RESUMEN
The effect of acetaldehyde on astrocytes have been investigated because not only do they play an important role in brain maturation but also recent reports have shown their delayed proliferation following both 'in vivo' and 'in vitro' ethanol exposure. Biochemical parameters related to apoptotic and necrotic processes were examined in primary cultures of rat astrocytes exposed for 4 days to acetaldehyde generated from ethanol by co-cultured alcohol dehydrogenase-transfected Chinese hamster ovary cells. Acetaldehyde levels in the culture media attained concentrations of approximately 450 microM. To study ethanol effects, alcohol oxidation was inhibited by 4-methylpyrazole (an inhibitor of alcohol dehydrogenase). Acetaldehyde but not ethanol increased intracellular calcium levels by 155%. Moreover, significant DNA fragmentation was detected using a random oligonucleotide primed synthesis assay, by flow cytometry and when using agar gel electrophoresis. Transglutaminase activity was elevated in the cells treated with acetaldehyde but when acetaldehyde formation was inhibited by 4-methylpyrazole the enzyme activity was unaffected. Nitrate levels in the culture media were unchanged. Additionally, microscopic examination of cell nuclei revealed chromatin condensation in astrocytes exposed to acetaldehyde. It can be concluded, that in 'in vitro' acetaldehyde exposed rat astrocytes apoptotic pathways are activated.
Asunto(s)
Acetaldehído/toxicidad , Apoptosis/efectos de los fármacos , Astrocitos/citología , Depsipéptidos , Péptidos , Alcohol Deshidrogenasa/genética , Animales , Animales Recién Nacidos , Antibacterianos/farmacología , Astrocitos/química , Astrocitos/enzimología , Células CHO , Calcio/análisis , Técnicas de Cultivo de Célula/métodos , División Celular/efectos de los fármacos , Células Cultivadas , Depresores del Sistema Nervioso Central/farmacología , Cromatografía Líquida de Alta Presión , Cricetinae , Medios de Cultivo/química , Fragmentación del ADN , Etanol/farmacología , Citometría de Flujo , Expresión Génica , Nitritos/análisis , Nitritos/metabolismo , Ratas , Transfección , Transglutaminasas/metabolismoRESUMEN
Paracetamol-induced toxicity is mainly due to the accumulation of its CYP450-mediated N-hydroxylation product - N-acetylimidoquinone. We examined cell viability, proliferation rates and intracellular calcium in PC12 cells and in a PC12 cell line transfected with cytochrome P4502E1 exposed to paracetamol. This drug had a concentration-related effect on cell survival and a LD(50) which was significantly different between both cell types. A 48% decrease of PC12 cells was found following application of 5 mmol/L paracetamol for 48 h. A total 73% decrease in cell numbers was found in cells metabolizing the drug. Culture protein levels were diminished in a similar manner. Paracetamol increased intracellular calcium (by 662%) only in CYP4502E1-transfected cells. The protective role of EGTA and verapamil modulating calcium homeostasis was more evident in CYP4502E1-transfected cells. These results suggest that biotransformation of paracetamol by CYP2E1 increases its cytotoxicity and that a calcium imbalance may have a key role in the initiation of cell injury.
RESUMEN
It has been shown that free radical damage may be involved in ethanol-induced cytotoxicity in cultured neural cells. Since changes in oxidative metabolism and the resulting lipid peroxidation readily modify biological membranes and alter cell functions we studied the effect of ethanol and its metabolite acetaldehyde on rat astroglial fatty acids profiles in the most common lipid classes of mitochondrial and microsomal membranes, i.e. phosphatidylcholine (PC) and phosphatidylethanolamine (PE). Rat astroglial cells were grown for 1 week in the presence of 50 m M or 100 m M ethanol. To examine acetaldehyde effects we used a 4-day co-culture model consisting of astroglial cells and alcohol dehydrogenase-transfected Chinese hamster ovary (CHO) cells. Acetaldehyde produced by these cells reached 172 mu M and 265 mu M, respectively, for ethanol concentrations of 10 and 20 m M. Mitochondrial and microsomal membranes were prepared by differential centrifugation, phosphatidylcholine and phosphatidylethanolamine were separated using thin layer chromatography and fatty acid quantitation was performed by GLC. Neither ethanol nor acetaldehyde changed the mitochondrial phosphatidylcholine or phosphatidylethanolamine profiles of total saturated, mono-unsaturated or polyunsaturated fatty acids. However, some significant alterations in particular fatty acids appeared especially after acetaldehyde but also after the highest ethanol dose. In microsomal phosphatidylcholine monounsaturated fatty acids were significantly increased after both, ethanol and acetaldehyde exposure. Among polyunsaturated fatty acids, arachidonic acid was found to be especially affected by both ethanol and acetaldehyde. Similar decreases were observed in adrenic, docosapentaenoic and docosahexaenoic acids in the groups treated with ethanol. In microsomal phosphatidylethanolamine, ethanol and acetaldehyde decreased monounsaturated and some polyunsaturated fatty acids. These data support the role of peroxidative processes in cultured rat astroglia exposed to ethanol and point to the role of acetaldehyde in this mechanism.
RESUMEN
Chlormethiazole is a sedative and anticonvulsive drug used in the treatment of alcohol withdrawal. Because it had been reported that chlormethiazole inhibits the alcohol-inducible cytochrome P450 2E1 in rat liver, we investigated the in vivo and in vitro effect of this drug on cytochrome P450 2E1 in human beings. The activity of this cytochrome was assessed using chlorzoxazone as a probe. The 6-hydroxychlorzoxazone-chlorzoxazone blood concentration ratio, reflecting the cytochrome P450 2E1 activity, was determined in 10 controls and in 24 alcoholic patients who had entered a hospital for detoxification. Alcoholic patients were administered either chlormethiazole (1.3-2.3 g/d) or chlorazepate (100-300 mg/d) as a sedative. Cytochrome P450 2E1 activity was significantly increased in alcoholic patients treated with chlorazepate (1.16 +/- 0.40 vs. 0.27 +/- 0.03, P < .05). In contrast, chlormethiazole treatment inhibited chlorzoxazone hydroxylation almost totally (0.046 +/- 0.03, P < .001). After 7-14 days of ethanol withdrawal, alcoholic patients treated with chlorazepate had ratio values similar to those of controls (0.31 +/- 0.05), whereas values from alcoholic patients treated with chlormethiazole remained low (0.049 +/- 0.01) even though chlormethiazole doses were gradually decreased. Pharmacokinetic studies in controls showed that chlormethiazole-mediated inhibition was present even when chlormethiazole was not detectable in the blood. In addition, the effect of chlormethiazole on cytochrome P450 2E1 was studied in vitro using human liver microsomes. Dixon plot analyses showed a noncompetitive inhibition (Ki = 12 micromol/L). These data clearly show that chlormethiazole is an efficient inhibitor of chlorzoxazone metabolism and thus of cytochrome P450 2E1 activity in human beings. Because cytochrome P450 2E1 induction after chronic ethanol consumption has detrimental effects on the liver through free radical formation, treatment of alcohol detoxification with chlormethiazole may be beneficial.
Asunto(s)
Clormetiazol/farmacología , Clorzoxazona/metabolismo , Inhibidores del Citocromo P-450 CYP2E1 , Inhibidores Enzimáticos/farmacología , Adulto , Anciano , Alcoholismo/tratamiento farmacológico , Femenino , Humanos , Hidroxilación , Masculino , Persona de Mediana Edad , Síndrome de Abstinencia a Sustancias/tratamiento farmacológicoRESUMEN
Rats selectively bred for high alcohol sensitivity (HAS) or low alcohol sensitivity (LAS) were tested for initial sensitivity to hypnotic doses of ethanol and a locomotor-altering dose of phenobarbital. Following 6 weeks of either a pair-fed control or 33% ethanol-derived calorie diet, animals were tested again for tolerance to ethanol and cross-tolerance to phenobarbital. HAS and LAS rats did not differ in baseline open field or Rotarod activity before chronic ethanol treatment. However, HAS rats were more sensitive to 50 mg/ kg phenobarbital relative to LAS rats. Both control- and ethanol-diet rats appeared to be less sensitive to phenobarbital after the 6-week treatment period. Chronic ethanol-exposed HAS and LAS rats demonstrated tolerance to ethanol and cross-tolerance to phenobarbital, and in particular LAS rats were even more active in the open field following phenobarbital relative to controls. In summary, significant differences in response to phenobarbital were observed between HAS and LAS rats. These observations suggest that initial sensitivity and tolerance to ethanol are associated with differences in phenobarbital sensitivity and are influenced by similar genes.
Asunto(s)
Etanol/farmacología , Moduladores del GABA/farmacología , Hipnóticos y Sedantes/farmacología , Fenobarbital/farmacología , Animales , Peso Corporal/efectos de los fármacos , Dieta , Interacciones Farmacológicas , Tolerancia a Medicamentos , Etanol/administración & dosificación , Etanol/sangre , Moduladores del GABA/administración & dosificación , Hipnóticos y Sedantes/administración & dosificación , Masculino , Actividad Motora/efectos de los fármacos , Fenobarbital/administración & dosificación , Ratas , Sueño/efectos de los fármacosRESUMEN
A panel of 14 human liver microsomal preparations metabolized at variable rates three symmetrical nitrosodialkylamines (N-nitroso-dipropyl, dibutyl and diamyl-amines, NDPA, NDBA, NDAA) into aldehydes and hydroxynitrosamines. Formation of linear aldehydes, convenient probes for alpha-hydroxylation of alkyl chain, and production of hydroxy metabolites of NDPA, NDBA and NDAA were simultaneously monitored by two specific HPLC detection methods. The longer the alkyl chain, the smaller the metabolic rate of the alpha-hydroxylation of the alkyl chain and the greater was the metabolic rate of the corresponding (omega-1)-hydroxy metabolite formation. Thus, the (omega-1)-hydroxylation of the alkyl chain was the major metabolic pathway of NDBA and NDAA in so far as it represented 3.3- and 86-fold of the alpha-hydroxylation. The balance between beta- to omega-hydroxylations and alpha-hydroxylation of carbon atoms of the alkyl chain depends upon its length and also upon the specific P450 isoform(s) involved. The hydroxylation site of the alkyl chain by P450 2E1 depends upon its length. For short alkyl chains, the main pathway was alpha-hydroxylation while for long alkyl chains, such as pentyl, (omega-1)-hydroxylation became the major pathway. The rate of alpha-hydroxylation was shown to be correlated with mutagenesis of 5 dialkylnitrosamines, as inferred from literature data, while the (omega-1)-hydroxylation was inversely correlated. Furthermore, other P450s than P450 2E1, such as P450 3A4 and 2C were shown to be involved in the metabolism of nitrosodialkylamines bearing long alkyl chains.
Asunto(s)
Sistema Enzimático del Citocromo P-450/metabolismo , Microsomas Hepáticos/metabolismo , Nitrosaminas/metabolismo , Adulto , Carcinógenos/metabolismo , Línea Celular , Cromatografía Líquida de Alta Presión , Sistema Enzimático del Citocromo P-450/genética , Femenino , Humanos , Hidroxilación , Masculino , Persona de Mediana Edad , Mutágenos/química , Mutágenos/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Relación Estructura-ActividadRESUMEN
Although a number of studies confirm the important role of metabolites in the cytotoxicity of acetaminophen, its precise mechanisms remain unknown. Acetaminophen is metabolized by microsomal enzymes. Cytochrome P4502E1 (CYP2E1) mediated N-hydroxylation results in the formation of N-acetyl-benzo-quinoneimine, a highly reactive intermediate. We examined biochemical parameters related to necrotic and apoptotic processes in acetaminophen-exposed PC12 cells is and in a PC12 cell line genetically engineered to express human CYP2E1. Both the [3H]thymidine incorporation test and the protein assay uniformly showed dose- and time-related significant growth retardation in both cell lines exposed to the drug. This was more evident in CYP2E1-transfected cells. Moreover, the cytotoxic effect of acetaminophen was increased as evidenced by lactate dehydrogenase activity in the culture medium. Both random oligonucleotide primed synthesis assay and enzyme-linked immunosorbent assay revealed significant DNA fragmentation in both cell lines, which was greater in transfected cells, reaching about 11% of total cellular DNA. These results were confirmed by flow cytometry and microscopic examination of cell nuclei. Intracellular calcium levels were increased only in transfected cells, approximately threefold when 5 mM acetaminophen was administered for 48 h. These results indicate the cytotoxic effects of acetaminophen via apoptosis, necrosis, and growth retardation. While the precise mechanism remains obscure, it seems that DNA fragmentation and apoptotic cascade represent a preliminary biochemical event in acute cell death, and that acetaminophen bio-transformation by CYP2E1 stimulates this pathway.
Asunto(s)
Acetaminofén/metabolismo , Acetaminofén/toxicidad , Citocromo P-450 CYP2E1/genética , Transfección , Animales , Apoptosis/efectos de los fármacos , Ciclo Celular/efectos de los fármacos , División Celular/efectos de los fármacos , Línea Celular , Fragmentación del ADN/efectos de los fármacos , Humanos , Células PC12 , RatasRESUMEN
Laurate and arachidonate omega and (omega-1)-hydroxylase activities, cytochrome P450 2E1 (CYP2E1), and CYP4A content were measured in 18 human kidney microsomal samples. The rates of laurate and arachidonate were found to be very different from those measured in human liver samples, with a laurate omega/omega-1 ratio of approximately 22 in human kidney vs 0.75 in human liver. Immunoblot analysis of the 18 human kidney microsomal samples identified 1 CYP4A electrophoretic band, but CYP2E1 was not detectable in human kidney, contrary to liver. Laurate and arachidonate omega-hydroxylase activities were significantly correlated with CYP4A content (r = 0.86 and 0.75, respectively). Polyclonal antirat CYP2E1 antibody did not affect omega-hydroxylase activity, whereas the polyclonal antirat CYP4A1 antibody inhibited it by 60%. These results suggest that, in contrast to other species, human kidney microsomes do not contain significant amounts of CYP2E1, but possess CYP4A and fatty acid omega-hydroxylase activity.
Asunto(s)
Citocromo P-450 CYP2E1/análisis , Sistema Enzimático del Citocromo P-450/análisis , Riñón/enzimología , Microsomas/enzimología , Oxigenasas de Función Mixta/análisis , Adulto , Anciano , Anciano de 80 o más Años , Ácido Araquidónico/metabolismo , Citocromo P-450 CYP4A , Femenino , Humanos , Hidroxilación , Cinética , Ácidos Láuricos/metabolismo , Masculino , Persona de Mediana EdadRESUMEN
Because of the important role of glial cells in brain maturation and reports on their delayed proliferation following ethanol exposure, it was considered of interest to investigate the mechanism of ethanol action on these cells. Biochemical parameters related to the apoptotic and necrotic processes in astroglial cells exposed for 1 week to 50 and 100 mM ethanol were examined. Ethanol increased intracellular calcium levels without changing transglutaminase activity and nitrite levels. Moreover, DNA fragmentation was noted with flow cytometry and with the random oligonucleotide primed synthesis assay but neither following agar gel electrophoresis nor in UV microscopy of cell nuclei. The DNA patterns obtained were different from these seen in programmed cell death. Additionally, immunocytochemical analysis showed greater fragility of astrocytes than oligodendrocytes to ethanol. These results support the hypothesis that astroglial cells in vitro exposed to ethanol die due to necrotic but not apoptotic mechanisms.
Asunto(s)
Apoptosis/efectos de los fármacos , Astrocitos/efectos de los fármacos , Encéfalo/efectos de los fármacos , Depsipéptidos , Etanol/toxicidad , Necrosis , Péptidos , Animales , Antibacterianos/toxicidad , Astrocitos/patología , Encéfalo/citología , Calcio/análisis , Calcio/metabolismo , Núcleo Celular/efectos de los fármacos , Núcleo Celular/patología , Células Cultivadas , Fragmentación del ADN , Femenino , Embarazo , RatasRESUMEN
Methadone, a synthetic drug, is one of the most widely used drugs for opiate dependency treatment. This drug has been demonstrated to be extensively metabolized by cytochrome P450 3A4 in human liver microsomes. Thus, the aim of this in vitro study was to determine if methadone is an inhibitor of other P450s characterized by their specific catalytic activities. Enzymatic activities specific to P450 2E1, P450 1A, P450 2B and P450 2C were not inhibited by methadone. Conversely, nifedipine oxidation, mediated by cytochrome P450 3A4, was potently inhibited by methadone by a mixed-type inhibition mechanism with a Ki of 100 microM. Fluvoxamine, a new antidepressant, was shown to be a potent mixed-type inhibitor of methadone N-demethylation with a Ki of 7 microM. Finally, methadone appears to be a mixed-type inhibitor and not a suicide inhibitor of cytochrome P450 3A family. Accordingly, caution should be advised in the clinical use of methadone when other drugs are administered that are able to induce or inhibit P450 3A4, such as rifampicin or nifedipine, diazepam and fluvoxamine.
Asunto(s)
Sistema Enzimático del Citocromo P-450/metabolismo , Sistema Enzimático del Citocromo P-450/farmacología , Metadona/metabolismo , Metadona/farmacología , Oxigenasas de Función Mixta/metabolismo , Oxigenasas de Función Mixta/farmacología , Unión Competitiva/efectos de los fármacos , Citocromo P-450 CYP1A1/análisis , Citocromo P-450 CYP2B1/análisis , Citocromo P-450 CYP3A , Inhibidores Enzimáticos del Citocromo P-450 , Dextrometorfano/metabolismo , Diazepam/farmacología , Fluvoxamina/farmacología , Humanos , Metadona/antagonistas & inhibidores , Microsomas Hepáticos/efectos de los fármacos , Microsomas Hepáticos/enzimología , Microsomas Hepáticos/metabolismo , Oxigenasas de Función Mixta/antagonistas & inhibidores , Nifedipino/farmacología , Rifampin/farmacologíaRESUMEN
There are numerous reports of the effect of ethanol on protein kinase C (PKC) in animals or with in vitro systems. However, the effect of ethanol on PKC in humans has not been extensively investigated despite the large number of studies involving PKC and human platelets. In this study, we administered ethanol to human volunteers and determined the level of PKC before and after a 0.4 g/kg dose of ethanol. We studied Native Americans and Caucasians of both sexes. There was an increases in PKC activity 60 min after ethanol administration. There were no ethnic, age, nor gender differences detected, nor was there any correlation between family history of alcoholism and the basal or stimulated platelet PKC levels. Neither was there any correlation of basal or stimulated PKC activity with the genotypes for ADH2, ADH3, ALDH2, CYP2E1, and CYP1A2.
Asunto(s)
Plaquetas/enzimología , Etanol/farmacología , Proteína Quinasa C/metabolismo , Adulto , Factores de Edad , Alcohol Deshidrogenasa/metabolismo , Aldehído Deshidrogenasa/metabolismo , Plaquetas/efectos de los fármacos , Citocromo P-450 CYP1A2/efectos de los fármacos , Citocromo P-450 CYP1A2/genética , Citocromo P-450 CYP1A2/metabolismo , Citocromo P-450 CYP2E1/efectos de los fármacos , Citocromo P-450 CYP2E1/genética , Citocromo P-450 CYP2E1/metabolismo , Etanol/administración & dosificación , Femenino , Genotipo , Humanos , Indígenas Norteamericanos/genética , Infusiones Intravenosas , Masculino , Persona de Mediana Edad , Proteína Quinasa C/efectos de los fármacos , Proteína Quinasa C/genética , Factores Sexuales , Estimulación Química , Población Blanca/genéticaRESUMEN
Caffeine metabolism by hepatic microsomal P450 enzymes is well documented in experimental animals and humans. However, its induction effect on P450 enzymes has not been thoroughly studied. In a preliminary experiment, the time-dependent incubation of 1 mM caffeine with rat hepatocyte culture resulted in an increase of its own metabolic rate. The dose-dependent expression of rat hepatic and renal cytochromes (CYP) 1A1/1A2 was then investigated after per os administration of caffeine. P450 expression was monitored by using specific enzymatic activities and Northern blot analysis. Caffeine caused a dose-dependent elevation of hepatic CYP1A1/1A2 activities in microsomal preparations, which ranged from 1.7- to 6-fold for ethoxyresorufin O-deethylase and 3- to 8.9-fold for methoxy-resorufin O-demethylase according to the dose regimen of 50 and 150 mg caffeine/kg/day for 3 days, respectively. Northern blot analysis demonstrated that caffeine treatment increased liver CYP1A1 and CYP1A2 mRNA levels over the dose regimen of 50-150 mg caffeine/kg/day for 3 days, respectively. The result of this study demonstrates that caffeine increases its own metabolism in a dose-dependent manner and induces CYP1A1/1A2 expression through either transcriptional activation or mRNA stabilization.
Asunto(s)
Citocromo P-450 CYP1A1/biosíntesis , Citocromo P-450 CYP1A2/biosíntesis , Animales , Células Cultivadas , Inducción Enzimática/efectos de los fármacos , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Riñón/enzimología , Masculino , Microsomas Hepáticos/enzimología , ARN Mensajero/genética , Ratas , Ratas WistarRESUMEN
Liver microsomal preparations from control and treated rats (cytochromes P450 1A, 2B, 3A and 2E1-induced) metabolized at variable metabolic rates three nitrosodialkylamines (N-nitroso-dipropyl, dibutyl and diamyl-amines) into aldehydes and hydroxy-nitrosamines. The longer the alkyl chain, the smaller was the metabolic rate of the alpha-hydroxylation of alkyl chain yielding aldehyde and the greater was the metabolic rate of the corresponding (omega-1)-hydroxyl metabolite formation. Thus, the (omega-1) hydroxylation of the alkyl chain was the major metabolic pathway of N-nitrosodiamylamine (NDAA) so far as it represented 22-fold the alpha-hydroxylation. The balance between beta to omega hydroxylation and alpha-hydroxylation depends upon the alkyl chain length and also on specific P450 isoform induction.
Asunto(s)
Carcinógenos/metabolismo , Microsomas Hepáticos/metabolismo , Nitrosaminas/metabolismo , Animales , Carbono/metabolismo , Carcinógenos/química , Hidroxilación , Nitrosaminas/química , RatasRESUMEN
Ethanol has been previously shown to reduce the unsaturated fatty acid content of cell membranes. It is not known, however, if the observed deleterious effects are due to ethanol itself or its metabolite, acetaldehyde. The present study was undertaken to assess the effect of acetaldehyde produced from ethanol by alcohol-deyhdrogenase-transfected Chinese hamster ovary Cells on the membrane lipids and the lipid peroxidation measured by free and bound malondialdehyde (MDA). The effects of ethanol alone was assessed in the presence of 4-methylpyrazole (4-MP), an inhibitor of alcohol dehydrogenase. After 8 days of incubation, total cellular lipids were extracted, subjected to TLC, and analyzed by gas chromatography. MDA concentration were determined by thiobarbituric acid reaction followed by HPLC detection. The level of acetaldehyde in the culture medium increased with concentration of ethanol from 5 to 20 mM as did the lipid peroxidation. Total cholesterol, phospholipids, and triglycerids all increased with increasing concentration of acetaldehyde. These effects were due to acetaldehyde as they were blocked by 4-MP. Some changes in fatty acid profiles were observed by effect of ethanol itself.
Asunto(s)
Acetaldehído/farmacología , Alcohol Deshidrogenasa/genética , Células CHO/enzimología , Etanol/metabolismo , Lípidos de la Membrana/metabolismo , Transfección , Acetaldehído/metabolismo , Alcohol Deshidrogenasa/antagonistas & inhibidores , Animales , Cromatografía Líquida de Alta Presión , Cricetinae , Inhibidores Enzimáticos/farmacología , Etanol/administración & dosificación , Ácidos Grasos/metabolismo , Fomepizol , Peroxidación de Lípido/efectos de los fármacos , Malondialdehído/metabolismo , Fosfolípidos/metabolismo , Pirazoles/farmacología , Sustancias Reactivas al Ácido Tiobarbitúrico/metabolismoRESUMEN
The metabolic dealkylation of nine nitrosodialkylamines, including five symmetrical (nitrosodimethylamine, nitrosodiethylamine, nitrosodipropylamine, nitrosodibutylamine and nitrosodiamylamine) and four asymmetrical nitrosodialkylamines (nitrosomethylethylamine, nitrosomethylpropylamine, nitrosomethylbutylamine and nitrosomethylamylamine), was investigated in 14 samples of human liver microsomes. All these nitrosodialkylamines were dealkytated to aldehydes that were separated by reversed phase HPLC and UV detected as dinitrophenylhydrazones. As the length of the alkyl chain increased from methyl to pentyl, dealkylation of symmetrical nitrosodialkylamines became less efficiently catalyzed by cytochrome P450. Conversely, oxidation of the methyl moiety of asymmetrical nitrosomethylalkylamines increased with the size of the alkyl moiety, while dealkylation of the longer alkyl group decreased. N-Dealkylase activities were significantly correlated with P450 activities measured in human liver microsomes. These catalytic activities involve CYP2A6 (coumarin 7-hydroxylation), CYP2C (mephenytoin 4-hydroxylation and tolbutamide hydroxylation), CYP2D6 (dextromethorphan O-demethylation), CYP2E1 (chlorzoxazone and p-nitrophenol hydroxylation) and CYP3A4 (nifedipine oxidation). By using 10 heterologously expressed P450s, it was shown that nitrosodimethylamine was mainly demethylated by CYP2E1. However, such enzyme specificity was lost with increasing size of the alkyl group. Therefore, the chain length of the alkyl group of nitrosodialkylamines determined the P450 involved in its oxidation. All these results emphasize that the catalytic site of P450 2EI has a geometric configuration such that only small molecules like nitrosodimethylamine fit favorably within the putative active site of the enzyme. Furthermore, there is good evidence that P450s other than P450 2E1, such as P450 2A6, 2C8/2C9/2C19 and 3A4, are involved in the metabolism of nitrosodialkylamines bearing bulky alkyl chains.
Asunto(s)
Hidrocarburo de Aril Hidroxilasas , Sistema Enzimático del Citocromo P-450/metabolismo , Microsomas Hepáticos/enzimología , Nitrosaminas/metabolismo , Adulto , Biotransformación , Citocromo P-450 CYP2A6 , Citocromo P-450 CYP2C19 , Citocromo P-450 CYP2D6/metabolismo , Citocromo P-450 CYP2E1/metabolismo , Citocromo P-450 CYP3A , Femenino , Humanos , Cinética , Masculino , Oxigenasas de Función Mixta/metabolismo , Proteínas Recombinantes/metabolismo , Especificidad por SustratoRESUMEN
Genetic polymorphisms of various cytochromes P450 have recently been described and could be implicated in the individual susceptibility of alcoholics to ethanol-related diseases. Rsal and Dral polymorphisms of CYP2E1 and Mspl polymorphism of CYP1A1 were studied in 260 controls and 511 alcoholic patients, without any clinical symptoms (n = 202) or with various ethanol-related diseases (n = 309), such as liver cirrhosis (n = 110), esophageal cancer (n = 62), upper aerodigestive tract cancer (n = 96), and other miscellaneous diseases (n = 41). Frequencies of the mutated alleles were found to be 2.5% (Rsal), 7.9% (Dral), and 8.7% (Mspl) in controls; 4%, 14.1%, and 12% in alcoholics without clinical symptoms; and 3.1%, 12.5%, and 11.2% in alcoholics with ethanol-related diseases. The only significant difference was found in the Dral polymorphism, whose frequency was enhanced in alcoholics with (p < 0.05) or without ethanol-related diseases (p < 0.01) when compared with controls. No differences were found between alcoholics without clinical symptoms and alcoholics with cirrhosis, esophageal cancer, or upper aerodigestive tract cancer. However, in liver cirrhosis and in ethanol-related cancers, the rare Dral-C allele was three times less frequent in patients under the age of 45 than in older patients, suggesting a protective role for this allele. In conclusion, our data indicate that the aforementioned mutations do not play a critical role in the development of cirrhosis, esophageal cancer, or upper aerodigestive tract cancers in Caucasians.
Asunto(s)
Alcoholismo/genética , Citocromo P-450 CYP1A1/genética , Citocromo P-450 CYP2E1/genética , Neoplasias Esofágicas/genética , Genotipo , Cirrosis Hepática/genética , Neoplasias de Oído, Nariz y Garganta/genética , Adulto , Alelos , Análisis Mutacional de ADN , Electroforesis en Gel de Poliacrilamida , Femenino , Frecuencia de los Genes , Humanos , Masculino , Persona de Mediana Edad , Reacción en Cadena de la Polimerasa , Polimorfismo de Longitud del Fragmento de Restricción , Factores de RiesgoRESUMEN
The formation of (omega-1)-hydroxylauric acid from lauric acid (LA) can be used as an indicator of the activity of cytochrome P450 2E1 (CYP2E1) in rat and human liver microsomes. A high-performance liquid chromatographic (HPLC) method that is capable of identifying and measuring the two main metabolites of lauric acid, (omega-1)- and omega-OH-LA, has been developed and used in the study of rat and human liver microsomes. Measurement of the enzymatic activities, based on the esterification of the metabolites and substrate with the fluorescent agent, 4-bromomethyl-6, 7-dimethoxycoumarin, is described using both radiometric and fluorimetric detection methods. Extraction efficiencies of metabolites and residual substrate were calculated using radioactivity and were greater than 85%. The assay is accurate and reproducible and has a detection limit of 75 pg (0.37 pmol). Additionally, a strong correlation between the two techniques was found in both human (r = 0.945, n = 15, p < 0.01) and rat (r = 0.949, n = 18, p < 0.01) livers, for the (omega-1)-hydroxylauric acid.
Asunto(s)
Cromatografía Líquida de Alta Presión/métodos , Cumarinas , Citocromo P-450 CYP2E1/metabolismo , Ácidos Láuricos/análisis , Ácidos Láuricos/metabolismo , Microsomas Hepáticos/química , Animales , Esterificación , Humanos , Masculino , Microsomas Hepáticos/enzimología , Radiometría , Ratas , Ratas Sprague-Dawley , Reproducibilidad de los Resultados , Espectrometría de FluorescenciaRESUMEN
Ethanol consumption combined with smoking increase the risk of cancer in many tissues. Such a mechanism implies the involvement of cytochrome P450 alcohol (CYP2E1), which is regulated by numerous xenobiotics. The combination of P450 2E1 inducers (acetone or pyridine) and 3-methylcholanthrene during rat treatment was shown to decrease the liver P450 2E1 content while it enhanced its expression in kidney. It is suggested that this differential tissue response helps explain the organotropy of nitrosamine carcinogenicity.
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Carcinógenos/farmacología , Sistema Enzimático del Citocromo P-450/genética , Expresión Génica/efectos de los fármacos , Riñón/enzimología , Hígado/enzimología , Pulmón/enzimología , Oxidorreductasas N-Desmetilantes/genética , Acetona/farmacología , Animales , Secuencia de Bases , Northern Blotting , Citocromo P-450 CYP2E1 , Masculino , Metilcolantreno/farmacología , Datos de Secuencia Molecular , Piridinas/farmacología , ARN Mensajero/metabolismo , Ratas , Ratas Sprague-DawleyRESUMEN
N-Nitrosamines eluted from reversed-phase HPLC were quantitatively photohydrolysed in a UV photoreactor in aqueous solution to give the nitrite ion which could be determined colorimetrically with the Griess reagent. The chromatographic behavior of N-nitroso compounds (including 19 volatile dialkyl and 7 non-volatile N-nitrosamines) was studied on three octadecylsilane columns. The capacity factor varies linearly with the number of carbons atom of the n-dialkyl chains. N-nitrosamines bearing di-n-alkyl chains with the same number of carbon atoms could be separated with a highly polar mobile phase. The yield of photohydrolysis depends upon pH and time of exposure under UV light. The response was shown to be linear in the 0-200 ng range with a limit of detection of 8 pmoles injected for N-dialkyl nitrosamines. This limit was 20 pmoles for N-nitrosamines bearing two phenyl groups. Although N-nitrosamines could be detected at 230 nm without post-column reaction, such a reaction enhances the specificity of detection in biological matrices such as gastric juice or alcoholic beverages.
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Cromatografía Líquida de Alta Presión/métodos , Nitrosaminas/análisis , Cerveza/análisis , Colorimetría , Jugo Gástrico/química , Humanos , Hidrólisis , Fotoquímica , Espectrofotometría UltravioletaRESUMEN
Methadone has become one of the most widely used drugs for opiate dependency treatment. This drug is extensively metabolized by the cytochrome P450 hepatic enzyme family in man, yielding an N-demethylated metabolite that cyclizes spontaneously into 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine. The specific forms of cytochrome P450 involved in this oxidative N-demethylation were examined in a panel of 20 human liver microsomal preparations previously characterized with respect to their P450 enzyme contents. Methadone was demethylated with an apparent Km of 545 +/- 258 microM (n = 3). The metabolic rates were 745 +/- 574 pmol/(min.mg of protein). This metabolic pathway was strongly correlated with estradiol 2-hydroxylation, testosterone 6 beta-hydroxylation, nifedipine oxidation, erythromycin N-demethylation, and toremifene N-demethylation, all of these monooxygenase activities being supported by P450 3A4. Furthermore, the total P450 3A content of liver microsomal samples, determined by immuno-quantification using a monoclonal anti-human P450 3A4 antibody, was correlated with methadone demethylation (r = 0.72; p < 0.003). Methadone metabolism was 60-72% inhibited either by three mechanism-based inhibitors of P450 3A4 (gestodene, TAO, and erythralosamine) or by four reversible inhibitors of P450 3A (ketoconazole, dihydroergotamine, quercetin, and diazepam with an apparent Ki of 50 microM) and by two nonspecific inhibitors (metyrapone and SKF-525A). Conversely, quinidine (inhibitor of P450 2D6), 7,8-benzoflavone (inhibitor of P450 1A), or sulfaphenazole (inhibitor of P450 2C) did not significantly inhibit, and may even have activated, methadone metabolism. Four heterologously expressed P450 proteins were able to catalyze the N-demethylation of methadone, namely, P450 2C8, P450 2C18, P450 2D6, and P450 3A4. However, referring to their relative liver content, it can be asserted that P450 3A4 is the major enzyme involved in the N-demethylation of methadone on average. Accordingly, caution should be advised in the clinical use of methadone when other drugs are also administered that induce or inhibit P450 3A4, such as rifampicin or diazepam, respectively.