RESUMEN
Doxorubicin (DOX) is a widely prescribed anthracycline antineoplastic drug for treating human solid tumors and leukemias. However, DOX therapy is limited by a cumulative, dose-dependent, and irreversible cardiomyopathy that occurs with repeated administration. Presumably, a pivotal initiating event of DOX-induced cardiotoxicity is the production of reactive oxygen species (ROS) and oxidation of lipids, DNA, and proteins. We recently identified activation of the Keap1/Nrf2-antioxidant response system-a major cellular defense mechanism against such oxidative stress-as an important response to acute DOX exposure in vitro. In the present study, we address the hypothesis that dysregulation of this pathway in cardiac tissue is also manifested in vivo following chronic DOX administration. Male, Sprague-Dawley rats received 6 weekly injections of 2 mg/kg (s.c.) DOX or saline followed by a 5-week drug-free period prior to analysis of cardiac tissue transcripts and proteins. In contrast to in vitro findings, the Keap1/Nrf2-antioxidant response system was suppressed in hearts of DOX-treated animals and consistent with the observed decrease in protein abundance for Nrf2 and PGAM5, both of which are substrates for Keap1. Although this shift in Keap1/Nrf2 suppresses the antioxidant pathway, the concurrent loss of PGAM5 could function as a signal for disposal of damaged mitochondria from the cell, thus removing the source of ROS. These findings identify the Keap1/Nrf2 and Keap1/PGAM5 pathways as important responses to DOX-induced cardiac injury in vivo; disruption of this system for mitochondrial hormesis may be an important contributing factor to cardiotoxicity after chronic drug administration.
Asunto(s)
Cardiopatías/metabolismo , Proteína 1 Asociada A ECH Tipo Kelch/metabolismo , Mitocondrias Cardíacas/metabolismo , Mitofagia , Miocitos Cardíacos/metabolismo , Factor 2 Relacionado con NF-E2/metabolismo , Animales , Cardiotoxicidad , Modelos Animales de Enfermedad , Doxorrubicina , Cardiopatías/inducido químicamente , Cardiopatías/genética , Cardiopatías/patología , Proteína 1 Asociada A ECH Tipo Kelch/genética , Masculino , Mitocondrias Cardíacas/patología , Proteínas Mitocondriales/genética , Proteínas Mitocondriales/metabolismo , Miocitos Cardíacos/patología , Factor 2 Relacionado con NF-E2/genética , Fosfoproteínas Fosfatasas/genética , Fosfoproteínas Fosfatasas/metabolismo , Ratas Sprague-Dawley , Especies Reactivas de Oxígeno/metabolismo , Transducción de SeñalRESUMEN
Doxorubicin (DOX) is a widely used treatment for human cancers, but increases the risk of life-threatening congestive heart failure (CHF). DOX-induced mitochondrial damage is cumulative and persistent, similar to that observed clinically for risk of CHF. Recent evidence suggests the persistent nature of this injury is caused by altered regulation of genes important to normal cardiac functioning. We hypothesize that chronic DOX therapy is associated with epigenetic modifications of DNA methylation status, particularly in critical regulators of mitochondrial function and capacity. Cardiac tissue from Sprague Dawley rats receiving injections of DOX (2 mg/kg, s.c.) or saline once a week for 6 weeks, followed by 5 weeks of drug-free holiday was used for Reduced Representation Bisulfite Sequencing to map specific sites of DNA methylation. Comparison of these methylomes indicated DOX exposure alters DNA methylation landscapes, and identified 14 genes with highly altered methylation status. Preliminary functional effects of DNA methylation changes were characterized by quantifying mRNA expression of selected targets (Rbm20, Nmnat2, Klhl29, Cacna1c, Scn5a.) Gene expression of Rbm20, Klhl29, and Nmnat2 were significantly altered in DOX treated animals; Klhl29 and Nmnat2 demonstrated significant decreases in protein expression corresponding to gene expression. Through an epigenotype-to-phenotype approach, this study identifies potential markers and molecular regulators of irreversible DOX-induced cardiovascular toxicity associated with clinically limiting CHF. However, none of the most prevalent genes identified directly relate to mitochondrial structure or function. Thus, the investigation fails to demonstrate a direct association between this altered methylome and persistent mitochondrionopathy associated with chronic doxorubicin cardiac toxicity.
Asunto(s)
Antibióticos Antineoplásicos/toxicidad , Metilación de ADN , Doxorrubicina/toxicidad , Animales , Crecimiento/efectos de los fármacos , Masculino , Estrés Oxidativo/efectos de los fármacos , Biosíntesis de Proteínas/efectos de los fármacos , Ratas , Ratas Sprague-Dawley , Pruebas de Toxicidad Crónica , Transcripción Genética/efectos de los fármacosRESUMEN
Doxorubicin (DOX) is a widely prescribed treatment for a broad scope of cancers, but clinical utility is limited by the cumulative, dose-dependent cardiomyopathy that occurs with repeated administration. DOX-induced cardiotoxicity is associated with the production of reactive oxygen species (ROS) and oxidation of lipids, DNA and proteins. A major cellular defense mechanism against such oxidative stress is activation of the Keap1/Nrf2-antioxidant response element (ARE) signaling pathway, which transcriptionally regulates expression of antioxidant genes such as Nqo1 and Gstp1. In the present study, we address the hypothesis that an initial event associated with DOX-induced oxidative stress is activation of the Keap1/Nrf2-dependent expression of antioxidant genes and that this is regulated through drug-induced changes in redox status of the Keap1 protein. Incubation of H9c2 rat cardiac myoblasts with DOX resulted in a time- and dose-dependent decrease in non-protein sulfhydryl groups. Associated with this was a near 2-fold increase in Nrf2 protein content and enhanced transcription of several of the Nrf2-regulated down-stream genes, including Gstp1, Ugt1a1, and Nqo1; the expression of Nfe2l2 (Nrf2) itself was unaltered. Furthermore, both the redox status and the total amount of Keap1 protein were significantly decreased by DOX, with the loss of Keap1 being due to both inhibited gene expression and increased autophagic, but not proteasomal, degradation. These findings identify the Keap1/Nrf2 pathway as a potentially important initial response to acute DOX-induced oxidative injury, with the primary regulatory events being the oxidation and autophagic degradation of the redox sensor Keap1 protein.
Asunto(s)
Doxorrubicina/toxicidad , Péptidos y Proteínas de Señalización Intracelular/fisiología , Mioblastos Cardíacos/efectos de los fármacos , Mioblastos Cardíacos/metabolismo , Estrés Oxidativo/efectos de los fármacos , Estrés Oxidativo/fisiología , Animales , Antibióticos Antineoplásicos/toxicidad , Línea Celular , Relación Dosis-Respuesta a Droga , Proteína 1 Asociada A ECH Tipo Kelch , Oxidación-Reducción/efectos de los fármacos , Ratas , Especies Reactivas de Oxígeno/metabolismoRESUMEN
The "methionine cycle" plays a critical role in the regulation of concentrations of (S)-adenosylmethionine (AdoMet), the major biological methyl donor. We set out to study sequence variation in genes encoding the enzyme that synthesizes AdoMet in liver, methionine adenosyltransferase 1A (MAT1A) and the major hepatic AdoMet using enzyme, glycine N-methyltransferase (GNMT), as well as functional implications of that variation. We resequenced MAT1A and GNMT using DNA from 288 subjects of three ethnicities, followed by functional genomic and genotype-phenotype correlation studies performed with 268 hepatic biopsy samples. We identified 44 and 42 polymorphisms in MAT1A and GNMT, respectively. Quantitative Western blot analyses for the human liver samples showed large individual variation in MAT1A and GNMT protein expression. Genotype-phenotype correlation identified two genotyped single-nucleotide polymorphisms (SNPs), reference SNP (rs) 9471976 (corrected p = 3.9 × 10(-10)) and rs11752813 (corrected p = 1.8 × 10(-5)), and 42 imputed SNPs surrounding GNMT that were significantly associated with hepatic GNMT protein levels (corrected p values < 0.01). Reporter gene studies showed that variant alleles for both genotyped SNPs resulted in decreased transcriptional activity. Correlation analyses among hepatic protein levels for methionine cycle enzymes showed significant correlations between GNMT and MAT1A (p = 1.5 × 10(-3)) and between GNMT and betaine homocysteine methyltransferase (p = 1.6 × 10(-7)). Our discovery of SNPs that are highly associated with hepatic GNMT protein expression as well as the "coordinate regulation" of methionine cycle enzyme protein levels provide novel insight into the regulation of this important human liver biochemical pathway.
Asunto(s)
Glicina N-Metiltransferasa/genética , Glicina N-Metiltransferasa/metabolismo , Hígado/enzimología , Metionina Adenosiltransferasa/genética , Metionina Adenosiltransferasa/metabolismo , Metionina/metabolismo , Polimorfismo de Nucleótido Simple , Negro o Afroamericano/genética , Pueblo Asiatico/genética , Biopsia , Western Blotting , Ácido Fólico/metabolismo , Regulación Enzimológica de la Expresión Génica , Genes Reporteros , Estudios de Asociación Genética , Genómica/métodos , Genotipo , Células HEK293 , Células Hep G2 , Humanos , Fenotipo , S-Adenosilmetionina/metabolismo , Análisis de Secuencia de ADN , Transfección , Población Blanca/genéticaRESUMEN
Methionine adenosyltransferase (MAT) catalyzes the synthesis of S-adenosylmethionine, the major biological methyl donor. MAT1A and MAT2A encode two distinct MAT isoforms in mammals. MAT2A is expressed in nonhepatic tissues, whereas MAT1A is expressed in the liver. A third gene, MAT2B, encodes a MAT2A regulatory protein. We resequenced MAT2A and MAT2B exons, splice junctions, and flanking regions using 288 DNA samples from three ethnic groups and also imputed additional single nucleotide polymorphisms (SNPs) across both genes using data from the 1000 Genomes Project. For MAT2A, resequencing identified 74 polymorphisms, including two nonsynonymous (ns) SNPs. Functional genomic studies of wild type and the two MAT2A variant allozymes (Val11 and Val205) showed that the Val11 allozyme had approximately 40% decreases in levels of enzyme activity and immunoreactive protein after COS-1 cell transfection. For MAT2B, 44 polymorphisms, 2 nonsynonymous, were identified during resequencing. Neither of the two MAT2B nsSNPs displayed alterations in levels of protein. Imputation using 1000 Genomes Project data resulted in 1730 additional MAT2A and 1997 MAT2B polymorphisms within ± 200 kilobases of each gene, respectively. Coexpression of MAT2A and MAT2B in COS-1 cells resulted in significantly increased MAT enzyme activity that correlated with increased MAT2A and MAT2B immunoreactive protein, apparently as a result of decreased degradation. Finally, studies of mRNA expression in lymphoblastoid cells showed that 7 SNPs in MAT2A and 16 SNPs in MAT2B were significantly associated with mRNA expression with p < 0.01. These observations provide a foundation for future mechanistic and clinical translational pharmacogenomic studies of MAT2A/2B.
Asunto(s)
Metionina Adenosiltransferasa/genética , Metionina Adenosiltransferasa/metabolismo , Animales , Células COS , Chlorocebus aethiops , Exones , Humanos , Metilación , Modelos Moleculares , Polimorfismo de Nucleótido Simple , Dominios y Motivos de Interacción de Proteínas , Análisis de Secuencia de ADN/métodosRESUMEN
Acetaminophen is the leading cause of acute hepatic failure in many developed nations. Acetaminophen hepatotoxicity is mediated by the reactive metabolite N-acetyl-p-benzoquinonimine (NAPQI). We performed a "discovery" genome-wide association study using a cell line-based model system to study the possible contribution of genomics to NAPQI-induced cytotoxicity. A total of 176 lymphoblastoid cell lines from healthy subjects were treated with increasing concentrations of NAPQI. Inhibiting concentration 50 values were determined and were associated with "glutathione pathway" gene single nucleotide polymorphisms (SNPs) and genome-wide basal messenger RNA expression, as well as with 1.3 million genome-wide SNPs. A group of SNPs in linkage disequilibrium on chromosome 3 was highly associated with NAPQI toxicity. The p value for rs2880961, the SNP with the lowest p value, was 1.88 × 10(-7). This group of SNPs mapped to a "gene desert," but chromatin immunoprecipitation assays demonstrated binding of several transcription factor proteins including heat shock factor 1 (HSF1) and HSF2, at or near rs2880961. These chromosome 3 SNPs were not significantly associated with variation in basal expression for any of the genome-wide genes represented on the Affymetrix U133 Plus 2.0 GeneChip. We have used a cell line-based model system to identify a SNP signal associated with NAPQI cytotoxicity. If these observations are validated in future clinical studies, this SNP signal might represent a potential biomarker for risk of acetaminophen hepatotoxicity. The mechanisms responsible for this association remain unclear.