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1.
J Neurochem ; 148(1): 136-156, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30269333

RESUMO

Histone hypoacetylation is associated with dopaminergic neurodegeneration in Parkinson's disease (PD), because of an imbalance in the activities of the enzymes responsible for histone (de)acetylation. Correction of this imbalance, with histone deacetylase (HDAC) inhibiting agents, could be neuroprotective. We therefore hypothesize that nicotinamide, being a selective inhibitor of HDAC class III as well as having modulatory effects on mitochondrial energy metabolism, would be neuroprotective in the lactacystin rat model of PD, which recapitulates the formation of neurotoxic accumulation of altered proteins within the substantia nigra to cause progressive dopaminergic cell death. Rats received nicotinamide for 28 days, starting 7 days after unilateral injection of the irreversible proteasome inhibitor, lactacystin, into the substantia nigra. Longitudinal motor behavioural testing and structural magnetic resonance imaging were used to track changes in this model of PD, and assessment of nigrostriatal integrity, histone acetylation and brain gene expression changes post-mortem used to quantify nicotinamide-induced neuroprotection. Counterintuitively, nicotinamide dose-dependently exacerbated neurodegeneration of dopaminergic neurons, behavioural deficits and structural brain changes in the lactacystin-lesioned rat. Nicotinamide treatment induced histone hyperacetylation and over-expression of numerous neurotrophic and anti-apoptotic factors in the brain, yet failed to result in neuroprotection, rather exacerbated dopaminergic pathology. These findings highlight the importance of inhibitor specificity within HDAC isoforms for therapeutic efficacy in PD, demonstrating the contrasting effects of HDAC class III inhibition upon cell survival in this animal model of the disease. OPEN SCIENCE BADGES: This article has received a badge for *Open Materials* because it provided all relevant information to reproduce the study in the manuscript. The complete Open Science Disclosure form for this article can be found at the end of the article. More information about the Open Practices badges can be found at https://cos.io/our-services/open-science-badges/.


Assuntos
Neurônios Dopaminérgicos/efeitos dos fármacos , Inibidores de Histona Desacetilases/farmacologia , Degeneração Neural/patologia , Niacinamida/farmacologia , Transtornos Parkinsonianos/patologia , Acetilação/efeitos dos fármacos , Acetilcisteína/análogos & derivados , Acetilcisteína/toxicidade , Animais , Morte Celular/efeitos dos fármacos , Modelos Animais de Doenças , Neurônios Dopaminérgicos/patologia , Masculino , Transtornos Parkinsonianos/induzido quimicamente , Ratos , Ratos Sprague-Dawley
2.
Neurotox Res ; 34(3): 706-716, 2018 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-30129004

RESUMO

Parkinson's disease (PD) is a neurodegenerative disorder of the central nervous system (CNS) caused by a progressive loss of nigrostriatal dopaminergic neurons. Dysfunction of the ubiquitin-proteasome system (UPS) plays an important role in the pathogenesis of PD. Intranigral administration of the UPS inhibitor lactacystin is used to obtain a valuable animal model for investigating putative neuroprotective treatments for PD. 1-Methyl-1,2,3,4-tetrahydroisoquinoline (1MeTIQ) is an endogenous amine that displays neuroprotective properties. This compound acts as a reversible monoamine oxidase (MAO) inhibitor and a natural free radical scavenger. In the present experiment, we investigated the effect of acute and chronic treatment with 1MeTIQ on locomotor activity and the release of dopamine as well as its metabolites in the striatum of unilaterally lactacystin-lesioned and sham-operated rats using in vivo microdialysis. Additionally, changes in the level of tyrosine hydroxylase (TH) in the substantia nigra were measured. Unilateral lactacystin injection into the substantia nigra caused significant impairment of dopamine release (approx. 45%) and a marked decline in the TH level. These effects were completely antagonized by multiple treatments with 1MeTIQ. The results obtained from the in vivo microdialysis study as well as from the ex vivo experiments suggest that multiple administration of 1MeTIQ protects dopaminergic neurons against the lactacystin-induced decline in TH concentration in the substantia nigra and prevents disturbances of dopamine release in the striatum. We have demonstrated that 1MeTIQ is capable of maintaining the physiological functions of the striatal dopamine neurons damaged by unilateral lactacystin lesion.


Assuntos
Lesões Encefálicas/metabolismo , Lesões Encefálicas/prevenção & controle , Encéfalo/efeitos dos fármacos , Dopamina/metabolismo , Fármacos Neuroprotetores/uso terapêutico , Tetra-Hidroisoquinolinas/uso terapêutico , Ácido 3,4-Di-Hidroxifenilacético/metabolismo , Acetilcisteína/análogos & derivados , Acetilcisteína/toxicidade , Análise de Variância , Animais , Encéfalo/metabolismo , Lesões Encefálicas/induzido quimicamente , Lesões Encefálicas/patologia , Inibidores de Cisteína Proteinase/toxicidade , Comportamento Exploratório/efeitos dos fármacos , Lateralidade Funcional/efeitos dos fármacos , Locomoção/efeitos dos fármacos , Masculino , Microdiálise , Ratos , Ratos Wistar
3.
Am J Physiol Renal Physiol ; 314(5): F956-F968, 2018 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-29357409

RESUMO

Oxidative stress and mitochondrial dysfunction exacerbate acute kidney injury (AKI), but their role in any associated progress to chronic kidney disease (CKD) remains unclear. Antioxidant therapies often benefit AKI, but their benefits in CKD are controversial since clinical and preclinical investigations often conflict. Here we examined the influence of the antioxidant N-acetyl-cysteine (NAC) on oxidative stress and mitochondrial function during AKI (20-min bilateral renal ischemia plus reperfusion/IR) and progression to chronic kidney pathologies in mice. NAC (5% in diet) was given to mice 7 days prior and up to 21 days post-IR (21d-IR). NAC treatment resulted in the following: prevented proximal tubular epithelial cell apoptosis at early IR (40-min postischemia), yet enhanced interstitial cell proliferation at 21d-IR; increased transforming growth factor-ß1 expression independent of IR time; and significantly dampened nuclear factor-like 2-initiated cytoprotective signaling at early IR. In the long term, NAC enhanced cellular metabolic impairment demonstrated by increased peroxisome proliferator activator-γ serine-112 phosphorylation at 21d-IR. Intravital multiphoton microscopy revealed increased endogenous fluorescence of nicotinamide adenine dinucleotide (NADH) in cortical tubular epithelial cells during ischemia, and at 21d-IR that was not attenuated with NAC. Fluorescence lifetime imaging microscopy demonstrated persistent metabolic impairment by increased free/bound NADH in the cortex at 21d-IR that was enhanced by NAC. Increased mitochondrial dysfunction in remnant tubular cells was demonstrated at 21d-IR by tetramethylrhodamine methyl ester fluorimetry. In summary, NAC enhanced progression to CKD following AKI not only by dampening endogenous cellular antioxidant responses at time of injury but also by enhancing persistent kidney mitochondrial and metabolic dysfunction.


Assuntos
Acetilcisteína/toxicidade , Lesão Renal Aguda/complicações , Antioxidantes/toxicidade , Rim/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Insuficiência Renal Crônica/induzido quimicamente , Lesão Renal Aguda/metabolismo , Lesão Renal Aguda/patologia , Lesão Renal Aguda/fisiopatologia , Animais , Apoptose/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Modelos Animais de Doenças , Progressão da Doença , Metabolismo Energético/efeitos dos fármacos , Rim/metabolismo , Rim/patologia , Rim/fisiopatologia , Masculino , Camundongos Endogâmicos C57BL , Microscopia de Fluorescência por Excitação Multifotônica , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Mitocôndrias/patologia , NAD/metabolismo , PPAR gama/metabolismo , Fosforilação , Insuficiência Renal Crônica/metabolismo , Insuficiência Renal Crônica/patologia , Insuficiência Renal Crônica/fisiopatologia , Transdução de Sinais/efeitos dos fármacos , Fatores de Tempo , Fator de Crescimento Transformador beta1/metabolismo
4.
Neurotox Res ; 34(1): 16-31, 2018 07.
Artigo em Inglês | MEDLINE | ID: mdl-29218504

RESUMO

Parkinson's disease (PD) is conventionally seen as resulting from single-system neurodegeneration affecting nigrostriatal dopaminergic neurons. However, accumulating evidence indicates multi-system degeneration and neurotransmitter deficiencies, including cholinergic neurons which degenerate in a brainstem nucleus, the pedunculopontine nucleus (PPN), resulting in motor and cognitive impairments. The neuropeptide galanin can inhibit cholinergic transmission, while being upregulated in degenerating brain regions associated with cognitive decline. Here we determined the temporal-spatial profile of progressive expression of endogenous galanin within degenerating cholinergic neurons, across the rostro-caudal axis of the PPN, by utilizing the lactacystin-induced rat model of PD. First, we show progressive neuronal death affecting nigral dopaminergic and PPN cholinergic neurons, reflecting that seen in PD patients, to facilitate use of this model for assessing the therapeutic potential of bioactive peptides. Next, stereological analyses of the lesioned brain hemisphere found that the number of PPN cholinergic neurons expressing galanin increased by 11%, compared to sham-lesioned controls, and increasing by a further 5% as the neurodegenerative process evolved. Galanin upregulation within cholinergic PPN neurons was most prevalent closest to the intra-nigral lesion site, suggesting that galanin upregulation in such neurons adapt intrinsically to neurodegeneration, to possibly neuroprotect. This is the first report on the extent and pattern of galanin expression in cholinergic neurons across distinct PPN subregions in both the intact rat CNS and lactacystin-lesioned rats. The findings pave the way for future work to target galanin signaling in the PPN, to determine the extent to which upregulated galanin expression could offer a viable treatment strategy for ameliorating PD symptoms associated with cholinergic degeneration.


Assuntos
Acetilcisteína/análogos & derivados , Colina O-Acetiltransferase/metabolismo , Inibidores de Cisteína Proteinase/toxicidade , Galanina/metabolismo , Neurônios/patologia , Doença de Parkinson , Núcleo Tegmental Pedunculopontino/patologia , Acetilcisteína/toxicidade , Análise de Variância , Animais , Modelos Animais de Doenças , Masculino , Neurônios/metabolismo , Doença de Parkinson/etiologia , Doença de Parkinson/metabolismo , Doença de Parkinson/patologia , Ratos , Ratos Sprague-Dawley , Substância Negra/efeitos dos fármacos , Substância Negra/patologia , Fatores de Tempo , Tirosina 3-Mono-Oxigenase/metabolismo
5.
Drug Chem Toxicol ; 41(1): 89-94, 2018 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-28504001

RESUMO

Diclofenac (DCF) adverse reactions involve diverse mechanisms in different models. We recently demonstrated that DCF-induced toxicity in HepaRG decreases as they express DCF-metabolizing enzymes. DCF metabolism promotes toxicity in Saccharomyces cerevisiae expressing heterologous cytochromes-P450. N-Acetylcysteine (NAC) is used to treat diverse medical conditions due to its multiple properties (antioxidant, metal chelator, thiol-disulfide disruption). The latter property accounts for its mucolytic effects and broadens its potential molecular targets to signal transduction proteins, ABC transporters and others. Interaction of NAC with DCF effects depends on the experimental model. This study aims to investigate NAC/DCF interaction and the involvement of ABC transporters in wild type and mutant Saccharomyces cerevisiae. DCF inhibited yeast growth in a dose- and time-dependent manner and the cells started adapting to DCF 24-h post-treatment. NAC potentiated DCF-induced toxicity if added prior or parallel to DCF. Pretreatment with NAC increased its potentiation effect and compromised cells adaption to DCF. Post-treatment with NAC potentiated DCF toxicity without compromising adaptation. Moreover, mutant strains in ABC transporters Pdr5, Yor1, Bpt1 or Pdr15, were more sensitive to DCF; while mutant strains in Pdr5, Vmr1 or Pdr12 were more sensitive to NAC/DCF interaction. DCF ± NAC elicited on the mutant strain in Yap1, an oxidative stress-related protein, the same effects as on the wild type. Therefore, oxidative stress does not seem to be key actor in DCF toxicity in our model. Our hypothesis is that NAC potentiation effect is at least due to its ability to disrupt disulfide bridge in proteins required to overcome DCF toxicity in yeast.


Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Acetilcisteína/toxicidade , Anti-Inflamatórios não Esteroides/toxicidade , Antioxidantes/toxicidade , Diclofenaco/toxicidade , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/efeitos dos fármacos , Transportadores de Cassetes de Ligação de ATP/genética , Anti-Inflamatórios não Esteroides/metabolismo , Diclofenaco/metabolismo , Dissulfetos/metabolismo , Relação Dose-Resposta a Droga , Sinergismo Farmacológico , Genótipo , Mutação , Estresse Oxidativo/efeitos dos fármacos , Fenótipo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crescimento & desenvolvimento , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Fatores de Tempo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
6.
Mol Neurobiol ; 55(1): 13-25, 2018 01.
Artigo em Inglês | MEDLINE | ID: mdl-28812231

RESUMO

Oxidative stress is a common feature in neurodegenerative diseases associated with neuroinflammation, and therefore, has been proposed as a key target for novel therapies for these diseases. Recently, adipose-derived stem cell (ASC)-based cell therapy has emerged as a novel strategy for neuroprotection. In this study, we evaluate the therapeutic role of ASC-conditioned medium (ASC-CM) against H2O2-induced neurotoxicity in a new in vitro model of ec23/brain-derived neurotrophic factor (BDNF)-differentiated human SH-SY5Y neuron-like cells (SH-SY5Yd). In the presence of ASC-CM, stressed SH-SY5Yd cells recover normal axonal morphology (with an almost complete absence of H2O2-induced axonal beading), electrophysiological features, and cell viability. This beneficial effect of ASC-CM was associated with its antioxidant capacity and the presence of growth factors, namely, BDNF, glial cell line-derived neurotrophic factor, and transforming growth factor ß1. Moreover, the neuroprotective effect of ASC-CM was very similar to that obtained from treatment with BDNF, an essential factor for SH-SY5Yd cell survival. Importantly, we also found that the addition of the antioxidant agent N-acetyl cysteine to ASC-CM abolished its restorative effect; this was associated with a strong reduction in reactive oxygen species (ROS), in contrast to the moderate decrease in ROS produced by ASC-CM alone. These results suggest that neuronal restorative effect of ASC-CM is associated with not only the release of essential neurotrophic factors, but also the maintenance of an appropriate redox state to preserve neuronal function.


Assuntos
Acetilcisteína/toxicidade , Adipócitos/efeitos dos fármacos , Meios de Cultivo Condicionados/farmacologia , Células-Tronco Mesenquimais/efeitos dos fármacos , Fármacos Neuroprotetores/farmacologia , Adipócitos/fisiologia , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/fisiologia , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/fisiologia , Células Cultivadas , Técnicas de Cocultura/métodos , Humanos , Células-Tronco Mesenquimais/fisiologia , Estresse Oxidativo/efeitos dos fármacos , Estresse Oxidativo/fisiologia , Espécies Reativas de Oxigênio/antagonistas & inibidores , Espécies Reativas de Oxigênio/metabolismo
7.
J Toxicol Environ Health A ; 81(1-3): 37-52, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29190187

RESUMO

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 Tecidual
8.
Dokl Biol Sci ; 476(1): 188-190, 2017 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-29101619

RESUMO

An experimental model of the preclinical stage of Parkinson's disease was induced by double intranasal administration of the proteasome inhibitor lactacystin. The results demonstrated signs of cognitive impairments expressed as impaired non-associative learning. This was related to degeneration of one-third of dopaminergic neurons in the ventral tegmental area of the midbrain and their axons in the dorsolateral prefrontal cortex. Impairment of non-associative learning may be an early non-motor marker of Parkinson's disease indicating the start of neurodegenerative processes in the dopaminergic mesocortical system of the brain.


Assuntos
Acetilcisteína/análogos & derivados , Disfunção Cognitiva/fisiopatologia , Aprendizagem/fisiologia , Doença de Parkinson Secundária/fisiopatologia , Acetilcisteína/administração & dosagem , Acetilcisteína/toxicidade , Animais , Axônios/efeitos dos fármacos , Axônios/fisiologia , Comportamento Animal/efeitos dos fármacos , Disfunção Cognitiva/induzido quimicamente , Modelos Animais de Doenças , Neurônios Dopaminérgicos/efeitos dos fármacos , Neurônios Dopaminérgicos/patologia , Humanos , Aprendizagem/efeitos dos fármacos , Mesencéfalo/efeitos dos fármacos , Mesencéfalo/fisiopatologia , Doença de Parkinson Secundária/induzido quimicamente , Córtex Pré-Frontal/efeitos dos fármacos , Córtex Pré-Frontal/fisiopatologia , Ratos
9.
Biochemistry (Mosc) ; 82(10): 1176-1182, 2017 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-29037138

RESUMO

Addition into the culture medium of the antioxidant N-acetylcysteine (NAC, 1 mM) in the presence of Cu2+ (0.0005-0.001 mM) induced intensive death of cultured rat cerebellar granule neurons, which was significantly decreased by the zinc ion chelator TPEN (N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine). However, the combined action of NAC and Zn2+ did not induce destruction of the neurons. Measurement of the relative intracellular concentration of Zn2+ with the fluorescent probe FluoZin-3 AM or of free radical production using a CellROX Green showed that incubation of the culture for 4 h with Cu2+ and NAC induced an intensive increase in the fluorescence of CellROX Green but not of FluoZin-3. Probably, the protective effect of TPEN in this case could be mediated by its ability to chelate Cu2+. Incubation of cultures in a balanced salt solution in the presence of 0.01 mM Cu2+ caused neuronal death already after 1 h if the NAC concentration in the solution was within 0.005-0.05 mM. NAC at higher concentrations (0.1-1 mM) together with 0.01 mM Cu2+ did not cause the death of neurons. These data imply that the antioxidant NAC can be potentially harmful to neurons even in the presence of nanomolar concentrations of variable valence metals.


Assuntos
Acetilcisteína/toxicidade , Apoptose/efeitos dos fármacos , Cobre/toxicidade , Neurônios/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Animais , Células Cultivadas , Cerebelo/citologia , Cerebelo/efeitos dos fármacos , Cerebelo/metabolismo , Quelantes/farmacologia , Cobre/química , Etilenodiaminas/farmacologia , Microscopia de Fluorescência , Neurônios/citologia , Neurônios/metabolismo , Compostos Policíclicos/química , Ratos , Ratos Wistar , Zinco/farmacologia
10.
Artigo em Alemão | MEDLINE | ID: mdl-28516258

RESUMO

The assessment of health risks resulting from the intake of genotoxic carcinogens in food depends essentially on a valid exposure assessment. The reliability of the external exposure estimation is restricted by various factors, e. g. inaccurate data from dietary protocols and variations of food contaminant contents. As an alternative, the individual internal exposure to genotoxic substances may be described by specific biomarkers in different matrices. For example, mercapturic acids formed after glutathione conjugation of electrophilic metabolites can be detected in the urine. This typically reflects the exposure to the parent compound over a period of one to two days. The determination of adducts in the blood proteins serum albumin (SA) and hemoglobin (Hb) allows for conclusions to be drawn about the external exposure within the last three weeks (SA) or within the last four months (Hb). Protein adducts are used routinely in occupational medicine as biomarkers of internal exposure to substances in the ambient air of the workplace. The availability of increasingly sensitive analytical techniques also makes it possible to detect numerous adducts in proteins from human blood samples that are formed after the continuous intake of very small doses of toxic substances from foods. Here, we present the current state of science exemplified by protein adducts of the food contaminants acrylamide, aflatoxin B1 and glycidol. The biomarker can be used in the future to investigate previously unknown relationships between internal exposure and disease incidences.


Assuntos
Biomarcadores/análise , Contaminação de Alimentos/análise , Doenças Transmitidas por Alimentos/diagnóstico , Medição de Risco , Acetilcisteína/análise , Acetilcisteína/toxicidade , Carcinógenos/análise , Carcinógenos/toxicidade , Adutos de DNA/análise , Adutos de DNA/toxicidade , Alemanha , Análise de Perigos e Pontos Críticos de Controle , Humanos , Testes de Mutagenicidade , Mutagênicos/análise , Mutagênicos/toxicidade
11.
Exp Brain Res ; 235(7): 2189-2202, 2017 07.
Artigo em Inglês | MEDLINE | ID: mdl-28439627

RESUMO

Proteinaceous inclusions, called Lewy bodies, are used as a pathological hallmark for Parkinson's disease (PD). Lewy bodies contain insoluble α-synuclein (aSyn) and many other ubiquitinated proteins, suggesting a role for protein degradation system failure in the PD pathogenesis. Indeed, proteasomal dysfunction has been linked to PD but commonly used in vivo toxin models, such as 6-OHDA or MPTP, do not have a significant effect on the proteasomal system or protein aggregation. Therefore, we wanted to study the characteristics of a proteasomal inhibitor, lactacystin, as a PD model on young and adult mice. To study this, we performed stereotactic microinjection of lactacystin above the substantia nigra pars compacta in young (2 month old) and adult (12-14 month old) C57Bl/6 mice. Motor behavior was measured by locomotor activity and cylinder tests, and the markers of neuroinflammation, aSyn, and dopaminergic system were assessed by immunohistochemistry and HPLC. We found that lactacystin induced a Parkinson's disease-like motor phenotype 5-7 days after injection in young and adult mice, and this was associated with widespread neuroinflammation based on glial cell markers, aSyn accumulation in substantia nigra, striatal dopamine decrease, and loss of dopaminergic cell bodies in the substantia nigra and terminals in the striatum. When comparing young and adult mice, adult mice were more sensitive for dopaminergic degeneration after lactacystin injection that further supports the use of adult mice instead of young when modeling neurodegeneration. Our data showed that lactacystin is useful in modeling various aspects of Parkinson's disease, and taken together, our findings emphasize the role of a protein degradation deficit in Parkinson's disease pathology, and support the use of proteasomal inhibitors as Parkinson's disease models.


Assuntos
Acetilcisteína/análogos & derivados , Inibidores de Cisteína Proteinase/toxicidade , Neuroglia/efeitos dos fármacos , Doença de Parkinson/etiologia , Doença de Parkinson/patologia , Substância Negra/efeitos dos fármacos , Acetilcisteína/toxicidade , Fatores Etários , Animais , Proteínas de Ligação ao Cálcio/metabolismo , Modelos Animais de Doenças , Membro Anterior/fisiopatologia , Proteína Glial Fibrilar Ácida/metabolismo , Glutamato Descarboxilase/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Proteínas dos Microfilamentos/metabolismo , Microinjeções , Neurotransmissores/metabolismo , Desempenho Psicomotor/efeitos dos fármacos , Sinucleínas/metabolismo , Tirosina 3-Mono-Oxigenase/metabolismo
12.
Environ Pollut ; 221: 256-265, 2017 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-27939626

RESUMO

Exposure to benzene is inevitable, and concerns regarding the adverse health effects of benzene have been raised. Most investigators found that benzene exposure induced hematotoxicity. In this regard, Our study aimed to explore a novel potential biomarker of adverse health effects following benzene exposure and the toxic mechanisms of benzene metabolites in vitro. This study consisted of 314 benzene-exposed workers and 288 control workers, an air benzene concentration of who were 2.64 ± 1.60 mg/m3 and 0.05 ± 0.01 mg/m3, respectively. In this population-based study, miR-34a expression was elevated in benzene-exposed workers. The correlation of miR-34a with the airborne benzene concentration, S-phenylmercapturic acid (S-PMA) and trans, trans-muconic acid (t, t-MA), all of which reflect benzene exposure, was found. Correlation analysis indicated that miR-34a was associated with peripheral blood count, alanine transaminase (ALT) and oxidative stress. Furthermore, multivariate analysis demonstrated that miR-34a expression was strongly associated with white blood cell count (structure loadings = 0.952). In population-based study, miR-34a had the largest contribution to altered peripheral blood counts, which reflect benzene-induced hematotoxicity. The role of miR-34a in benzene toxicity was assessed using lentiviral vector transfection. Results revealed that 1,4-benzoquinone induced abnormal cell apoptosis and simultaneously upregulated miR-34a accompanied with decreased Bcl-2. Finally, inhibition of miR-34a elevated Bcl-2 and decreased 1,4-benzoquinone-induced apoptosis. In conclusion, miR-34a was observed to be involved in benzene-induced hematotoxicity by targeting Bcl-2 and could be regarded as a potential novel biomarker for benzene toxicity.


Assuntos
Benzeno/toxicidade , Benzoquinonas/toxicidade , Genes bcl-2 , MicroRNAs/metabolismo , Acetilcisteína/análogos & derivados , Acetilcisteína/toxicidade , Apoptose/genética , Apoptose/fisiologia , Biomarcadores/metabolismo , Humanos , Ácido Sórbico/análogos & derivados , Ácido Sórbico/toxicidade , Regulação para Cima
13.
Neurosci Lett ; 636: 83-89, 2017 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-27818354

RESUMO

Lewy bodies, the histopathological hallmarks of Parkinson's disease (PD), contain insoluble and aggregated α-synuclein (aSyn) and many other proteins, proposing a role for failure in protein degradation system in the PD pathogenesis. Proteasomal dysfunction has indeed been linked to PD and aSyn oligomers have been shown to inhibit proteasomes and autophagy. Our recent studies have shown that inhibitors of prolyl oligopeptidase (PREP) can prevent the aggregation and enhance the clearance of accumulated aSyn, and therefore, we wanted to study if PREP inhibition can overcome the aSyn aggregation and toxicity induced by lactacystin, a proteasomal inhibitor. The cells overexpressing human A30P or A53T mutated aSyn were incubated with lactacystin and a PREP inhibitor, KYP-2047, for 48h. Theafter, the cells were fractioned, and the effects of lactacystin with/without 1µM KYP-2047 on aSyn aggregation and ubiquitin accumulation, cell viability and on autophagic markers (p62, Beclin1 and LC3BII) were studied. We found that KYP-2047 attenuated lactacystin-induced cell death in mutant aSyn overexpressing cells but not in non-overexpressing control cells. KYP-2047 reduced significantly SDS-insoluble high-molecular-weight aSyn oligomers that were in line with the cell viability results. In addition, significant reduction in protein accumulation marker, p62, was seen in SDS fraction while LC3BII, a marker for autophagosome formation, was increased, indicating to enhanced autophagy. Our results further streghten the possibilities for PREP inhibitors as a potential drug therapy against synucleinopathies and other protein aggregating diseases.


Assuntos
Acetilcisteína/análogos & derivados , Prolina/análogos & derivados , Inibidores de Proteassoma/toxicidade , Serina Endopeptidases/metabolismo , Inibidores de Serino Proteinase/farmacologia , alfa-Sinucleína/metabolismo , Acetilcisteína/toxicidade , Autofagia , Linhagem Celular Tumoral , Sobrevivência Celular , Humanos , Mutação , Prolina/farmacologia , Agregados Proteicos , alfa-Sinucleína/genética
14.
Exp Neurol ; 290: 15-28, 2017 04.
Artigo em Inglês | MEDLINE | ID: mdl-28024798

RESUMO

Zonisamide (ZNS), an anticonvulsant drug exhibiting symptomatic effects in Parkinson's disease (PD), was recently reported to exert neuroprotection in rodent models. One of the proposed neuroprotective mechanisms involves increased protein expression of xCT, the specific subunit of the cystine/glutamate antiporter system xc-, inducing glutathione (GSH) synthesis. Here, we investigated the outcome of ZNS treatment in a mouse model of PD based on intranigral proteasome inhibition, and whether the observed effects would be mediated by system xc-. The proteasome inhibitor lactacystin (LAC) was administered intranigrally to male C57BL/6J mice receiving repeated intraperitoneal injections of either ZNS 30mgkg-1 or vehicle. Drug administration was initiated three days prior to stereotaxic LAC injection and was maintained until six days post-surgery. One week after lesion, mice were behaviorally assessed and investigated in terms of nigrostriatal neurodegeneration and molecular changes at the level of the basal ganglia, including expression levels of xCT. ZNS reduced the loss of nigral dopaminergic neurons following LAC injection and the degree of sensorimotor impairment. ZNS failed, however, to modulate xCT expression in basal ganglia of lesioned mice. In a separate set of experiments, the impact of ZNS treatment on system xc- was investigated in control conditions in vivo as well as in vitro. Similarly, ZNS did not influence xCT or glutathione levels in naive male C57BL/6J mice, nor did it alter system xc- activity or glutathione content in vitro. Taken together, these results demonstrate that ZNS treatment provides neuroprotection and behavioral improvement in a PD mouse model based on proteasome inhibition via system xc- independent mechanisms.


Assuntos
Acetilcisteína/análogos & derivados , Sistema y+ de Transporte de Aminoácidos/efeitos dos fármacos , Inibidores de Cisteína Proteinase/toxicidade , Isoxazóis/farmacologia , Fármacos Neuroprotetores/farmacologia , Doença de Parkinson Secundária/induzido quimicamente , Doença de Parkinson Secundária/prevenção & controle , Acetilcisteína/administração & dosagem , Acetilcisteína/antagonistas & inibidores , Acetilcisteína/toxicidade , Animais , Gânglios da Base/efeitos dos fármacos , Gânglios da Base/metabolismo , Gânglios da Base/patologia , Comportamento Animal/efeitos dos fármacos , Inibidores de Cisteína Proteinase/administração & dosagem , Neurônios Dopaminérgicos/efeitos dos fármacos , Neurônios Dopaminérgicos/patologia , Glutationa/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Microinjeções , Atividade Motora/efeitos dos fármacos , Doença de Parkinson Secundária/psicologia , Equilíbrio Postural/efeitos dos fármacos , Técnicas Estereotáxicas , Substância Negra , Zonisamida
15.
Neurochem Res ; 41(3): 554-67, 2016 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-26318862

RESUMO

Mitochondrial dysfunction, ubiquitin-proteasomal system impairment and excitotoxicity occur during the injury and death of neurons in neurodegenerative conditions. The aim of this work was to elucidate the cellular mechanisms that are universally altered by these conditions. Through overlapping expression profiles of rotenone-, lactacystin- and N-methyl-D-aspartate-treated cortical neurons, we have identified three affected biological processes that are commonly affected; oxidative stress, dysfunction of calcium signalling and inhibition of the autophagic-lysosomal pathway. These data provides many opportunities for therapeutic intervention in neurodegenerative conditions, where mitochondrial dysfunction, proteasomal inhibition and excitotoxicity are evident.


Assuntos
Autofagia , Sinalização do Cálcio , Lisossomos/metabolismo , Neurônios/metabolismo , Estresse Oxidativo , Acetilcisteína/análogos & derivados , Acetilcisteína/toxicidade , Animais , Humanos , Análise em Microsséries , Doenças Neurodegenerativas/metabolismo , Doenças Neurodegenerativas/patologia , Neurônios/efeitos dos fármacos , Praguicidas/toxicidade , Complexo de Endopeptidases do Proteassoma/metabolismo , Inibidores de Proteassoma/toxicidade , Receptores de N-Metil-D-Aspartato/metabolismo , Rotenona/toxicidade , Ubiquitina/metabolismo
16.
PLoS Genet ; 11(12): e1005710, 2015 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-26681446

RESUMO

Degeneration of nigrostriatal dopaminergic system is the principal lesion in Parkinson's disease. Because glial cell line-derived neurotrophic factor (GDNF) promotes survival of dopamine neurons in vitro and in vivo, intracranial delivery of GDNF has been attempted for Parkinson's disease treatment but with variable success. For improving GDNF-based therapies, knowledge on physiological role of endogenous GDNF at the sites of its expression is important. However, due to limitations of existing genetic model systems, such knowledge is scarce. Here, we report that prevention of transcription of Gdnf 3'UTR in Gdnf endogenous locus yields GDNF hypermorphic mice with increased, but spatially unchanged GDNF expression, enabling analysis of postnatal GDNF function. We found that increased level of GDNF in the central nervous system increases the number of adult dopamine neurons in the substantia nigra pars compacta and the number of dopaminergic terminals in the dorsal striatum. At the functional level, GDNF levels increased striatal tissue dopamine levels and augmented striatal dopamine release and re-uptake. In a proteasome inhibitor lactacystin-induced model of Parkinson's disease GDNF hypermorphic mice were protected from the reduction in striatal dopamine and failure of dopaminergic system function. Importantly, adverse phenotypic effects associated with spatially unregulated GDNF applications were not observed. Enhanced GDNF levels up-regulated striatal dopamine transporter activity by at least five fold resulting in enhanced susceptibility to 6-OHDA, a toxin transported into dopamine neurons by DAT. Further, we report how GDNF levels regulate kidney development and identify microRNAs miR-9, miR-96, miR-133, and miR-146a as negative regulators of GDNF expression via interaction with Gdnf 3'UTR in vitro. Our results reveal the role of GDNF in nigrostriatal dopamine system postnatal development and adult function, and highlight the importance of correct spatial expression of GDNF. Furthermore, our results suggest that 3'UTR targeting may constitute a useful tool in analyzing gene function.


Assuntos
Dopamina/genética , Neurônios Dopaminérgicos/metabolismo , Fator Neurotrófico Derivado de Linhagem de Célula Glial/genética , Doença de Parkinson Secundária/genética , Substância Negra/metabolismo , Acetilcisteína/análogos & derivados , Acetilcisteína/toxicidade , Animais , Sistema Nervoso Central/metabolismo , Sistema Nervoso Central/patologia , Modelos Animais de Doenças , Dopamina/metabolismo , Neurônios Dopaminérgicos/patologia , Regulação da Expressão Gênica no Desenvolvimento , Fator Neurotrófico Derivado de Linhagem de Célula Glial/biossíntese , Humanos , Rim/crescimento & desenvolvimento , Rim/metabolismo , Camundongos , Neostriado/metabolismo , Neostriado/patologia , Fármacos Neuroprotetores/metabolismo , Doença de Parkinson Secundária/induzido quimicamente , Doença de Parkinson Secundária/patologia , Substância Negra/patologia
17.
Anal Chim Acta ; 894: 54-64, 2015 Sep 24.
Artigo em Inglês | MEDLINE | ID: mdl-26423628

RESUMO

Mercapturic acid metabolites from dietary acrylamide are important short-term exposure biomarkers for evaluating the in vivo toxicity of acrylamide. Most of studies have focused on the measurement of two metabolites, N-acetyl-S-(2-carbamoylethyl)-L-cysteine (AAMA) and N-acetyl-S-(2-carbamoyl-2-hydroxyethyl)-L-cysteine (GAMA). Thus, the comprehensive profile of acrylamide urinary metabolites cannot be fully understood. We developed an isotope dilution ultra-high performance liquid chromatography tandem mass spectrometry (UHPLC-MS/MS) method for the simultaneous determination of all four mercapturic acid adducts of acrylamide and its primary metabolite glycidamide under the electroscopy ionization negative (ESI-) mode in the present study. The limit of detection (LOD) and limit of quantification (LOQ) of the analytes ranged 0.1-0.3 ng/mL and 0.4-1.0 ng/mL, respectively. The recovery rates with low, intermediate and high spiking levels were calculated as 95.5%-105.4%, 98.2%-114.0% and 92.2%-108.9%, respectively. Acceptable within-laboratory reproducibility (RSD<7.0%) substantially supported the use of current method for robust analysis. Rapid pretreatment procedures and short run time (8 min per sample) ensured good efficiency of metabolism profiling, indicating a wide application for investigating short-term internal exposure of dietary acrylamide. Our proposed UHPLC-MS/MS method was successfully applied to the toxicokinetic study of acrylamide in rats. Meanwhile, results of human urine analysis indicated that the levels of N-acetyl-S-(2-carbamoylethyl)-L-cysteine-sulfoxide (AAMA-sul), which did not appear in the mercapturic acid metabolites in rodents, were more than the sum of GAMA and N-acetyl-S-(1-carbamoyl-2-hydroxyethyl)-L-cysteine (iso-GAMA). Thus, AAMA-sul may alternatively become a specific biomarker for investigating the acrylamide exposure in humans. Current proposed method provides a substantial methodology support for comprehensive profiling of toxicokinetics and daily internal exposure evaluations of acrylamide in vivo.


Assuntos
Acetilcisteína/química , Acrilamida/química , Biomarcadores/análise , Técnicas de Química Analítica , Cromatografia Líquida de Alta Pressão , Isótopos/química , Espectrometria de Massas em Tandem , Acetilcisteína/toxicidade , Acetilcisteína/urina , Animais , Biomarcadores/química , Dieta , Feminino , Humanos , Masculino , Ratos , Ratos Sprague-Dawley , Toxicocinética , Adulto Jovem
18.
J Neural Transm (Vienna) ; 122(12): 1645-57, 2015 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-26253900

RESUMO

The leucine-rich repeat kinase 2 (LRRK2) mutation G2019S is one of the most common genetic causes in Parkinson's disease (PD). The penetrance of G2019S LRRK2 is incomplete and is age-dependent, therefore, it has been speculated that environmental toxins and aging could contribute to G2019S LRRK2-related PD pathogenesis. To prove this speculation, we performed a longitudinal investigation in mice bearing G2019S LRRK2 mutation. BAC G2019S LRRK2 transgenic (Tg) mice and their wildtype (Wt) littermates were treated with lactacystin, a specific proteasome inhibitor. The susceptibilities of mice to lactacystin-induced nigrostriatal dopaminergic (DAergic) degeneration were evaluated, at 5 and 12 months of age. We found that lactacystin treatment caused a greater decline of striatal DA content in the Tg mice at either 5 or 12 months of age than their age-matched Wt littermates. Moreover, the lactacystin-treated Tg or Wt mice at 12 months of age lose much more nigral tyrosine hydroxylase (TH)-positive neurons than the mice at 5 months of age, indicating an age-associated DAergic neurotoxicity. Additionally, stereotactic injection of lactacystin induced a dramatic increase of activated microglia in substantia nigra of mice at 12 months of age, compared with mice at 5 months of age. In summary, our study suggests that expression of the G2019S mutation in the mouse LRRK2 gene confers an age-associated high susceptibility to proteasome inhibition-induced nigrostriatal DAergic degeneration.


Assuntos
Envelhecimento/fisiologia , Corpo Estriado/fisiopatologia , Doenças Neurodegenerativas/genética , Doenças Neurodegenerativas/fisiopatologia , Proteínas Serina-Treonina Quinases/metabolismo , Substância Negra/fisiopatologia , Acetilcisteína/análogos & derivados , Acetilcisteína/toxicidade , Envelhecimento/genética , Animais , Corpo Estriado/patologia , Inibidores de Cisteína Proteinase/toxicidade , Modelos Animais de Doenças , Neurônios Dopaminérgicos/patologia , Neurônios Dopaminérgicos/fisiologia , Predisposição Genética para Doença , Camundongos Transgênicos , Microglia/patologia , Microglia/fisiologia , Atividade Motora/fisiologia , Mutação , Doenças Neurodegenerativas/patologia , Proteínas Serina-Treonina Quinases/genética , Substância Negra/patologia , Tirosina 3-Mono-Oxigenase/metabolismo
19.
Environ Sci Pollut Res Int ; 22(22): 18267-77, 2015 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-26210583

RESUMO

Quantum dots (QDs) are engineered semiconductor nanocrystals with promising application in biomedicine, which have potential toxic effect on biomacromolecules by direct interaction and indirect impact in the body. In this work, the effect of N-acetyl-L-cysteine-capped CdTe quantum dots with fluorescence emission peak at 612 nm (QDs-612) on copper-zinc superoxide dismutase (Cu/ZnSOD) at molecular and cellular level was investigated using isothermal titration calorimetry, spectroscopic techniques, cell counting kit-8, and total SOD assay. The hydrophobic interaction between Cu/ZnSOD and QDs-612 caused static fluorescence quenching of the protein, which was spontaneous with binding constant calculated to be 3.28 × 10(5) L mol(-1). The microenvironment of tyrosine residues, skeleton, and secondary structure of Cu/ZnSOD were changed with adding QDs-612. The molecular Cu/ZnSOD activity was inhibited at different concentrations of QDs-612 as well as the intracellular Cu/ZnSOD activity after 2-h exposure. Compared with the cell viability of hepatocytes and nephrocytes (decreased markedly of the initial level) with higher concentrations of QDs-612 in the absence of vitamin C, the cell viability of these two primary cells increased in the presence of vitamin C, indicating the oxidative damage induced by QDs-612. Therefore, the inhibition of Cu/ZnSOD activity in these two primary cells may be caused by the oxidative damage of massive ROS or direct interaction with QDs-612. This work establishes a new approach to investigate the biological toxicity of CdTe QDs to biomacromolecule from both molecular and cellular perspectives and obtains experimental evidence to thoroughly study the toxicity of CdTe QDs in vivo.


Assuntos
Acetilcisteína/toxicidade , Compostos de Cádmio/toxicidade , Hepatócitos/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Pontos Quânticos/toxicidade , Superóxido Dismutase/efeitos dos fármacos , Telúrio/toxicidade , Animais , Células Cultivadas , Rim/citologia , Camundongos
20.
Oxid Med Cell Longev ; 2015: 363827, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26180585

RESUMO

Polyphenolic extracts from the edible part of artichoke (Cynara scolymus L.) have been shown to be potential chemopreventive and anticancer dietary compounds. High doses of polyphenolic extracts (AEs) induce apoptosis and decrease the invasive potential of the human breast cancer cell line, MDA-MB231. However, the molecular mechanism underlying AEs antiproliferative effects is not completely understood. We demonstrate that chronic and low doses of AEs treatment at sublethal concentrations suppress human breast cancer cell growth via a caspases-independent mechanism. Furthermore, AEs exposure induces a significant increase of senescence-associated ß-galactosidase (SA-ß-gal) staining and upregulation of tumour suppressor genes, p16(INK4a) and p21(Cip1/Waf1) in MDA-MB231 cells. AEs treatment leads to epigenetic alterations in cancer cells, modulating DNA hypomethylation and lysine acetylation levels in total proteins. Cell growth arrest correlates with increased reactive oxygen species (ROS) production in AEs treated breast cancer cells. Inhibition of ROS generation by N-acetylcysteine (NAC) attenuates the antiproliferative effect. These findings demonstrate that chronic AEs treatment inhibits breast cancer cell growth via the induction of premature senescence through epigenetic and ROS-mediated mechanisms. Our results suggest that artichoke polyphenols could be a promising dietary tool either in cancer chemoprevention or/and in cancer treatment as a nonconventional, adjuvant therapy.


Assuntos
Senescência Celular/efeitos dos fármacos , Cynara scolymus/química , Polifenóis/toxicidade , Acetilcisteína/toxicidade , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Inibidor p16 de Quinase Dependente de Ciclina/metabolismo , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Cynara scolymus/metabolismo , Feminino , Células HCT116 , Humanos , Polifenóis/química , Espécies Reativas de Oxigênio/metabolismo
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