RESUMEN
The leading cause of drug-induced liver injury in the developed world is overdose with N-acetyl-p-aminophenol (APAP). A comparative metabonomic approach was applied to the study of both xenobiotic and endogenous metabolic profiles reflective of in vivo exposure to APAP (300 mg/kg) and its structural isomer N-acetyl-m-aminophenol (AMAP; 300 mg/kg) in C57BL/6J mice, which was anchored with histopathology. Liver and urine samples were collected at 1 h, 3 h and 6 h post-treatment and analyzed by 1H nuclear magnetic resonance (NMR) spectroscopy and gas chromatography-mass spectrometry (liver only). Histopathology revealed the presence of centrilobular necrosis from 3 h post-APAP treatment, while an AMAP-mediated necrotic endpoint was not observed within the timescale of this study, yet two of five treated mice showed minimal centrilobular eosinophilia. The 1H-NMR xenobiotic metabolic profile of APAP-treated animals comprised of mercapturate (urine and liver) and glutathionyl (liver) conjugates detected at 1 h post-treatment. This finding corroborated the hepatic endogenous metabolic profile which showed depletion of glutathione from 1 h onwards. In contrast, AMAP glutathionyl conjugates were not detected, nor was AMAP-induced depletion of hepatic glutathione observed. APAP administration induced significant endogenous hepatic metabolic perturbations, primarily linked to oxidative and energetic stress, and perturbation of amino acid metabolism. Early depletion of glutathione was followed by depletion of additional sulfur-containing metabolites, while altered levels of mitochondrial and glycolytic metabolites indicated a disruption of energy homeostasis. In contrast, AMAP administration caused minimal, transient, distinct metabolic perturbations and by 6 h the metabolic profiles of AMAP-treated mice were indistinguishable from those of controls.
Asunto(s)
Acetaminofén/efectos adversos , Analgésicos no Narcóticos/efectos adversos , Enfermedad Hepática Inducida por Sustancias y Drogas/metabolismo , Hígado/efectos de los fármacos , Acetaminofén/análogos & derivados , Acetaminofén/química , Acetaminofén/farmacocinética , Acetilcisteína/análogos & derivados , Acetilcisteína/metabolismo , Acetilcisteína/orina , Analgésicos no Narcóticos/química , Analgésicos no Narcóticos/metabolismo , Analgésicos no Narcóticos/farmacocinética , Animales , Biomarcadores/sangre , Biomarcadores/metabolismo , Biomarcadores/orina , Biotransformación , Enfermedad Hepática Inducida por Sustancias y Drogas/diagnóstico por imagen , Enfermedad Hepática Inducida por Sustancias y Drogas/patología , Enfermedad Hepática Inducida por Sustancias y Drogas/fisiopatología , Metabolismo Energético/efectos de los fármacos , Eosinofilia/etiología , Glutatión/análogos & derivados , Glutatión/antagonistas & inhibidores , Glutatión/metabolismo , Isomerismo , Hígado/diagnóstico por imagen , Hígado/metabolismo , Hígado/patología , Imagen por Resonancia Magnética , Masculino , Metabolómica/métodos , Ratones Endogámicos C57BL , Necrosis , Estrés Oxidativo/efectos de los fármacos , Organismos Libres de Patógenos Específicos , Distribución TisularRESUMEN
Idiosyncratic hepatotoxicity has been associated with the oral tyrosine kinase inhibitor lapatinib, which is used in metastatic breast cancer therapy. Lapatinib is extensively metabolized by cytochrome P450 3A4/5 to yield an O-debenzylated metabolite, which can undergo further oxidation to a reactive quinone imine. A recent clinical study reported that concomitant use of lapatinib with dexamethasone increased the incidence of hepatotoxicity in metastatic breast cancer patients treated with lapatinib, and so we hypothesized that induction of CYP3A enhances the bioactivation of lapatinib to reactive intermediates that contribute to hepatotoxicity. Therefore, we examined the effect of CYP3A4 induction on the cytotoxicity and metabolism of lapatinib in the HepaRG human hepatic cell line. Differentiated HepaRG cells were pretreated with dexamethasone (100 µM) or the prototypical CYP3A4 inducer rifampicin (4 µM) for 72 hours, followed by incubation with lapatinib (0-100 µM) for 24 hours. Cell viability was monitored using WST-1 assays, and metabolites were quantified by liquid chromatography coupled to tandem mass spectrometry. Induction of CYP3A4 by dexamethasone or rifampicin enhanced lapatinib-induced cytotoxicity, compared with treatment with lapatinib alone. A direct comparison of the cytotoxicity of lapatinib versus O-debenzylated lapatinib demonstrated that the O-debenzylated metabolite was significantly more cytotoxic than lapatinib itself. Furthermore, pretreatment with 25 µM l-buthionine sulfoximine to deplete intracellular glutathione markedly enhanced lapatinib cytotoxicity. Cytotoxicity was correlated with increased formation of O-debenzylated lapatinib and cysteine adducts of the putative quinone imine intermediate. Collectively, these data suggest that CYP3A4 induction potentiates lapatinib-induced hepatotoxicity via increased reactive metabolite formation.
Asunto(s)
Biotransformación/efectos de los fármacos , Citocromo P-450 CYP3A/metabolismo , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Cisteína/metabolismo , Dexametasona/farmacología , Glutatión/metabolismo , Hepatocitos/efectos de los fármacos , Hepatocitos/enzimología , Hepatocitos/metabolismo , Humanos , Lapatinib , Hígado/efectos de los fármacos , Hígado/enzimología , Hígado/metabolismo , Proteínas Tirosina Quinasas/metabolismo , Quinazolinas/farmacología , Rifampin/farmacologíaRESUMEN
Cytochrome b(5) (cyt b(5)) is one of the key components in the microsomal cytochrome P450 monooxygenase system. Consensus has not been reached about the underlying mechanism of cyt b(5) modulation of CYP catalysis. Both cyt b(5) and apo b(5) are reported to stimulate the activity of several P450 isoforms. In this study, the surface interactions of both holo and apo b(5) with CYP3A4 were investigated and compared for the first time. Chemical cross-linking coupled with mass spectrometric analysis was used to identify the potential electrostatic interactions between the protein surfaces. Subsequently, the models of interaction of holo/apo b(5) with CYP3A4 were built using the identified interacting sites as constraints. Both cyt b(5) and apo b(5) were predicted to bind to the same groove on CYP3A4 with close contacts to the B-B' loop of CYP3A4, a substrate recognition site. Mutagenesis studies further confirmed that the interacting sites on CYP3A4 (Lys96, Lys127, and Lys421) are functionally important. Mutation of these residues reduced or abolished cyt b(5) binding affinity. The critical role of Arg446 on CYP3A4 in binding to cyt b(5) and/or cytochrome P450 reductase was also discovered. The results indicated that electrostatic interactions on the interface of the two proteins are functionally important. The results indicate that apo b(5) can dock with CYP3A4 in a manner analogous to that of holo b(5), so electron transfer from cyt b(5) is not required for its effects.
Asunto(s)
Citocromo P-450 CYP3A/química , Citocromos b5/química , Sitios de Unión , Reactivos de Enlaces Cruzados/química , Citocromo P-450 CYP2E1/metabolismo , Citocromo P-450 CYP3A/genética , Citocromo P-450 CYP3A/metabolismo , Citocromos b5/metabolismo , Etildimetilaminopropil Carbodiimida/química , Espectrometría de Masas , Modelos Moleculares , Mutagénesis Sitio-Dirigida , Esteroide Hidroxilasas/metabolismo , Testosterona/metabolismoRESUMEN
Vitamin D(3) is critical for the regulation of calcium and phosphate homeostasis. In some individuals, mineral homeostasis can be disrupted by long-term therapy with certain antiepileptic drugs and the antimicrobial agent rifampin, resulting in drug-induced osteomalacia, which is attributed to vitamin D deficiency. We now report a novel CYP3A4-dependent pathway, the 4-hydroxylation of 25-hydroxyvitamin D(3) (25OHD(3)), the induction of which may contribute to drug-induced vitamin D deficiency. The metabolism of 25OHD(3) was fully characterized in vitro. CYP3A4 was the predominant source of 25OHD(3) hydroxylation by human liver microsomes, with the formation of 4ß,25-dihydroxyvitamin D(3) [4ß,25(OH)(2)D(3)] dominating (V(max)/K(m) = 0.85 ml · min(-1) · nmol enzyme(-1)). 4ß,25(OH)(2)D(3) was found in human plasma at concentrations comparable to that of 1α,25-dihydroxyvitamin D(3), and its formation rate in a panel of human liver microsomes was strongly correlated with CYP3A4 content and midazolam hydroxylation activity. Formation of 4ß,25(OH)(2)D(3) in primary human hepatocytes was induced by rifampin and inhibited by CYP3A4-specific inhibitors. Short-term treatment of healthy volunteers (n = 6) with rifampin selectively induced CYP3A4-dependent 4ß,25(OH)(2)D(3), but not CYP24A1-dependent 24R,25-dihydroxyvitamin D(3) formation, and altered systemic mineral homeostasis. Our results suggest that CYP3A4-dependent 25OHD(3) metabolism may play an important role in the regulation of vitamin D(3) in vivo and in the etiology of drug-induced osteomalacia.
Asunto(s)
Citocromo P-450 CYP3A/metabolismo , Vitamina D/metabolismo , Cromatografía Líquida de Alta Presión , Humanos , Microsomas Hepáticos/enzimología , Espectrometría de Masas en TándemRESUMEN
Mitochondria are the primary locus for the generation of reactive nitrogen species including peroxynitrite and subsequent protein tyrosine nitration. Protein tyrosine nitration may have important functional and biological consequences such as alteration of enzyme catalytic activity. In the present study, mouse liver mitochondria were incubated with peroxynitrite, and the mitochondrial proteins were separated by 1D and 2D gel electrophoresis. Nitrotyrosinylated proteins were detected with an anti-nitrotyrosine antibody. One of the major proteins nitrated by peroxynitrite was carbamoyl phosphate synthetase 1 (CPS1) as identified by LC-MS protein analysis and Western blotting. The band intensity of nitration normalized to CPS1 was increased in a peroxynitrite concentration-dependent manner. In addition, CPS1 activity was decreased by treatment with peroxynitrite in a peroxynitrite concentration- and time-dependent manner. The decreased CPS1 activity was not recovered by treatment with reduced glutathione, suggesting that the decrease of the CPS1 activity is due to tyrosine nitration rather than cysteine oxidation. LC-MS analysis of in-gel digested samples, and a Popitam-based modification search located 5 out of 36 tyrosine residues in CPS1 that were nitrated. Taken together with previous findings regarding CPS1 structure and function, homology modeling of mouse CPS1 suggested that nitration at Y1450 in an α-helix of allosteric domain prevents activation of CPS1 by its activator, N-acetyl-l-glutamate. In conclusion, this study demonstrated the tyrosine nitration of CPS1 by peroxynitrite and its functional consequence. Since CPS1 is responsible for ammonia removal in the urea cycle, nitration of CPS1 with attenuated function might be involved in some diseases and drug-induced toxicities associated with mitochondrial dysfunction.
Asunto(s)
Carbamoil-Fosfato Sintasa (Amoniaco)/química , Mitocondrias Hepáticas/enzimología , Proteínas Mitocondriales/química , Nitratos/química , Tirosina/química , Regulación Alostérica , Secuencia de Aminoácidos , Animales , Ratones , Modelos Moleculares , Datos de Secuencia Molecular , Ácido Peroxinitroso/química , Estructura Secundaria de Proteína , Estructura Terciaria de ProteínaRESUMEN
The hypothesis that N-acetyl-m-aminophenol (AMAP), the meta isomer of acetaminophen, will covalently bind to and inhibit human CYP2E1 in a time- and NADPH-dependent manner was investigated. Liquid chromatography/electrospray ionization-mass spectrometry analysis indicated that AMAP metabolites (i.e., AMAP*) selectively and covalently modified CYP2E1 apoprotein in a ratio of 1.4:1 (AMAP*/CYP2E1) in a reconstituted system. The deconvoluted spectra of CYP2E1 apoprotein from incubations containing NADPH and AMAP displayed mass shifts of 167.2 ± 7.1 and 334.4 ± 6.5 Da, suggesting the addition of one and two hydroxylated AMAP metabolites to CYP2E1, respectively. Mass shifts in cytochrome P450 reductase, cytochrome b(5), and heme from these samples were not observed. CYP2E1 inhibition by AMAP increased with time in the presence of NADPH; a reversible inhibition component was also observed. The results support a bioactivation process that involves formation of a hydroquinone metabolite that undergoes further oxidation to a quinone, which reacts with CYP2E1 nucleophilic residues. The data are consistent with evidence from previous studies that identified hydroxylated AMAP glutathione conjugates collected from mice and indicate that cysteine residues are the most likely sites for adduct formation. This study reports the first direct evidence of AMAP-derived hydroquinone metabolites bound to human CYP2E1.
Asunto(s)
Acetaminofén/farmacología , Inhibidores del Citocromo P-450 CYP2E1 , Cromatografía Líquida de Alta Presión , Humanos , Isomerismo , NADP/metabolismo , Espectrometría de Masa por Ionización de ElectrosprayRESUMEN
Lapatinib, an oral tyrosine kinase inhibitor used for breast cancer, has been reported to cause idiosyncratic hepatotoxicity. Recently, it has been found that lapatinib forms a metabolite-inhibitor complex (MIC) with CYP3A4 via the formation of an alkylnitroso intermediate. Because CYP3A5 is highly polymorphic compared with CYP3A4 and also oxidizes lapatinib, we investigated the interactions of lapatinib with CYP3A5. Lapatinib inactivated CYP3A5 in a time-, concentration-, and NADPH-dependent manner using testosterone as a probe substrate with K(I) and k(inact) values of 0.0376 mM and 0.0226 min(-1), respectively. However, similar results were not obtained when midazolam was used as the probe substrate, suggesting that inactivation of CYP3A5 by lapatinib is site-specific. Poor recovery of CYP3A5 activity postdialysis and the lack of a Soret peak confirmed that lapatinib does not form a MIC with CYP3A5. The reduced CO difference spectrum further suggested that a large fraction of the reactive metabolite of lapatinib is covalently adducted to the apoprotein of CYP3A5. GSH trapping of a reactive metabolite of lapatinib formed by CYP3A5 confirmed the formation of a quinoneimine-GSH adduct derived from the O-dealkylated metabolite of lapatinib. In silico docking studies supported the preferential formation of an O-dealkylated metabolite of lapatinib by CYP3A5 compared with an N-hydroxylation reaction that is predominantly catalyzed by CYP3A4. In conclusion, lapatinib appears to be a mechanism-based inactivator of CYP3A5 via adduction of a quinoneimine metabolite.
Asunto(s)
Citocromo P-450 CYP3A/metabolismo , Inhibidores de Proteínas Quinasas/farmacología , Quinazolinas/farmacología , Inhibidores del Citocromo P-450 CYP3A , Remoción de Radical Alquila , Humanos , Hidroxilación , Lapatinib , Midazolam/farmacología , NADP/metabolismo , Testosterona/metabolismoRESUMEN
Pulegone is the major constituent of pennyroyal oil, a folkloric abortifacient that is associated with hepatotoxicity and, in severe cases, death. Cytochrome P450-mediated oxidation of pulegone generates menthofuran, which is further oxidized to form electrophilic reactive intermediates, menthofuran epoxide and the ring-opened γ-ketoenal, both of which can form adducts to hepatocellular proteins. Modification of hepatocellular proteins by the electrophilic reactive intermediates of menthofuran has been implicated in hepatotoxicity caused by pennyroyal oil. Herein, we describe the identification of several proteins that are the likely targets of menthofuran-derived reactive metabolites. These proteins were isolated from the livers of rats treated with a hepatotoxic dose of menthofuran by two-dimensional gel electrophoresis (2D-gel) separation and detected by Western blot analysis using an antiserum developed to detect protein adducts resulting from menthofuran bioactivation. The antibody-reacting proteins were excised from the 2D-gel and subjected to tryptic digestion for analysis of peptide fragments by LC-MS/MS. Although 10 spots were detected by Western blot analysis, only 4 were amenable to characterization by LC-MS/MS: serum albumin, mitochondrial aldehyde dehydrogenase (ALDH2), cytoplasmic malate dehydrogenase (MDH1), and mitochondrial ATP synthase subunit d. No direct adduct was detected, and, therefore, we complemented our analysis with enzyme activity determination. ALDH2 activity decreased by 88%, and ATP synthase complex V activity decreased by 34%, with no activity changes to MDH1. Although the relationship between these reactive metabolite adducted proteins and hepatotoxicity is not clear, these targeted enzymes are known to play critical roles in maintaining cellular homeostasis.
Asunto(s)
Aldehído Deshidrogenasa/metabolismo , Hígado/efectos de los fármacos , Hígado/metabolismo , Malato Deshidrogenasa/metabolismo , Proteínas Mitocondriales/metabolismo , ATPasas de Translocación de Protón Mitocondriales/metabolismo , Monoterpenos/farmacología , Albúmina Sérica/metabolismo , Aldehído Deshidrogenasa/antagonistas & inhibidores , Aldehído Deshidrogenasa Mitocondrial , Animales , Citoplasma/efectos de los fármacos , Citoplasma/enzimología , Citoplasma/metabolismo , Relación Dosis-Respuesta a Droga , Hígado/química , Malato Deshidrogenasa/antagonistas & inhibidores , Masculino , Mitocondrias/efectos de los fármacos , Mitocondrias/enzimología , Mitocondrias/metabolismo , Proteínas Mitocondriales/antagonistas & inhibidores , ATPasas de Translocación de Protón Mitocondriales/antagonistas & inhibidores , Estructura Molecular , Ratas , Ratas Sprague-Dawley , Relación Estructura-ActividadRESUMEN
The uricosuric diuretic agent tienilic acid (TA) is a thiophene-containing compound that is metabolized by P450 2C9 to 5-OH-TA. A reactive metabolite of TA also forms a covalent adduct to P450 2C9 that inactivates the enzyme and initiates immune-mediated hepatic injury in humans, purportedly through a thiophene-S-oxide intermediate. The 3-thenoyl regioisomer of TA, tienilic acid isomer (TAI), is chemically very similar and is reported to be oxidized by P450 2C9 to a thiophene-S-oxide, yet it is not a mechanism-based inactivator (MBI) of P450 2C9 and is reported to be an intrinsic hepatotoxin in rats. The goal of the work presented in this article was to identify the reactive metabolites of TA and TAI by the characterization of products derived from P450 2C9-mediated oxidation. In addition, in silico approaches were used to better understand both the mechanisms of oxidation of TA and TAI and/or the structural rearrangements of oxidized thiophene compounds. Incubation of TA with P450 2C9 and NADPH yielded the well-characterized 5-OH-TA metabolite as the major product. However, contrary to previous reports, it was found that TAI was oxidized to two different types of reactive intermediates that ultimately lead to two types of products, a pair of hydroxythiophene/thiolactone tautomers and an S-oxide dimer. Both TA and TAI incorporated ¹8O from ¹8O2 into their respective hydroxythiophene/thiolactone metabolites indicating that these products are derived from an arene oxide pathway. Intrinsic reaction coordinate calculations of the rearrangement reactions of the model compound 2-acetylthiophene-S-oxide showed that a 1,5-oxygen migration mechanism is energetically unfavorable and does not yield the 5-OH product but instead yields a six-membered oxathiine ring. Therefore, arene oxide formation and subsequent NIH-shift rearrangement remains the favored mechanism for formation of 5-OH-TA. This also implicates the arene oxide as the initiating factor in TA induced liver injury via covalent modification of P450 2C9. Finally, in silico modeling of P450 2C9 active site ligand interactions with TA using the catalytically active iron-oxo species revealed significant differences in the orientations of TA and TAI in the active site, which correlated well with experimental results showing that TA was oxidized only to a ring carbon hydroxylated product, whereas TAI formed both ring carbon hydroxylated products and an S-oxide.
Asunto(s)
Hidrocarburo de Aril Hidroxilasas/metabolismo , Diuréticos/metabolismo , Ticrinafeno/metabolismo , Animales , Citocromo P-450 CYP2C9 , Diuréticos/química , Humanos , NADP/metabolismo , Oxidación-Reducción , Ratas , Estereoisomerismo , Ticrinafeno/químicaRESUMEN
An NMR-based metabonomic approach was applied to study the systems level metabolic effects of two closely related thiophene compounds, tienilic acid (TA) and tienilic acid isomer (TAI). The metabonomic data were anchored with traditional clinical chemistry and histopathologic analyses. TA was removed from the market as a result of suspected immune-mediated hepatotoxicity, whereas TAI is an intrinsic hepatotoxin. Equimolar doses of TA and TAI were administered to Sprague-Dawley rats, and sampling was conducted at 2, 6, and 24 h post-treatment. Histopathologic analyses revealed development of a significant hepatic lesion 24 h post-TAI treatment with a parallel increase in plasma alanine aminotransferase (ALT) activity. In contrast, TA was not associated with the development of a hepatic lesion or an increase in plasma ALT activity. High-resolution NMR spectral metabolic profiles were generated for liver extracts, plasma, and urine at multiple time points. Multivariate statistical tools were applied to model the metabolic profiles and identify discriminatory metabolites that reflected both the adaptation to TA administration and the onset and progression of TAI-induced hepatotoxicity. TAI was shown to induce marked metabolic effects on the metabolome at all time points, with dramatic metabolic perturbations at 24 h post-treatment correlating with the histopathologic and clinical chemistry evidence of a hepatic lesion. The TAI-induced metabolic perturbations provided evidence for the generation of electrophilic reactive metabolites and a significant impairment of bioenergetic metabolic pathways. TA induced early metabolic perturbations that were largely resolved by 24 h post-treatment, suggesting the reestablishment of metabolic homeostasis and the ability to adapt to the intervention, with hepatic hypotaurine potentially representing a means of assessment of hepatic adaptation. This comparative metabonomic approach enabled the discrimination of metabolic perturbations that were common to both treatments and were interpreted as nontoxic thiophene-induced perturbations. Importantly, this approach enabled the identification of temporal metabolic perturbations that were unique to TAI or TA treatment and hence were of relevance to the development of toxicity or the ability to adapt. This approach is applicable to the future study of pharmacologically and structurally similar compounds and represents a refined means of identification of biomarkers of toxicity.
Asunto(s)
Ticrinafeno/metabolismo , Animales , Química Clínica , Determinación de Punto Final , Hígado/química , Hígado/metabolismo , Hígado/patología , Espectroscopía de Resonancia Magnética , Masculino , Estructura Molecular , Fenotipo , Ratas , Ratas Sprague-Dawley , Estereoisomerismo , Ticrinafeno/administración & dosificación , Ticrinafeno/química , Pruebas de ToxicidadRESUMEN
Human cytochrome P450 3A4 (CYP3A4) metabolizes a significant portion of clinically relevant drugs and often exhibits complex steady-state kinetics that can involve homotropic and heterotropic cooperativity between bound ligands. In previous studies, the hydroxylation of the sedative midazolam (MDZ) exhibited homotropic cooperativity via a decrease in the ratio of 1'-OH-MDZ to 4-OH-MDZ at higher drug concentrations. In this study, MDZ exhibited heterotropic cooperativity with the antiepileptic drug carbamazepine (CBZ) with characteristic decreases in the 1'-OH-MDZ to 4-OH-MDZ ratios. To unravel the structural basis of MDZ cooperativity, we probed MDZ and CBZ bound to CYP3A4 using longitudinal T(1) nuclear magnetic resonance (NMR) relaxation and molecular docking with AutoDock 4.2. The distances calculated from longitudinal T(1) NMR relaxation were used during simulated annealing to constrain the molecules to the substrate-free X-ray crystal structure of CYP3A4. These simulations revealed that either two MDZ molecules or an MDZ molecule and a CBZ molecule assume a stacked configuration within the CYP3A4 active site. In either case, the proton at position 4 of the MDZ molecule was closer to the heme than the protons of the 1'-CH(3) group. In contrast, molecular docking of a single molecule of MDZ revealed that the molecule was preferentially oriented with the 1'-CH(3) position closer to the heme than position 4. This study provides the first detailed molecular analysis of heterotropic and homotropic cooperativity of a human cytochrome P450 from an NMR-based model. Cooperativity of ligand binding through direct interaction between stacked molecules may represent a common motif for homotropic and heterotropic cooperativity.
Asunto(s)
Citocromo P-450 CYP3A/metabolismo , Hipnóticos y Sedantes/metabolismo , Midazolam/metabolismo , Algoritmos , Anticonvulsivantes/química , Anticonvulsivantes/metabolismo , Biocatálisis , Carbamazepina/química , Carbamazepina/metabolismo , Dominio Catalítico , Simulación por Computador , Citocromo P-450 CYP3A/química , Citocromo P-450 CYP3A/genética , Humanos , Hidroxilación , Hipnóticos y Sedantes/química , Cinética , Ligandos , Midazolam/análogos & derivados , Midazolam/química , Modelos Moleculares , Conformación Molecular , Resonancia Magnética Nuclear Biomolecular , Unión Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismoRESUMEN
Lapatinib, an oral breast cancer drug, has recently been reported to be a mechanism-based inactivator of cytochrome P450 (P450) 3A4 and also an idiosyncratic hepatotoxicant. It was suggested that formation of a reactive quinoneimine metabolite was involved in mechanism-based inactivation (MBI) and/or hepatotoxicity. We investigated the mechanism of MBI of P450 3A4 by lapatinib. Liquid chromatography-mass spectrometry analysis of P450 3A4 after incubation with lapatinib did not show any peak corresponding to irreversible modifications. The enzymatic activity inactivated by lapatinib was completely restored by the addition of potassium ferricyanide. These results indicate that the mechanism of MBI by lapatinib is quasi-irreversible and mediated via metabolic intermediate complex (MI complex) formation. This finding was verified by the increase in a signature Soret absorbance at approximately 455 nm. Two amine oxidation products of the metabolism of lapatinib by P450 3A4 were characterized: N-hydroxy lapatinib (M3) and the oxime form of N-dealkylated lapatinib (M2), suggesting that a nitroso or another related intermediate generated from M3 is involved in MI complex formation. In contrast, P450 3A5 was much less susceptible to MBI by lapatinib via MI complex formation than P450 3A4. In addition, P450 3A5 had a significantly lower ability than 3A4 to generate M3, consistent with N-hydroxylation as the initial step in the pathway to MI complex formation. In conclusion, our results demonstrate that the primary mechanism for MBI of P450 3A4 by lapatinib is not irreversible modification by the quinoneimine metabolite, but quasi-irreversible MI complex formation mediated via oxidation of the secondary amine group of lapatinib.
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Antineoplásicos/metabolismo , Inhibidores del Citocromo P-450 CYP3A , Quinazolinas/metabolismo , Antineoplásicos/toxicidad , Enfermedad Hepática Inducida por Sustancias y Drogas/enzimología , Enfermedad Hepática Inducida por Sustancias y Drogas/etiología , Cromatografía Liquida , Citocromo P-450 CYP3A/genética , Citocromo P-450 CYP3A/metabolismo , Activación Enzimática , Escherichia coli/genética , Ferricianuros/farmacología , Humanos , Lapatinib , Espectrometría de Masas , Microsomas Hepáticos/efectos de los fármacos , Microsomas Hepáticos/enzimología , Estructura Molecular , Oxidación-Reducción , Unión Proteica , Quinazolinas/toxicidadRESUMEN
Cytochrome P450s promote a variety of rearrangement reactions both as a consequence of the nature of the radical and other intermediates generated during catalysis, and of the neighboring structures in the substrate that can interact either with the initial radical intermediates or with further downstream products of the reactions. This article will review several kinds of previously published cytochrome P450-catalyzed rearrangement reactions, including changes in stereochemistry, radical clock reactions, allylic rearrangements, "NIH" and related shifts, ring contractions and expansions, and cyclizations that result from neighboring group interactions. Although most of these reactions can be carried out by many members of the cytochrome P450 superfamily, some have only been observed with select P450s, including some reactions that are catalyzed by specific endoperoxidases and cytochrome P450s found in plants.
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Sistema Enzimático del Citocromo P-450/metabolismo , Animales , Ciclización , Humanos , Hidroxilación , Estereoisomerismo , Especificidad por SustratoRESUMEN
Comparative proteomic analysis following treatment with acetaminophen (APAP) was performed on two different models of APAP-mediated hepatocellular injury in order to both identify common targets for adduct formation and track drug-induced changes in protein expression. Male C57BL/6 mice were used as a model for APAP-mediated liver injury in vivo, and TAMH cells were used as a model for APAP-mediated cytotoxicity in vitro. SEQUEST was unable to identify the precise location of sites of adduction following treatment with APAP in either system. However, semiquantitative analysis of the proteomic data sets using spectral counting revealed a downregulation of P450 isoforms associated with APAP bioactivation and an upregulation of proteins related to the electron transport chain by APAP compared to the control. Both mechanisms are likely compensatory in nature as decreased P450 expression is likely to attenuate toxicity associated with N-acetyl-p-quinoneimine (NAPQI) formation, whereas APAP-induced electron transport chain component upregulation may be an attempt to promote cellular bioenergetics.
Asunto(s)
Acetaminofén/toxicidad , Mitocondrias/metabolismo , Proteínas Mitocondriales/metabolismo , Proteómica , Animales , Línea Celular , Enfermedad Hepática Inducida por Sustancias y Drogas/metabolismo , Cromatografía Líquida de Alta Presión , Sistema Enzimático del Citocromo P-450/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Isoformas de Proteínas/metabolismo , Espectrometría de Masas en TándemRESUMEN
Fatalities stemming from hepatotoxicity associated with the clinical use of lapatinib (Tykerb), an oral dual tyrosine kinase inhibitor (ErbB-1 and ErbB-2) used in the treatment of metastatic breast cancer, have been reported. We investigated the inhibition of CYP3A4 by lapatinib as a possible cause of its idiosyncratic toxicity. Inhibition of CYP3A4 was time-, concentration-, and NADPH-dependent, with k(inact) = 0.0202 min(-1) and K(i) = 1.709 µM. The partition ratio was approximately 50.9. Addition of GSH did not affect the rate of inactivation. Testosterone protected CYP3A4 from inactivation by lapatinib. The characteristic Soret peak associated with a metabolite-intermediate complex was not observed for lapatinib during spectral difference scanning. However, reduced carbon monoxide (CO)-difference spectroscopy did reveal a 43% loss of the spectrally detectable CYP3A4-CO complex in the presence of lapatinib. Incubation of either lapatinib or its dealkylated metabolite with human liver microsomes in the presence of GSH resulted in the formation of a reactive metabolite (RM)-GSH adduct derived from the O-dealkylated metabolite of lapatinib. In addition, coincubation of lapatinib with ketoconazole inhibited the formation of the RM-GSH adduct. In conclusion, we demonstrated for the first time that lapatinib is a mechanism-based inactivator of CYP3A4, most likely via the formation and further oxidation of its O-dealkylated metabolite to a quinoneimine that covalently modifies the CYP3A4 apoprotein and/or heme moiety.
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Inhibidores del Citocromo P-450 CYP3A , Citocromo P-450 CYP3A/metabolismo , Quinazolinas/metabolismo , Quinazolinas/farmacología , Relación Dosis-Respuesta a Droga , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/metabolismo , Inhibidores Enzimáticos/farmacología , Humanos , Lapatinib , Microsomas Hepáticos/efectos de los fármacos , Microsomas Hepáticos/enzimología , Quinazolinas/químicaRESUMEN
Menthofuran is a monoterpene present in mint plants that is oxidized by mammalian cytochrome P450 (CYP) to hepatotoxic metabolites. Evidence has been presented that p-cresol and other unusual oxidative products are metabolites of menthofuran in rats and that p-cresol may be responsible in part for the hepatotoxicity caused by menthofuran [ Madyastha, K. M. and Raj, C. P. (1992) Drug Metab. Dispos. 20, 295 - 301]. In the present study, several oxidative metabolites of menthofuran were characterized in rat and human liver microsomes and in rat liver slices exposed to cytotoxic concentrations of menthofuran. Metabolites that were identified were monohydroxylation products of the furanyl and cyclohexyl groups, mintlactones and hydroxymintlactones, a reactive γ-ketoenal, and a glutathione conjugate. A similar spectrum of metabolites was found in urine 24 h after the administration of hepatotoxic doses of menthofuran to rats. In no case was p-cresol (or any of the other reported unusual oxidative metabolites of menthofuran) detected above background concentrations that were well below concentrations of p-cresol that cause cytotoxicity in rat liver slices. Thus, the major metabolites responsible for the hepatotoxic effects of menthofuran appear to be a γ-ketoenal and/or epoxides formed by oxidation of the furan ring.
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Hígado/metabolismo , Monoterpenos/metabolismo , Animales , Cresoles/metabolismo , Cresoles/toxicidad , Sistema Enzimático del Citocromo P-450/metabolismo , Cromatografía de Gases y Espectrometría de Masas , Humanos , Hígado/efectos de los fármacos , Hígado/patología , Microsomas Hepáticos/metabolismo , Monoterpenos/toxicidad , Monoterpenos/orina , RatasRESUMEN
In order to speed up the process of cross-linked peptide identification and characterization, we have previously reported the development of Pro-CrossLink, a suite of software tools consisting of three programs, DetectShift, IdentifyXLink and AssignXLink for mass spectrometric data analysis. Since its public disclosure, Pro-CrossLink has been downloaded by 101 research groups. Pro-CrossLink users have provided us with valuable feedback on the use of the DetectShift program. Here we assess some reasons for the generation of false positives by DetectShift. In addition, we provide users with suggestions on optimal parameter setting and efficient use of the software program.
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Marcaje Isotópico/métodos , Péptidos/análisis , Programas Informáticos , Reactivos de Enlaces Cruzados/química , Espectrometría de Masas/métodos , Isótopos de Oxígeno/análisis , Isótopos de Oxígeno/química , Mapeo Peptídico , Procesamiento de Señales Asistido por ComputadorRESUMEN
Previously, S-(1,1,2,2-tetrafluoroethyl)-l-cysteine (TFEC) was shown to mediate cytotoxicity by covalently modifying a well-defined group of intramitochondrial proteins including aconitase, alpha-ketoglutarate dehydrogenase (alphaKGDH) subunits, heat shock protein 60 (HSP60) and mitochondrial HSP70 (mtHSP70). To investigate the cellular responses to this mitochondrial damage, microarray analysis of TFEC treated murine hepatocytes of the TAMH cell line was carried out. Results of these studies revealed a HSP response that was significantly stronger than other well-characterized hepatotoxicants including acetaminophen, diquat and rotenone. Specifically, cytosolic HSP25, HSP40, HSP70, HSP105 and microsomal HSP32 (HO-1) were strongly upregulated within the first few hours of TFEC treatment, while little change was observed among other HSPs that are predominantly localized in the mitochondria and endoplasmic reticulum (ER). Post-translational modification of HSP25 was also observed with the appearance of a unique DTT-resistant immunoreactive band at about 50kDa, a putative dimer. The biological significance of HSP responses to TFEC-induced toxicity were subsequently demonstrated using the "gain of function" pretreatment: heat shock. Overall, we report an atypical HSP induction profile that does not conform to changes expected of a classical temperature shock. Furthermore, despite a well-defined intramitochondrial origin of toxicity, TFEC rapidly evokes an early and strong upregulation of cytosolic stress proteins. The cytoprotective effects of such HSP responses suggest a plausible role in modulating the progression of TFEC-induced cellular injury.
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Cisteína/análogos & derivados , Proteínas de Choque Térmico/biosíntesis , Hemo-Oxigenasa 1/biosíntesis , Hidrocarburos Fluorados/toxicidad , Mitocondrias Hepáticas/efectos de los fármacos , Línea Celular , Supervivencia Celular/efectos de los fármacos , Cisteína/toxicidad , Citosol/metabolismo , Dimerización , Expresión Génica/efectos de los fármacos , Silenciador del Gen , Proteínas de Choque Térmico/genética , Hepatocitos/efectos de los fármacos , Hepatocitos/metabolismo , Mitocondrias Hepáticas/metabolismo , Análisis por Matrices de Proteínas/métodos , Procesamiento Proteico-Postraduccional/efectos de los fármacos , ARN Mensajero/metabolismo , ARN Interferente Pequeño/farmacología , Transfección , Regulación hacia ArribaRESUMEN
S-(1,1,2,2-Tetrafluoroethyl)-L-cysteine (TFEC), a major metabolite of the industrial gas tetrafluoroethylene, has been shown to mediate nephrotoxicity by necrosis. TFEC-induced cell death is associated with an early covalent modification of specific intramitochondrial proteins; including aconitase, alpha-ketoglutarate dehydrogenase (KGDH) subunits, HSP60 and HSP70. Previous studies have indicated that the TAMH line accurately models TFEC-induced in vivo cell death with dose- and time-dependent inhibitions of both KGDH and aconitase activities. Here, we show that the molecular pathway leading to TFEC-mediated cell death is associated with an early cytosolic to mitochondrial translocation of BAX, a pro-apoptotic member of the BCL-2 family. Immunoblot analyses indicated movement of BAX (21 kDa) to the mitochondrial fraction after exposure to a cytotoxic concentration of TFEC (250 microM). Subsequent cytochrome c release from mitochondria was also demonstrated, but only a modest increase in caspase activities was observed, suggesting a degeneration of early apoptotic signals into secondary necrosis. Significantly, TAMH cells overexpressing BCL-xL preserved cell viability even to supratoxicological concentrations of TFEC (< or =600 microM), and this cytoprotection was associated with decreased HSP70i upregulation, indicating suppression of TFEC-induced proteotoxicity. Hence, TFEC-induced necrotic cell death in the TAMH cell line is mediated by BAX and antagonized by the anti-apoptotic BCL-2 family member, BCL-xL.
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Fluorocarburos/metabolismo , Fluorocarburos/farmacología , Mitocondrias/efectos de los fármacos , Proteínas Proto-Oncogénicas c-bcl-2/biosíntesis , Proteínas Proto-Oncogénicas c-bcl-2/fisiología , Muerte Celular/efectos de los fármacos , Muerte Celular/genética , Línea Celular , Cisteína/metabolismo , Cisteína/farmacología , Regulación de la Expresión Génica/efectos de los fármacos , Regulación de la Expresión Génica/fisiología , Humanos , Mitocondrias/genética , Proteína bcl-XRESUMEN
Tetrafluoroethylcysteine (TFEC), a metabolite of the industrial gas tetrafluoroethylene, can cause both nephrotoxicity and limited hepatotoxicity in animal models, and this is associated with the covalent modification of specific intramitochondrial proteins including heat shock protein 60 (HSP60), mitochondrial HSP70 (mtHSP70), aspartate aminotransferase (AST), aconitase, and alpha-ketoglutarate dehydrogenase (alphaKGDH). Using the murine TAMH cell line as a useful in vitro model for TFEC toxicity, we demonstrate a rapid and sustained induction of Nrf2, a member of the "cap-and-collar" transcription factor family, following exposure to cytotoxic concentrations of TFEC. A functional correlate was also established with the rapid translocation of cytosolic Nrf2 into the nucleus. In addition, transcriptional and translational upregulation of known Nrf2 regulated genes including glutamate cysteine ligase (GCL), both catalytic and modulatory subunits, heme oxygenase-1, and glutathione S-transferase (GST) isoforms were detected. While Nrf2 activation is often linked to perturbation of cellular thiol status and/or oxidative stress, we were unable to detect any significant depletion of cellular glutathione or oxidation of mitochondrial membrane cardiolipin or increases in reactive oxygen species (ROS). These data suggest Nrf2 activation is likely independent of classical oxidative stress or, at best, a result of a transient, low-level redox stress. Moreover, supporting evidence indicates an early endoplasmic reticular (ER) stress response after TFEC treatment, with a time-dependent upregulation of the ER responsive genes gadd34, gadd45, gadd153, and ndr1 . These findings suggest an alternative pathway for Nrf2 activation, i.e., Nrf2 phosphorylation through ER-mediated protein kinases such as PKR-like endoplasmic reticular kinase (PERK). Overall, the results implicate a role for Nrf2 in the cellular response to TFEC toxicity and suggest a previously unrecognized role for the ER in this model of mitochondrially initiated cytotoxicity.