RESUMO
Acrylamide (AA) is classified as a probable human carcinogen and is ubiquitous in foods processed at high temperatures. The carcinogenicity of AA has been attributed to its active metabolite, glycidamide (GA). Both AA and GA can spontaneously or enzymatically conjugate with glutathione (GSH) to form their corresponding GSH conjugates. Profiling AA-glutathione conjugate (AA-GSH) and GA-glutathione conjugates (2 isomers: GA2-GSH and GA3-GSH) in serum would better illustrate AA detoxification compared with urinary metabolite analysis. However, the lack of AA-, GA2, and GA3-GSH study remains a critical data gap. Our study aimed to investigate the toxicokinetics of AA-, GA2-and GA3-GSH in Sprague Dawley rats treated with 0.1 mg/kg, 1.0 mg/kg, or 5.0 mg/kg AA. Blood samples were collected for LC-MS/MS analysis of the GSH conjugate products. Within 24 h of treatment, we observed rapid formation, elimination, and linear kinetics of AA-, GA2-and GA3-GSH. The ∑GA-GSH AUC/AA-GSH AUC ratios were 0.14-0.29, similar to ∑GA/AA AUC in serum but different from ∑GA/AA-derived urinary mercapturic acids in rodents. Our analysis of AA- and GA-GSHs values represents direct detoxification of AA and GA in vivo. This study advances our understanding of sex and inter-species differences in AA detoxification and may refine the existing kinetic models for a more relevant risk extrapolation.
Assuntos
Acrilamida/toxicidade , Glutationa/análogos & derivados , Acrilamida/química , Acrilamida/metabolismo , Animais , Carcinógenos/química , Carcinógenos/metabolismo , Carcinógenos/toxicidade , Compostos de Epóxi/química , Compostos de Epóxi/metabolismo , Compostos de Epóxi/toxicidade , Feminino , Glutationa/metabolismo , Glutationa/toxicidade , Humanos , Masculino , Redes e Vias Metabólicas , Modelos Biológicos , Ratos , Ratos Sprague-Dawley , ToxicocinéticaRESUMO
Heart transplantation is a life-saving therapy for end-stage organ failure. Organ deterioration during transportation limits storage to 4 hours, limiting hearts available. Approaches ameliorating organ damage could increase the number of hearts acceptable for transplantation. Prior studies show that adipose-derived stem/stromal cell secretome (ASC-S) rescues tissues from postischemic damage in vivo. This study tested whether ASC-S preserved the function of mouse hearts and human induced pluripotent stem cell-derived cardiomyocytes (iCM) exposed to organ transportation and transplantation conditions. Hearts were subjected to cold University of Wisconsin (UW) cardioplegic solution ± ASC-S for 6 hours followed by analysis using the Langendorff technique. In parallel, the effects of ASC-S on the recovery of iCM from UW solution were examined when provided either during or after cold cardioplegia. Exposure of hearts and iCM to UW deteriorated contractile activity and caused cell apoptosis, worsening in iCM as a function of exposure time; these were ameliorated by augmenting with ASC-S. Silencing of superoxide dismutase 3 and catalase expression prior to secretome generation compromised the ASC-S cardiomyocyte-protective effects. In this study, a novel in vitro iCM model was developed to complement a rodent heart model in assessing efficacy of approaches to improve cardiac preservation. ASC-S displays strong cardioprotective activity on iCM either with or following cold cardioplegia. This effect is associated with ASC-S-mediated cellular clearance of reactive oxygen species. The effect of ASC-S on the temporal recovery of iCM function supports the possibility of lengthening heart storage by augmenting cardioplegic transport solution with ASC-S, expanding the pool of hearts for transplantation.
Assuntos
Soluções Cardioplégicas/toxicidade , Células-Tronco Pluripotentes Induzidas/metabolismo , Células-Tronco Mesenquimais/metabolismo , Miócitos Cardíacos/metabolismo , Soluções para Preservação de Órgãos/toxicidade , Recuperação de Função Fisiológica/fisiologia , Adenosina/toxicidade , Alopurinol/toxicidade , Animais , Glutationa/toxicidade , Humanos , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , Insulina/toxicidade , Preparação de Coração Isolado/métodos , Masculino , Células-Tronco Mesenquimais/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Miócitos Cardíacos/efeitos dos fármacos , Rafinose/toxicidade , Recuperação de Função Fisiológica/efeitos dos fármacosRESUMO
Objectives: l-Glutathione (GSH) is an endogenous tripeptide with super antioxidant properties. In this study, preformulation parameters of GSH and its degradation products were fully investigated.Significance: To date, no experimental preformulation data is available for GSH. Therefore, to the author's knowledge, this is the first study to experimentally determine the preformulation parameters of GSH, which can be considered more reliable for further studies.Methods: An HPLC method for GSH was optimized and validated to accurately quantify the GSH amount in solution, used to investigate GSH's solubility and Log P. Differential Scanning Calorimeter and Thermogravimetric Analyzer were used to evaluate the thermal properties of GSH. Polarized microscope and Fourier-transform Infrared Spectroscopy were used to determine GSH's crystal habits and functional groups, respectively. Forced degradation kinetics and the degradation products were investigated and identified by LC-MS, respectively. GSH's cellular cytotoxicity on fibroblasts was investigated by MTT assay.Results: It was determined that GSH has high aqueous solubility (252.7 mg/mL), low Log P (-3.1), a melting endotherm of 195 °C and decomposition at 210°C, negligible moisture content, and a rectangular/cylindrical-shaped crystalline form. Seven degradation products were identified; one of the major degradation products of GSH under different conditions is first order kinetic oxidation into glutathione disulfide. No cytotoxicity was observed when fibroblasts were treated with GSH (0.005-10.000 mg/mL).Conclusions: Precise preformulation parameters of GSH were obtained, and these are imperative for the development and optimization of advanced GSH formulations.
Assuntos
Química Farmacêutica/métodos , Citotoxinas/química , Citotoxinas/toxicidade , Glutationa/química , Glutationa/toxicidade , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/fisiologia , Células Cultivadas , Fenômenos Químicos/efeitos dos fármacos , Citotoxinas/análise , Relação Dose-Resposta a Droga , Composição de Medicamentos/métodos , Fibroblastos/efeitos dos fármacos , Fibroblastos/patologia , Glutationa/análise , Humanos , Cinética , Espectrometria de Massas em Tandem/métodos , Difração de Raios X/métodosRESUMO
A progressive aggregation-induced emission (AIE) strategy is established based on two diverse stimulus-responsive patterns of copper nanoclusters (CuNCs) for imaging of aluminum ions (Al3+) in cellular microenvironment. The non-emissive CuNCs were facilely synthesized with l-glutathione (GSH) as both stabilizing agent and reducing agent, and demonstrated the excellent AIE characteristics in the ethanol/water mixture. Moreover, the dispersed CuNCs can be aggregated to give the AIE behavior in aqueous solutions by reducing the pH value, and could be further aggregated with 94-fold reinforce by introducing Al3+ ascribe to the strong coordination ability between Al3+ and the functional groups of GSH, demonstrating the progressive AIE process. Under endocytosis, the progressive AIE strategy can be employed to distinguish the Al3+ in the locations of lysosome against other organelles due to the acidic microenvironment of lysosome. The progressive AIE advantages of CuNCs provide a new concept for signal transduction, and have the promising applications in decoding the functions of intracellular biomolecules.
Assuntos
Alumínio/metabolismo , Microambiente Celular , Cobre/química , Glutationa/química , Nanoestruturas/química , Cobre/toxicidade , Glutationa/toxicidade , Células HeLa , Humanos , Lisossomos/metabolismo , Microscopia Confocal , Nanoestruturas/toxicidadeRESUMO
The potential mammalian hepatotoxicity of a new class of GSH-responsive cyclodextrin-based nanosponges loaded with the anticancer drug doxorubicin (Dox-GSH-NS) was investigated. Previous studies showed that these nanosponges can release medicaments preferentially in cells having high GSH content, a common feature of chemoresistant cells, and showed enhanced anti-tumoral activity compared to free Dox in vitro and in vivo in cells with high GSH content. Following these promising results, we investigated here the Dox-GSH-NS hepatotoxicity in human HepG2 cells (in vitro) and in the organotypic cultures of rat precision-cut liver slices (PCLS, ex vivo), while their accumulation in rat liver was assessed in vivo. Moreover, the transport in Dox uptake, as well as its efflux, was studied in vitro. Overall, benefiting of the integration of different investigational models, a good safety profile of Dox-GSH-NSs was evidenced, and their hepatotoxicity resulted to be comparable with respect to free Dox both in vitro and ex vivo. Furthermore, in vivo studies showed that the hepatic accumulation of the Dox loaded in the NS is comparable with respect to the free drug. In addition, Dox-GSH-NSs are taken up by active mechanisms, and can escape the efflux drug pump, thus, contributing to overcoming drug resistance.
Assuntos
Antineoplásicos/administração & dosagem , Ciclodextrinas/administração & dosagem , Doxorrubicina/administração & dosagem , Sistemas de Liberação de Medicamentos , Glutationa/administração & dosagem , Nanoestruturas/administração & dosagem , Animais , Antineoplásicos/química , Antineoplásicos/toxicidade , Sobrevivência Celular/efeitos dos fármacos , Cumarínicos/administração & dosagem , Cumarínicos/química , Cumarínicos/toxicidade , Ciclodextrinas/química , Ciclodextrinas/toxicidade , Doxorrubicina/química , Doxorrubicina/toxicidade , Glutationa/química , Glutationa/toxicidade , Células Hep G2 , Humanos , Fígado/metabolismo , Masculino , Nanoestruturas/química , Nanoestruturas/toxicidade , Ratos WistarRESUMO
In this paper, a new strategy was presented for fluorescence labeling and imaging Al3+ in live cells with excess aluminum ions using thiolated fluorescence gold nanoclusters (Au NCs). The glutathione (GSH)-capped Au NCs were prepared via a green, facile one-pot method in aqueous solution and displayed excellent stability, ultrasmall size, monodispersity, and larger Stokes shift, which exhibits a relatively weak fluorescence at 650â¯nm Al3+-induced fluorescence enhancement of the GSH-Au NCs can be observed due to Al3+-triggered aggregation-induced emission (AIE) effect, which allows the role of GSH-Au NCs as a ï¬uorescence light-up probe for detection of Al3+. Moreover, it was demonstrated that the ï¬uorescence probe for Al3+ showed a wide detection range from 100 to 600⯵M and good selectivity against other metal ions and common biomolecule. Furthermore, due to the advantages of excellent biocompatibility, low toxicity, red emission and high specificity, the proposed GSH-Au NCs fluorescence probes are suitable for the imaging of high concentrations of aluminum ions in cells, which can be applied to the diagnosis of cellular aluminum poisoning.
Assuntos
Alumínio/análise , Técnicas Biossensoriais/métodos , Corantes Fluorescentes/química , Glutationa/química , Ouro/química , Nanopartículas Metálicas/química , Linhagem Celular , Fluorescência , Corantes Fluorescentes/efeitos da radiação , Corantes Fluorescentes/toxicidade , Glutationa/toxicidade , Ouro/efeitos da radiação , Ouro/toxicidade , Humanos , Nanopartículas Metálicas/efeitos da radiação , Nanopartículas Metálicas/toxicidade , Raios UltravioletaRESUMO
Vildagliptin (VG), a dipeptidyl peptidase-4 inhibitor, is used for treating type 2 diabetes. On rare occasions, VG causes liver injury as an adverse reaction. One case report suggested the involvement of immune responses in the hepatotoxicity, but the underlying mechanisms are unknown. We recently reported that VG binds covalently in vitro to l-cysteine to produce a thiazoline acid metabolite, M407, implying that the covalent binding may trigger the immune-mediated hepatotoxicity. There was no evidence, however, that such a thiazoline acid metabolite was formed in vivo. In the present study, we administered a single oral dose of VG to male Sprague-Dawley rats, and detected M407 in plasma. The sum of urinary and fecal excretions of M407 reached approximately 2% of the dose 48 hours postdosing. Using bile duct-cannulated rats, we demonstrated that M407 was secreted into bile as a glucuronide, designated as M583. Another newly identified thiazoline metabolite of VG, the cysteinylglycine conjugate M464, was detected in urine, feces, and bile. The formation of M464 was confirmed by in vitro incubation of VG with glutathione even in the absence of metabolic enzymes. A glutathione adduct against the nitrile moiety M611 was also detected in vitro but not in vivo. In summary, we found three new thiazoline-containing thiol adduct metabolites in VG-administered rats. Nonenzymatic covalent binding of VG would likely occur in humans, and it may be relevant to predicting adverse reactions.
Assuntos
Cisteína/metabolismo , Inibidores da Dipeptidil Peptidase IV/farmacocinética , Glutationa/metabolismo , Compostos de Sulfidrila/metabolismo , Vildagliptina/farmacocinética , Animais , Doença Hepática Induzida por Substâncias e Drogas/etiologia , Doença Hepática Induzida por Substâncias e Drogas/prevenção & controle , Cisteína/química , Cisteína/toxicidade , Diabetes Mellitus Tipo 2/tratamento farmacológico , Diabetes Mellitus Tipo 2/metabolismo , Inibidores da Dipeptidil Peptidase IV/administração & dosagem , Inibidores da Dipeptidil Peptidase IV/efeitos adversos , Glutationa/química , Glutationa/toxicidade , Humanos , Masculino , Modelos Animais , Ratos , Ratos Sprague-Dawley , Compostos de Sulfidrila/química , Compostos de Sulfidrila/toxicidade , Vildagliptina/administração & dosagem , Vildagliptina/efeitos adversosRESUMO
BACKGROUND: Storage of donor hearts in cardioplegic solutions supplemented with conditioning agents activating endogenous mitochondrial protective signaling enhanced their postreperfusion recovery. The present study investigates the role of timing and duration of cardiac exposure to cyclosporine A (CsA), another putative mitochondrial protectant, on cardiac functional recovery and potential mechanisms of CsA action in an isolated working rat heart model of donor heart retrieval and storage. METHODS: After measurement of baseline function, hearts were arrested and stored for 6 hours at 4°C in either Celsior alone or Celsior + CsA (0.2 µM), then reperfused for 45 minutes in Krebs solution, when functional recovery was assessed. Two additional groups of Celsior-alone stored hearts were exposed to 0.2 µM CsA for the initial 15 minutes (nonworking period) or the full 45-minute period of reperfusion. Coronary effluent was collected pre- and poststorage for assessment of lactate dehydrogenase release. Tissue samples were collected at the end of each study for immunoblotting and histological studies. RESULTS: CsA supplementation during cold storage or the first 15-minute reperfusion significantly improved functional recovery and significantly increased phospho-AMPKαThr172 and phospho-ULK-1Ser757. Hearts exposed to CsA for 45 minutes at reperfusion recovered poorly with no phospho-AMP-activated protein kinase α activation, decreased phospho-eNOSSer633, and decreased mitochondrial cytochrome c content with increased lactate dehydrogenase release. CONCLUSIONS: Inclusion of CsA during cold storage is cardioprotective. Effects of CsA addition to the perfusate during reperfusion were time dependent, with benefits at 15 minutes but not 45 minutes of reperfusion. The toxic effect with the presence of CsA for the full 45-minute reperfusion is associated with impaired mitochondrial integrity and decreased eNOS phosphorylation.
Assuntos
Soluções Cardioplégicas/farmacologia , Ciclosporina/farmacologia , Transplante de Coração , Coração/efeitos dos fármacos , Soluções para Preservação de Órgãos/farmacologia , Preservação de Órgãos , Proteínas Quinases Ativadas por AMP/metabolismo , Animais , Proteína Homóloga à Proteína-1 Relacionada à Autofagia/metabolismo , Soluções Cardioplégicas/toxicidade , Isquemia Fria , Ciclosporina/toxicidade , Dissacarídeos/farmacologia , Dissacarídeos/toxicidade , Eletrólitos/farmacologia , Eletrólitos/toxicidade , Glutamatos/farmacologia , Glutamatos/toxicidade , Glutationa/farmacologia , Glutationa/toxicidade , Coração/fisiopatologia , Transplante de Coração/efeitos adversos , Histidina/farmacologia , Histidina/toxicidade , Preparação de Coração Isolado , Masculino , Manitol/farmacologia , Manitol/toxicidade , Mitocôndrias Cardíacas/efeitos dos fármacos , Mitocôndrias Cardíacas/metabolismo , Mitocôndrias Cardíacas/patologia , Óxido Nítrico Sintase Tipo III/metabolismo , Soluções para Preservação de Órgãos/toxicidade , Fosforilação , Ratos Wistar , Recuperação de Função Fisiológica , Fatores de TempoRESUMO
Aminochrome induces neurotoxic alpha-synuclein oligomer formation relevant to the etiology of Parkinson's disease. Oxidative stress produces aminochrome from dopamine, but conjugation with glutathione catalyzed by glutathione transferase M2-2 significantly decreases aminochrome-induced toxicity and alpha-synuclein oligomer formation. Notably, in the presence of the aminochrome-glutathione conjugate, previously unknown species of alpha-synuclein oligomers are formed. These aminochrome-glutathione oligomers of alpha-synuclein differ from formerly characterized oligomers and (i) have high molecular weight, and are stable and SDS-resistant, as determined by the Western blot method, (ii) show positive NBT-quinone-protein staining, which indicates the formation of alpha-synuclein adducts containing aminochrome. Furthermore, aminochrome-glutathione alpha-synuclein oligomers (iii) have distinctive shape and size, as determined by transmission electron microscopy, and (iv) are not toxic in U373MG cells. In conclusion, glutathione conjugated with aminochrome induces a new type of alpha-synuclein oligomers of a different size and shape, which have no demonstrable toxicity.
Assuntos
Glutationa/metabolismo , Indolquinonas/metabolismo , Estresse Oxidativo/fisiologia , alfa-Sinucleína/metabolismo , Morte Celular/efeitos dos fármacos , Morte Celular/fisiologia , Linhagem Celular Tumoral , Glutationa/toxicidade , Glutationa Transferase/metabolismo , Glutationa Transferase/toxicidade , Humanos , Indolquinonas/toxicidade , alfa-Sinucleína/toxicidadeRESUMO
BACKGROUND: The misconception about dietary supplements being safe has led many into the in-patient wards. Cellgevity® (CGV) is a Max International premiere antioxidant supplement formula used by a large population. This study evaluated the effects of therapeutic and supra-therapeutic doses of CGV on reproductive function and biochemical indices in Wistar rats. METHODS: Seventy-two Wistar rats weighing 130 ± 15.8 g were grouped into two categories (male or female) of six rats per group. Control group received distilled water (10 ml/kg). Others received therapeutic (14.3 mg/kg or 28.6 mg/kg) and supra-therapeutic CGV doses (1000, 2000 or 3000 mg/kg) body weight per oral respectively. RESULTS: After 60 days, supra-therapeutic doses of CGV reduced sperm motility (p < 0.05) by 31.8%, 31.3% and 34.5% respectively and increased (p < 0.05) abnormality in sperms by 200%, 241% and 141.3% respectively. CGV altered male (luteinizing, follicle stimulating hormones and testosterone) and female reproductive hormones (luteinizing, follicle stimulating hormones estrogen and progesterone) respectively. Therapeutic doses of CGV elevated reduced glutathione, superoxide dismutase, catalase and glutathione S-transferase, although, this was exceeded by supra-therapeutic doses and more in females than male rats. Supra-therapeutic dose (3000 mg/kg CGV) decreased body weight in both male and female rats by 50% (F(1.5, 30) = 1.2, p = 0.041) and 62.7% (F(2.1, 30) = 0.38, p = 0.038) respectively in treated rats. Supratherapeutic (3000 mg/kg) dose of CGV increased (p < 0.05) creatinine level by 99.1% while serum total protein was reduced (p < 0.05) by 60.1% (2000 mg/kg) and 57.2% (3000 mg/kg) respectively in male animals. In Female rats, supra-therapeutic doses of CGV elevated creatinine levels by 72.2% (1000 mg/kg), 60.2% (2000 mg/kg) and 124.8% (3000 mg/kg) respectively and 3000 mg/kg produces elevated serum low density lipoprotein by 34.6% in treated rats. Serum cholesterol, triglycerides, albumin, alkaline phosphatase were unaltered by CGV dosing. Histology shows seminiferous tubules with reduced spermatogenic cells. Also, female rat kidney revealed acute tubular necrosis at highest dose used in this study. CONCLUSION: Overall, these data suggest that pro-oxidant potential of the supra-therapeutic CGV doses is evident. Hence, it is necessary that its administration be done with caution using appropriate doses.
Assuntos
Antioxidantes/toxicidade , Glutationa/toxicidade , Animais , Antioxidantes/administração & dosagem , Catalase/metabolismo , Feminino , Glutationa/administração & dosagem , Glutationa Transferase/metabolismo , Hormônios Esteroides Gonadais/metabolismo , Rim/efeitos dos fármacos , Rim/patologia , Masculino , Ovário/efeitos dos fármacos , Ovário/metabolismo , Ratos Wistar , Motilidade dos Espermatozoides/efeitos dos fármacos , Espermatozoides/anormalidades , Espermatozoides/efeitos dos fármacos , Superóxido Dismutase/metabolismo , Testículo/efeitos dos fármacos , Testículo/metabolismo , Testículo/patologiaRESUMO
Electromechanical coupling studies have described the intervention of nitric oxide and S-nitrosylation processes in Ca2+ release induced by stretch, with heterogeneous findings. On the other hand, ion channel function activated by stretch is influenced by nitric oxide, and concentration-dependent biphasic effects upon several cellular functions have been described. The present study uses isolated and perfused rabbit hearts to investigate the changes in mechanoelectric feedback produced by two different concentrations of the nitric oxide carrier S-nitrosoglutathione. Epicardial multielectrodes were used to record myocardial activation at baseline and during and after left ventricular free wall stretch using an intraventricular device. Three experimental series were studied: (a) control (n = 10); (b) S-nitrosoglutathione 10 µM (n = 11); and (c) S-nitrosoglutathione 50 µM (n = 11). The changes in ventricular fibrillation (VF) pattern induced by stretch were analyzed and compared. S-nitrosoglutathione 10 µM did not modify VF at baseline, but attenuated acceleration of the arrhythmia (15.6 ± 1.7 vs. 21.3 ± 3.8 Hz; p < 0.0001) and reduction of percentile 5 of the activation intervals (42 ± 3 vs. 38 ± 4 ms; p < 0.05) induced by stretch. In contrast, at baseline using the 50 µM concentration, percentile 5 was shortened (38 ± 6 vs. 52 ± 10 ms; p < 0.005) and the complexity index increased (1.77 ± 0.18 vs. 1.27 ± 0.13; p < 0.0001). The greatest complexity indices (1.84 ± 0.17; p < 0.05) were obtained during stretch in this series. S-nitrosoglutathione 10 µM attenuates the effects of mechanoelectric feedback, while at a concentration of 50 µM the drug alters the baseline VF pattern and accentuates the increase in complexity of the arrhythmia induced by myocardial stretch.
Assuntos
Antiarrítmicos/toxicidade , Glutationa/análogos & derivados , Mecanorreceptores/metabolismo , Mecanotransdução Celular , Doadores de Óxido Nítrico/toxicidade , Nitrocompostos/toxicidade , Fibrilação Ventricular/induzido quimicamente , Fibrilação Ventricular/prevenção & controle , Potenciais de Ação/efeitos dos fármacos , Animais , Sinalização do Cálcio , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Retroalimentação Fisiológica , Glutationa/metabolismo , Glutationa/toxicidade , Frequência Cardíaca/efeitos dos fármacos , Preparação de Coração Isolado , Óxido Nítrico/metabolismo , Doadores de Óxido Nítrico/metabolismo , Nitrocompostos/metabolismo , Coelhos , Fibrilação Ventricular/metabolismo , Fibrilação Ventricular/fisiopatologiaRESUMO
Trichloroethylene (TCE) is a ubiquitous environmental toxicant that is a liver and kidney carcinogen. Conjugation of TCE with glutathione (GSH) leads to formation of nepthrotoxic and mutagenic metabolites postulated to be critical for kidney cancerdevelopment; however, relatively little is known regarding their tissue levels as previous analytical methods for their detection lacked sensitivity. Here, an LC-MS/MS-based method for simultaneous detection of S-(1,2-dichlorovinyl)-glutathione (DCVG), S-(1,2-dichlorovinyl)-L-cysteine (DCVC), and N-acetyl-S-(1,2-dichlorovinyl)-L-cysteine (NAcDCVC) in multiple mouse tissues was developed. This analytical method is rapid, sensitive (limits of detection (LOD) 3-30 fmol across metabolites and tissues), and robust to quantify all three metabolites in liver, kidneys, and serum. The method was used to characterize inter-tissue and inter-strain variability in formation of conjugative metabolites of TCE. Single oral dose of TCE (24, 240 or 800 mg/kg) was administered to male mice from 20 inbred strains of Collaborative Cross. Inter-strain variability in the levels of DCVG, DCVC, and NAcDCVC (GSD = 1.6-2.9) was observed. Whereas NAcDCVC was distributed equally among analyzed tissues, highest levels of DCVG were detected in liver and DCVC in kidneys. Evidence indicated that inter-strain variability in conjugative metabolite formation of TCE might affect susceptibility to adverse health effects and that this method might aid in filling data gaps in human health assessment of TCE.
Assuntos
Acetilcisteína/análogos & derivados , Cisteína/análogos & derivados , Glutationa/análogos & derivados , Glutationa/metabolismo , Glutationa/toxicidade , Tricloroetileno/metabolismo , Tricloroetileno/toxicidade , Acetilcisteína/metabolismo , Acetilcisteína/toxicidade , Animais , Cisteína/metabolismo , Cisteína/toxicidade , Fígado/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos , Razão Sinal-Ruído , Distribuição TecidualRESUMO
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
Gold nanoclusters are emerging as new materials for biomedical applications because of promises offered by their ultrasmall size and excellent biocompatibility. Here, the synthesis and optical and biological characterizations of a highly luminescent folate-functionalized Au22 cluster (Au22 -FA) are reported. The Au22 -FA clusters are synthesized by functionalizing the surface of Au22 (SG)18 clusters, where SG is glutathione, with benzyl chloroformate and folate. The functionalized clusters are highly water-soluble and exhibit remarkably bright luminescence with a quantum yield of 42%, significantly higher than any other water-soluble gold clusters protected with thiolate ligands. The folate groups conjugated to the gold cluster give rise to additional luminescence enhancement by energy transfer sensitization. The brightness of Au22 -FA is found to be 4.77 mM-1 cm-1 , nearly 8-fold brighter than that of Au22 (SG)18 . Further biological characterizations have revealed that the Au22 -FA clusters are well-suited for bioimaging. The Au22 -FA clusters exhibit excellent photostability and low toxicity; nearly 80% cell viability at 1000 ppm of the cluster. Additionally, the Au22 -FA clusters show target specificity to folate-receptor positive cells. Finally, the time-course in vivo luminescence images of intravenous-injected mice show that the Au22 -FA clusters are renal-clearable, leaving only 8% of them remained in the body after 24 h post-injection.
Assuntos
Ácido Fólico/química , Ouro/química , Substâncias Luminescentes/química , Nanopartículas Metálicas/química , Imagem Molecular/métodos , Animais , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Ácido Fólico/farmacocinética , Glutationa/química , Glutationa/farmacocinética , Glutationa/toxicidade , Ouro/farmacocinética , Humanos , Substâncias Luminescentes/farmacocinética , Substâncias Luminescentes/toxicidade , Nanopartículas Metálicas/toxicidade , Camundongos , NanomedicinaRESUMO
Recently, epidemiological studies revealed a positive relationship between an outbreak of occupational cholangiocarcinoma and exposure to organic solvents containing 1,2-dichloropropane (1,2-DCP). In 1,2-DCP-administered animal models, we previously found biliary excretion of potentially oncogenic metabolites consisting of glutathione- (GSH-) conjugated forms of 1,2-DCP (GS-DCPs); however, the GS-DCP production pathway remains unknown. To enhance the understanding of 1,2-DCP-related risks to human health, we examined the reactivity of GSH with 1,2-DCP in vitro and compared it to that with dichloromethane (DCM), the other putative substance responsible for occupational cholangiocarcinoma. Our results showed that 1,2-DCP was spontaneously conjugated with GSH, whereas this spontaneous reaction was hardly detected between DCM and GSH. Further analysis revealed that glutathione S-transferase theta 1 (GSTT1) exhibited less effect on the 1,2-DCP reaction as compared with that observed for DCM. Although GSTT1-mediated bioactivation of dihaloalkanes could be a plausible explanation for the production of reactive metabolites related to carcinogenesis based on previous studies, this catalytic pathway might not mainly contribute to 1,2-DCP-related occupational cholangiocarcinoma. Considering the higher catalytic activity of GSTT1 on DCM as compared with that on 1,2-DCP, our findings suggested differences in the activation processes associated with 1,2-DCP and DCM metabolism.
Assuntos
Neoplasias dos Ductos Biliares , Colangiocarcinoma , Glutationa Transferase/metabolismo , Glutationa , Proteínas de Neoplasias/metabolismo , Propano/análogos & derivados , Neoplasias dos Ductos Biliares/induzido quimicamente , Neoplasias dos Ductos Biliares/enzimologia , Neoplasias dos Ductos Biliares/patologia , Colangiocarcinoma/induzido quimicamente , Colangiocarcinoma/enzimologia , Colangiocarcinoma/patologia , Glutationa/análogos & derivados , Glutationa/toxicidade , Humanos , Propano/toxicidadeRESUMO
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
Many pyrrolizidine alkaloids (PAs) are hepatotoxic, genotoxic, and carcinogenic phytochemicals. Metabolism of PAs in vivo generates four (±)-6,7-dihydro-7-hydroxy-1-hydroxymethyl-5H-pyrrolizine (DHP)-DNA adducts that have been proposed to be responsible for PA-induced liver tumor formation in rats. In this present study, we determined that the same set of DHP-DNA adducts was formed upon the incubation of 7-glutathione-DHP and 7-cysteine-DHP with cultured human hepatocarcinoma HepG2 cells. These results suggest that 7-glutathione-DHP and 7-cysteine-DHP are reactive metabolites of PAs that can bind to cellular DNA to form DHP-DNA adducts in HepG2 cells, and can potentially initiate liver tumor formation.
Assuntos
Carcinógenos/toxicidade , Cisteína/análogos & derivados , Glutationa/análogos & derivados , Pirróis/toxicidade , Alcaloides de Pirrolizidina/toxicidade , Animais , Cisteína/metabolismo , Cisteína/toxicidade , Adutos de DNA , Glutationa/metabolismo , Glutationa/toxicidade , Alcaloides de Pirrolizidina/metabolismo , Ratos , Ratos Endogâmicos F344RESUMO
Non-invasive imaging of gastrointestinal (GI) tract using novel but efficient contrast agents is of the most important issues in the diagnosis and prognosis of GI diseases. Here, for the first time, we reported the design and synthesis of biothiol-decorated lead sulfide nanodots, as well as their usages in functional dual-modality imaging of GI tract in vivo. Due to the presence of glutathione on the surface of the nanodots, these well-prepared contrast agents could decrease the unwanted ion leakage, withstand the harsh conditions in GI tract, and avoid the systemic absorption after oral administration. Compared with clinical barium meal and iodine-based contrast agents, these nanodots exhibited much more significant enhancement in contrast efficiency during both 2D X-ray imaging and 3D CT imaging. Different from some conventional invasive imaging modalities, such as gastroscope and enteroscope, non-invasive imaging strategy by using glutathione modified PbS nanodots as contrast agents could reduce the painfulness towards patients, facilitate the imaging procedure, and economize the manipulation period. Moreover, long-term toxicity and bio-distribution of these nanodots after oral administration were evaluated in detail, which indicated their overall safety. Based on our present study, these nanodots could act as admirable contrast agents to integrate X-ray imaging and CT imaging for the direct visualization of GI tract.
Assuntos
Meios de Contraste/química , Trato Gastrointestinal/diagnóstico por imagem , Glutationa/química , Chumbo/química , Pontos Quânticos/química , Sulfetos/química , Animais , Meios de Contraste/síntese química , Meios de Contraste/toxicidade , Glutationa/síntese química , Glutationa/toxicidade , Células HeLa , Humanos , Chumbo/toxicidade , Camundongos , Pontos Quânticos/toxicidade , Pontos Quânticos/ultraestrutura , Ratos Wistar , Sulfetos/síntese química , Sulfetos/toxicidade , Tomografia Computadorizada por Raios X/métodosRESUMO
Due to eutrophication processes in our water bodies, cyanobacterial blooms can develop worldwide. Most of these blooms are toxic. The most prominent cyanobacterial toxins are the group of the microcystins, which are cyclic heptapeptides, currently with more than 100 congeners known. The biotransformation of microcystins starts with the conjugation to the cell internal tripeptide glutathione, catalysed by glutathione S-transferase enzymes. This conjugate is further broken down to a cysteine conjugate, enhancing the cell internal transport and excretion of the conjugated toxin from the organisms. Still many questions remain open, thinking on an obviously good working detoxification system on the one side and the often seen negative effects up to the death of humans on the other sides.
Assuntos
Toxinas Bacterianas/metabolismo , Cianobactérias/metabolismo , Glutationa/metabolismo , Toxinas Marinhas/metabolismo , Redes e Vias Metabólicas , Microcistinas/metabolismo , Animais , Toxinas Bacterianas/toxicidade , Biotransformação , Toxinas de Cianobactérias , Sistema Enzimático do Citocromo P-450/metabolismo , Glutationa/toxicidade , Humanos , Toxinas Marinhas/toxicidade , Microcistinas/toxicidadeRESUMO
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.