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Methods for assessing compound identification confidence in metabolomics and related studies have been debated and actively researched for the past two decades. The earliest effort in 2007 focused primarily on mass spectrometry and nuclear magnetic resonance spectroscopy and resulted in four recommended levels of metabolite identification confidence - the Metabolite Standards Initiative (MSI) Levels. In 2014, the original MSI Levels were expanded to five levels (including two sublevels) to facilitate communication of compound identification confidence in high resolution mass spectrometry studies. Further refinement in identification levels have occurred, for example to accommodate use of ion mobility spectrometry in metabolomics workflows, and alternate approaches to communicate compound identification confidence also have been developed based on identification points schema. However, neither qualitative levels of identification confidence nor quantitative scoring systems address the degree of ambiguity in compound identifications in context of the chemical space being considered, are easily automated, or are transferable between analytical platforms. In this perspective, we propose that the metabolomics and related communities consider identification probability as an approach for automated and transferable assessment of compound identification and ambiguity in metabolomics and related studies. Identification probability is defined simply as 1/N, where N is the number of compounds in a reference library or chemical space that match to an experimentally measured molecule within user-defined measurement precision(s), for example mass measurement or retention time accuracy, etc. We demonstrate the utility of identification probability in an in silico analysis of multi-property reference libraries constructed from the Human Metabolome Database and computational property predictions, provide guidance to the community in transparent implementation of the concept, and invite the community to further evaluate this concept in parallel with their current preferred methods for assessing metabolite identification confidence.
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Since his graduate studies on alcohol induction of a novel cytochrome P450 (P450) enzyme, through his postdoctoral work on hormonal regulation of sexually differentiated P450s, the author has maintained an interest in the regulation of P450 (P450) drug metabolizing enzymes. This article is a recounting of his scientific career and focusses on his laboratory's work on inflammatory regulation of P450 enzymes that formed the basis for the Bernard B. Brodie Award. Key findings and publications are identified and discussed that contributed to the elucidation of some important principles: 1) Inflammatory stimuli generally downregulate P450 enzymes, resulting in reduced metabolism of substrate drugs; 2) The main mechanism for this downregulation is transcriptional and involves both the activation of negatively acting transcription factors and the suppression of positive transcription factors; 3) Inflammatory cytokines such as interleukin 1 (IL1), interleukin 6 (IL6) and tumor necrosis factor α (TNFα) act on hepatocytes to mediate this regulation; 4) These cytokines selectively regulate different P450 enzymes, and therefore different P450s may be downregulated in different inflammatory diseases or disease models; 5) Nitric oxide (NO) formed by inducible nitric oxide synthase 2 (NOS2) reacts with P450s in an enzyme-specific manner to stimulate their proteolytic degradation; and 6) Both tyrosine nitration and heme nitrosylation are likely required for this NO-stimulated degradation. Finally, findings from clinical studies are discussed that shine a light on the importance of P450 regulation by inflammation for drug development, clinical practice, and personalized medicine. Significance Statement This article discusses the key publications and findings in the author's lab that helped to identify inflammation as an important factor contributing to interindividual variation in drug metabolism.
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This article was solicited to commemorate the 50th anniversary of Drug Metabolism and Disposition (DMD) and features perspectives from five former editors spanning the years 1994 to 2020. During that time frame the journal underwent significant changes in manuscript submission and processing as well as multiple generational changes in the composition of the editorial board and associate editors. A constant, however, has been the commitment to be the premier journal for publications of articles in the areas of drug metabolism, absorption, distribution, excretion, and pharmacokinetics. Advances in some of those areas during the past 3 decades have been monumental. Two cases in point involve cytochromes P450 and drug transporters. In 1994 rigorous characterization of human cytochrome P450 enzymes was in its infancy, there were no proven selective inhibitors, and the idea of solving a human P450 X-ray crystal structure was just a fantasy. Likewise, little was known about individual drug transporters. Today, detailed knowledge of individual human P450 enzymes and drug transporters is integral in drug design and drug discovery and in avoiding drug interactions. In the face of these huge advances in knowledge, each editor has been charged with maintaining the caliber and significance of the journal and its financial solvency while serving the needs of individual authors. We present 5 individual perspectives on the challenges and rewards of serving as DMD editor and hope that, by humanizing the job, we will encourage others to assume positions of responsibility in publication of society journals. SIGNIFICANCE STATEMENT: The 5 most recent former editors of DMD describe their experiences and perspectives on the position in the context of constantly changing scientific emphases, technology, and publishing practices. The article offers subscribers, authors, and future editors and editorial board members valuable insights into the inner workings of the journal.
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Inativação Metabólica , HumanosRESUMO
Precision medicine and exposomics require methods to assess xenobiotic metabolism in human metabolomic analyses, including the identification of known and undocumented drug and chemical exposures as well as their metabolites. Recent work demonstrated the use of high-throughput generation of xenobiotic metabolites with human liver S-9 fractions for their detection in human plasma and urine. Here, we tested whether a panel of lentivirally transduced human hepatoma cell lines (Huh7) that express individual cytochrome P450 (P450) enzymes could be used to generate P450-specific metabolites in a high-throughput manner, while simultaneously identifying the enzymes responsible. Cell-line activities were verified using P450-specific probe substrates. To increase analytical throughput, we used a pooling strategy where 36 chemicals were grouped into 12 unique mixtures, each mixture containing 6 randomly selected compounds, and each compound being present in two separate mixtures. Each mixture was incubated with 8 different P450 cell lines for 0 and 2 hours and extracts were analyzed using liquid chromatography-high-resolution mass spectrometry. Cell lines selectively metabolized test substrates, e.g., pazopanib, bupropion, and ß-naphthoflavone with expected substrate-enzyme specificities. Predicted metabolites from the remaining 33 compounds as well as many unidentified m/z features were detected. We also showed that a specific bupropion metabolite generated by CYP2B6 cells, but not detected in the S9 system, was identified in human samples. Our data show that the chemical mixtures approach accelerated characterization of xenobiotic chemical space, while simultaneously identifying enzyme sources that can be used for scalable generation of metabolites for their identification in human metabolomic analyses. SIGNIFICANCE STATEMENT: High-resolution mass spectrometry (HRMS) enables the detection of exposures to drugs and other xenobiotics in human samples, but chemical identification can be difficult for several reasons. This paper demonstrates the utility of a panel of engineered cytochrome P450-expressing hepatoma cells in a scalable workflow for production of xenobiotic metabolites, which will facilitate their use as surrogate standards to validate xenobiotic detection by HRMS in human metabolomic studies.
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Carcinoma Hepatocelular , Neoplasias Hepáticas , Bupropiona , Linhagem Celular , Sistema Enzimático do Citocromo P-450/metabolismo , Humanos , XenobióticosRESUMO
Advances in genomics have revealed many of the genetic underpinnings of human disease, but exposomics methods are currently inadequate to obtain a similar level of understanding of environmental contributions to human disease. Exposomics methods are limited by low abundance of xenobiotic metabolites and lack of authentic standards, which precludes identification using solely mass spectrometry-based criteria. Here, we develop and validate a method for enzymatic generation of xenobiotic metabolites for use with high-resolution mass spectrometry (HRMS) for chemical identification. Generated xenobiotic metabolites were used to confirm identities of respective metabolites in mice and human samples based upon accurate mass, retention time and co-occurrence with related xenobiotic metabolites. The results establish a generally applicable enzyme-based identification (EBI) for mass spectrometry identification of xenobiotic metabolites and could complement existing criteria for chemical identification.
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Sistema Enzimático do Citocromo P-450/metabolismo , Espectrometria de Massas/métodos , Microssomos Hepáticos/enzimologia , Xenobióticos/metabolismo , Animais , Sistema Enzimático do Citocromo P-450/genética , Expressão Gênica , Humanos , Isoenzimas/genética , Isoenzimas/metabolismo , Marcação por Isótopo , Fígado/enzimologia , Desintoxicação Metabólica Fase I/genética , Desintoxicação Metabólica Fase II/genética , CamundongosRESUMO
Many hepatic cytochrome P450 enzymes and their associated drug metabolizing activities are down-regulated in disease states, and much of this has been associated with inflammatory cytokines and their signaling pathways. One such pathway is the induction of inducible nitric oxide synthase (NOS2) and generation of nitric oxide (NO) in many tissues and cells including the liver and hepatocytes. Experiments in the 1990s demonstrated that NO could bind to and inhibit P450 enzymes, and suggested that inhibition of NOS could attenuate, and NO generation could mimic, the down-regulation by inflammatory stimuli of not only P450 catalytic activities but also of mRNA expression and protein levels of certain P450 enzymes. This review will summarize and examine the evidence that NO functionally inhibits and down-regulates P450 enzymes in vivo and in vitro, with a particular focus on the mechanisms by which these effects are achieved.
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Sistema Enzimático do Citocromo P-450/genética , Sistema Enzimático do Citocromo P-450/metabolismo , Inflamação/enzimologia , Fígado/enzimologia , Óxido Nítrico/metabolismo , Animais , Inibidores das Enzimas do Citocromo P-450/farmacologia , Sistema Enzimático do Citocromo P-450/efeitos dos fármacos , Regulação da Expressão Gênica , Humanos , Fígado/metabolismo , Transdução de SinaisRESUMO
Human cytochrome P450 (P450) CYP2B6 undergoes nitric oxide (NO)-dependent proteasomal degradation in response to the NO donor dipropylenetriamine NONOate (DPTA) and biologic NO in HeLa and HuH7 cell lines. CYP2B6 is also downregulated by NO in primary human hepatocytes. We hypothesized that NO or derivative reactive nitrogen species may generate adducts of tyrosine and/or cysteine residues, causing CYP2B6 downregulation, and selected Tyr and Cys residues for mutation based on predicted solvent accessibility. CYP2B6V5-Y317A, -Y380A, and -Y190A mutant proteins expressed in HuH7 cells were less sensitive than wild-type (WT) enzyme to degradation evoked by DPTA, suggesting that these tyrosines are targets for NO-dependent downregulation. The Y317A or Y380A mutants did not show increases in high molecular mass (HMM) species after treatment with DPTA or bortezomib + DPTA, in contrast to the WT enzyme. Carbon monoxide-releasing molecule 2 treatment caused rapid suppression of 2B6 enzyme activity, significant HMM species generation, and ubiquitination of CYP2B6 protein but did not stimulate CYP2B6 degradation. The CYP2B6 inhibitor 4-(4-chlorophenyl)imidazole blocked NO-dependent CYP2B6 degradation, suggesting that NO access to the active site is important. Molecular dynamics simulations predicted that tyrosine nitrations of CYP2B6 would cause significant destabilizing perturbations of secondary structure and remove correlated motions likely required for enzyme function. We propose that cumulative nitrations of Y190, Y317, and Y380 by reactive nitrogen species cause destabilization of CYP2B6, which may act synergistically with heme nitrosylation to target the enzyme for degradation. SIGNIFICANCE STATEMENT: This work provides novel insight into the mechanisms by which nitric oxide, which is produced in hepatocytes in response to inflammation, triggers the ubiquitin-dependent proteasomal degradation of the cytochrome P450 (P450) enzyme CYP2B6. Our data demonstrate that both nitration of specific tyrosine residues and interaction of nitric oxide (NO) with the P450 heme are necessary for NO to trigger ubiquitination and protein degradation.
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Citocromo P-450 CYP2B6/química , Citocromo P-450 CYP2B6/metabolismo , Doadores de Óxido Nítrico/farmacologia , Tirosina/química , Linhagem Celular , Citocromo P-450 CYP2B6/genética , Regulação para Baixo , Células HeLa , Hepatócitos/citologia , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Humanos , Cultura Primária de Células , ProteóliseRESUMO
Several cytochrome P450 enzymes are known to be down-regulated by nitric oxide (NO). CYP2A6 is responsible for the metabolism of nicotine and several other xenobiotics, but its susceptibility to down-regulation by NO has not been reported. To address this question, we used Huh7 human hepatoma cell lines to express CYP2A6 with a C-terminal V5 tag (CYP2A6V5). NO donor treatment [dipropylenetriamine NONOate (DPTA)] down-regulated CYP2A6 protein to approximately 40% of control levels in 4 hours. An NO scavenging agent protected CYP2A6 from down-regulation by DPTA in a concentration-dependent manner, demonstrating that the down-regulation is NO-dependent. Experiments with the protein synthesis inhibitor cycloheximide showed that CYP2A6 protein down-regulation occurs posttranslationally. In the presence of proteasome inhibitors MG132 or bortezomib, NO-treated cells showed an accumulation of a high molecular mass signal, whereas autophagy inhibitors chloroquine and 3-methyladenine and the lysosomal and calpain inhibitor E64d had no effect. Immunoprecipitation of CYP2A6 followed by Western blotting with an antiubiquitin antibody showed that the high molecular mass species contain polyubiquitinated CYP2A6 protein. This suggests that NO led to the degradation of protein via the ubiquitin-proteasome pathway. The down-regulation by NO was blocked by the reversible CYP2A6 inhibitor pilocarpine but not by the suicide inhibitor methoxsalen, demonstrating that down-regulation requires NO access to the active site but does not require catalytic activity of the enzyme. These findings provide novel insights toward the regulation of CYP2A6 in a human cell line and can influence our understanding of CYP2A6-related drug metabolism. SIGNIFICANCE STATEMENT: This study demonstrates that the nicotine metabolizing enzyme CYP2A6 is down-regulated by nitric oxide, a molecule produced in large amounts in the context of inflammation and that is also inhaled from cigarette smoke. This occurs via ubiquitination and proteasomal degradation, and does not require catalytic activity of the enzyme. This work adds to the growing knowledge of the selective effect and mechanism of action of nitric oxide (NO) on cytochrome P450 enzymes and suggests a possible novel mode of interaction between nicotine and NO in cigarette smokers.
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Citocromo P-450 CYP2A6/antagonistas & inibidores , Óxido Nítrico/farmacologia , Complexo de Endopeptidases do Proteassoma/metabolismo , Linhagem Celular Tumoral , Fumar Cigarros/metabolismo , Citocromo P-450 CYP2A6/metabolismo , Regulação para Baixo/efeitos dos fármacos , Regulação para Baixo/fisiologia , Humanos , Nicotina/metabolismo , Óxido Nítrico/metabolismo , Inibidores de Proteassoma/farmacologia , Inibidores da Síntese de Proteínas/farmacologia , Proteólise/efeitos dos fármacos , Ubiquitinação/efeitos dos fármacos , Ubiquitinação/fisiologiaRESUMO
BACKGROUND: Given the central importance of anti-malarial drugs in the treatment of malaria, there is a need to understand the effect of Plasmodium infection on the broad spectrum of drug metabolizing enzymes. Previous studies have shown reduced clearance of quinine, a treatment for Plasmodium infection, in individuals with malaria. METHODS: The hepatic expression of a large panel of drug metabolizing enzymes was studied in the livers of mice infected with the AS strain of Plasmodium chabaudi chabaudi, a nonlethal parasite in most strains of mice with several features that model human Plasmodium infections. C57BL/6J mice were infected with P. chabaudi by intraperitoneal injection of infected erythrocytes and sacrificed at different times after infection. Relative hepatic mRNA levels of various drug metabolizing enzymes, cytokines and acute phase proteins were measured by reverse transcriptase-real time PCR. Relative levels of cytochrome P450 proteins were measured by Western blotting with IR-dye labelled antibodies. Pharmacokinetics of 5 prototypic cytochrome P450 substrate drugs were measured by cassette dosing and high-resolution liquid chromatography-mass spectrometry. The results were analysed by MANOVA and post hoc univariate analysis of variance. RESULTS: The great majority of enzyme mRNAs were down-regulated, with the greatest effects occurring at the peak of parasitaemia 8 days post infection. Protein levels of cytochrome P450 enzymes in the Cyp 2b, 2c, 2d, 2e, 3a and 4a subfamilies were also down-regulated. Several distinct groups differing in their temporal patterns of regulation were identified. The cassette dosing study revealed that at the peak of parasitaemia, the clearances of caffeine, bupropion, tolbutamide and midazolam were markedly reduced by 60-70%. CONCLUSIONS: These findings in a model of uncomplicated human malaria suggest that changes in drug clearance in this condition may be of sufficient magnitude to cause significant alterations in exposure and response of anti-malarial drugs and co-medications.
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Antimaláricos/farmacocinética , Sistema Enzimático do Citocromo P-450/metabolismo , Regulação para Baixo , Fígado/enzimologia , Malária/parasitologia , Plasmodium chabaudi/fisiologia , Proteínas de Fase Aguda/metabolismo , Animais , Citocinas/metabolismo , Eritrócitos/parasitologia , Feminino , Inativação Metabólica , Camundongos , Camundongos Endogâmicos C57BL , RNA Mensageiro/metabolismoRESUMO
Nitric oxide (NO) is an essential signaling molecule in the body, regulating numerous biological processes. Beside its physiological roles, NO affects drug metabolism by modulating the activity and/or expression of cytochrome P450 enzymes. Previously, our lab showed that NO generation caused by inflammatory stimuli results in CYP2B6 degradation via the ubiquitin-proteasome pathway. In the current study, we tested the NO-mediated regulation of CYP2J2 that metabolizes arachidonic acids to bioactive epoxyeicosatrienoic acids, as well as therapeutic drugs such as astemizole and ebastine. To investigate the effects of NO on CYP2J2 expression and activity, Huh7 cells stably transduced with CYP2J2 with a C-terminal V5 tag were treated with dipropylenetriamine-NONOate (DPTA), a NO donor. The level of CYP2J2 proteins were decreased in a time- and concentration-dependent manner, and the activity was also rapidly inhibited. However, mRNA expression was not altered and the protein synthesis inhibitor cycloheximide did not attenuate DPTA-mediated downregulation of CYP2J2. Removal of DPTA from the culture media quickly restored the activity of remaining CYP2J2, and no further CYP2J2 degradation occurred. To determine the mechanism of CYP2J2 down-regulation by NO, cells were treated with DPTA in the presence or absence of protease inhibitors including proteasomal, lysosomal and calpain inhibitors. Remarkably, the down-regulation of CYP2J2 by NO was attenuated by calpeptin, a calpain inhibitor. However, other calpain inhibitors or calcium chelator show no inhibitory effects on the degradation. The proteasome inhibitor bortezomib showed small but significant restoration of CYP2J2 levels although stimulated ubiquitination of CYP2J2 was not detected. In conclusion, these data suggest that NO regulates CYP2J2 posttranslationally and NO-evoked CYP2J2 degradation undergoes ubiquitin-independent proteasomal degradation pathway unlike CYP2B6.
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Carcinoma Hepatocelular/patologia , Sistema Enzimático do Citocromo P-450/metabolismo , Regulação da Expressão Gênica , Neoplasias Hepáticas/patologia , Óxido Nítrico/farmacologia , Processamento de Proteína Pós-Traducional , Antineoplásicos/farmacologia , Bortezomib/farmacologia , Carcinoma Hepatocelular/tratamento farmacológico , Carcinoma Hepatocelular/metabolismo , Citocromo P-450 CYP2J2 , Sistema Enzimático do Citocromo P-450/genética , Regulação para Baixo , Sequestradores de Radicais Livres/farmacologia , Humanos , Neoplasias Hepáticas/tratamento farmacológico , Neoplasias Hepáticas/metabolismo , Doadores de Óxido Nítrico/farmacologia , Complexo de Endopeptidases do Proteassoma/efeitos dos fármacos , Células Tumorais CultivadasRESUMO
This article is a report on a symposium entitled "Physiological Regulation of Drug Metabolism and Transport" sponsored by the American Society for Pharmacology and Experimental Therapeutics and held at the Experimental Biology 2017 meeting in Chicago, IL. The contributions of physiologic and pathophysiological regulation of drug-metabolizing enzymes and transporters to interindividual variability in drug metabolism are increasingly recognized but in many cases are not well understood. The presentations herein discuss the phenomenology, consequences, and mechanism of such regulation. CYP2D6 transgenic mice were used to provide insights into the mechanism of regulation of this enzyme in pregnancy, via hepatocyte nuclear factor 4α, small heterodimer partner, and retinoids. Regulation of intestinal and hepatic drug-processing enzymes by the intestinal microbiota via tryptophan and its metabolites was investigated. The potential impact of parasitic infections on human drug metabolism and clearance was assessed in mice infected with Schistosoma mansoni or Plasmodium chabaudi chabaudi AS, both of which produced widespread and profound effects on murine hepatic drug-metabolizing enzymes. Finally, the induction of Abcc drug efflux transporters by fasting was investigated. This was demonstrated to occur via a cAMP, protein kinase A/nuclear factor-E2-related factor 2/Sirtuin 1 pathway via antioxidant response elements on the Abcc genes.
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Transporte Biológico/fisiologia , Jejum/fisiologia , Inativação Metabólica/fisiologia , Inflamação/fisiopatologia , Microbiota/fisiologia , Animais , Elementos de Resposta Antioxidante/fisiologia , Citocromo P-450 CYP2D6/metabolismo , Jejum/metabolismo , Feminino , Microbioma Gastrointestinal/fisiologia , Fator 4 Nuclear de Hepatócito/metabolismo , Humanos , Inflamação/metabolismo , Fígado/metabolismo , Malária/metabolismo , Malária/fisiopatologia , Masculino , Proteínas de Membrana Transportadoras/metabolismo , Taxa de Depuração Metabólica/fisiologia , Camundongos , Camundongos Transgênicos , Plasmodium chabaudi/patogenicidade , Gravidez , Esquistossomose mansoni/metabolismo , Esquistossomose mansoni/fisiopatologia , Triptofano/metabolismoRESUMO
Nitric oxide (NO) is known to down-regulate drug-metabolizing cytochrome P450 enzymes in an enzyme-selective manner. Ubiquitin-proteasome-dependent and -independent pathways have been reported. Here, we studied the regulation of expression of human CYP51A1, the lanosterol 14α-demethylase required for synthesis of cholesterol and other sterols in mammals, which is found in every kingdom of life. In Huh7 human hepatoma cells, treatment with NO donors caused rapid post-translational down-regulation of CYP51A1 protein. Human NO synthase (NOS)-dependent down-regulation was also observed in cultured human hepatocytes treated with a cytokine mixture and in Huh7 cells expressing human NOS2 under control of a doxycycline-regulated promoter. This down-regulation was partially attenuated by proteasome inhibitors, but only trace levels of ubiquitination could be found. Further studies with inhibitors of other proteolytic pathways suggest a possible role for calpains, especially when the proteasome is inhibited. NO donors also down-regulated CYP51A1 mRNA in Huh7 cells, but to a lesser degree, than the down-regulation of the protein.
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Sequência Conservada , Lanosterol/metabolismo , Óxido Nítrico/farmacologia , Proteólise/efeitos dos fármacos , Esterol 14-Desmetilase/metabolismo , Calpaína/metabolismo , Linhagem Celular Tumoral , Regulação para Baixo/efeitos dos fármacos , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Humanos , Doadores de Óxido Nítrico/farmacologia , Inibidores de Proteassoma/farmacologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Esterol 14-Desmetilase/genética , Ubiquitinação/efeitos dos fármacosRESUMO
We showed previously that rat cytochrome P450 CYP2B1 undergoes NO-dependent proteasomal degradation in response to inflammatory stimuli, and that the related human enzyme CYP2B6 is also down-regulated by NO in primary human hepatocytes. To investigate the mechanism of CYP2B6 down-regulation, we made several cell lines (HeLa and HuH7 cells) in which native CYP2B6 or CYP2B6 with a C-terminal V5 tag (CYP2B6V5) are expressed from a lentiviral vector with a cytomegalovirus promoter. Native CYP2B6 protein was rapidly down-regulated in HeLa cells within 3h of treatment with the NO donor (Z)-1-[2-(2-Aminoethyl)-N-(2-ammonioethyl)amino]diazen-1-ium-1,2-diolate, while its mRNA level was not down-regulated. Treatment of the cells with the NO donor (Z)-1-[N-(3-aminopropyl)-N-(3-ammoniopropyl)amino]diazen-1-ium-1,2-diolate also resulted in rapid down-regulation of CYP2B6 activity, measured as the formation of 7-hydroxy-4-trifluoromethylcoumarin, as well as 2B6 protein in the CYP2B6 HeLa cell line. CYP2B6V5 was also down-regulated by NO donors in HuH7 cells. Down-regulation was observed in the presence of cycloheximide, demonstrating that this occurs via a post-translational mechanism. We generated a HeLa cell line expressing both CYP2B6V5 and human nitric oxide synthase 2 (NOS2), the latter under positive control by tetracycline. The cellular NO produced by doxycycline treatment also effectively down-regulated CYP2B6 protein, which was blocked by the co-treatment with the NOS2 competitive inhibitor L-NG-nitroarginine methyl ester (L-NAME). We next investigated the proteolytic enzymes responsible for NO-dependent CYP2B6 degradation. Neither calpain inhibitors (N-Acetyl-L-leucyl-L-leucyl-L-norleucinal, carbobenzoxy-valinyl-phenylalaninal), nor lysosomal protease inhibitors (3-methyladenine and chloroquine) inhibited the NO dependent CYP2B6V5 down-regulation. The proteasome inhibitors MG132 and bortezomib attenuated, but did not completely block the NO-induced down-regulation in the HuH7 cell line. However, when cells were co-treated with NO donor and proteasome inhibitors, high molecular mass species could be detected on native CYP2B6 as well as CYP2B6V5 Western blots. Further investigation demonstrated that CYP2B6 protein was polyubiquitinated and this was dramatically enhanced by co-treatment with NO donor and bortezomib. Taken together, our data demonstrate that CYP2B6 is down-regulated in an NO-dependent manner via ubiquitination and proteasomal degradation.
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Citocromo P-450 CYP2B6/metabolismo , Hepatócitos/fisiologia , Óxido Nítrico/metabolismo , Proteólise/efeitos dos fármacos , Adenina/análogos & derivados , Adenina/metabolismo , Animais , Bortezomib/farmacologia , Cumarínicos/metabolismo , Citocromo P-450 CYP2B6/genética , Regulação para Baixo , Células HeLa , Humanos , NG-Nitroarginina Metil Éster/farmacologia , Doadores de Óxido Nítrico/farmacologia , Óxido Nítrico Sintase Tipo II/antagonistas & inibidores , Óxido Nítrico Sintase Tipo II/genética , Óxido Nítrico Sintase Tipo II/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Ratos , Ubiquitina/metabolismo , UbiquitinaçãoRESUMO
Variations in drug metabolism may alter drug efficacy and cause toxicity; better understanding of the mechanisms and risks shall help to practice precision medicine. At the 21st International Symposium on Microsomes and Drug Oxidations held in Davis, California, USA, in October 2-6, 2016, a number of speakers reported some new findings and ongoing studies on the regulation mechanisms behind variable drug metabolism and toxicity, and discussed potential implications to personalized medications. A considerably insightful overview was provided on genetic and epigenetic regulation of gene expression involved in drug absorption, distribution, metabolism, and excretion (ADME) and drug response. Altered drug metabolism and disposition as well as molecular mechanisms among diseased and special populations were presented. In addition, the roles of gut microbiota in drug metabolism and toxicology as well as long non-coding RNAs in liver functions and diseases were discussed. These findings may offer new insights into improved understanding of ADME regulatory mechanisms and advance drug metabolism research.
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UNLABELLED: Beyond the well-defined role of the Eph (erythropoietin-producing hepatocellular) receptor tyrosine kinases in developmental processes, cell motility, cell trafficking/adhesion, and cancer, nothing is known about their involvement in liver pathologies. During blood-stage rodent malaria infection we have found that EphB2 transcripts and proteins were up-regulated in the liver, a result likely driven by elevated surface expression on immune cells including macrophages. This was significant for malaria pathogenesis because EphB2(-/-) mice were protected from malaria-induced liver fibrosis despite having a similar liver parasite burden compared with littermate control mice. This protection was correlated with a defect in the inflammatory potential of hepatocytes from EphB2(-/-) mice resulting in a reduction in adhesion molecules, chemokine/chemokine receptor RNA levels, and infiltration of leukocytes including macrophages/Kupffer cells, which mediate liver fibrosis during rodent malaria infections. These observations are recapitulated in the well-established carbon tetrachloride model of liver fibrosis in which EphB2(-/-) carbon tetrachloride-treated mice showed a significant reduction of liver fibrosis compared to carbon tetrachloride-treated littermate mice. Depletion of macrophages by clodronate-liposomes abrogates liver EphB2 messenger RNA and protein up-regulation and fibrosis in malaria-infected mice. CONCLUSION: During rodent malaria, EphB2 expression promotes malaria-associated liver fibrosis; to our knowledge, our data are the first to implicate the EphB family of receptor tyrosine kinases in liver fibrosis or in the pathogenesis of malaria infection.
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Movimento Celular/imunologia , Hepatócitos/enzimologia , Cirrose Hepática/enzimologia , Cirrose Hepática/patologia , Receptor EphB2/metabolismo , Animais , Movimento Celular/fisiologia , Sobrevivência Celular , Células Cultivadas , Modelos Animais de Doenças , Progressão da Doença , Feminino , Hepatócitos/metabolismo , Células de Kupffer/metabolismo , Macrófagos/metabolismo , Malária/patologia , Malária/fisiopatologia , Camundongos , Camundongos Endogâmicos C57BL , Receptores de Quimiocinas/metabolismo , Regulação para CimaRESUMO
We studied the impact of administering XPro1595, a novel antagonist of soluble tumor necrosis factor-α (TNFα), on the regulation of hepatic cytochrome P450 enzymes in the C. rodentium model of infectious colitis. XPro1595 was administered subcutaneously every three days throughout the infection, or as a single injection near the peak of infection. When given throughout the infection, XPro1595 selectively blocked the down-regulation of Cyp3a11 and 3a25 mRNAs, as well as the induction of Cyp2a4/5, without affecting the down-regulation of Cyp4a10, Cyp4a14, Cyp2b10 or flavin-mooxygenase-3. Induction of Cyp3a11, Cyp3a25, Cyp2c29 and Cyp3a13 mRNAs were observed only in XPro1595-treated mice. Administration of a single dose of XPro1595 was relatively ineffective. These results a) confirm the role of soluble TNFα in hepatic Cyp3a regulation during infectious colitis deduced from studies in TNFα receptor-1 knockout mice; b) indicate the potential for soluble TNFα-specific antagonists to cause disease-dependent drug-drug interactions; and, c) suggest a novel mechanism by which an anti-inflammatory therapeutic protein can produce an opposite effect to that of the disease by selectively neutralizing one of multiple signals regulating drug-metabolizing enzyme expression. More research is needed to determine whether or not this is applicable to other diseases or disease models.
RESUMO
Various disease models have been shown to alter hepatic drug-metabolizing enzyme (DME) and transporter expression and to induce cholestasis through altered enzyme and transporter expression. Previously, we detailed the regulation of hepatic DMEs during infectious colitis caused by Citrobacter rodentium infection. We hypothesized that this infection would also modulate hepatic drug transporter expression and key genes of bile acid (BA) synthesis and transport. Mice lacking Toll-like receptor 4 (TLR4), interleukin-6 (IL-6), or interferon-gamma (IFNγ) and appropriate wild-type animals were orally infected with C. rodentium and sacrificed 7 days later. In two wild-type strains, drug transporter mRNA expression was significantly decreased by infection for Slc22a4, Slco1a1, Slco1a4, Slco2b1, and Abcc6, whereas the downregulation of Abcc2, Abcc3, and Abcc4 were strain-dependent. In contrast, mRNA expressions of Slco3a1 and Abcb1b were increased in a strain-dependent manner. Expression of Abcb11, Slc10a1, the two major hepatic BA transporters, and Cyp7a1, the rate-limiting enzyme of BA synthesis, was also significantly decreased in infected animals. None of the above effects were caused by bacterial lipopolysaccharide, since they still occurred in the absence of functional TLR4. The downregulation of Slc22a4 and Cyp7a1 was absent in IFNγ-null mice, and the downregulation of Slco1a1 was abrogated in IL-6-null mice, indicating in vivo roles for these cytokines in transporter regulation. These data indicate that C. rodentium infection modulates hepatic drug processing through alteration of transporter expression as well as DMEs. Furthermore, this infection downregulates important genes of BA synthesis and transport and may increase the risk for cholestasis.
Assuntos
Ácidos e Sais Biliares/metabolismo , Proteínas de Transporte/genética , Colite/metabolismo , Citocinas/imunologia , Infecções por Enterobacteriaceae/metabolismo , Fígado/metabolismo , Animais , Citrobacter/patogenicidade , Colite/imunologia , Colite/microbiologia , Infecções por Enterobacteriaceae/imunologia , Infecções por Enterobacteriaceae/microbiologia , Feminino , Regulação da Expressão Gênica , Homeostase , Fígado/imunologia , Camundongos , Camundongos Endogâmicos C3H , Camundongos Endogâmicos C57BLRESUMO
Murine hepatic Cyp4a mRNAs are markedly downregulated during inflammation. Here, we investigated the roles of Cyp4a10 and Cyp4a14 in the response to infection with C. rodentium. Absence of either Cyp4a gene attenuated or abrogated the changes in spleen weight, colon crypt length, hepatic cytokine, and acute phase protein mRNAs, and serum acute phase proteins and cytokines caused by infection. Cyp4a10(-/-) mice on a low-salt diet had a similar hepatic acute phase response as those mice on a high-salt diet, suggesting that hypertension associated with this genotype is not the cause of their altered inflammatory response. In contrast, wild-type, Cyp4a10(-/-), and Cyp4a14(-/-) mice showed similar responses to injected LPS. These results implicate Cyp4a10 and Cyp4a14 in the regulation of the host inflammatory response to enteropathogenic bacterial infection but not to acute aseptic inflammation. Understanding the mechanism of this role may lead to novel therapeutic approaches in some inflammatory diseases.
Assuntos
Citrobacter rodentium/imunologia , Sistema Enzimático do Citocromo P-450/genética , Infecções por Enterobacteriaceae/imunologia , Inflamação/imunologia , Animais , Família 4 do Citocromo P450 , Citocinas/biossíntese , Citocinas/sangue , Feminino , Hipertensão/imunologia , Inflamação/microbiologia , Lipopolissacarídeos/imunologia , Masculino , Camundongos , Camundongos Knockout , RNA Mensageiro/genética , Sódio na Dieta/farmacologiaRESUMO
Inflammation and infection downregulate the activity and expression of cytochrome P450s (P450s) and other drug metabolizing enzymes (DMEs) involved in hepatic drug clearance. Schistosoma mansoni infection was reported to cause a downregulation of hepatic P450-dependent activities in mouse liver, but little is known about the specific enzymes affected or whether phase II DMEs are also affected. Here we describe the effect of murine schistosomiasis on the expression of hepatic P450s, NADPH-cytochrome P450 reductase (Cpr), phase II drug metabolizing enzymes, and nuclear receptors at 30 and 45 days postinfection (dpi). Although the hepatic expression of some of these genes was altered at 30 dpi, we observed substantial changes in the expression of the majority of P450 mRNAs and proteins measured, Cpr protein, as well as many of the UDP-glucuronosyltransferases and sulfotransferases at 45 dpi. S. mansoni infection also altered nuclear receptor expression, inducing mRNA levels at 30 dpi and depressing levels at 45 dpi. S. mansoni evoked a T helper 2 (Th2) inflammatory response at 45 dpi, as indicated by the induction of hepatic Th2 cytokine mRNAs [interleukins 4, 5, and 13], whereas the hepatic proinflammatory response was relatively weak. Thus, chronic schistosomiasis markedly and selectively alters the expression of multiple DMEs, which may be associated with Th2 cytokine release. This would represent a novel mechanism of DME regulation in disease states. These findings have important implications for drug testing in infected mice, whereas the relevance to humans with schistosomiasis needs to be determined.