RESUMO
The impact of transport proteins in the disposition of chemicals is becoming increasingly evident. Alteration in disposition can cause altered pharmacokinetic and pharmacodynamic parameters, potentially leading to reduced efficacy or overt toxicity. We have developed a quantitative in silico model, based upon literature and experimentally derived data, to model the disposition of carboxydichlorofluroscein (CDF), a substrate for the SLCO1A/B and ABCC subfamilies of transporters. Kinetic parameters generated by the in silico model closely match both literature and experimentally derived kinetic values, allowing this model to be used for the examination of transporter action in primary rat hepatocytes. In particular, we show that the in silico model is suited to the rapid, accurate determination of K(i) values, using 3-[[3-[2-(7-chloroquinolin-2-yl)vinyl]phenyl]-(2-dimethylcarbamoylethylsulfanyl)methylsulfanyl] propionic acid (MK571) as a prototypical pan-ABCC inhibitor. In vitro-derived data are often used to predict in vivo response, and we have examined how differences in protein expression levels between these systems may affect chemical disposition. We show that ABCC2 and ABCC3 are overexpressed in sandwich culture hepatocytes by 3.5- and 2.3-fold, respectively, at the protein level. Correction for this in markedly different disposition of CDF, with the area under the concentration versus time curve and C(max) of intracellular CDF increasing by 365 and 160%, respectively. Finally, using kinetic simulations we show that ABCC2 represents a fragile node within this pathway, with alterations in ABCC2 having the most prominent effects on both the K(m) and V(max) through the pathway. This is the first demonstration of the utility of modeling approaches to estimate the impact of drug transport processes on chemical disposition.
Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Transporte Biológico/fisiologia , Fluoresceínas/metabolismo , Hepatócitos/metabolismo , Transportadores de Cassetes de Ligação de ATP/genética , Animais , Transporte Biológico/efeitos dos fármacos , Células Cultivadas , Fluoresceínas/análise , Expressão Gênica , Taxa de Depuração Metabólica , Modelos Biológicos , Preparações Farmacêuticas/metabolismo , Fenômenos Fisiológicos , RatosRESUMO
The mood stabilizing agents lithium chloride (LiCl) and sodium valproate (VPA) have recently gained interest as potential neuroprotective therapeutics. However, exploitation of these therapeutic applications is hindered by both a lack of molecular understanding of the mode of action, and a number of sub-optimal properties, including a relatively small therapeutic window and variable patient response. Human neuroblastoma cells (SH-SY5Y) were exposed to 1 mM lithium chloride or 1 mM sodium valproate for 6 h or 72 h, and transcriptomes measured by Affymetrix U133A/B microarray. Statistically significant gene expression changes were identified using SAM software, with selected changes confirmed at transcript (TaqMan) and protein (Western blotting) levels. Finally, anti-apoptotic action was measured by an in vitro fluorescent assay. Exposure of SH-SY5Y cells to therapeutically relevant concentrations of either lithium chloride or sodium valproate elicited 936 statistically significant changes in gene expression. Amongst these changes we observed a large (maximal 31.3-fold) increase in the expression of the homeodomain protein Six1, and have characterized the time- and dose-dependent up-regulation of this gene in response to both drugs. In addition, we demonstrate that, like LiCl or VPA treatment, Six1 over-expression protects SH-SY5Y cells from staurosporine-induced apoptosis via the blockade of caspsase-3 activation, whereas removal of Six1 protein via siRNA antagonises the ability of LiCl and VPA to protect SH-SY5Y cells from STS-induced apoptosis. These results provide a novel mechanistic rationale underlying the neuroprotective mechanism of LiCl and VPA, suggesting exciting possibilities for the development of novel therapeutic agents against neurodegenerative diseases such as Alzheimer's or Parkinsonism.
Assuntos
Antimaníacos/farmacologia , Proteínas de Homeodomínio/metabolismo , Cloreto de Lítio/farmacologia , Regulação para Cima , Ácido Valproico/farmacologia , Antimaníacos/metabolismo , Apoptose/efeitos dos fármacos , Apoptose/fisiologia , Linhagem Celular Tumoral , Perfilação da Expressão Gênica , Inativação Gênica , Proteínas de Homeodomínio/genética , Humanos , Cloreto de Lítio/metabolismo , Neuroblastoma/metabolismo , Estaurosporina/farmacologia , Ácido Valproico/metabolismoRESUMO
CYP3A4 is the most abundant cytochrome P450 in human liver, comprising approximately 30% of the total liver P450 content. This enzyme has an important role in endogenous processes, most notably steroid catabolism, and also plays a fundamental role in the metabolism of more than half of the clinically used drugs currently prescribed. The majority of CYP3A substrates are also capable of upregulating CYP3A activity, mainly through transcriptional activation. The molecular mechanisms that underlie the transcriptional activation of CYP3A4 are complex, with many steroid hormone nuclear receptors, including GR, PXR, VDR and CAR, playing a role in these mechanisms. However, the net result of transcriptional activation is an increase in the metabolism of the inducing compounds and, therefore, increased clearance. An important side effect of this transcriptional activation is that co-administered chemicals metabolized by CYP3A may also have their pharmacokinetics altered. Such changes can result in reduced clinical efficacy of drugs, resulting in poor patient response, or the development of an adverse drug response. This review will examine examples of established interactions caused through transcriptional activation of CYP3A4, and speculate on whether such effects are clinically important and should be considered during the design of treatment regimes or, alternatively, are relatively minor and cause little physiological effects.
Assuntos
Marcadores Genéticos , Farmacogenética/métodos , Animais , Células Cultivadas , Doença Hepática Induzida por Substâncias e Drogas/genética , Biologia Computacional , Expressão Gênica , Cardiopatias/induzido quimicamente , Cardiopatias/genética , Hepatócitos/efeitos dos fármacos , Humanos , Nefropatias/induzido quimicamente , Nefropatias/genética , Camundongos , Degeneração Neural/induzido quimicamente , Degeneração Neural/genética , Ratos , Reprodução/efeitos dos fármacosRESUMO
DNA microarrays and related tools offer promise for identification of pathways involved in toxic responses to xenobiotics. To be useful for risk assessment, experimental data must be challenged for reliability and interlaboratory reproducibility. Toward this goal, the Hepatotoxicity Working Group of the International Life Sciences Institute (ILSI) Health and Environmental Sciences Institute (HESI) Technical Committee on Application of Genomics to Mechanism-Based Risk Assessment evaluated and compared biological and gene expression responses in rats exposed to two model hepatotoxins--clofibrate and methapyrilene. This collaborative effort provided an unprecedented opportunity for the working group to evaluate and compare multiple biological, genomic, and toxicological parameters across different laboratories and microarray platforms. Many of the results from this collaboration are presented in accompanying articles in this mini-monograph, whereas others have been published previously. (Italic)In vivo(/Italic) studies for both compounds were conducted in two laboratories using a standard experimental protocol, and RNA samples were distributed to 16 laboratories for analysis on six microarray platforms. Histopathology, clinical chemistry, and organ weight changes were consistent with reported effects. Gene expression results demonstrated reasonable agreement between laboratories and across platforms. Discrepancies in expression profiles of some individual genes were largely due to platform differences and approaches to data analysis rather than to biological or interlaboratory variability. Despite these discrepancies there was overall agreement in the biological pathways affected by these compounds, demonstrating that transcriptional profiling is reproducible between laboratories and can reliably identify affected pathways necessary to provide mechanistic insight. This effort represents an important first step toward the use of transcriptional profiling in risk assessment.
Assuntos
Perfilação da Expressão Gênica , Fígado/efeitos dos fármacos , Fígado/patologia , Análise de Sequência com Séries de Oligonucleotídeos , Animais , Antialérgicos/toxicidade , Clofibrato/toxicidade , Coleta de Dados , Hipolipemiantes/toxicidade , Masculino , Metapirileno/toxicidade , Variações Dependentes do Observador , Ratos , Ratos Sprague-Dawley , Reprodutibilidade dos Testes , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Medição de RiscoRESUMO
A CYP3A4 promoter-reporter gene construct has been used to assess the ability of 16 known (in vivo) and putative (in vitro) inducers to transactivate a CYP3A4 reporter gene in HepG2 cells. With the exception of pravastatin, the remaining 15 compounds transactivated the CYP3A4 reporter gene with differing inductive abilities (I(max):EC(50)) over two orders of magnitude, ranging from 1.1 (phenytoin) to 222.9 (lovastatin) in a receptor-supplemented system and it is proposed that the lack of response to pravastatin is due to loss of the known hepatic uptake transporter in HepG2 cells. In addition, reporter gene assays were used to investigate two promoter mutants namely a T to C change at -191 bp in the hepatic nuclear factor 3 binding site (HNF-3, -187 to -194 bp) and an A to G change at -205 bp in the oestrogen response element (ERE, -202 to -212 bp), which conferred differential responsiveness to steroid and xenobiotic inducers.
Assuntos
Sistema Enzimático do Citocromo P-450/genética , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Células Cultivadas , Citocromo P-450 CYP3A , Indução Enzimática/efeitos dos fármacos , Estradiol/farmacologia , Genes Reporter/genética , Humanos , Hidrocortisona/farmacologia , Mutação/genética , Receptor de Pregnano X , Receptores Citoplasmáticos e Nucleares/genética , Receptores Citoplasmáticos e Nucleares/fisiologia , Receptores de Glucocorticoides/genética , Receptores de Glucocorticoides/fisiologia , Receptores de Esteroides/genética , Receptores de Esteroides/fisiologia , TransfecçãoRESUMO
The nuclear receptor superfamily of ligand-activated transcription factors plays a central role in the regulation of cellular responses to chemical challenge. Nuclear receptors are activated by a wide range of both endogenous and exogenous chemicals, and their target genes include those involved in the metabolism and transport of the activating chemical. Such target gene activation, thus, acts to remove the stimulating xenobiotic or to maintain homeostatic levels of endogenous chemicals. Given the dual nature of this system it is important to understand how these two roles are balanced, such that xenobiotics are efficiently removed while not impacting negatively on homeostasis of endogenous chemicals. Using DNA microarray technology we have examined the transcriptome response of primary rat hepatocytes to two nuclear receptor ligands: Pregnenalone-16α-carbonitrile (PCN), a xenobiotic PXR agonist, and lithocholic acid, an endogenous mixed PXR/VDR/FXR agonist. We demonstrate that despite differences in the profile of activated nuclear receptors, transcriptome responses for these two ligands are broadly similar at lower concentrations, indicating a conserved general response. However, as concentrations of stimulating ligand rises, the transcriptome responses diverge, reflecting a need for specific responses to the two stimulating chemicals. Finally, we demonstrate a novel feed-back loop for PXR, whereby ligand-activation of PXR suppresses transcription of the PXR gene, acting to attenuate PXR protein expression levels at higher ligand concentrations. Through in silico simulation we demonstrate that this feed-back loop is an important factor to prevent hyperexpression of PXR target genes such as CYP3A and confirm these findings in vitro. This novel insight into the regulation of the PXR-mediated regulatory signal networks provides a potential mechanistic rationale for the robustness in steroid homeostasis within the cell.
Assuntos
Citocromo P-450 CYP3A/genética , Retroalimentação Fisiológica , Carbonitrila de Pregnenolona/farmacologia , Receptores de Esteroides/fisiologia , Animais , Células Cultivadas , Análise por Conglomerados , Citocromo P-450 CYP3A/metabolismo , Relação Dose-Resposta a Droga , Regulação para Baixo/efeitos dos fármacos , Retroalimentação Fisiológica/efeitos dos fármacos , Retroalimentação Fisiológica/fisiologia , Perfilação da Expressão Gênica , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Hepatócitos/efeitos dos fármacos , Hepatócitos/enzimologia , Hepatócitos/metabolismo , Hepatócitos/fisiologia , Análise em Microsséries , Modelos Biológicos , Receptor de Pregnano X , Ratos , Ratos Sprague-Dawley , Receptores de Esteroides/agonistas , Receptores de Esteroides/metabolismo , Xenobióticos/farmacologiaRESUMO
This review addresses the general structure and function of nuclear receptors and places specific emphasis on their role in xenosensing, resulting in the activation of a battery of genes mediating drug metabolism, conjugation, and transport. The pregnane-X receptor is a nuclear receptor that functions to control a battery of genes predominantly involved in drug metabolism and we place emphasis on how this important cellular mediator is transcriptionally activated. We have identified both positive and negative regulatory elements in the PXR promoter, the balance of which dictates the steady state expression of the PXR gene.
Assuntos
Receptores Citoplasmáticos e Nucleares/genética , Receptores de Esteroides/genética , Regulação da Expressão Gênica/fisiologia , Humanos , Ligantes , PPAR gama/metabolismo , Receptor de Pregnano X , Receptores Citoplasmáticos e Nucleares/metabolismo , Transcrição GênicaRESUMO
Recent data have made it increasingly clear that the gene expression profile of a cell system, and its alteration in response to external stimuli, is highly dependent on both the higher order chromatin structure of the genome and the interaction of gene products in interpreting stimuli. To further explore this phenomenon, we have examined the role of both of these factors in controlling xenobiotic-mediated gene expression changes in primary and transformed human hepatocytes (HuH7). Using quantitative polymerase chain reaction, expression levels of several transcription factors implicated in the liver-specific regulation of the CYP3A gene family were examined in human adult and fetal liver RNA samples. These expression profiles were then compared with those obtained from both primary and transformed human hepatocytes, showing that, in general, cultured cells exhibit a distinct profile compared with either the fetal or adult samples. Transcriptome profiles before and after exposure to the CYP3A transcriptional activators rifampicin, dexamethasone, pregnane-16alpha-carbonitrile, and phenobarbital were subsequently examined. Whereas exposure to these compounds elicited a dose-dependent increase in CYP3A transcription in primary hepatocytes, no alteration in expression levels was observed for the hepatoma cell line HuH7. Alteration in the expression levels of pregnane X receptor and chicken ovalbumin upstream promoter transcription factor I, and the disruption of higher order chromatin within HuH7 cells altered CYP3A expression and/or activation by xenobiotics toward that observed in primary hepatocytes. These data provide potential roles for these two processes in regulating CYP3A expression in vivo.
Assuntos
Hidrocarboneto de Aril Hidroxilases/biossíntese , Cromatina/química , Cromatina/metabolismo , Perfilação da Expressão Gênica , Regulação Enzimológica da Expressão Gênica/fisiologia , Hepatócitos/enzimologia , Oxirredutases N-Desmetilantes/biossíntese , Fatores de Transcrição/biossíntese , Adulto , Idoso , Hidrocarboneto de Aril Hidroxilases/genética , Linhagem Celular , Cromatina/genética , Citocromo P-450 CYP3A , Feminino , Feto/enzimologia , Perfilação da Expressão Gênica/métodos , Humanos , Masculino , Pessoa de Meia-Idade , Oxirredutases N-Desmetilantes/genética , Conformação Proteica , Fatores de Transcrição/genéticaRESUMO
Cytochrome P450 3A4 (CYP3A4) is the major cytochrome P450 present in adult human liver and is involved in the metabolism of over 50% of therapeutic compounds currently in use. Since expression levels of CYP3A4 are regulated by many of these compounds, this raises the potential for drug-drug interactions and subsequent altered efficacy or toxicity of the individual compounds at the dose prescribed. Hence, understanding the molecular mechanisms of CYP3A4 regulation is of key importance in predicting and understanding such interactions. To examine this we have used DNase I footprinting and bioinformatic analysis to identify putative transcription factor binding sites within the 250 base pairs of promoter proximal to the transcription start site. We identified several protected fragments within this region that corresponded to putative binding sites for Sp1, AP2, CCAAT/enhancer binding protein (C/EBPalpha), and hepatic nuclear factor-3 (HNF3), as well as confirming previously identified C/EBPalpha, pregnane X receptor (PXR), and HNF3 binding sites. Sequential site-directed mutagenesis of C/EBPalpha, Sp1, HNF3, and PXR binding sites was next used to examine the role of these sites in basal CYP3A4 expression. Disruption of the C/EBPalpha, HNF3, and PXR binding sites all affected basal expression. Finally, the role of these sites was examined in activation of CYP3A4 expression by rifampicin, metyrapone, clotrimazole, and phenobarbital. Disruption of any of these sites either led to an altered pattern of activation by the xenobiotic, as altered maximal activation, or altered the EC(50) value of activation. Such effects were xenobiotic-specific, with each disrupted site playing a role in the activation of some of the xenobiotics.
Assuntos
Proteína alfa Estimuladora de Ligação a CCAAT/fisiologia , Sistema Enzimático do Citocromo P-450/genética , Sistema Enzimático do Citocromo P-450/metabolismo , Proteínas de Ligação a DNA/fisiologia , Proteínas Nucleares/fisiologia , Receptores Citoplasmáticos e Nucleares/fisiologia , Receptores de Esteroides/fisiologia , Fator de Transcrição Sp1/fisiologia , Fatores de Transcrição/fisiologia , Xenobióticos/farmacologia , Sequência de Bases/genética , Sítios de Ligação/efeitos dos fármacos , Sítios de Ligação/fisiologia , Linhagem Celular Tumoral , Citocromo P-450 CYP3A , Relação Dose-Resposta a Droga , Fator 3-alfa Nuclear de Hepatócito , Fator 3-beta Nuclear de Hepatócito , Humanos , Dados de Sequência Molecular , Receptor de Pregnano XRESUMO
CYP3A4 is the most abundant cytochrome P450 (P450) in human liver, comprising approximately 30% of the total liver P450 content. This enzyme has an important role in steroid catabolism and metabolism of foreign compounds, with the majority of pharmaceutical compounds being substrates for CYP3A4. The molecular mechanisms that underlie transcriptional activation of CYP3A4 are complex with many steroid hormone nuclear receptors, including glucocorticoid receptor, pregnane X receptor (PXR), vitamin D receptor, and constitutive androstane receptor, playing roles. Nowhere is this more evident than in the induction of CYP3A4 gene expression by glucocorticoids. CYP3A genes lack a consensus glucocorticoid receptor response element and yet are highly induced by classical glucocorticoids such as hydrocortisone and dexamethasone. Recent evidence has demonstrated that glucocorticoids are ligands for the orphan nuclear receptor PXR, and induction of CYP3A genes by glucocorticoids may occur primarily through PXR interactions. In this paper, we present a mutant that disrupts a hepatocyte-nuclear-factor-3/CCAAT-enhancer binding protein alpha binding site in the CYP3A4 proximal promoter. This mutation disrupts induction of a reporter gene construct by the glucocorticoids dexamethasone and hydrocortisone; yet induction by the potent PXR ligand rifampicin is unaffected. Such data provides strong evidence that glucocorticoids induce CYP3A4 gene expression both through the established PXR-dependent pathway but also through a PXR-independent pathway.
Assuntos
Sistema Enzimático do Citocromo P-450/biossíntese , DNA/metabolismo , Glucocorticoides/farmacologia , Receptores Citoplasmáticos e Nucleares/genética , Receptores de Esteroides/genética , Elementos de Resposta , Fatores de Transcrição , Sítios de Ligação , Proteína alfa Estimuladora de Ligação a CCAAT/genética , Proteína alfa Estimuladora de Ligação a CCAAT/metabolismo , Citocromo P-450 CYP3A , Sistema Enzimático do Citocromo P-450/genética , Pegada de DNA , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Desoxirribonuclease I , Dexametasona/farmacologia , Ensaio de Desvio de Mobilidade Eletroforética , Indução Enzimática , Fator 3-alfa Nuclear de Hepatócito , Humanos , Hidrocortisona/farmacologia , Ligantes , Mutação , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Receptor de Pregnano X , Receptores Citoplasmáticos e Nucleares/metabolismo , Receptores de Esteroides/metabolismo , Rifampina/farmacologia , Células Tumorais CultivadasRESUMO
Leydig cell tumours (LCTs) are frequently observed during rodent carcinogenicity studies, however, the significance of this effect to humans remains a matter of debate. Many chemicals that produce LCTs also induce hepatic cytochromes P450 (CYPs), but it is unknown whether these two phenomena are causally related. Our aim was to investigate the existence of a liver-testis axis wherein microsomal enzyme inducers enhance testosterone metabolic clearance, resulting in a drop in circulating hormone levels and a consequent hypertrophic response from the hypothalamic-pituitary-testis axis. Lansoprazole was selected as the model compound as it induces hepatic CYPs and produces LCTs in rats. Male Sprague-Dawley rats were dosed with lansoprazole (150 mg/kg/day) or vehicle for 14 days. Lansoprazole treatment produced effects on the liver consistent with an enhanced metabolic capacity, including significant increases in relative liver weights, total microsomal CYP content, individual CYP protein levels, and enhanced CYP-dependent testosterone metabolism in vitro. Following intravenous administration of [14C]testosterone, lansoprazole-treated rats exhibited a significantly smaller area under the curve and significantly higher plasma clearance. Significant reductions in plasma and testicular testosterone levels were observed, confirming the ability of this compound to perturb androgen homeostasis. No significant changes in plasma LH, FSH, or prolactin levels were detected under our experimental conditions. Lansoprazole treatment exerted no marked effects on testicular testosterone metabolism. In summary, lansoprazole treatment induced hepatic CYP-dependent testosterone metabolism in vitro and enhanced plasma clearance of radiolabelled testosterone in vivo. These effects may contribute to depletion of circulating testosterone levels and hence play a role in the mode of LCT induction in lansoprazole-treated rats.