an NADPH-supported microsome-catalyzed reaction. Using GLC-mass spectrometry, 1-nitrobutane (an N-oxidation product) and butyronitrile (a dehydration product) were identified as metabolites from microsomal incubations of n-BO. However, neither of these metabolic products inhibited AIDH directly or in the presence of liver microsomes and NADPH. We conclude that another NADPH-dependent, cytochrome P450-catalyzed metabolic product of n-BO is responsible for the inhibition of AIDH by n-BO.
Sujet(s)
Aldehyde dehydrogenase/antagonistes et inhibiteurs , Cytochrome P-450 enzyme system/métabolisme , Microsomes du foie/enzymologie , Oximes/métabolisme , Acétaldéhyde/sang , Animaux , Biotransformation , Hydroxylamine , Hydroxylamines/pharmacologie , NADP , Nitriles/métabolisme , Oximes/pharmacologie , Oxygène , RatsRÉSUMÉ
Cytochrome P450 (CYP450) 2E1 (CYP2E1) is induced by pure ethanol following its chronic administration, and commercial alcoholic beverages, whose major constituent is ethanol, are generally assumed to have a similar effect on this isoform of CYP450. Recently, we serendipitously discovered that beer administered to rats for six weeks had only a minimal inductive effect on hepatic microsomal CYP2E1 activity, while rats on 10% ethanol had CYP2E1 levels five-fold greater than controls. The daily ethanol intake levels for the beer fed and 10% ethanol fed rats were equivalent. In addition, CYP450 spectral features of microsomes from beer fed and ethanol fed rats were markedly different. Spectral examination of microsomes from beer fed rats revealed that about 40% of the total CYP450 content existed in the form of a metabolic intermediate (MI) complex, while no evidence was found for MI complex formation in microsomes of ethanol fed rats. We conclude that beer contains an unidentified component(s) that apparently blocks the typical ethanol induction of CYP2E1 and form an MI complex with CYP450.
Sujet(s)
Consommation d'alcool , Bière , Cytochrome P-450 enzyme system/biosynthèse , Éthanol/pharmacologie , Microsomes du foie/enzymologie , Oxidoreductases, (N-demethylating)/biosynthèse , Animaux , Cytochrome P-450 CYP2E1 , Induction enzymatique , Mâle , Microsomes du foie/effets des médicaments et des substances chimiques , Rats , Rat Sprague-Dawley , Valeurs de référenceSujet(s)
Charbon de bois , Microsomes du foie/effets des médicaments et des substances chimiques , NADPH-ferrihemoprotéine reductase/métabolisme , Phénobarbital/pharmacologie , Animaux , Benzfétamine/pharmacologie , Sites de fixation , Induction enzymatique/effets des médicaments et des substances chimiques , Éthylmorphine/pharmacologie , Mâle , Microsomes du foie/enzymologie , Oxydoréduction , Phénobarbital/isolement et purification , Rats , Facteurs temps , Conservation de tissuRÉSUMÉ
In the presence of ATP hepatic microsomes sequester calcium. This sequestration is thought to be important in the modulation of free cytosolic calcium concentration. We find that on the addition of NADPH the uptake of calcium by the hepatic microsomes is inhibited 27-85%. This inhibition is reversed by the addition of 1 mM reduced glutathione (85-91% of control), incubation under a nitrogen atmosphere (112% of control), or incubation in a 80% carbon monoxide/20% oxygen atmosphere (75% of control). Superoxide dismutase had no effect on the inhibition, while catalase reversed the inhibition by 35%. The addition of 1 mM reduced glutathione at 2 and 5 min after the addition of NADPH led to uptakes of calcium which paralleled the uptake seen when the reduced glutathione was added at the beginning of the incubation. The effect of reduced glutathione showed saturation kinetics with a Km of 10 microM. Together these data suggest that cytochrome P-450 reduces the activity of the microsomal ATP-dependent calcium pump both by the production of hydrogen peroxide and by the direct oxidation of the protein thiols. The reversal of this effect by reduced glutathione appears to be enzymatically catalyzed.
Sujet(s)
Calcium-Transporting ATPases/métabolisme , Calcium/métabolisme , Cytochrome P-450 enzyme system/métabolisme , Glutathion/métabolisme , Microsomes du foie/enzymologie , Animaux , Transport biologique actif , Catalase/métabolisme , Acide édétique/pharmacologie , Cinétique , Mâle , Mannitol/métabolisme , NADP/pharmacologie , Oxydoréduction , Oxygène/physiologie , Rats , Superoxide dismutase/métabolismeRÉSUMÉ
The hepatic, microsomal, thiol:protein disulfide oxidoreductase catalyzes the glutathione (GSH) reduction of protein disulfides to sulfhydryl groups. In the presence of physiological concentrations of glucagon this activity increased from 2.3 to 6.4 fold in isolated microsomes. The stimulation had a P50 for glucagon of 7.8 X 10(-10) M which was only observed at microsomal protein concentrations of less than 100 micrograms/ml and in the presence of a GSH reducing system. This latter observation suggests that the stimulation may be inhibited by the presence of oxidized glutathione. These data support the hypothesis that glucagon may act in part by stimulating the reduction of protein disulfides by the thiol:protein disulfide oxidoreductase.
Sujet(s)
Glucagon/pharmacologie , Microsomes du foie/enzymologie , Oxidoreductases/métabolisme , Protein-disulfide reductase (glutathione)/métabolisme , Animaux , Relation dose-effet des médicaments , Activation enzymatique , Glutathion/métabolisme , Insuline/métabolisme , Microsomes du foie/effets des médicaments et des substances chimiques , NADP/métabolisme , Rats , Thiols/métabolismeRÉSUMÉ
Weanling, male Sprague-Dawley rats given 10% ethanol in the drinking water and food ad lib. for up to 8 weeks consumed 17% of their calories as ethanol. The alanine aminotransferase (ALT), aspartate aminotransferase (AST), and liver histology by light microscopy were unaffected by this treatment. Similarly, hepatic microsomal NADPH-cytochrome c reductase, ethylmorphine N-demethylase and benzphetamine N-demethylase activities were also not affected by ethanol consumption. On the other hand, cytochrome P-450 content, aniline hydroxylase activity and acetaminophen metabolism as measured by both the cysteine conjugate and the [3H]acetaminophen covalently-bound to microsomal protein were increased significantly by ethanol consumption. The maximal effect was seen by 6 weeks. The 2- to 3-fold increase in aniline and acetaminophen metabolism, the absence of liver damage, and the similarity in weight gains and caloric intakes for controls and treated animals suggest that the rat on 10% ethanol in the drinking water is a reasonable model for studies of the effect of moderate alcohol consumption on specific biochemical pathways.
Sujet(s)
Éthanol/pharmacologie , Microsomes du foie/enzymologie , Préparations pharmaceutiques/métabolisme , Acétaminophène/métabolisme , Alanine transaminase/métabolisme , Aniline hydroxylase/métabolisme , Animaux , Aspartate aminotransferases/métabolisme , Poids/effets des médicaments et des substances chimiques , Cytochrome P-450 enzyme system/métabolisme , Ration calorique , Ethylmorphine-N-demethylase/métabolisme , Mâle , Microsomes du foie/effets des médicaments et des substances chimiques , NADH, NADPH oxidoreductases/métabolisme , NADPH-ferrihemoprotéine reductase/métabolisme , Oxidoreductases, (N-demethylating)/métabolisme , Rats , Lignées consanguines de ratsRÉSUMÉ
Chronic alcohol consumption significantly increases the risk of drug interactions. We have described its effects on hamster microsomal monooxygenases. Male Syrian hamsters (85 g) were given 10% ethanol in water and food ad lib for up to 6 weeks. Microsomal electron transport components and metabolism of ethylmorphine, benzphetamine, aniline, and acetaminophen were measured. At 4 weeks, SDS-PAGE of ethanol microsomes showed an induced band with an Mr of 53,900 daltons and there was a 2-3 fold stimulation of aniline and acetaminophen metabolism. Cytochrome P-450 increase was not significant. For the six week period, Caloric intake (3 weeks, p less than 0.001), liquid consumption (3 weeks, p less than 0.05) and body weights (6 weeks, p less than 0.05) of ethanol animals were significantly greater than controls; kidney weights were significantly less (p less than 0.05). Ethanol consumption increased from 20% of the daily caloric intake (week 1) to 31% (week 6). Induction of specific substrate metabolism without apparent deleterious physiological changes establishes hamsters fed 10% ethanol in drinking water as a biochemical model for the study of chronic alcohol consumption and specific drug interactions.
Sujet(s)
Éthanol/administration et posologie , Microsomes du foie/effets des médicaments et des substances chimiques , Mixed function oxygenases/analyse , Animaux , Poids/effets des médicaments et des substances chimiques , Cricetinae , Électrophorèse sur gel de polyacrylamide , Ration calorique/effets des médicaments et des substances chimiques , Éthanol/pharmacologie , Mâle , Mesocricetus , Microsomes du foie/enzymologieRÉSUMÉ
We have modified our radiometric assay for ethylmorphine N-demethylase to examine the metabolism of codeine. We find that the current assay gives excellent separation of metabolites with a zero time activity of 0.08%. The N- and O-demethylations are linear for up to 30 min with up to 1 mg/ml of microsomal protein. The pH profiles show slightly different maxima (N-demethylation, pH 8.0; O-demethylation, pH 7.8). The kinetic parameters for N-demethylation were markedly higher (Vmax = 5.6 nmol/min/mg protein; KM = 714 microM) than those for O-demethylase (Vmax = 0.75 nmol/min/mg protein; KM = 149 microM). These data suggest that the HCHO results primarily from the N-demethylase. Further, the differences in the kinetic parameters and the pH profile suggest that these two activities are catalyzed by different enzymatic systems.
Sujet(s)
Codéine/métabolisme , Animaux , Désalkylation , Techniques in vitro , Foie/métabolisme , Mâle , Rats , Lignées consanguines de rats , Facteurs tempsRÉSUMÉ
Cimetidine has been reported to decrease plasma clearance of drugs in humans and animals. This reduction in hepatic drug metabolism could be due to cimetidine's intrinsic H2-receptor blocking activity. Alternatively, the imidazole ring structure of cimetidine could explain these observations because imidazole derivatives have been reported to be potent inhibitors of hepatic microsomal drug metabolism. Rat and human hepatic microsomal drug metabolism in the presence of cimetidine and ranitidine, a nonimidazole H2-receptor antagonist, have been studied. High binding affinity of cimetidine for cytochrome P 450(Ks = 31 micro M) was seen, while no evidence for ranitidine binding to cytochrome P450- was observed. Cimetidine inhibited meperidine and pentobarbital metabolism by both rat and human hepatic microsomes while ranitidine did not affect these two cytochrome P450-medicated biotransformation reactions. Conjugation of morphine, a reaction not mediated by cytochrome P450, was unaffected by either cimetidine or ranitidine. The imidazole structure of cimetidine rather than its H2-receptor blocking activity is primarily responsible for cimetidine-induced inhibition of hepatic drug metabolism.
Sujet(s)
Cimétidine/pharmacologie , Furanes/pharmacologie , Guanidines/pharmacologie , Antihistaminiques des récepteurs H2/pharmacologie , Microsomes du foie/effets des médicaments et des substances chimiques , Animaux , Cytochrome P-450 enzyme system/métabolisme , Humains , Techniques in vitro , Mâle , Péthidine/métabolisme , Microsomes du foie/métabolisme , Morphine/métabolisme , Ranitidine , Rats , Lignées consanguines de rats , Récepteur histaminergique H2/effets des médicaments et des substances chimiques , Relation structure-activitéRÉSUMÉ
1. NADPH-cytochrome c reductase was solubilized with bromelain and purified about 400-fold from sucrose/pyrophosphate-washed microsomal fractions from southern armyworm (Spodoptera eridania) larval midguts. 2. The enzyme has a mol.wt. of 70 035 +/- 1300 and contained 2 mol of flavin/mol of enzyme consisting of almost equimolar amounts of FMN and FAD. 3. Aerobic titration of the enzyme with NADPH caused the formation of a stable half-reduced state at 0.5 mol of NADPH/mol of flavin. 4. Kinetic analysis showed that the reduction of cytochrome c proceeded by a Bi Bi Ping Pong mechanism. 5. Apparent Km values for NADPH and cytochrome c and Ki values for NADP+ and 2'-AMP were considerably higher for the insect reductase than for the mammalian liver enzyme. 6. These are discussed in relation to possible differences in the active sites of the enzymes.