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1.
Cell Death Dis ; 11(3): 192, 2020 Mar 17.
Artigo em Inglês | MEDLINE | ID: mdl-32184394

RESUMO

Conditions of impaired adrenal function and tissue destruction, such as in Addison's disease, and treatment resistance of adrenocortical carcinoma (ACC) necessitate improved understanding of the pathophysiology of adrenal cell death. Due to relevant oxidative processes in the adrenal cortex, our study investigated the role of ferroptosis, an iron-dependent cell death mechanism and found high adrenocortical expression of glutathione peroxidase 4 (GPX4) and long-chain-fatty-acid CoA ligase 4 (ACSL4) genes, key factors in the initiation of ferroptosis. By applying MALDI mass spectrometry imaging to normal and neoplastic adrenocortical tissue, we detected high abundance of arachidonic and adrenic acid, two long chain polyunsaturated fatty acids which undergo peroxidation during ferroptosis. In three available adrenal cortex cell models (H295R, CU-ACC1 and CU-ACC-2) a high susceptibility to GPX4 inhibition with RSL3 was documented with EC50 values of 5.7 × 10-8, 8.1 × 10-7 and 2.1 × 10-8 M, respectively, while all non-steroidogenic cells were significantly less sensitive. Complete block of GPX4 activity by RSL3 led to ferroptosis which was completely reversed in adrenal cortex cells by inhibition of steroidogenesis with ketoconazole but not by blocking the final step of cortisol synthesis with metyrapone. Mitotane, the only approved drug for ACC did not induce ferroptosis, despite strong induction of lipid peroxidation in ACC cells. Together, this report is the first to demonstrate extraordinary sensitivity of adrenal cortex cells to ferroptosis dependent on their active steroid synthetic pathways. Mitotane does not induce this form of cell death in ACC cells.

2.
Nature ; 575(7784): 693-698, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31634899

RESUMO

Ferroptosis is an iron-dependent form of necrotic cell death marked by oxidative damage to phospholipids1,2. To date, ferroptosis has been thought to be controlled only by the phospholipid hydroperoxide-reducing enzyme glutathione peroxidase 4 (GPX4)3,4 and radical-trapping antioxidants5,6. However, elucidation of the factors that underlie the sensitivity of a given cell type to ferroptosis7 is crucial to understand the pathophysiological role of ferroptosis and how it may be exploited for the treatment of cancer. Although metabolic constraints8 and phospholipid composition9,10 contribute to ferroptosis sensitivity, no cell-autonomous mechanisms have been identified that account for the resistance of cells to ferroptosis. Here we used an expression cloning approach to identify genes in human cancer cells that are able to complement the loss of GPX4. We found that the flavoprotein apoptosis-inducing factor mitochondria-associated 2 (AIFM2) is a previously unrecognized anti-ferroptotic gene. AIFM2, which we renamed ferroptosis suppressor protein 1 (FSP1) and which was initially described as a pro-apoptotic gene11, confers protection against ferroptosis elicited by GPX4 deletion. We further demonstrate that the suppression of ferroptosis by FSP1 is mediated by ubiquinone (also known as coenzyme Q10, CoQ10): the reduced form, ubiquinol, traps lipid peroxyl radicals that mediate lipid peroxidation, whereas FSP1 catalyses the regeneration of CoQ10 using NAD(P)H. Pharmacological targeting of FSP1 strongly synergizes with GPX4 inhibitors to trigger ferroptosis in a number of cancer entities. In conclusion, the FSP1-CoQ10-NAD(P)H pathway exists as a stand-alone parallel system, which co-operates with GPX4 and glutathione to suppress phospholipid peroxidation and ferroptosis.


Assuntos
Proteínas Reguladoras de Apoptose/metabolismo , Ferroptose/genética , Glutationa/metabolismo , Proteínas Mitocondriais/metabolismo , Animais , Proteínas Reguladoras de Apoptose/genética , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica , Técnicas de Inativação de Genes , Humanos , Peroxidação de Lipídeos/genética , Camundongos , Proteínas Mitocondriais/genética , Ubiquinona/análogos & derivados , Ubiquinona/metabolismo
3.
J Med Chem ; 62(1): 266-275, 2019 01 10.
Artigo em Inglês | MEDLINE | ID: mdl-29688708

RESUMO

Glutathione peroxidase 4 (GPX4) is essential for cell membrane repair, inflammation suppression, and ferroptosis inhibition. GPX4 upregulation provides unique drug discovery opportunities for inflammation and ferroptosis-related diseases. However, rational design of protein activators is challenging. Until now, no compound has been reported to activate the enzyme activity of GPX4. Here, we identified a potential allosteric site in GPX4 and successfully found eight GPX4 activators using a novel computational strategy and experimental studies. Compound 1 from the virtual screen increased GPX4 activity, suppressed ferroptosis, reduced pro-inflammatory lipid mediator production, and inhibited NF-κB pathway activation. Further chemical synthesis and structure-activity relationship studies revealed seven more activators. The strongest compound, 1d4, increased GPX4 activity to 150% at 20 µM in the cell-free assay and 61 µM in cell extracts. Therefore, we demonstrated that GPX4 can be directly activated using chemical compounds to suppress ferroptosis and inflammation. Meanwhile, the discovery of GPX4 activators verified the possibility of rational design of allosteric activators.


Assuntos
Apoptose , Glutationa Peroxidase/química , Sulfonamidas/química , Regulação Alostérica , Sítio Alostérico , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Eicosanoides/biossíntese , Glutationa Peroxidase/genética , Glutationa Peroxidase/metabolismo , Humanos , Ligação de Hidrogênio , Simulação de Dinâmica Molecular , Mutagênese , NF-kappa B/metabolismo , Fosfolipídeo Hidroperóxido Glutationa Peroxidase , Piperazinas/farmacologia , Estrutura Terciária de Proteína , Transdução de Sinais/efeitos dos fármacos , Relação Estrutura-Atividade , Sulfonamidas/metabolismo , Sulfonamidas/farmacologia
4.
J Clin Invest ; 128(8): 3341-3355, 2018 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-29939160

RESUMO

High-risk neuroblastoma is a devastating malignancy with very limited therapeutic options. Here, we identify withaferin A (WA) as a natural ferroptosis-inducing agent in neuroblastoma, which acts through a novel double-edged mechanism. WA dose-dependently either activates the nuclear factor-like 2 pathway through targeting of Kelch-like ECH-associated protein 1 (noncanonical ferroptosis induction) or inactivates glutathione peroxidase 4 (canonical ferroptosis induction). Noncanonical ferroptosis induction is characterized by an increase in intracellular labile Fe(II) upon excessive activation of heme oxygenase-1, which is sufficient to induce ferroptosis. This double-edged mechanism might explain the superior efficacy of WA as compared with etoposide or cisplatin in killing a heterogeneous panel of high-risk neuroblastoma cells, and in suppressing the growth and relapse rate of neuroblastoma xenografts. Nano-targeting of WA allows systemic application and suppressed tumor growth due to an enhanced accumulation at the tumor site. Collectively, our data propose a novel therapeutic strategy to efficiently kill cancer cells by ferroptosis.


Assuntos
Apoptose/efeitos dos fármacos , Neuroblastoma/tratamento farmacológico , Vitanolídeos/farmacologia , Animais , Linhagem Celular Tumoral , Embrião de Galinha , Heme Oxigenase-1/metabolismo , Humanos , Proteína 1 Associada a ECH Semelhante a Kelch/metabolismo , Camundongos , Proteínas de Neoplasias/metabolismo , Neuroblastoma/metabolismo , Neuroblastoma/patologia , Ensaios Antitumorais Modelo de Xenoenxerto
5.
Trends Pharmacol Sci ; 38(5): 489-498, 2017 05.
Artigo em Inglês | MEDLINE | ID: mdl-28363764

RESUMO

The past decade has yielded tremendous insights into how cells die. This has come with our understanding that several distinct forms of cell death are encompassed under the umbrella term necrosis. Among these distinct forms of regulated necrotic cell death, ferroptosis has attracted considerable attention owing to its putative involvement in diverse pathophysiological processes. A key feature of the ferroptosis process is the requirement of phospholipid peroxidation, a process that has been linked with several human pathologies. Now with the establishment of a connection between lipid peroxidation and a distinctive cell death pathway, the search for new small molecules able to suppress lipid peroxidation has gained momentum and may yield novel cytoprotective strategies. We review here advances in our understanding of the ferroptotic process and summarize the development of lipid peroxidation inhibitors with the ultimate goal of suppressing ferroptosis-relevant cell death and related pathologies.


Assuntos
Ácidos Graxos/farmacologia , Peroxidação de Lipídeos/efeitos dos fármacos , Animais , Apoptose/fisiologia , Humanos , Ferro/fisiologia
6.
Free Radic Biol Med ; 108: 86-93, 2017 07.
Artigo em Inglês | MEDLINE | ID: mdl-28323132

RESUMO

UVA light is hardly absorbed by the DNA molecule, but recent works point to a direct mechanism of DNA lesion by these wavelengths. UVA light also excite endogenous chromophores, which causes DNA damage through ROS. In this study, DNA samples were irradiated with UVA light in different conditions to investigate possible mechanisms involved in the induction of DNA damage. The different types of DNA lesions formed after irradiation were determined through the use of endonucleases, which recognize and cleave sites containing oxidized bases and cyclobutane pyrimidine dimers (CPDs), as well as through antibody recognition. The formation of 8-oxo-7,8-dihydro-2'-deoxyguanine (8-oxodG) was also studied in more detail using electrochemical detection. The results show that high NaCl concentration and concentrated DNA are capable of reducing the induction of CPDs. Moreover, concerning damage caused by oxidative stress, the presence of sodium azide and metal chelators reduce their induction, while deuterated water increases the amounts of oxidized bases, confirming the involvement of singlet oxygen in the generation of these lesions. Curiously, however, high concentrations of DNA also enhanced the formation of oxidized bases, in a reaction that paralleled the increase in the formation of singlet oxygen in the solution. This was interpreted as being due to an intrinsic photosensitization mechanism, depending directly on the DNA molecule to absorb UVA and generate singlet oxygen. Therefore, the DNA molecule itself may act as a chromophore for UVA light, locally producing a damaging agent, which may lead to even greater concerns about the deleterious impact of sunlight.


Assuntos
Dano ao DNA , DNA/química , Desoxiguanosina/análogos & derivados , Oxigênio Singlete/química , Timo/fisiologia , 8-Hidroxi-2'-Desoxiguanosina , Animais , Anticorpos Antinucleares/metabolismo , Bovinos , Sistema Livre de Células , DNA/imunologia , DNA/efeitos da radiação , Desoxiguanosina/química , Desoxiguanosina/metabolismo , Estresse Oxidativo , Transtornos de Fotossensibilidade , Dímeros de Pirimidina/química , Cloreto de Sódio/metabolismo , Luz Solar , Raios Ultravioleta/efeitos adversos
7.
Nat Chem Biol ; 13(1): 81-90, 2017 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-27842066

RESUMO

Enigmatic lipid peroxidation products have been claimed as the proximate executioners of ferroptosis-a specialized death program triggered by insufficiency of glutathione peroxidase 4 (GPX4). Using quantitative redox lipidomics, reverse genetics, bioinformatics and systems biology, we discovered that ferroptosis involves a highly organized oxygenation center, wherein oxidation in endoplasmic-reticulum-associated compartments occurs on only one class of phospholipids (phosphatidylethanolamines (PEs)) and is specific toward two fatty acyls-arachidonoyl (AA) and adrenoyl (AdA). Suppression of AA or AdA esterification into PE by genetic or pharmacological inhibition of acyl-CoA synthase 4 (ACSL4) acts as a specific antiferroptotic rescue pathway. Lipoxygenase (LOX) generates doubly and triply-oxygenated (15-hydroperoxy)-diacylated PE species, which act as death signals, and tocopherols and tocotrienols (vitamin E) suppress LOX and protect against ferroptosis, suggesting a homeostatic physiological role for vitamin E. This oxidative PE death pathway may also represent a target for drug discovery.


Assuntos
Ácido Araquidônico/metabolismo , Ácidos Graxos Insaturados/metabolismo , Fosfolipídeos/metabolismo , Animais , Ácido Araquidônico/antagonistas & inibidores , Morte Celular/efeitos dos fármacos , Linhagem Celular , Coenzima A Ligases/antagonistas & inibidores , Coenzima A Ligases/deficiência , Coenzima A Ligases/metabolismo , Ácidos Graxos Insaturados/antagonistas & inibidores , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout
8.
Nat Chem Biol ; 13(1): 91-98, 2017 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-27842070

RESUMO

Ferroptosis is a form of regulated necrotic cell death controlled by glutathione peroxidase 4 (GPX4). At present, mechanisms that could predict sensitivity and/or resistance and that may be exploited to modulate ferroptosis are needed. We applied two independent approaches-a genome-wide CRISPR-based genetic screen and microarray analysis of ferroptosis-resistant cell lines-to uncover acyl-CoA synthetase long-chain family member 4 (ACSL4) as an essential component for ferroptosis execution. Specifically, Gpx4-Acsl4 double-knockout cells showed marked resistance to ferroptosis. Mechanistically, ACSL4 enriched cellular membranes with long polyunsaturated ω6 fatty acids. Moreover, ACSL4 was preferentially expressed in a panel of basal-like breast cancer cell lines and predicted their sensitivity to ferroptosis. Pharmacological targeting of ACSL4 with thiazolidinediones, a class of antidiabetic compound, ameliorated tissue demise in a mouse model of ferroptosis, suggesting that ACSL4 inhibition is a viable therapeutic approach to preventing ferroptosis-related diseases.


Assuntos
Apoptose , Coenzima A Ligases/metabolismo , Glutationa Peroxidase/metabolismo , Neoplasias Mamárias Experimentais/metabolismo , Animais , Morte Celular/efeitos dos fármacos , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Coenzima A Ligases/antagonistas & inibidores , Coenzima A Ligases/deficiência , Feminino , Glutationa Peroxidase/deficiência , Humanos , Hipoglicemiantes/farmacologia , Neoplasias Mamárias Experimentais/tratamento farmacológico , Neoplasias Mamárias Experimentais/patologia , Camundongos , Camundongos Knockout , Necrose , Fosfolipídeo Hidroperóxido Glutationa Peroxidase , Tiazolidinedionas/farmacologia
9.
Nat Rev Drug Discov ; 15(5): 348-66, 2016 05.
Artigo em Inglês | MEDLINE | ID: mdl-26775689

RESUMO

The discovery of regulated cell death presents tantalizing possibilities for gaining control over the life-death decisions made by cells in disease. Although apoptosis has been the focus of drug discovery for many years, recent research has identified regulatory mechanisms and signalling pathways for previously unrecognized, regulated necrotic cell death routines. Distinct critical nodes have been characterized for some of these alternative cell death routines, whereas other cell death routines are just beginning to be unravelled. In this Review, we describe forms of regulated necrotic cell death, including necroptosis, the emerging cell death modality of ferroptosis (and the related oxytosis) and the less well comprehended parthanatos and cyclophilin D-mediated necrosis. We focus on small molecules, proteins and pathways that can induce and inhibit these non-apoptotic forms of cell death, and discuss strategies for translating this understanding into new therapeutics for certain disease contexts.


Assuntos
Doença , Terapia de Alvo Molecular , Necrose , Bibliotecas de Moléculas Pequenas/farmacologia , Animais , Humanos , Transdução de Sinais
10.
Free Radic Biol Med ; 84: 246-253, 2015 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-25843658

RESUMO

Vitamin E (VE) deficiency results in pronounced muscle weakness and atrophy but the cell biological mechanism of the pathology is unknown. We previously showed that VE supplementation promotes membrane repair in cultured cells and that oxidants potently inhibit repair. Here we provide three independent lines of evidence that VE is required for skeletal muscle myocyte plasma membrane repair in vivo. We also show that when another lipid-directed antioxidant, glutathione peroxidase 4 (Gpx4), is genetically deleted in mouse embryonic fibroblasts, repair fails catastrophically, unless cells are supplemented with VE. We conclude that lipid-directed antioxidant activity provided by VE, and possibly also Gpx4, is an essential component of the membrane repair mechanism in skeletal muscle. This work explains why VE is essential to muscle health and identifies VE as a requisite component of the plasma membrane repair mechanism in vivo.


Assuntos
Antioxidantes/farmacologia , Membrana Celular/metabolismo , Músculo Esquelético/fisiologia , Vitamina E/farmacologia , Animais , Membrana Celular/efeitos dos fármacos , Células Cultivadas , Glutationa Peroxidase/metabolismo , Masculino , Camundongos , Fibras Musculares Esqueléticas/efeitos dos fármacos , Fibras Musculares Esqueléticas/fisiologia , Músculo Esquelético/citologia , Músculo Esquelético/efeitos dos fármacos , Fosfolipídeo Hidroperóxido Glutationa Peroxidase , Ratos Sprague-Dawley
11.
Antioxid Redox Signal ; 22(11): 938-50, 2015 Apr 10.
Artigo em Inglês | MEDLINE | ID: mdl-25647640

RESUMO

AIMS: Mitochondrial thioredoxin reductase (Txnrd2) is a central player in the control of mitochondrial hydrogen peroxide (H2O2) abundance by serving as a direct electron donor to the thioredoxin-peroxiredoxin axis. In this study, we investigated the impact of targeted disruption of Txnrd2 on tumor growth. RESULTS: Tumor cells with a Txnrd2 deficiency failed to activate hypoxia-inducible factor-1α (Hif-1α) signaling; it rather caused PHD2 accumulation, Hif-1α degradation and decreased vascular endothelial growth factor (VEGF) levels, ultimately leading to reduced tumor growth and tumor vascularization. Increased c-Jun NH2-terminal Kinase (JNK) activation proved to be the molecular link between the loss of Txnrd2, an altered mitochondrial redox balance with compensatory upregulation of glutaredoxin-2, and elevated PHD2 expression. INNOVATION: Our data provide compelling evidence for a yet-unrecognized mitochondrial Txnrd-driven, regulatory mechanism that ultimately prevents cellular Hif-1α accumulation. In addition, simultaneous targeting of both the mitochondrial thioredoxin and glutathione systems was used as an efficient therapeutic approach in hindering tumor growth. CONCLUSION: This work demonstrates an unexpected regulatory link between mitochondrial Txnrd and the JNK-PHD2-Hif-1α axis, which highlights how the loss of Txnrd2 and the resulting altered mitochondrial redox balance impairs tumor growth as well as tumor-related angiogenesis. Furthermore, it opens a new avenue for a therapeutic approach to hinder tumor growth by the simultaneous targeting of both the mitochondrial thioredoxin and glutathione systems.


Assuntos
Proliferação de Células , Prolina Dioxigenases do Fator Induzível por Hipóxia/genética , Mitocôndrias/metabolismo , Neovascularização Patológica/metabolismo , Tiorredoxina Redutase 2/genética , Animais , Células Cultivadas , Técnicas de Silenciamento de Genes , Xenoenxertos , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Prolina Dioxigenases do Fator Induzível por Hipóxia/metabolismo , Camundongos , Camundongos Transgênicos , Transplante de Neoplasias , Espécies Reativas de Oxigênio/metabolismo
12.
Antioxid Redox Signal ; 22(18): 1667-80, 2015 Jun 20.
Artigo em Inglês | MEDLINE | ID: mdl-25566681

RESUMO

SIGNIFICANCE: An ancient anionic phospholipid, cardiolipin (CL), ubiquitously present in prokaryotic and eukaryotic membranes, is essential for several structural and functional purposes. RECENT ADVANCES: The emerging role of CLs in signaling has become the focus of many studies. CRITICAL ISSUES: In this work, we describe two major pathways through which mitochondrial CLs may fulfill the signaling functions via utilization of their (i) asymmetric distribution across membranes and translocations, leading to the surface externalization and (ii) ability to undergo oxidation reactions to yield the signature products recognizable by the executionary machinery of cells. FUTURE DIRECTIONS: We present a concept that CLs and their oxidation/hydrolysis products constitute a rich communication language utilized by mitochondria of eukaryotic cells for diversified regulation of cell physiology and metabolism as well as for inter-cellular interactions.


Assuntos
Cardiolipinas/metabolismo , Membranas Mitocondriais/metabolismo , Oxirredução , Transdução de Sinais , Animais , Apoptose , Cardiolipinas/química , Humanos , Hidrólise , Metabolismo dos Lipídeos , Mitocôndrias/metabolismo , Células Procarióticas/química , Células Procarióticas/metabolismo
13.
Biochim Biophys Acta ; 1850(8): 1566-74, 2015 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-25450170

RESUMO

BACKGROUND: During maturation and storage, spermatozoa generate substantial amounts of reactive oxygen species (ROS) and are thus forced to cope with an increasingly oxidative environment that is both needed and detrimental to their biology. Such a janus-faceted intermediate needs to be tightly controlled and this is done by a wide array of redox enzymes. These enzymes not only have to prevent unspecific modifications of essential cellular biomolecules by quenching undesired ROS, but they are also required and often directly involved in critical protein modifications. SCOPE OF REVIEW: The present review is conceived to present an update on what is known about critical roles of redox enzymes, whereby special emphasis is put on the family of glutathione peroxidases, which for the time being presents the best characterized tasks during gametogenesis. MAJOR CONCLUSIONS: We therefore demonstrate that understanding the function of (seleno)thiol-based oxidases/reductases is not a trivial task and relevant knowledge will be mainly gained by using robust systems, as exemplified by several (conditional) knockout studies. We thus stress the importance of using such models for providing unequivocal evidence in the molecular understanding of redox regulatory mechanisms in sperm maturation. GENERAL SIGNIFICANCE: ROS are not merely detrimental by-products of metabolism and their proper generation and usage by specific enzymes is essential for vital functions as beautifully exemplified during male gametogenesis. As such, lessons learnt from thiol-based oxidases/reductases in male gametogenesis could be used as a general principle for other organs as it is most likely not only restricted to this developmental phase. This article is part of a Special Issue entitled Redox regulation of differentiation and de-differentiation.


Assuntos
Espécies Reativas de Oxigênio/metabolismo , Espermatogênese , Espermatozoides/metabolismo , Compostos de Sulfidrila/metabolismo , Animais , Glutationa Peroxidase/metabolismo , Humanos , Masculino , Modelos Biológicos , Oxirredução , Fosfolipídeo Hidroperóxido Glutationa Peroxidase , Espermatozoides/citologia
14.
J Toxicol Environ Health A ; 76(6): 345-53, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23557233

RESUMO

The chemotherapeutic agent cisplatin (cDDP) is widely used to treat a variety of solid and hematological tumors. However, cDDP exerts severe side effects, such as nephrotoxicity, neurotoxicity, and bone-marrow suppression. The use of some dietary compounds to protect organs that are not targets in association with chemotherapy has been encouraged. This study evaluated the protective effects of chlorophyll b (CLb) on DNA damage induced by cDDP by use of single-cell gel electrophoresis (SCGE) assays. Further, this investigation also determined platinum (Pt) and magnesium (Mg) bioaccumulation in mice tissues after treatment with CLb alone and/or in association of cDDP (simultaneous treatment) by inductively coupled plasma-mass spectroscopy (ICP-MS). All parameters were studied in peripheral blood cells (PBC), kidneys, and liver of mice after administration of CLb (0.2 or 0.5 mg/kg of body weight [b.w.]), cDDP (6 mg/kg b.w.), and the combination CLb 0.2 plus cDDP or CLb 0.5 plus cDDP. Pt accumulation in liver and kidneys was higher than that found in PBC, while DNA damage was higher in kidneys and liver than in PBC. Further, treatment with CLb alone did not induce DNA damage. Evidence indicates that genotoxic effects produced by cDDP may not be related to Pt accumulation and distribution. In combined treatments, CLb decreased DNA damage in tissues, but the PT contents did not change and these treatments also showed that CLb may be an important source of Mg. Thus, our results indicate that consumption of CLb-rich foods may diminish the adverse health effects induced by cDDP exposure.


Assuntos
Antimutagênicos/farmacologia , Antineoplásicos/toxicidade , Clorofila/farmacologia , Cisplatino/toxicidade , Dano ao DNA/efeitos dos fármacos , Animais , Antineoplásicos/farmacocinética , Cisplatino/farmacocinética , Ensaio Cometa , Feminino , Rim/efeitos dos fármacos , Rim/metabolismo , Fígado/efeitos dos fármacos , Fígado/metabolismo , Compostos de Magnésio/metabolismo , Masculino , Camundongos , Compostos de Platina/metabolismo
15.
Neurochem Int ; 62(5): 738-49, 2013 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-23333338

RESUMO

Oxidative stress is a major common hallmark of many neurodegenerative disease such as Alzheimer's disease (AD), Parkinson's disease (PD), amyotrophic lateral sclerosis (ALS), and stroke. Novel concepts in our understanding of oxidative stress indicate that a perturbed redox circuitry could be strongly linked with the onset of such diseases. In this respect, glutathione and thioredoxin dependent antioxidant enzymes play a central role as key regulators due to the fact that a slight dysfunction of any of these enzymes leads to sustained reactive oxygen species (ROS) production. Apart from their classical role as ROS scavengers, some of these enzymes are also able to control post-translational modifications. Therefore, efficient control of ROS production and reversibility of post-translational modifications are critical as improper control of such events may lead to the activation of pathological redox circuits that eventually culminate in neuronal cell death. To dissect the apparently opposing functions of ROS in cell physiology and pathophysiology, a proper working toolkit is mandatory. In vivo modeling is an absolute requirement due to the complexity of redox signaling systems that often contradict data obtained from in vitro approaches. Hence, inducible/conditional knockout mouse models for key redox enzymes are emerging as powerful tools to perturb redox circuitries in a temporal and spatial manner. In this review we address the basics of ROS generation, chemistry and detoxification as well as examples in where applications of mouse models of important enzymes have been successfully applied in the study of neurodegenerative processes. We also highlight the importance of new models to overcome present technical limitations in order to advance in the study of redox processes in the role of neurodegeneration.


Assuntos
Glutationa/metabolismo , Doenças Neurodegenerativas/metabolismo , Tiorredoxinas/metabolismo , Animais , Encéfalo/metabolismo , Morte Celular , Camundongos , Doenças Neurodegenerativas/genética , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais
16.
Mutat Res ; 726(2): 109-15, 2011 Dec 24.
Artigo em Inglês | MEDLINE | ID: mdl-21820078

RESUMO

Aim of this study was to investigate the cytotoxic and genotoxic properties of inorganic and organic mercury compounds, i.e., HgCl(2) and methylmercury (MeHg). In addition, the DNA-protective and antioxidant effects of the flavonoid quercetin (QC) were studied. All experiments were conducted with human-derived liver cells (HepG2), which possess antioxidant and drug-metabolizing enzymes in an inducible form. 8-Hydroxydeoxyguanosine (8-OHdG) and comet formation were monitored as endpoints of DNA damage. The impact of the metal compounds on the redox status was also investigated, since it is assumed that their toxic effects are due to oxidative damage. A number of biochemical parameters related to oxidative stress, namely glutathione, malondialdehyde, protein carbonyl and formation of reactive oxygen species (ROS) were measured after treatment of the cells with the mercury compounds in the presence and absence of quercetin. To elucidate the mechanisms that underlie the effects of QC, three protocols (pre-, simultaneous and post-treatment) were used. Both mercury compounds (range 0.1-5.0µM) caused induction of DNA migration and formation of 8-OHdG. In combination with the flavonoid (range 0.1-5.0µM), DNA-protective effects of QC were observed after pre- and simultaneous treatment but not when the flavonoid was added after treatment with the metal compounds. Exposure to the metal compounds led also to substantial changes of all parameters of the redox status and co-treatment experiments with QC showed that these alterations are reversed by the flavonoid. Taken together, the results of our experiments indicate that these two mercury compounds cause DNA damage and oxidative stress in human-derived liver cells and that the flavonoid reduces these effects. Since the concentrations of the metals and of the flavonoids used in the present work reflect human exposure, our findings can be taken as an indication that QC may protect humans against the adverse effects caused by the metal.


Assuntos
Antimutagênicos/farmacologia , Antioxidantes/farmacologia , Dano ao DNA/efeitos dos fármacos , Cloreto de Mercúrio/toxicidade , Compostos de Metilmercúrio/toxicidade , Oxirredução/efeitos dos fármacos , Quercetina/farmacologia , 8-Hidroxi-2'-Desoxiguanosina , Sobrevivência Celular , Desoxiguanosina/análogos & derivados , Desoxiguanosina/análise , Células Hep G2 , Humanos , Estresse Oxidativo/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo
17.
Arch Toxicol ; 85(9): 1151-7, 2011 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-21286687

RESUMO

Aim of the study was to find out whether consumption of quercetin (QC), an abundant flavonoid in the human diet, protects against DNA damage caused by exposure to organic mercury. Therefore, rats were treated orally with methylmercury (MeHg) and the flavonoid with doses that reflect the human exposure. The animals received MeHg (30 µg/kg/bw/day), QC (0.5-50 mg/kg/bw/day), or combinations of both over 45 days. Subsequently, the glutathione levels (GSH) and the activities of glutathione peroxidase (GPx) and catalase (CAT) were determined, and DNA damage was measured in hepatocytes and peripheral leukocytes in single cell gel electrophoresis assays. MeHg decreased the concentration of GSH and the activity of GPx by 17 and 12%, respectively and caused DNA damage to liver and blood cells, while with QC no such effects were seen. When the flavonoid was given in combination with MeHg, the intermediate and the highest concentrations (5.0 and 50.0 mg/kg/bw/day) were found to cause DNA protection; DNA migration was reduced by 54 and 65% in the hepatocytes and by 27 and 36% in the leukocytes; furthermore, the reduction in GSH and GPx levels caused by MeHg treatment was restored. In summary, our results indicate that consumption of QC-rich foods may protect Hg-exposed humans against the adverse health effects of the metal.


Assuntos
Antioxidantes/farmacologia , Dano ao DNA/efeitos dos fármacos , Compostos de Metilmercúrio/toxicidade , Mutagênicos/toxicidade , Estresse Oxidativo/efeitos dos fármacos , Quercetina/farmacologia , Animais , Catalase/metabolismo , Ensaio Cometa , Glutationa/metabolismo , Glutationa Peroxidase/metabolismo , Hepatócitos/efeitos dos fármacos , Hepatócitos/enzimologia , Hepatócitos/metabolismo , Leucócitos/efeitos dos fármacos , Leucócitos/enzimologia , Leucócitos/metabolismo , Fígado/efeitos dos fármacos , Fígado/enzimologia , Fígado/metabolismo , Masculino , Compostos de Metilmercúrio/sangue , Compostos de Metilmercúrio/farmacocinética , Mutagênicos/farmacocinética , Ratos , Ratos Wistar
18.
Ecotoxicol Environ Saf ; 74(3): 487-93, 2011 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-20970192

RESUMO

The present study evaluates a possible protective effect of fish oil against oxidative damage promoted by methylmercury (MeHg) in sub-chronically exposed rats. Reduced glutathione peroxidase and catalase enzyme activity and reduced glutathione levels were observed in MeHg-exposed animals compared to controls. Methylmercury exposure was also associated with DNA damage. Administration of fish oil to the methylmercury-exposed animals did not ameliorate enzyme activity or glutathione levels. On the other hand, a significant DNA protective effect (about 30%) was observed with fish oil treatment. There were no differences in the total mercury concentration in rat liver, kidney, heart or brain after MeHg administration with or without fish oil co-administration. Histopathological analyses showed a significant leukocyte infiltration in rat tissues after MeHg exposure, but this effect was significantly reduced after co-administration of fish oil. Taken together, our findings demonstrate oxidative damage even after low-level MeHg exposure and the protective effect of fish oil. This protection seems not to be related to antioxidant defenses or mercury re-distribution in rat tissues. It is probably due to the anti-inflammatory effects of fish oil.


Assuntos
Antioxidantes/farmacologia , Óleos de Peixe/farmacologia , Compostos de Metilmercúrio/toxicidade , Estresse Oxidativo/efeitos dos fármacos , Animais , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Encéfalo/patologia , Catalase/metabolismo , Dano ao DNA/efeitos dos fármacos , Glutationa Peroxidase/metabolismo , Rim/metabolismo , Fígado/metabolismo , Masculino , Mercúrio/metabolismo , Mercúrio/toxicidade , Miocárdio/metabolismo , Miocárdio/patologia , Ratos , Ratos Wistar
19.
São Paulo; s.n; 2011. 233 p. ilus, tab, graf.
Tese em Português | LILACS | ID: lil-691571

RESUMO

O estudo do processo da peroxidação de lipídios tem aumentado nos últimos anos, principalmente devido à implicação dos hidroperóxidos de lipídios (LOOH) em diversos processos patológicos. A decomposição destes LOOH é capaz de gerar subprodutos capazes de promover danos em biomoléculas, incluindo proteínas e DNA. No presente trabalho, utilizando hidroperóxidos de ácido linoléico isotopicamente marcado com átomo de oxigênio-18 (LA18O18OH), fomos capazes de demonstrar que estas moléculas gerararam oxigênio singlete marcado [18(1O2)] em células em cultura. A detecção de tal espécie foi possível através da utilização de uma nova metodologia utilizando um derivado antracenico. Para este propósito foi utilizado o derivado de antraceno 3,3'-(9,10-antracenodiil) bisacrilato (DADB), cujo produto especifico da reação com o 1O2 (o endoperóxido do DADB DADBO2) do pode ser facilmente detectado por HPLC-MS/MS. De forma a expandir a compreensão dos efeitos tóxicos desses LOOH, investigamos o efeito destes compostos gerados intracelularmente. Para tal, foi utilizado o Rosa bengala (RB), um fotosensibilizador que tem afinidade por espaços apolares como membranas e lisossomos. A fotosenssibilização deste composto foi capaz de induzir a morte celular, e esta morte estaria relacionada a uma maior formação de 1O2 e a um maior acumulo de peróxidos. Nestes estudos foi possível demonstrar que carotenóides e sistemas antioxidantes dependentes de glutationa foram capazes de proteger contra os efeitos tóxicos da fotosensibilização na presença de RB. Adicionalmente foram avaliados os efeitos da hemoglobina (Hb) e do hidroperóxido do ácido linoléico (LAOOH) em uma série de parâmetros toxicológicos, como citotoxicidade, estado redox, a peroxidação lipídica e dano ao DNA. Nós demonstramos que a pré-incubação das células com Hb e sua posterior exposição à LAOOH (Hb + LAOOH) levou a um aumento na morte celular, a oxidação do DCFH, formação de malonaldeído e fragmentação do DNA e que esses...


The study of the process of lipid peroxidation has increased in recent years, mainly due to the involvement of lipid hydroperoxide (LOOH) in a series of pathological processes. The decomposition of LOOH is able to generate products that can promote damage to biomolecules, including proteins and DNA. In the present work, using linoleic acid hydroperoxide isotopically labeled with 18O2 (LA18O18OH), we demonstrate that these molecules were able to generate labeled singlet oxygen [18(1O2)] in cultured cells. The detection of such species was possible using a new methodology using an anthracene derivative .For this purpose we used the anthracene derivative of 3,3'-(9,10-antracendiil) bisacrilate (DADB), whose specific reaction product with 1O2 (DADB endoperoxide DADBO2) can be easily detected by HPLC-MS/MS. In order to expand the understanding of the toxic effects of LOOH, we investigated the effect of these compounds generated intracellularly. For this porpoise, we used Rose Bengal (RB), a photosensitizer that has affinity for apolar spaces such as membranes and lysosomes. The photosensitization of this compound was able to induce cell death, and this death was related to increased formation of 1O2 and a higher accumulation of peroxides. In these studies we have shown that carotenoids and glutathione-dependent antioxidant systems were capable of protecting against the toxic effects of photosensitization in the presence of RB. Additionally, we evaluated the effects of hemoglobin (Hb) and linoleic acid hydroperoxide (LAOOH) in a series of toxicological endpoints such as cytotoxicity, redox status, lipid peroxidation and DNA damage. We demonstrated that preincubation of cells with Hb and its subsequent exposure to LAOOH (Hb + LAOOH) led to an increase in cell death, DCFH oxidation, formation of malonaldehyde and DNA fragmentation, and that these effects were related to the peroxide and the heme group. It was demonstrated that cells incubated with LAOOH and Hb showed...


Assuntos
Oxigênio Singlete/química , Oxigênio Singlete/sangue , Peroxidação de Lipídeos/genética , Antracenos/análise , Antracenos/química , Fenômenos Bioquímicos , Cromatografia Líquida de Alta Pressão , Genotoxicidade/análise , Hemoglobinas/química , Fármacos Fotossensibilizantes , Fenômenos Genéticos/efeitos da radiação
20.
Mutagenesis ; 25(3): 223-9, 2010 May.
Artigo em Inglês | MEDLINE | ID: mdl-20032005

RESUMO

Silybin (SB), a constituent of the medicinal plant Silybum marianum, is reported to be a potent hepatoprotective agent, but little is currently known regarding its genotoxicity, mutagenicity and potential chemopreventive properties. In this study, we evaluated the ability of SB to induce DNA migration and micronuclei (MN) formation in human hepatoma cells (HepG2). Also, possible preventive effects of SB on MN formation induced by three different mutagens, bleomycin (BLEO), benzo[a]pyrene (B[a]P) and aflatoxin B(1) (AFB(1)), were studied. To clarify the possible mechanism of SB antimutagenicity, three treatment protocols were applied: pretreatment, in which SB was added before the application of the mutagens; simultaneous treatment, in which SB was added during treatment and post-treatment, in which SB was added after the application of the mutagens. At concentrations up to 100 microM, SB was non-genotoxic, while at a concentration of 200 microM, SB induced DNA migration, generated oxidized DNA bases, reduced cell viability, decreased the replicative index of the cells and induced oxidative stress. It is noteworthy that SB was able to reduce the genotoxic effect induced by B[a]P, BLEO and AFB(1) in pretreatment and simultaneous treatments but had no significant effect on DNA damage induction in post-treatment. Taken together, our findings indicate that SB presents anti-genotoxic activity in vitro, which suggests potential use as a chemopreventive agent.


Assuntos
Carcinoma Hepatocelular/patologia , Neoplasias Hepáticas/patologia , Mutagênicos/toxicidade , Silimarina/toxicidade , Aflatoxina B1/toxicidade , Benzo(a)pireno/toxicidade , Bleomicina/toxicidade , Morte Celular/efeitos dos fármacos , Divisão Celular/efeitos dos fármacos , Linhagem Celular Tumoral , DNA/metabolismo , Dano ao DNA , DNA-Formamidopirimidina Glicosilase/metabolismo , Endonucleases/metabolismo , Proteínas de Escherichia coli/metabolismo , Humanos , Espaço Intracelular/efeitos dos fármacos , Espaço Intracelular/metabolismo , Micronúcleos com Defeito Cromossômico/efeitos dos fármacos , Mutagênicos/química , Espécies Reativas de Oxigênio/metabolismo , Silibina , Silimarina/química
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