RESUMEN
Endosulfan is an organochlorine pesticide commonly used in agriculture. Endosulfan has affects on vertebrate xenobiotic metabolism pathways that may be mediated, in part, by its ability to activate the pregnane X receptor (PXR) and/or the constitutive androstane receptor (CAR) which can elevate expression of cytochrome P450 (CYP) enzymes. This study examined the dose-dependency and receptor specificity of CYP induction in vitro and in vivo. The HepG2 cell line was transiently transfected with CYP2B6- and CYP3A4-luciferase promoter reporter plasmids along with human PXR (hPXR) or hCAR expression vectors. In the presence of hPXR, endosulfan-alpha exposure caused significant induction of CYP2B6 (16-fold) and CYP3A4 (11-fold) promoter activities over control at 10 microM. The metabolite endosulfan sulfate also induced CYP2B6 (12-fold) and CYP3A4 (6-fold) promoter activities over control at 10 microM. In the presence of hCAR-3, endosulfan-alpha induced CYP2B6 (2-fold) promoter activity at 10 microM, but not at lower concentrations. These data indicate that endosulfan-alpha significantly activates hPXR strongly and hCAR weakly. Using western blot analysis of human hepatocytes, the lowest concentrations at which CYP2B6 and CYP3A4 protein levels were found to be significantly elevated by endosulfan-alpha were 1.0 microM and 10 microM, respectively. In mPXR-null/hPXR-transgenic mice, endosulfan-alpha exposure (2.5mg/kg/day) caused a significant reduction of tribromoethanol-induced sleep times by approximately 50%, whereas no significant change in sleep times was observed in PXR-null mice. These data support the role of endosulfan-alpha as a strong activator of PXR and inducer of CYP2B6 and CYP3A4, which may impact metabolism of CYP2B6 or CYP3A4 substrates.
Asunto(s)
Hidrocarburo de Aril Hidroxilasas/biosíntesis , Citocromo P-450 CYP3A/biosíntesis , Endosulfano/toxicidad , Hepatocitos/efectos de los fármacos , Insecticidas/toxicidad , Oxidorreductasas N-Desmetilantes/biosíntesis , Receptores de Esteroides/agonistas , Anestésicos/metabolismo , Anestésicos/farmacología , Animales , Apoptosis/efectos de los fármacos , Hidrocarburo de Aril Hidroxilasas/genética , Receptor de Androstano Constitutivo , Citocromo P-450 CYP2B6 , Citocromo P-450 CYP3A/genética , Sistema Enzimático del Citocromo P-450/biosíntesis , Relación Dosis-Respuesta a Droga , Inducción Enzimática , Etanol/análogos & derivados , Etanol/metabolismo , Etanol/farmacología , Genes Reporteros , Células Hep G2 , Hepatocitos/enzimología , Humanos , Luciferasas/biosíntesis , Luciferasas/genética , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Oxidorreductasas N-Desmetilantes/genética , Receptor X de Pregnano , Regiones Promotoras Genéticas/efectos de los fármacos , ARN Mensajero/metabolismo , Receptores Citoplasmáticos y Nucleares/agonistas , Receptores Citoplasmáticos y Nucleares/genética , Receptores Citoplasmáticos y Nucleares/metabolismo , Receptores de Esteroides/genética , Receptores de Esteroides/metabolismo , Sueño/efectos de los fármacos , Factores de Tiempo , TransfecciónRESUMEN
Agrochemicals and other xenobiotics are metabolized by xenobiotic-metabolizing enzymes (XMEs) to products that may be more or less toxic than the parent chemical. In this regard, phase-I XMEs such as cytochrome P450s (CYPs) are of primary importance. Interactions at the level of metabolism may take place via either inhibition or induction of XMEs. Such interactions have often been investigated, in vitro, in experimental animals, using subcellular fractions such as liver microsomes, but seldom in humans or at the level of individual XME isoforms. The authors have been investigating the metabolism of a number of agrochemicals by human liver microsomes and recombinant CYP isoforms and have recently embarked on studies of the induction of XMEs in human hepatocytes. The insecticides chlorpyrifos, carbaryl, carbofuran and fipronil, as well as the repellant DEET, are all extensively metabolized by human liver microsomes and, although a number of CYP isoforms may be involved, CYP2B6 and CYP3A4 are usually the most important. Permethrin is hydrolyzed by esterase(s) present in both human liver microsomes and cytosol. A number of metabolic interactions have been observed. Chlorpyrifos and other phosphorothioates are potent inhibitors of the CYP-dependent metabolism of both endogenous substrates, such as testosterone and estradiol, and exogenous substrates, such as carbaryl, presumably as a result of the interaction of highly reactive sulfur, released during the oxidative desulfuration reaction, with the heme iron of CYP. The hydrolysis of permethrin in human liver can be inhibited by chlorpyrifos oxon and by carbaryl. Fipronil can inhibit testosterone metabolism by CYP3A4 and is an effective inducer of CYP isoforms in human hepatocytes.
Asunto(s)
Interacciones Farmacológicas , Insecticidas/metabolismo , Microsomas Hepáticos/enzimología , Contaminantes Ambientales/metabolismo , HumanosRESUMEN
Xenobiotics, including drugs and environmental chemicals, can influence cytochrome P450 (CYP) levels by altering the transcription of CYP genes. To minimize potential drug-pesticide and pesticide-pesticide interactions it is important to evaluate the potential of pesticides to induce CYP isoforms and to cause cytotoxicity in humans. The present study was designed to examine chlorpyrifos and DEET mediated induction of CYP isoforms and also to characterize their potential cytotoxic effects on primary human hepatocytes. DEET significantly induced CYP3A4, CYP2B6, CYP2A6 and CYP1A2 mRNA expression while chlorpyrifos induced CYP1A1, CYP1A2 and CYP3A4 mRNA, and to a lesser extent, CYP1B1 and CYP2B6 mRNA in primary human hepatocytes. Chlorpyrifos and DEET also mediated the expression of CYP isoforms, particularly CYP3A4, CYP2B6 and CYP1A1, as shown by CYP3A4-specific protein expression, testosterone metabolism and CYP1Al-specific activity assays. DEET is a mild, while chlorpyrifos is a relatively potent, inducer of adenylate kinase and caspase-3/7, an indicator of apoptosis, while inducing 15-20% and 25-30% cell death, respectively. Therefore, DEET and chlorpyrifos mediated induction of CYP mRNA and functional CYP isoforms together with their cytotoxic potential in human hepatocytes suggests that exposure to chlorpyrifos and/or DEET should be considered in human health impact analysis.
Asunto(s)
Cloropirifos/toxicidad , DEET/toxicidad , Hepatocitos/efectos de los fármacos , Repelentes de Insectos/toxicidad , Insecticidas/toxicidad , Adenilato Quinasa/metabolismo , Caspasa 3/metabolismo , Caspasa 7/metabolismo , Muerte Celular/efectos de los fármacos , Células Cultivadas , Citocromo P-450 CYP1A1/metabolismo , Citocromo P-450 CYP3A/metabolismo , Sistema Enzimático del Citocromo P-450/biosíntesis , Sistema Enzimático del Citocromo P-450/genética , Relación Dosis-Respuesta a Droga , Inducción Enzimática/efectos de los fármacos , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Hepatocitos/enzimología , Humanos , Isoenzimas/biosíntesis , Isoenzimas/genética , ARN Mensajero/genética , Células Tumorales CultivadasRESUMEN
Deltamethrin [(S)-alpha-cyano-3-phenoxybenzyl-cis-(1 R,3R)-3(2,2-dibromovinyl)(2,2-dimethyl-cyclopropane-carboxylate] and permethrin [3-phenoxybenzyl(1RS)-cis,trans-3-(2,2-dichlorovinyl)-2,2-dimethyl-cyclopropanecarboxylate] are pyrethroid insecticides used in agriculture, public health and military deployments. Pyrethroids are known to be capable of inducing cytochrome P450 (CYP) 2B1/2B2, CYP1A1 and overall CYP content in rat liver. The objectives of this study were to evaluate the potential of deltamethrin and permethrin to cause cytotoxicity and to induce CYP isoforms in human hepatocytes. Permethrin and deltamethrin showed dose-dependent effects on adenylate kinase activity in HepG2 cells, in which 50 and 100 microM doses, respectively, induced a 3-5 fold increase in activity, and also induced adenylate kinase activity in primary human hepatocytes. An approximately 3-fold induction was noted at 200 microM deltamethrin and a 4-fold induction at 100 microM permethrin. Cytotoxicity was noted in HepG2 cells following 48-72 h exposure to 100 or 200 microM deltamethrin and permethrin, respectively. Dose-dependent induction of caspase-3/7 was initiated by 12.5 microM deltamethrin or by 3.125 microM permethrin. Actinomycin D, a positive control for induction of caspase 3/7, induced caspase-3/7, an effect completely abrogated by the specific inhibitor Z-DEVD-FMK. At 100 microM deltamethrin 2-3 fold induction of CYP1A1 and CYP2B6 mRNA was observed, while at the same time an approximately 25-fold induction of CYP3A4 was noted. Permethrin-mediated CYP induction was much less potent, 4-fold or less for CYP1A1, CYP3A4, CYP3A5, CYP2B6 and CYP2A6. It has also been shown that these pyrethroids are ligands for the pregnane X receptor (PXR).
Asunto(s)
Sistema Enzimático del Citocromo P-450/biosíntesis , Hepatocitos/efectos de los fármacos , Insecticidas/toxicidad , Nitrilos/toxicidad , Permetrina/toxicidad , Piretrinas/toxicidad , Adenilato Quinasa/metabolismo , Adulto , Caspasa 3/metabolismo , Caspasa 7/metabolismo , Muerte Celular/efectos de los fármacos , Células Cultivadas , Citocromo P-450 CYP3A/metabolismo , Sistema Enzimático del Citocromo P-450/genética , Relación Dosis-Respuesta a Droga , Inducción Enzimática/efectos de los fármacos , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Hepatocitos/enzimología , Humanos , Isoenzimas/biosíntesis , Isoenzimas/genética , Receptor X de Pregnano , ARN Mensajero/genética , Receptores de Esteroides/metabolismo , Células Tumorales CultivadasRESUMEN
Investigations utilizing recombinant human xenobiotic-metabolizing enzymes as well as human hepatocytes have revealed a number of interactions not only between different environmental chemicals (ECs) but also between ECs and endogenous metabolites. Organophosphorus insecticides (OPs) are potent inhibitors of the human metabolism of carbaryl, carbofuran, DEET and fipronil, as well as the jet fuel components, nonane and naphthalene. OPs are potent irreversible inhibitors of testosterone metabolism by cytochrome P450 (CYP) 3A4 and of estradiol metabolism by CYP3A4 and CYP1A2. All of these CYP inhibitions are believed to be due to the release of reactive sulfur during CYP-catalyzed oxidative desulfuration. It has also been shown that the esterase(s) responsible for the initial step in permethrin metabolism in human liver is inhibited by both chlorpyrifos oxon and carbaryl. A number of pesticides, including chlorpyrifos, fipronil and permethrin, and the repellent, DEET, have been shown to be inducers of CYP isoforms in human hepatocytes, with fipronil being the most potent. Several agrochemicals, including fipronil and the pyrethroids, permethrin and deltamethrin, show toxicity toward human hepatocytes with fipronil being the most potent in this regard. Endosulfan-alpha, which has shown promise as a model substrate for phenotyping CYP3A4 and CYP2B6 in human liver microsomes, is also an inducer of CYP2B6, acting through the PXR receptor.
Asunto(s)
Inhibidores de la Colinesterasa/metabolismo , Sistema Enzimático del Citocromo P-450/metabolismo , Contaminantes Ambientales/metabolismo , Plaguicidas/metabolismo , Inhibidores Enzimáticos del Citocromo P-450 , Sistema Enzimático del Citocromo P-450/genética , Contaminantes Ambientales/química , Inducción Enzimática , Estradiol/metabolismo , Femenino , Humanos , Isoenzimas/metabolismo , Hígado/citología , Hígado/enzimología , Masculino , Microsomas Hepáticos/enzimología , Proteínas Recombinantes/antagonistas & inhibidores , Proteínas Recombinantes/metabolismo , Testosterona/antagonistas & inhibidores , Testosterona/metabolismoRESUMEN
Previous studies have established that chlorpyrifos (CPS), fipronil, and nonane can all be metabolized by human liver microsomes (HLM) and a number of cytochrome P450 (CYP) isoforms. However, metabolic interactions between these three substrates have not been described. In this study the effect of either coincubation or preincubation of CPS with HLM or CYP isoforms with either fipronil or nonane as substrate was investigated. In both co- and preincubation experiments, CPS significantly inhibited the metabolism of fipronil or nonane by HLM although CPS inhibited the metabolism of fipronil more effectively than that of nonane. CPS significantly inhibited the metabolism of fipronil by CYP3A4 as well as the metabolism of nonane by CYP2B6. In both cases, preincubation with CPS caused greater inhibition than coincubation, suggesting that the inhibition is mechanism based.
Asunto(s)
Alcanos/farmacología , Hidrocarburo de Aril Hidroxilasas/metabolismo , Cloropirifos/farmacología , Sistema Enzimático del Citocromo P-450/metabolismo , Insecticidas/farmacología , Microsomas Hepáticos/enzimología , Oxidorreductasas N-Desmetilantes/metabolismo , Pirazoles/farmacología , Hidrocarburo de Aril Hidroxilasas/antagonistas & inhibidores , Citocromo P-450 CYP2B6 , Citocromo P-450 CYP3A , Inhibidores Enzimáticos del Citocromo P-450 , Activación Enzimática/efectos de los fármacos , Femenino , Humanos , Isoenzimas/antagonistas & inhibidores , Isoenzimas/metabolismo , Masculino , Oxidorreductasas N-Desmetilantes/antagonistas & inhibidoresRESUMEN
Chlorpyrifos-oxon (CPO), a metabolite of chlorpyrifos, is a potent inhibitor of acetylcholinesterase and, although the neurotoxicological impact of this organophosphorus compound has been broadly studied both in vitro and in vivo, there are few studies of metabolic interactions of CPO with other xenobiotics. CPO significantly activated the production of 1-naphthol (5-fold), 2-naphthol (10-fold), trans-1,2-dihydro-1,2-naphthalenediol (1.5-fold), and 1,4-naphthoquinone from naphthalene by human liver microsomes (HLM). It was further demonstrated that the production of naphthalene metabolites by CYP2C8, 2C9*(1), 2C19, 2D6*(1), 3A4, 3A5, and 3A7 was activated by CPO, while the production of naphthalene metabolites by CYP1A1, 1A2, 1B1, and 2B6 was inhibited by CPO. CPO inhibited CYP1A2 production of naphthalene metabolites, while activating their production by CYP3A4. Similarly, CPO inhibited the production of N,N-diethyl-m-hydroxymethylbenzamide (BALC) from DEET by human liver microsomes, but activated the production of N-ethyl-m-toluamide (ET) from this substrate. CYP2B6, the most efficient isoform for BALC production, was inhibited by CPO, while CYP3A4, the most efficient isoform for ET production, was activated by CPO. CPO inhibited CYP2B6 production of both BALC and ET from DEET, but activated CYP3A4 production of ET, while inhibiting CYP3A4 BALC production. CPO appears to facilitate the binding of naphthalene to CYP3A4. This metabolic activation is independent of cytochrome b5, suggesting that activation of CYP3A4 by CPO is associated with a conformational change of the isoform rather than facilitating electron transfer.
Asunto(s)
Cloropirifos/análogos & derivados , Sistema Enzimático del Citocromo P-450/metabolismo , DEET/metabolismo , Insecticidas/toxicidad , Microsomas Hepáticos/metabolismo , Naftalenos/metabolismo , Células Cultivadas , Cloropirifos/toxicidad , Citocromo P-450 CYP1A2/metabolismo , Citocromo P-450 CYP3A/metabolismo , Depresión Química , Activación Enzimática , HumanosRESUMEN
Cytochrome P450 (CYP) 2B6 (CYP2B6) is a human CYP isoform found in variable amounts in the liver and other organs. It is known to be inducible and polymorphic and has a wide range of xenobiotic substrates. Studies of CYP2B6 to date have concentrated heavily on clinical drugs. In the present communication, however, we concentrate on its role in the metabolism of environmental xenobiotics. The term environment is used, in its broadest sense, to include natural ecosystems and agroecosystems as well as the industrial and indoor domestic environments. In essence, this excludes only clinical drugs and drugs of abuse. Many of these chemicals, including agrochemicals and industrial chemicals, can serve as substrates, inhibitors and/or inducers of CYP2B6, these activities being often modified by the existence of polymorphic variants. Metabolism-based interactions between environmental chemicals are discussed, as well as the emerging possibility of metabolic interactions between environmental chemicals and clinical drugs.
Asunto(s)
Hidrocarburo de Aril Hidroxilasas/metabolismo , Oxidorreductasas N-Desmetilantes/metabolismo , Xenobióticos/metabolismo , Agroquímicos/metabolismo , Citocromo P-450 CYP2B6 , Contaminantes Ambientales/metabolismo , HumanosRESUMEN
The metabolism of chlorpyrifos (CPS) and chlorpyrifos oxon (CPO) by human hepatocytes and human liver S9 fractions was investigated using LC-MS/MS. Cytochrome P450 (CYP)-dependent and phase II-related products were determined following incubation with CPS and CPO. CYP-related products, 3,5,6-trichloro-2-pyridinol (TCP), diethyl thiophosphate, and dealkylated CPS, were found following CPS treatment and dealkylated CPO following CPO treatment. Diethyl phosphate was not identified because of its high polarity and lack of retention with the chromatographic conditions employed. Phase II-related conjugates, including O- and S-glucuronides as well as 11 GSH-derived metabolites, were identified in CPS-treated human hepatocytes, although the O-sulfate of TCP conjugate was found only when human liver S9 fractions were used as the enzyme source. O-Glucuronide of TCP was also identified in CPO-treated hepatocytes. CPS and CPO were identified using HPLC-UV after CPS metabolism by the human liver S9 fraction. However, CPO was not found following treatment of human hepatocytes with either CPS or CPO. These results suggest that human liver plays an important role in detoxification, rather than activation, of CPS.
Asunto(s)
Cloropirifos/análogos & derivados , Hepatocitos/efectos de los fármacos , Hepatocitos/metabolismo , Adulto , Biotransformación , Cloropirifos/análisis , Cloropirifos/química , Cloropirifos/metabolismo , Cloropirifos/toxicidad , Cromatografía Líquida de Alta Presión , Femenino , Humanos , Masculino , Espectrometría de Masas , Persona de Mediana Edad , Fracciones SubcelularesRESUMEN
Recent studies have demonstrated the potential of pesticides to either inhibit or induce xenobiotic metabolizing enzymes in humans. Exposure of human hepatocytes to doses of fipronil (5-amino-1-[2,6-dichloro-4-(trifluoromethyl)phenyl]-4-[(trifluoromethyl) sulfinyl]-1H-pyrazole-3-carbonitrile) ranging from 0.1 to 25 microM resulted in a dose dependent increase in CYP1A1 mRNA expression (3.5 to approximately 55-fold) as measured by the branched DNA assay. In a similar manner, CYP3A4 mRNA expression was also induced (10-30-fold), although at the higher doses induction returned to near control levels. CYP2B6 and 3A5 were also induced by fipronil, although at lower levels (2-3-fold). Confirmation of bDNA results were sought through western blotting and/or enzyme activity assays. Western blots using CYP3A4 antibody demonstrated a dose responsive increase from 0.5 to 1 microM followed by decreasing responses at higher concentrations. Similar increases and decreases were observed in CYP3A4-specific activity levels as measured using 6beta-hydroxytestosterone formation following incubation with testosterone. Likewise, activity levels for a CYP1A1-specific substrate, luciferin CEE, demonstrated that CYP1A1 enzyme activities were maximally induced by 1 microM fipronil followed by dramatically declining activity measurements at 10 and 25 microM. Cytotoxic effects of fipronil and fipronil sulfone were examined using the adenylate kinase and the trypan blue exclusion assays in HepG2 cells and human hepatocytes. The results indicate both that HepG2 cells and primary human hepatocytes are sensitive to the cytotoxic effects of fipronil. The maximum induction of adenylate kinase was ca. 3-fold greater than the respective controls in HepG2 and 6-10-fold in the case of primary hepatocytes. A significant time- and dose-dependent induction of adenylate kinase activity in HepG2 cells was noted from 0.1 to 12.5 microM fipronil followed by decreasing activities at 25 and 50 microM. For fipronil sulfone, cytotoxic effects increased throughout the dose range. The trypan blue assay indicated that cytotoxic effects contributing to an increase of greater than 10% of control values was indicated at doses above 12.5 microM. However, fipronil sulfone induced cytotoxic effects at lower doses. The possibility that cytotoxic effects were due to apoptosis was indicated by significant time- and dose-dependent induction of caspase-3/7 activity in both HepG2 cells and human hepatocytes. Fipronil mediated activation of caspase-3/7 in concurrence with compromised ATP production and viability are attributed to apoptotic cell death.
Asunto(s)
Sistema Enzimático del Citocromo P-450/metabolismo , Hepatocitos/efectos de los fármacos , Hepatocitos/enzimología , Pirazoles/farmacología , Caspasa 3/metabolismo , Caspasa 7/metabolismo , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Sistema Enzimático del Citocromo P-450/genética , Inducción Enzimática/efectos de los fármacos , Regulación de la Expresión Génica , Humanos , Isoenzimas/genética , Isoenzimas/metabolismo , Pirazoles/química , ARN Mensajero/genética , Azufre/química , Transcripción Genética/genética , Receptor fas/metabolismoRESUMEN
High-resolution radar images reveal near-Earth asteroid (66391) 1999 KW4 to be a binary system. The approximately 1.5-kilometer-diameter primary (Alpha) is an unconsolidated gravitational aggregate with a spin period approximately 2.8 hours, bulk density approximately 2 grams per cubic centimeter, porosity approximately 50%, and an oblate shape dominated by an equatorial ridge at the object's potential-energy minimum. The approximately 0.5-kilometer secondary (Beta) is elongated and probably is denser than Alpha. Its average orbit about Alpha is circular with a radius approximately 2.5 kilometers and period approximately 17.4 hours, and its average rotation is synchronous with the long axis pointed toward Alpha, but librational departures from that orientation are evident. Exotic physical and dynamical properties may be common among near-Earth binaries.
RESUMEN
Endosulfan-alpha is metabolized to a single metabolite, endosulfan sulfate, in pooled human liver microsomes (Km = 9.8 microM, Vmax = 178.5 pmol/mg/min). With the use of recombinant cytochrome P450 (P450) isoforms, we identified CYP2B6 (Km = 16.2 microM, Vmax = 11.4 nmol/nmol P450/min) and CYP3A4 (Km = 14.4 microM, Vmax = 1.3 nmol/nmol P450/min) as the primary enzymes catalyzing the metabolism of endosulfan-alpha, although CYP2B6 had an 8-fold higher intrinsic clearance rate (CL(int) = 0.70 microl/min/pmol P450) than CYP3A4 (CL(int) = 0.09 microl/min/pmol P450). Using 16 individual human liver microsomes (HLMs), a strong correlation was observed with endosulfan sulfate formation and S-mephenytoin N-demethylase activity of CYP2B6 (r(2) = 0.79), whereas a moderate correlation with testosterone 6 beta-hydroxylase activity of CYP3A4 (r(2) = 0.54) was observed. Ticlopidine (5 microM), a potent CYP2B6 inhibitor, and ketoconazole (10 microM), a selective CYP3A4 inhibitor, together inhibited approximately 90% of endosulfan-alpha metabolism in HLMs. Using six HLM samples, the percentage total normalized rate (% TNR) was calculated to estimate the contribution of each P450 in the total metabolism of endosulfan-alpha. In five of the six HLMs used, the percentage inhibition with ticlopidine and ketoconazole in the same incubation correlated with the combined % TNRs for CYP2B6 and CYP3A4. This study shows that endosulfan-alpha is metabolized by HLMs to a single metabolite, endosulfan sulfate, and that it has potential use, in combination with inhibitors, as an in vitro probe for CYP2B6 and 3A4 catalytic activities.
Asunto(s)
Hidrocarburo de Aril Hidroxilasas/metabolismo , Sistema Enzimático del Citocromo P-450/metabolismo , Endosulfano/metabolismo , Microsomas Hepáticos/metabolismo , Oxidorreductasas N-Desmetilantes/metabolismo , Hidrocarburo de Aril Hidroxilasas/antagonistas & inhibidores , Biotransformación/efectos de los fármacos , Cromatografía Líquida de Alta Presión , Citocromo P-450 CYP2B6 , Citocromo P-450 CYP3A , Inhibidores Enzimáticos del Citocromo P-450 , Endosulfano/química , Endosulfano/farmacocinética , Inhibidores Enzimáticos/farmacología , Humanos , Insecticidas/química , Insecticidas/metabolismo , Insecticidas/farmacocinética , Cetoconazol/farmacología , Cinética , Microsomas Hepáticos/efectos de los fármacos , Microsomas Hepáticos/enzimología , Estructura Molecular , Oxidorreductasas N-Desmetilantes/antagonistas & inhibidores , Oxigenasas/metabolismo , Esteroide Hidroxilasas/metabolismo , Sulfatos/metabolismo , Ticlopidina/farmacologíaRESUMEN
Cytochromes P450 (P450s) are major catalysts in the metabolism of xenobiotics and endogenous substrates such as estradiol (E2). It has previously been shown that E2 is predominantly metabolized in humans by CYP1A2 and CYP3A4 with 2-hydroxyestradiol (2-OHE2) the major metabolite. This study examines effects of deployment-related and other chemicals on E2 metabolism by human liver microsomes (HLM) and individual P450 isoforms. Kinetic studies using HLM, CYP3A4, and CYP1A2 showed similar affinities (Km) for E2 with respect to 2-OHE2 production. Vmax and CLint values for HLM are 0.32 nmol/min/mg protein and 7.5 microl/min/mg protein; those for CYP3A4 are 6.9 nmol/min/nmol P450 and 291 microl/min/nmol P450; and those for CYP1A2 are 17.4 nmol/min/nmol P450 and 633 microl/min/nmol P450. Phenotyped HLM use showed that individuals with high levels of CYP1A2 and CYP3A4 have the greatest potential to metabolize E2. Preincubation of HLM with a variety of chemicals, including those used in military deployments, resulted in varying levels of inhibition of E2 metabolism. The greatest inhibition was observed with organophosphorus compounds, including chlorpyrifos and fonofos, with up to 80% inhibition for 2-OHE2 production. Carbaryl, a carbamate pesticide, and naphthalene, a jet fuel component, inhibited ca. 40% of E2 metabolism. Preincubation of CYP1A2 with chlorpyrifos, fonofos, carbaryl, or naphthalene resulted in 96, 59, 84, and 87% inhibition of E2 metabolism, respectively. Preincubation of CYP3A4 with chlorpyrifos, fonofos, deltamethrin, or permethrin resulted in 94, 87, 58, and 37% inhibition of E2 metabolism. Chlorpyrifos inhibition of E2 metabolism is shown to be irreversible.
Asunto(s)
Inhibidores de la Colinesterasa/toxicidad , Inhibidores del Citocromo P-450 CYP1A2 , Inhibidores Enzimáticos del Citocromo P-450 , Estradiol/análogos & derivados , Hígado/efectos de los fármacos , Carbaril/toxicidad , Cloropirifos/toxicidad , Citocromo P-450 CYP1A2/genética , Citocromo P-450 CYP1A2/metabolismo , Citocromo P-450 CYP3A , Sistema Enzimático del Citocromo P-450/genética , Sistema Enzimático del Citocromo P-450/metabolismo , Relación Dosis-Respuesta a Droga , Estradiol/metabolismo , Femenino , Fonofos/toxicidad , Humanos , Técnicas In Vitro , Cinética , Hígado/enzimología , Masculino , Microsomas Hepáticos , Medicina Militar , Proteínas Recombinantes/antagonistas & inhibidores , Proteínas Recombinantes/metabolismoRESUMEN
Marsupials are born ectothermic and gradually become endothermic during pouch occupancy. In order to study the timing of this transition, we measured the metabolic rates of eight pouch-young eastern barred bandicoots (Perameles gunnii) at 25 degrees C (the thermoneutral zone of adults), at 35 degrees C (pouch temperature), and after injection with norepinephrine. From 5 to 7 wk of age, oxygen consumption (VO(2)) was higher at 35 degrees C than at 25 degrees C. At 8 wk, VO(2) was significantly higher at 25 degrees C than at 35 degrees C. Norepinephrine administration at 5 and 6 wk had no effect on metabolic rate, but at 8 wk, near pouch vacation, it resulted in a significant increase in metabolic rate. Our results indicate that signs of thermoregulation for P. gunnii begin at 7 wk, and at 8 wk the species is endothermic, that is, able to increase heat production in response to both cold and norepinephrine. The appearance of fur, first seen at the age of 6 wk and fully developed at 8 wk, coincides with the onset of endothermy.
Asunto(s)
Regulación de la Temperatura Corporal/fisiología , Marsupiales/fisiología , Factores de Edad , Animales , Metabolismo Basal/fisiología , Frío , Femenino , Masculino , Marsupiales/metabolismo , Norepinefrina/farmacología , Consumo de Oxígeno/fisiología , Análisis de Regresión , TasmaniaRESUMEN
Although it has been known for some time that organophosphate chemicals containing the P = S moiety are irreversible inhibitors of cytochrome P450, this knowledge has not been generally applied to the human metabolism of xenobiotics. Recent studies have demonstrated that organophosphate insecticides containing this moiety are potent inhibitors of the metabolism of both xenobiotics and endogenous substrates by human liver microsomes and by specific human cytochrome P450 isoforms.
Asunto(s)
Compuestos Organofosforados/farmacología , Esteroides/metabolismo , Xenobióticos/metabolismo , Animales , Carbaril/metabolismo , Cloropirifos/metabolismo , Cloropirifos/farmacología , DEET/metabolismo , Depresión Química , Estrógenos/metabolismo , Femenino , Humanos , Repelentes de Insectos/metabolismo , Insecticidas/metabolismo , Insecticidas/farmacología , Masculino , Testosterona/metabolismoRESUMEN
The polycyclic aromatic hydrocarbon naphthalene is an environmental pollutant, a component of jet fuel, and, since 2000, has been reclassified as a potential human carcinogen. Few studies of the in vitro human metabolism of naphthalene are available, and these focus primarily on lung metabolism. The current studies were performed to characterize naphthalene metabolism by human cytochromes P450. Naphthalene metabolites from pooled human liver microsomes (pHLMs) were trans-1,2-dihydro-1,2-naphthalenediol (dihydrodiol), 1-naphthol, and 2-naphthol. Metabolite production generated Km values of 23, 40, and 116 microM And Vmax values of 2860, 268, and 22 pmol/mg protein/min, respectively. P450 isoform screening of naphthalene metabolism identified CYP1A2 as the most efficient isoform for producing dihydrodiol and 1-naphthol, and CYP3A4 as the most effective for 2-naphthol production. Metabolism of the primary metabolites of naphthalene was also studied to identify secondary metabolites. Whereas 2-naphthol was readily metabolized by pHLMs to produce 2,6- and 1,7-dihydroxynaphthalene, dihydrodiol and 1-naphthol were inefficient substrates for pHLMs. A series of human p450 isoforms was used to further explore the metabolism of dihydrodiol and 1-naphthol. 1,4-Naphthoquinone and four minor unknown metabolites from 1-naphthol were observed, and CYP1A2 and 2D6*1 were identified as the most active isoforms for the production of 1,4-naphthoquinone. Dihydrodiol was metabolized by P450 isoforms to three minor unidentified metabolites with CYP3A4 and CYP2A6 having the greatest activity toward this substrate. The metabolism of dihydrodiol by P450 isoforms was lower than that of 1-naphthol. These studies identify primary and secondary metabolites of naphthalene produced by pHLMs and P450 isoforms.
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Sistema Enzimático del Citocromo P-450/metabolismo , Microsomas Hepáticos/metabolismo , Naftalenos/metabolismo , Humanos , Isoenzimas/metabolismo , Cinética , Microsomas Hepáticos/química , Estructura Molecular , Naftalenos/química , Naftalenos/farmacocinética , Naftoquinonas/química , Naftoquinonas/metabolismoAsunto(s)
Agricultura , Encuestas Epidemiológicas , Plaguicidas/toxicidad , Animales , Humanos , Insecticidas/toxicidadRESUMEN
Nonane, a component of jet-propulsion fuel 8 (JP-8), is metabolized to 2-nonanol and 2-nonanone by pooled human liver microsomes (pHLM). Cytochrome P450 (CYP) isoforms 1A2, 2B6 and 2E1 metabolize nonane to 2-nonanol, whereas alcohol dehydrogenase, CYPs 2B6 and 2E1 metabolize 2-nonanol to 2-nonanone. Nonane and 2-nonanol showed no significant effect on the metabolism of testosterone, estradiol or N,N-diethyl-m-toluamide (DEET), but did inhibit carbaryl metabolism. JP-8 showed modest inhibition of testosterone, estradiol and carbaryl metabolism, but had a more significant effect on the metabolism of DEET. JP-8 was shown to inhibit CYPs 1A2 and 2B6 mediated metabolism of DEET, suggesting that at least some of the components of JP-8 might be metabolized by CYPs 1A2and/or 2B6.
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Alcohol Deshidrogenasa/metabolismo , Sistema Enzimático del Citocromo P-450/metabolismo , Combustibles Fósiles , Hidrocarburos/farmacocinética , Hidrocarburos/toxicidad , Microsomas Hepáticos/enzimología , Biotransformación , Cromatografía Líquida de Alta Presión , Inhibidores Enzimáticos del Citocromo P-450 , DEET/farmacocinética , Estradiol/metabolismo , Humanos , Isoenzimas/antagonistas & inhibidores , Isoenzimas/metabolismo , Microsomas Hepáticos/efectos de los fármacos , Testosterona/metabolismoRESUMEN
It has been suggested that chemicals and, more specifically, chemical interactions, are involved as causative agents in deployment-related illnesses. Unfortunately, this hypothesis has proven difficult to test, because toxicological investigations of deployment-related chemicals are usually carried out on surrogate animals and are difficult to extrapolate to humans. Other parts of the problem, such as the definition of variation within human populations and the development of methods for designating groups or individuals at significantly greater risk, cannot be carried out on surrogate animals, and the data must be derived from humans. The relatively recent availability of human cell.fractions, such as microsomes, cytosol, etc., human cells such as primary hepatocytes, recombinant human enzymes, and their isoforms and polymorphic variants has enabled a significant start to be made in developing the human data needed. These initial studies have examined the human metabolism by cytochrome P450, other phase I enzymes, and their isoforms and, in some cases, their polymorphic variants of compounds such as chlorpyrifos, carbaryl, DEET, permethrin, and pyridostigmine bromide, and, to a lesser extent, other chemicals from the same chemical and use classes, including solvents, jet fuel components, and sulfur mustard metabolites. A number of interactions at the metabolic level have been described both with respect to other xenobiotics and to endogenous metabolites. Probably the most dramatic have been seen in the ability of chlorpyrifos to inhibit not only the metabolism of other xenobiotics such as carbaryl and DEET but also to inhibit the metabolism of steroid hormones.