RESUMEN
The human testis is sensitive to toxicant-induced injury but current methods for detecting adverse effects are limited, insensitive and unreliable. Animal studies use sensitive histopathological endpoints to assess toxicity, but require testicular tissue that is not available during human clinical trials. More sensitive and reliable molecular biomarkers of testicular injury are needed to better monitor testicular toxicity in both clinical and preclinical. Adult male Wistar Han rats were exposed for 4weeks to compounds previously associated with testicular injury, including cisplatin (0, 0.2, 0.3, or 0.4mg/kg/day), BI665915 (0, 20, 70, 100mg/kg/d), BI665636 (0, 20, 100mg/kg/d) or BI163538 (0, 70, 150, 300mg/kg/d) to evaluate reproductive toxicity and assess changes in sperm mRNA levels. None of the compounds resulted in any significant changes in body, testis or epididymis weights, nor were there decreases in testicular homogenization resistant spermatid head counts. Histopathological evaluation found that only BI665915 treatment caused any testicular effects, including minor germ cell loss and disorganization of the seminiferous tubule epithelium, and an increase in the number of retained spermatid heads. A custom PCR-array panel was used to assess induced changes in sperm mRNA. BI665915 treatment resulted in a significant increase in clusterin (Clu) levels and decreases in GTPase, IMAP family member 4 (Gimap4), prostaglandin D2 synthase (Ptgds) and transmembrane protein with EGF like and two follistatin like domains 1 (Tmeff1) levels. Correlation analysis between transcript levels and quantitative histopathological endpoints found a modest association between Clu with retained spermatid heads. These results demonstrate that sperm mRNA levels are sensitive molecular indicators of testicular injury that can potentially be translated into a clinical setting.
Asunto(s)
Acetamidas/toxicidad , Cisplatino/toxicidad , Oxadiazoles/toxicidad , ARN Mensajero/biosíntesis , Espermatozoides/efectos de los fármacos , Espermatozoides/metabolismo , Testículo/efectos de los fármacos , Testículo/metabolismo , Animales , Relación Dosis-Respuesta a Droga , Evaluación Preclínica de Medicamentos/métodos , Masculino , Tamaño de los Órganos/efectos de los fármacos , Tamaño de los Órganos/fisiología , Ratas , Ratas Wistar , Espermatogénesis/efectos de los fármacos , Espermatogénesis/fisiología , Espermatozoides/patología , Testículo/patologíaRESUMEN
Although histopathology is considered the gold standard for assessing testicular toxicity in the nonclinical setting, identification of noninvasive biomarkers for testicular injury are desirable to improve safety monitoring capabilities for clinical trials. Inhibin B has been investigated as a noninvasive biomarker for testicular toxicity. This study investigates the correlation of Inhibin B in Wistar Han rats with the onset and reversibility of testicular histopathology from classical testicular toxicants carbendazim, cetrorelix acetate (CTX), and 1,2-dibromo-3-chloropropane (DBCP). The dose regimen included Interim (day 8), Drug (day 29), and nondosing Recovery (day 58) Phases. Inhibin B was not effective at predicting the onset of carbendazim- or CTX-mediated testicular pathology in rats. Inhibin B was reduced by DBCP administration at the end of the Drug Phase only, acting as a leading indicator of the onset of testicular toxicity before the onset of germ cell depletion. However, since Inhibin B was only decreased at the end of the Dosing Phase and not at the Recovery Phase, when the onset of testicular pathology occurred, it is unclear if monitoring Inhibin B would provide sufficient advanced warning for the onset of testicular pathology. Furthermore, follicle stimulating hormone was decreased following CTX and DBCP administration in the Interim Phase and CTX in the Drug Phase. Inhibin B has limited predictive capacity as a leading testicular biomarker in rats.
Asunto(s)
Bencimidazoles/toxicidad , Carbamatos/toxicidad , Hormona Liberadora de Gonadotropina/análogos & derivados , Inhibinas/sangre , Propano/análogos & derivados , Testículo/patología , Animales , Bencimidazoles/administración & dosificación , Biomarcadores/sangre , Peso Corporal/efectos de los fármacos , Carbamatos/administración & dosificación , Hormona Folículo Estimulante/sangre , Hormona Liberadora de Gonadotropina/administración & dosificación , Hormona Liberadora de Gonadotropina/toxicidad , Hormona Luteinizante/sangre , Masculino , Tamaño de los Órganos/efectos de los fármacos , Propano/administración & dosificación , Propano/toxicidad , Ratas , Ratas Wistar , Análisis de Supervivencia , Testículo/efectos de los fármacos , Testículo/metabolismoRESUMEN
The Developmental and Reproductive Toxicity Technical Committee of the Health and Environmental Sciences Institute hosted a working consortium of companies to evaluate a new commercially available analytic assay for Inhibin B in rat serum or plasma. After demonstrating that the kit was stable and robust, the group performed a series of independent pathogenesis studies (23 different compound/investigator combinations) designed to examine the correlation between the appearance of lesions in the testis and changes in circulating levels of Inhibin B. These studies were reported individually in the previous articles in this series (this issue), and are discussed in this paper. For roughly half of these exposures, lesions appeared well before Inhibin B changed. A few of the studies showed a good correlation between seminiferous tubule damage and reduced circulating Inhibin B levels, while for seven exposures, circulating Inhibin B was reduced with no detectable alteration in testis histology. Whether this indicates a prodromal response or a false-positive signal will require further investigation. These exceptions could plausibly suggest some value of circulating Inhibin B as a useful biomarker in some circumstances. However, for roughly half of these exposures, Inhibin B appeared to be a lagging biomarker, requiring significant damage to the seminiferous tubules before a consistent and credible reduction in circulating levels of Inhibin B was observed.
Asunto(s)
Ecología , Salud , Inhibinas/sangre , Testículo/metabolismo , Testículo/patología , Animales , Biomarcadores/sangre , Masculino , Ratas , Ratas Sprague-Dawley , Ratas WistarRESUMEN
The nonclinical safety assessments for gene therapies are evolving, leveraging over 20 years of experimental and clinical experience. Despite the growing experience with these therapeutics, there are no approved harmonized global regulatory documents for developing gene therapies with only the ICH (International Council for Harmonization of Technical Requirements for Pharmaceuticals for Human Use) S12 guidance on nonclinical biodistribution currently under discussion. Several health authorities have issued guidance over the last 15 years on the nonclinical safety aspects for gene therapy products, but many of the recommendations are limited to high-level concepts on nonclinical safety aspects or altogether silent on key topics. Historically, this approach was appropriately vague given our relatively small dataset of nonclinical experience, where a comprehensive and detailed regulatory guidance approach was unlikely to be appropriate to address all scenarios. However, harmonization of key considerations and assumptions can provide a consistent basis for developing the appropriate nonclinical safety development plans for individual programs, reducing uncertainty across regulatory regions and unnecessary animal use. Several key areas of nonclinical safety testing are nearing maturation for a harmonized approach, including species selection, certain aspects of study design, study duration, and unintended genomic integration risks. Furthermore, several emerging topics are unaddressed in current regulatory guidance for gene therapy products, which will become key areas of differentiation for the next generation of therapeutics. These topics include redosing, juvenile/pediatric safety, and reproductive/developmental safety testing, where relevant experience from other modalities can be applied. The rationale and potential study design considerations for these topics will be proposed, acknowledging that certain aspects of gene therapy development are not considered appropriate for harmonization. This article provides an overview of the current nonclinical safety regulatory landscape, summarizes typical nonclinical safety study designs, highlights areas of uncertainty, and discusses emerging topics that warrant consideration. Specific recommendations and perspectives are provided to inform future regulatory discussions and harmonization efforts.
Asunto(s)
Terapia Genética , Humanos , Animales , Niño , Distribución Tisular , Terapia Genética/efectos adversosRESUMEN
BACKGROUND: Testicular toxicity (TT) is a sporadic and challenging issue in pharmaceutical drug development. Efforts to develop TT screening assays or biomarkers have been overshadowed by consortium efforts to predict drug-induced toxicities such as hepatic injury, which are encountered more frequently. METHODS: To gauge the current state of the field and to prioritize future TT activities, the International Life Sciences Institute-Health and Environmental Sciences Institute Developmental and Reproductive Toxicology (DART) Technical Committee sponsored a survey to better understand the incidence and nature of TT findings encountered during drug development. RESULTS: Highlights from the 16 survey respondents include: (1) Although preclinical TT was encountered relatively infrequently, half of the participants observed repeated problems with TT during pharmaceutical development, (2) despite control measures such as use of sexually mature animals to diminish confounding effects of spurious lesions, interpretation of TT remains a challenge, (3) "traditional" evaluation tools such as hormonal monitoring and newer approaches such as -omics are utilized to investigate testicular changes, and (4) an understanding of the risk and relevance of TT findings is achieved through joint consideration of factors such as species specificity, potential mode of action, and safety margins. CONCLUSIONS: TT remains a relatively uncommon but persistent challenge in pharmaceutical development. Although current preclinical TT approaches appear to be effective in limiting the occurrence of pharmaceutical candidate attrition in clinical trials, improved biomarker or screening platforms would allow companies to identify TT at an earlier stage, thus decreasing the time and resources expended on safety evaluation of pharmaceutical candidates.
Asunto(s)
Diseño de Fármacos , Efectos Colaterales y Reacciones Adversas Relacionados con Medicamentos , Preparaciones Farmacéuticas/metabolismo , Testículo/efectos de los fármacos , Pruebas de Toxicidad , Animales , Evaluación Preclínica de Medicamentos , Humanos , MasculinoRESUMEN
The Developmental and Reproductive Toxicology Technical Committee of the ILSI Health and Environmental Sciences Institute has undertaken a project to address the impact of juvenile animal studies on pediatric drug development. A workshop, sponsored and organized by the Health and Environmental Sciences Institute Developmental and Reproductive Toxicity Technical Committee, was held on May 5-6, 2010, in Washington, DC, to discuss the outcome of a global survey and the value of juvenile animal studies in the development of drugs intended for use in pediatric patients. During this workshop, summary data from the 2009-2010 survey were presented, and breakout sessions were used to discuss specific case studies to try to assess the impact of juvenile animal studies performed to support specific pediatric drug development. The objectives of the Workshop on The Value of Juvenile Animal Studies were to (1) provide a forum for scientists representing industry, academia, and regulatory agencies to discuss the impact of juvenile animal studies on pediatric drug development, (2) evaluate summary data from the survey to understand how the juvenile study data are being used and their impact in labeling and risk assessment, (3) discuss selected case studies from the survey to highlight key findings, and (4) identify the areas of improvement for the designs of juvenile animal studies. The take home message that resonated from the workshop discussions was that well-designed juvenile animal studies have demonstrated value in support of certain pediatric drug development programs. However, it was also clear that a juvenile animal study is not always warranted.
Asunto(s)
Animales de Laboratorio/crecimiento & desarrollo , Evaluación Preclínica de Medicamentos , Modelos Animales , Animales , Medición de Riesgo , Pruebas de ToxicidadRESUMEN
The recent approval of aducanumab for Alzheimer's disease has heightened the interest in therapies targeting the amyloid hypothesis. Our research has focused on identification of novel compounds to improve amyloid processing by modulating gamma secretase activity, thereby addressing a significant biological deficit known to plague the familial form of the disease. Herein, we describe the design, synthesis, and optimization of new gamma secretase modulators (GSMs) based on previously reported oxadiazine 1. Potency improvements with a focus on predicted and measured properties afforded high-quality compounds further differentiated via robust Aß42 reductions in both rodents and nonhuman primates. Extensive preclinical profiling, efficacy studies, and safety studies resulted in the nomination of FRM-024, (+)-cis-5-(4-chlorophenyl)-6-cyclopropyl-3-(6-methoxy-5-(4-methyl-1H-imidazole-1-yl)pyridin-2-yl)-5,6-dihydro-4H-1,2,4-oxadiazine, as a GSM preclinical candidate for familial Alzheimer's disease.
Asunto(s)
Secretasas de la Proteína Precursora del Amiloide/metabolismo , Encéfalo/metabolismo , Descubrimiento de Drogas , Inhibidores y Moduladores de Gamma Secretasa/farmacología , Péptidos beta-Amiloides/metabolismo , Animales , Área Bajo la Curva , Perros , Inhibidores y Moduladores de Gamma Secretasa/farmacocinética , Semivida , Haplorrinos , Humanos , Ratones , Fragmentos de Péptidos/metabolismo , RatasRESUMEN
The Raf kinase inhibitory protein 1 (RKIP-1) and its orthologs are conserved throughout evolution and widely expressed in eukaryotic organisms. In its non-phosphorylated form RKIP-1 negatively regulates the Raf/MEK/ERK pathway by interfering with the activity of Raf-1. In its phosphorylated state, RKIP-1 dissociates from Raf-1 and inhibits GRK-2, a negative regulator of G-protein coupled receptors (GPCRs). Available data indicate that the phosphorylation of RKIP-1 by PKC can stimulate both the Raf/MEK/ERK and GPCR pathways. RKIP-1 has also been implicated as a negative regulator of the NF-kappaB pathway. Recent studies have shown that phosphorylated RKIP-1 binds to the centrosomal and kinetochore regions of metaphase chromosomes, where it may be involved in regulating the partitioning of chromosomes and the progression through mitosis. The collective evidence indicates that RKIP-1 regulates the activity and mediates the crosstalk between several important cellular signaling pathways. A variety of ablative interventions suggest that reduced RKIP-1 function may influence metastasis, angiogenesis, resistance to apoptosis, and genome integrity. Attenuation of RKIP-1 may also affect cardiac and neurological functions, spermatogenesis, sperm decapacitation, and reproductive behavior. In this review, the role of RKIP-1 in cellular signaling, and especially its functions revealed using a mouse knockout model, are discussed.
Asunto(s)
Proteínas de Unión a Fosfatidiletanolamina/fisiología , Transducción de Señal/fisiología , Enfermedad de Alzheimer/fisiopatología , Animales , Sistema Nervioso Central/fisiología , Ratones , Ratones Noqueados , Neoplasias/fisiopatología , Proteínas de Unión a Fosfatidiletanolamina/genética , Reproducción/fisiología , Espermatogénesis/fisiologíaRESUMEN
The purpose of this study was to examine arm and trunk actions in overhead forehand strokes used in badminton games across skill levels. The participants were 80 students (40 boys, 40 girls) who were randomly selected from video recordings of 300 students ages 16 to 19 years. The videotaped performances of overhead forehand strokes were coded based on three steps of arm action (elbow flexion, elbow and humeral flexion, and upward backswing) and three steps of trunk action (no trunk action, forward-backward movement, and trunk rotation). Students across the four skill levels exhibited different patterns of arm and trunk actions. Students at advanced levels used more mature arm and trunk actions.
Asunto(s)
Envejecimiento/fisiología , Brazo/fisiología , Rendimiento Atlético/fisiología , Fenómenos Biomecánicos/fisiología , Destreza Motora/fisiología , Movimiento/fisiología , Deportes de Raqueta/fisiología , Logro , Adolescente , Adulto , Niño , Desarrollo Infantil/fisiología , Femenino , Humanos , Masculino , Contracción Muscular/fisiología , Distribución por SexoRESUMEN
The purpose of this study was to describe skills and offensive tactics frequently used in pick-up basketball games. 65 participants were recruited from public basketball courts. An observational instrument was developed to analyze the performances of pick-up games. Participants' performances were videotaped and coded. Results indicated that the passing skills most frequently observed in the games were chest pass, overhead pass, and bounce pass. For dribbling, crossover dribble and change-of-pace dribble were frequently observed. Jump shot, set shot, and layup were also frequently used. The offensive tactics frequently used included drive, cut, and set screen. The study may be beneficial for helping young people prepare to play pick-up basketball games.
Asunto(s)
Baloncesto/fisiología , Conocimiento , Destreza Motora/fisiología , Adolescente , Adulto , Conducta Competitiva/fisiología , Femenino , Humanos , Masculino , Persona de Mediana Edad , Aptitud Física/fisiología , Encuestas y Cuestionarios , Grabación en VideoRESUMEN
Raf kinase inhibitor protein-1 (RKIP-1) belongs to the phosphatidyl ethanolamine-binding family of proteins (PEBP), which are highly conserved throughout evolution and widely expressed in tissues of mammalian organisms. RKIP-1 is a modulator of extracellular signal-regulated kinase (ERK), nuclear factor-kappa B (NF-kappaB), and G protein coupled receptor (GPCR) signaling cascades and is implicated as a factor in numerous physiological processes and disease states including metastasis. Testicular germ cells also express high levels of RKIP mRNA during spermatogenesis, particularly from late pachytene spermatocytes through step 15 elongate spermatids. Therefore, the sensitivity of spermatogenesis to injury was compared in wild-type and RKIP-1(-/-) mice. Unlike what has been described with tumor suppressors such as p53, RKIP-1(-/-) and wild-type mice were equally sensitive to germ cell toxicity by x-irradiation as assessed by terminal deoxynucleotidyl transferase biotin-deoxyuridine triphosphate (dUTP) nick end labeling (TUNEL) positivity 9 hours after a 5 Gy exposure and testicular spermatid head counts 15.5 days after 0.5 Gy exposure. Recent findings also indicate that RKIP is a decapacitation factor receptor on sperm. The present study demonstrates that sperm from RKIP-deficient mice are precociously capacitated compared with their wild-type counterparts. Data from mating experiments indicate decreased reproduction rates between crosses of RKIP-1(-/-) male mice and either heterozygous or RKIP-1(-/-) females. Furthermore, RKIP immunolocalization of epididymal sperm supports transfer of the protein from germ cell cytoplasm to the sperm via the cytoplasmic droplet during epididymal transport. Overall, these studies indicate an important role for RKIP in reproduction as a modulator of capacitation but not in the regulation of testicular injury.
Asunto(s)
Infertilidad Masculina/genética , Proteínas de Unión a Fosfatidiletanolamina/deficiencia , Capacitación Espermática/fisiología , Testículo/lesiones , Animales , Epidídimo/patología , Masculino , Ratones , Ratones Noqueados , Proteínas de Unión a Fosfatidiletanolamina/genética , Recuento de Espermatozoides , Cabeza del Espermatozoide , Espermatozoides/citología , Espermatozoides/patologíaRESUMEN
Mice pretreated with the peroxisome proliferator clofibrate (CFB) are resistant to acetaminophen (APAP) hepatotoxicity. Whereas the mechanism of protection is not entirely known, CFB decreases protein adducts formed by the reactive metabolite of APAP, N-acetyl-p-benzoquinone imine (NAPQI). NAD(P)H:quinone oxidoreductase 1 (NQO1) is an enzyme with antioxidant properties that is responsible for the reduction of cellular quinones. We hypothesized that CFB increases NQO1 activity, which in turn enhances the conversion of NAPQI back to the parent APAP. This could explain the decreases in APAP covalent binding and glutathione depletion produced by CFB without affecting APAP bioactivation to NAPQI. Administration of CFB (500mg/kg, i.p.) to male CD-1 mice for 5 or 10 days increased NQO1 protein and activity levels. To evaluate the capacity of NQO1 to reduce NAPQI back to APAP, we utilized a microsomal activating system. Cytochrome P450 enzymes present in microsomes bioactivate APAP to NAPQI, which binds the electrophile trapping agent, N-acetyl cysteine (NAC). We analyzed the formation of APAP-NAC metabolite in the presence of human recombinant NQO1. Results indicate that NQO1 is capable of reducing NAPQI. The capacity of NQO1 to amelioriate APAP toxicity was then evaluated in primary hepatocytes. Primary hepatocytes isolated from mice dosed with CFB are resistant to APAP toxicity. These hepatocytes were also exposed to ES936, a high affinity, and irreversible inhibitor of NQO1 in the presence of APAP. Concentrations of ES936 that resulted in over 94% inhibition of NQO1 activity did not increase the susceptibility of hepatocytes from CFB treated mice to APAP. Whereas NQO1 is mechanistically capable of reducing NAPQI, CFB-mediated hepatoprotection does not appear to be dependent upon enhanced expression of NQO1.
Asunto(s)
Acetaminofén/toxicidad , Analgésicos no Narcóticos/toxicidad , Enfermedad Hepática Inducida por Sustancias y Drogas/enzimología , Clofibrato/farmacología , NAD(P)H Deshidrogenasa (Quinona)/metabolismo , Proliferadores de Peroxisomas/farmacología , Animales , Benzoquinonas/metabolismo , Western Blotting , Enfermedad Hepática Inducida por Sustancias y Drogas/etiología , Enfermedad Hepática Inducida por Sustancias y Drogas/prevención & control , Interacciones Farmacológicas , Inhibidores Enzimáticos/farmacología , Hepatocitos/efectos de los fármacos , Hepatocitos/enzimología , Iminas/metabolismo , Indolquinonas/farmacología , L-Lactato Deshidrogenasa/metabolismo , Masculino , Ratones , Microsomas Hepáticos/efectos de los fármacos , Microsomas Hepáticos/enzimología , NAD(P)H Deshidrogenasa (Quinona)/antagonistas & inhibidoresRESUMEN
Herein we describe the design, synthesis, and evaluation of a novel series of oxadiazine-based gamma secretase modulators obtained via isosteric amide replacement and critical consideration of conformational restriction. Oxadiazine lead 47 possesses good in vitro potency with excellent predicted CNS drug-like properties and desirable ADME/PK profile. This lead compound demonstrated robust Aß42 reductions and subsequent Aß37 increases in both rodent brain and CSF at 30 mg/kg dosed orally.
Asunto(s)
Enfermedad de Alzheimer/tratamiento farmacológico , Secretasas de la Proteína Precursora del Amiloide/metabolismo , Péptidos beta-Amiloides/antagonistas & inhibidores , Diseño de Fármacos , Oxazinas/química , Oxazinas/farmacología , Fragmentos de Péptidos/antagonistas & inhibidores , Enfermedad de Alzheimer/metabolismo , Péptidos beta-Amiloides/metabolismo , Animales , Encéfalo/efectos de los fármacos , Encéfalo/metabolismo , Línea Celular , Humanos , Macaca fascicularis , Ratones , Oxazinas/farmacocinética , Fragmentos de Péptidos/metabolismo , Ratas WistarRESUMEN
Sertoli cells are the primary cellular target for a number of pharmaceutical and environmental testicular toxicants, including 2,5-hexanedione, carbendazim, and mono-(2-ethylhexyl) phthalate. Exposure to these individual compounds can result in impaired Sertoli cell function and subsequent germ cell loss. The loss of testicular function is marked by histopathological changes in seminiferous tubule diameter, seminiferous epithelial sloughing, vacuolization, spermatid head retention, germ cell apoptosis, and altered microtubule assembly. The present study investigates dose-response relationships for these classic Sertoli cell toxicants using histopathology endpoints. Understanding the relationship between the Sertoli cell toxicant dose and its histopathologic manifestations will help establish the sensitivity of these endpoints as markers of testicular injury. The results indicate that no single histopathology endpoint was sensitive on its own in identifying altered testicular morphology resulting from toxicant exposure. However, when multiple endpoints were combined dose-response relationships could be associated with incremental alterations in histopathology. The data generated from these experiments will be useful in further investigating the effects of Sertoli cell toxicant exposure in animal toxicity studies. In addition, this work is fundamental to a planned investigation of the histopathologic and gene expression changes associated with testicular toxicant co-exposures, which may occur both occupationally and environmentally.
Asunto(s)
Bencimidazoles/toxicidad , Carbamatos/toxicidad , Dietilhexil Ftalato/análogos & derivados , Hexanonas/toxicidad , Células de Sertoli/efectos de los fármacos , Animales , Apoptosis/efectos de los fármacos , Peso Corporal/efectos de los fármacos , Dietilhexil Ftalato/toxicidad , Relación Dosis-Respuesta a Droga , Masculino , Tamaño de los Órganos/efectos de los fármacos , ARN Mensajero/análisis , Ratas , Ratas Endogámicas F344 , Testículo/efectos de los fármacos , Testículo/metabolismo , Testículo/patología , Tubulina (Proteína)/genéticaRESUMEN
Pretreatment of mice with the peroxisome proliferator clofibrate (CFB) protects against acetaminophen (APAP)-induced hepatotoxicity. Previous studies have shown that activation of the nuclear peroxisome proliferator activated receptor-alpha (PPARalpha) is required for this effect. The present study utilizes gene expression profile analysis to identify potential pathways contributing to PPARalpha-mediated hepatoprotection. Gene expression profiles were compared between wild type and PPARalpha-null mice pretreated with vehicle or CFB (500 mg/kg, i.p., daily for 10 days) and then challenged with APAP (400 mg/kg, p.o.). Total hepatic RNA was isolated 4 h after APAP treatment and hybridized to Affymetrix Mouse Genome MGU74 v2.0 GeneChips. Gene expression analysis was performed utilizing GeneSpring software. Our analysis identified 53 genes of interest including vanin-1, cell cycle regulators, lipid-metabolizing enzymes, and aldehyde dehydrogenase 2, an acetaminophen binding protein. Vanin-1 could be important for CFB-mediated hepatoprotection because this protein is involved in the synthesis of cysteamine and cystamine. These are potent antioxidants capable of ameliorating APAP toxicity in rodents and humans. HPLC-ESI/MS/MS analysis of liver extracts indicates that enhanced vanin-1 gene expression results in elevated cystamine levels, which could be mechanistically associated with CFB-mediated hepatoprotection.
Asunto(s)
Clofibrato/farmacología , Perfilación de la Expresión Génica/métodos , Hígado/efectos de los fármacos , PPAR alfa/genética , Acetaminofén/administración & dosificación , Acetaminofén/toxicidad , Acil-CoA Oxidasa/genética , Acil-CoA Oxidasa/metabolismo , Amidohidrolasas , Animales , Anticolesterolemiantes/farmacología , Anticolesterolemiantes/uso terapéutico , Moléculas de Adhesión Celular/genética , Moléculas de Adhesión Celular/metabolismo , Enfermedad Hepática Inducida por Sustancias y Drogas , Clofibrato/uso terapéutico , Análisis por Conglomerados , Cistamina/química , Cistamina/metabolismo , Cisteamina/química , Cisteamina/metabolismo , Enoil-CoA Hidratasa/genética , Enoil-CoA Hidratasa/metabolismo , Proteínas Ligadas a GPI , Hígado/metabolismo , Hígado/patología , Hepatopatías/genética , Hepatopatías/prevención & control , Malato Deshidrogenasa/genética , Malato Deshidrogenasa/metabolismo , Masculino , Ratones , Ratones Endogámicos , Ratones Noqueados , Análisis de Secuencia por Matrices de Oligonucleótidos/métodos , PPAR alfa/metabolismo , Panteteína/química , Panteteína/metabolismo , Ácido Pantoténico/química , Ácido Pantoténico/metabolismo , Proliferadores de Peroxisomas/metabolismo , Complejo de la Endopetidasa Proteasomal/genética , Complejo de la Endopetidasa Proteasomal/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Hepatic transporters play a vital role in the disposition of endogenous compounds and xenobiotics in the liver. The current study investigates the expression and regulation of hepatic efflux transporters in response to treatment with the peroxisome proliferator-activated receptor (PPAR)alpha agonist clofibrate (CFB). Changes in mRNA and protein levels for several hepatic transporters were assessed in male CD-1 mice after 10 days of CFB dosing (500 mg/kg i.p.). Administration of CFB up-regulated mRNA levels for breast cancer resistance protein (Bcrp) and multidrug resistance-associated proteins 3 and 4 (Mrp3 and Mrp4, respectively). Western blot analysis confirmed that CFB enhances protein expression of liver Bcrp, Mrp3, and Mrp4 in CD-1 mice. To further characterize the regulation of these hepatic transporters, CFB-mediated changes in transporter mRNA levels were assessed in wild-type (sv/129) and PPARalpha-null male mice. Wild-type mice treated with CFB showed similar changes in mRNA levels for all of these transporters, whereas the PPARalpha-null mice did not. Although protein expression of Mrp3 and Mrp4 in the wild-type mice correlated well with changes in mRNA levels, Bcrp protein was not up-regulated by CFB treatment. These results show that PPARalpha activation by CFB coordinately regulates the hepatic efflux transporters Mrp3 and Mrp4. Induction of Mrp3 and Mrp4 by CFB may alter the disposition of toxicants and xenobiotics that are substrates for these transporters.