Assuntos
Leucemia Mielogênica Crônica BCR-ABL Positiva , Leucemia Mieloide , Humanos , Sequenciamento de Nucleotídeos em Larga Escala , Leucemia Mielogênica Crônica BCR-ABL Positiva/genética , Mutação , Doença Crônica , Proteínas de Fusão bcr-abl/genética , Resistencia a Medicamentos Antineoplásicos/genética , Inibidores de Proteínas Quinases/farmacologiaRESUMO
OBJECTIVE: A prominent, safe and efficient therapy for patients with chronic myeloid leukemia (CML) is inhibiting oncogenic protein BCR::ABL1 in a targeted manner with imatinib, a tyrosine kinase inhibitor. A substantial part of patients treated with imatinib report skeletomuscular adverse events affecting their quality of life. OCTN2 membrane transporter is involved in imatinib transportation into the cells. At the same time, the crucial physiological role of OCTN2 is cellular uptake of carnitine which is an essential co-factor for the mitochondrial ß-oxidation pathway. This work investigates the impact of imatinib treatment on carnitine intake and energy metabolism of muscle cells. METHODS: HTB-153 (human rhabdomyosarcoma) cell line and KCL-22 (CML cell line) were used to study the impact of imatinib treatment on intracellular levels of carnitine and vice versa. The energy metabolism changes in cells treated by imatinib were quantified and compared to changes in cells exposed to highly specific OCTN2 inhibitor vinorelbine. Mouse models were used to test whether in vitro observations are also achieved in vivo in thigh muscle tissue. The analytes of interest were quantified using a Prominence HPLC system coupled with a tandem mass spectrometer. RESULTS: This work showed that through the carnitine-specific transporter OCTN2, imatinib and carnitine intake competed unequally and intracellular carnitine concentrations were significantly reduced. In contrast, carnitine preincubation did not influence imatinib cell intake or interfere with leukemia cell targeting. Blocking the intracellular supply of carnitine with imatinib significantly reduced the production of most Krebs cycle metabolites and ATP. However, subsequent carnitine supplementation rescued mitochondrial energy production. Due to specific inhibition of OCTN2 activity, the influx of carnitine was blocked and mitochondrial energy metabolism was impaired in muscle cells in vitro and in thigh muscle tissue in a mouse model. CONCLUSIONS: This preclinical experimental study revealed detrimental effect of imatinib on carnitine-mediated energy metabolism of muscle cells providing a possible molecular background of the frequently occurred side effects during imatinib therapy such as fatigue, muscle pain and cramps.
RESUMO
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.
Assuntos
Carcinógenos Ambientais/toxicidade , Dano ao DNA/efeitos dos fármacos , Contaminação de Alimentos , Alimentos/toxicidade , beta-Naftoflavona/farmacologia , Animais , Carcinógenos Ambientais/isolamento & purificação , Citocromo P-450 CYP1A1/antagonistas & inibidores , Citocromo P-450 CYP1A1/genética , Citocromo P-450 CYP1A1/metabolismo , Avaliação Pré-Clínica de Medicamentos , Indução Enzimática/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Contaminação de Alimentos/análise , Intestino Delgado/efeitos dos fármacos , Intestino Delgado/enzimologia , Intestino Delgado/metabolismo , Fígado/efeitos dos fármacos , Fígado/enzimologia , Fígado/metabolismo , Masculino , Testes de Mutagenicidade , Ratos , Ratos WistarRESUMO
OBJECTIVES: The aim of this study was to assess the effect of various flavonoids on the NADPH:cytochrome P450 oxidoreductase (CYPOR) activity in respect of the reduction of different electron acceptors as well as to study an impact of flavonoids on monooxygenation of a model substrate of cytochrome P450 (CYP). DESIGN: The modulation of CYPOR activity was determined spectrophotometrically based on the time course of the reduction of different electron acceptors. The CYP reduction was monitored via its complex formation with CO, having pronounced the absorption maximum at 450 nm. Finally, effect of CYPOR stimulation by 7,8benzoflavone (ANF) on 7pentoxyresorufin Odepentylation was assayed in the microsomal monooxygenation system using the fluorimetric detection of formed resorufin. RESULTS: The stimulation of CYPOR activity via ANF was found to be associated with following electron acceptors: cytochrome c, potassium ferricyanide, cytochrome b5, but not with CYP. Surprisingly, 5,6benzoflavone, a position isomer of ANF, was ineffective in the CYPOR stimulation as well as the other flavonoids tested. In microsomal preparations, ANF did not markedly enhance the reaction rate of monooxygenation of CYP2B4 model substrate. CONCLUSION: Our results document that among all of the tested flavonoids only ANF is able to stimulate CYPOR activity, however, the ANF-mediated stimulation of CYPOR has no impact on the oxidative metabolism catalyzed by CYP system.
Assuntos
Sistema Enzimático do Citocromo P-450/metabolismo , Flavonoides/farmacologia , NADPH-Ferri-Hemoproteína Redutase/metabolismo , Animais , Antioxidantes/farmacologia , Benzoflavonas/química , Benzoflavonas/metabolismo , Monóxido de Carbono/metabolismo , Citocromos b5/metabolismo , Citocromos c/metabolismo , Ferricianetos/metabolismo , Flavonoides/química , Fluorometria , Masculino , Microssomos/efeitos dos fármacos , Microssomos/enzimologia , Microssomos/metabolismo , Oxazinas/metabolismo , Oxirredução/efeitos dos fármacos , Coelhos , Espectrofotometria , Ácido Tióctico/farmacologia , Fatores de Tempo , beta-Naftoflavona/química , beta-Naftoflavona/metabolismoAssuntos
Crise Blástica , Imidazóis , Leucemia Mielogênica Crônica BCR-ABL Positiva , Mutação , Piridazinas , Piridazinas/uso terapêutico , Piridazinas/administração & dosagem , Leucemia Mielogênica Crônica BCR-ABL Positiva/tratamento farmacológico , Leucemia Mielogênica Crônica BCR-ABL Positiva/genética , Leucemia Mielogênica Crônica BCR-ABL Positiva/patologia , Imidazóis/uso terapêutico , Imidazóis/administração & dosagem , Humanos , Crise Blástica/genética , Crise Blástica/tratamento farmacológico , Crise Blástica/patologia , Animais , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Camundongos , Proteínas de Fusão bcr-abl/genética , Inibidores de Proteínas Quinases/uso terapêutico , Inibidores de Proteínas Quinases/farmacologia , Niacinamida/análogos & derivados , PirazóisRESUMO
OBJECTIVES: Since flavonoids and other natural compounds exert beneficial effects on human health, their consumption rapidly increases. However, they can modulate the activity of xenobiotic-metabolizing enzymes involved in activation and detoxification of food and environmental carcinogens. Thus, their potential negative effects should be examined. METHODS: The induction effects of selected chemopreventive compounds, administered per orally by gastric gavages to rats, on cytochrome P450 (CYP) 1A and 2B were determined in liver and small intestine using Western blotting analysis and specific metabolic activity assays. RESULTS: Comparing CYPs expression along small intestine, the highest induction was observed in the proximal part near pylorus with rapid decrease towards the distal part. In response to chemopreventive compounds, the marked induction of CYP1A and CYP2B in liver was observed after diallyl sulphide and flavone treatment. In small intestine, beta-naphthoflavone, diallyl sulphide and curcumin induced CYP1A1 and CYP2B1. In both tissues, resveratrol did not significantly affect CYPs expression. The results of Western blotting detection of CYPs correlate well with their specific enzymatic activities. CONCLUSIONS: Presented data indicate ambiguous impact of chemopreventive compounds on cytochromes P450, main xenobiotic-metabolizing enzymes. Thus, the question of safety and unlimited consumption of these compounds arises.
Assuntos
Anticarcinógenos/farmacologia , Sistema Enzimático do Citocromo P-450/biossíntese , Flavonoides/farmacologia , Intestino Delgado/enzimologia , Compostos Alílicos/farmacologia , Animais , Western Blotting , Curcumina/farmacologia , Citocromo P-450 CYP1A1/biossíntese , Citocromo P-450 CYP1A1/genética , Citocromo P-450 CYP2B1/biossíntese , Citocromo P-450 CYP2B1/genética , Fígado/efeitos dos fármacos , Fígado/enzimologia , Masculino , Ratos , Ratos Wistar , Resveratrol , Estilbenos/farmacologia , Sulfetos/farmacologia , Xenobióticos/metabolismo , beta-Naftoflavona/farmacologiaRESUMO
OBJECTIVES: Flavonoids are widely accepted as health promoting phytochemicals, however, some flavonoids show ability of direct interaction with DNA and/or enhance carcinogen activation into DNA modifying agents. Thus, their potential harmful effect on the human body should be examined in detail. METHODS: Direct interaction of flavonoids (quercetin, rutin) with DNA was examined using square wave voltammetry on carbon paste electrode. Induction effect of selected flavonoids on content of cytochrome P450 1A1, carcinogens activating enzyme, in colon and liver microsomal samples of animals exposed to flavonoids was determined by Western blotting, using anti-cytochrome P450 1A1 specific antibody. RESULTS: Of the natural flavonoids tested, induction of CYP1A1 was elicited by the typical citrus flavonoid naringenin in the colon, as well as by flavone in the liver. Moreover, synthetic beta-naphthoflavone and naturally occurring chrysin, quercetin and diosmin induced CYP1A1 in both tissues. The oxidation signals of guanine and adenine in the DNA molecule were decreased in the presence of flavonoids. CONCLUSIONS: Although flavonoids are often considered to be safe because of their "plant origin", ingestion of flavonoids should be taken with caution. Enhanced expression of CYP1A1 in colon tissue might be responsible for increasing incidence of colorectal carcinoma in humans. Electrochemistry can be used to study the interactions of flavonoids and DNA.
Assuntos
DNA de Cadeia Simples/metabolismo , Flavonoides/metabolismo , Flavonoides/toxicidade , Adenina/metabolismo , Animais , Colo/efeitos dos fármacos , Colo/metabolismo , Citocromo P-450 CYP1A1/metabolismo , Eletroquímica , Ativação Enzimática/efeitos dos fármacos , Flavonoides/farmacologia , Guanidina/metabolismo , Masculino , Microssomos Hepáticos/efeitos dos fármacos , Microssomos Hepáticos/metabolismo , Modelos Biológicos , RatosRESUMO
In recent years, the consumption and use of dietary supplements containing concentrated phytochemicals (e.g. flavonoids) increased dramatically. Flavonoids, as foreign compounds (xenobiotics), have great potential to modulate the activity of cytochrome P450s (CYPs), xenobiotic-metabolizing enzymes involved in the activation and detoxification of food and environmental carcinogens. Thus, the aim of this study was to investigate the effects of model glycosylated and deglycosylated flavonoids on CYPs in rat liver and small intestine, as the two main organs responsible for xenobiotic metabolism, after p.o. administration by gastric gavages. The effects of two glycosylated flavonoids (isoquercitrin, rutin) and their aglycone (quercetin) on CYPs were determined using Western blotting technique and specific activity assays with alkyl-resorufin derivatives. In liver microsomes, a considerable increase of all the measured marker activities (EROD, MROD, PROD) was observed only after isoquercitrin treatment. To evaluate the effects of flavonoids on CYPs along small intestine, the tissue was dissected into proximal (near pylorus), middle and distal parts. Of all the tested compounds, isoquercitrin was the most efficient CYP inducer, namely in the middle part of small intestine. Obtained data demonstrate the different effects of flavonoid glycosides and aglycone on CYP expression in rat liver and small intestine. Since these phytochemicals are xenobiotics, and thus they can increase the human risk of cancer development, their consumption in large quantities should be carefully considered.