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BACKGROUND: Ambient air quality monitoring is a governmental duty that is widely carried out in order to detect non-biological ("chemical") components in ambient air, such as particles of < 10 µm (PM10, PM2.5), ozone, sulphur dioxide, and nitrogen oxides. These monitoring networks are publicly funded and air quality data are open to the public. The situation for biological particles that have detrimental effects on health, as is the case of pollen and fungal spores, is however very different. Most pollen and spore monitoring networks are not publicly funded and data are not freely available. The information regarding which biological particle is being monitored, where and by whom, is consequently often not known, even by aerobiologists themselves. This is a considerable problem, as local pollen data are an important tool for the prevention of allergic symptoms. OBJECTIVE: The aim of this study was to review pollen monitoring stations throughout the world and to create an interactive visualization of their distribution. METHODS: The method employed to collect information was based on: (a) a review of the recent and historical bibliography related to pollen and fungal spore monitoring, and (b) personal surveys of the managers of national and regional monitoring networks. The interactive application was developed using the R programming language. RESULTS: We have created an inventory of the active pollen and spore monitoring stations in the world. There are at least 879 active pollen monitoring stations in the world, most of which are in Europe (> 500). The prevalent monitoring method is based on the Hirst principle (> 600 stations). The inventory is visualised as an interactive and on-line map. It can be searched, its appearance can be adjusted to the users' needs and it is updated regularly, as new stations or changes to those that already exist can be submitted online. CONCLUSIONS: The map shows the current situation of pollen and spore monitoring and facilitates collaboration among those individuals who are interested in pollen and spore counts. It might also help to improve the monitoring of biological particles up to the current level employed for non-biological components.
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BACKGROUND: B cells play a central role in IgE-mediated allergies. In damaged airway epithelium, they are exposed directly to aeroallergens. We aimed to assess whether direct exposure of B cells to pollen constituents affects allergic sensitization. METHODS: B cells from murine splenocytes and from blood samples of healthy donors were incubated for 8 days under Th2-like conditions with aqueous ragweed pollen extracts (Amb-APE) or its constituents. Secreted total IgM, IgG, and IgE was quantified by ELISA. Additionally, birch, grass, or pine-pollen extracts were tested. The number of viable cells was evaluated by ATP measurements. B-cell proliferation was measured by CFSE staining. IgE class switch was analyzed by quantitation of class switch transcripts. In an OVA/Alum i.p.-sensitization mouse model, Amb-APE was intranasally instilled for 11 consecutive days. RESULTS: Upon Th2 priming of murine B cells, ragweed pollen extract caused a dose-dependent increase in IgE production, while IgG and IgM were not affected. The low-molecular-weight fraction and phytoprostane E1 (PPE1) increased IgE production, while Amb a 1 did not. PPE1 enhanced IgE also in human memory B cells. Under Th1 conditions, Amb-APE did not influence immunoglobulin secretion. The IgE elevation was not ragweed specific. It correlated with proliferation of viable B cells, but not with IgE class switch. In vivo, Amb-APE increased total IgE and showed adjuvant activity in allergic airway inflammation. CONCLUSIONS: Aqueous pollen extracts, the protein-free fraction of Amb-APE, and the pollen-contained substance PPE1 specifically enhance IgE production in Th2-primed B cells. Thus, pollen-derived nonallergenic substances might be responsible for B-cell-dependent aggravation of IgE-mediated allergies.
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Alérgenos/inmunología , Formación de Anticuerpos/inmunología , Linfocitos B/inmunología , Inmunoglobulina E/inmunología , Polen/inmunología , Células Th2/inmunología , Ambrosia/inmunología , Animales , Antígenos de Plantas/inmunología , Linfocitos B/metabolismo , Femenino , Humanos , Inmunización , Memoria Inmunológica , Activación de Linfocitos/inmunología , Ratones , Ovalbúmina/inmunología , Extractos Vegetales/inmunología , Neumonía/inmunología , Neumonía/metabolismo , Neumonía/patología , Células Th2/metabolismoRESUMEN
Pollen is routinely monitored, but it is unknown whether pollen counts represent allergen exposure. We therefore simultaneously determined olive pollen and Ole e 1 in ambient air in Córdoba, Spain, and Évora, Portugal, using Hirst-type traps for pollen and high-volume cascade impactors for allergen. Pollen from different days released 12-fold different amounts of Ole e 1 per pollen (both locations P < 0.001). Average allergen release from pollen (pollen potency) was much higher in Córdoba (3.9 pg Ole e 1/pollen) than in Évora (0.8 pg Ole e 1/pollen, P = 0.004). Indeed, yearly olive pollen counts in Córdoba were 2.4 times higher than in Évora, but Ole e 1 concentrations were 7.6 times higher. When modeling the origin of the pollen, >40% of Ole e 1 exposure in Évora was explained by high-potency pollen originating from the south of Spain. Thus, olive pollen can vary substantially in allergen release, even though they are morphologically identical.
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Alérgenos/análisis , Antígenos de Plantas/análisis , Exposición a Riesgos Ambientales/análisis , Proteínas de Plantas/análisis , Polen , Exposición a Riesgos Ambientales/estadística & datos numéricos , Monitoreo del Ambiente , Ensayo de Inmunoadsorción Enzimática , Modelos Estadísticos , Portugal , Estaciones del Año , España , Tiempo (Meteorología)RESUMEN
BACKGROUND: Polymorphous light eruption (PLE) is the most common chronic and idiopathic photodermatosis. PLE is assumed to represent an immunological hypersensitivity reaction to a radiation-induced cutaneous antigen involving reactive oxygen species (ROS) on the basis of a genetic predisposition. Among others, cellular protection against ROS is provided by glutathione S-transferases (GSTs). Different variants of the GST enzymes may influence the activity and efficiency of detoxification and biotransformation of unknown UV-induced skin-antigens and other factors that may play an important role in the pathogenesis of PLE. METHODS: In this study the relationship between isoenzymes of the GST genes GSTM1, GSTT1 and GSTP1 and possible protective or predisposing effects on PLE was examined in 29 patients and 144 controls. Diagnosis of PLE was based on the presence of characteristic clinical features. RESULTS: No association between the functional polymorphisms of the GST gene family and PLE was found. Prevalence of certain GST isoenzymes or polymorphisms in patients with PLE did not differ from healthy controls. CONCLUSION: Our data do not support prevalence of GST isoenzymes or polymorphisms as a protective effect against PLE. Especially a higher carrier frequency of GSTP1 Val(105) as a protective factor against PLE which has been published before could not be proved. The GST genotypes GSTM1, GSTT1 and GSTP1 (including SNPs) seem to have no relevant association with PLE.
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Gutatión-S-Transferasa pi/genética , Glutatión Transferasa/genética , Trastornos por Fotosensibilidad/genética , Adulto , Anciano , Anciano de 80 o más Años , Secuencia de Bases , Cartilla de ADN , Femenino , Humanos , Masculino , Persona de Mediana Edad , Trastornos por Fotosensibilidad/enzimología , Adulto JovenRESUMEN
Ketamine is an anesthetic and analgesic regularly used in veterinary patients. As ketamine is almost always administered in combination with other drugs, interactions between ketamine and other drugs bear the risk of either adverse effects or diminished efficacy. Since cytochrome P450 enzymes (CYPs) play a pivotal role in the phase I metabolism of the majority of all marketed drugs, drug-drug interactions often occur at the active site of these enzymes. CYPs have been thoroughly examined in humans and laboratory animals, but little is known about equine CYPs. The characterization of equine CYPs is essential for a better understanding of drug metabolism in horses. We report annotation, cloning and heterologous expression of the equine CYP2B6 in V79 Chinese hamster fibroblasts. After computational annotation of all CYP2B genes, the coding sequence (CDS) of equine CYP2B6 was amplified by RT-PCR from horse liver total RNA and revealed an amino acid sequence identity of 77% and a similarity of 93.7% to its human ortholog. A non-synonymous variant c.226G>A in exon 2 of the equine CYP2B6 was detected in 97 horses. The mutant A-allele showed an allele frequency of 82%. Two further variants in exon 3 were detected in one and two horses of this group, respectively. Transfected V79 cells were incubated with racemic ketamine and norketamine as probe substrates to determine metabolic activity. The recombinant equine CYP2B6 N-demethylated ketamine to norketamine and produced metabolites of norketamine, such as hydroxylated norketamines and 5,6-dehydronorketamine. V(max) for S-/and R-norketamine formation was 0.49 and 0.45nmol/h/mg cellular protein and K(m) was 3.41 and 2.66µM, respectively. The N-demethylation of S-/R-ketamine was inhibited concentration-dependently with clopidogrel showing an IC(50) of 5.63 and 6.26µM, respectively. The functional importance of the recorded genetic variants remains to be explored. Equine CYP2B6 was determined to be a CYP enzyme involved in ketamine and norketamine metabolism, thus confirming results from inhibition studies with horse liver microsomes. Clopidogrel seems to be a feasible inhibitor for equine CYP2B6. The specificity still needs to be established with other single equine CYPs. Heterologous expression of single equine CYP enzymes opens new possibilities to substantially improve the understanding of drug metabolism and drug interactions in horses.
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Hidrocarburo de Aril Hidroxilasas/biosíntesis , Hidrocarburo de Aril Hidroxilasas/genética , Fibroblastos/enzimología , Regulación Enzimológica de la Expresión Génica , Genómica , Ketamina/farmacología , Oxidorreductasas N-Desmetilantes/biosíntesis , Oxidorreductasas N-Desmetilantes/genética , Animales , Cricetinae , Cricetulus , Citocromo P-450 CYP2B6 , Femenino , Fibroblastos/efectos de los fármacos , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Variación Genética/efectos de los fármacos , Variación Genética/fisiología , Caballos , HumanosRESUMEN
UNLABELLED: Outdoor particulate matter (PM(10)) is associated with detrimental health effects. However, individual PM(10) exposure occurs mostly indoors. We therefore compared the toxic effects of classroom, outdoor, and residential PM(10). Indoor and outdoor PM(10) was collected from six schools in Munich during teaching hours and in six homes. Particles were analyzed by scanning electron microscopy and X-ray spectroscopy (EDX). Toxicity was evaluated in human primary keratinocytes, lung epithelial cells and after metabolic activation by several human cytochromes P450. We found that PM(10) concentrations during teaching hours were 5.6-times higher than outdoors (117 ± 48 µg/m(3) vs. 21 ± 15 µg/m(3), P < 0.001). Compared to outdoors, indoor PM contained more silicate (36% of particle number), organic (29%, probably originating from human skin), and Ca-carbonate particles (12%, probably originating from paper). Outdoor PM contained more Ca-sulfate particles (38%). Indoor PM at 6 µg/cm(2) (10 µg/ml) caused toxicity in keratinocytes and in cells expressing CYP2B6 and CYP3A4. Toxicity by CYP2B6 was abolished with the reactive oxygen species scavenger N-acetylcysteine. We concluded that outdoor PM(10) and indoor PM(10) from homes were devoid of toxicity. Indoor PM(10) was elevated, chemically different and toxicologically more active than outdoor PM(10). Whether the effects translate into a significant health risk needs to be determined. Until then, we suggest better ventilation as a sensible option. PRACTICAL IMPLICATIONS: Indoor air PM(10) on an equal weight base is toxicologically more active than outdoor PM(10). In addition, indoor PM(10) concentrations are about six times higher than outdoor air. Thus, ventilation of classrooms with outdoor air will improve air quality and is likely to provide a health benefit. It is also easier than cleaning PM(10) from indoor air, which has proven to be tedious.
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Contaminación del Aire Interior/efectos adversos , Contaminación del Aire Interior/análisis , Material Particulado/análisis , Material Particulado/toxicidad , Hidrocarburo de Aril Hidroxilasas/metabolismo , Biotransformación , Carbonato de Calcio/análisis , Carbonato de Calcio/toxicidad , Línea Celular , Células Cultivadas , Niño , Citocromo P-450 CYP2B6 , Citocromo P-450 CYP3A/metabolismo , Alemania , Vivienda , Humanos , Queratinocitos/efectos de los fármacos , Queratinocitos/metabolismo , Microscopía Electrónica de Rastreo , Oxidorreductasas N-Desmetilantes/metabolismo , Tamaño de la Partícula , Instituciones Académicas , Silicio/análisis , Silicio/toxicidad , Azufre/análisis , Azufre/toxicidadRESUMEN
BACKGROUND: Proof is lacking that pollen count is representative for allergen exposure, also because allergens were found in nonpollen-bearing fractions of ambient air. OBJECTIVE: We monitored simultaneously birch pollen and the major birch pollen allergen Bet v 1 in different size fractions of ambient air from 2004 till 2007 in Munich, Germany. METHODS: Air was sampled with a ChemVol high-volume cascade impactor equipped with stages for particulate matter (PM)>10 microm, 10 microm>PM>2.5 microm, and 2.5 microm>PM>0.12 microm. Allergen was determined with a Bet v 1-specific ELISA. Pollen count was assessed with a Burkard pollen trap. We also measured the development of allergen in pollen during ripening. RESULTS: About 93 +/- 3% of Bet v 1 was found in the PM > 10 microm fraction, the fraction containing birch pollen. We did not measure any Bet v 1 in 2.5 microm > PM > 0.12 microm. Either in Munich no allergen was in this fraction or the allergen was absorbed to diesel soot particles that also deposit in this fraction. Pollen released 115% more Bet v 1 in 2007 than in 2004. Also within 1 year, the release of allergen from the same amount of pollen varied more than 10-fold between different days. This difference was explained by a rapidly increasing expression of Bet v 1 in pollen in the week just before pollination. Depending on the day the pollen is released during ripening, its potency varies. CONCLUSION: In general, pollen count and allergen in ambient air follow the same temporal trends. However, because a 10-fold difference can exist in allergen potency of birch pollen, symptoms might be difficult to correlate with pollen counts, but perhaps better with allergen exposure.
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Aire/análisis , Antígenos de Plantas/análisis , Betula , Monitoreo del Ambiente/métodos , Polen , Antígenos de Plantas/inmunología , Betula/inmunología , Ensayo de Inmunoadsorción Enzimática , Alemania , Polen/inmunología , Rinitis Alérgica Estacional/inmunologíaRESUMEN
HISTORY AND ADMISSION FINDINGS: Seventeen East-European workers with a suspected lead-intoxication presented themselves to the Department of Toxicology. All of them had worked on the renovation of pylons of a high-tension line. The old paint, known to contain lead was removed with needle descalers. The patients had blood lead concentrations between 325 and 1124 microg/l, but no specific symptoms. The workers neglected the protective measures at their working-place. INVESTIGATIONS: 12 of 17 workers had lead-concentrations above 400 microg/l (Reference < 90 microg/l). 10 of 17 patients showed an increased level of free protoporphyrins and all workers showed a decreased activity of delta-aminolaevulinacid-dehydratase (ALAD). TREATMENT AND COURSE: Patients with lead-concentration above 700 microg/l were treated with the chelating agent meso-2,3-dimercaptosuccinic acid (DMSA) 3 x 200 mg/d for nine days. The patients with lead concentrations between 400 and 700 microg/l were treated which DMSA 3 x 100 mg/d. After the DMSA-treatment the lead-concentrations had dropped (p < 0.001). During the DMSA-therapy one patient had to be treated in the hospital because of a generalised allergic exanthema. CONCLUSION: We report seventeen patients with high lead concentration in their blood due to occupational exposure. The high blood lead levels showed that the workers had not been protected adequately. This examplifies that occupational lead exposure still occurs, also in Germany. By patients with unspecific symptoms connected with lead exposure a biomonitoring for lead is necessary.
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Quelantes/uso terapéutico , Intoxicación por Plomo/epidemiología , Enfermedades Profesionales/epidemiología , Exposición Profesional , Succímero/uso terapéutico , Antídotos/uso terapéutico , Alemania/epidemiología , Humanos , Cinética , Plomo/sangre , Dolor/inducido químicamente , Dolor/etiología , Porfobilinógeno Sintasa/sangreAsunto(s)
Cichorium intybus/efectos adversos , Dermatitis Alérgica por Contacto/etiología , Dermatitis Profesional/etiología , Manipulación de Alimentos , Hipersensibilidad a los Alimentos/etiología , Dermatosis de la Mano/inducido químicamente , Adulto , Dermatitis Alérgica por Contacto/diagnóstico , Dermatitis Profesional/diagnóstico , Hipersensibilidad a los Alimentos/diagnóstico , Dermatosis de la Mano/diagnóstico , Humanos , Masculino , Pruebas del Parche/métodosRESUMEN
Hybridomas were isolated that produce 13 monoclonal antibodies (mAbs) that are specific and highly inhibitory to members of the human P450 2C subfamily, 2C8, 2C9, 2C9*2, and 2C19. Many of the mAbs to P450 2C8, 2C9, and 2C19 are specific and exhibit potent inhibitory activity (85-95%). mAb 281-1-1 specifically binds, immunoblots, and strongly inhibits the activity of P450 2C8. mAb 763-15-5 specifically binds and strongly inhibits the activity of P450 2C9. mAb 1-7-4-8 specifically binds and strongly inhibits the activity of P450 2C19. The other mAbs bind and inhibit sets and subsets of the P450 2C family. The single and the combinatorial use of the mAbs can "reaction phenotype", i.e., determine the metabolic contribution and interindividual variation of a P450 isoform for the metabolism of a drug or nondrug xenobiotic in human liver microsomes. The utility of the mAb-based analytic system was examined with the model substrates Taxol (paclitaxel), diazepam, tolbutamide, diclofenac, mephenytoin, and imipramine. The mAb system can identify drugs metabolized by a common P450 or several P450s and polymorphic P450s. The mAb system identifies drugs or drug metabolic pathways that are catalyzed by a single P450 and thus may be used for in vivo phenotyping. The mAb system can identify whether a particular drug is metabolized by a single P450 that may exhibit polymorphic expression in humans. The mAb system offers large potential for studies of cytochrome P450 function useful in drug discovery and reduces the possibility of adverse drug reactions due to polymorphisms and drug interactions.
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Anticuerpos Monoclonales/farmacología , Especificidad de Anticuerpos , Hidrocarburo de Aril Hidroxilasas , Inhibidores Enzimáticos del Citocromo P-450 , Sistema Enzimático del Citocromo P-450/inmunología , Inhibidores Enzimáticos/farmacología , Oxigenasas de Función Mixta/antagonistas & inhibidores , Oxigenasas de Función Mixta/inmunología , Esteroide 16-alfa-Hidroxilasa , Esteroide Hidroxilasas/antagonistas & inhibidores , Esteroide Hidroxilasas/inmunología , Animales , Anticuerpos Monoclonales/aislamiento & purificación , Anticuerpos Monoclonales/metabolismo , Sitios de Unión de Anticuerpos , Citocromo P-450 CYP2C19 , Sistema Enzimático del Citocromo P-450/metabolismo , Inhibidores Enzimáticos/metabolismo , Humanos , Hibridomas , Ratones , Ratones Endogámicos BALB C , Microsomas Hepáticos/enzimología , Microsomas Hepáticos/inmunología , Microsomas Hepáticos/metabolismo , Oxigenasas de Función Mixta/metabolismo , Esteroide Hidroxilasas/metabolismoRESUMEN
Humans are exposed to polycyclic aromatic hydrocarbons (PAHs) through many environmental pollutants, especially cigarette smoke. These chemicals cause a variety of tumors and immunotoxic effects, as a consequence of bioactivation by P-450 cytochromes to dihydrodiol epoxides. The recently identified cytochrome P4501B1 (CYP1B1) bioactivates PAHs but is also a physiological regulator, as evidenced by linkage of CYP1B1 deficiency to congenital human glaucoma. This investigation demonstrates that CYP1B1 null mice are almost completely protected from the acute bone marrow cytotoxic and preleukemic effects of the prototypic PAH 7,12-dimethylbenz[a]anthracene (DMBA). CYP1B1 null mice did not produce the appreciable amounts of bone marrow DMBA dihydrodiol epoxide DNA adducts present in wild-type mice, despite comparable hepatic inductions of the prominent PAH-metabolizing P-450 cytochrome, CYP1A1. Wild-type mice constitutively expressed low levels of bone marrow CYP1B1. These findings suggest that CYP1B1 is responsible for the formation of DMBA dihydrodiol epoxides in the bone marrow. Furthermore, this study substantiates the importance of DMBA dihydrodiol epoxide generation at the site of cancer initiation and suggests that tissue-specific constitutive CYP1B1 expression may contribute to cancer susceptibility in the human population.
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9,10-Dimetil-1,2-benzantraceno/farmacocinética , 9,10-Dimetil-1,2-benzantraceno/toxicidad , Hidrocarburo de Aril Hidroxilasas , Células de la Médula Ósea/patología , Sistema Enzimático del Citocromo P-450/metabolismo , Leucemia Experimental/patología , Preleucemia/patología , Animales , Apoptosis/efectos de los fármacos , Células de la Médula Ósea/efectos de los fármacos , Carcinógenos/farmacocinética , Carcinógenos/toxicidad , Cruzamientos Genéticos , Citocromo P-450 CYP1A1/biosíntesis , Citocromo P-450 CYP1B1 , Sistema Enzimático del Citocromo P-450/deficiencia , Sistema Enzimático del Citocromo P-450/genética , Inducción Enzimática/efectos de los fármacos , Humanos , Leucemia Experimental/inducido químicamente , Leucemia Experimental/enzimología , Hígado/efectos de los fármacos , Hígado/enzimología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Preleucemia/inducido químicamente , Preleucemia/enzimologíaRESUMEN
We previously demonstrated that murine bone marrow stromal cells express high levels of cytochrome P4501B1 (CYP1B1) that metabolizes 7,12-dimethylbenza[a]anthracene (DMBA), and that DMBA activates the Ah receptor (AhR) in these cells in vitro. More recently, we reported that CYP1B1 is required for DMBA-induced lymphoblastoma formation in vivo. In this study, we addressed the hypothesis that bone marrow stromal cell CYP1B1, and not AhR activation, is required for DMBA-induced pre-B-cell apoptosis. Although DMBA did not directly cause apoptosis in pre-B cells, dose-dependent apoptosis of pre-B cells was observed when they were cocultured with a bone marrow stromal cell line. The DMBA 3,4-dihydrodiol metabolite was more potent in effecting pre-B-cell apoptosis than DMBA, whereas the potent AhR agonist 2,3,7,8-tetrachlorodibenzo-p-dioxin was inactive. Both pre-B cells and bone marrow stromal cells contained DMBA-diol-epoxide DNA adducts, indicating that reactive metabolites were transferred from stromal cells to pre-B cells. DMBA caused apoptosis when cocultured with primary bone marrow stromal cells isolated from AhR-null mice but not CYP1B1-null mice. When cocultured with AhR-null primary bone marrow stromal cells, DMBA induced approximately 50% of the pre-B-cell apoptosis seen with stromal cells from AhR-heterozygous mice. This reduced level of apoptosis parallels the decreased CYP1B1 expression in AhR-null mouse bone marrow stromal cells. These findings provide convincing evidence that bone marrow stromal cell CYP1B1 metabolism of DMBA, but not AhR activation, is required for DMBA-induced pre-B-cell apoptosis.
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9,10-Dimetil-1,2-benzantraceno/farmacología , Apoptosis , Hidrocarburo de Aril Hidroxilasas , Linfocitos B/efectos de los fármacos , Células de la Médula Ósea/enzimología , Carcinógenos/farmacología , Sistema Enzimático del Citocromo P-450/fisiología , 9,10-Dimetil-1,2-benzantraceno/metabolismo , Animales , Linfocitos B/patología , Células de la Médula Ósea/citología , Carcinógenos/metabolismo , Citocromo P-450 CYP1B1 , Aductos de ADN/metabolismo , Ratones , Receptores Colinérgicos/metabolismo , Células del Estroma/enzimología , Células Tumorales CultivadasAsunto(s)
Sistema Enzimático del Citocromo P-450/genética , Hidrocarburos Policíclicos Aromáticos/farmacología , Animales , Cricetinae , Sistema Enzimático del Citocromo P-450/biosíntesis , Sistema Enzimático del Citocromo P-450/metabolismo , Inducción Enzimática , Ingeniería Genética , Hidrocarburos Policíclicos Aromáticos/química , Hidrocarburos Policíclicos Aromáticos/metabolismoRESUMEN
CYP1B1-null mice, created by targeted gene disruption in embryonic stem cells, were born at the expected frequency from heterozygous matings with no observable phenotype, thus establishing that CYP1B1 is not required for mouse development. CYP1B1 was not detectable in cultured embryonic fibroblast (EF) or in different tissues, such as lung, of the CYP1B1-null mouse treated with the aryl hydrocarbon receptor agonist 2,3,7,8-tetrachlorodibenzo-p-dioxin whereas the equivalent wild-type EF cells express basal and substantial inducible CYP1B1 and lung expresses inducible CYP1B1. CYP1A1 is induced to far higher levels than CYP1B1 in liver, kidney, and lung in wild-type mice and is induced to a similar extent in CYP1B1-null mice. 7,12-dimethylbenz[a]anthracene (DMBA) was toxic in wild-type EFs that express CYP1B1 but not CYP1A1. These cells effectively metabolized DMBA, consistent with CYP1B1 involvement in producing the procarcinogenic 3,4-dihydrodiol as a major metabolite, whereas CYP1B1-null EF showed no significant metabolism and were resistant to DMBA-mediated toxicity. When wild-type mice were administered high levels of DMBA intragastrically, 70% developed highly malignant lymphomas whereas only 7.5% of CYP1B1-null mice had lymphomas. Skin hyperplasia and tumors were also more frequent in wild-type mice. These results establish that CYP1B1, located exclusively at extrahepatic sites, mediates the carcinogenicity of DMBA. Surprisingly, CYP1A1, which has a high rate of DMBA metabolism in vitro, is not sufficient for this carcinogenesis, which demonstrates the importance of extrahepatic P450s in determining susceptibility to chemical carcinogens and validates the search for associations between P450 expression and cancer risk in humans.
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Hidrocarburo de Aril Hidroxilasas , Sistema Enzimático del Citocromo P-450/genética , Regulación Enzimológica de la Expresión Génica , Predisposición Genética a la Enfermedad , Linfoma/genética , Neoplasias Experimentales/genética , 9,10-Dimetil-1,2-benzantraceno/farmacocinética , Animales , Biotransformación , Carcinógenos , Células Cultivadas , Quimera , Citocromo P-450 CYP1B1 , Embrión de Mamíferos , Femenino , Fibroblastos/enzimología , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Biblioteca Genómica , Pulmón/enzimología , Linfoma/inducido químicamente , Ratones , Ratones Noqueados , Neoplasias Experimentales/inducido químicamente , Especificidad de Órganos , Dibenzodioxinas Policloradas/farmacología , Mapeo Restrictivo , Células MadreRESUMEN
Acetaminophen (APAP) hepatotoxicity is due to its biotransformation to a reactive metabolite, N-acetyl-p-benzoquinone imine (NAPQI), that is capable of binding to cellular macromolecules. At least two forms of cytochrome P450, CYP2E1 and CYP1A2, have been implicated in this reaction in mice. To test the combined roles of CYP1A2 and CYP2E1 in an intact animal model, a double-null mouse line lacking functional expression of CYP1A2 and CYP2E1 was produced by cross-breeding Cyp1a2-/- mice with Cyp2e1-/- mice. Animals deficient in the expression of both P450s developed normally and exhibited no obvious phenotypic abnormalities. Comparison of the dose-response to APAP (200-1200 mg/kg) indicated that double-null animals were highly resistant to APAP-induced toxicity whereas the wild-type animals were sensitive. Administration of 600 to 800 mg/kg of this drug to male wild-type animals resulted in increased plasma concentrations of liver enzymes (alanine aminotransferase, sorbitol dehydrogenase), lipidosis, hepatic necrosis, and renal tubular necrosis. In contrast, when APAP of equivalent or higher dose was administered to the double-null mice, plasma levels of liver enzymes and liver histopathology were normal. However, administration of 1200 mg of APAP/kg to the double-null mice resulted in infrequent liver lipidosis and mild kidney lesions. Consistent with the protection from hepatotoxicity, the expected depletion of hepatic glutathione (GSH) content was significantly retarded and APAP covalent binding to hepatic cytosolic proteins was not detectable in the double-null mice. Likewise, in vitro activation of APAP by liver microsomes from the double-null mice was approximately one tenth of that in microsomes from wild-type mice. Thus, the protection against APAP toxicity afforded by deletion of both CYP2E1 and CYP1A2 likely reflects greatly diminished production of the toxic electrophile, NAPQI.
Asunto(s)
Acetaminofén/toxicidad , Analgésicos no Narcóticos/toxicidad , Citocromo P-450 CYP1A2/fisiología , Citocromo P-450 CYP2E1/fisiología , Túbulos Renales/efectos de los fármacos , Hígado/efectos de los fármacos , Microsomas Hepáticos/efectos de los fármacos , Alanina Transaminasa/metabolismo , Animales , Citocromo P-450 CYP1A2/genética , Citocromo P-450 CYP2E1/genética , ADN/análisis , Relación Dosis-Respuesta a Droga , Eliminación de Gen , Genotipo , Glutatión/metabolismo , Túbulos Renales/patología , L-Iditol 2-Deshidrogenasa/metabolismo , Lipidosis/metabolismo , Hígado/enzimología , Hígado/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Microsomas Hepáticos/enzimología , Necrosis , Compuestos de Sulfhidrilo/metabolismo , Tasa de SupervivenciaRESUMEN
The human cytochrome P450 2B6 metabolizes, among numerous other substrates, diazepam, 7-ethoxycoumarin, testosterone, and phenanthrene. A recombinant baculovirus containing the human 2B6 cDNA was constructed and used to express 2B6 in Sf9 insect cells. The 2B6 was present at 1.8 +/- 0.4% of the total cellular protein and was purified to a specific content of 13.3 nmol/mg protein. Mice were immunized with the purified 2B6, and a total of 811 hybridomas were obtained from the fusion of NS-1 myeloma cells and spleen cells of the immunized mice. Monoclonal antibodies (MAbs) from 24 of the hybrids exhibited immunobinding to 2B6 as determined by ELISA. One of the MAbs, 49-10-20, showed a strong immunoblotting activity and was highly inhibitory to 2B6 enzyme activity. MAb 49-10-20 inhibited cDNA-expressed 2B6-catalyzed metabolism of diazepam, phenanthrene, 7-ethoxycoumarin, and testosterone by 90-91%. MAb 49-10-20 showed extremely high specificity for 2B6 and did not bind to 17 other human and rodent P450s or inhibit the metabolism of phenanthrene catalyzed by human 1A2, 2A6, 2C8, 2C9, 2D6, 2E1, 3A4, and 3A5. MAb 49-10-20 was used to determine the contribution of 2B6 to the metabolism of phenanthrene and diazepam in human liver. In ten liver samples, MAb 49-10-20 inhibited phenanthrene metabolism variably by a wide range of 8-42% and diazepam demethylation by 1-23%. The degree of inhibition by the 2B6 specific MAb 49-10-20 defines the contribution of 2B6 to phenanthrene and diazepam metabolism in each human liver. This technique using inhibitory MAb 49-10-20 determines the contribution of 2B6 to the metabolism of its substrates in a human tissue containing multiple P450s. This study is a prototype for the use of specific and highly inhibitory MAbs to determine individual P450 function.
Asunto(s)
Anticuerpos Monoclonales/farmacología , Hidrocarburo de Aril Hidroxilasas , Inhibidores Enzimáticos del Citocromo P-450 , Oxidorreductasas N-Desmetilantes/antagonistas & inhibidores , Animales , Línea Celular , Citocromo P-450 CYP2B6 , Sistema Enzimático del Citocromo P-450/metabolismo , Diazepam/metabolismo , Humanos , Hígado/enzimología , Ratones , Ratones Endogámicos BALB C , Oxidorreductasas N-Desmetilantes/metabolismo , Fenantrenos/metabolismo , Proteínas Recombinantes/antagonistas & inhibidores , Spodoptera , Especificidad por SustratoRESUMEN
Acquired resistance to paclitaxel can be mediated by P-glycoprotein or by alterations involving tubulin. We report two paclitaxel-resistant sublines derived from 1A9 human ovarian carcinoma cells. Single-step paclitaxel selection with verapamil yielded two clones that are resistant to paclitaxel and collaterally sensitive to vinblastine. The resistant sublines are not paclitaxel-dependent, and resistance remained stable after 3 years of drug-free culture. All cell lines accumulate [3H]paclitaxel equally, and no MDR-1 mRNA was detected by polymerase chain reaction following reverse transcription. Total tubulin content is similar, but the polymerized fraction increased in parental but not in resistant cells following the paclitaxel addition. Purified tubulin from parental cells demonstrated paclitaxel-driven increased polymerization, in contrast to resistant cell tubulin, which did not polymerize under identical conditions. In contrast, epothilone B, an agent to which the resistant cells retained sensitivity, increased assembly. Comparable expression of beta-tubulin isotypes was found in parental and resistant cells, with predominant expression of the M40 and beta2 isotypes. Sequence analysis demonstrated acquired mutations in the M40 isotype at nucleotide 810 (T --> G; Phe270 --> Val) in 1A9PTX10 cells and nucleotide 1092 (G --> A; Ala364 --> Thr) in 1A9PTX22 cells. These results identify residues beta270 and beta364 as important modulators of paclitaxel's interaction with tubulin.
Asunto(s)
Antineoplásicos Fitogénicos/uso terapéutico , Epotilonas , Neoplasias Ováricas/tratamiento farmacológico , Paclitaxel/uso terapéutico , Polímeros/metabolismo , Tubulina (Proteína)/genética , Sitios de Unión , Resistencia a Antineoplásicos/genética , Compuestos Epoxi/metabolismo , Femenino , Humanos , Modelos Moleculares , Hibridación de Ácido Nucleico , Neoplasias Ováricas/genética , Fenotipo , Reacción en Cadena de la Polimerasa , Tiazoles/metabolismo , Tubulina (Proteína)/metabolismo , Células Tumorales CultivadasRESUMEN
Heterologous expression using baculovirus vectors has become a popular method for the production of catalytically active cytochrome P450s (CYPs). We have systematically optimized the multiplicity of infection (MOI) for a coinfection approach for the coexpression of CYP2A6 (viral vector designated v2A6) and NADPH-P450 oxidoreductase (OR; viral vector designated vOR) using Sf9 insect cells. A 3000-fold range of MOI was examined in stationary culture and stirred suspension culture. Surprisingly, our results indicate that the best CYP2A6 catalytic activity (850-1300 pmol/ min/mg total lysate protein as measured by coumarin 7-hydroxylase activity) was obtained only when using a low MOI of v2A6 (1.5-3 x 10(-2)) and a vOR of 10- to 20-fold less. This activity was approximately 7- to 11-fold higher than the best activity obtained when infecting cells with v2A6 alone. At this level of coinfection, the P450 content ranged from 180 to 250 pmol/mg total lysate protein, and the NADPH cytochrome c reductase activity ranged from 350 to 520 nmol/min/mg total lysate protein. Increasing the MOI of both viruses to 50-fold higher resulted in lower overall activity with the optimum (250 pmol/min/mg total lysate protein) being seen earlier postinfection (60 vs. 72 hr). Increasing the MOI of vOR to levels comparable with those of v2A6, decreased coumarin 7-hydroxylase activity 14-fold. These results suggest that the best CYP2A6 catalytic activity depends on properly posttranslationally modified proteins accumulating in a right ratio as a result of primary, secondary, and possibly tertiary infection of both viruses. These results also suggest that high OR expression results in degradation of P450.
Asunto(s)
Hidrocarburo de Aril Hidroxilasas , Baculoviridae/genética , Sistema Enzimático del Citocromo P-450/genética , Oxigenasas de Función Mixta/genética , NADPH-Ferrihemoproteína Reductasa/genética , Animales , Catálisis , Línea Celular , Clonación Molecular , Citocromo P-450 CYP2A6 , Sistema Enzimático del Citocromo P-450/metabolismo , Humanos , Oxigenasas de Función Mixta/metabolismo , SpodopteraRESUMEN
The anticarcinogenicity of some flavonoids has been attributed to modulation of the cytochrome P450 enzymes, which metabolize procarcinogens to their activated forms. However, the mechanism by which flavonoids inhibit some P450-mediated activities while activating others is a longstanding, intriguing question. We employed flash photolysis to measure carbon monoxide binding to P450 as a rapid kinetic technique to probe the interaction of the prototype flavonoid alpha-naphthoflavone with human cytochrome P450s 1A1 and 3A4, whose benzo[a]pyrene hydroxylation activities are respectively inhibited and stimulated by this compound. This flavonoid inhibited P450 1A1 binding to benzo[a]pyrene via a classical competitive mechanism. In contrast, alpha-naphthoflavone stimulated P450 3A4 by selectively binding and activating an otherwise inactive subpopulation of this P450 and promoting benzo[a]pyrene binding to the latter. These data indicate that flavonoids enhance activity by increasing the pool of active P450 molecules within this P450 macrosystem. Activators in other biological systems may similarly exert their effect by expanding the population of active receptor molecules.