RESUMEN
A key step in drug discovery, common to many disease areas, is preclinical demonstration of efficacy in a mouse model of disease. However, this demonstration and its translation to the clinic can be impeded by mouse-specific pathways of drug metabolism. Here, we show that a mouse line extensively humanized for the cytochrome P450 gene superfamily ("8HUM") can circumvent these problems. The pharmacokinetics, metabolite profiles, and magnitude of drug-drug interactions of a test set of approved medicines were in much closer alignment with clinical observations than in wild-type mice. Infection with Mycobacterium tuberculosis, Leishmania donovani, and Trypanosoma cruzi was well tolerated in 8HUM, permitting efficacy assessment. During such assessments, mouse-specific metabolic liabilities were bypassed while the impact of clinically relevant active metabolites and DDI on efficacy were well captured. Removal of species differences in metabolism by replacement of wild-type mice with 8HUM therefore reduces compound attrition while improving clinical translation, accelerating drug discovery.
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Enfermedades Transmisibles , Descubrimiento de Drogas , Ratones , Animales , Interacciones Farmacológicas , Modelos Animales de Enfermedad , Sistema Enzimático del Citocromo P-450/metabolismo , AceleraciónRESUMEN
Caveolae are small flask-shaped invaginations of the surface membrane which are proposed to recruit and co-localize signaling molecules. The distinctive caveolar shape is achieved by the oligomeric structural protein caveolin, of which three isoforms exist. Aside from the finding that caveolin-3 is specifically expressed in muscle, functional differences between the caveolin isoforms have not been rigorously investigated. Caveolin-3 is relatively cysteine-rich compared to caveolins 1 and 2, so we investigated its cysteine post-translational modifications. We find that caveolin-3 is palmitoylated at 6 cysteines and becomes glutathiolated following redox stress. We map the caveolin-3 palmitoylation sites to a cluster of cysteines in its C terminal membrane domain, and the glutathiolation site to an N terminal cysteine close to the region of caveolin-3 proposed to engage in protein interactions. Glutathiolation abolishes caveolin-3 interaction with heterotrimeric G protein alpha subunits. Our results indicate that a caveolin-3 oligomer contains up to 66 palmitates, compared to up to 33 for caveolin-1. The additional palmitoylation sites in caveolin-3 therefore provide a mechanistic basis by which caveolae in smooth and striated muscle can possess unique phospholipid and protein cargoes. These unique adaptations of the muscle-specific caveolin isoform have important implications for caveolar assembly and signaling.
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Caveolina 3 , Cisteína , Músculo Esquelético , Procesamiento Proteico-Postraduccional , Isoformas de ProteínasRESUMEN
The aryl hydrocarbon receptor (AHR) recognizes xenobiotics as well as natural compounds such as tryptophan metabolites, dietary components and microbiota-derived factors, and it is important for maintenance of homeostasis at mucosal surfaces. AHR activation induces cytochrome P4501 (CYP1) enzymes, which oxygenate AHR ligands, leading to their metabolic clearance and detoxification. Thus, CYP1 enzymes have an important feedback role that curtails the duration of AHR signalling, but it remains unclear whether they also regulate AHR ligand availability in vivo. Here we show that dysregulated expression of Cyp1a1 in mice depletes the reservoir of natural AHR ligands, generating a quasi AHR-deficient state. Constitutive expression of Cyp1a1 throughout the body or restricted specifically to intestinal epithelial cells resulted in loss of AHR-dependent type 3 innate lymphoid cells and T helper 17 cells and increased susceptibility to enteric infection. The deleterious effects of excessive AHR ligand degradation on intestinal immune functions could be counter-balanced by increasing the intake of AHR ligands in the diet. Thus, our data indicate that intestinal epithelial cells serve as gatekeepers for the supply of AHR ligands to the host and emphasize the importance of feedback control in modulating AHR pathway activation.
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Retroalimentación Fisiológica , Intestinos/inmunología , Receptores de Hidrocarburo de Aril/metabolismo , Transducción de Señal , Animales , Citrobacter rodentium/inmunología , Colon/citología , Colon/inmunología , Colon/metabolismo , Colon/microbiología , Citocromo P-450 CYP1A1/metabolismo , Femenino , Inmunidad Innata , Mucosa Intestinal/metabolismo , Intestinos/citología , Intestinos/microbiología , Ligandos , Masculino , Ratones , Células Th17/inmunologíaRESUMEN
Age-related diseases account for a high proportion of the total global burden of disease. Despite recent advances in understanding their molecular basis, there is a lack of suitable early biomarkers to test selected compounds and accelerate their translation to clinical trials. We have investigated the utility of in vivo stress reporter systems as surrogate early biomarkers of the degenerative disease progression. We hypothesized that cellular stress observed in models of human degenerative disease preceded overt cellular damage and at the same time will identify potential cytoprotective pathways. To test this hypothesis, we generated novel accelerated ageing (progeria) reporter mice by crossing the LmnaG609G mice into our oxidative stress/inflammation (Hmox1) and DNA damage (p21) stress reporter models. Histological analysis of reporter expression demonstrated a time-dependent and tissue-specific activation of the reporters in tissues directly associated with Progeria, including smooth muscle cells, the vasculature and gastrointestinal tract. Importantly, reporter expression was detected prior to any perceptible deleterious phenotype. Reporter expression can therefore be used as an early marker of progeria pathogenesis and to test therapeutic interventions. This work also demonstrates the potential to use stress reporter approaches to study and find new treatments for other degenerative diseases.
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Progeria , Ratones , Humanos , Animales , Progeria/genética , Progeria/metabolismo , Daño del ADN/genética , Estrés Oxidativo , Biomarcadores/metabolismo , FenotipoRESUMEN
BACKGROUND: Simultaneous inhibition of multiple components of the BRAF-MEK-ERK cascade (vertical inhibition) has become a standard of care for treating BRAF-mutant melanoma. However, the molecular mechanism of how vertical inhibition synergistically suppresses intracellular ERK activity, and consequently cell proliferation, are yet to be fully elucidated. METHODS: We develop a mechanistic mathematical model that describes how the mutant BRAF inhibitor, dabrafenib, and the MEK inhibitor, trametinib, affect BRAFV600E-MEK-ERK signalling. The model is based on a system of chemical reactions that describes cascade signalling dynamics. Using mass action kinetics, the chemical reactions are re-expressed as ordinary differential equations that are parameterised by in vitro data and solved numerically to obtain the temporal evolution of cascade component concentrations. RESULTS: The model provides a quantitative method to compute how dabrafenib and trametinib can be used in combination to synergistically inhibit ERK activity in BRAFV600E-mutant melanoma cells. The model elucidates molecular mechanisms of vertical inhibition of the BRAFV600E-MEK-ERK cascade and delineates how elevated BRAF concentrations generate drug resistance to dabrafenib and trametinib. The computational simulations further suggest that elevated ATP levels could be a factor in drug resistance to dabrafenib. CONCLUSIONS: The model can be used to systematically motivate which dabrafenib-trametinib dose combinations, for treating BRAFV600E-mutated melanoma, warrant experimental investigation.
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Quinasas MAP Reguladas por Señal Extracelular/antagonistas & inhibidores , Sistema de Señalización de MAP Quinasas , Quinasas de Proteína Quinasa Activadas por Mitógenos/antagonistas & inhibidores , Modelos Biológicos , Modelos Químicos , Proteínas Proto-Oncogénicas B-raf/antagonistas & inhibidores , Quinasas MAP Reguladas por Señal Extracelular/química , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Humanos , Imidazoles/química , Imidazoles/farmacología , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Melanoma/tratamiento farmacológico , Melanoma/enzimología , Melanoma/genética , Quinasas de Proteína Quinasa Activadas por Mitógenos/química , Quinasas de Proteína Quinasa Activadas por Mitógenos/metabolismo , Mutación , Oximas/química , Oximas/farmacología , Inhibidores de Proteínas Quinasas/química , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Proto-Oncogénicas B-raf/química , Proteínas Proto-Oncogénicas B-raf/genética , Proteínas Proto-Oncogénicas B-raf/metabolismo , Piridonas/química , Piridonas/farmacología , Pirimidinonas/química , Pirimidinonas/farmacologíaRESUMEN
The aryl hydrocarbon receptor (AhR), a transcription factor known for mediating xenobiotic toxicity, is expressed in B cells, which are known targets for environmental pollutants. However, it is unclear what the physiological functions of AhR in B cells are. We show here that expression of Ahr in B cells is up-regulated upon B-cell receptor (BCR) engagement and IL-4 treatment. Addition of a natural ligand of AhR, FICZ, induces AhR translocation to the nucleus and transcription of the AhR target gene Cyp1a1, showing that the AhR pathway is functional in B cells. AhR-deficient (Ahr-/-) B cells proliferate less than AhR-sufficient (Ahr+/+) cells following in vitro BCR stimulation and in vivo adoptive transfer models confirmed that Ahr-/- B cells are outcompeted by Ahr+/+ cells. Transcriptome comparison of AhR-deficient and AhR-sufficient B cells identified cyclin O (Ccno), a direct target of AhR, as a top candidate affected by AhR deficiency.
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Linfocitos B/fisiología , Proliferación Celular , Receptores de Hidrocarburo de Aril/metabolismo , Ciclinas/metabolismo , Citocromo P-450 CYP1A1/biosíntesis , Perfilación de la Expresión Génica , Interleucina-4/metabolismo , Receptores de Antígenos de Linfocitos B/metabolismo , Receptores de Hidrocarburo de Aril/deficiencia , Transcripción GenéticaRESUMEN
Pyrrolizidine alkaloids (PAs) are widely occurring phytotoxins which can induce severe liver damage in humans and other mammalian species by mechanisms that are not fully understood. Therefore, we investigated the development of PA hepatotoxicity in vivo, using an acutely toxic dose of the PA senecionine in mice, in combination with intravital two-photon microscopy, histology, clinical chemistry, and in vitro experiments with primary mouse hepatocytes and liver sinusoidal endothelial cells (LSECs). We observed pericentral LSEC necrosis together with elevated sinusoidal marker proteins in the serum of senecionine-treated mice and increased sinusoidal platelet aggregation in the damaged tissue regions. In vitro experiments showed no cytotoxicity to freshly isolated LSECs up to 500 µM senecionine. However, metabolic activation of senecionine by preincubation with primary mouse hepatocytes increased the cytotoxicity to cultivated LSECs with an EC50 of approximately 22 µM. The cytochrome P450 (CYP)-dependency of senecionine bioactivation was confirmed in CYP reductase-deficient mice where no PA-induced hepatotoxicity was observed. Therefore, toxic metabolites of senecionine are generated by hepatic CYPs, and may be partially released from hepatocytes leading to destruction of LSECs in the pericentral region of the liver lobules. Analysis of hepatic bile salt transport by intravital two-photon imaging revealed a delayed uptake of a fluorescent bile salt analogue from the hepatic sinusoids into hepatocytes and delayed elimination. This was accompanied by transcriptional deregulation of hepatic bile salt transporters like Abcb11 or Abcc1. In conclusion, senecionine destroys LSECs although the toxic metabolite is formed in a CYP-dependent manner in the adjacent pericentral hepatocytes.
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Colestasis/inducido químicamente , Sistema Enzimático del Citocromo P-450/metabolismo , Hígado/citología , Alcaloides de Pirrolicidina/toxicidad , Animales , Células Cultivadas , Colestasis/patología , Sistema Enzimático del Citocromo P-450/genética , Células Endoteliales/efectos de los fármacos , Regulación de la Expresión Génica/efectos de los fármacos , Hepatocitos/efectos de los fármacos , Hígado/efectos de los fármacos , Hígado/patología , Masculino , Ratones Endogámicos C57BL , Ratones Noqueados , Necrosis/inducido químicamente , Agregación Plaquetaria/efectos de los fármacos , Alcaloides de Pirrolicidina/farmacocinética , Pruebas de Toxicidad/métodosRESUMEN
The rapid rise in popularity of Electronic Nicotine Delivery Systems (ENDS), or vaping, has not eluded the athletic population. Increasing use of youth sports participants, as well as collegiate and professionals, means sports medicine providers and coaches alike should be familiar with the effects of these devices. Current evidence demonstrates an impact on both the cardiovascular and pulmonary systems. These effects appear less significant than smoking tobacco cigarettes, but the extent and longevity of the consequences of ENDS use is unclear. Mechanisms for recovery, such as sleep and injury healing, also are affected. Lastly, the emergence of life-threatening pulmonary disease associated with ENDS use may have devastating effects on an athlete. This article will highlight the recent literature on the impact of ENDS on athletes and athletic performance, as well as the potentially life-threatening consequences of use.
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Atletas , Sistemas Electrónicos de Liberación de Nicotina , Vapeo , Humanos , Medicina DeportivaRESUMEN
The constitutive androstane receptor (CAR) is a xenobiotic sensor expressed in hepatocytes that activates genes involved in drug metabolism, lipid homeostasis, and cell proliferation. Much progress has been made in understanding the mechanism of activation of human CAR by drugs and xenobiotics. However, many aspects of the activation pathway remain to be elucidated. In this report, we have used viral constructs to express human CAR, its splice variants, and mutant CAR forms in hepatocytes from Car-/- mice in vitro and in vivo. We demonstrate CAR expression rescued the ability of Car-/- hepatocytes to respond to a wide range of CAR activators including phenobarbital. Additionally, two major splice isoforms of human CAR, CAR2 and CAR3, were inactive with almost all the agents tested. In contrast to the current model of CAR activation, ectopic CAR1 is constitutively localized in the nucleus and is loaded onto Cyp2b10 gene in the absence of an inducing agent. In studies to elucidate the role of threonine T38 in CAR regulation, we found that the T38D mutant was inactive even in the presence of CAR activators. However, the T38A mutant was activated by CAR inducers, showing that T38 is not essential for CAR activation. Also, using the inhibitor erlotinib, we could not confirm a role for the epidermal growth factor receptor in CAR regulation. Our data suggest that CAR is constitutively bound to gene regulatory regions and is regulated by exogenous agents through a mechanism which involves protein phosphorylation in the nucleus.
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Cromatina/genética , Hepatocitos/fisiología , Receptores Citoplasmáticos y Nucleares/genética , Activación Transcripcional/genética , Animales , Núcleo Celular/efectos de los fármacos , Núcleo Celular/genética , Células Cultivadas , Receptor de Androstano Constitutivo , Receptores ErbB/genética , Clorhidrato de Erlotinib/farmacología , Femenino , Hepatocitos/efectos de los fármacos , Humanos , Ratones , Ratones Endogámicos C57BL , Fenobarbital/farmacología , Isoformas de Proteínas/genética , Activación Transcripcional/efectos de los fármacosRESUMEN
The anticancer drug ellipticine exerts its genotoxic effects after metabolic activation by cytochrome P450 (CYP) enzymes. The present study has examined the role of cytochrome P450 oxidoreductase (POR) and cytochrome b5 (Cyb5), electron donors to P450 enzymes, in the CYP-mediated metabolism and disposition of ellipticine in vivo. We used Hepatic Reductase Null (HRN) and Hepatic Cytochrome b5/P450 Reductase Null (HBRN) mice. HRN mice have POR deleted specifically in hepatocytes; HBRN mice also have Cyb5 deleted in the liver. Mice were treated once with 10â¯mg/kg body weight ellipticine (nâ¯=â¯4/group) for 24â¯h. Ellipticine-DNA adduct levels measured by 32P-postlabelling were significantly lower in HRN and HBRN livers than in wild-type (WT) livers; however no significant difference was observed between HRN and HBRN livers. Ellipticine-DNA adduct formation in WT, HRN and HBRN livers correlated with Cyp1a and Cyp3a enzyme activities measured in hepatic microsomes in the presence of NADPH confirming the importance of P450 enzymes in the bioactivation of ellipticine in vivo. Hepatic microsomal fractions were also utilised in incubations with ellipticine and DNA in the presence of NADPH, cofactor for POR, and NADH, cofactor for Cyb5 reductase (Cyb5R), to examine ellipticine-DNA adduct formation. With NADPH adduct formation decreased as electron donors were lost which correlated with the formation of the reactive metabolites 12- and 13-hydroxy-ellipticine in hepatic microsomes. No difference in adduct formation was observed in the presence of NADH. Our study demonstrates that Cyb5 contributes to the P450-mediated bioactivation of ellipticine in vitro, but not in vivo.
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Antineoplásicos/metabolismo , Citocromo-B(5) Reductasa/deficiencia , Citocromos b5/deficiencia , Elipticinas/metabolismo , Hepatocitos/enzimología , Hígado/enzimología , Activación Metabólica , Animales , Antineoplásicos/farmacología , Hidrocarburo de Aril Hidroxilasas/metabolismo , Citocromo P-450 CYP3A , Sistema Enzimático del Citocromo P-450/metabolismo , Citocromo-B(5) Reductasa/genética , Citocromos b5/genética , Aductos de ADN/metabolismo , Elipticinas/farmacología , Genotipo , Ratones Endogámicos C57BL , Ratones Noqueados , Microsomas Hepáticos/enzimología , NADPH-Ferrihemoproteína Reductasa/metabolismo , FenotipoRESUMEN
Evidence exists that humans are exposed to plastic microparticles via diet. Data on intestinal particle uptake and health-related effects resulting from microplastic exposure are scarce. Aim of the study was to analyze the uptake and effects of microplastic particles in human in vitro systems and in rodents in vivo. The gastrointestinal uptake of microplastics was studied in vitro using the human intestinal epithelial cell line Caco-2 and thereof-derived co-cultures mimicking intestinal M-cells and goblet cells. Different sizes of spherical fluorescent polystyrene (PS) particles (1, 4 and 10 µm) were used to study particle uptake and transport. A 28-days in vivo feeding study was conducted to analyze transport at the intestinal epithelium and oxidative stress response as a potential consequence of microplastic exposure. Male reporter gene mice were treated three times per week by oral gavage with a mixture of 1 µm (4.55 × 107 particles), 4 µm (4.55 × 107 particles) and 10 µm (1.49 × 106 particles) microplastics at a volume of 10 mL/kg/bw. Effects of particles on macrophage polarization were investigated using the human cell line THP-1 to detect a possible impact on intestinal immune cells. Altogether, the results of the study demonstrate the cellular uptake of a minor fraction of particles. In vivo data show the absence of histologically detectable lesions and inflammatory responses. The particles did not interfere with the differentiation and activation of the human macrophage model. The present results suggest that oral exposure to PS microplastic particles under the chosen experimental conditions does not pose relevant acute health risks to mammals.
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Macrófagos/efectos de los fármacos , Microplásticos/toxicidad , Estrés Oxidativo/efectos de los fármacos , Poliestirenos/administración & dosificación , Administración Oral , Animales , Transporte Biológico , Células CACO-2 , Línea Celular , Técnicas de Cocultivo , Células Caliciformes/metabolismo , Humanos , Absorción Intestinal , Mucosa Intestinal/metabolismo , Masculino , Ratones , Tamaño de la Partícula , Poliestirenos/farmacocinética , Poliestirenos/toxicidadRESUMEN
KEY POINTS: Haem oxygenase 1 (Hmox1) is a cytoprotective enzyme with anti-inflammatory and anti-oxidant properties that is induced in response to multiple noxious environmental stimuli and disease states. Tools to enable its expression to be monitored in vivo have been unavailable until now. In a new Hmox1 reporter model we provide high-fidelity, single-cell resolution blueprints for Hmox1 expression throughout the body of mice. We show for the first time that Hmox1 is constitutively expressed at barrier tissues at the interface between the internal and external environments, and that it is highly induced in muscle cells during systemic inflammation. These data suggest novel biological insights into the role of Hmox1 and pave the way for the use of the model to study the role of environmental stress in disease pathology. ABSTRACT: Hmox1 protein holds great promise as a biomarker of in vivo stress responses as it is highly induced in stressed or damaged cells. However, Hmox1 expression patterns have thus far only been available in simple model organisms with limited relevance to humans. We now report a new Hmox1 reporter line that makes it possible to obtain this information in mice, a premiere model system for studying human disease and toxicology. Using a state-of-the-art strategy, we expressed multiple complementary reporter molecules from the murine Hmox1 locus, including firefly luciferase, to allow long-term, non-invasive imaging of Hmox1 expression, and ß-galactosidase for high-resolution mapping of expression patterns post-mortem. We validated the model by confirming the fidelity of reporter expression, and its responsiveness to oxidative and inflammatory stimuli. In addition to providing blueprints for Hmox1 expression in mice that provide novel biological insights, this work paves the way for the broad application of this model to establish cellular stresses induced by endogenous processes and those resulting from exposure to drugs and environmental agents. It will also enable studies on the role of oxidative stress in the pathogenesis of disease and its prevention.
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Genes Reporteros , Hemo-Oxigenasa 1/metabolismo , Inflamación/diagnóstico , Proteínas de la Membrana/metabolismo , Estrés Oxidativo , Animales , Femenino , Hemo-Oxigenasa 1/genética , Inflamación/genética , Inflamación/metabolismo , Luciferasas/metabolismo , Masculino , Proteínas de la Membrana/genética , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , beta-Galactosidasa/metabolismoRESUMEN
Benzo[a]pyrene (BaP) is an environmental pollutant that, based on evidence largely from in vitro studies, exerts its genotoxic effects after metabolic activation by cytochrome P450s. In the present study, Hepatic Reductase Null (HRN) and Hepatic Cytochrome b 5 /P450 Reductase Null (HBRN) mice have been used to study the role of P450s in the metabolic activation of BaP in vivo. In HRN mice, cytochrome P450 oxidoreductase (POR), the electron donor to P450, is deleted specifically in hepatocytes. In HBRN mice the microsomal haemoprotein cytochrome b 5 , which can also act as an electron donor from cytochrome b 5 reductase to P450s, is also deleted in the liver. Wild-type (WT), HRN and HBRN mice were treated by i.p. injection with 125 mg/kg body weight BaP for 24 h. Hepatic microsomal fractions were isolated from BaP-treated and untreated mice. In vitro incubations carried out with BaP-pretreated microsomal fractions, BaP and DNA resulted in significantly higher BaP-DNA adduct formation with WT microsomal fractions compared to those from HRN or HBRN mice. Adduct formation (i.e. 10-(deoxyguanosin-N2-yl)-7,8,9-trihydroxy-7,8,9,10-tetrahydro-BaP [dG-N2-BPDE]) correlated with observed CYP1A activity and metabolite formation (i.e. BaP-7,8-dihydrodiol) when NADPH or NADH was used as enzymatic cofactors. BaP-DNA adduct levels (i.e. dG-N2-BPDE) in vivo were significantly higher (~ sevenfold) in liver of HRN mice than WT mice while no significant difference in adduct formation was observed in liver between HBRN and WT mice. Our results demonstrate that POR and cytochrome b 5 both modulate P450-mediated activation of BaP in vitro. However, hepatic P450 enzymes in vivo appear to be more important for BaP detoxification than its activation.
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Benzo(a)pireno/metabolismo , Citocromo-B(5) Reductasa/metabolismo , Aductos de ADN/metabolismo , Hepatocitos/enzimología , NADPH-Ferrihemoproteína Reductasa/metabolismo , Animales , Ratones , Ratones Noqueados , Microsomas Hepáticos/enzimologíaRESUMEN
This corrects the article DOI: 10.1038/bjc.2016.363.
RESUMEN
Ponatinib, a pan-BCR-ABL tyrosine kinase inhibitor for the treatment of chronic myeloid leukemia (CML), causes severe side effects including vascular occlusions, pancreatitis, and liver toxicity, although the underlying mechanisms remain unclear. Modifications of critical proteins through reactive metabolites are thought to be responsible for a number of adverse drug reactions. In vitro metabolite screening of ponatinib with human liver microsomes and glutathione revealed unambiguous signals of ponatinib-glutathione (P-GSH) adducts. Further profiling of human cytochrome P450 (P450) indicated that CYP1A1 was the predominant P450 enzyme driving this reaction. P-GSH conjugate formation paralleled the disappearance of hydroxylated ponatinib metabolites, suggesting the initial reaction was epoxide generation. Mouse glutathione S-transferase p1 (mGstp1) further enhanced P-GSH adduct formation in vitro. Ponatinib pharmacokinetics were determined in vivo in wild-type (WT) mice and mice humanized for CYP1A1/2 and treated with the CYP1A1 inducers 2,3,7,8-tetrachlorodibenzodioxin or 3-methylcholanthrene. Ponatinib exposure was significantly decreased in treated mice compared with controls (7.7- and 2.2-fold for WT and humanized CYP1A1/2, respectively). Interestingly, the P-GSH conjugate was only found in the feces of CYP1A1-induced mice, but not in control animals. Protein adducts were also identified by liquid chromatography-tandem mass spectrometry analysis of mGstp1 tryptic digests. These results indicate that not only could CYP1A1 be involved in ponatinib disposition, which has not been previously reported, but also that electrophilic intermediates resulting from CYP1A1 metabolism in normal tissues may contribute to ponatinib toxicity. These data are consistent with a recent report that CML patients who smoke are at greater risk of disease progression and premature death.
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Biocatálisis , Citocromo P-450 CYP1A1/metabolismo , Imidazoles/metabolismo , Piridazinas/metabolismo , Animales , Glutatión/metabolismo , Gutatión-S-Transferasa pi/metabolismo , Humanos , Imidazoles/toxicidad , Masculino , Ratones , Piridazinas/toxicidad , Proteínas Recombinantes/metabolismoRESUMEN
Tamoxifen is an estrogen receptor antagonist used in the treatment of breast cancer. It is a prodrug that is converted by several cytochrome P450 enzymes to a primary metabolite, N-desmethyltamoxifen (NDT), which is then further modified by CYP2D6 to a pharmacologically potent secondary metabolite, 4-hydroxy-N-desmethyltamoxifen (endoxifen). Antidepressants (ADs), which are often coprescribed to patients receiving tamoxifen, are also metabolized by CYP2D6 and evidence suggests that a drug-drug interaction between these agents adversely affects the outcome of tamoxifen therapy by inhibiting endoxifen formation. We evaluated this potentially important drug-drug interaction in vivo in mice humanized for CYP2D6 (hCYP2D6). The rate of conversion of NDT to endoxifen by hCYP2D6 mouse liver microsomes (MLMs) in vitro was similar to that of the most active members of a panel of 13 individual human liver microsomes. Coincubation with quinidine, a CYP2D6 inhibitor, ablated endoxifen generation by hCYP2D6 MLMs. The NDT-hydroxylation activity of wild-type MLMs was 7.4 times higher than that of hCYP2D6, whereas MLMs from Cyp2d knockout animals were inactive. Hydroxylation of NDT correlated with that of bufuralol, a CYP2D6 probe substrate, in the human liver microsome panel. In vitro, ADs of the selective serotonin reuptake inhibitor class were, by an order of magnitude, more potent inhibitors of NDT hydroxylation by hCYP2D6 MLMs than were compounds of the tricyclic class. At a clinically relevant dose, paroxetine pretreatment inhibited the generation of endoxifen from NDT in hCYP2D6 mice in vivo. These data demonstrate the potential of ADs to affect endoxifen generation and, thereby, the outcome of tamoxifen therapy.
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Antidepresivos/farmacología , Antineoplásicos Hormonales/metabolismo , Citocromo P-450 CYP2D6/metabolismo , Microsomas Hepáticos/metabolismo , Tamoxifeno/análogos & derivados , Animales , Antineoplásicos Hormonales/sangre , Biotransformación , Cromatografía Liquida , Citocromo P-450 CYP2D6/genética , Interacciones Farmacológicas , Femenino , Técnicas de Sustitución del Gen , Humanos , Técnicas In Vitro , Fase I de la Desintoxicación Metabólica , Ratones , Microsomas Hepáticos/enzimología , Tamoxifeno/sangre , Tamoxifeno/metabolismo , Espectrometría de Masas en TándemRESUMEN
Many of the enzymes involved in xenobiotic metabolism are maintained at a low basal level and are only synthesized in response to activation of upstream sensor/effector proteins. This induction can have implications in a variety of contexts, particularly during the study of the pharmacokinetics, pharmacodynamics, and drug-drug interaction profile of a candidate therapeutic compound. Previously, we combined in vivo SILAC material with a targeted high resolution single ion monitoring (tHR/SIM) LC-MS/MS approach for quantification of 197 peptide pairs, representing 51 drug metabolism enzymes (DME), in mouse liver. However, as important enzymes (for example, cytochromes P450 (Cyp) of the 1a and 2b subfamilies) are maintained at low or undetectable levels in the liver of unstimulated metabolically labeled mice, quantification of these proteins was unreliable. In the present study, we induced DME expression in labeled mice through synchronous ligand-mediated activation of multiple upstream nuclear receptors, thereby enhancing signals for proteins including Cyps 1a, 2a, 2b, 2c, and 3a. With this enhancement, 115 unique, lysine-containing, Cyp-derived peptides were detected in the liver of a single animal, as opposed to 56 in a pooled sample from three uninduced animals. A total of 386 peptide pairs were quantified by tHR/SIM, representing 68 Phase I, 30 Phase II, and eight control proteins. This method was employed to quantify changes in DME expression in the hepatic cytochrome P450 reductase null (HRN) mouse. We observed compensatory induction of several enzymes, including Cyps 2b10, 2c29, 2c37, 2c54, 2c55, 2e1, 3a11, and 3a13, carboxylesterase (Ces) 2a, and glutathione S-transferases (Gst) m2 and m3, along with down-regulation of hydroxysteroid dehydrogenases (Hsd) 11b1 and 17b6. Using DME-enhanced in vivo SILAC material with tHR/SIM, therefore, permits the robust analysis of multiple DME of importance to xenobiotic metabolism, with improved utility for the study of drug pharmacokinetics, pharmacodynamics, and of chemically treated and genetically modified mouse models.
Asunto(s)
Aminoácidos/metabolismo , Enzimas/aislamiento & purificación , Marcaje Isotópico/métodos , Hígado/enzimología , Modelos Biológicos , Animales , Técnicas de Cultivo de Célula , Cromatografía Liquida , Grupo Citocromo a/aislamiento & purificación , Masculino , Ratones , Ratones Endogámicos C57BL , NADPH-Ferrihemoproteína Reductasa/genética , Espectrometría de Masas en TándemRESUMEN
Bile acids are synthesized from cholesterol in the liver and subjected to multiple metabolic biotransformations in hepatocytes, including oxidation by cytochromes P450 (CYPs) and conjugation with taurine, glycine, glucuronic acid, and sulfate. Mice and rats can hydroxylate chenodeoxycholic acid (CDCA) at the 6ß-position to form α-muricholic acid (MCA) and ursodeoxycholic acid (UDCA) to form ß-MCA. However, MCA is not formed in humans to any appreciable degree and the mechanism for this species difference is not known. Comparison of several Cyp-null mouse lines revealed that α-MCA and ß-MCA were not detected in the liver samples from Cyp2c-cluster null (Cyp2c-null) mice. Global bile acid analysis further revealed the absence of MCAs and their conjugated derivatives, and high concentrations of CDCA and UDCA in Cyp2c-null mouse cecum and feces. Analysis of recombinant CYPs revealed that α-MCA and ß-MCA were produced by oxidation of CDCA and UDCA by Cyp2c70, respectively. CYP2C9-humanized mice have similar bile acid metabolites as the Cyp2c-null mice, indicating that human CYP2C9 does not oxidize CDCA and UDCA, thus explaining the species differences in production of MCA. Because humans do not produce MCA, they lack tauro-ß-MCA, a farnesoid X receptor antagonist in mouse that modulates obesity, insulin resistance, and hepatosteatosis.
Asunto(s)
Ácidos Cólicos/biosíntesis , Sistema Enzimático del Citocromo P-450/fisiología , Animales , Expresión Génica , Células Hep G2 , Humanos , Cinética , Hígado/enzimología , Masculino , Ratones Endogámicos C57BL , Ratones Noqueados , Oxidación-Reducción , Especificidad de la EspecieRESUMEN
BACKGROUND: Although the nuclear factor-erythroid 2-related factor 2 (NRF2) pathway is one of the most frequently dysregulated in cancer, it is not clear whether mutational status is a good predictor of NRF2 activity. Here we utilise four members of the aldo-keto reductase (AKR) superfamily as biomarkers to address this question. METHODS: Twenty-three cell lines of diverse origin and NRF2-pathway mutational status were used to determine the relationship between AKR expression and NRF2 activity. AKR expression was evaluated in lung cancer biopsies and Cancer Genome Atlas (TCGA) and Oncomine data sets. RESULTS: AKRs were expressed at a high basal level in cell lines carrying mutations in the NRF2 pathway. In non-mutant cell lines, co-ordinate induction of AKRs was consistently observed following activation of NRF2. Immunohistochemical analysis of lung tumour biopsies and interrogation of TCGA data revealed that AKRs are enriched in both squamous cell carcinomas (SCCs) and adenocarcinomas that contain somatic alterations in the NRF2 pathway but, in the case of SCC, AKRs were also enriched in most other tumours. CONCLUSIONS: An AKR biomarker panel can be used to determine NRF2 status in tumours. Hyperactivation of the NRF2 pathway is far more prevalent in lung SCC than previously predicted by genomic analyses.
Asunto(s)
Aldehído Reductasa/metabolismo , Biomarcadores/metabolismo , Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Neoplasias Pulmonares/metabolismo , Factor 2 Relacionado con NF-E2/metabolismo , Aldo-Ceto Reductasas , Carcinoma de Pulmón de Células no Pequeñas/patología , Línea Celular Tumoral , Humanos , Neoplasias Pulmonares/patologíaRESUMEN
This symposium summary, sponsored by the ASPET, was held at Experimental Biology 2015 on March 29, 2015, in Boston, Massachusetts. The symposium focused on: 1) the interactions of cytochrome P450s (P450s) with their redox partners; and 2) the role of the lipid membrane in their orientation and stabilization. Two presentations discussed the interactions of P450s with NADPH-P450 reductase (CPR) and cytochrome b5. First, solution nuclear magnetic resonance was used to compare the protein interactions that facilitated either the hydroxylase or lyase activities of CYP17A1. The lyase interaction was stimulated by the presence of b5 and 17α-hydroxypregnenolone, whereas the hydroxylase reaction was predominant in the absence of b5. The role of b5 was also shown in vivo by selective hepatic knockout of b5 from mice expressing CYP3A4 and CYP2D6; the lack of b5 caused a decrease in the clearance of several substrates. The role of the membrane on P450 orientation was examined using computational methods, showing that the proximal region of the P450 molecule faced the aqueous phase. The distal region, containing the substrate-access channel, was associated with the membrane. The interaction of NADPH-P450 reductase (CPR) with the membrane was also described, showing the ability of CPR to "helicopter" above the membrane. Finally, the endoplasmic reticulum (ER) was shown to be heterogeneous, having ordered membrane regions containing cholesterol and more disordered regions. Interestingly, two closely related P450s, CYP1A1 and CYP1A2, resided in different regions of the ER. The structural characteristics of their localization were examined. These studies emphasize the importance of P450 protein organization to their function.