RESUMO
Human pregnane X receptor (PXR) is critical for regulating the expression of key drug-metabolizing enzymes such as CYP3A and CYP2C. Our recent study revealed that treatment with rodent-specific PXR agonist pregnenolone-16α-carbonitrile (PCN) significantly induced hepatomegaly and promoted liver regeneration after two-thirds partial hepatectomy (PHx) in mice. However, it remains unclear whether PXR activation induces hepatomegaly and liver regeneration and simultaneously promotes metabolic function of the liver. Here, we investigated the metabolism activity of CYP1A2, CYP3A1/2 and CYP2C6/11 during PXR activation-induced liver enlargement and regeneration in rats after cocktail dosing of CYP probe drugs. For PCN-induced hepatomegaly, a notable increase in the metabolic activity of CYP3A1/2 and CYP2C6/11, as evidenced by the plasma exposure of probe substrates and the AUC ratios of the characteristic metabolites to its corresponding probe substrates. The metabolic activity of CYP1A2, CYP3A1/2 and CYP2C6/11 decreased significantly after PHx. However, PCN treatment obviously enhanced the metabolic activity of CYP2C6/11 and CYP3A1/2 in PHx rats. Furthermore, the protein expression levels of CYP3A1/2 and CYP2C6/11 in liver were up-regulated. Taken together, this study demonstrates that PXR activation not only induces hepatomegaly and liver regeneration in rats, but also promotes the protein expression and metabolic activity of the PXR downstream metabolizing enzymes such as CYP3A1/2 and CYP2C6/11 in the body.
Assuntos
Citocromo P-450 CYP3A , Hepatomegalia , Regeneração Hepática , Fígado , Receptor de Pregnano X , Carbonitrila de Pregnenolona , Animais , Receptor de Pregnano X/metabolismo , Receptor de Pregnano X/genética , Regeneração Hepática/efeitos dos fármacos , Masculino , Citocromo P-450 CYP3A/metabolismo , Carbonitrila de Pregnenolona/farmacologia , Fígado/metabolismo , Fígado/enzimologia , Fígado/efeitos dos fármacos , Ratos , Hepatomegalia/metabolismo , Hepatomegalia/patologia , Hidrocarboneto de Aril Hidroxilases/metabolismo , Hidrocarboneto de Aril Hidroxilases/genética , Família 2 do Citocromo P450/metabolismo , Família 2 do Citocromo P450/genética , Ratos Sprague-Dawley , Citocromo P-450 CYP1A2/metabolismo , Citocromo P-450 CYP1A2/genética , Esteroide 16-alfa-Hidroxilase/metabolismo , Esteroide 16-alfa-Hidroxilase/genética , Esteroide 12-alfa-Hidroxilase/metabolismo , Esteroide 12-alfa-Hidroxilase/genética , HepatectomiaRESUMO
To evaluate the effect of imrecoxib on CYP2C11 enzyme activity, mRNA, and protein expression, a UPLC method was established. Tolbutamide was selected as the CYP2C11 enzyme-specific probe drug and incubated with imrecoxib in rat liver microsomes. The yield of 4-hydroxytolbutamide was measured using UPLC to investigate the effect of imrecoxib on CYP2C11 enzyme activity. Imrecoxib (10 mg/kg) was administered intragastrically twice daily. After 1, 7, and 14 days of administration, the liver tissues were analyzed. The expression of CYP2C11 enzyme mRNA was determined using reverse transcription-polymerase chain reaction, and its protein expression was determined using Western blot analysis. Imrecoxib concentration was inversely proportional to the production of 4-hydroxytolbutamide in liver microsomes. Imrecoxib demonstrated a dose-dependent inhibitory effect on CYP2C11 activity with IC50 = 74.77 µM. After administration, reverse transcription-polymerase chain reaction showed CYP2C11 enzyme mRNA expressions were 65% (P < 0.05), 35%, and 34% of the control group, respectively (P < 0.01). Western blot analysis showed CYP2C11 enzyme protein expressions were 80, 37, and 34% of the control group, respectively (P < 0.01). Imrecoxib can reduce mRNA and protein expression of CYP2C11 enzyme in rat liver and inhibit the activity of CYP2C11 enzyme in a dose-dependent manner. However, it does not produce clinically significant drug interactions.
Assuntos
Hidrocarboneto de Aril Hidroxilases , Pirróis , Animais , Hidrocarboneto de Aril Hidroxilases/metabolismo , Família 2 do Citocromo P450/genética , Família 2 do Citocromo P450/metabolismo , Microssomos Hepáticos/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Mensageiro/farmacologia , Ratos , Esteroide 16-alfa-Hidroxilase/genética , Esteroide 16-alfa-Hidroxilase/metabolismo , SulfetosRESUMO
Paeoniflorin is the major constituent in extracts of the paeony root, the purpose of the present study was to assess the effects of paeoniflorin on the activities and mRNA expression of the rat hepatic drug-metabolizing enzymes cytochrome P450 (CYP1A2), CYP2C11 and CYP3A1 in vivo.Sprague-Dawley (SD) male rats were treated with paeoniflorin at the dosage of 25, 50 and 100 mg/kg or 0.9% sodium chloride solution by intragastric administration for 7 days, then were given probe drugs phenacetin (CYP1A2), tolbutamide (CYP2C11), or midazolam (CYP3A1) orally on the eighth day. Blood samples were collected at various times, and the plasma concentrations of the probe drugs were estimated with ultra-high-performance liquid chromatography. The mRNA expression levels of rat hepatic CYP1A2, CYP2C11 and CYP3A1 were analysed with real-time PCR.The pharmacokinetic results indicated that paeoniflorin inhibits the activities of CYP1A2, CYP2C11 and CYP3A1 in vivo. The effect was most pronounced on CYP3A1, according to the United States Food and Drug Administration classification of inhibitors of CYP3A, it reached the category of moderate inhibition. The mRNA expression levels of 3 CYP enzymes were also tended to be inhibited.We conclude that paeoniflorin can inhibit the activities of CYP1A2, CYP2C11 and CYP3A1 in vivo, which may affect the metabolism of drugs that are primarily dependent on these pathways.
Assuntos
Hidrocarboneto de Aril Hidroxilases , Citocromo P-450 CYP1A2 , Animais , Citocromo P-450 CYP1A2/genética , Família 2 do Citocromo P450/genética , Glucosídeos/farmacologia , Masculino , Monoterpenos , RNA Mensageiro/genética , Ratos , Ratos Sprague-Dawley , Esteroide 16-alfa-Hidroxilase/genéticaRESUMO
1. CYP2C11 is the most abundant isoform of cytochrome P450s (CYPs) in male rats and is considered the main enzyme for warfarin metabolism. 2. To further access the in vivo function of CYP2C11 in warfarin metabolism and efficacy, a CYP2C11-null rat model was used to study warfarin metabolism with both in vitro and in vivo approaches. Prothrombin time (PT) of warfarin was also determined. 3. The maximum rate of metabolism (Vmax) and intrinsic clearance (CLint) of liver microsomes from CYP2C11-null males were reduced by 37 and 64%, respectively, compared to those in Sprague Dawley (S-D) rats. The Km of liver microsomes from CYP2C11-null males was increased by 73% compared to that of S-D rats. The time to reach the maximum plasma concentration (Tmax) of warfarin in CYP2C11-null males was significantly delayed compared to that in S-D males, and the CL rate was also reduced. The PT of CYP2C11-null rats was moderately longer than that of S-D rats. 4. In conclusion, the clearance rate of warfarin was mildly decreased and its anticoagulant effect was moderately increased in male rats following CYP2C11 gene knockout. CYP2C11 played a certain role in the clearance and efficacy of warfarin, while it did not seem to be essential.
Assuntos
Hidrocarboneto de Aril Hidroxilases/genética , Família 2 do Citocromo P450/genética , Esteroide 16-alfa-Hidroxilase/genética , Varfarina/farmacocinética , Animais , Anticoagulantes/farmacocinética , Hidrocarboneto de Aril Hidroxilases/metabolismo , Família 2 do Citocromo P450/metabolismo , Feminino , Técnicas de Inativação de Genes , Masculino , Microssomos Hepáticos/efeitos dos fármacos , Microssomos Hepáticos/metabolismo , Ratos Sprague-Dawley , Esteroide 16-alfa-Hidroxilase/metabolismoRESUMO
Primary prostate cancer can have extensive microheterogeneity, but its contribution to the later emergence of metastatic castration-resistant prostate cancer (mCRPC) remains unclear. In this study, we microdissected residual prostate cancer foci in radical prostatectomies from 18 men treated with neoadjuvant-intensive androgen deprivation therapy (leuprolide, abiraterone acetate, and prednisone) and analyzed them for resistance mechanisms. Transcriptome profiling showed reduced but persistent androgen receptor (AR) activity in residual tumors, with no increase in neuroendocrine differentiation. Proliferation correlated negatively with AR activity but positively with decreased RB1 expression, and whole-exome sequencing (WES) further showed enrichment for RB1 genomic loss. In 15 cases where 2 or 3 tumor foci were microdissected, WES confirmed a common clonal origin but identified multiple oncogenic alterations unique to each focus. These findings show that subclones with oncogenic alterations found in mCRPC are present in primary prostate cancer and are selected for by neoadjuvant-intense androgen deprivation therapy. In particular, this study indicates that subclonal RB1 loss may be more common than previously appreciated in intermediate- to high-risk primary prostate cancer and may be an early event, independent of neuroendocrine differentiation, in the development of mCRPC. Comprehensive molecular analyses of primary prostate cancer may detect aggressive subclones and possibly inform adjuvant strategies to prevent recurrence.Significance: Neoadjuvant androgen deprivation therapy for prostate cancer selects for tumor foci with subclonal genomic alterations, which may comprise the origin of metastatic castration-resistant prostate cancer. Cancer Res; 78(16); 4716-30. ©2018 AACR.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/administração & dosagem , Resistencia a Medicamentos Antineoplásicos/genética , Recidiva Local de Neoplasia/tratamento farmacológico , Neoplasias de Próstata Resistentes à Castração/tratamento farmacológico , Acetato de Abiraterona/administração & dosagem , Idoso , Antagonistas de Androgênios/administração & dosagem , Hidrocarboneto de Aril Hidroxilases/genética , Biomarcadores Tumorais , Carcinogênese , Evolução Clonal , Família 2 do Citocromo P450/genética , Humanos , Masculino , Pessoa de Meia-Idade , Terapia Neoadjuvante , Recidiva Local de Neoplasia/genética , Recidiva Local de Neoplasia/patologia , Prostatectomia , Neoplasias de Próstata Resistentes à Castração/genética , Neoplasias de Próstata Resistentes à Castração/patologia , Receptores Androgênicos/genética , Esteroide 16-alfa-Hidroxilase/genéticaRESUMO
Potato (Solanum tuberosum) is a major food crop, while the most tissues of potato accumulates steroidal glycoalkaloids (SGAs) α-solanine and α-chaconine. Since SGAs confer a bitter taste on human and show the toxicity against various organisms, reducing the SGA content in the tubers is requisite for potato breeding. However, generation of SGA-free potato has not been achieved yet, although silencing of several SGA biosynthetic genes led a decrease in SGAs. Here, we show that the knockout of St16DOX encoding a steroid 16α-hydroxylase in SGA biosynthesis causes the complete abolition of the SGA accumulation in potato hairy roots. Nine candidate guide RNA (gRNA) target sequences were selected from St16DOX by in silico analysis, and the two or three gRNAs were introduced into a CRISPR/Cas9 vector designated as pMgP237-2A-GFP that can express multiplex gRNAs based on the pre-tRNA processing system. To establish rapid screening of the candidate gRNAs that can efficiently mutate the St16DOX gene, we used a potato hairy root culture system for the introduction of the pMgP237 vectors. Among the transgenic hairy roots, two independent lines showed no detectable SGAs but accumulated the glycosides of 22,26-dihydroxycholesterol, which is the substrate of St16DOX. Analysis of the two lines with sequencing exhibited the mutated sequences of St16DOX with no wild-type sequences. Thus, generation of SGA-free hairy roots of tetraploid potato was achieved by the combination of the hairy root culture and the pMgP237-2A-GFP vector. This experimental system is useful to evaluate the efficacy of candidate gRNA target sequences in the short-term.
Assuntos
Sistemas CRISPR-Cas/genética , Edição de Genes/métodos , Genes de Plantas/genética , Raízes de Plantas/genética , Solanina/metabolismo , Solanum tuberosum/genética , Esteroide 16-alfa-Hidroxilase/genética , Técnicas de Inativação de Genes/métodos , Raízes de Plantas/metabolismo , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/metabolismo , Análise de Sequência de DNA , Solanum tuberosum/metabolismo , Esteroide 16-alfa-Hidroxilase/metabolismoRESUMO
CYP2C11 is involved in the metabolism of many drugs in rats. To assess the roles of CYP2C11 in physiology and drug metabolism, a CYP2C11-null rat model was generated using the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9method. A 2-base pair insertion was added to exon 6 of CYP2C11 in Sprague-Dawley rats. CYP2C11 was not detected by western blotting in liver microsomes of CYP2C11-null rats. No off-target effects were found at 11 predicted sites of the knockout model. The CYP2C11-null rats were viable and had no obvious abnormalities, with the exception of reduced fertility. Puberty in CYP2C11-null rats appeared to be delayed by â¼20 days, and the average litter size fell by 43%. Tolbutamide was used as a probe in this drug metabolism study. In the liver microsomes of CYP2C11-null rats, the Vmax and intrinsicclearance values decreased by 22% and 47%, respectively, compared with those of wild-type rats. The Km values increased by 47% compared with that of wild types. However, our pharmacokinetics study showed no major differences in any parameters between the two strains, in both males and females. In conclusion, a CYP2C11-null rat model was successfully generated and is a valuable tool to study the in vivo function of CYP2C11.
Assuntos
Hidrocarboneto de Aril Hidroxilases/genética , Sistemas CRISPR-Cas/genética , Família 2 do Citocromo P450/genética , Esteroide 16-alfa-Hidroxilase/genética , Animais , Sistemas CRISPR-Cas/efeitos dos fármacos , Feminino , Inativação Metabólica/fisiologia , Masculino , Microssomos Hepáticos/efeitos dos fármacos , Modelos Animais , Ratos , Ratos Sprague-Dawley , Tolbutamida/farmacologiaRESUMO
1. Aspirin (ASA) and clopidogrel (CLP) are used in combination as dual antiplatelet therapy (DAPT) for acute coronary syndrome based on their complementary mechanisms for platelet aggregation inhibition. However, the pharmacokinetics of such drug combination usage has not been thoroughly investigated. 2. In the current study, an LC-MS/MS method was developed to simultaneously determine the plasma concentrations of ASA and its metabolite salicylic acid (SA) with CLP and its metabolites, clopidogrel carboxylic acid (CLPM) and clopidogrel active metabolite derivative (CAMD). The pharmacokinetics of ASA, SA, CLP, CLPM and CAMD in rats receiving two-week DAPT with ASA and CLP were then determined. 3. After two-week DAPT with ASA and CLP in rats, the activities of aspirin esterase and rCyp2c11, enzymes mediating rat metabolism of ASA and CLP, respectively, in prepared rat liver microsomes were measured followed by further determination of rCyp2c11 mRNA expressions. The results demonstrated that DAPT led to minimal impact on aspirin esterase activity but significant decrease in rCyp2c11 activity and mRNA expression. 4. In conclusion, our findings on impairment in rCyp2C11 activity and mRNA expression by DAPT in rats could provide guidance on its safe clinical use with other CYP 2C19 substrates.
Assuntos
Hidrocarboneto de Aril Hidroxilases/metabolismo , Aspirina/farmacocinética , Família 2 do Citocromo P450/metabolismo , Fígado/efeitos dos fármacos , Inibidores da Agregação Plaquetária/farmacologia , Esteroide 16-alfa-Hidroxilase/metabolismo , Ticlopidina/análogos & derivados , Animais , Hidrocarboneto de Aril Hidroxilases/genética , Aspirina/sangue , Cromatografia Líquida/métodos , Clopidogrel , Família 2 do Citocromo P450/genética , Inativação Metabólica , Fígado/metabolismo , Masculino , Inibidores da Agregação Plaquetária/farmacocinética , Ratos Sprague-Dawley , Ácido Salicílico/metabolismo , Esteroide 16-alfa-Hidroxilase/genética , Espectrometria de Massas em Tandem/métodos , Ticlopidina/sangue , Ticlopidina/farmacocinéticaRESUMO
NEW FINDINGS: What is the central question of this study? Is there a beneficial effect and what are the mechanisms of acute and multiple hyperbaric oxygenation (HBO2 ) exposures on the outcome of cerebral tissue injury induced by a transient middle cerebral artery occlusion model in diabetic female rats? Are 20-hydroxyeicosatetreanoic acid and epoxyeicosatrienoic acids involved? What is the main finding and its importance? Equal reduction of cortical and total infarct size in rats treated with HBO2 and HET0016 (20-hydroxyeicosatetreanoic acid production inhibitor) and significant mRNA upregulation of epoxyeicosatrienoic acid-producing enzymes (Cyp2J3 and Cyp2C11) in treated groups suggest that HBO2 and HET0016 are highly effective stroke treatments and that cytochrome P450 metabolites are involved in this therapeutic effect. We evaluated the effects of acute and repetitive hyperbaric oxygenation (HBO2 ), 20-hydroxyeicosatetreanoic acid (20-HETE) inhibition by N-hydroxy-N'-(4-butyl-2methylphenyl)-formamidine (HET0016) and their combination on experimental stroke outcomes. Streptozotocin-induced type 1 diabetic Sprague-Dawley female rats (n = 42; n = 7 per group), were subjected to 30 min of transient middle cerebral artery occlusion (t-MCAO)-reperfusion and divided into the following groups: (1) control group, without treatment; and groups exposed to: (2) HBO2 ; (3) multiple HBO2 (HBO2 immediately and second exposure 12 h after t-MCAO); (4) HET0016 pretreatment (1 mg kg-1 , 3 days before t-MCAO) combined with HBO2 after t-MCAO; (5) HET0016 treatment (1 h before, during and for 6 h after t-MCAO); and (6) HET0016 treatment followed by HBO2 after t-MCAO. Messenger RNA expression of CYP2J3, CYP2C11, CYP4A1, endothelial nitric oxide synthase and epoxide hydrolase 2 was determined by real-time qPCR. Cortical infarct size and total infarct size were equally and significantly reduced in HBO2 - and HET0016-treated rats. Combined treatment with HET0016 and HBO2 provided no significant additive effect compared with HET0016 treatment only. Messenger RNA of Cyp2J3 was significantly increased in all study groups, and mRNA of Cyp2C11 was significantly increased in the multiple HBO2 group and the HET0016 treatment followed by HBO2 group, compared with the control group. Expression of endothelial nitric oxide synthase was significantly increased after HBO2 treatments, and expression of epoxide hydrolase 2 was increased in all groups compared with the control group. In diabetic female Sprague-Dawley rats, HBO2 and HET0016 are highly effective stroke treatments, suggesting the involvement of cytochrome P450 metabolites and the NO pathway in this therapeutic effect.
Assuntos
Amidinas/farmacologia , Encéfalo/efeitos dos fármacos , Diabetes Mellitus Experimental/terapia , Diabetes Mellitus Tipo 1/terapia , Ácidos Hidroxieicosatetraenoicos/metabolismo , Oxigenoterapia Hiperbárica , Infarto da Artéria Cerebral Média/terapia , Fármacos Neuroprotetores/farmacologia , Traumatismo por Reperfusão/prevenção & controle , Animais , Hidrocarboneto de Aril Hidroxilases/genética , Hidrocarboneto de Aril Hidroxilases/metabolismo , Encéfalo/metabolismo , Encéfalo/patologia , Terapia Combinada , Sistema Enzimático do Citocromo P-450/genética , Sistema Enzimático do Citocromo P-450/metabolismo , Família 2 do Citocromo P450/genética , Família 2 do Citocromo P450/metabolismo , Família 4 do Citocromo P450/genética , Família 4 do Citocromo P450/metabolismo , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Experimental/fisiopatologia , Diabetes Mellitus Tipo 1/metabolismo , Diabetes Mellitus Tipo 1/fisiopatologia , Epóxido Hidrolases/genética , Epóxido Hidrolases/metabolismo , Feminino , Regulação Enzimológica da Expressão Gênica , Infarto da Artéria Cerebral Média/metabolismo , Infarto da Artéria Cerebral Média/patologia , Infarto da Artéria Cerebral Média/fisiopatologia , Óxido Nítrico Sintase Tipo III/genética , Óxido Nítrico Sintase Tipo III/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos Sprague-Dawley , Traumatismo por Reperfusão/metabolismo , Traumatismo por Reperfusão/patologia , Traumatismo por Reperfusão/fisiopatologia , Esteroide 16-alfa-Hidroxilase/genética , Esteroide 16-alfa-Hidroxilase/metabolismo , Fatores de TempoRESUMO
The sexually dimorphic expression of cytochromes P450 (CYP) drug-metabolizing enzymes has been reported in all species examined. These sex differences are only expressed during adulthood and are solely regulated by sex differences in circulating growth hormone (GH) profiles. Once established, however, the different male- and female-dependent CYP isoform profiles are permanent and immutable, suggesting that adult CYP expression requires imprinting. As the hormone that regulates an adult function is likely the same hormone that imprints the function, we selectively blocked GH secretion in some newborn male rats, whereas others received concurrent physiologic replacement of rat GH. The results demonstrate that adult male GH activation of the signal transduction pathway regulating expression of the principal CYP2C11 isoform is obligatorily dependent on perinatal GH imprinting, without which CYP2C11 and drug metabolism would be permanently and profoundly suppressed. As there are other adult metabolic functions also regulated by GH, pediatric drug therapy known to disrupt GH secretion could unintentionally impair adult health.
Assuntos
Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Hormônio do Crescimento/farmacologia , Hepatócitos/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Animais , Hidrocarboneto de Aril Hidroxilases/genética , Hidrocarboneto de Aril Hidroxilases/metabolismo , Família 2 do Citocromo P450/genética , Família 2 do Citocromo P450/metabolismo , Feminino , Hormônio do Crescimento/sangue , Hepatócitos/metabolismo , Masculino , Ratos , Esteroide 16-alfa-Hidroxilase/genética , Esteroide 16-alfa-Hidroxilase/metabolismo , Proteínas Supressoras da Sinalização de Citocina/genética , Proteínas Supressoras da Sinalização de Citocina/metabolismoRESUMO
Several drug-metabolizing cytochrome P450 (CYP) enzymes exhibit sexual dimorphism depending on the pituitary growth hormone (GH) secretory patterns. However, the mechanism underlying CYP sexual dimorphism remains unclear. We previously established a transgenic (Alb-DsRed2 Tg) rat that expressed red fluorescent DsRed2 protein, particularly in hepatocytes, to visualize cell differentiation and multiplication and found that hepatic DsRed2 expression exhibited sexual dimorphism that was limited to adult males. In this study, we compared the expression patterns between sexual dimorphic Cyps and DsRed2 in Tg rats after experimentally reversing the GH secretory patterns in males and females. Postnatal day 1 male and female Tg rats were gonadectomized and then testosterone propionate (0.25 mg/rat) was subcutaneously administered to ovariectomized females immediately after surgery. Cyp mRNA and DsRed2 expression levels were quantified using RT-PCR and an in vivo imaging system, respectively. GH-dependent Cyps and hepatic DsRed2 expression patterns were reversed in males and females at 9 weeks after birth and were significantly correlated (P<0.05). This suggested that DsRed2 expression in these Tg rats depended on GH secretory patterns. Based on DsRed2 fluorescence, this Tg rat model could become a tool to readily and effectively evaluate changes in GH-dependent Cyp expression.
Assuntos
Hidrocarboneto de Aril Hidroxilases/genética , Família 2 do Citocromo P450/genética , Expressão Gênica , Hormônio do Crescimento/metabolismo , Esteroide 16-alfa-Hidroxilase/genética , Esteroide Hidroxilases/genética , Animais , Hidrocarboneto de Aril Hidroxilases/metabolismo , Família 2 do Citocromo P450/metabolismo , Feminino , Fígado/metabolismo , Proteínas Luminescentes/química , Masculino , Modelos Animais , RNA Mensageiro/metabolismo , Ratos , Ratos Transgênicos , Ratos Wistar , Caracteres Sexuais , Esteroide 16-alfa-Hidroxilase/metabolismo , Esteroide Hidroxilases/metabolismo , Proteína Vermelha FluorescenteRESUMO
Chronic renal failure (CRF) impedes renal excretion of drugs and their metabolism by reducing the expression of liver cytochrome P450 (P450). Uremic serum contains factors, such as parathyroid hormone (PTH), that decrease liver P450s. The P450s are also involved in the metabolism of xenobiotics in the brain. This study investigates: 1) the effects of CRF on rat brain P450, 2) the role of PTH in the downregulation of brain P450s in CRF rats, and 3) the effects of PTH on P450s in astrocytes. Protein and mRNA expression of P450s were assessed in the brain of CRF and control (CTL) rats, as well as from CTL or CRF rats that underwent parathyroidectomy (PTX) 1 week before nephrectomy. CYP3A activity was measured using 3-[(3, 4-difluorobenzyl) oxy]-5, 5-dimethyl-4-[4-methylsulfonyl) phenyl] furan-2(5H)-1 metabolism in brain microsomal preparation. CYP3A protein expression was assessed in primary cultured astrocytes incubated with serum obtained from CRF or CTL rats or with PTH. Significant downregulations (≥40%) of CYP1A, CYP2C11, and CYP3A proteins were observed in microsomes from CRF rat brains. CYP3A activity reduction was also observed. CYP3A expression and activity were unaffected in PTX-pretreated CRF rats. Serum of PTX-treated CRF rats had no impact on CYP3A levels in astrocytes compared with that of untreated CRF rats. Finally, PTH addition to normal calf serum induced a reduction in CYP3A protein similar to CRF serum, suggesting that CRF-induced hyperparathyroidism is associated with a significant decrease in P450 drug-metabolizing enzymes in the brain, which may have implications in drug response.
Assuntos
Encéfalo/enzimologia , Sistema Enzimático do Citocromo P-450/metabolismo , Falência Renal Crônica/enzimologia , Animais , Hidrocarboneto de Aril Hidroxilases/genética , Hidrocarboneto de Aril Hidroxilases/metabolismo , Astrócitos/enzimologia , Células Cultivadas , Citocromo P-450 CYP1A1/genética , Citocromo P-450 CYP1A1/metabolismo , Citocromo P-450 CYP2E1/genética , Citocromo P-450 CYP2E1/metabolismo , Citocromo P-450 CYP3A/genética , Citocromo P-450 CYP3A/metabolismo , Sistema Enzimático do Citocromo P-450/genética , Família 2 do Citocromo P450/genética , Família 2 do Citocromo P450/metabolismo , Modelos Animais de Doenças , Regulação Enzimológica da Expressão Gênica , Falência Renal Crônica/genética , Masculino , Nefrectomia , Hormônio Paratireóideo/metabolismo , Paratireoidectomia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos Sprague-Dawley , Esteroide 16-alfa-Hidroxilase/genética , Esteroide 16-alfa-Hidroxilase/metabolismoRESUMO
Our recent work showed that the brain serotonergic system negatively regulated liver cytochrome P450. The aim of our present research was to study the effect of damage to the serotonergic innervation of the paraventricular (PVN) or arcuate nuclei (ARC) of the hypothalamus on the neuroendocrine regulation of cytochrome P450 (CYP). Male rats received bilateral injections of the serotonergic neurotoxin 5,7-dihydroxytryptamine (5,7-DHT) into the PVN or ARC. One week after the injection brain neurotransmitters, serum hormones (growth hormone, testosterone, corticosterone, thyroid hormones), pituitary somatostatin and liver cytochrome P450 expression and activity were measured. Lesion of the serotonergic innervation of the PVN decreased serotonin level in the hypothalamic area containing the PVN, causing an increase in growth hormone and testosterone concentrations in the blood and, subsequently, an increase in the expression (mRNA and protein level) and activity of isoform CYP2C11 in the liver. In contrast, damage to the serotonergic innervation of the ARC, which caused a decrease in serotonin level in the hypothalamic area containing the ARC, reduced the concentration of growth hormone and the expression and activity of CYP2C11. In conclusion, the obtained results show a reverse effect of the serotonergic innervation of the hypothalamic paraventricular (a negative effect) and arcuate nuclei (a positive effect) on growth hormone secretion and growth hormone-dependent CYP2C11 expression. They also suggest that CYP2C11 expression may be changed by drugs acting via the serotonergic system, their effect depending on their mechanism of action, route of administration (intracerebral, peripheral) and distribution pattern within the hypothalamus.
Assuntos
5,7-Di-Hidroxitriptamina/farmacologia , Núcleo Arqueado do Hipotálamo/efeitos dos fármacos , Hidrocarboneto de Aril Hidroxilases/genética , Família 2 do Citocromo P450/genética , Fígado/efeitos dos fármacos , Núcleo Hipotalâmico Paraventricular/efeitos dos fármacos , Serotonina/metabolismo , Esteroide 16-alfa-Hidroxilase/genética , Animais , Núcleo Arqueado do Hipotálamo/metabolismo , Masculino , Núcleo Hipotalâmico Paraventricular/metabolismo , Hormônios Hipofisários/sangue , Ratos WistarRESUMO
16-dehydropregnenolone (DHP) is a promising novel antihyperlipidemic agent developed and patented by Central Drug Research Institute (CDRI), India. The purpose of the present study was to investigate whether DHP influences the activities and mRNA expression of hepatic drug-metabolizing cytochrome P450 (CYP) enzymes (CYP1A2, CYP2C11, CYP2D2, CYP2E1 and CYP3A1) in Sprague-Dawley (SD) rats. A cocktail suspension of CYP probe substrates which contained caffeine (CYP1A2), tolbutamide (CYP2C11), dextromethorphan (CYP2D2), chlorzoxazone (CYP2E1) and dapsone (CYP3A1) was administered orally on eighth- or fifteenth-day to rats pre-treated with DHP intragastrically at a dose of 36 and 72mg/kg for one week and two weeks. The concentrations of probe drugs in plasma were estimated by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Alongside, the effect of DHP on CYPs activity and mRNA expression levels were assayed in isolated rat liver microsomes and by real-time reverse transcription-polymerase chain reaction (RT-PCR), respectively. DHP had significant inducing effects on CYP1A2, 2C11, 2D2 and 2E1 with no effect on CYP3A1 in dose- and time-dependent manner, as revealed from the pharmacokinetic profiles of the probe drugs in rats. In-vitro microsomal activities and mRNA expression results were in good agreement with the in-vivo pharmacokinetic results. Collectively, the results unveiled that DHP is an inducer of rat hepatic CYP enzymes. Hence, intense attention should be paid when DHP is co-administered with drugs metabolized by CYP1A2, 2C11, 2D2 and 2E1, which might result in drug-drug interactions and therapeutic failure.
Assuntos
Hidrocarboneto de Aril Hidroxilases/genética , Citocromo P-450 CYP1A2/genética , Citocromo P-450 CYP2E1/genética , Citocromo P-450 CYP3A/genética , Família 2 do Citocromo P450/genética , Hipolipemiantes/farmacocinética , Pregnenolona/análogos & derivados , Esteroide 16-alfa-Hidroxilase/genética , Administração Oral , Animais , Hidrocarboneto de Aril Hidroxilases/metabolismo , Cafeína/metabolismo , Cafeína/farmacologia , Clorzoxazona/metabolismo , Clorzoxazona/farmacologia , Citocromo P-450 CYP1A2/metabolismo , Citocromo P-450 CYP2E1/metabolismo , Citocromo P-450 CYP3A/metabolismo , Família 2 do Citocromo P450/metabolismo , Dapsona/metabolismo , Dapsona/farmacologia , Dextrometorfano/metabolismo , Dextrometorfano/farmacologia , Regulação da Expressão Gênica , Hipolipemiantes/administração & dosagem , Fígado/efeitos dos fármacos , Fígado/enzimologia , Masculino , Microssomos Hepáticos/efeitos dos fármacos , Microssomos Hepáticos/enzimologia , Pregnenolona/administração & dosagem , Pregnenolona/farmacocinética , Ratos , Ratos Sprague-Dawley , Esteroide 16-alfa-Hidroxilase/metabolismo , Tolbutamida/metabolismo , Tolbutamida/farmacologiaRESUMO
Sunitinib (SUN) is a multi-targeted tyrosine kinase inhibitor that was recently approved for the treatment of gastrointestinal tract and renal cancers. To date, very little is known about the effects of SUN on the expression of hepatic and renal xenobiotic-metabolizing enzymes (XMEs) and transporters. The present study was designed to investigate the capacity of chronic SUN treatment to modulate the mRNA and protein expression levels of phase I cytochrome P450 (CYP), phase II conjugating enzymes, and phase III transporters in rat liver and kidneys. For this purpose, SUN (25, 50 and 100 mg/kg) was injected IP into Wistar albino rats for 4 weeks; thereafter, the mRNA and protein expression levels of several XMEs and transporters were determined by RT-PCR and Western blot analysis, respectively. Real-time PCR analysis showed that SUN significantly induced the hepatic and renal CYP1A1, 1A2, 1B1, 2E1 and 4F4, whereas it inhibited CYP2C11 and 4A2. Furthermore, SUN specifically induced renal, but not hepatic, CYP2J3 and 3A2, while it induced only hepatic CYP4A1. With regard to phase II, SUN induced hepatic GSTA1 and UGT1A and renal NQO1 and UGT1A mRNA levels, whereas it inhibited renal GST1A expression. On the other hand, both renal and hepatic P-gp, MRP2 and BCRP transporters were significantly induced by SUN at the mRNA and protein expression levels. Importantly, these differential effects were associated with changes in oxidative stress genes and lipid peroxidation levels. In conclusion, SUN can serve as XME and transporters modulator, which potentially may counteract the efficacy of the treatment, adverse reactions and drug interactions in SUN treatment.
Assuntos
Indóis/farmacologia , Rim/efeitos dos fármacos , Fígado/efeitos dos fármacos , Pirróis/farmacologia , Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP/genética , Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP/metabolismo , Transportadores de Cassetes de Ligação de ATP/genética , Transportadores de Cassetes de Ligação de ATP/metabolismo , Animais , Hidrocarboneto de Aril Hidroxilases/antagonistas & inibidores , Hidrocarboneto de Aril Hidroxilases/genética , Hidrocarboneto de Aril Hidroxilases/metabolismo , Citocromo P-450 CYP1A1/genética , Citocromo P-450 CYP1A1/metabolismo , Citocromo P-450 CYP1A2/genética , Citocromo P-450 CYP1A2/metabolismo , Citocromo P-450 CYP1B1/genética , Citocromo P-450 CYP1B1/metabolismo , Citocromo P-450 CYP2E1/genética , Citocromo P-450 CYP2E1/metabolismo , Sistema Enzimático do Citocromo P-450/genética , Sistema Enzimático do Citocromo P-450/metabolismo , Família 2 do Citocromo P450/antagonistas & inibidores , Família 2 do Citocromo P450/genética , Família 2 do Citocromo P450/metabolismo , Família 4 do Citocromo P450/genética , Família 4 do Citocromo P450/metabolismo , Glucuronosiltransferase/genética , Glucuronosiltransferase/metabolismo , Glutationa Transferase/genética , Glutationa Transferase/metabolismo , Isoenzimas/genética , Isoenzimas/metabolismo , Rim/enzimologia , Peroxidação de Lipídeos/efeitos dos fármacos , Fígado/enzimologia , Masculino , NAD(P)H Desidrogenase (Quinona)/genética , NAD(P)H Desidrogenase (Quinona)/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Ratos Wistar , Esteroide 16-alfa-Hidroxilase/antagonistas & inibidores , Esteroide 16-alfa-Hidroxilase/genética , Esteroide 16-alfa-Hidroxilase/metabolismo , Sunitinibe , Transcriptoma , Xenobióticos/metabolismoRESUMO
Glibenclamide and thymoquinone plasma concentrations were analysed using a sensitive RP-HPLC method, and non-compartmental model pharmacokinetic parameters were calculated. The maximum reduction in blood glucose level was observed 3 hours following glibenclamide administration, which reached 47.4% of baseline, whereas it was reduced by 53.0% to 56.2% when co-administrated with thymoquinone. Plasma concentration of glibenclamide was increased by 13.4% and 21.8% by the co-administration of thymoquinone as single and multiple doses, respectively (P<0.05). The AUC and TI/2 of glibenclamide were also increased respectively by 32.0% and 17.4% with a thymoquinone single dose, and by 52.5% and 92.8% after chronic treatment. Furthermore, diabetic rats treated with thymoquinone demonstrated a marked decrease in hepatic protein expressions of CYP3A2 and CYP2C 11 enzymes that are responsible for the metabolism of glibenclamide. The current data suggest that thymoquinone exhibits a synergistic effect with glibenclamide on glucose level, which could be explained by reducing CYP450 activity at the protein level.
Assuntos
Benzoquinonas/administração & dosagem , Diabetes Mellitus Experimental/tratamento farmacológico , Glibureto/farmacocinética , Hipoglicemiantes/farmacocinética , Nigella/química , Extratos Vegetais/administração & dosagem , Animais , Hidrocarboneto de Aril Hidroxilases/genética , Hidrocarboneto de Aril Hidroxilases/metabolismo , Glicemia/metabolismo , Citocromo P-450 CYP3A/genética , Citocromo P-450 CYP3A/metabolismo , Família 2 do Citocromo P450 , Diabetes Mellitus Experimental/enzimologia , Diabetes Mellitus Experimental/genética , Diabetes Mellitus Experimental/metabolismo , Modelos Animais de Doenças , Interações Medicamentosas , Humanos , Masculino , Ratos , Ratos Wistar , Esteroide 16-alfa-Hidroxilase/genética , Esteroide 16-alfa-Hidroxilase/metabolismoRESUMO
Perinatal exposure of rats and mice to the typically reported 4mg/g bd wt dose of monosodium glutamate (MSG) results in a complete block in GH secretion as well as obesity, growth retardation and a profound suppression of several cytochrome P450s, including CYP2C11, the predominant male-specific isoform--all irreversible effects. In contrast, we have found that a lower dose of the food additive, 2mg/g bd wt on alternate days for the first 9days of life results in a transient neonatal depletion of plasma GH, a subsequent permanent overexpression of CYP2C11 as well as subnormal (mini) GH pulse amplitudes in an otherwise normal adult masculine episodic GH profile. The overexpressed CYP2C11 was characterized by a 250% increase in mRNA, but only a 40 to 50% increase in CYP2C11 protein and its catalytic activity. Using freshly isolated hepatocytes as well as primary cultures exposed to the masculine-like episodic GH profile, we observed normal induction, activation, nuclear translocation and binding to the CYP2C11 promoter of the GH-dependent signal transducers required for CYP2C11 transcription. The disproportionately lower expression levels of CYP2C11 protein were associated with dramatically high expression levels of an aberrant, presumably nontranslated CYP2C11 mRNA, a 200% increase in CYP2C11 ubiquitination and a 70-80% decline in miRNAs associated, at normal levels, with a suppression of CYP2C expression. Whereas the GH-responsiveness of CYP2C7 and CYP2C6 as well as albumin was normal in the MSG-derived hepatocytes, the abnormal expression of CYP2C11 was permanent and irreversible.
Assuntos
Hidrocarboneto de Aril Hidroxilases/biossíntese , Aromatizantes/toxicidade , Hepatócitos/efeitos dos fármacos , Glutamato de Sódio/toxicidade , Esteroide 16-alfa-Hidroxilase/biossíntese , Transcrição Gênica/efeitos dos fármacos , Transporte Ativo do Núcleo Celular , Fatores Etários , Albuminas/metabolismo , Animais , Animais Recém-Nascidos , Hidrocarboneto de Aril Hidroxilases/genética , Sítios de Ligação , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Células Cultivadas , Sistema Enzimático do Citocromo P-450/genética , Sistema Enzimático do Citocromo P-450/metabolismo , Família 2 do Citocromo P450 , Indução Enzimática , Feminino , Hormônio do Crescimento/sangue , Hepatócitos/enzimologia , Masculino , MicroRNAs/metabolismo , Regiões Promotoras Genéticas , RNA Mensageiro/biossíntese , Ratos Sprague-Dawley , Fator de Transcrição STAT5/metabolismo , Caracteres Sexuais , Fatores Sexuais , Transdução de Sinais/efeitos dos fármacos , Esteroide 16-alfa-Hidroxilase/genética , Esteroide 21-Hidroxilase/genética , Esteroide 21-Hidroxilase/metabolismo , Proteínas Supressoras da Sinalização de Citocina/metabolismo , Fatores de Tempo , UbiquitinaçãoRESUMO
Previously, a facilitating effect of hyperbaric oxygenation (HBO2) on aortic ring responses to angiotensin-(1-7) in healthy rats was reported, with epoxyeicosatrienoic acids (EETs) possibly playing an important role. The aim of this study was to assess whether HBO2 exerts similar effects in diabetic rats and to further explore the role of specific cytochrome P450 (CYP) enzymes in changes induced by HBO2. Aortic relaxation to angiotensin-(1-7) was significantly higher in HBO2 diabetic rats compared to control diabetic rats, while HBO2 had no effect on angiotensin II contraction. N-methylsulphonyl-6-(2-propargyloxyphenyl/hexanamide inhibited the facilitation of angiotensin-(1-7) responses in HBO2 rats, suggesting an important role of EETs in this modulation. mRNA expression of CYP2J3 and protein expression of CYP2C11 were significantly upregulated in HBO2 diabetic rats, whereas CYP4A1, CYP4A2 and CYP4A3 mRNA and CYP2J3 protein expression was similar between groups. Mean arterial pressure, ferric reducing ability of plasma and Thiobarbituric Acid Reactive Substances levels and serum angiotensin-(1-7) concentrations were not significantly changed.
Assuntos
Ácido 8,11,14-Eicosatrienoico/análogos & derivados , Angiotensina I/farmacologia , Diabetes Mellitus Tipo 1/terapia , Angiopatias Diabéticas/prevenção & controle , Oxigenoterapia Hiperbárica , Fragmentos de Peptídeos/farmacologia , Resistência Vascular/efeitos dos fármacos , Vasodilatadores/farmacologia , Ácido 8,11,14-Eicosatrienoico/metabolismo , Amidas/farmacologia , Angiotensina I/sangue , Angiotensina II/farmacologia , Animais , Aorta Torácica/efeitos dos fármacos , Aorta Torácica/metabolismo , Aorta Torácica/fisiopatologia , Hidrocarboneto de Aril Hidroxilases/antagonistas & inibidores , Hidrocarboneto de Aril Hidroxilases/genética , Hidrocarboneto de Aril Hidroxilases/metabolismo , Sistema Enzimático do Citocromo P-450/química , Sistema Enzimático do Citocromo P-450/genética , Sistema Enzimático do Citocromo P-450/metabolismo , Família 2 do Citocromo P450 , Diabetes Mellitus Tipo 1/complicações , Diabetes Mellitus Tipo 1/metabolismo , Diabetes Mellitus Tipo 1/fisiopatologia , Indução Enzimática , Inibidores Enzimáticos/farmacologia , Oxigenoterapia Hiperbárica/efeitos adversos , Masculino , Estresse Oxidativo , Fragmentos de Peptídeos/sangue , Ratos Sprague-Dawley , Esteroide 16-alfa-Hidroxilase/antagonistas & inibidores , Esteroide 16-alfa-Hidroxilase/genética , Esteroide 16-alfa-Hidroxilase/metabolismo , Vasoconstrição/efeitos dos fármacos , Vasodilatação/efeitos dos fármacos , Vasodilatadores/sangueRESUMO
AIM: Combined use of herbs and drugs may result in clinically important herb-drug interactions. The majorities of these interactions are thought to be metabolism-based and involve induction or inhibition of cytochrome P450 (CYP). The current study was designed to investigate the effect of some commonly used herbs on rat CYP2C11 gene expression and metabolic activity. METHODS: Wistar rats were treated for 7 days with increasing doses of 3 herbs; Nigella sativa, Trigonella foenum-graecum, and Ferula asafoetida. Thereafter, CYP2C11 mRNA and protein levels were determined by real-time polymerase chain reaction (RT-PCR) and western blot analyses, respectively. In vitro metabolic activity of CYP2C11 was performed on rat hepatic microsomes using tolbutamide as specific substrate. RESULTS: Our results showed that all the 3 herbs significantly inhibited the mRNA and protein expression levels of CYP2C11 in a dose-dependent manner. Furthermore, the in vitro enzyme metabolic activity study showed a significant decrease in the formation of 4-hyroxy-tolbutamide, a tolbutamide metabolite, at the higher doses. The inhibitory effects of the investigated herbs on rat CYP2C11 was in the order: Nigella Sativa > Trigonella foenum-graecum > Ferula asafoetida. CONCLUSIONS: The 3 herbs are strong inhibitor of CYP2C11 expression, which can lead to an undesirable pharmacological effect of clinically used CYP2C11 substrate drugs with a low therapeutic index.
Assuntos
Hidrocarboneto de Aril Hidroxilases/biossíntese , Ferula/efeitos adversos , Expressão Gênica/efeitos dos fármacos , Interações Ervas-Drogas , Fígado/metabolismo , Nigella sativa/efeitos adversos , Esteroide 16-alfa-Hidroxilase/biossíntese , Trigonella/efeitos adversos , Animais , Hidrocarboneto de Aril Hidroxilases/análise , Hidrocarboneto de Aril Hidroxilases/genética , Família 2 do Citocromo P450 , Relação Dose-Resposta a Droga , Masculino , Microssomos Hepáticos/metabolismo , Ratos , Esteroide 16-alfa-Hidroxilase/análise , Esteroide 16-alfa-Hidroxilase/genética , Tolbutamida/metabolismoRESUMO
BACKGROUND & AIMS: It has recently been reported that anti-inflammatory lipid mediators are increased in the late phase of acute inflammation, whereas proinflammatory lipid mediators are regulated at the initiation of inflammation. The purpose of this study was to evaluate changes of hepatic lipid mediators due to high-fat diet (HFD) feeding in endotoxemic rats. METHODS: Male Wistar rats were fed either HFD or control diet for 12 weeks, and were then killed 0, 1.5, and 6 h after lipopolysaccharide (LPS) injection. Analyses included lipidomics assessment of mediators using liquid chromatography-electrospray ionization/multi-stage mass spectrometry; measuring expression of hepatic polyunsaturated fatty acid (PUFA)-oxidizing enzyme, tumor necrosis factor (TNF)-α, interleukin (IL)-6, and inducible nitric oxide synthase mRNA levels; blood biochemical tests; and liver histology. RESULTS: HFD feeding worsened liver injury, increased expression of TNF-α and IL-6 mRNA, and increased oxidative stress after LPS injection. PUFA-oxidizing enzymes were higher in HFD-fed rats after LPS injection. The proinflammatory prostaglandin (PG)E2 and thromboxane B2 were increased 1.5 h after LPS injection, and had decreased by 6 h in HFD-fed rats. In contrast, potent pro-resolving resolvins derived from eicosapentaenoic acid and docosahexaenoic acid were not detected, but anti-inflammatory epoxyeicosatrienoic acids, lipoxin A4, and 15-deoxy-PGJ2 were increased after LPS injection in HFD-fed rats. CONCLUSIONS: HFD feeding for 12 weeks enhanced proinflammatory lipid mediators 1.5 h after LPS injection suggesting relation to liver injury.