RESUMEN
OBJECTIVES: The aim of this study was to investigate a role of cytochrome P450 (CYP) and peroxidase in ellipticine oxidative activation in two mouse strains differing in expression of NADPH:CYP reductase (POR) [the HRN (Hepatic Cytochrome P450 Reductase Null) mice, in which POR is deleted in hepatocytes and its wild-type (WT) counterpart], and in levels of CYP1A1/2 and cytochrome b5 that were modulated by treatment of these mouse models with a CYP1A inducer, benzo[a]pyrene (BaP). METHODS: Ellipticine-DNA adducts were detected by 32P-postlabeling. HPLC was employed for the separation and characterization of ellipticine metabolites. RESULTS: Hepatic microsomes of HRN and WT mice activate ellipticine to form ellipticine-derived DNA adducts. A 2.2- and 10.4-fold increase in amounts of ellipticine-derived DNA adducts formed by liver microsomes was caused by exposure of HRN and WT mice to BaP, respectively. The results found and utilization of NADPH and arachidonic acid, cofactors of CYP- and cyclooxygenase (COX)-dependent enzyme systems, respectively, as well as inhibitors of CYP1A1/2 and 3A, demonstrate that the CYP1A and 3A enzymes play a major role in ellipticine activation in liver microsomes. In addition, the COX enzyme is important in ellipticine activation in liver of HRN mice. CONCLUSION: The CYP1A and 3A enzymes activate ellipticine mainly in liver of WT mice, whereas peroxidase COX plays this role in liver of HRN mice. Treatment of mice with BaP increases an impact of CYP1A on ellipticine activation. A pattern of expression levels of these enzymes plays a crucial role in their impact on this process.
Asunto(s)
Antineoplásicos/farmacocinética , Benzo(a)pireno/farmacología , Elipticinas/farmacocinética , Animales , Biotransformación/efectos de los fármacos , Citocromo P-450 CYP1A1/metabolismo , Citocromo P-450 CYP1A2/metabolismo , Sistema Enzimático del Citocromo P-450/metabolismo , Evaluación Preclínica de Medicamentos , Interacciones Farmacológicas , Femenino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Microsomas Hepáticos/efectos de los fármacos , Microsomas Hepáticos/metabolismoRESUMEN
BACKGROUND AND AIMS: Gene flow by seed and pollen largely shapes the genetic structure within and among plant populations. Seed dispersal is often strongly spatially restricted, making gene flow primarily dependent on pollen dispersal within and into populations. To understand distance-dependent pollination success, pollen dispersal and gene flow were studied within and into a population of the alpine monocarpic perennial Campanula thyrsoides. METHODS: A paternity analysis was performed on sampled seed families using microsatellites, genotyping 22 flowering adults and 331 germinated offspring to estimate gene flow, and pollen analogues were used to estimate pollen dispersal. The focal population was situated among 23 genetically differentiated populations on a subalpine mountain plateau (<10 km(2)) in central Switzerland. KEY RESULTS: Paternity analysis assigned 110 offspring (33·2 %) to a specific pollen donor (i.e. 'father') in the focal population. Mean pollination distance was 17·4 m for these offspring, and the pollen dispersal curve based on positive LOD scores of all 331 offspring was strongly decreasing with distance. The paternal contribution from 20-35 offspring (6·0-10·5 %) originated outside the population, probably from nearby populations on the plateau. Multiple potential fathers were assigned to each of 186 offspring (56·2 %). The pollination distance to 'mother' plants was negatively affected by the mothers' degree of spatial isolation in the population. Variability in male mating success was not related to the degree of isolation of father plants. CONCLUSIONS: Pollen dispersal patterns within the C. thyrsoides population are affected by spatial positioning of flowering individuals and pollen dispersal may therefore contribute to the course of evolution of populations of this species. Pollen dispersal into the population was high but apparently not strong enough to prevent the previously described substantial among-population differentiation on the plateau, which may be due to the monocarpic perenniality of this species.
Asunto(s)
Campanulaceae/fisiología , Flujo Génico , Genética de Población , Polen/fisiología , Campanulaceae/genética , ADN de Plantas/genética , Demografía , Flores/genética , Flores/fisiología , Estructuras Genéticas , Variación Genética , Genotipo , Repeticiones de Microsatélite/genética , Polen/genética , Polinización , Reproducción , Semillas/genética , Semillas/fisiología , SuizaRESUMEN
OBJECTIVES: Valproic acid (VPA) and trichostatin A (TSA) exert antitumor activity as histone deacetylase inhibitors, whereas ellipticine action is based mainly on DNA intercalation, inhibition of topoisomerase II and formation of cytochrome P450 (CYP)- and peroxidase-mediated covalent DNA adducts. This is the first report on the molecular mechanism of combined treatment of human neuroblastoma UKF-NB-3 and UKF-NB-4 cells with these compounds. METHODS: HPLC with UV detection was employed for the separation and characterization of ellipticine metabolites formed by microsomes and peroxidases. Covalent DNA modifications by ellipticine in neuroblastoma cells and in incubations with microsomes and peroxidases were detected by 32P-postlabeling. Expression of CYP enzymes, peroxidases and cytochrome b5 was examined by Western blot. RESULTS: The cytotoxicity of ellipticine to neuroblastomas was increased by pre-treating these cells with VPA or TSA. A higher sensitivity of cells to ellipticine correlated with an increase in formation of covalent ellipticine-derived DNA adducts in these cells. To evaluate the mechanisms of this finding, we investigated the modulation by VPA and TSA of CYP- and peroxidase-mediated ellipticine-derived DNA adduct formation in vitro. The effects of ellipticine in the presence of VPA and TSA on expression of CYPs and peroxidases relevant for ellipticine activation and levels of cytochrome b5 and P-glycoprotein in neuroblastoma cells were also investigated. Based on these studies, we attribute most of the enhancing effects of VPA and TSA on ellipticine cytotoxicity to enhanced ellipticine-DNA adduct formation caused by an increase in levels of cytochrome b5, CYP3A4 and CYP1A1 in neuroblastoma cells. A lower sensitivity of UKF-NB-4 cells to combined effects of ellipticine with VPA and TSA than of UKF-NB-3 cells is also attributable to high levels of P-glycoprotein expressed in this cell line. CONCLUSION: The results found here warrant further studies and may help in the design of new protocols geared to the treatment of high risk neuroblastomas.
Asunto(s)
Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Neoplasias Encefálicas/tratamiento farmacológico , Daño del ADN , Elipticinas/administración & dosificación , Ácidos Hidroxámicos/administración & dosificación , Neuroblastoma/tratamiento farmacológico , Ácido Valproico/administración & dosificación , Animales , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/patología , Relación Dosis-Respuesta a Droga , Evaluación Preclínica de Medicamentos , Elipticinas/farmacología , Inhibidores de Histona Desacetilasas/administración & dosificación , Inhibidores de Histona Desacetilasas/farmacología , Humanos , Ácidos Hidroxámicos/farmacología , Microsomas Hepáticos/efectos de los fármacos , Microsomas Hepáticos/metabolismo , Modelos Biológicos , Neuroblastoma/genética , Neuroblastoma/patología , Ratas , Resultado del Tratamiento , Células Tumorales Cultivadas , Ácido Valproico/farmacologíaRESUMEN
OBJECTIVES: Benzo[a]pyrene (BaP) and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) are carcinogens, which frequently occur in the human diet. Their metabolic activation to reactive species binding to DNA is mediated by cytochromes P450 (CYPs) 1A1 and 1A2. Thus, levels and activities of these CYPs are crucial for initiation of BaP- and PhPI-mediated carcinogenesis. Here, the effect of CYP1A1/2 induction due to their prototype flavonoid inducer, ß-naphthoï¬avone (BNF), on BaP- and PhPI-derived DNA adduct formation in rats was examined. METHODS: Male rats pretreated with BNF were treated with a single dose of either carcinogen by oral gavage. Nuclease P1 version of 32P-postlabeling assay and online column-switching liquid chromatography-electrospray ionization-tandem mass spectrometry were used to detect and quantify covalent DNA adducts formed by BaP and PhIP in-vivo, respectively. Expression of CYP1A1/2 enzymes was examined by Western blot. Enzymatic activities of CYP1A1/2 were assessed using their marker substrates (ethoxyresorufin and methoxyresorufin). RESULTS: Treatment of rats with a single dose of BNF produced an increase in levels CYP1A1/2 and CYP1A1 proteins in liver and small intestine, respectively. An increase in CYP1A1 protein expression found in both organs correlated well with specific activities of these CYPs. The CYP1A1 expression levels and its specific activity in small intestine decreased along the length of the organ, being highest in its proximal part and lowest in its distal part. The BNF induction of CYP1A1/2 resulted in a significant increase in the formation of BaP- and PhIP-DNA adducts in liver and in the distal part of the small intestine, respectively. Thus, pretreatment of rats with BNF did not prevent the PhIP and BaP activation, but vice versa, enhanced their genotoxicity. CONCLUSIONS: The results of this study demonstrate that the administration of only a single dose of CYP-inducing flavonoid prior to the intake of food carcinogens may increase the risk of a tumor formation.
Asunto(s)
Carcinógenos Ambientales/toxicidad , Daño del ADN/efectos de los fármacos , Contaminación de Alimentos , Alimentos/toxicidad , beta-naftoflavona/farmacología , Animales , Carcinógenos Ambientales/aislamiento & purificación , Citocromo P-450 CYP1A1/antagonistas & inhibidores , Citocromo P-450 CYP1A1/genética , Citocromo P-450 CYP1A1/metabolismo , Evaluación Preclínica de Medicamentos , Inducción Enzimática/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Contaminación de Alimentos/análisis , Intestino Delgado/efectos de los fármacos , Intestino Delgado/enzimología , Intestino Delgado/metabolismo , Hígado/efectos de los fármacos , Hígado/enzimología , Hígado/metabolismo , Masculino , Pruebas de Mutagenicidad , Ratas , Ratas WistarRESUMEN
OBJECTIVE: The herbal drug aristolochic acid (AA) derived from Aristolochia species has been shown to be the cause of aristolochic acid nephropathy (AAN), Balkan endemic nephropathy (BEN) and their urothelial malignancies. One of the common features of AAN and BEN is that not all individuals exposed to AA suffer from nephropathy and tumor development. One cause for these different responses may be individual differences in the activities of the enzymes catalyzing the biotransformation of AA. Thus, the identification of enzymes principally involved in the metabolism of AAI, the major toxic component of AA, and detailed knowledge of their catalytic specificities is of major importance. Therefore, the present study has been designed to evaluate the cytochrome P450 (CYP)-mediated oxidative detoxification and reductive activation of AAI in a rat model. METHODS: DNA adduct formation was investigated by the nuclease P1 version of the 32P-postlabeling method. The CYP-mediated formation of a detoxication metabolite of AAI, 8-hydroxyaristolochic acid I (AAIa), in vitro in rat hepatic microsomes was determined by HPLC. RESULTS: Rat hepatic CYPs both detoxicate AAI by its oxidation to AAIa and reductively activate this carcinogen to a cyclic N-acylnitrenium ion forming AAI-DNA adducts in vitro. To define the role of hepatic CYPs in AAI demethylation and activation, the modulation of AAIa and AAI-DNA adduct formation by CYP inducers and selective CYP inhibitors was investigated. Based on these studies, we attribute the major role of CYP1A1 and 1A2 in AAI detoxication by its demethylation to AAIa, and, under hypoxic conditions also to AAI activation to species forming DNA adducts. Using microsomes of Baculovirus transfected insect cells (Supersomes™) containing recombinantly expressed rat CYPs, NADPH:CYP reductase and/or cytochrome b5, a major role of CYP1A1 and 1A2 in both reactions in vitro was confirmed. CONCLUSION: Based on the results found in this and former studies we propose that AAI activation and detoxication in rats are dictated mainly by AAI binding affinity to CYP1A1/2 or NADPH(P)H:quinone oxidoreductase, by their turnover and by the balance between oxidation and reduction of AAI by CYP1A.
Asunto(s)
Ácidos Aristolóquicos/farmacocinética , Sistema Enzimático del Citocromo P-450/fisiología , Hígado/efectos de los fármacos , Animales , Biotransformación , Carcinógenos/farmacocinética , Citocromo P-450 CYP1A1/genética , Citocromo P-450 CYP1A1/metabolismo , Citocromo P-450 CYP1A2 , Sistema Enzimático del Citocromo P-450/genética , Sistema Enzimático del Citocromo P-450/metabolismo , Citocromos/genética , Citocromos/metabolismo , Aductos de ADN/metabolismo , Evaluación Preclínica de Medicamentos , Inactivación Metabólica , Hígado/enzimología , Hígado/metabolismo , Masculino , Microsomas Hepáticos/efectos de los fármacos , Microsomas Hepáticos/metabolismo , Modelos Biológicos , Ratas , Ratas WistarRESUMEN
Exposure to aristolochic acid I (AAI) is associated with aristolochic acid nephropathy, Balkan endemic nephropathy, and urothelial cancer. Individual differences in xenobiotic-metabolizing enzyme activities are likely to be a reason for interindividual susceptibility to AA-induced disease. We evaluated the reductive activation and oxidative detoxication of AAI by cytochrome P450 (P450) 1A1 and 1A2 using the Cyp1a1(-/-) and Cyp1a2(-/-) single-knockout and Cyp1a1/1a2(-/-) double-knockout mouse lines. Incubations with hepatic microsomes were also carried out in vitro. P450 1A1 and 1A2 were found to (i) activate AAI to form DNA adducts and (ii) detoxicate it to 8-hydroxyaristolochic acid I (AAIa). AAI-DNA adduct formation was significantly higher in all tissues of Cyp1a1/1a2(-/-) than Cyp1a(+/+) wild-type (WT) mice. AAI-DNA adduct levels were elevated only in selected tissues from Cyp1a1(-/-) versus Cyp1a2(-/-) mice, compared with those in WT mice. In hepatic microsomes, those from WT as well as Cyp1a1(-/-) and Cyp1a2(-/-) mice were able to detoxicate AAI to AAIa, whereas Cyp1a1/1a2(-/-) microsomes were less effective in catalyzing this reaction, confirming that both mouse P450 1A1 and 1A2 are both involved in AAI detoxication. Under hypoxic conditions, mouse P450 1A1 and 1A2 were capable of reducing AAI to form DNA adducts in hepatic microsomes; the major roles of P450 1A1 and 1A2 in AAI-DNA adduct formation were further confirmed using selective inhibitors. Our results suggest that, in addition to P450 1A1 and 1A2 expression levels in liver, in vivo oxygen concentration in specific tissues might affect the balance between AAI nitroreduction and demethylation, which in turn would influence tissue-specific toxicity or carcinogenicity.
Asunto(s)
Ácidos Aristolóquicos/farmacocinética , Carcinógenos/farmacocinética , Citocromo P-450 CYP1A1/metabolismo , Citocromo P-450 CYP1A2/metabolismo , Medicamentos Herbarios Chinos/farmacocinética , Animales , Ácidos Aristolóquicos/orina , Nefropatía de los Balcanes/enzimología , Biotransformación , Citocromo P-450 CYP1A1/deficiencia , Citocromo P-450 CYP1A1/genética , Citocromo P-450 CYP1A2/deficiencia , Citocromo P-450 CYP1A2/genética , Aductos de ADN , Susceptibilidad a Enfermedades , Femenino , Riñón/enzimología , Hígado/enzimología , Pulmón/enzimología , Ratones , Ratones Noqueados , Microsomas/enzimología , Neoplasias Urológicas/enzimologíaRESUMEN
Aristolochic acid (AA), a naturally occurring nephrotoxin and rodent carcinogen, has recently been associated with the development of urothelial cancer in humans. Understanding which enzymes are involved in AA activation and/or detoxication is important in the assessment of an individual susceptibility to this natural carcinogen. We examined the ability of enzymes of rat renal and hepatic cytosolic fractions to activate AA to metabolites forming DNA adducts by the nuclease P1-enhanced version of the (32)P-postlabeling assay. Cytosolic fractions of both these organs generated AA-DNA adduct patterns reproducing those found in renal tissues from humans exposed to AA. 7-(Deoxyadenosin-N(6)-yl)aristolactam I, 7-(deoxyguanosin-N(2)-yl)aristolactam I and 7-(deoxyadenosin-N(6)-yl)aristolactam II were identified as AA-DNA adducts formed from AAI and 7-(deoxyguanosin-N(2)-yl)aristolactam II and 7-(deoxyadenosin-N(6)-yl)aristolactam II were generated from AAII by hepatic cytosol. Qualitatively the same AA-DNA adduct patterns were observed, although at lower levels, upon incubation of AAs with renal cytosol. To define the role of cytosolic reductases in the reductive activation of AA, we investigated the modulation of AA-DNA adduct formation by cofactors, specific inducers or selective inhibitors of the cytosolic reductases, DT-diaphorase, xanthine oxidase (XO) and aldehyde oxidase. The role of the enzymes in AA activation was also investigated by correlating the DT-diaphorase- and XO-dependent catalytic activities in cytosolic sample with the levels of AA-DNA adducts formed by the same cytosolic sample. On the basis of these studies, we attribute most of the cytosolic activation of AA to DT-diaphorase, although a role of cytosolic XO cannot be ruled out. With purified DT-diaphorase, the participation of this enzyme in the formation of AA-DNA adducts was confirmed. The binding orientation of AAI in the active site of DT-diaphorase was predicted by computer modeling based on published X-ray structures. The results presented here are the first report demonstrating a reductive activation of carcinogenic AAs by DT-diaphorase.