RESUMO
Although all-trans-retinoic acid (ATRA) provides protection against a variety of conditions in vivo, particularly ischemia, the molecular mechanisms underpinning these effects remain unclear. The present studies were designed to assess potential mechanisms by which ATRA affords cytoprotection against renal toxicants in LLC-PK1 cells. Pretreatment of LLC-PK1 cells with ATRA (25 µM) for 24 h afforded cytoprotection against oncotic cell death induced by p-aminophenol (PAP), 2-(glutathion-S-yl)hydroquinone (MGHQ), and iodoacetamide but not against apoptotic cell death induced by cisplatin. Inhibition of protein synthesis with cycloheximide blunted ATRA protection, indicating essential cell survival pathways must be engaged before toxicant exposure to provide cytoprotection. Interestingly, ATRA did not prevent the PAP-induced generation of reactive oxygen species (ROS) nor did it alter glutathione levels. Moreover, ATRA had no significant effect on Nrf2 protein expression, and the Nrf2 inducers sulforaphane and MG132 did not influence ATRA cytoprotection, suggesting cytoprotective pathways beyond those that influence ROS levels contribute to ATRA protection. In contrast, ATRA rapidly (15 min) induced levels of the cellular stress kinases p-ERK and p-AKT at concentrations of ATRA (10 and 25 µM) required for cytoprotection. Consistent with a role for p-ERK in ATRA-mediated cytoprotection, inhibition of p-ERK with PD98059 reduced the ability of ATRA to afford protection against PAP toxicity. Collectively, these data suggest that p-ERK and its downstream targets, independent of ROS and antioxidant signaling, are important contributors to the cytoprotective effects of ATRA against oncotic cell death.
Assuntos
Células Epiteliais/efeitos dos fármacos , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Rim/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo , Tretinoína/farmacologia , Aminofenóis/toxicidade , Animais , Apoptose/efeitos dos fármacos , Cisplatino/toxicidade , Citoproteção , Ativação Enzimática , Células Epiteliais/enzimologia , Células Epiteliais/patologia , Glutationa/análogos & derivados , Glutationa/toxicidade , Iodoacetamida/toxicidade , Rim/enzimologia , Rim/patologia , Células LLC-PK1 , Necrose , Fosforilação , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais/efeitos dos fármacos , Suínos , Fatores de TempoRESUMO
Increased activity of B-Raf has been identified in approximately 7% of human cancers. Treatment of Eker rats (Tsc-2(EK/+) ), bearing a mutation in one allele of the tuberous sclerosis-2 (Tsc-2) gene, with the nephrocarcinogen 2,3,5-tris-(glutathion-S-yl) hydroquinone (TGHQ) results in loss of the wild-type allele of Tsc-2 in renal preneoplastic lesions and tumors. These tumors have increased protein expression of B-Raf, C-Raf (Raf-1), and increased expression and activity of ERK kinase. Similar changes are observed in Raf kinases following TGHQ-mediated transformation of primary renal epithelial cells derived from Tsc-2(EK/+) rats (QTRRE cells), cells that are also null for tuberin. Herein, we utilized LC-MS/MS to identify constitutive phosphorylation of S345 and S483 in both 100- and 95-kDa forms of B-Raf in QTRRE cells. Using microRotofor liquid-phase isoelectric focusing, we identified four fractions of B-Raf that contain different post-translational modification profiles in QTRRE cells. Amplification of the kinase domain of B-Raf from QTRRE cells, outer-stripe of the outer medulla of 8-month TGHQ- or vehicle-treated Tsc-2(+/+) and Tsc-2(EK/+) rats, as well as tumors excised from 8-month TGHQ-treated Tsc-2(EK/+) rats revealed three splice variants of B-Raf within the kinase domain. These splice variants differed by approximately 340, 544, and 600 bp; confirmed by sequencing. No point mutations within the kinase domain of B-Raf were identified. In addition, B-Raf/Raf-1/14-3-3 complex formation in the QTRRE cells was decreased by sorafenib, with concomitant selective decreases in p-ERK levels. Transcriptional and post-translational characterization of critical kinases, such as B-Raf, may contribute to the progression of tuberous sclerosis RCC. (246/250) © 2015 Wiley Periodicals, Inc.
Assuntos
Carcinoma de Células Renais/metabolismo , Glutationa/análogos & derivados , Hidroquinonas/toxicidade , Neoplasias Renais/metabolismo , Proteínas Proto-Oncogênicas B-raf/genética , Proteínas Proto-Oncogênicas B-raf/metabolismo , Esclerose Tuberosa/metabolismo , Animais , Carcinoma de Células Renais/induzido quimicamente , Carcinoma de Células Renais/genética , Linhagem Celular , Transformação Celular Neoplásica/induzido quimicamente , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/metabolismo , Glutationa/toxicidade , Humanos , Neoplasias Renais/induzido quimicamente , Neoplasias Renais/genética , Masculino , Neoplasias Experimentais , Fosforilação , Domínios Proteicos , Processamento de Proteína Pós-Traducional , Proteínas Proto-Oncogênicas B-raf/química , Proteínas Proto-Oncogênicas c-raf , Splicing de RNA/efeitos dos fármacos , Ratos , Esclerose Tuberosa/induzido quimicamente , Esclerose Tuberosa/genética , Proteína 2 do Complexo Esclerose Tuberosa , Proteínas Supressoras de Tumor/deficiênciaRESUMO
Methylglyoxal (MG) is a highly reactive dicarbonyl compound involved in the formation of advanced glycation endproducts (AGE). Levels of MG are elevated in patients with type-2 diabetes mellitus (T2DM), and AGE have been implicated in the progression of diabetic complications. The antihyperglycemic drug metformin (MF) has been suggested to be a scavenger of MG. The present work examined and characterized unequivocally the resulting scavenged product from the metformin-MG reaction. The primary product was characterized by (1)H, (13)C, 2D-HSQC, and HMBC NMR and tandem mass spectrometry. X-ray diffraction analysis determined the structure of the metformin and MG-derived imidazolinone compound as (E)-1,1-dimethyl-2-(5-methyl-4-oxo-4,5-dihydro-1H-imidazol-2-yl)guanidine (IMZ). A LC-MS/MS multiple reaction monitoring method was developed to detect and quantify the presence of IMZ in metformin-treated T2DM patients. Urine from >90 MF-treated T2DM patients was analyzed, with increased levels of MF directly correlating with elevations in IMZ. Urinary MF was detected in the range of 0.17 µM to 23.0 mM, and simultaneous measurement of IMZ concentrations were in the range of 18.8 nM to 4.3 µM. Since plasma concentrations of MG range from 40 nM to 4.5 µM, the level of IMZ production may be of therapeutic significance. Thus, in addition to lowering hepatic gluconeogenesis, metformin also scavenges the highly reactive MG in vivo, thereby reducing potentially detrimental MG protein adducts, with subsequent reductions in diabetic complications.
Assuntos
Hipoglicemiantes/metabolismo , Metformina/metabolismo , Aldeído Pirúvico/química , Adulto , Idoso , Idoso de 80 Anos ou mais , Cromatografia Líquida de Alta Pressão , Cristalografia por Raios X , Diabetes Mellitus Tipo 2/tratamento farmacológico , Diabetes Mellitus Tipo 2/patologia , Feminino , Humanos , Hipoglicemiantes/química , Hipoglicemiantes/uso terapêutico , Imidazolinas/urina , Masculino , Metformina/química , Metformina/uso terapêutico , Pessoa de Meia-Idade , Conformação Molecular , Aldeído Pirúvico/sangue , Espectrometria de Massas em Tandem , Adulto JovemRESUMO
BACKGROUND: Proteomic approaches identifying biomarkers have been applied to asthma to only a very limited extent. METHODS: With an antibody array (RayBiotech, Norcross, GA, USA), the relative intensity and rank differences of 444 proteins were compared in 24 plasma samples obtained at age 3, 11 from children with and 12 without asthma diagnoses at ages 5 and 9. Protein candidates identified by antibody array were quantitated by ELISA in an enlarged sample. Proteins found to differentiate children with and without asthma were also examined for association with known Year 1 asthma risk factors, eczema, and wheeze. RESULTS: In the antibody array, four proteins had rank differences between asthma and non-asthma groups (FDR <0.1). By ELISA, mean log (±s.e.m.) erythropoietin (EPO) level (IU/l) was lower (0.750 ± 0.048 vs. 0.898 ± 0.035; p = 0.006) and mean (±s.e.m.) soluble GP130 (sGP130) level (ng/ml) was higher in the asthma vs. the non-asthma group (302 ± 13 vs. 270 ± 8; p = 0.041). The other 2 array proteins (galactin-3 and eotaxin-3) did not differ by ELISA by asthma. EPO related to the asthma risk factor, first year eczema, whereas sGP130 related to first year wheeze. CONCLUSIONS: Through two independent assessments, age 3 plasma levels of EPO and sGP130 were found related to childhood asthma.
Assuntos
Asma/diagnóstico , Biomarcadores/sangue , Receptor gp130 de Citocina/sangue , Eritropoetina/sangue , Análise Serial de Proteínas/métodos , Anticorpos/metabolismo , Criança , Pré-Escolar , Estudos de Coortes , Eczema , Feminino , Seguimentos , Humanos , Masculino , Proteômica , Sons Respiratórios , Fatores de RiscoRESUMO
Increasing evidence identifies dicarbonyl stress from reactive glucose metabolites, such as methylglyoxal (MG), as a major pathogenic link between hyperglycemia and complications of diabetes. MG covalently modifies arginine residues, yet the site specificity of this modification has not been thoroughly investigated. Sites of MG adduction in the plasma proteome were identified using LC-MS/MS analysis in vitro following incubation of plasma proteins with MG. Treatment of plasma proteins with MG yielded 14 putative MG hotspots from five plasma proteins (albumin [nine hotspots], serotransferrin, haptoglobin [2 hotspots], hemopexin, and Ig lambda-2 chain C regions). The search results revealed two versions of MG-arginine modification, dihydroxyimidazolidine (R+72) and hydroimidazolone (R+54) adducts. One of the sites identified was R257 in human serum albumin, which is a critical residue located in drug binding site I. This site was validated as a target for MG modification by a fluorescent probe displacement assay, which revealed significant drug dissociation at 300 µM MG from a prodan-HSA complex (75 µM). Moreover, twelve human plasma samples (six male, six female, with two type 2 diabetic subjects from both genders) were analyzed using multiple reaction monitoring (MRM) tandem mass spectrometry and revealed the presence of the MG-modified albumin R257 peptide. These data provide insights into the nature of the site-specificity of MG modification of arginine, which may be useful for therapeutic treatments that aim to prevent MG-mediated adverse responses in patients.
Assuntos
Proteínas Sanguíneas/metabolismo , Diabetes Mellitus Tipo 2/sangue , Processamento de Proteína Pós-Traducional , Proteômica , Aldeído Pirúvico/sangue , Arginina , Sítios de Ligação , Biomarcadores/sangue , Cromatografia Líquida de Alta Pressão , Diabetes Mellitus Tipo 2/diagnóstico , Feminino , Ensaios de Triagem em Larga Escala , Humanos , Masculino , Mapeamento de Peptídeos , Ligação Proteica , Carbonilação Proteica , Proteômica/métodos , Albumina Sérica/metabolismo , Albumina Sérica Humana , Espectrometria de Massas por Ionização por Electrospray , Espectrometria de Massas em TandemRESUMO
Acetaminophen (APAP) hepatotoxicity is protected by S-adenosyl-l-methionine (SAMe) treatment 1hour (h) after APAP in C57/Bl6 mice. This study examined protein carbonylation as well as mitochondrial and cytosolic protein adduction by 4-hydroxynonenal (4-HNE) using mass spectrometry (MS) analysis. Additional studies investigated the leakage of mitochondrial proteins and 4-HNE adduction of these proteins. Male C57/Bl6 mice (n=5/group) were divided into the following groups and treated as indicated: Veh (15ml/kg water, ip), SAMe (1.25mmol/kg, ip), APAP (250mg/kg), and SAMe given 1h after APAP (S+A). APAP toxicity was confirmed by an increase (p<0.05) in plasma ALT (U/l) and liver weight/10g body weight relative to the Veh, SAMe and S+A groups 4h following APAP treatment. SAMe administered 1h post-APAP partially corrected APAP hepatotoxicity as ALT and liver weight/10g body weights were lower in the S+A group compared the APAP group. APAP induced leakage of the mitochondrial protein, carbamoyl phosphate synthase-1 (CPS-1) into the cytosol and which was reduced in the S+A group. SAMe further reduced the extent of APAP mediated 4-HNE adduction of CPS-1. MS analysis of hepatic and mitochondrial subcellular fractions identified proteins from APAP treated mice. Site specific 4-HNE adducts were identified on mitochondrial proteins sarcosine dehydrogenase and carbamoyl phosphate synthase-1 (CPS-1). In summary, APAP is associated with 4-HNE adduction of proteins as identified by MS analysis and that CPS-1 leakage was greater in APAP treated mice. SAMe reduced the extent of 4-HNE adduction of proteins as well as leakage of CPS-1.
Assuntos
Acetaminofen , Aldeídos/metabolismo , Antioxidantes/farmacologia , Doença Hepática Induzida por Substâncias e Drogas/prevenção & controle , Fígado/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , S-Adenosilmetionina/farmacologia , Espectrometria de Massas em Tandem , Animais , Carbamoil-Fosfato Sintase (Amônia)/metabolismo , Doença Hepática Induzida por Substâncias e Drogas/etiologia , Doença Hepática Induzida por Substâncias e Drogas/metabolismo , Cromatografia Líquida , Citoproteção , Modelos Animais de Doenças , Fígado/metabolismo , Masculino , Camundongos Endogâmicos BALB C , Mitocôndrias Hepáticas/efeitos dos fármacos , Mitocôndrias Hepáticas/metabolismo , Carbonilação Proteica , Processamento de Proteína Pós-Traducional , Sarcosina Desidrogenase/metabolismoRESUMO
BACKGROUND: HDL carries a rich protein cargo and examining HDL protein composition promises to improve our understanding of its functions. Conventional mass spectrometry methods can be lengthy and difficult to extend to large populations. In addition, without prior enrichment of the sample, the ability of these methods to detect low abundance proteins is limited. Our objective was to develop a high-throughput approach to examine HDL protein composition applicable to diabetes and cardiovascular disease (CVD). METHODS: We optimized two multiplexed assays to examine HDL proteins using a quantitative immunoassay (Multi-Analyte Profiling- MAP) and mass spectrometric-based quantitative proteomics (Multiple Reaction Monitoring-MRM). We screened HDL proteins using human xMAP (90 protein panel) and MRM (56 protein panel). We extended the application of these two methods to HDL isolated from a group of participants with diabetes and prior cardiovascular events and a group of non-diabetic controls. RESULTS: We were able to quantitate 69 HDL proteins using MAP and 32 proteins using MRM. For several common proteins, the use of MRM and MAP was highly correlated (p < 0.01). Using MAP, several low abundance proteins implicated in atherosclerosis and inflammation were found on HDL. On the other hand, MRM allowed the examination of several HDL proteins not available by MAP. CONCLUSIONS: MAP and MRM offer a sensitive and high-throughput approach to examine changes in HDL proteins in diabetes and CVD. This approach can be used to measure the presented HDL proteins in large clinical studies.
Assuntos
Lipoproteínas HDL/sangue , Espectrometria de Massas em Tandem , Idoso , Proteínas Sanguíneas/metabolismo , Doenças Cardiovasculares/sangue , Estudos de Casos e Controles , Diabetes Mellitus/sangue , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Proteoma/metabolismo , Estatísticas não ParamétricasRESUMO
Biologically reactive intermediates formed as endogenous products of various metabolic processes are considered important factors in a variety of human diseases, including Parkinson's disease and other neurological disorders, diabetes and complications thereof, and other inflammatory-associated diseases. Chemical-induced toxicities are also frequently mediated via the bioactivation of relatively stable organic molecules to reactive electrophilic metabolites. Indeed, chemical-induced toxicities have long been known to be associated with the ability of electrophilic metabolites to react with a variety of targets within the cell, including their covalent adduction to nucleophilic residues in proteins, and nucleotides within DNA. Although we possess considerable knowledge of the various biochemical mechanisms by which chemicals undergo metabolic bioactivation, we understand far less about the processes that couple bioactivation to toxicity. Identifying specific sites within a protein, which are targets for adduction, can provide the initial information necessary to determine whether such adventitious posttranslational modifications significantly alter either protein structure and/or function. To address this problem, we have developed mass spectrometry (MS)-based approaches to identify specific amino acid targets of electrophile adduction (electrophile-binding motifs), coupled with molecular modeling of such adducts, to determine the potential structural and functional consequences. Where appropriate, functional assays are subsequently conducted to assess protein function.
Assuntos
Espectrometria de Massas/métodos , Proteínas/química , Proteínas/metabolismo , Proteômica/métodos , Animais , Humanos , Camundongos , Modelos Moleculares , Compostos Orgânicos/química , Compostos Orgânicos/metabolismo , Ligação Proteica , Testes de ToxicidadeRESUMO
Exposure of human bladder urothelial cells (UROtsa) to 50 nM of the arsenic metabolite, monomethylarsonous acid (MMA(III)), for 12 weeks results in irreversible malignant transformation. The ability of continuous, low-level MMA(III) exposure to cause an increase in genotoxic potential by inhibiting repair processes necessary to maintain genomic stability is unknown. Following genomic insult within cellular systems poly(ADP-ribose) polymerase-1 (PARP-1), a zinc finger protein, is rapidly activated and recruited to sites of DNA strand breaks. When UROtsa cells are continuously exposed to 50 nM MMA(III), PARP-1 activity does not increase despite the increase in MMA(III)-induced DNA single-strand breaks through 12 weeks of exposure. When UROtsa cells are removed from continuous MMA(III) exposure (2 weeks), PARP-1 activity increases coinciding with a subsequent decrease in DNA damage levels. Paradoxically, PARP-1 mRNA expression and protein levels are elevated in the presence of continuous MMA(III) indicating a possible mechanism to compensate for the inhibition of PARP-1 activity in the presence of MMA(III). The zinc finger domains of PARP-1 contain vicinal sulfhydryl groups which may act as a potential site for MMA(III) to bind, displace zinc ion, and render PARP-1 inactive. Mass spectrometry analysis demonstrates the ability of MMA(III) to bind a synthetic peptide representing the zinc-finger domain of PARP-1, and displace zinc from the peptide in a dose-dependent manner. In the presence of continuous MMA(III) exposure, continuous 4-week zinc supplementation restored PARP-1 activity levels and reduced the genotoxicity associated with MMA(III). Zinc supplementation did not produce an overall increase in PARP-1 protein levels, decrease the levels of MMA(III)-induced reactive oxygen species, or alter Cu-Zn superoxide dismutase levels. Overall, these results present two potential interdependent mechanisms in which MMA(III) may increase the susceptibility of UROtsa cells to genotoxic insult and/or malignant transformation: elevated levels of MMA(III)-induced DNA damage through the production of reactive oxygen species, and the direct MMA(III)-induced inhibition of PARP-1.
Assuntos
Carcinógenos/toxicidade , Transformação Celular Neoplásica/efeitos dos fármacos , Dano ao DNA/efeitos dos fármacos , Compostos Organometálicos/toxicidade , Inibidores de Poli(ADP-Ribose) Polimerases , Espécies Reativas de Oxigênio/metabolismo , Bexiga Urinária/efeitos dos fármacos , Urotélio/efeitos dos fármacos , Western Blotting , Linhagem Celular , Transformação Celular Neoplásica/metabolismo , Cloretos/farmacologia , Ensaio Cometa , Citometria de Fluxo , Humanos , Poli(ADP-Ribose) Polimerase-1 , Poli(ADP-Ribose) Polimerases/metabolismo , Bexiga Urinária/citologia , Neoplasias da Bexiga Urinária/induzido quimicamente , Urotélio/citologia , Compostos de Zinco/farmacologiaRESUMO
The analysis of self-assembled protein microarrays, using matrix-assisted laser desorption/ionization (MALDI) mass spectrometry, combines two high-throughput platforms for investigation of the proteome. In this article, we describe the fabrication in situ of protein arrays optimized for MALDI characterization. Using the green fluorescent protein (GFP) both as an epitope for immobilization and as a gauge for relative protein expression, we were able to generate amounts of protein on the array slides sufficient for MALDI identification. In addition, expression of N-terminal protein constructs fused to GFP demonstrated mass shifts consistent with that of the full-length protein. We envision this technology to be important for the functional screening of protein interactions.
Assuntos
Análise Serial de Proteínas/métodos , Proteínas/metabolismo , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Proteínas Imobilizadas/química , Proteínas Imobilizadas/genética , Proteínas Imobilizadas/metabolismo , Biossíntese de Proteínas , Proteínas/química , Proteínas/genética , Transcrição GênicaRESUMO
Quantitative methods for estimation of cancer risk have been developed for daily, lifetime human exposures. There are a variety of studies or methodologies available to address less-than-lifetime exposures. However, a common framework for evaluating risk from less-than-lifetime exposures (including short-term and/or intermittent exposures) does not exist, which could result in inconsistencies in risk assessment practice. To address this risk assessment need, a committee of the International Life Sciences Institute (ILSI) Health and Environmental Sciences Institute conducted a multisector workshop in late 2009 to discuss available literature, different methodologies, and a proposed framework. The proposed framework provides a decision tree and guidance for cancer risk assessments for less-than-lifetime exposures based on current knowledge of mode of action and dose-response. Available data from rodent studies and epidemiological studies involving less-than-lifetime exposures are considered, in addition to statistical approaches described in the literature for evaluating the impact of changing the dose rate and exposure duration for exposure to carcinogens. The decision tree also provides for scenarios in which an assumption of potential carcinogenicity is appropriate (e.g., based on structural alerts or genotoxicity data), but bioassay or other data are lacking from which a chemical-specific cancer potency can be determined. This paper presents an overview of the rationale for the workshop, reviews historical background, describes the proposed framework for assessing less-than-lifetime exposures to potential human carcinogens, and suggests next steps.
Assuntos
Carcinógenos/toxicidade , Exposição Ambiental/normas , Mutagênicos/toxicidade , Bioensaio/métodos , Carcinógenos/administração & dosagem , Bases de Dados Factuais , Árvores de Decisões , Relação Dose-Resposta a Droga , Determinação de Ponto Final , Contaminação de Alimentos/análise , Guias como Assunto , Produtos Domésticos/efeitos adversos , Humanos , Mutagênicos/administração & dosagem , National Institute of Environmental Health Sciences (U.S.) , Neoplasias/induzido quimicamente , Praguicidas/efeitos adversos , Medição de Risco , Fatores de Tempo , Estados Unidos , United States Environmental Protection Agency , United States Food and Drug AdministrationRESUMO
Matrix assisted laser desorption ionization (MALDI) mass spectrum images are created from an array of mass spectra collected over a tissue surface. We have increased the mass range of proteins that can be detected in tissue sections from kidneys, heart, lung and brain of different rodent species by a modification of the sandwich technique, which involves co-crystallizing matrix with analyte. A tissue section is placed upon a drop of sinapinic acid matrix dissolved in 90% ethanol and 0.5% Triton X-100. Once the matrix has dried, a seed layer of sinapinic crystals is added as a dispersion in xylene. Additional layers of sinapinic acid are added as solutions in 90% ethanol followed by 50% acetonitrile. Numerous peaks with signal to noise ratio of four or greater are observed between 25 kDa to 50 kDa. This represents approximately 10 times as many peaks as are detected using traditional matrix spotting and spraying.
Assuntos
Diagnóstico por Imagem/métodos , Processamento de Imagem Assistida por Computador/métodos , Rim/química , Proteínas/análise , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Análise de Variância , Animais , Química Encefálica , Pulmão/química , Camundongos , Miocárdio/química , Ratos , Sensibilidade e EspecificidadeRESUMO
Quinones represent an important class of endogenous compounds such as neurotransmitters and coenzyme Q10, electrophilic xenobiotics, and environmental toxicants that have known reactivity based on their ability to redox cycle and generate oxidative stress, as well as to alkylate target proteins. It is likely that topological, chemical, and physical features combine to determine which proteins become targets for chemical adduction. Chemical-induced post-translational modification of certain critical proteins causes a change in structure/function that contributes to the toxicological response to chemical exposure. In this study, we have identified a number of proteins that are modified by quinone-thioethers after administration of 2-(glutathion-S-yl)HQ. Parallel one-dimensional gel electrophoresis was performed, and the Coomassie-stained gel was aligned with the corresponding Western blot, which was probed for adductions. Immunopositive bands were then subjected to trypsin digestion and analyzed via liquid chromatography/tandem mass spectrometry. The proteins that were subsequently identified contained a higher than average (9.7 versus 5.5%) lysine content and numerous stretches of lysine run-ons, which is a presumed electrophile binding motif. Approximately 50% of these proteins have also been identified as targets for electrophilic adduction by a diverse group of chemicals by other investigators, implying overlapping electrophile adductomes. By identifying a motif targeted by electrophiles it becomes possible to make predictions of proteins that may be targeted for adduction and possible sites on these proteins that are adducted. An understanding of proteins targeted for adduction is essential to unraveling the toxicity produced by these electrophiles.
Assuntos
Lisina/química , Quinonas/química , Motivos de Aminoácidos/efeitos dos fármacos , Sequência de Aminoácidos , Animais , Western Blotting , Cromatografia Líquida , Masculino , Espectrometria de Massas , Estrutura Molecular , Ligação Proteica/efeitos dos fármacos , Processamento de Proteína Pós-Traducional , Proteínas/química , Quinonas/farmacologia , RatosRESUMO
3,4-Methylenedioxymethamphetamine (MDMA, Ecstasy) is a widely misused synthetic amphetamine derivative and a serotonergic neurotoxicant in animal models and possibly humans. The underlying mechanism of neurotoxicity involves the formation of reactive oxygen species although their source remains unclear. It has been postulated that MDMA-induced neurotoxicity is mediated via the formation of bioreactive metabolites. In particular, the primary catechol metabolites, 3,4-dihydroxymethamphetamine (HHMA) and 3,4-dihydroxyamphetamine (HHA), subsequently cause the formation of glutathione and N-acetylcysteine conjugates, which retain the ability to redox cycle and are serotonergic neurotoxicants in rats. Although the presence of such metabolites has been recently demonstrated in rat brain microdialysate, their formation in humans has not been reported. The present study describes the detection of 5-(N-acetylcystein-S-yl)-3,4-dihydroxymethamphetamine (N-Ac-5-Cys-HHMA) and 5-(N-acetylcystein-S-yl)-3,4-dihydroxyamphetamine (N-Ac-5-Cys-HHA) in human urine of 15 recreational users of MDMA (1.5 mg/kg) in a controlled setting. The results reveal that in the first 4 h after MDMA ingestion approximately 0.002% of the administered dose was recovered as thioether adducts. Genetic polymorphisms in CYP2D6 and catechol-O-methyltransferase expression, the combination of which are major determinants of steady-state levels of HHMA and 4-hydroxy-3-methoxyamphetamine, probably explain the interindividual variability seen in the recovery of N-Ac-5-Cys-HHMA and N-Ac-5-Cys-HHA. In summary, the formation of neurotoxic thioether adducts of MDMA has been demonstrated for the first time in humans. The findings lend weight to the hypothesis that the bioactivation of MDMA to neurotoxic metabolites is a relevant pathway to neurotoxicity in humans.
Assuntos
N-Metil-3,4-Metilenodioxianfetamina/isolamento & purificação , N-Metil-3,4-Metilenodioxianfetamina/metabolismo , Sulfetos/química , Urina/química , Adulto , Ingestão de Alimentos , Feminino , Humanos , Masculino , Espectrometria de Massas , Taxa de Depuração Metabólica , Adulto JovemRESUMO
3, 4-Methylenedioxymethamphetamine (MDMA) is a hallucinogenic amphetamine derivative. The acute effects of MDMA are hyperthermia, hyperactivity, and behavioral changes, followed by long-term serotonergic neurotoxicity in rats and primates. However, the underlying mechanisms of MDMA neurotoxicity remain elusive. We reported that pretreatment of rats with Ro 4-1284, a reversible inhibitor of the vesicular monoamine transporter 2 (VMAT2), reduced MDMA-induced hyperactivity in rats, abolished the hyperthermic response, and the long-term neurotoxicity. Current studies focused on the effects of co- and/or postinhibition of VMAT2 on the acute and chronic effects of MDMA and on the dose-response relationship between MDMA-induced elevations in body temperature and subsequent reductions in indolamine concentrations. Sprague Dawley rats were treated with MDMA (20, 25, or 27.5 mg/kg sc), and either co- and/or posttreatment with the VMAT2 inhibitor (10 mg/kg ip). Rats simultaneously treated with Ro 4-1284 and MDMA exhibited a more rapid increase in body temperature compared to just MDMA. However, the duration of the elevated body temperature was significantly shortened (approximately 3 h vs approximately 8 h, respectively). A similar body temperature response was observed in rats posttreated (7 h after MDMA) with Ro 4-1284. Despite decreases in the area under the curve (Δtemp X time) of body temperature caused by Ro 4-1284, there were no significant differences in the degree of indolamine depletion between any of the MDMA-treated groups. The results suggest that the neuroprotective effects of VMAT2 inhibition is likely due to the indirect monoamine depleting effects of the Ro 4-1284 pretreatment, rather than by the direct inhibition of VMAT2 function.
Assuntos
2-etil-1,3,4,6,7,11b-hexaidro-3-isobutil-9,10-dimetoxi-2H-benzo(a)quinolizin-2-ol/farmacologia , N-Metil-3,4-Metilenodioxianfetamina/toxicidade , Síndromes Neurotóxicas/prevenção & controle , Proteínas Vesiculares de Transporte de Monoamina/antagonistas & inibidores , Animais , Temperatura Corporal/efeitos dos fármacos , Febre/induzido quimicamente , Febre/tratamento farmacológico , Indóis/metabolismo , Masculino , Síndromes Neurotóxicas/etiologia , Ratos , Ratos Sprague-DawleyRESUMO
2,3,5-Tris-(glutathion-S-yl)hydroquinone (TGHQ) is a nephrotoxic and nephrocarcinogenic metabolite of hydroquinone. TGHQ generates reactive oxygen species (ROS), causing DNA-strand breaks, hyperactivation of PARP-1, increases in intracellular calcium ([Ca2+]i), and cell death. PARP-1 catalyzes the attachment of ADP-ribose polymers (PAR) to target proteins. In human kidney proximal tubule cells, ROS-mediated PARP-1 hyperactivation and elevations in [Ca2+]i are reciprocally coupled. The molecular mechanism of this interaction is unclear. The aim of the present study was to identify ROS-induced PAR-associated proteins to further understand their potential role in cell death. PAR-associated proteins were enriched by immunoprecipitation, identified by LC-MS/MS, and relative abundance was obtained by spectral counting. A total of 356 proteins were PAR-modified following TGHQ treatment. A total of 13 proteins exhibited gene ontology annotations related to calcium. Among these proteins, the general transcription factor II-I (TFII-I) is directly involved in the modulation of [Ca2+]i. TFII-I binding to phospholipase C (PLC) leads to calcium influx via the TRPC3 channel. However, inhibition of TRPC3 or PLC had no effect on TGHQ-mediated cell death, suggesting that their loss of function may be necessary but insufficient to cause cell death. Nevertheless, TGHQ promoted a time-dependent translocation of TFII-I from the nucleus to the cytosol concomitant with a decrease in tyrosine phosphorylation in α/ß-TFII-I. Therefore it is likely that ROS have an important impact on the function of TFII-I, such as regulation of transcription, and DNA translesion synthesis. Our data also shed light on PAR-mediated signaling during oxidative stress, and contributes to the development of strategies to prevent PAR-dependent cell death.
RESUMO
2,3,5-tris(Glutathion-S-yl)hydroquinone (TGHQ), a potent nephrotoxic and nephroncarcinogenic metabolite of benzene and hydroquinone, retains the ability to redox cycle and create oxidative stress. We have previously detected that TGHQ induces ROS-dependent necrotic or apoptotic cell death in renal epithelial HK-2 and human leukemic HL-60â¯cells respectively. Herein, we sought to determine the nature of the Nrf2 regulation in HK-2 and HL-60â¯cells undergoing TGHQ-mediated ROS-dependent cell death, due to the key role of Nrf2 in oxidative stress. Intriguingly, Nrf2 was upregulated in HK-2, but not in HL-60â¯cells, despite the ROS-dependent nature of cell death in both cell types. The possibility that TGHQ targeted the GSK3ß-dependent Nrf2 stabilization pathway in HL-60â¯cells was discounted, whereas TGHQ-induced decreases in Nrf2 phosphorylation at Ser40 site appears to partially underlie the inability of TGHQ to up-regulate Nrf2 expression in HL-60â¯cells. Moreover, whereas the TGHQ-induced post-translational stabilization of Nrf2 in HK-2â¯cells resulted in the expected upregulation of HO1 and NQO1 mRNA, TGHQ actually decreased Nrf2 mRNA in HL-60â¯cells, with a concomitant decrease in NQO1, but not HO1 mRNA. In summary, we define differences between the two cell types that might contribute to the engagement of the Nrf2 signaling pathways. By extension, these data provide evidence that Nrf2 is not necessarily activated in ROS-dependent cell death, and further delve into the knowledge that Nrf2 regulation sensing by cells might be achieved at solely transcriptional level, not related to its degradation.
Assuntos
Apoptose/efeitos dos fármacos , Glutationa/análogos & derivados , Hidroquinonas/farmacologia , Fator 2 Relacionado a NF-E2/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Linhagem Celular , Glutationa/química , Glutationa/farmacologia , Glicogênio Sintase Quinase 3 beta/metabolismo , Células HL-60 , Heme Oxigenase-1/genética , Heme Oxigenase-1/metabolismo , Humanos , Peróxido de Hidrogênio/farmacologia , Hidroquinonas/química , Leupeptinas/farmacologia , NAD(P)H Desidrogenase (Quinona)/genética , NAD(P)H Desidrogenase (Quinona)/metabolismo , Fosforilação/efeitos dos fármacos , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteína Quinase C/metabolismo , Regulação para Cima/efeitos dos fármacosRESUMO
The serotonergic neurotoxicity of 3,4-(+/-)-methylenedioxymethamphetamine (MDMA) appears dependent upon systemic metabolism because direct injection of MDMA into the brain fails to reproduce the neurotoxicity. MDMA is demethylenated to the catechol metabolite N-methyl-alpha-methyldopamine (N-Me-alpha-MeDA). Thioether (glutathione and N-acetylcysteine) metabolites of N-Me-alpha-MeDA are neurotoxic and are present in rat brain following s.c. injection of MDMA. Because multidose administration of MDMA is typical of drug intake during rave parties, the present study was designed to determine the effects of multiple doses of MDMA on the concentration of neurotoxic thioether metabolites in rat brain. Administration of MDMA (20 mg/kg s.c.) at 12-h intervals for a total of four injections led to a significant accumulation of the N-Me-alpha-MeDA thioether metabolites in striatal dialysate. The area under the curve (AUC)(0-300 min) for 5-(glutathion-S-yl)-N-Me-alpha-MeDA increased 33% between the first and fourth injections and essentially doubled for 2,5-bis-(glutathion-S-yl)-N-Me-alpha-MeDA. Likewise, accumulation of the mercapturic acid metabolites was reflected by increases in the AUC(0-300 min) for both 5-(N-acetylcystein-S-yl)-N-Me-alpha-MeDA (35%) and 2,5-bis-(N-acetylcystein-S-yl)-N-Me-alpha-MeDA (85%), probably because processes for their elimination become saturated. Indeed, the elimination half-life of 5-(N-acetylcystein-S-yl)-N-Me-alpha-MeDA and 2,5-bis-(N-acetylcystein-S-yl)-N-Me-alpha-MeDA increased by 53 and 28%, respectively, between the first and third doses. Finally, although the C(max) values for the monothioether conjugates were essentially unchanged after each injection, the values increased by 38 and approximately 50% for 2,5-bis-(glutathion-S-yl)-N-Me-alpha-MeDA and 2,5-bis-(N-acetylcystein-S-yl)-N-Me-alpha-MeDA, respectively, between the first and fourth injections. The data indicate that neurotoxic metabolites of MDMA may accumulate in brain after multiple dosing.
Assuntos
3,4-Metilenodioxianfetamina/farmacocinética , Encéfalo/metabolismo , Serotoninérgicos/farmacocinética , Sulfetos/metabolismo , 3,4-Metilenodioxianfetamina/farmacologia , Animais , Encéfalo/efeitos dos fármacos , Ácido Hidroxi-Indolacético/metabolismo , Masculino , Ratos , Ratos Sprague-Dawley , Serotonina/metabolismo , Serotoninérgicos/farmacologiaRESUMO
2,3,5-tris(Glutathion-S-yl)hydroquinone, a potent nephrotoxic and nephrocarcinogenic metabolite of benzene and hydroquinone, generates reactive oxygen species (ROS) causing DNA strand breaks and the subsequent activation of DNA repair enzymes, including poly(ADP-ribose) polymerase (PARP)-1. Under robust oxidative DNA damage, PARP-1 is hyperactivated, resulting in the depletion of NAD+ and ATP with accompanying elevations in intracellular calcium concentrations (iCa2+), and ultimately necrotic cell death. The role of Ca2+ during PARP-dependent necrotic cell death remains unclear. We therefore sought to determine the relationship between Ca2+ and PARP-1 during ROS-induced necrotic cell death in human renal proximal tubule epithelial cells (HK-2). Our experiments suggest that store-operated Ca2+ channel (SOC) entry contributes to the coupling of PARP-1 activation to increases in iCa2+ during ROS-induced cell death. Poly(ADP-ribose)glycohydrolase (PARG), which catalyzes the degradation of PARs to yield free ADP-ribose (ADPR), is known to activate Ca2+ channels such as TRPM2. However, siRNA knockdown of PARG did not restore cell viability, indicating that free ADPR is not responsible for SOC activation in HK-2 cells. The data indicate that PARP-1 and iCa2+ are coupled through activation of SOC mediated Ca2+ entry in an apparently ADPR-independent fashion; alternative PAR-mediated signaling likely contributes to PARP-dependent necrotic cell death, perhaps via PAR-mediated signaling proteins that regulate iCa2+ homeostasis.
Assuntos
Cálcio/metabolismo , Morte Celular , Glicosídeo Hidrolases/metabolismo , Poli(ADP-Ribose) Polimerase-1/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Linhagem Celular , Células Cultivadas , Glutationa/análogos & derivados , Glutationa/toxicidade , Humanos , Hidroquinonas/toxicidade , Transporte de Íons , Necrose , OxirreduçãoRESUMO
Synucleinopathies, including Parkinson's disease (PD), are neurodegenerative diseases characterized by accumulation of α-synuclein (SYN), a small neuronal protein with prion like properties that plays a central role in PD pathogenesis. SYN can misfold and generate toxic oligomers/aggregates, which can be cytotoxic. Environmental arsenic (As)-containing pesticide use correlates with increased incidence of PD. Moreover, because As exposure can lead to inhibition of autophagic flux we hypothesize that As can facilitate the accumulation of toxic SYN oligomers/aggregates and subsequent increases in markers of autophagy. We therefore examined the role of As in the oligomerization of SYN, and the consequences thereof. Chronic exposure of SH-SY5Y cells overexpressing SYN to As caused a dose-dependent oligomerization of SYN, with concomitant increases in protein ubiquitination and expression of other stress markers (protein glutathione binding, γ-GCS, light chain 3 (LC3)-I/II, P62, and NAD(P)H dehydrogenase quinone 1), indicative of an increased proteotoxic stress. Immunocytochemical analyses revealed an accumulation of SYN, and it's colocalization with LC3, a major autophagic protein. Mice exposed to As (100 ppb) for 1 month, exhibited elevated SYN accumulation in the cortex and striatum, and elevations in protein ubiquitination and LC3-I and II levels. However, tyrosine hydroxylase (TH), an indicator of dopaminergic cell density, was upregulated in the As exposed animals. Because SYN can inhibit TH function, and As can decrease monoamine levels, As exposure possibly leads to compensatory mechanisms leading to an increase in TH expression. Our findings suggest that susceptible individuals may be at higher risk of developing synucleinopathies and/or neurodegeneration due to environmental As exposure.