RESUMEN
UNLABELLED: (99m)Tc-sestamibi is a widely used radiopharmaceutical agent for myocardial and oncologic imaging. Because of its unique role as a P-glycoprotein (Pgp)-specific substrate, this compound can be used to examine Pgp functional activity in vitro and in vivo under pathologic conditions. Our objective was to use (99m)Tc-sestamibi as a tool to investigate whether systemic inflammation induced by Escherichia coli lipopolysaccharide (LPS) would affect in vivo Pgp function in the brain, heart, liver, and kidneys of rats. Moreover, we also wanted to examine LPS-mediated effects in the placenta of pregnant rats because of the limited amount of in vivo data on this tissue. METHODS: Rats were injected intraperitoneally with LPS or an equal volume of saline as controls. After certain time periods (6 or 24 h), animals were administered 20 MBq of (99m)Tc-sestamibi intravenously, and then images were taken at 0.5, 1, 2, and 3 h. Tissues of rats were excised for (99m)Tc-sestamibi biodistribution analysis by gamma-counting and messenger RNA (mRNA) analysis by reverse transcription-polymerase chain reaction. Western blot analysis with antibody C-219 was used to detect Pgp levels. RESULTS: LPS treatment for 6 h caused a significant downregulation of mdr1a mRNA levels in the brain, heart, and liver, whereas 24 h of LPS treatment significantly reduced mdr1a mRNA levels only in the liver. A significant downregulation of mdr1a mRNA was seen in the brain, heart, and liver within 6 h after LPS administration. Imaging and biodistribution studies demonstrated a higher accumulation of (99m)Tc-sestamibi in the brain, heart, and liver of LPS-treated rats. In the brain, LPS-imposed downregulation of mdr1a mRNA levels was transient, with significant suppression at 4, 6, and 12 h, and the levels recovered to nearly normal by 24 h. This time-dependent downregulation of mRNA correlated with protein levels determined by Western blot analysis. Biodistribution studies of pregnant rats demonstrated a 3.5-fold-higher accumulation of (99m)Tc-sestamibi in the fetal tissues of LPS-treated pregnant rats than in saline-treated control rats. Furthermore, placental mdr1a and mdr1b mRNA levels were also significantly downregulated by LPS treatment. CONCLUSION: Our results indicate that LPS-induced systemic inflammation caused an increased retention of (99m)Tc-sestamibi in the brain, heart, liver, and fetal tissues. These results correlated with a reduction in mdr1a mRNA levels in each organ.
Asunto(s)
Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/metabolismo , Endotoxemia/diagnóstico por imagen , Endotoxemia/metabolismo , Feto/diagnóstico por imagen , Feto/metabolismo , Preñez/metabolismo , Tecnecio Tc 99m Sestamibi/farmacocinética , Animales , Endotoxemia/embriología , Femenino , Masculino , Tasa de Depuración Metabólica , Especificidad de Órganos , Embarazo , Cintigrafía , Radiofármacos/farmacocinética , Ratas , Ratas Sprague-Dawley , Distribución TisularRESUMEN
The commonest mitochondrial diseases are probably those impairing the function of complex I of the respiratory electron transport chain. Such complex I impairment may contribute to various neurodegenerative disorders e.g. Parkinson's disease. In the following, using hepatocytes as a model cell, we have shown for the first time that the cytotoxicity caused by complex I inhibition by rotenone but not that caused by complex III inhibition by antimycin can be prevented by coenzyme Q (CoQ1) or menadione. Furthermore, complex I inhibitor cytotoxicity was associated with the collapse of the mitochondrial membrane potential and reactive oxygen species (ROS) formation. ROS scavengers or inhibitors of the mitochondrial permeability transition prevented cytotoxicity. The CoQ1 cytoprotective mechanism required CoQ1 reduction by DT-diaphorase (NQO1). Furthermore, the mitochondrial membrane potential and ATP levels were restored at low CoQ1 concentrations (5 microM). This suggests that the CoQ1H2 formed by NQO1 reduced complex III and acted as an electron bypass of the rotenone block. However cytoprotection still occurred at higher CoQ1 concentrations (>10 microM), which were less effective at restoring ATP levels but readily restored the cellular cytosolic redox potential (i.e. lactate: pyruvate ratio) and prevented ROS formation. This suggests that CoQ1 or menadione cytoprotection also involves the NQO1 catalysed reoxidation of NADH that accumulates as a result of complex I inhibition. The CoQ1H2 formed would then also act as a ROS scavenger.
Asunto(s)
Hepatocitos/efectos de los fármacos , Mitocondrias Hepáticas/efectos de los fármacos , NAD(P)H Deshidrogenasa (Quinona)/metabolismo , Estrés Oxidativo/efectos de los fármacos , Ubiquinona/farmacología , Animales , Membrana Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Citoprotección , Glutatión/metabolismo , Hepatocitos/enzimología , Masculino , Potenciales de la Membrana/efectos de los fármacos , Mitocondrias Hepáticas/enzimología , NAD/metabolismo , Oxidación-Reducción , Ratas , Ratas Sprague-Dawley , Especies Reactivas de Oxígeno/metabolismo , Rotenona/toxicidad , Desacopladores/toxicidad , Vitamina K 3/farmacologíaRESUMEN
The cytotoxicity of alkenals towards hepatocytes was related to their electrophilicity not their hydrophobicity as cytotoxicity decreased as the chain length increased from acrolein to hexenal and then cytotoxicity increased from hexenal to nonenal. The sequence of events found was rapid glutathione depletion, lipid peroxidation, and inhibition of respiration before cell lysis occurred. Cytotoxicity markedly increased if glutathione was depleted beforehand. Although acrolein-induced cytotoxicity was only delayed by antioxidants or glycolytic substrates (e.g. fructose), it was prevented by NADH generators (e.g. xylitol and sorbitol) due to increased metabolism by ADH1. Cytotoxicity induced by trans,trans-2,4-decadienal (decadienal), on the other hand, was prevented by antioxidants and/or glycolytic substrates but was not prevented by NADH generators. Decadienal-induced cytotoxicity was also more increased by mitochondrial ALDH2 inhibitors than acrolein and was more increased by decreasing mitochondrial NAD+ with rotenone or decreased by increasing mitochondrial NAD+ with oxaloacetate. This suggests that the high electrophilicity of acrolein makes acrolein a more promiscuous inhibitor than decadienal. This results in the inactivation of more enzymes required for cell viability including the cytosolic and mitochondrial ALDHs as well as other enzymes (e.g. mitochondrial) making the reductive detoxication of acrolein by ADH1 more important than the oxidative detoxification by ALDHs. Decadienal is detoxified by all cytosolic and mitochondrial ALDHs and is less dependent on ADH1 for detoxication. There was also marked cytotoxic synergism between acrolein and decadienal presumably because of ALDH inactivation by acrolein.
Asunto(s)
Hepatocitos/efectos de los fármacos , Aldehído Deshidrogenasa/metabolismo , Aldehídos/farmacología , Animales , Hepatocitos/enzimología , Masculino , Ratas , Ratas Sprague-DawleyRESUMEN
The potential impact of tesamorelin on CYP3A activity was investigated by examining its effect on the pharmacokinetics of simvastatin and ritonavir. In two randomized, two-way crossover studies, subjects were administered 2 mg tesamorelin on Days 1-7 with 80 mg simvastatin or 100 mg ritonavir co-administered on Day 6 (Treatment A), and a single dose of simvastatin or ritonavir alone on Day 6 (Treatment B). Pharmacokinetic samples were collected on Day 6 to measure simvastatin, ritonavir and tesamorelin plasma concentrations. For simvastatin, A/B ratios of least squares geometric means and corresponding 90% confidence intervals (CIs) for AUC0-t , AUC0-inf and Cmax were contained within the usual no effect range of 80-125%. For ritonavir, ratios and 90% CIs for AUCs were within this acceptance range, but the lower CI for Cmax was 74.8%, suggesting a decreased rate of exposure. However, since the A/B ratios for AUCs and Cmax parameters were approximately 90%, these were minor decreases and no dose adjustment of ritonavir is required in the presence of tesamorelin. These studies showed that the impact of tesamorelin on CYP3A activity appears to be minimal, if any. Either medication may be co-administered with tesamorelin in patients without changing their original dosing regimen.
RESUMEN
Membrane transporters play a critical role in the absorption, distribution, and elimination of both endogenous substrates and xenobiotics. Defects in transporter function can lead to altered drug disposition including toxicity or loss of efficacy. Inflammation is one condition during which variable drug response has been demonstrated, and this can be attributed, at least in part, to changes in the expression of transporter genes. Thus, knowledge of the mechanisms behind transporter regulation can significantly contribute to our ability to predict variations in drug disposition among individuals and during inflammatory disease. The discovery of several xenobiotic-activated nuclear hormone receptors during the past decade including the pregnane X receptor, constitutive androstane receptor, and farnesoid X receptor has contributed greatly toward this endeavor. These receptors regulate the expression of transporters such as P-glycoprotein, MRP2, MRP3, BCRP, and OATP2 (Oatp1a1/OATP1B1), all of which undergo altered expression during an inflammatory response. Nuclear receptors may therefore play an important role in mediating this effect. This review presents what is currently known about the role of nuclear receptors in transporter regulation during inflammation. The use of this knowledge toward understanding interindividual variation in drug response and drug interactions during inflammation as well toward the development of therapeutics to treat transporter-related diseases will also be discussed.
Asunto(s)
Regulación de la Expresión Génica , Proteínas de Transporte de Membrana/metabolismo , Receptores Citoplasmáticos y Nucleares/metabolismo , Animales , Transporte Biológico , Humanos , InflamaciónRESUMEN
Inflammation-induced changes in the pharmacokinetics and dynamics of numerous drugs have been reported. Altered drug disposition during inflammatory disease has traditionally been ascribed primarily to changes in drug metabolism and protein binding. Emerging evidence within the last decade, however, has demonstrated that the inflammatory response affects the expression of several important drug transporters and these changes significantly impact the disposition and activity of drug substrates.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/metabolismo , Proteínas de Transporte de Catión/metabolismo , Infecciones/metabolismo , Inflamación/metabolismo , Reacción de Fase Aguda , Animales , Encéfalo/metabolismo , Femenino , Humanos , Infecciones/tratamiento farmacológico , Inflamación/tratamiento farmacológico , Mucosa Intestinal/metabolismo , Riñón/metabolismo , Hígado/metabolismo , Modelos Biológicos , Placenta/metabolismo , Embarazo , Receptores Citoplasmáticos y Nucleares/metabolismoRESUMEN
In humans, indirect evidence suggests that hypoxia reduces the rate of biotransformation of drugs cleared by cytochrome P450 (P450) subfamilies CYP1A, 2B, and 2C. The aim of this study was to assess whether acute moderate hypoxia modulates the expression of CYP2B4, 2C5, and 2C16 in vivo, and to determine whether the changes in hepatic P450 are conveyed by serum mediators. Moreover, because hypoxia increases the expression of P-glycoprotein in vitro, we examined whether in vivo acute moderate hypoxia modulates the expression of several membrane transporters in the liver. Rabbits and rats were exposed to a fractional concentration of oxygen of 8% for 48 h to generate a stable arterial partial pressure of O2 of 34 +/- 1 mm Hg. Compared with rabbits breathing room air, hypoxia in rabbits reduced the amount of CYP1A1, 1A2, 2B4, 2C5, and 2C16 proteins and increased the expression of CYP3A6. Sera of rabbits with hypoxia were fractionated by size exclusion chromatography, the fractions were tested for their ability to modify the expression of P450 isoforms, and serum mediators were identified through neutralization experiments. The serum mediators responsible for the down-regulation of P450 isoforms were interferon-gamma, interleukin-1beta (IL-1beta), and IL-2. In vivo, in rats, hypoxia increased the mRNA and protein expression of P-glycoprotein but did not affect the mRNA of breast cancer resistance protein and organic anion-transporting polypeptide 2. It is concluded that in vivo, hypoxia down-regulates rabbit hepatic CYP1A1, 1A2, 2B4, 2C5, and 2C16 and up-regulates CYP3A6. CYP3A11 and P-glycoprotein were up-regulated in the livers of hypoxic rats.
Asunto(s)
Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/metabolismo , Hidrocarburo de Aril Hidroxilasas/metabolismo , Citocromo P-450 CYP1A1/metabolismo , Hígado/metabolismo , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/genética , Enfermedad Aguda , Animales , Hidrocarburo de Aril Hidroxilasas/genética , Hipoxia de la Célula , Células Cultivadas , Cromatografía Líquida de Alta Presión , Citocromo P-450 CYP1A1/genética , Citocromo P-450 CYP3A , Regulación hacia Abajo/efectos de los fármacos , Hepatocitos/citología , Hepatocitos/efectos de los fármacos , Hepatocitos/metabolismo , Hipoxia/fisiopatología , Immunoblotting , Isoenzimas/genética , Isoenzimas/metabolismo , Hígado/efectos de los fármacos , Hígado/enzimología , Masculino , Modelos Animales , Oxígeno/metabolismo , Oxígeno/farmacología , Consumo de Oxígeno , Receptor X de Pregnano , ARN Mensajero/genética , ARN Mensajero/metabolismo , Conejos , Receptores de Esteroides/genética , Receptores de Esteroides/metabolismo , Regulación hacia Arriba/efectos de los fármacosRESUMEN
Activation of the pregnane X receptor (PXR) mediates the induction of several drug transporters and -metabolizing enzymes. In vitro studies have reported that several of these genes are induced after exposure to the hepatocarcinogen, 2-acetylaminofluorene (2-AAF). Thus, we hypothesized that PXR may play a role in the in vivo induction of gene expression by 2-AAF. We examined the expression of the drug-metabolizing enzymes CYP1A2 and CYP3A11 and the drug transporters breast cancer resistance protein (BCRP), MRP2, and OATP2. Wild-type (PXR+/+) and PXR-null (PXR-/-) C57BL/6 mice were injected daily for 7 days with 150 or 300 mg/kg 2-AAF suspended in corn oil (i.p.), whereas the control group received corn oil vehicle. Levels of mRNA isolated from liver were measured by reverse transcription-polymerase chain reaction and normalized to beta-actin. Treatment of PXR+/+ mice resulted in a dose-dependent 2- to 4-fold induction (p<0.001) of MRP2, OATP2, BCRP, CYP3A11, and CYP1A2, but no induction was observed in PXR-/- mice. Induction of PXR mRNA was observed in the 2-AAF-treated PXR+/+ mice. Furthermore, a dose-dependent increase in CYP3A4 promoter construct activity was observed in HepG2 cells cotransfected with human or rat PXR, indicating that 2-AAF does indeed activate PXR. These results suggest that PXR is responsible for 2-AAF-mediated induction of drug efflux transporters and biotransformation enzymes in the liver. Moreover, novel findings demonstrate that PXR plays a role in regulation of the drug efflux transporter, BCRP, in mice.
Asunto(s)
2-Acetilaminofluoreno/farmacología , Transportadoras de Casetes de Unión a ATP/biosíntesis , Sistema Enzimático del Citocromo P-450/biosíntesis , Hígado/enzimología , Receptores Citoplasmáticos y Nucleares/fisiología , Receptores de Esteroides/fisiología , Animales , Transporte Biológico , Línea Celular Tumoral , Inducción Enzimática , Humanos , Inactivación Metabólica , Hígado/efectos de los fármacos , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Receptor X de Pregnano , Receptores Citoplasmáticos y Nucleares/genética , Receptores de Esteroides/genéticaRESUMEN
Inflammation and proinflammatory cytokines suppress the expression of several hepatic transporters and metabolic enzymes, often resulting in cholestatic liver disease. However, mechanism(s) of this down-regulation have not been fully elucidated. As the pregnane X receptor (PXR) is involved in inducing many of these hepatic proteins, it is possible that PXR is also involved in their down-regulation during inflammation. Thus, we compared the effect of inflammation on hepatic gene regulation in wild-type (PXR(+/+)) versus PXR-null (PXR(-/-)) mice. Treatment of PXR(+/+) but not PXR(-/-) mice with the PXR activators 5-pregnen-3beta-ol-20-one-16alpha-carbonitrile (PCN) or 17beta-hydroxy-11beta-[4-dimethylamino phenyl]-17alpha-[1-propynyl] estra-4,9-dien-3-one (RU486) resulted in increased mRNA levels of bsep, mdr1a, mrp2, mrp3, oatp2, and cyp3a11, indicating involvement of PXR in their regulation. Significantly lower mRNA levels of bsep, mdr2, mrp2, mrp3, ntcp, oatp2, and cyp3a11 were found in endotoxin-treated PXR(+/+) mice. In endotoxin-treated PXR(-/-) mice, the extent of mrp2 suppression was significantly diminished. Changes in MRP2 expression were supported by Western blot analysis. Although interleukin (IL)-6 imposed significant decreases in the expression of bsep, mrp2, and cyp3a11 in PXR(+/+) mice, this was not observed in PXR(-/-) mice. Of note, significantly lower levels of PXR mRNA and protein were detected in endotoxin- and IL-6-treated PXR(+/+) mice. In addition, endotoxin and IL-6 were also able to suppress PCN-mediated induction of bsep, mrp2, cyp3a11, and PXR. Taken together, our results suggest that PXR plays a role in the down-regulation of several hepatic proteins during inflammation.
Asunto(s)
Regulación de la Expresión Génica/fisiología , Inflamación/genética , Hígado/metabolismo , Receptores Citoplasmáticos y Nucleares/fisiología , Receptores de Esteroides/fisiología , Animales , Hidrocarburo de Aril Hidroxilasas/biosíntesis , Ácidos y Sales Biliares/metabolismo , Proteínas Portadoras/metabolismo , Citocromo P-450 CYP3A , Citocinas/metabolismo , Endotoxinas/farmacología , Regulación de la Expresión Génica/efectos de los fármacos , Interleucina-6/metabolismo , Hígado/efectos de los fármacos , Hígado/enzimología , Masculino , Glicoproteínas de Membrana/metabolismo , Proteínas de la Membrana , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Oxidorreductasas N-Desmetilantes/biosíntesis , Receptor X de Pregnano , Carbonitrilo de Pregnenolona/farmacología , Receptores Citoplasmáticos y Nucleares/genética , Receptores de Esteroides/genéticaRESUMEN
The pregnane X receptor (PXR) mediates the induction of various genes by xenobiotics, including several ATP-binding cassette transporters. PXR is also activated by bile acids likely to prevent their accumulation to toxic levels; however, the role of PXR in the regulation of MRP3, an important bile acid efflux transporter, has not been elucidated. The impact of PXR activators on the hepatic expression of MRP3 was examined in vivo and in vitro. The human hepatoma cell lines HuH7 and HepG2 were treated with PXR activators including clotrimazole, rifampicin, 17beta-hydroxy-11beta-[4-dimethylamino phenyl]-17alpha-[1-propynyl]estra-4,9-dien-3-one (RU486), metyrapone, nifedipine, lithocholic acid, and 5-pregnen-3beta-ol-20-one-16alpha-carbonitrile (PCN). Levels of MRP3 mRNA, as determined by reverse transcription-polymerase chain reaction, were induced 1.6- to 8-fold in a dose-dependent manner (p < 0.05). Corresponding decreases in the multidrug resistance-associated protein-dependent cellular retention of 5-carboxyfluorescein were also seen in the treated HuH7 cells. In vivo studies demonstrated increased PXR mRNA and induction of MRP3 mRNA in the livers of wild-type mice treated with the PXR activator RU486. On the other hand, MRP3 induction was not seen in the RU486-treated PXR-null mice. These results suggest that PXR activation may play a role in the regulation of MRP3 expression.
Asunto(s)
Proteínas Asociadas a Resistencia a Múltiples Medicamentos/biosíntesis , Receptores Citoplasmáticos y Nucleares/agonistas , Receptores Citoplasmáticos y Nucleares/metabolismo , Receptores de Esteroides/agonistas , Receptores de Esteroides/metabolismo , Animales , Línea Celular Tumoral , Humanos , Hígado/efectos de los fármacos , Hígado/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Mifepristona/farmacología , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/genética , Receptor X de Pregnano , ARN Mensajero/biosíntesis , ARN Mensajero/genéticaRESUMEN
The introduction includes a literature review of DNA reactive species and DNA adduct formation that results from aromatic amine N-oxidation catalyzed by hepatic cytochrome P450 vs. that catalyzed by nonhepatic peroxidases. Experimental evidence is then described for a novel oxidative stress mechanism involving prooxidant N-cation radical formation by both oxidases, which is proposed as a contributing mechanism for aromatic amine induced cytotoxicity and carcinogenesis. Aromatic amine N-cation radicals formed by peroxidases were found to cooxidize GSH or NADH and form reactive oxygen species. The latter could explain the reported DNA oxidative damage found in vivo following methylaminoazobenzene administration [Hirano et al. Analyses of Oxidative DNA Damage and Its Repair Activity in the Livers of 3'-Methyl-4-dimethylaminoazobenzene-Treated Rodents. Jpn. J. Cancer Res. 2000, 91, 681-685]. It was also found that the prooxidant activity of the aromatic amine increased as its redox potential, i.e., ease of oxidation decreased with o-anisidine and aminofluorene being the most effective at forming reactive oxygen species. This suggests that the rate-limiting step in the cooxidation is the rate of arylamine oxidation by the peroxidase. Incubation of hepatocytes with aromatic amines caused a decrease in the mitochondrial membrane potential before cytotoxicity ensued. The CYP1A2-induced hepatocytes isolated from 3-methylcholanthrene administered rats were much more susceptible to some arylamines and were protected by CYP1A2 inhibitors. Hepatocyte GSH was also depleted by all arylamines tested and extensive GSH oxidation occurred with o-anisidine and aminofluorene, which was prevented by CYP1A2 inhibitors. This suggests that in intact hepatocytes CYP1A2 may also catalyze a one-electron oxidation of some arylamines to form prooxidant cation radicals, which cooxidize GSH to form the reactive oxygen species.