RESUMO
Vitamin D deficiency is common in patients with chronic liver diseases. However, vitamin D status in persons with chronic hepatitis B virus (HBV) infection is not consistently reported. Specifically, the impact of liver dysfunction on vitamin D status has not been well addressed.We recruited a group of patients (n = 345) with chronic hepatitis B (n = 115), hepatitis B related cirrhosis (n = 115), and age- and gender-matched healthy controls (n = 115). Serum 25-hydroxyvitamin D3 [25(OH)D3], its related metabolic enzymes, intact parathyroid hormone were measured. Calcium, magnesium, and phosphorus were obtained from medical record.Serum 25(OH)D3 levels in chronic hepatitis B patients (7.83â±â3.47âng/mL) were significantly lower than that in healthy controls (9.76â±â4.36âng/mL, Pâ<â0.001), but significantly higher than that in hepatitis B-related cirrhotic patients (5.21â±â3.67âng/mL, Pâ<â0.001). Furthermore, 25(OH)D3 decreased stepwise with higher Child-Pugh classification. However, there were no significant differences in 25(OH)D3 levels between (1) hepatitis B e antigen (HBeAg +) and HBeAg(-) persons, or (2) among persons with different HBV viral load, or (3) between treatment naïve and patients on antiviral therapy. Multiple logistic regression analyses confirmed that higher Child-Pugh score was independently associated with 25(OH)D3 deficiency (<10âng/mL) with an odds ratio of 1.20 (confidence interval 1.03-1.39, P = 0.016). Levels of cytochrome P450 (CYP) 27A1 were significantly decreased, whereas levels of CYP24A1 were significantly elevated in cirrhotic patients.These results suggest that decreasing vitamin D levels are likely to be a result, rather than a cause, of liver dysfunction and irrespective of HBV viral load. Reduction in 25(OH)D3 levels is possibly due to downregulation of the synthetic hydroxylase CYP27A1 and concurrent upregulation of degrading CYP24A1 in patients with liver cirrhosis.
Assuntos
Hepatite B Crônica/sangue , Hepatite B Crônica/virologia , Carga Viral , Vitamina D/sangue , Estudos Transversais , Feminino , Hepatite B Crônica/complicações , Hepatite B Crônica/enzimologia , Humanos , Cirrose Hepática/sangue , Cirrose Hepática/enzimologia , Cirrose Hepática/etiologia , Masculino , Pessoa de Meia-IdadeRESUMO
Valuing faculty as educators is essential for medical schools to fulfill their unique mission of educating physicians. The 2006 Consensus Conference on Educational Scholarship, sponsored by the Association of American Medical Colleges (AAMC) Group on Educational Affairs, provided educators seeking academic promotion with a portfolio-based format for documenting activities in five domains, using evidence of quantity, quality, a scholarly approach, and educational scholarship. Yet, the lack of a rigorous, widely accepted system to assess educator portfolio submissions during the promotion and tenure process continues to impede the ability to fully value educators and educational scholars.The AAMC Task Force on Educator Evaluation was formed in 2010 to establish consensus guidelines for use by those responsible for the rigorous evaluation of the educational contributions of faculty. The task force delineated the educational contributions currently valued by institutions and then fulfilled its charge by creating the Toolbox for Evaluating Educators, a resource which contains explicit evidence-based criteria to evaluate faculty in each of the five domains of educator activity. Adoption of such criteria is now the rate-limiting step in using a fair process to recognize educators through academic promotion. To inform institutional review and implementation of these criteria, this article describes the iterative, evidence- and stakeholder-based process to establish the criteria. The authors advocate institutional adoption of these criteria so that faculty seeking academic promotion as educators, like their researcher colleagues, can be judged and valued using established standards for the assessment of their work.
Assuntos
Mobilidade Ocupacional , Docentes de Medicina/normas , Faculdades de Medicina , Guias como Assunto , HumanosRESUMO
Inflammation is a major factor shaping outcome during the early, acute phase of traumatic spinal cord injury (SCI). It is known that pro-inflammatory signaling within the injured spinal cord drives pathological alterations in neurosensory processing and shapes functional outcome early after injury. However, it is unclear whether inflammation persists into the chronic phase of injury or shapes sensory processing long after injury. To investigate these possibilities, we have performed biochemical and behavioral assessments 9 months after moderate thoracic spinal contusion injury in the rat. We have found that levels of the pro-inflammatory lipid mediators leukotriene B4 and prostaglandin E2 are elevated in the chronic spinal cord lesion site. Additionally, using metabolomic profiling, we have detected elevated levels of pro-oxidative and inflammatory metabolites, along with alterations in multiple biological pathways within the chronic lesion site. We found that 28 d treatment of chronically injured rats with the dual COX/5-LOX inhibitor licofelone elevated levels of endogenous anti-oxidant and anti-inflammatory metabolites within the lesion site. Furthermore, licofelone treatment reduced hypersensitivity of hindpaws to mechanical, but not thermal, stimulation, indicating that mechanical sensitivity is modulated by pro-inflammatory signaling in the chronic phase of injury. Together, these findings provide novel evidence of inflammation and oxidative stress within spinal cord tissue far into the chronic phase of SCI, and demonstrate a role for inflammatory modulation of mechanical sensitivity in the chronic phase of injury.
Assuntos
Inibidores de Ciclo-Oxigenase/farmacologia , Hiperalgesia/tratamento farmacológico , Inflamação/tratamento farmacológico , Pirróis/farmacologia , Traumatismos da Medula Espinal/tratamento farmacológico , Animais , Anti-Inflamatórios não Esteroides/uso terapêutico , Cromatografia Líquida de Alta Pressão , Dinoprostona/metabolismo , Ensaio de Imunoadsorção Enzimática , Feminino , Cromatografia Gasosa-Espectrometria de Massas , Membro Posterior/fisiologia , Temperatura Alta , Hiperalgesia/etiologia , Hiperalgesia/fisiopatologia , Inflamação/fisiopatologia , Leucotrieno B4/metabolismo , Locomoção/efeitos dos fármacos , Metabolômica , Estresse Oxidativo/fisiologia , Estimulação Física , Ratos , Ratos Sprague-Dawley , Traumatismos da Medula Espinal/complicações , Traumatismos da Medula Espinal/fisiopatologiaRESUMO
INTRODUCTION: Cytochromes P450 (P450) and associated monooxygenases are a family of heme proteins involved in metabolism of endogenous compounds (arachidonic acid, eicosanoids and prostaglandins) as also xenobiotics including drugs and environmental chemicals. Liver is the major organ involved in P450-mediated metabolism and hepatic enzymes have been characterized. Extrahepatic organs, such as lung, kidney and brain have the capability for biotransformation through P450 enzymes. Brain, including human brain, expresses P450 enzymes that metabolize xenobiotics and endogenous compounds. AREAS COVERED: An overview of P450-mediated metabolism in brain is presented focusing on distinct differences seen in expression of P450 enzymes, generation of unique P450 enzymes in brain through alternate splicing and their consequences in terms of metabolism of psychoactive drugs and inflammatory prompts, such as leukotrienes, thus modulating inflammatory response. EXPERT OPINION: The brain possesses unique P450s that metabolize drugs and endogenous compounds through pathways that are markedly different from that seen in liver indicating that extrapolation directly from liver to brain is not appropriate. It is therefore necessary to characterize the unique brain P450s and their ability to metabolize xenobiotics and endogenous compounds to better understand the functions of this important class of enzymes in brain, especially human brain.
Assuntos
Encéfalo/enzimologia , Sistema Enzimático do Citocromo P-450/metabolismo , Processamento Alternativo , Ácido Araquidônico/metabolismo , Biotransformação , Eicosanoides/metabolismo , Expressão Gênica , Humanos , Fígado/enzimologia , Oxigenases de Função Mista/metabolismo , Prostaglandinas/metabolismo , Xenobióticos/metabolismoRESUMO
Although the mechanisms that regulate CYP4F genes have been and are currently being studied in a number of laboratories, the specific mechanisms for the regulation of these genes are not yet fully understood. This study shows that nuclear factor κB of the light-chain-enhancer in activated B cells (NF-κB) can inhibit CYP4F11 expression in human liver carcinoma cell line (HepG2) as summarized below. Tumor necrosis factor-α (TNF-α), a proinflammatory cytokine, has been shown to activate NF-κB signaling while also activating the c-Jun NH(2)-terminal kinase (JNK) signaling pathway. Other studies have reported that JNK signaling can up-regulate CYP4F11 expression. The results of this study demonstrate that in the presence of TNF-α and the specific NF-κB translocation inhibitor N-[3,5-bis(trifluoromethyl)phenyl]-5-chloro-2-hydroxybenzamide (IMD-0354), there is a greater increase in CYP4F11 expression than that elicited by TNF-α alone, indicating that NF-κB plays an inhibitory role. Moreover, NF-κB stimulation by overexpression of mitogen-activated protein kinase kinase kinase inhibited CYP4F11 promoter expression. CYP4F11 promoter inhibition can also be rescued in the presence of TNF-α when p65, a NF-κB protein, is knocked down. Thus, NF-κB signaling pathways negatively regulate the CYP4F11 gene.
Assuntos
Sistema Enzimático do Citocromo P-450/metabolismo , NF-kappa B/fisiologia , Sequência de Bases , Sítios de Ligação/fisiologia , Sistema Enzimático do Citocromo P-450/fisiologia , Família 4 do Citocromo P450 , Células Hep G2 , Humanos , Dados de Sequência Molecular , Transdução de Sinais/fisiologiaRESUMO
Inflammatory processes are involved in the pathogenesis and/or progression of acute central nervous system (CNS) infection, traumatic brain injury and neurodegenerative disorders among others indicating the need for novel strategies to limit neuroinflammation. Eicosanoids including leukotrienes, particularly leukotriene B(4) (LTB(4)) are principle mediator(s) of inflammatory response, initiating and amplifying the generation of cytokines and chemokines. Cytochrome P450 (Cyp), a family of heme proteins mediate metabolism of xenobiotics and endogenous compounds, such as eicosanoids and leukotrienes. Cytochrome P4504F (Cyp4f) subfamily includes five functional enzymes in mouse. We cloned and expressed the mouse Cyp4f enzymes, assayed their relative expression in brain and examined their ability to hydroxylate the inflammatory cascade prompt LTB(4) to its inactive 20-hydroxylated product. We then examined the role of Cyp4fs in regulating inflammatory response in vitro, in microglial cells and in vivo, in mouse brain using lipopolysacharide (LPS), as a model compound to generate inflammatory response. We demonstrate that mouse brain Cyp4fs are expressed ubiquitously in several cell types in the brain, including neurons and microglia, and modulate inflammatory response triggered by LPS, in vivo and in microglial cells, in vitro through metabolism of LTB(4) to the inactive 20-hydroxy LTB(4). Chemical inhibitor or shRNA to Cyp4fs enhance and inducer of Cyp4fs attenuates inflammatory response. Further, induction of Cyp4f expression lowers LTB(4) levels and affords neuroprotection in microglial cells or mice exposed to LPS. Thus, catalytic activity of Cyp4fs is a novel target for modulating neuroinflammation through hydroxylation of LTB(4).
Assuntos
Encéfalo/enzimologia , Inibidores das Enzimas do Citocromo P-450 , Neurite (Inflamação)/enzimologia , Animais , Encéfalo/efeitos dos fármacos , Encéfalo/patologia , Células Cultivadas , Clonagem Molecular , Sistema Enzimático do Citocromo P-450/efeitos dos fármacos , Sistema Enzimático do Citocromo P-450/genética , Sistema Enzimático do Citocromo P-450/metabolismo , Família 4 do Citocromo P450 , Modelos Animais de Doenças , Inibidores Enzimáticos/farmacologia , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Lipopolissacarídeos/farmacologia , Linfotoxina-beta/genética , Linfotoxina-beta/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Microglia/efeitos dos fármacos , Microglia/enzimologia , Microglia/patologia , Neurite (Inflamação)/tratamento farmacológico , Neurite (Inflamação)/patologia , Fármacos Neuroprotetores/farmacologia , RNA Mensageiro/metabolismoRESUMO
Mechanisms regulating CYP4F genes remain under investigation, although characterization of CYP4F regulatory modalities would facilitate the discovery of new drug targets. This present study shows that all-trans- and 9-cis-retinoic acids can inhibit CYP4F11 expression in human keratinocyte-derived HaCaT cells. Transrepression of many genes by retinoic acids is mediated by interactions between retinoid receptors and the activator protein 1 (AP-1) complex. Proinflammatory cytokines tumor necrosis factor alpha (TNF-alpha) and interleukin 1beta, which can activate the AP-1 complex, induce CYP4F11 transcription in HaCaT cells. The c-Jun N-terminal kinase (JNK)-specific inhibitor 1,9-pyrazoloanthrone (SP600125) blocked the induction of CYP4F11 by both cytokines, indicating involvement of the JNK pathway. Furthermore, TNF-alpha failed to induce CYP4F11 transcription when HaCaT cells were preincubated with retinoic acids. Retinoic acids are ligands for the retinoic acid receptors (RARs) and the retinoid X receptors (RXRs). The RXR agonist 6-(1(3,5,5,8,8-pentamethyl-5,6,7,8-tetrahydronaphthalen-2-yl)cyclopropyl) nicotinic acid (LG268) greatly induced CYP4F11 transcription, whereas the RAR agonist 4-(2-(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)-1-propenyl)benzoic acid (TTNPB) markedly inhibited CYP4F11 transcription, indicating that down-regulation of CYP4F11 transcription by retinoic acid is mediated by RARs and may also be related to ligand competition for RXRs. Thus, the CYP4F11 gene is positively regulated by multiple signaling pathways in HaCaT keratinocytes, including RXR and JNK signaling pathways.
Assuntos
Sistema Enzimático do Citocromo P-450/genética , Regulação da Expressão Gênica/genética , Queratinócitos/metabolismo , Alitretinoína , Antracenos/farmacologia , Benzoatos/farmacologia , Linhagem Celular , Sistema Enzimático do Citocromo P-450/metabolismo , Família 4 do Citocromo P450 , Expressão Gênica/efeitos dos fármacos , Expressão Gênica/genética , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Interleucina-1alfa/farmacologia , Proteínas Quinases JNK Ativadas por Mitógeno/antagonistas & inibidores , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Queratinócitos/efeitos dos fármacos , Ácidos Nicotínicos/farmacologia , Fosforilação/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Receptores do Ácido Retinoico/agonistas , Receptores do Ácido Retinoico/genética , Receptores do Ácido Retinoico/metabolismo , Receptor alfa de Ácido Retinoico , Receptor X Retinoide alfa/agonistas , Receptor X Retinoide alfa/genética , Receptor X Retinoide alfa/metabolismo , Receptor X Retinoide beta/agonistas , Receptor X Retinoide beta/genética , Receptor X Retinoide beta/metabolismo , Retinoides/farmacologia , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Tetra-Hidronaftalenos/farmacologia , Tretinoína/farmacologia , Fator de Necrose Tumoral alfa/farmacologia , Receptor gama de Ácido RetinoicoRESUMO
Metabolic inactivation of leukotriene B4 (LTB4) is an innate mechanism to resolve tissue inflammation. We studied the nine Cyp4f genes in the mouse genome, measuring cutaneous transcript levels by real-time polymerase chain reaction, and LTB4 metabolism in mouse and human skin. Transcripts arising from Cyp4f13 and 4f16 ranked most abundant, Cyp4f14, 4f17, and 4f37 ranked least abundant, and Cyp4f18 and 4f39 ranked intermediate. Those from Cyp4f15 and Cyp4f40 were highly variable or too low to measure in some animals. Retinoic acid exposure induced microsomal LTB4 hydroxylation activities in mouse and human skin cells. Two NADPH-dependent LTB4 metabolites eluted identically with 20-OH and 20-COOH LTB4 reference standards. Collision induced dissociation of the precursor ion m/z 351 confirmed that LTB4 products from CYP4F3A and human epidermal keratinocytes are identical structurally to 20-OH LTB4. We conclude 20-hydroxylation is the major CYP-dependent LTB4 inactivation pathway in skin; this retinoid-inducible metabolic pathway has capacity to modulate tissue levels of pro-inflammatory lipids.
Assuntos
Citocromos/metabolismo , Leucotrieno B4/antagonistas & inibidores , Retinoides/farmacologia , Pele/metabolismo , Animais , Cromatografia Líquida de Alta Pressão , Humanos , Hidroxilação , Leucotrieno B4/metabolismo , Espectrometria de Massas , Camundongos , Reação em Cadeia da Polimerase , Pele/citologiaRESUMO
We reported previously that infection of C3H/HeOuJ (HeOu) mice with the murine intestinal pathogen Citrobacter rodentium caused a selective modulation of hepatic cytochrome P450 (P450) gene expression in the liver that was independent of the Toll-like receptor 4. However, HeOu mice are much more sensitive to the pathogenic effects of C. rodentium infection, and the P450 down-regulation was associated with significant morbidity in the animals. Here, we report that oral infection of C57BL/6 mice with C. rodentium, which produced only mild clinical signs and symptoms, produced very similar effects on hepatic P450 expression in this strain. As in HeOu mice, CYP4A mRNAs and proteins were among the most sensitive to down-regulation, whereas CYP4F18 was induced. CYP2D9 mRNA was also induced 8- to 9-fold in the C57BL/6 mice. The time course of P450 regulation followed that of colonic inflammation and bacterial colonization, peaking at 7 to 10 days after infection and returning to normal at 15 to 24 days as the infection resolved. These changes also correlated with the time course of significant elevations in the serum of the proinflammatory cytokines interleukin (IL)-6 and tumor necrosis factor-alpha, as well as of interferon-gamma and IL-2, with serum levels of IL-6 being markedly higher than those of the other cytokines. Intraperitoneal administration of C. rodentium produced a rapid down-regulation of P450 enzymes that was quantitatively and qualitatively different from that of oral infection, although CYP2D9 was induced in both models, suggesting that the effects of oral infection on the liver are not due to bacterial translocation.
Assuntos
Citrobacter rodentium , Sistema Enzimático do Citocromo P-450/metabolismo , Infecções por Enterobacteriaceae/enzimologia , Regulação Enzimológica da Expressão Gênica , Microssomos Hepáticos/enzimologia , Sepse/metabolismo , Animais , Fenômenos Biológicos , Sistema Enzimático do Citocromo P-450/genética , Modelos Animais de Doenças , Infecções por Enterobacteriaceae/genética , Infecções por Enterobacteriaceae/metabolismo , Feminino , Mucosa Intestinal/metabolismo , Intestinos/microbiologia , Intestinos/patologia , Fígado/enzimologia , Fígado/metabolismo , Fígado/microbiologia , Camundongos , Camundongos Endogâmicos BALB C , Microssomos Hepáticos/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Sepse/enzimologiaRESUMO
Cytochrome P450 (CYP) 4Fs constitute a subgroup of the cytochrome P450 superfamily and are involved in cellular protection and metabolism of numerous molecules, including drugs, toxins, and eicosanoids. CYP4Fs are widely distributed in rat brain with each isoform having a unique distribution pattern throughout different brain regions. The present study shows that traumatic brain injury (TBI) triggers inflammation and elicits changes in mRNA expression of CYP4Fs in the frontal and occipital lobes and the hippocampus. At 24 h post-injury, almost all CYP4F mRNA expression is suppressed compared with sham control throughout these three regions, while at 2 weeks post-injury, all CYP4F mRNAs increase, reaching levels higher than those at 24 h post-injury or uninjured controls. These changes in CYP4F levels inversely correlate with levels of leukotriene B4 (LTB4) levels in the brain following injury at the same time points. TBI also causes changes in CYP4F protein expression and localization around the injury site. CYP4F1 and CYP4F6 immunoreactivity increases in surrounding astrocytes, while CYP4F4 immunoreactivity shifts from endothelia of cerebral vessels to astrocytes.
Assuntos
Lesões Encefálicas/metabolismo , Encéfalo/metabolismo , Sistema Enzimático do Citocromo P-450/genética , Leucotrieno B4/metabolismo , Animais , Encéfalo/patologia , Encéfalo/fisiopatologia , Lesões Encefálicas/patologia , Lesões Encefálicas/fisiopatologia , Sistema Enzimático do Citocromo P-450/metabolismo , Família 4 do Citocromo P450 , Regulação para Baixo/genética , Encefalite/metabolismo , Encefalite/patologia , Encefalite/fisiopatologia , Regulação da Expressão Gênica/fisiologia , Isoenzimas/genética , Isoenzimas/metabolismo , Masculino , RNA Mensageiro/metabolismo , Ratos , Ratos Sprague-Dawley , Fatores de Tempo , Regulação para Cima/genéticaRESUMO
Cytochrome P450 (P450) is a super-family of drug metabolizing enzymes. P450 enzymes have dual function; they can metabolize drugs to pharmacologically inactive metabolites facilitating their excretion or biotransform them to pharmacologically active metabolites which may have longer half-life than the parent drug. The variable pharmacological response to psychoactive drugs typically seen in population groups is often not accountable by considering dissimilarities in hepatic metabolism. Metabolism in brain specific nuclei may play a role in pharmacological modulation of drugs acting on the CNS and help explain some of the diverse response to these drugs seen in patient population. P450 enzymes are also present in brain where drug metabolism can take place and modify therapeutic action of drugs at the site of action. We have earlier demonstrated an intrinsic difference in the biotransformation of alprazolam (ALP) in brain and liver, relatively more alpha-hydroxy alprazolam (alpha-OHALP) is formed in brain as compared to liver. In the present study we show that recombinant CYP3A43 metabolizes ALP to both alpha-OHALP and 4-hydroxy alprazolam (4-OHALP) while CYP3A4 metabolizes ALP predominantly to its inactive metabolite, 4-OHALP. The expression of CYP3A43 mRNA in human brain samples correlates with formation of relatively higher levels of alpha-OH ALP indicating that individuals who express higher levels of CYP3A43 in the brain would generate larger amounts of alpha-OHALP. Further, the expression of CYP3A43 was relatively higher in brain as compared to liver across different ethnic populations. Since CYP3A enzymes play a prominent role in the metabolism of drugs, the higher expression of CYP3A43 would generate metabolite profile of drugs differentially in human brain and thus impact the pharmacodynamics of psychoactive drugs at the site of action.
Assuntos
Alprazolam/farmacocinética , Ansiolíticos/farmacocinética , Hidrocarboneto de Aril Hidroxilases/metabolismo , Encéfalo/enzimologia , Hidrocarboneto de Aril Hidroxilases/genética , Humanos , Hibridização in Situ Fluorescente , RNA Mensageiro/genética , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Leukotrienes are implicated in the pathogenesis of diverse, inflammation-driven diseases. Metabolic inactivation of leukotriene signaling is an innate response to resolve inflammation, yet little is known of mechanisms regulating disposition of leukotrienes in peripheral tissues afflicted in common inflammatory diseases. We studied leukotriene hydroxylases (CYP4F gene products) in human skin, a common target of inflammation and adverse drug reactions. Epidermal keratinocytes express at least six CYP4F enzymes; the most highly expressed and highly regulated is CYP4F3A-the main neutrophil leukotriene hydroxylase. Differentiation-specific factors and retinoids are positive CYP4F regulators in vitro, effecting increased leukotriene B4 hydroxylation (inactivation). CYP4F expression is up-regulated in situ in hyperproliferative dermatoses-an innate mechanism to repair and restore epidermal barrier competency-and after retinoid therapy. Enhanced CYP4F-mediated inactivation of leukotriene signaling is a previously unrecognized antiinflammatory property of therapeutic retinoids mediated by preferential interactions between retinoid X receptors and CYP4F promoter elements in epidermal cells.
Assuntos
Leucotrienos/metabolismo , Receptores X de Retinoides/metabolismo , Transdução de Sinais , Diferenciação Celular/efeitos dos fármacos , Células Cultivadas , Sistema Enzimático do Citocromo P-450/genética , Sistema Enzimático do Citocromo P-450/metabolismo , Células Epidérmicas , Epiderme/metabolismo , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Humanos , Inflamação/genética , Inflamação/metabolismo , Inflamação/patologia , Tretinoína/farmacologia , Regulação para Cima/efeitos dos fármacosRESUMO
We investigated expression of the CYP4F subfamily in human leukocytes by flow cytometry using anti-CYP4F3A antibody and quantitative reverse transcription-polymerase chain reaction (QRT-PCR). More than 90% of CD11b, CD13, CD14, CD33, and eosinophil marker-positive cells expressed CYP4F3A. mRNA for CYP4F3A was found in neutrophils, monocytes, and eosinophils. CYP4F12 mRNA was detected in eosinophils and neutrophils. In eosinophils, transcription of the CYP4F12 gene was started from two sites at 49 and 85 nucleotides upstream from the 3' end of exon I. Recombinant CYP4F12 expressed in yeast cell microsomes catalyzed the omega-hydroxylation of leukotriene B4 (LTB4) and 6-trans-LTB4. In contrast, the CYP4F12 did not show any activity toward eicosanoids such as lipoxin A4 and 12-HETE, which are substrates for CYP4F3A, indicating that the physiological roles of CYP4F3A and CYP4F12 in eosinophils are different.
Assuntos
Sistema Enzimático do Citocromo P-450/metabolismo , Eosinófilos/enzimologia , Regulação Enzimológica da Expressão Gênica , Isoenzimas/metabolismo , Região 5'-Flanqueadora , Antígenos CD/metabolismo , Sequência de Bases , Biomarcadores/metabolismo , Sistema Enzimático do Citocromo P-450/genética , Família 4 do Citocromo P450 , Humanos , Isoenzimas/genética , Leucotrieno B4/química , Leucotrieno B4/metabolismo , Dados de Sequência Molecular , Alinhamento de SequênciaRESUMO
CYP1A1, a cytochrome P450 enzyme, metabolizes polycyclic aromatic hydrocarbons to genotoxic metabolite(s) that bind to DNA and initiate carcinogenesis. RT-PCR amplification of the complete open reading frame of CYP1A1 generated an amplicon of 1593 bp having deletion of 87 bp of exon-6 that translated into functional P450 enzyme. Unlike wild type CYP1A1, exon 6 del CYP1A1 did not metabolize polycyclic aromatic hydrocarbons such as, benzo(a)pyrene to genotoxic, ultimate carcinogens that form DNA adducts. Exon 6 del CYP1A1 metabolized ethoxyresorufin (the classical substrate for CYP1A1) less efficiently compared with wild type CYP1A1 while pentoxy and benzyloxyresorufin (classical substrates for CYP2B) were dealkylated more efficiently. In silico docking showed alteration of the substrate access channel in exon 6 del CYP1A1 such that benzo(a)pyrene does not bind in any orientation that would permit the formation of carcinogenic metabolites. Genotyping revealed that the splice variant was not generated due to differences in genomic DNA sequence and the variant was present only in brain but not in liver, kidney, lung, or heart from the same individual. We provide evidence that unique P450 enzymes, generated by alternate splicing in a histiospecific manner can modify genotoxic potential of carcinogens such as benzo(a)pyrene by altering their biotransformation pathway.
Assuntos
Processamento Alternativo/genética , Encéfalo/enzimologia , Citocromo P-450 CYP1A1/genética , DNA/metabolismo , Hidrocarbonetos Policíclicos Aromáticos/metabolismo , Polimorfismo Genético/genética , Adolescente , Adulto , Idoso , Benzo(a)pireno/metabolismo , Biotransformação/genética , Carcinógenos/metabolismo , Criança , DNA/genética , Adutos de DNA/metabolismo , Dano ao DNA/genética , Feminino , Humanos , Inativação Metabólica/genética , Masculino , Pessoa de Meia-Idade , Mutação/genética , Oxazinas/metabolismoRESUMO
Cytochrome P450 (CYP) 4F mediated leukotriene B(4) (LTB(4)) metabolism modulates inflammation during injury and infection. Here we show that in addition to LTB(4), the recombinant rat CYP4Fs catalyze omega-hydroxylations of lipoxin A(4), and hydroxyeicosatetraeonic acids. CYP4F gene regulation studies in primary hepatocytes reveal that pro-inflammatory cytokines interleukin (IL) -1beta, IL-6 and tumor necrosis factor (TNF) -alpha produce a general inductive response whereas IL-10, an anti-inflammatory cytokine, suppresses CYP4F expression. The molecular mechanism behind IL-6 related induction of CYP4F4 and 4F5 is partially signal transducer and activator of transcription 3 (STAT3) dependent. When hepatocytes are subjected to high concentrations of LTB(4) or prostaglandin E(2), lipid mediators of inflammation, only an increase in CYP4F5 mRNA expression is observed. Collectively, the results from isozyme activity and substrate driven CYP4F induction do not support the notion that an autoregulatory pathway could control the excessive concentrations of LTB(4) during an inflammatory challenge to hepatocytes.
Assuntos
Domínio Catalítico , Sistema Enzimático do Citocromo P-450/metabolismo , Citocinas/fisiologia , Hepatócitos/enzimologia , Animais , Células Cultivadas , Inibidores das Enzimas do Citocromo P-450 , Sistema Enzimático do Citocromo P-450/biossíntese , Família 4 do Citocromo P450 , Regulação para Baixo/genética , Indução Enzimática/genética , Hepatócitos/patologia , Mediadores da Inflamação/fisiologia , Interleucina-6/genética , Isoenzimas/biossíntese , Isoenzimas/genética , Isoenzimas/metabolismo , Leucotrieno B4/antagonistas & inibidores , Leucotrieno B4/metabolismo , Masculino , Ratos , Ratos Endogâmicos F344 , Fator de Transcrição STAT3/fisiologia , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/genética , Transdução de Sinais/genética , Regulação para Cima/genéticaRESUMO
With the release of the chimpanzee genomic database, much work has been accomplished to understand more fully the closest related species to humans. This study investigates the cytochrome P450 3A (CYP3A) subfamily and examines differences which may be expected between chimpanzees and humans in regards to CYP3A metabolism. A previous publication had reported the presence of five putative chimpanzee CYP3A isoforms, as compared to the four in humans (Williams ET et al., Mol Phylogenet Evol 33, 300-8). Based on the previous report, the chimpanzee CYP3A5 should have had a different C-terminus than its human counterpart; therefore, CYP3A5 and CYP3A67 were cloned. The CYP3A5 clone obtained disputes the previous prediction and confirms that the nucleotide similarity between the two species is 99.7%. While CYP3A67 is most closely related to CYP3A7, with significant differences in the amino acid sequences. Also, the mRNA expression of CYP3A67 can rival the expression of CYP3A4 in the tissues analyzed. CYP3A7 was not found to be expressed in any chimpanzee tissue examined. Total CYP3A protein expression was not significantly different between chimpanzees and humans. Metabolism assays using benzphetamine and erythromycin with chimpanzee liver microsomes did not reveal major differences between chimpanzees and humans. In conclusion, adult CYP3A metabolism may not be significantly different between chimpanzees and humans.
RESUMO
The CYP3As are broad-spectrum drug-metabolizing enzymes that are collectively responsible for more than 50% of xenobiotic metabolism. Unlike other CYP3As, murine CYP3A44 is expressed predominantly in the female liver, with much lower levels in male livers and no detectable expression in brain or kidney in either gender. In this study, we examined the role of nuclear hormone receptors in the regulation of Cyp3a44 gene expression. Interestingly, we observed differential effects of pregnane X receptor (PXR) and constitutive androstane receptor (CAR) -mediated activation of Cyp3a44 gene expression, which was gender-specific. For example, activation of PXR by pregnenolone-16alpha-carbonitrile (PCN) and dexamethasone (DEX) induced CYP3A44 mRNA levels in a PXR-dependent fashion in male mice, whereas no induction was detected in female mice. In contrast, PCN and DEX down-regulated CYP3A44 expression in female PXR null animals. Similar to PXR, CAR activation also showed a male-specific induction with no effect on CYP3A44 levels in females. When PXR knockout mice were challenged with the CAR activator phenobarbital, a significant up-regulation of male CYP3A44 levels was observed, whereas levels in females remained unchanged. We conclude that gender has a critical impact on PXR- and CAR-mediated effects of CYP3A44 expression.
Assuntos
Sistema Enzimático do Citocromo P-450/metabolismo , Receptores Citoplasmáticos e Nucleares/metabolismo , Receptores de Esteroides/metabolismo , Fatores de Transcrição/metabolismo , Animais , Receptor Constitutivo de Androstano , Citocromo P-450 CYP3A , Sistema Enzimático do Citocromo P-450/genética , Feminino , Fígado/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Receptor de Pregnano X , RNA Mensageiro/metabolismo , Receptores Citoplasmáticos e Nucleares/deficiência , Receptores Citoplasmáticos e Nucleares/genética , Receptores de Esteroides/deficiência , Receptores de Esteroides/genética , Fatores Sexuais , Testosterona/metabolismo , Fatores de Transcrição/deficiência , Fatores de Transcrição/genéticaRESUMO
Traumatic brain injury is known to cause several secondary effects, which lead to multiple organ dysfunction syndrome. An acute systemic inflammatory response seems to play an integral role in the development of such complications providing the potential for massive secondary injury. We show that a contusion injury to the rat brain causes large migration of inflammatory cells (especially macrophages and neutrophils) in the major airways and alveolar spaces at 24 h post-injury, which is associated with enhanced pulmonary leukotriene B4 (LTB4) production within the lung. However, by 2 weeks after injury, a temporal switch occurs and the resolution of inflammation is underway. We provide evidence that 5-lipoxygenase and Cytochrome P450 4Fs (CYP4Fs), the respective enzymes responsible for LTB4 synthesis and breakdown, play crucial roles in setting the cellular concentration of LTB4. Activation of LTB4 breakdown via induction of CYP4Fs, predominantly in the lung tissue, serves as an endogenous signal to ameliorate further secondary damage. In addition, we show that CYP4Fs are localized primarily in the airways and pulmonary endothelium. Given the fact that adherence to the microvascular endothelium is an initial step in neutrophil diapedesis, the temporally regulated LTB4 clearance in the endothelium presents a novel focus for treatment of pulmonary inflammation after injury.
Assuntos
Lesões Encefálicas/complicações , Lesões Encefálicas/enzimologia , Sistema Enzimático do Citocromo P-450/fisiologia , Pneumopatias/enzimologia , Pneumopatias/etiologia , Pneumonia/enzimologia , Pneumonia/etiologia , Acidentes de Trânsito , Animais , Barreira Hematoencefálica/fisiologia , Western Blotting , Lesões Encefálicas/patologia , Líquido da Lavagem Broncoalveolar/citologia , Feminino , Humanos , Técnicas Imunoenzimáticas , Imuno-Histoquímica , Leucotrieno B4/análise , Leucotrieno B4/metabolismo , Pulmão/metabolismo , Pulmão/patologia , Pneumopatias/patologia , Masculino , Microscopia Confocal , Pneumonia/patologia , Proteínas/química , Proteínas/metabolismo , RNA/biossíntese , RNA/genética , Ratos , Ratos Long-Evans , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
The cytochrome P450 4F (CYP4F) subfamily has over the last few years come to be recognized for its dual role in modulating the concentrations of eicosanoids during inflammation as well as in the metabolism of clinically significant drugs. The first CYP4F was identified because it catalyzed the hydroxylation of leukotriene B(4) (LTB(4)) and since then many additional members of this subfamily have been documented for their distinct catalytic roles and functional significance. Recent evidence emerging in relation to the temporal change of CYP4F expression in response to injury and infection supports an important function for these isozymes in curtailing inflammation. Their tissue-dependent expression, isoform-based catalytic competence and unique response to the external stimuli imply a critical role for them to regulate organ-specific functions. From this standpoint variations in relative CYP4F levels in humans may have direct influence on the metabolic outcome through their ability to generate and/or degrade bioactive eicosanoids or therapeutic agents. This review covers the enzymatic characteristics and regulatory properties of human and rodent CYP4F isoforms and their physiological relevance to major pathways in eicosanoid and drug metabolism.
Assuntos
Sistema Enzimático do Citocromo P-450/metabolismo , Eicosanoides/metabolismo , Preparações Farmacêuticas/metabolismo , Animais , Sistema Enzimático do Citocromo P-450/genética , Regulação Enzimológica da Expressão Gênica/genética , Regulação Enzimológica da Expressão Gênica/fisiologia , Humanos , Rim/enzimologia , Leucotrienos/metabolismo , Fígado/enzimologia , Camundongos , Prostaglandinas/metabolismo , RatosRESUMO
The human cytochrome P450 (CYP) superfamily of enzymes regulate hepatic phase 1 drug metabolism and subsequently play a significant role in pharmacokinetics, drug discovery and drug development. Alternative splicing of the cytochrome CYP gene transcripts enhances gene diversity and may play a role in transcriptional regulation of certain CYP proteins. Tissue-specific alternative splicing of CYPs is significant for its potential to add greater dimension to differential drug metabolism in hepatic and extrahepatic tissues, such as the brain, and to our understanding of the CYP family. This review provides an overview of tissue-specific splicing patterns, splicing types, regulation and the functional diversities between liver and splice variant CYP proteins and further explores the relevance of tissue-specific alternative splicing of CYPs in the nervous system.