RESUMO
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
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), 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
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
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.
Assuntos
Arsênio/farmacologia , Doenças Neurodegenerativas/metabolismo , Doença de Parkinson/metabolismo , alfa-Sinucleína/metabolismo , Animais , Linhagem Celular , Feminino , CamundongosRESUMO
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
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
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
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
3,4-(±)-Methylenedioxymethamphetamine (MDMA, Ecstasy) is a ring-substituted amphetamine derivative with potent psychostimulant properties. The neuropharmacological effects of MDMA are biphasic in nature, initially causing synaptic monoamine release, primarily of serotonin (5-HT). Conversely, the long-term effects of MDMA manifest as prolonged depletions in 5-HT, and reductions in 5-HT reuptake transporter (SERT), indicative of serotonergic neurotoxicity. MDMA-induced 5-HT efflux relies upon disruption of vesicular monoamine storage, which increases cytosolic 5-HT concentrations available for release via a carrier-mediated mechanism. The vesicular monoamine transporter 2 (VMAT2) is responsible for packaging monoamine neurotransmitters into cytosolic vesicles. Thus, VMAT2 is a molecular target for a number of psychostimulant drugs, including methamphetamine and MDMA. We investigated the effects of depressed VMAT2 activity on the adverse responses to MDMA, via reversible inhibition of the VMAT2 protein with Ro4-1284. A single dose of MDMA (20 mg/kg, subcutaneous) induced significant hyperthermia in rats. Ro4-1284 (10 mg/kg, intraperitoneal) pretreatment prevented the thermogenic effects of MDMA, instead causing a transient decrease in body temperature. MDMA-treated rats exhibited marked increases in horizontal velocity and rearing behavior. In the presence of Ro4-1284, MDMA-mediated horizontal hyperlocomotion was delayed and attenuated, whereas rearing activity was abolished. Finally, Ro4-1284 prevented deficits in 5-HT content in rat cortex and striatum, and reduced depletions in striatal SERT staining, 7 days after MDMA administration. In summary, acute inhibition of VMAT2 by Ro4-1284 protected against MDMA-mediated hyperthermia, hyperactivity, and serotonergic neurotoxicity. The data suggest the involvement of VMAT2 in the thermoregulatory, behavioral, and neurotoxic effects of MDMA.
Assuntos
Encéfalo/efeitos dos fármacos , Alucinógenos , N-Metil-3,4-Metilenodioxianfetamina , Síndromes Neurotóxicas/prevenção & controle , Neurônios Serotoninérgicos/efeitos dos fármacos , Serotonina/metabolismo , Proteínas Vesiculares de Transporte de Monoamina/antagonistas & inibidores , 2-etil-1,3,4,6,7,11b-hexaidro-3-isobutil-9,10-dimetoxi-2H-benzo(a)quinolizin-2-ol/farmacologia , Animais , Comportamento Animal/efeitos dos fármacos , Regulação da Temperatura Corporal/efeitos dos fármacos , Encéfalo/metabolismo , Encéfalo/patologia , Encéfalo/fisiopatologia , Modelos Animais de Doenças , Febre/induzido quimicamente , Febre/fisiopatologia , Febre/prevenção & controle , Hipercinese/induzido quimicamente , Hipercinese/prevenção & controle , Hipercinese/psicologia , Masculino , Atividade Motora/efeitos dos fármacos , Síndromes Neurotóxicas/metabolismo , Síndromes Neurotóxicas/fisiopatologia , Síndromes Neurotóxicas/psicologia , Ratos Sprague-Dawley , Neurônios Serotoninérgicos/metabolismo , Neurônios Serotoninérgicos/patologia , Fatores de Tempo , Proteínas Vesiculares de Transporte de Monoamina/metabolismoRESUMO
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
The generation of reactive oxygen species (ROS) has been implicated in the pathogenesis of renal ischemia/reperfusion injury, and many other pathological conditions. DNA strand breaks caused by ROS lead to the activation of poly(ADP-ribose)polymerase-1 (PARP-1), the excessive activation of which can result in cell death. We have utilized a model in which 2,3,5-tris(glutathion-S-yl)hydroquinone (TGHQ), a nephrotoxic and nephrocarcinogenic metabolite of hydroquinone, causes ROS-dependent cell death in human renal proximal tubule epithelial cells (HK-2), to further elucidate the role of PARP-1 in ROS-dependent cell death. TGHQ-induced ROS generation, DNA strand breaks, hyperactivation of PARP-1, rapid depletion of nicotinamide adenine dinucleotide (NAD), elevations in intracellular Ca(2+) concentrations, and subsequent nonapoptotic cell death in both a PARP- and Ca(2+)-dependent manner. Thus, inhibition of PARP-1 with PJ34 completely blocked TGHQ-mediated accumulation of poly(ADP-ribose) polymers and NAD consumption, and delayed HK-2 cell death. In contrast, chelation of intracellular Ca(2+) with BAPTA completely abrogated TGHQ-induced cell death. Ca(2+) chelation also attenuated PARP-1 hyperactivation. Conversely, inhibition of PARP-1 modulated TGHQ-mediated changes in Ca(2+) homeostasis. Interestingly, PARP-1 hyperactivation was not accompanied by the translocation of apoptosis-inducing factor (AIF) from mitochondria to the nucleus, a process usually associated with PARP-dependent cell death. Thus, pathways coupling PARP-1 hyperactivation to cell death are likely to be context-dependent, and therapeutic strategies designed to target PARP-1 need to recognize such variability. Our studies provide new insights into PARP-1-mediated nonapoptotic cell death, during which PARP-1 hyperactivation and elevations in intracellular Ca(2+) are reciprocally coupled to amplify ROS-induced nonapoptotic cell death.
Assuntos
Cálcio/metabolismo , Células Epiteliais/efeitos dos fármacos , Glutationa/análogos & derivados , Hidroquinonas/toxicidade , Poli(ADP-Ribose) Polimerases/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Técnicas de Cultura de Células , Morte Celular/efeitos dos fármacos , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Glutationa/toxicidade , Humanos , Túbulos Renais Proximais/citologia , Poli(ADP-Ribose) Polimerase-1RESUMO
Metabolism of 3,4-(±)-methylenedioxymethamphetamine (MDMA) is necessary to elicit its neurotoxic effects. Perturbations in phase I and phase II hepatic enzymes can alter the neurotoxic profile of systemically administered MDMA. In particular, catechol-O-methyltransferase (COMT) plays a critical role in determining the fraction of MDMA that is converted to potentially neurotoxic metabolites. Thus, cytochrome P450 mediated demethylenation of MDMA, or its N-demethylated metabolite, 3,4-(±)-methylenedioxyamphetamine, give rise to the catechols, N-methyl-α-methyldopamine and α-methyldopamine, respectively. Methylation of these catechols by COMT limits their oxidation and conjugation to glutathione, a process that ultimately gives rise to neurotoxic metabolites. We therefore determined the effects of modulating COMT, a critical enzyme involved in determining the fraction of MDMA that is converted to potentially neurotoxic metabolites, on MDMA-induced toxicity. Pharmacological inhibition of COMT in the rat potentiated MDMA-induced serotonin deficits and exacerbated the acute MDMA-induced hyperthermic response. Using a genetic mouse model of COMT deficiency, in which mice lack a functional COMT gene, such mice displayed greater reductions in dopamine concentrations relative to their wild-type (WT) counterparts. Neither WT nor COMT deficient mice were susceptible to MDMA-induced decreases in serotonin concentrations. Interestingly, mice devoid of COMT were far more susceptible to the acute hyperthermic effects of MDMA, exhibiting greater increases in body temperature that ultimately resulted in death. Our findings support the view that COMT plays a pivotal role in determining the toxic response to MDMA.
Assuntos
3,4-Metilenodioxianfetamina/toxicidade , Catecol O-Metiltransferase/metabolismo , Animais , Sequência de Bases , Benzofenonas/farmacologia , Cromatografia Líquida de Alta Pressão , Primers do DNA , Técnicas Eletroquímicas , Feminino , Febre/induzido quimicamente , Fígado/efeitos dos fármacos , Fígado/enzimologia , Camundongos , Camundongos Endogâmicos C57BL , Norepinefrina/sangue , Ratos , Ratos Sprague-DawleyRESUMO
3,4-(±)-Methylenedioxymethamphetamine (MDMA) is a ring-substituted amphetamine derivative with potent psychostimulant properties. The neuropharmacological effects of MDMA are biphasic in nature, initially causing synaptic monoamine release, primarily of serotonin (5-HT), inducing thermogenesis and hyperactivity (5-HT syndrome). The long-term effects of MDMA manifest as a prolonged depletion in 5-HT, and structural damage to 5-HT nerve terminals. MDMA toxicity is in part mediated by an ability to inhibit the presynaptic 5-HT reuptake transporter (SERT). Using a SERT-knockout (SERT-KO) rat model, we determined the impact of SERT deficiency on thermoregulation, locomotor activity, and neurotoxicity in SERT-KO or Wistar-based wild-type (WT) rats exposed to MDMA. WT and SERT-KO animals exhibited the highest thermogenic responses to MDMA (four times 10 mg/kg, sc at 12 h intervals) during the diurnal (first and third) doses according to peak body temperature and area under the curve (∑°C × h) analysis. Although no differences in peak body temperature were observed between MDMA-treated WT and SERT-KO animals, ∑°C × h following the first MDMA dose was reduced in SERT-KO rats. Exposure to a single dose of MDMA stimulated horizontal velocity in both WT and SERT-KO rats, however, this effect was delayed and attenuated in the KO animals. Finally, SERT-KO rats were insensitive to MDMA-induced long-term (7 days) depletions in 5-HT and its metabolite, 5-hydroxyindole acetic acid, in both cortex and striatum. In conclusion, SERT deficiency modulated MDMA-mediated thermogenesis, hyperactivity and neurotoxicity in KO rats. The data confirm that the SERT is essential for the manifestation of the acute and long-term toxicities of MDMA.
Assuntos
Regulação da Temperatura Corporal/efeitos dos fármacos , Atividade Motora/efeitos dos fármacos , N-Metil-3,4-Metilenodioxianfetamina/toxicidade , Síndromes Neurotóxicas/etiologia , Proteínas da Membrana Plasmática de Transporte de Serotonina/deficiência , Serotonina/metabolismo , Animais , Temperatura Corporal/efeitos dos fármacos , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Técnicas de Silenciamento de Genes , Ácido Hidroxi-Indolacético/metabolismo , Masculino , Síndromes Neurotóxicas/metabolismo , Síndromes Neurotóxicas/fisiopatologia , Ratos Wistar , Proteínas da Membrana Plasmática de Transporte de Serotonina/genética , Fatores de TempoRESUMO
OBJECTIVE: Our objective was to examine the role of hypertriglyceridemia on the capacity of HDL to facilitate ABCA-1 mediated cholesterol efflux in type 2 diabetes (T2DM). METHODS: HDL mediated cholesterol efflux through the ABCA-1 transporter was measured using BHK cell lines in samples of 71 participants with T2DM in the presence or absence of high triglyceride levels (TG). Additionally, HDL mediated efflux was measured in 13 diabetic and non-diabetic participants fasting and four hours after a high-fat test challenge. RESULTS: HDL mediated cholesterol efflux function was increased in participants with T2DM with hypertriglyceridemia when compared to participants with T2DM without hypertriglyceridemia (efflux ratio mean±standard deviation (SD), T2DM+TG: 1.17±0.25 vs. T2DM - TG: 1.03±0.19, p=0.0098). In the fat challenge study, we observed a significant increase in ABCA-1 mediated cholesterol efflux capacity following an ingestion of high-fat test meal by participants in both groups of T2DM (n=6, efflux ratio, mean±SD, pre: 0.86±0.4 vs. post: 1.34±0.6, p=0.01) and non-diabetic participants (n=7, efflux ratio mean±SD pre: 1.24±0.31 vs. post: 1.39±0.42, p=0.04) that was partly explained by the difference in CETP activity (r=0.6, p=0.03). CONCLUSION: Our study suggests that high triglyceride levels facilitate ABCA-1 mediated efflux function of HDL in part by activating CETP.
Assuntos
Transportador 1 de Cassete de Ligação de ATP/fisiologia , Colesterol/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Hipertrigliceridemia/metabolismo , Lipoproteínas HDL/metabolismo , Transportador 1 de Cassete de Ligação de ATP/metabolismo , Idoso , Animais , Transporte Biológico , Células Cultivadas , Proteínas de Transferência de Ésteres de Colesterol/metabolismo , Cricetinae , Estudos Transversais , Diabetes Mellitus Tipo 2/complicações , Feminino , Humanos , Hipertrigliceridemia/etiologia , Masculino , Pessoa de Meia-IdadeRESUMO
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
3,4-(±)-Methylenedioxymethamphetamine (MDMA) and 3,4-(±)-methylenedioxyamphetamine (MDA), a primary metabolite of MDMA, are phenylethylamine derivatives that cause serotonergic neurotoxicity. Although several phenylethylamine derivatives activate microglia, little is known about the effects of MDMA on glial cells, and evidence of MDMA-induced microglial activation remains ambiguous. We initially determined microglial occupancy status of the parietal cortex in rats at various time points following a single neurotoxic dose of MDMA (20mg/kg, SC). A biphasic microglial response to MDMA was observed, with peak microglial occupancy occurring 12- and 72-h post-MDMA administration. Because direct injection of MDMA into the brain does not produce neurotoxicity, the glial response to MDMA metabolites was subsequently examined in vivo and in vitro. Rats were treated with MDA (20mg/kg, SC) followed by ex vivo biopsy culture to determine the activation of quiescent microglia. A reactive microglial response was observed 72 h after MDA administration that subsided by 7 days. In contrast, intracerebroventricular (ICV) administration of MDA failed to produce a microglial response. However, thioether metabolites of MDA derived from α-methyldopamine (α-MeDA) elicited a robust microglial response following icv injection. We subsequently determined the direct effects of various MDMA metabolites on primary cultures of E18 hippocampal mixed glial and neuronal cells. 5-(Glutathion-S-yl)-α-MeDA, 2,5-bis-(glutathion-S-yl)-α-MeDA, and 5-(N-acetylcystein-S-yl)-α-MeDA all stimulated the proliferation of glial fibrillary acidic protein-positive astrocytes at a dose of 10 µM. The findings indicate that glial cells are activated in response to MDMA/MDA and support a role for thioether metabolites of α-MeDA in the neurotoxicity.
Assuntos
3,4-Metilenodioxianfetamina/toxicidade , Microglia/efeitos dos fármacos , N-Metil-3,4-Metilenodioxianfetamina/toxicidade , Síndromes Neurotóxicas/metabolismo , 3,4-Metilenodioxianfetamina/metabolismo , Animais , Astrócitos/efeitos dos fármacos , Astrócitos/metabolismo , Astrócitos/patologia , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Técnicas de Cocultura , Relação Dose-Resposta a Droga , Proteína Glial Fibrilar Ácida/metabolismo , Hipocampo/efeitos dos fármacos , Hipocampo/metabolismo , Hipocampo/patologia , Imuno-Histoquímica , Injeções Intraventriculares , Masculino , Microglia/metabolismo , Microglia/patologia , N-Metil-3,4-Metilenodioxianfetamina/metabolismo , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Neurônios/patologia , Síndromes Neurotóxicas/etiologia , Síndromes Neurotóxicas/patologia , Ratos , Ratos Sprague-Dawley , SulfetosRESUMO
The oxidative modification of apolipoprotein A-I 's methionine148(M148) is associated with defective HDL function in vitro. Multiple Reaction Monitoring (MRM) is a mass spectrometric technique that can be used to quantitate post-translational modifications. In this study, we developed an MRM assay to monitor the abundance ratio of the peptide containing oxidized M148 to the native peptide in Apo A-I. Measurement of the oxidized-to-unoxidized-M148 ratio was reproducible (CV<5%). The extent of methionine M148 oxidation in the HDL of healthy controls, and type 2 diabetic participants with and without prior cardiovascular events (CVD) were then examined. The results suggest a significant increase in the relative ratio of the peptide containing oxidized M148 to the unmodified peptide in the HDL of participants with diabetes and CVD (p<0.001), compared to participants without CVD. Monitoring the abundance ratio of the peptides containing oxidized and unoxidized M148 by MRM provides a means of examining the relationship between M148 oxidation and vascular complications in CVD.
RESUMO
Type 2 diabetes mellitus (T2DM) is an important risk factor for cardiovascular disease (CVD)--the leading cause of death in the United States. Yet not all subjects with T2DM are at equal risk for CVD complications; the challenge lies in identifying those at greatest risk. Therapies directed toward treating conventional risk factors have failed to significantly reduce this residual risk in T2DM patients. Thus newer targets and markers are needed for the development and testing of novel therapies. Herein we review two complementary MS-based approaches--mass spectrometric immunoassay (MSIA) and MS/MS as MRM--for the analysis of plasma proteins and PTMs of relevance to T2DM and CVD. Together, these complementary approaches allow for high-throughput monitoring of many PTMs and the absolute quantification of proteins near the low picomolar range. In this review article, we discuss the clinical relevance of the high density lipoprotein (HDL) proteome and Apolipoprotein A-I PTMs to T2DM and CVD as well as provide illustrative MSIA and MRM data on HDL proteins from T2DM patients to provide examples of how these MS approaches can be applied to gain new insight regarding cardiovascular risk factors. Also discussed are the reproducibility, interpretation, and limitations of each technique with an emphasis on their capacities to facilitate the translation of new biomarkers into clinical practice.