RESUMO
Heavy metals are both integral parts of cells and environmental toxicants, and their deregulation is associated with severe cellular dysfunction and various diseases. Here we show that the Hippo pathway plays a critical role in regulating heavy metal homeostasis. Hippo signalling deficiency promotes the transcription of heavy metal response genes and protects cells from heavy metal-induced toxicity, a process independent of its classic downstream effectors YAP and TAZ. Mechanistically, the Hippo pathway kinase LATS phosphorylates and inhibits MTF1, an essential transcription factor in the heavy metal response, resulting in the loss of heavy metal response gene transcription and cellular protection. Moreover, LATS activity is inhibited following heavy metal treatment, where accumulated zinc directly binds and inhibits LATS. Together, our study reveals an interplay between the Hippo pathway and heavy metals, providing insights into this growth-related pathway in tissue homeostasis and stress response.
Assuntos
Cádmio/metabolismo , Proteínas de Ligação a DNA/metabolismo , Via de Sinalização Hippo/fisiologia , Proteínas Serina-Treonina Quinases/metabolismo , Fatores de Transcrição/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Zinco/metabolismo , Cádmio/toxicidade , Linhagem Celular Tumoral , Regulação da Expressão Gênica/genética , Células HEK293 , Células HeLa , Homeostase/genética , Humanos , Inativação Metabólica/fisiologia , Fosforilação , Proteínas Serina-Treonina Quinases/genética , Estresse Fisiológico/fisiologia , Transcrição Gênica/genética , Proteínas Supressoras de Tumor/genética , Zinco/toxicidade , Fator MTF-1 de TranscriçãoRESUMO
Diphenylamine NSAIDs are taken frequently for chronic pain conditions, yet their use may potentiate hepatotoxicity risks through poorly characterized metabolic mechanisms. Our previous work revealed that seven marketed or withdrawn diphenylamine NSAIDs undergo bioactivation into quinone-species metabolites, whose reaction specificities depended on halogenation and the type of acidic group on the diphenylamine. Herein, we identified cytochromes P450 responsible for those bioactivations, determined reaction specificities, and estimated relative contributions of enzymes to overall hepatic bioactivations and detoxifications. A qualitative activity screen revealed CYP2C8, 2C9, 2C19, and 3A4 played roles in drug bioactivation. Subsequent steady-state studies with recombinant CYPs recapitulated the importance of halogenation and acidic group type on bioactivations but importantly, showed patterns unique to each CYP. CYP2C9, 2C19 and 3A4 bioactivated all NSAIDs with CYP2C9 dominating all possible bioactivation pathways. For each CYP, specificities for overall oxidative metabolism were not impacted significantly by differences in NSAID structures but the values themselves differed among the enzymes such that CYP2C9 and 3A4 were more efficient than others. When considering hepatic CYP abundance, CYP2C9 almost exclusively accounted for diphenylamine NSAID bioactivations, whereas CYP3A4 provided a critical counterbalance favoring their overall detoxification. Preference for either outcome would depend on molecular structures favoring metabolism by the CYPs as well as the influence of clinical factors altering their expression and/or activity. While focused on NSAIDs, these findings have broader implications on bioactivation risks given the expansion of the diphenylamine scaffold to other drug classes such as targeted cancer therapeutics.
Assuntos
Anti-Inflamatórios não Esteroides/metabolismo , Citocromo P-450 CYP2C9/metabolismo , Citocromo P-450 CYP3A/metabolismo , Difenilamina/metabolismo , Microssomos Hepáticos/efeitos dos fármacos , Microssomos Hepáticos/metabolismo , Anti-Inflamatórios não Esteroides/toxicidade , Difenilamina/toxicidade , Humanos , Inativação Metabólica/efeitos dos fármacos , Inativação Metabólica/fisiologia , Especificidade por Substrato/efeitos dos fármacos , Especificidade por Substrato/fisiologiaRESUMO
Exemestane (EXE) is a hormonal therapy used to treat estrogen receptor-positive breast cancer by inhibiting the final step of estrogen biosynthesis catalyzed by the enzyme aromatase. Cysteine conjugates of EXE and its active metabolite 17ß-dihydro-EXE (DHE) are the major metabolites found in both the urine and plasma of patients taking EXE. The initial step in cysteine conjugate formation is glutathione conjugation catalyzed by the glutathione S-transferase (GST) family of enzymes. The goal of the present study was to identify cytosolic hepatic GSTs active in the GST-mediated metabolism of EXE and 17ß-DHE. Twelve recombinant cytosolic hepatic GSTs were screened for their activity against EXE and 17ß-DHE, and glutathionylated EXE and 17ß-DHE conjugates were detected by ultra-performance liquid chromatography tandem mass spectrometry. GST α (GSTA) isoform 1, GST µ (GSTM) isoform 3 and isoform 1 were active against EXE, whereas only GSTA1 exhibited activity against 17ß-DHE. GSTM1 exhibited the highest affinity against EXE with a Michaelis-Menten constant (KM) value that was 3.8- and 7.1-fold lower than that observed for GSTA1 and GSTM3, respectively. Of the three GSTs, GSTM3 exhibited the highest intrinsic clearance against EXE (intrinsic clearance = 0.14 nl·min-1·mg-1). The KM values observed for human liver cytosol against EXE (46 µM) and 17ß-DHE (77 µM) were similar to those observed for recombinant GSTA1 (53 and 30 µM, respectively). Western blot analysis revealed that GSTA1 and GSTM1 composed 4.3% and 0.57%, respectively, of total protein in human liver cytosol; GSTM3 was not detected. These data suggest that GSTA1 is the major hepatic cytosolic enzyme involved in the clearance of EXE and its major active metabolite, 17ß-DHE. SIGNIFICANCE STATEMENT: Most previous studies related to the metabolism of the aromatase inhibitor exemestane (EXE) have focused mainly on phase I metabolic pathways and the glucuronidation phase II metabolic pathway. However, recent studies have indicated that glutathionylation is the major metabolic pathway for EXE. The present study is the first to characterize hepatic glutathione S-transferase (GST) activity against EXE and 17ß-dihydro-EXE and to identify GST α 1 and GST µ 1 as the major cytosolic GSTs involved in the hepatic metabolism of EXE.
Assuntos
Androstadienos/farmacocinética , Neoplasias da Mama , Glutationa Transferase/metabolismo , Inativação Metabólica/fisiologia , Fígado/enzimologia , Antineoplásicos Hormonais/farmacocinética , Inibidores da Aromatase/farmacocinética , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/metabolismo , Cromatografia Líquida , Cisteína/metabolismo , Citosol/metabolismo , Estrogênios/biossíntese , Glutationa Transferase/química , Eliminação Hepatobiliar/fisiologia , Humanos , Isoformas de Proteínas , Receptores de EstrogênioRESUMO
Most drugs are administered to children orally. An information gap remains on the protein abundance of small intestinal drug-metabolizing enzymes (DMEs) and drug transporters (DTs) across the pediatric age range, which hinders precision dosing in children. To explore age-related differences in DMEs and DTs, surgical leftover intestinal tissues from pediatric and adult jejunum and ileum were collected and analyzed by targeted quantitative proteomics for apical sodium-bile acid transporter, breast cancer resistance protein (BCRP), monocarboxylate transporter 1 (MCT1), multidrug resistance protein 1 (MDR1), multidrug resistance-associated protein (MRP) 2, MRP3, organic anion-transporting polypeptide 2B1, organic cation transporter 1, peptide transporter 1 (PEPT1), CYP2C19, CYP3A4, CYP3A5, UDP glucuronosyltransferase (UGT) 1A1, UGT1A10, and UGT2B7. Samples from 58 children (48 ileums, 10 jejunums, age range: 8 weeks to 17 years) and 16 adults (8 ileums, 8 jejunums) were analyzed. When comparing age groups, BCRP, MDR1, PEPT1, and UGT1A1 abundance was significantly higher in adult ileum as compared with the pediatric ileum. Jejunal BCRP, MRP2, UGT1A1, and CYP3A4 abundance was higher in the adults compared with children 0-2 years of age. Examining the data on a continuous age scale showed that PEPT1 and UGT1A1 abundance was significantly higher, whereas MCT1 and UGT2B7 abundance was lower in adult ileum as compared with the pediatric ileum. Our data contribute to the deeper understanding of the ontogeny of small intestinal drug-metabolizing enzymes and drug transporters and shows DME-, DT-, and intestinal location-specific, age-related changes. SIGNIFICANCE STATEMENT: This is the first study that describes the ontogeny of small intestinal DTs and DMEs in human using liquid chromatography with tandem mass spectrometry-based targeted quantitative proteomics. The current analysis provides a detailed picture about the maturation of DT and DME abundances in the human jejunum and ileum. The presented results supply age-related DT and DME abundance data for building more accurate PBPK models that serve to support safer and more efficient drug dosing regimens for the pediatric population.
Assuntos
Inativação Metabólica/fisiologia , Intestino Delgado , Proteínas de Membrana Transportadoras/metabolismo , Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP/metabolismo , Adulto , Fatores Etários , Transporte Biológico Ativo , Criança , Cromatografia Líquida/métodos , Citocromo P-450 CYP3A/metabolismo , Ensaios Enzimáticos/métodos , Ontologia Genética , Glucuronosiltransferase/metabolismo , Humanos , Intestino Delgado/efeitos dos fármacos , Intestino Delgado/enzimologia , Intestino Delgado/metabolismo , Taxa de Depuração Metabólica , Proteína 2 Associada à Farmacorresistência Múltipla/metabolismo , Proteínas de Neoplasias/metabolismo , Transportador 1 de Peptídeos/metabolismo , Espectrometria de Massas em Tandem/métodosRESUMO
Trapping assays are conducted at lead optimization stages to detect reactive metabolites (RMs) that can contribute to drug toxicity. The commonly used dansyl glutathione (dGSH) provides a sensitive analysis owing to the fluorescent label, however, it captures only soft electrophilic RMs. TRs for hard electrophilic RMs, few of which are labeled fluorescently, can detect hard electrophilic aldehydes only by forming unstable imine derivatives. In this study, we aimed to develop novel fluorescently labeled TRs that detect both soft and hard electrophilic RMs and form stable ring structures with aldehydes. We designed four dansylated TRs based on cysteine, which has both soft and hard nucleophilic groups. To evaluate the reactivity of the TRs, we incubated them with several substrates and found that one of the TRs (CysGlu-Dan) detected all the soft and hard electrophilic RMs. We also examined the inhibition potential of each TR for seven major CYPs involved in drug metabolism and found that CysGlu-Dan showed an inhibitory profile similar to that of dGSH. In conclusion, CysGlu-Dan can be used to evaluate the risk of RMs in drug discovery.
Assuntos
Cisteína , Efeitos Colaterais e Reações Adversas Relacionados a Medicamentos , Inativação Metabólica/fisiologia , Indicadores e Reagentes , Marcação por Isótopo/métodos , Elementos de Resposta Antioxidante , Biomarcadores Farmacológicos/análise , Cromatografia/métodos , Cisteína/química , Cisteína/metabolismo , Sistema Enzimático do Citocromo P-450/metabolismo , Efeitos Colaterais e Reações Adversas Relacionados a Medicamentos/diagnóstico , Efeitos Colaterais e Reações Adversas Relacionados a Medicamentos/metabolismo , Efeitos Colaterais e Reações Adversas Relacionados a Medicamentos/prevenção & controle , Humanos , Indicadores e Reagentes/química , Indicadores e Reagentes/metabolismo , Espectrometria de Massas/métodos , Medição de RiscoRESUMO
3,3'-Diindolylmethane (DIM), a major phytochemical derived from ingestion of cruciferous vegetables, is also a dietary supplement. In preclinical models, DIM is an effective cancer chemopreventive agent and has been studied in a number of clinical trials. Previous pharmacokinetic studies in preclinical and clinical models have not reported DIM metabolites in plasma or urine after oral dosing, and the pharmacological actions of DIM on target tissues is assumed to be solely via the parent compound. Seven subjects (6 males and 1 female) ranging from 26-65 years of age, on a cruciferous vegetable-restricted diet prior to and during the study, took 2 BioResponse DIM 150-mg capsules (45.3 mg DIM/capsule) every evening for one week with a final dose the morning of the first blood draw. A complete time course was performed with plasma and urine collected over 48 hours and analyzed by UPLC-MS/MS. In addition to parent DIM, two monohydroxylated metabolites and 1 dihydroxylated metabolite, along with their sulfate and glucuronide conjugates, were present in both plasma and urine. Results reported here are indicative of significant phase 1 and phase 2 metabolism and differ from previous pharmacokinetic studies in rodents and humans, which reported only parent DIM present after oral administration. 3-((1H-indole-3-yl)methyl)indolin-2-one, identified as one of the monohydroxylated products, exhibited greater potency and efficacy as an aryl hydrocarbon receptor agonist when tested in a xenobiotic response element-luciferase reporter assay using Hepa1 cells. In addition to competitive phytochemical-drug adverse reactions, additional metabolites may exhibit pharmacological activity highlighting the importance of further characterization of DIM metabolism in humans. SIGNIFICANCE STATEMENT: 3,3'-Diindolylmethane (DIM), derived from indole-3-carbinol in cruciferous vegetables, is an effective cancer chemopreventive agent in preclinical models and a popular dietary supplement currently in clinical trials. Pharmacokinetic studies to date have found little or no metabolites of DIM in plasma or urine. In marked contrast, we demonstrate rapid appearance of mono- and dihydroxylated metabolites in human plasma and urine as well as their sulfate and glucuronide conjugates. The 3-((1H-indole-3-yl)methyl)indolin-2-one metabolite exhibited significant aryl hydrocarbon receptor agonist activity, emphasizing the need for further characterization of the pharmacological properties of DIM metabolites.
Assuntos
Indóis , Administração Oral , Anticarcinógenos/sangue , Anticarcinógenos/farmacocinética , Anticarcinógenos/urina , Cápsulas , Suplementos Nutricionais , Desenvolvimento de Medicamentos , Vias de Eliminação de Fármacos , Feminino , Humanos , Inativação Metabólica/fisiologia , Indóis/sangue , Indóis/farmacocinética , Indóis/urina , Masculino , Pessoa de Meia-Idade , Compostos Fitoquímicos/sangue , Compostos Fitoquímicos/farmacocinética , Compostos Fitoquímicos/urinaRESUMO
Variability in individual capacity for hepatic elimination of therapeutic drugs is well recognized and is associated with variable expression and activity of liver enzymes and transporters. Although genotyping offers some degree of stratification, there is often large variability within the same genotype. Direct measurement of protein expression is impractical due to limited access to tissue biopsies. Hence, determination of variability in hepatic drug metabolism and disposition using liquid biopsy (blood samples) is an attractive proposition during drug development and in clinical practice. This study used a multi-"omic" strategy to establish a liquid biopsy technology intended to assess hepatic capacity for metabolism and disposition in individual patients. Plasma exosomal analysis (n = 29) revealed expression of 533 pharmacologically relevant genes at the RNA level, with 147 genes showing evidence of expression at the protein level in matching liver tissue. Correction of exosomal RNA expression using a novel shedding factor improved correlation against liver protein expression for 97 liver-enriched genes. Strong correlation was demonstrated for 12 key drug-metabolizing enzymes and 4 drug transporters. The developed test allowed reliable patient stratification, and in silico trials demonstrated utility in adjusting drug dose to achieve similar drug exposure between patients with variable hepatic elimination. Accordingly, this approach can be applied in characterization of volunteers prior to enrollment in clinical trials and for patient stratification in clinical practice to achieve more precise individual dosing.
Assuntos
Transporte Biológico/fisiologia , Sistema Enzimático do Citocromo P-450/metabolismo , Fígado/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Exossomos/metabolismo , Feminino , Humanos , Inativação Metabólica/fisiologia , Biópsia Líquida/métodos , Masculino , Taxa de Depuração Metabólica/fisiologia , Pessoa de Meia-Idade , Adulto JovemRESUMO
BACKGROUND AND AIMS: Constitutive androstane receptor (CAR) agonists, such as 1,4-bis [2-(3,5-dichloropyridyloxy)] benzene (TCPOBOP), are known to cause robust hepatocyte proliferation and hepatomegaly in mice along with induction of drug metabolism genes without any associated liver injury. Yes-associated protein (Yap) is a key transcription regulator that tightly controls organ size, including that of liver. Our and other previous studies suggested increased nuclear localization and activation of Yap after TCPOBOP treatment in mice and the potential role of Yap in CAR-driven proliferative response. Here, we investigated a direct role of Yap in CAR-driven hepatomegaly and hepatocyte proliferation using hepatocyte-specific Yap-knockout (KO) mice. APPROACH AND RESULTS: Adeno-associated virus 8-thyroxine binding globulin promoter-Cre recombinase vector was injected to Yap-floxed mice for achieving hepatocyte-specific Yap deletion followed by TCPOBOP treatment. Yap deletion did not decrease protein expression of CAR or CAR-driven induction of drug metabolism genes (including cytochrome P450 [Cyp] 2b10, Cyp2c55, and UDP-glucuronosyltransferase 1a1 [Ugt1a1]). However, Yap deletion substantially reduced TCPOBOP-induced hepatocyte proliferation. TCPOBOP-driven cell cycle activation was disrupted in Yap-KO mice because of delayed (and decreased) induction of cyclin D1 and higher expression of p21, resulting in decreased phosphorylation of retinoblastoma protein. Furthermore, the induction of other cyclins, which are sequentially involved in progression through cell cycle (including cyclin E1, A2, and B1), and important mitotic regulators (such as Aurora B kinase and polo-like kinase 1) was remarkably reduced in Yap-KO mice. Microarray analysis revealed that 26% of TCPOBOP-responsive genes that were mainly related to proliferation, but not to drug metabolism, were altered by Yap deletion. Yap regulated these proliferation genes through alerting expression of Myc and forkhead box protein M1, two critical transcriptional regulators of CAR-mediated hepatocyte proliferation. CONCLUSIONS: Our study revealed an important role of Yap signaling in CAR-driven hepatocyte proliferation; however, CAR-driven induction of drug metabolism genes was independent of Yap.
Assuntos
Proliferação de Células/fisiologia , Receptor Constitutivo de Androstano/fisiologia , Hepatócitos/fisiologia , Inativação Metabólica/genética , Proteínas de Sinalização YAP/fisiologia , Animais , Ciclo Celular , Feminino , Regulação da Expressão Gênica , Genes/genética , Hepatócitos/metabolismo , Humanos , Inativação Metabólica/fisiologia , Regeneração Hepática , Camundongos Knockout , TranscriptomaRESUMO
Elucidating the mechanisms for circadian expression of drug-metabolizing enzymes is essential for a better understanding of dosing time-dependent drug metabolism and pharmacokinetics. CYP2B6 (Cyp2b10 in mice) is an important enzyme responsible for metabolism and detoxification of approximately 10% of drugs. Here, we aimed to investigate a potential role of nuclear receptor co-repressor RIP140 in circadian regulation of Cyp2b10 in mice.We first uncovered diurnal rhythmicity in hepatic RIP140 mRNA and protein with peak values at ZT10 (ZT, zeitgeber time). RIP140 ablation up-regulated Cyp2b10 expression and blunted its rhythm in mice and in AML-12 cells. Consistent with a negative regulatory effect, overexpression of RIP140 inhibited Cyp2b10 promoter activity and reduced cellular Cyp2b10 expression.Furthermore, RIP140 suppressed Car- and Pxr-mediated transactivation of Cyp2b10, and the suppressive effects were attenuated when the RIP140 gene was silenced. Chromatin immunoprecipitation assays revealed that recruitment of RIP140 protein to the Cyp2b10 promoter was circadian time-dependent in wild-type mice. More extensive recruitment was observed at ZT10 than at ZT2 consistent with the rhythmic pattern of RIP140 protein. However, the time-dependency of RIP140 recruitment was lost in RIP140-/- mice.Additionally, we identified a D-box and a RORE cis-element in RIP140 promoter. D-box- and RORE-acting clock components such as Dbp, E4bp4, Rev-erbα/ß and Rorα transcriptionally regulated RIP140, potentially accounting for its rhythmic expression.In conclusion, RIP140 regulates diurnal expression of Cyp2b10 in mouse liver through periodical repression of Car- and Pxr-mediated transactivation. This co-regulator-driven mechanism represents a novel source of diurnal rhythmicity in drug-metabolizing enzymes.
Assuntos
Família 2 do Citocromo P450/metabolismo , Inativação Metabólica/fisiologia , Correpressor 1 de Receptor Nuclear/genética , Animais , Ritmo Circadiano , Sistema Enzimático do Citocromo P-450 , Fígado/metabolismo , Camundongos , Ativação TranscricionalRESUMO
BACKGROUND: Therapeutic drug monitoring (TDM) can identify patients with subtherapeutic asparaginase (ASNase) activity [silent inactivation (SI)] and prospectively guide therapeutic adaptation. However, limited intra-individual variability is a precondition for targeted dosing and the diagnosis of SI. METHODS: In the AIEOP-BFM acute lymphoblastic leukemia (ALL) 2009 trial, 2771 children with ALL were included and underwent ASNase-TDM in a central laboratory in Münster. Two biweekly administrations of pegylated ASNase during induction and a third dose during reinduction or the high-risk block, which was administered several weeks later, were monitored. We calculated (1) the incidence of SI; and (2) the predictivity of SI for SI after the subsequent administration. ASNase activities monitored during induction were categorized into percentiles at the respective sampling time points. These percentiles were used to calculate the intra-individual range of percentiles as a surrogate for intrapatient variability and to evaluate the predictivity of ASNase activity for the subsequent administration. RESULTS: The overall incidence of SI was low (4.9%). The positive predictive value of SI identified by one sample was ≤21%. Confirmation of SI by a second sample indicated a high positive predictive value of 100% for biweekly administrations, but not for administration more than 17 weeks later. Sampling and/or documentation errors were risks for misdiagnosis of SI. High intra-individual variability in ASNase activities, with ranges of percentiles over more than 2 quartiles and low predictivity, was observed in approximately 25% of the patients. These patients were likely to fail dose individualization based on TDM data. CONCLUSIONS: To use TDM as a basis for clinical decisions, standardized clinical procedures are required and high intra-individual variability should be taken into account. Details of the treatment are available in the European Clinical Trials Database at https://www.clinicaltrialsregister.eu/ctr-search/trial/2007-004270-43/DE.
Assuntos
Asparaginase/sangue , Monitoramento de Medicamentos/métodos , Polietilenoglicóis/uso terapêutico , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamento farmacológico , Adolescente , Asparaginase/administração & dosagem , Asparaginase/uso terapêutico , Asparagina/sangue , Criança , Pré-Escolar , Relação Dose-Resposta a Droga , Feminino , Humanos , Inativação Metabólica/fisiologia , Lactente , Masculino , Polietilenoglicóis/administração & dosagemRESUMO
BACKGROUND: Cancer is one of the most serious diseases threatening human health with high morbidity and mortality in the world. For the treatment of cancer, chemotherapy is one of the most widely used strategies, for almost all kinds of tumors and diverse stages of tumor development. The efficacy of chemotherapy not only depends on the activity of the drug administrated but also on whether the compound could reach the effective therapeutic concentration in tumor cells. Therefore, expression and activity of drug-metabolizing enzymes (DMEs) in tumor tissues and metabolic organs of cancer patients are important for the dispositional behavior of anticancer drugs as well as the clinical response of chemotherapy. METHODS: This review summarizes the recent advancement of the DMEs expression and activity in various cancers, as well as the potential regulatory mechanisms of major DMEs in cancer and cancer therapy. RESULTS: Compared to normal tissues, expression and activity of major DMEs are significantly dysregulated in patients by various factors including epigenetic modification, ligand-activated transcriptional regulation and signaling pathways. Additionally, DMEs play an important role in anticancer drug efficacy, chemoresistance as well as the activation of prodrugs. CONCLUSION: This review reinforces a more comprehensive understanding of DMEs in cancer and cancer therapy, and provides more opportunities for cancer therapy.
Assuntos
Antineoplásicos/metabolismo , Antineoplásicos/uso terapêutico , Inativação Metabólica/efeitos dos fármacos , Inativação Metabólica/fisiologia , Neoplasias/tratamento farmacológico , Neoplasias/metabolismo , Animais , Humanos , Transdução de Sinais/efeitos dos fármacos , Transcrição Gênica/efeitos dos fármacosRESUMO
Mycenarubinâ C, a previously unknown red pyrroloquinoline alkaloid, was isolated from fruiting bodies of the mushroom Mycena rosea and its structure was elucidated mainly by NMR spectroscopy and mass spectrometry. Unlike mycenarubinâ A, the major pyrroloquinoline alkaloid in fruiting bodies of M.â rosea, mycenarubinâ C, contains an eight-membered ring with an additional C1 unit that is hitherto unprecedented for pyrroloquinoline alkaloids known in nature. Incubation of mycenarubinâ A with an excess of formaldehyde revealed that mycenarubinâ C was generated nearly quantitatively from mycenarubinâ A. An investigation into the formaldehyde content of fresh fruiting bodies of M.â rosea showed the presence of considerable amounts of formaldehyde, with values of 5â µg per gram of fresh weight in fresh fruiting bodies. Although mycenarubinâ C did not show bioactivity against selected bacteria and fungi, formaldehyde inhibits the growth of the mycoparasite Spinellus fusiger at concentrations present in fruiting bodies of M.â rosea. Therefore, formaldehyde might play an ecological role in the chemical defence of M.â rosea against S.â fusiger. In turn, S.â fusiger produces gallic acid-presumably to detoxify formaldehyde by reaction of this aldehyde with amino acids and gallic acid to Mannich adducts.
Assuntos
Agaricales/química , Alcaloides/farmacologia , Formaldeído/farmacologia , Carpóforos/química , Mucorales/efeitos dos fármacos , Pirróis/farmacologia , Quinolinas/farmacologia , Agaricales/imunologia , Agaricales/metabolismo , Alcaloides/biossíntese , Aminoácidos/metabolismo , Antibiose , Formaldeído/metabolismo , Carpóforos/imunologia , Carpóforos/metabolismo , Ácido Gálico/metabolismo , Inativação Metabólica/fisiologia , Espectroscopia de Ressonância Magnética , Estrutura Molecular , Mucorales/metabolismo , Pirróis/metabolismo , Quinolinas/metabolismoRESUMO
Deoxynivalenol (DON) cannot be totally removed due to its stable chemical characteristics and chronic exposure to low doses of DON causes significant toxic effects in humans and animals. However, the potential hazard of such low-dose exposure in target organs still remains not completely understood, especially in liver, which is mainly responsible for detoxification of DON. In the present study, we demonstrated for the first time that estimated human daily DON exposure (25 µg/kg bw) for 30 and 90 days caused low-grade inflammatory infiltration around hepatic centrilobular veins, elevated systemic IL-1ß, IL-6 and TNF-α and impaired liver function evidenced by increased serum ALT activity. At the molecular level, expressions of autophagy-related proteins as well as Cleaved Caspase-3 and Cleaved Caspase-7 were upregulated during DON exposure, which indicated the activation of autophagy and apoptosis. Importantly, AAV-mediated liver-specific overexpression of HO-1 reversed DON-induced liver damages, upregulated autophagy and attenuated apoptosis in liver, while AAV-mediated HO-1 silence aggravated DON-induced liver damages, inhibited autophagy and increased apoptosis. Furthermore, in vitro experiments demonstrated that lentivirus-mediated HO-1 overexpression in Hepa 1-6 cells prolonged the duration of autophagy and delayed the onset of apoptosis. HO-1 silence in Hepa 1-6 cells inhibited activation of autophagy and accelerated occurrence of apoptosis, and these could be recovered by CO pre-treatment. Therefore, we suppose that HO-1 might be a potential research target to protect human and animal from liver injuries induced by low dose of DON exposure.
Assuntos
Doença Hepática Induzida por Substâncias e Drogas/etiologia , Heme Oxigenase-1/metabolismo , Proteínas de Membrana/metabolismo , Tricotecenos/toxicidade , Animais , Apoptose/efeitos dos fármacos , Autofagia/efeitos dos fármacos , Monóxido de Carbono/farmacologia , Linhagem Celular Tumoral , Doença Hepática Induzida por Substâncias e Drogas/metabolismo , Doença Hepática Induzida por Substâncias e Drogas/patologia , Relação Dose-Resposta a Droga , Heme Oxigenase-1/genética , Inativação Metabólica/efeitos dos fármacos , Inativação Metabólica/fisiologia , Testes de Função Hepática , Masculino , Proteínas de Membrana/genética , Camundongos Endogâmicos C57BL , Tricotecenos/administração & dosagemRESUMO
Protein kinase inhibitors (PKI) are a growing class of anticancer agents. They are prescribed with flat doses, and their oral administration is associated with interindividual variability in exposure. Patients can be over- or underexposed, due to numerous factors. We reviewed key pharmacokinetic concepts and mechanisms by which PKIs prescription could be altered. Challenging situations that could lead to increased toxicity or to therapeutic failure are described and recommendation for clinicians are proposed. Finally, the interest of therapeutic drug monitoring and indications for its use in daily practice is discussed.
Assuntos
Padrões de Prática Médica/normas , Inibidores de Proteínas Quinases/farmacocinética , Inibidores de Proteínas Quinases/uso terapêutico , Área Sob a Curva , Biomarcadores Farmacológicos/análise , Relação Dose-Resposta a Droga , Interações Medicamentosas , Monitoramento de Medicamentos/métodos , Humanos , Inativação Metabólica/fisiologia , Farmacologia Clínica , Padrões de Prática Médica/estatística & dados numéricos , Inibidores de Proteínas Quinases/farmacologia , Distribuição TecidualRESUMO
Neonicotinoid insecticides have been widely used throughout the world over the last two decades. In the present study, we investigated the degradation of neonicotinoid insecticides nitenpyram (NIT) and dinotefuran (DIN) by the white-rot fungus Phanerochaete sordida YK-624. While NIT was completely degraded by P. sordida YK-624 under ligninolytic conditions, only a 20% decrease was observed under nonligninolytic conditions. On the other hand, P. sordida YK-624 degraded 31% of DIN under ligninolytic conditions after a 20-day incubation, while it did not degrade DIN under nonligninolytic conditions. We found that cytochromes P450 played a key role in the biotransformation of NIT and DIN by P. sordida YK-624. A novel NIT metabolite (E)-N-((6-chloropyridin-3-yl)methyl)-N-ethyl-N'-hydroxy acetimidamide (CPMHA) and a novel DIN metabolite N-((4aS,7aS,E)-1-methylhexahydrofuro[2,3-d]pyrimidin-2(1H)-ylidene)nitramide (PHPF) were identified in this study. In addition, to evaluate neurotoxicity, the effects of NIT, DIN and their metabolites on the viability of human neuroblastoma cells SH-SY5Y were determined. PHPF showed higher neurological toxicity than DIN, whereas the metabolite of NIT, CPMHA, showed no toxic effect. Our results indicated that the neurological toxicity of NIT could be effectively removed by P. sordida YK-624.
Assuntos
Biodegradação Ambiental , Guanidinas/metabolismo , Inativação Metabólica/fisiologia , Inseticidas/metabolismo , Neonicotinoides/metabolismo , Nitrocompostos/metabolismo , Phanerochaete/metabolismo , Linhagem Celular Tumoral , Sistema Enzimático do Citocromo P-450/metabolismo , Humanos , Neurotoxinas/metabolismoRESUMO
The Eucalyptus cultivation acreage was large in Guangxi provinces. Guanglin 9 (Eucalyptus grandis × Eucalyptus urophylla) is a widely cultivated Eucalyptus species and has been found to grow normally in soils contaminated by heavy metals such as arsenic (As), but the detoxification mechanisms are not clear yet. Through hydroponic experiment, the adsorption and detoxification of As in Eucalyptus were studied from three aspects, namely subcellular distribution of As, chemical forms of As, and the response of sulfhydryl substances. The subcellular distribution data showed that in the Eucalyptus roots, As was mainly present in the soluble fraction (66.3-79.9%), in leaves in the soluble fraction (50.6-53.8%), and the cell wall fraction (35.6-40.0%) under As stress. The chemical form data showed that in roots, As was mainly present in ethanol extraction state (29.5-40.0%), deionized water extraction state (28.3-31.7%), and sodium chloride extraction state (24.1-33.8%). As stress can induce the formation of non-protein thiols (NPT), glutathione (GSH), and phytochelatins (PCs). With the increasing As concentration, the NPT (maximum increase 55.9%) and GSH (maximum increase 79.9%) contents in roots significantly increased, while the PC content significantly increased and then significantly decreased. It is concluded that the As detoxification mechanisms of Eucalyptus are mainly vacuolar compartmentalization and the chelation of sulfhydryl substances, while cell wall adsorption and As chemical forms have limited effects on As detoxification.
Assuntos
Arsênio/toxicidade , Eucalyptus/fisiologia , Poluentes do Solo/toxicidade , Arsênio/análise , Parede Celular/metabolismo , Quelantes/farmacologia , China , Eucalyptus/efeitos dos fármacos , Glutationa/metabolismo , Hidroponia , Inativação Metabólica/fisiologia , Metais Pesados/análise , Fitoquelatinas/metabolismo , Folhas de Planta/metabolismo , Raízes de Plantas/metabolismo , Solo/química , Poluentes do Solo/análise , Compostos de Sulfidrila/metabolismoRESUMO
The bone marrow (BM) microenvironment contributes to drug resistance in acute myeloid leukaemia (AML) and multiple myeloma (MM). We have shown that the critical drug metabolizing enzymes cytochrome P450 (CYP) 3A4 and cytidine deaminase (CDA) are highly expressed by BM stroma, and play an important role in this resistance to chemotherapy. However, what factors influence the chemoprotective capacity of the BM microenvironment, specifically related to CYP3A4 and CDA expression, are unknown. In this study, we found that the presence of AML cells decreases BM stromal expression of CYP3A4 and CDA, and this effect appears to be at least partially the result of cytokines secreted by AML cells. We also observed that stromal CYP3A4 expression is up-regulated by drugs commonly used in AML induction therapy, cytarabine, etoposide and daunorubicin, resulting in cross-resistance. Cytarabine also up-regulated CDA expression. The up-regulation of CYP3A4 associated with disease control was reversed by clarithromycin, a potent inhibitor of CYP3A4. Our data suggest that minimal residual disease states are characterized by high levels of stromal drug metabolizing enzymes and thus, strong microenvironment-mediated drug resistance. These results further suggest a potential role for clinically targeting drug metabolizing enzymes in the microenvironment.
Assuntos
Medula Óssea/metabolismo , Microambiente Tumoral/fisiologia , Células da Medula Óssea/metabolismo , Linhagem Celular , Linhagem Celular Tumoral , Citarabina/metabolismo , Citarabina/uso terapêutico , Citocromo P-450 CYP3A/metabolismo , Daunorrubicina/metabolismo , Daunorrubicina/uso terapêutico , Resistencia a Medicamentos Antineoplásicos/fisiologia , Etoposídeo/metabolismo , Etoposídeo/uso terapêutico , Humanos , Inativação Metabólica/fisiologia , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/metabolismo , Células Estromais/metabolismo , Regulação para Cima/fisiologiaRESUMO
The metabolism of antitumor-active 5-diethylaminoethylamino-8-hydroxyimidazoacridinone (C-1311) has been investigated widely over the last decade but some aspects of molecular mechanisms of its metabolic transformation are still not explained. In the current work, we have reported a direct and rapid analytical tool for better prediction of C-1311 metabolism which is based on electrochemistry (EC) coupled on-line with electrospray ionization mass spectrometry (ESI-MS). Simulation of the oxidative phase I metabolism of the compound was achieved in a simple electrochemical thin-layer cell consisting of three electrodes (ROXY™, Antec Leyden, the Netherlands). We demonstrated that the formation of the products of N-dealkylation reactions can be easily simulated using purely instrumental approach. Newly reported products of oxidative transformations like hydroxylated or oxygenated derivatives become accessible. Structures of the electrochemically generated metabolites were elucidated on the basis of accurate mass ion data and tandem mass spectrometry experiments. In silico prediction of main sites of C-1311 metabolism was performed using MetaSite software. The compound was evaluated for cytochrome P450 1A2-, 3A4-, and 2D6-mediated reactions. The results obtained by EC were also compared and correlated with those of reported earlier for conventional in vitro enzymatic studies in the presence of liver microsomes and in the model peroxidase system. The in vitro experimental approach and the in silico metabolism findings showed a quite good agreement with the data from EC/ESI-MS analysis. Thus, we conclude here that the electrochemical technique provides the promising platform for the simple evaluation of drug metabolism and the reaction mechanism studies, giving first clues to the metabolic transformation of pharmaceuticals in the human body.
Assuntos
Aminoacridinas/metabolismo , Antineoplásicos/metabolismo , Fenômenos Bioquímicos/fisiologia , Simulação por Computador , Sistema Enzimático do Citocromo P-450/metabolismo , Técnicas Eletroquímicas/métodos , Eletroquímica/métodos , Eletrodos , Humanos , Inativação Metabólica/fisiologia , Microssomos Hepáticos/metabolismo , Oxirredução , Espectrometria de Massas por Ionização por Electrospray/métodos , Espectrometria de Massas em Tandem/métodosRESUMO
Physiologically based pharmacokinetic (PBPK) models often include several sets of correlated parameters, such as organ volumes and blood flows. Because of recent advances in proteomics, it has been demonstrated that correlations are also present between abundances of drug-metabolising enzymes in the liver. As the focus of population PBPK has shifted the emphasis from the average individual to theoretically conceivable extremes, reliable estimation of the extreme cases has become paramount. We performed a simulation study to assess the impact of the correlation between the abundances of two enzymes on the pharmacokinetics of drugs that are substrate of both, under assumptions of presence or lack of such correlations. We considered three semi-physiological models representing the cases of: (1) intravenously administered drugs metabolised by two enzymes expressed in the liver; (2) orally administered drugs metabolised by CYP3A4 expressed in the liver and gut wall; (3) intravenously administered drugs that are substrates of CYP3A4 and OATP1B1 in the liver. Finally, the impact of considering or ignoring correlation between enzymatic abundances on global sensitivity analysis (GSA) was investigated using variance based GSA on a reduced PBPK model for repaglinide, substrate of CYP3A4 and CYP2C8. Implementing such correlations can increase the confidence interval for population pharmacokinetic parameters (e.g., AUC, bioavailability) and impact the GSA results. Ignoring these correlations could lead to the generation of implausible parameters combinations and to an incorrect estimation of pharmacokinetic related parameters. Thus, known correlations should always be considered in building population PBPK models.
Assuntos
Preparações Farmacêuticas/metabolismo , Disponibilidade Biológica , Carbamatos/metabolismo , Simulação por Computador , Citocromo P-450 CYP2C8/metabolismo , Citocromo P-450 CYP3A/metabolismo , Humanos , Inativação Metabólica/fisiologia , Fígado/metabolismo , Transportador 1 de Ânion Orgânico Específico do Fígado/metabolismo , Modelos Biológicos , Farmacocinética , Piperidinas/metabolismoRESUMO
The distribution of a drug within the body should be considered as involving movement of unbound drug between the various aqueous spaces of the body. At true steady state, even for a compound of restricted lipoidal permeability, unbound concentrations in all aqueous compartments (blood, extracellular, and intracellular) are considered identical, unless a compartment has a clearance/transport process. In contrast, total drug concentrations may differ greatly, reflecting binding or partitioning into constituents of each compartment. For most highly lipid permeable drugs, this uniform unbound concentration is expected to apply. However, many compounds have restricted lipoidal permeability and are subjected to transport/clearance processes causing a gradient between intracellular and extracellular unbound concentrations even at steady state. Additional concerns arise where the drug target resides in a site of limited vascularity. Many misleading assumptions about drug concentrations and access to drug targets are based on total drug. Correction, if made, is usually by measuring tissue binding, but this is limited by the lack of homogenicity of the organ or compartment. Rather than looking for technology to measure the unbound concentration it may be better to focus on designing high lipoidal permeable molecules with a high chance of achieving a uniform unbound drug concentration. It is hoped this paper will stimulate greater understanding of the path from circulation to cell interior, and thereby in part avoid or minimize the need to provide the experimentally very determining, and sometimes still questionable, answer to this problem.