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1.
Oncogene ; 43(22): 1701-1713, 2024 May.
Artigo em Inglês | MEDLINE | ID: mdl-38600165

RESUMO

Triple-negative breast cancer (TNBC) is a very aggressive and heterogeneous group of tumors. In order to develop effective therapeutic strategies, it is therefore essential to identify the subtype-specific molecular mechanisms underlying disease progression and resistance to chemotherapy. TNBC cells are highly dependent on exogenous cystine, provided by overexpression of the cystine/glutamate antiporter SLC7A11/xCT, to fuel glutathione synthesis and promote an oxidative stress response consistent with their high metabolic demands. Here we show that TNBC cells of the mesenchymal stem-like subtype (MSL) utilize forced cystine uptake to induce activation of the transcription factor NRF2 and promote a glutathione-independent mechanism to defend against oxidative stress. Mechanistically, we demonstrate that NRF2 activation is mediated by direct cysteinylation of the inhibitor KEAP1. Furthermore, we show that cystine-mediated NRF2 activation induces the expression of important genes involved in oxidative stress response, but also in epithelial-to-mesenchymal transition and stem-like phenotype. Remarkably, in survival analysis, four upregulated genes (OSGIN1, RGS17, SRXN1, AKR1B10) are negative prognostic markers for TNBC. Finally, expression of exogenous OSGIN1, similarly to expression of exogenous NRF2, can prevent cystine depletion-dependent death of MSL TNBC cells. The results suggest that the cystine/NRF2/OSGIN1 axis is a potential target for effective treatment of MSL TNBCs.


Assuntos
Fator 2 Relacionado a NF-E2 , Estresse Oxidativo , Neoplasias de Mama Triplo Negativas , Fator 2 Relacionado a NF-E2/metabolismo , Fator 2 Relacionado a NF-E2/genética , Neoplasias de Mama Triplo Negativas/metabolismo , Neoplasias de Mama Triplo Negativas/patologia , Neoplasias de Mama Triplo Negativas/genética , Humanos , Feminino , Linhagem Celular Tumoral , Proteína 1 Associada a ECH Semelhante a Kelch/metabolismo , Proteína 1 Associada a ECH Semelhante a Kelch/genética , Cisteína/metabolismo , Transição Epitelial-Mesenquimal/genética , Sistema y+ de Transporte de Aminoácidos/metabolismo , Sistema y+ de Transporte de Aminoácidos/genética , Regulação Neoplásica da Expressão Gênica , Sobrevivência Celular/genética
2.
Cell Cycle ; 22(17): 1827-1853, 2023 09.
Artigo em Inglês | MEDLINE | ID: mdl-37522842

RESUMO

Background: Desipramine a representative of tricyclic antidepressants (TCAs) promotes recovery of depressed patients by inhibition of reuptake of neurotransmitters serotonin (SER) and norepinephrine (NE) in the presynaptic membrane by directly blocking their respective transporters SERT and NET.Aims: To study the effect of desipramine on programmed erythrocyte death (eryptosis) and explore the underlying mechanisms.Methods: Phosphatidylserine (PS) exposure on the cell surface as marker of cell death was estimated from annexin-V-binding, cell volume from forward scatter in flow cytometry. Hemolysis was determined photometrically, and intracellular glutathione [GSH]i from high performance liquid chromatography.Results: Desipramine dose-dependently significantly enhanced the percentage of annexin-V-binding cells and didn´t impact glutathione (GSH) synthesis. Desipramine-induced eryptosis was significantly reversed by pre-treatment of erythrocytes with either nitric oxide (NO) donor sodium nitroprusside (SNP) or N-acetyl-L-cysteine (NAC). The highest inhibitory effect was obtained by using both inhibitors together. Calcium (Ca2+) depletion aggravated desipramine-induced eryptosis. Changing the order of treatment, i.e. desipramine first followed by inhibitors, could not influence the inhibitory effect of SNP or NAC.Conclusion: Antidepressants-caused intoxication can be treated by SNP and NAC, respectively. B) Patients with chronic hypocalcemia should not be treated with tricyclic anti-depressants or their dose should be noticeably reduced.


Assuntos
Eriptose , Doadores de Óxido Nítrico , Humanos , Doadores de Óxido Nítrico/farmacologia , Doadores de Óxido Nítrico/metabolismo , Nitroprussiato/farmacologia , Nitroprussiato/metabolismo , Cálcio/metabolismo , Acetilcisteína/farmacologia , Desipramina/farmacologia , Desipramina/metabolismo , Eritrócitos/metabolismo , Glutationa/metabolismo , Glutationa/farmacologia , Anexinas/metabolismo , Anexinas/farmacologia , Fosfatidilserinas/metabolismo , Tamanho Celular , Ceramidas/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Estresse Oxidativo
3.
Antioxidants (Basel) ; 12(5)2023 May 13.
Artigo em Inglês | MEDLINE | ID: mdl-37237960

RESUMO

Glutathione (GSH) has special antioxidant properties due to its high intracellular concentration, ubiquity, and high reactivity towards electrophiles of the sulfhydryl group of its cysteine moiety. In most diseases where oxidative stress is thought to play a pathogenic role, GSH concentration is significantly reduced, making cells more susceptible to oxidative damage. Therefore, there is a growing interest in determining the best method(s) to increase cellular glutathione for both disease prevention and treatment. This review summarizes the major strategies for successfully increasing cellular GSH stores. These include GSH itself, its derivatives, NRf-2 activators, cysteine prodrugs, foods, and special diets. The possible mechanisms by which these molecules can act as GSH boosters, their related pharmacokinetic issues, and their advantages and disadvantages are discussed.

4.
Cells ; 11(22)2022 11 18.
Artigo em Inglês | MEDLINE | ID: mdl-36429096

RESUMO

Nitisinone (NTBC) was recently approved to treat alkaptonuria (AKU), but there is no information on its impact on oxidative stress and inflammation, which are observed in AKU. Therefore, serum samples collected during the clinical studies SONIA1 (40 AKU patients) and SONIA2 (138 AKU patients) were tested for Serum Amyloid A (SAA), CRP and IL-8 by ELISA; Advanced Oxidation Protein Products (AOPP) by spectrophotometry; and protein carbonyls by Western blot. Our results show that NTBC had no significant effects on the tested markers except for a slight but statistically significant effect for NTBC, but not for the combination of time and NTBC, on SAA levels in SONIA2 patients. Notably, the majority of SONIA2 patients presented with SAA > 10 mg/L, and 30 patients in the control group (43.5%) and 40 patients (58.0%) in the NTBC-treated group showed persistently elevated SAA > 10 mg/L at each visit during SONIA2. Higher serum SAA correlated with lower quality of life and higher morbidity. Despite no quantitative differences in AOPP, the preliminary analysis of protein carbonyls highlighted patterns that deserve further investigation. Overall, our results suggest that NTBC cannot control the sub-clinical inflammation due to increased SAA observed in AKU, which is also a risk factor for developing secondary amyloidosis.


Assuntos
Alcaptonúria , Humanos , Alcaptonúria/tratamento farmacológico , Alcaptonúria/complicações , Alcaptonúria/metabolismo , Produtos da Oxidação Avançada de Proteínas/metabolismo , Produtos da Oxidação Avançada de Proteínas/uso terapêutico , Qualidade de Vida , Biomarcadores/metabolismo , Proteína Amiloide A Sérica/metabolismo , Inflamação/metabolismo , Estresse Oxidativo
5.
J Appl Toxicol ; 42(12): 1948-1961, 2022 12.
Artigo em Inglês | MEDLINE | ID: mdl-35854198

RESUMO

Indoxyl sulphate (IS) is a uremic toxin accumulating in the plasma of chronic kidney disease (CKD) patients. IS accumulation induces side effects in the kidneys, bones and cardiovascular system. Most studies assessed IS effects on cell lines by testing higher concentrations than those measured in CKD patients. Differently, we exposed a human microvascular endothelial cell line (HMEC-1) to the IS concentrations measured in the plasma of healthy subjects (physiological) or CKD patients (pathological). Pathological concentrations reduced cell proliferation rate but did not increase long-term oxidative stress level. Indeed, total protein thiols decreased only after 24 h of exposure in parallel with an increased Nrf-2 protein expression. IS induced actin cytoskeleton rearrangement with formation of stress fibres. Proteomic analysis supported this hypothesis as many deregulated proteins are related to actin filaments organization or involved in the endothelial to mesenchymal transition. Interestingly, two proteins directly linked to cardiovascular diseases (CVD) in in vitro and in vivo studies underwent deregulation: COP9 signalosome complex subunit 9 and thrombomodulin. Future experiments will be needed to investigate the role of these proteins and the signalling pathways in which they are involved to clarify the possible link between CKD and CVD.


Assuntos
Doenças Cardiovasculares , Insuficiência Renal Crônica , Humanos , Indicã/toxicidade , Indicã/metabolismo , Toxinas Urêmicas , Células Endoteliais/metabolismo , Proteômica , Doenças Cardiovasculares/metabolismo
6.
Antioxidants (Basel) ; 11(7)2022 Jul 14.
Artigo em Inglês | MEDLINE | ID: mdl-35883857

RESUMO

SARS-CoV-2 infection can cause a severe respiratory distress syndrome with inflammatory and thrombotic complications, the severity of which increases with patients' age and presence of comorbidity. The reasons for an age-dependent increase in the risk of severe COVID-19 could be many. These include defects in the homeostatic processes that control the cellular redox and its pivotal role in sustaining the immuno-inflammatory response to the host and the protection against oxidative stress and tissue degeneration. Pathogens may take advantage of such age-dependent abnormalities. Alterations of the thiol redox balance in the lung tissue and lining fluids may influence the risk of infection, and the host capability to respond to pathogens and to avoid severe complications. SARS-CoV-2, likewise other viruses, such as HIV, influenza, and HSV, benefits in its replication cycle of pro-oxidant conditions that the same viral infection seems to induce in the host cell with mechanisms that remain poorly understood. We recently demonstrated that the pro-oxidant effects of SARS-CoV-2 infection are associated with changes in the cellular metabolism and transmembrane fluxes of Cys and GSH. These appear to be the consequence of an increased use of Cys in viral protein synthesis and to ER stress pathway activation that interfere with transcription factors, as Nrf2 and NFkB, important to coordinate the metabolism of GSH with other aspects of the stress response and with the pro-inflammatory effects of this virus in the host cell. This narrative review article describes these cellular and molecular aspects of SARS-CoV-2 infection, and the role that antivirals and cytoprotective agents such as N-acetyl cysteine may have to limit the cytopathic effects of this virus and to recover tissue homeostasis after infection.

7.
J Pineal Res ; 73(1): e12806, 2022 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-35524288

RESUMO

Melatonin (MLT) is a cytoprotective agent holding potential to prevent cadmium (Cd) toxicity and its impact in testicular function and fertility. In this study, we explored such potential in porcine pre-pubertal Sertoli cells (SCs). Cd toxicity resulted in impaired SC viability and function, abnormal cellular H2 O2 generation and efflux, and induction of reductive stress by the upregulation of Nrf2 expression and activity, cystine uptake and glutathione biosynthesis, glutathione-S-transferase P (GSTP) expression, and protein glutathionylation inhibition. Cd toxicity also stimulated the activity of cellular kinases (MAPK-ERK1/2 and Akt) and NFkB transcription factor, and cJun expression was increased. MLT produced a potent cytoprotective effect when co-administered with Cd to SCs; its efficacy and the molecular mechanism behind its cytoprotective function varied according to Cd concentrations. However, a significant restoration of cell viability and function, and of H2 O2 levels, was observed both at 5 and 10 µM Cd. Mechanistically, these effects of MLT were associated with a significant reduction of the Cd-induced activation of Nrf2 and GSTP expression at all Cd concentrations. CAT and MAPK-ERK1/2 activity upregulation was associated with these effects at 5 µM Cd, whereas glutathione biosynthesis and efflux were involved at 10 µM Cd together with an increased expression of the cystine transporter xCT, of cJun and Akt and NFkB activity. MLT protects SCs from Cd toxicity reducing its H2 O2 generation and reductive stress effects. A reduced activity of Nrf2 and the modulation of other molecular players of MLT signaling, provide a mechanistic rational for the cytoprotective effect of this molecule in SCs.


Assuntos
Melatonina , Fator 2 Relacionado a NF-E2 , Animais , Cádmio/farmacologia , Cistina/metabolismo , Cistina/farmacologia , Glutationa/metabolismo , Masculino , Melatonina/metabolismo , Melatonina/farmacologia , Fator 2 Relacionado a NF-E2/metabolismo , Estresse Oxidativo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Células de Sertoli/metabolismo , Suínos
8.
Int J Mol Sci ; 23(5)2022 Mar 05.
Artigo em Inglês | MEDLINE | ID: mdl-35269995

RESUMO

Thiols (sulfhydryl groups) are effective antioxidants that can preserve the correct structure of proteins, and can protect cells and tissues from damage induced by oxidative stress. Abnormal levels of thiols have been measured in the blood of patients with moderate-to-severe chronic kidney disease (CKD) compared to healthy subjects, as well as in end-stage renal disease (ESRD) patients on haemodialysis or peritoneal dialysis. The levels of protein thiols (a measure of the endogenous antioxidant capacity inversely related to protein oxidation) and S-thiolated proteins (mixed disulphides of protein thiols and low molecular mass thiols), and the protein thiolation index (the molar ratio of the S-thiolated proteins to free protein thiols in plasma) have been investigated in the plasma or red blood cells of CKD and ESRD patients as possible biomarkers of oxidative stress. This type of minimally invasive analysis provides valuable information on the redox status of the less-easily accessible tissues and organs, and of the whole organism. This review provides an overview of reversible modifications in protein thiols in the setting of CKD and renal replacement therapy. The evidence suggests that protein thiols, S-thiolated proteins, and the protein thiolation index are promising biomarkers of reversible oxidative stress that could be included in the routine monitoring of CKD and ESRD patients.


Assuntos
Falência Renal Crônica , Insuficiência Renal Crônica , Antioxidantes/metabolismo , Biomarcadores/metabolismo , Humanos , Falência Renal Crônica/terapia , Oxirredução , Estresse Oxidativo , Proteínas/metabolismo , Insuficiência Renal Crônica/terapia , Compostos de Sulfidrila/química
9.
Arch Biochem Biophys ; 717: 109137, 2022 03 15.
Artigo em Inglês | MEDLINE | ID: mdl-35090868

RESUMO

Alkaptonuria (AKU) is an ultra-rare genetic disease caused by a deficient activity of the enzyme homogentisate 1,2-dioxygenase (HGD) leading to the accumulation of homogentisic acid (HGA) on connective tissues. Even though AKU is a multi-systemic disease, osteoarticular cartilage is the most affected system and the most damaged tissue by the disease. In chondrocytes, HGA causes oxidative stress dysfunctions, which induce a series of not fully characterized cellular responses. In this study, we used a human chondrocytic cell line as an AKU model to evaluate, for the first time, the effect of HGA on autophagy, the main homeostasis system in articular cartilage. Cells responded timely to HGA treatment with an increase in autophagy as a mechanism of protection. In a chronic state, HGA-induced oxidative stress decreased autophagy, and chondrocytes, unable to restore balance, activated the chondroptosis pathway. This decrease in autophagy also correlated with the accumulation of ochronotic pigment, a hallmark of AKU. Our data suggest new perspectives for understanding AKU and a mechanistic model that rationalizes the damaging role of HGA.


Assuntos
Alcaptonúria/prevenção & controle , Autofagia/efeitos dos fármacos , Biomarcadores/metabolismo , Homogentisato 1,2-Dioxigenase/metabolismo , Ácido Homogentísico/metabolismo , Alcaptonúria/metabolismo , Apoptose/efeitos dos fármacos , Cartilagem Articular/efeitos dos fármacos , Linhagem Celular , Condrócitos/citologia , Ácido Homogentísico/farmacologia , Humanos , Ocronose/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Transdução de Sinais
10.
Amino Acids ; 54(4): 675-686, 2022 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-34129091

RESUMO

S-glutathionylated proteins (GSSP), i.e., protein-mixed disulfides with glutathione (GSH), are considered a suitable biomarker of oxidative stress. In fact, they occur within cells at low level and their concentration increases markedly under pro-oxidant conditions. Plasma is something different, since it is physiologically rich in S-thiolated proteins (RSSP), i.e., protein-mixed disulfides with various types of low molecular mass thiols (LMM-SH). However, albumin, which is largely the most abundant plasma protein, possesses a cysteine residue at position 34 that is mostly reduced (about 60%) under physiological conditions, but easily involved in the formation of additional RSSP in the presence of oxidants. The quantification of GSSP requires special attention to sample handling, since their level can be overestimated as a result of artefactual oxidation of GSH. We have developed the present protocol to avoid this methodological problem. Samples should be treated as soon as possible after their collection with the alkylating agent N-ethylmaleimide that masks -SH groups and prevents their oxidation. The GSH released from mixed disulfides by reduction with dithiothreitol is then labeled with the fluorescent probe monobromobimane and quantified by HPLC. The method can be applied to many different biological samples, comprising blood components, red blood cell plasma membrane, cultured cells, and solid organs from animal models.


Assuntos
Dissulfetos , Glutationa , Animais , Compostos Bicíclicos com Pontes , Cromatografia Líquida de Alta Pressão , Cisteína/química , Dissulfetos/química , Glutationa/metabolismo , Oxirredução , Proteínas/metabolismo , Compostos de Sulfidrila/metabolismo
11.
Int J Mol Sci ; 24(1)2022 Dec 30.
Artigo em Inglês | MEDLINE | ID: mdl-36614132

RESUMO

Urea is the uremic toxin accumulating with the highest concentration in the plasma of chronic kidney disease (CKD) patients, not being completely cleared by dialysis. Urea accumulation is reported to exert direct and indirect side effects on the gastrointestinal tract, kidneys, adipocytes, and cardiovascular system (CVS), although its pathogenicity is still questioned since studies evaluating its side effects lack homogeneity. Here, we investigated the effects of physiological and pathological urea concentrations on a human endothelial cell line from the microcirculation (Human Microvascular Endothelial Cells-1, HMEC-1). Urea (5 g/L) caused a reduction in the proliferation rate after 72 h of exposure and appeared to be a potential endothelial-to-mesenchymal transition (EndMT) stimulus. Moreover, urea induced actin filament rearrangement, a significant increase in matrix metalloproteinases 2 (MMP-2) expression in the medium, and a significant up- or down-regulation of other EndMT biomarkers (keratin, fibrillin-2, and collagen IV), as highlighted by differential proteomic analysis. Among proteins whose expression was found to be significantly dysregulated following exposure of HMEC-1 to urea, dimethylarginine dimethylaminohydrolase (DDAH) and vasorin turned out to be down-regulated. Both proteins have been directly linked to cardiovascular diseases (CVD) by in vitro and in vivo studies. Future experiments will be needed to deepen their role and investigate the signaling pathways in which they are involved to clarify the possible link between CKD and CVD.


Assuntos
Doenças Cardiovasculares , Insuficiência Renal Crônica , Humanos , Células Endoteliais/metabolismo , Ureia/farmacologia , Proteômica , Diálise Renal , Insuficiência Renal Crônica/metabolismo , Proteínas/metabolismo , Doenças Cardiovasculares/metabolismo
12.
Redox Biol ; 45: 102041, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34146958

RESUMO

Viral infections sustain their replication cycle promoting a pro-oxidant environment in the host cell. In this context, specific alterations of the levels and homeostatic function of the tripeptide glutathione have been reported to play a causal role in the pro-oxidant and cytopathic effects (CPE) of the virus. In this study, these aspects were investigated for the first time in SARS-CoV2-infected Vero E6 cells, a reliable and well-characterized in vitro model of this infection. SARS-CoV2 markedly decreased the levels of cellular thiols, essentially lowering the reduced form of glutathione (GSH). Such an important defect occurred early in the CPE process (in the first 24 hpi). Thiol analysis in N-acetyl-Cys (NAC)-treated cells and membrane transporter expression data demonstrated that both a lowered uptake of the GSH biosynthesis precursor Cys and an increased efflux of cellular thiols, could play a role in this context. Increased levels of oxidized glutathione (GSSG) and protein glutathionylation were also observed along with upregulation of the ER stress marker PERK. The antiviral drugs Remdesivir (Rem) and Nelfinavir (Nel) influenced these changes at different levels, essentially confirming the importance or blocking viral replication to prevent GSH depletion in the host cell. Accordingly, Nel, the most potent antiviral in our in vitro study, produced a timely activation of Nrf2 transcription factor and a GSH enhancing response that synergized with NAC to restore GSH levels in the infected cells. Despite poor in vitro antiviral potency and GSH enhancing function, Rem treatment was found to prevent the SARS-CoV2-induced glutathionylation of cellular proteins. In conclusion, SARS-CoV2 infection impairs the metabolism of cellular glutathione. NAC and the antiviral Nel can prevent such defect in vitro.


Assuntos
COVID-19 , Glutationa , Glutationa/metabolismo , Dissulfeto de Glutationa/metabolismo , Humanos , Oxirredução , RNA Viral , SARS-CoV-2
13.
Redox Biol ; 41: 101902, 2021 05.
Artigo em Inglês | MEDLINE | ID: mdl-33662873

RESUMO

SARS-CoV-2 (COVID-19) infection can cause a severe respiratory distress syndrome. The risk of severe manifestations and mortality characteristically increase in the elderly and in the presence of non-COVID-19 comorbidity. We and others previously demonstrated that the low molecular weight (LMW) and protein thiol/disulfide ratio declines in human plasma with age and such decline is even more rapid in the case of inflammatory and premature aging diseases, which are also associated with the most severe complications of COVID-19 infection. The same decline with age of the LMW thiol/disulfide ratio observed in plasma appears to occur in the extracellular fluids of the respiratory tract and in association with many pulmonary diseases that characteristically reduce the concentrations and adaptive stress response of the lung glutathione. Early evidence in literature suggests that the thiol to disulfide balance of critical Cys residues of the COVID-19 spike protein and the ACE-2 receptor may influence the risk of infection and the severity of the disease, with a more oxidizing environment producing the worst prognosis. With this hypothesis paper we propose that the age-dependent decline of LMW thiol/disulfide ratio of the extracellular fluids, could play a role in promoting the physical (protein-protein) interaction of CoV-2 and the host cell in the airways. Therefore, this redox-dependent interaction is expected to affect the risk of severe infection in an age-dependent manner. The hypothesis can be verified in experimental models of in vitro CoV-2 infection and at the clinical level in that LMW thiols and protein thiolation can now be investigated with standardized, reliable and versatile laboratory protocols. Presenting the verification strategy of our hypothesis, we also discuss available nutritional and ancillary pharmacological strategies to intervene on the thiol/disulfide ratio of extracellular fluids of subjects at risk of infection and COVID-19 patients.


Assuntos
COVID-19 , Compostos de Sulfidrila , Idoso , Dissulfetos , Humanos , Oxirredução , SARS-CoV-2
14.
Appl Physiol Nutr Metab ; 46(9): 1133-1142, 2021 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-33740389

RESUMO

The effect of oral glutathione (GSH) supplementation was studied in obese subjects with and without type 2 diabetes (T2DM) on measures of glucose homeostasis and markers of oxidative stress. Twenty subjects (10 patients with T2DM and 10 obese subjects) were recruited for the study, and randomized in a double-blinded placebo-controlled manner to consume either 1000 mg GSH per day or placebo for 3 weeks. Before and after the 3 weeks insulin sensitivity was measured with the hyperinsulinemic-euglycemic clamp and a muscle biopsy was obtained to measure GSH and skeletal muscle mitochondrial hydrogen peroxide (H2O2) emission rate. Whole body insulin sensitivity increased significantly in the GSH group. Skeletal muscle GSH was numerically increased (∼19%) in the GSH group; no change was seen in GSH to glutathione disulfide ratio. Skeletal muscle mitochondrial H2O2 emission rate did not change in response to the intervention and neither did the urinary excretion of the RNA oxidation product 8-oxo-7,8-dihydroguanosine or the DNA oxidation product 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG), although 8-oxodG decreased as a main effect of time. Oral GSH supplementation improves insulin sensitivity in obese subjects with and without T2DM, although it does not alter markers of oxidative stress. The study has been registered in clinicaltrials.gov (NCT02948673). Novelty: Reduced glutathione supplementation increases insulin sensitivity in obese subjects with and without T2DM. H2O2 emission rate from skeletal muscle mitochondria was not affected by GSH supplementation.


Assuntos
Diabetes Mellitus Tipo 2/fisiopatologia , Suplementos Nutricionais , Glutationa/administração & dosagem , Resistência à Insulina/fisiologia , Obesidade/fisiopatologia , Administração Oral , Biomarcadores/metabolismo , Glicemia/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Suplementos Nutricionais/efeitos adversos , Teste de Tolerância a Glucose , Glutationa/efeitos adversos , Glutationa/sangue , Glutationa/metabolismo , Dissulfeto de Glutationa/metabolismo , Humanos , Peróxido de Hidrogênio/metabolismo , Pessoa de Meia-Idade , Mitocôndrias Musculares/metabolismo , Músculo Esquelético/metabolismo , Obesidade/metabolismo , Estresse Oxidativo , Consumo de Oxigênio
15.
Int J Mol Sci ; 22(2)2021 Jan 09.
Artigo em Inglês | MEDLINE | ID: mdl-33435325

RESUMO

Oxidative stress plays a key role in the pathophysiology of retinal diseases, including age-related macular degeneration (AMD) and diabetic retinopathy, which are the major causes of irreversible blindness in developed countries. An excess of reactive oxygen species (ROS) can directly cause functional and morphological impairments in retinal pigment epithelium (RPE), endothelial cells, and retinal ganglion cells. Antioxidants may represent a preventive/therapeutic strategy and reduce the risk of progression of AMD. Among antioxidants, N-acetyl-L-cysteine (NAC) is widely studied and has been proposed to have therapeutic benefit in treating AMD by mitigating oxidative damage in RPE. Here, we demonstrate that N-acetyl-L-cysteine ethyl ester (NACET), a lipophilic cell-permeable cysteine derivative, increases the viability in oxidative stressed RPE cells more efficiently than NAC by reacting directly and more rapidly with oxidizing agents, and that NACET, but not NAC, pretreatment predisposes RPE cells to oxidative stress resistance and increases the intracellular reduced glutathione (GSH) pool available to act as natural antioxidant defense. Moreover, we demonstrate the ability of NACET to increase GSH levels in rats' eyes after oral administration. In conclusion, even if experiments in AMD animal models are still needed, our data suggest that NACET may play an important role in preventing and treating retinal diseases associated with oxidative stress, and may represent a valid and more efficient alternative to NAC in therapeutic protocols in which NAC has already shown promising results.


Assuntos
Acetilcisteína/farmacologia , Antioxidantes/farmacologia , Cisteína/análogos & derivados , Estresse Oxidativo/efeitos dos fármacos , Epitélio Pigmentado da Retina/efeitos dos fármacos , Acetilcisteína/análogos & derivados , Animais , Antioxidantes/química , Linhagem Celular , Cisteína/química , Cisteína/farmacologia , Humanos , Masculino , Ratos Sprague-Dawley , Espécies Reativas de Oxigênio/metabolismo , Epitélio Pigmentado da Retina/citologia , Epitélio Pigmentado da Retina/metabolismo
16.
Oxid Med Cell Longev ; 2020: 2975256, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33299524

RESUMO

Accumulating evidence indicates that oxidative stress plays a role in the pathophysiology of chronic kidney disease (CKD) and its progression; during renal replacement therapy, oxidative stress-derived oxidative damage also contributes to the development of CKD systemic complications, such as cardiovascular disease, hypertension, atherosclerosis, inflammation, anaemia, and impaired host defence. The main mechanism underlying these events is the retention of uremic toxins, which act as a substrate for oxidative processes and elicit the activation of inflammatory pathways targeting endothelial and immune cells. Due to the growing worldwide spread of CKD, there is an overwhelming need to find oxidative damage biomarkers that are easy to measure in biological fluids of subjects with CKD and patients undergoing renal replacement therapy (haemodialysis, peritoneal dialysis, and kidney transplantation), in order to overcome limitations of invasive monitoring of CKD progression. Several studies investigated biomarkers of protein oxidative damage in CKD, including plasma protein carbonyls (PCO), the most frequently used biomarker of protein damage. This review provides an up-to-date overview on advances concerning the correlation between plasma protein carbonylation in CKD progression (from stage 1 to stage 5) and the possibility that haemodialysis, peritoneal dialysis, and kidney transplantation improve plasma PCO levels. Despite the fact that the role of plasma PCO in CKD is often underestimated in clinical practice, emerging evidence highlights that plasma PCO can serve as good biomarkers of oxidative stress in CKD and substitutive therapies. Whether plasma PCO levels merely serve as biomarkers of CKD-related oxidative stress or whether they are associated with the pathogenesis of CKD complications deserves further evaluation.


Assuntos
Biomarcadores/sangue , Estresse Oxidativo/fisiologia , Diálise Renal , Insuficiência Renal Crônica/terapia , Terapia de Substituição Renal , Humanos , Transplante de Rim/efeitos adversos , Transplante de Rim/métodos , Oxirredução , Insuficiência Renal Crônica/sangue
17.
Oxid Med Cell Longev ; 2020: 3562972, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33062138

RESUMO

AIMS: Anethole dithiolethione (ADT) is a marketed drug to treat xerostomia. Its mechanism of action is still unknown, but several preclinical studies indicate that it is able to increase intracellular glutathione (GSH) and protect against oxidative stress. Here, we investigated the molecular mechanisms behind these effects. RESULTS: Oral treatment of rats confirmed the GSH enhancing properties of ADT; among the different organs examined in this study, only the kidney showed a significant GSH increase that was already observed at low-dose treatments. The increase in GSH correlated with a decrease in γ-glutamyltranspeptidase (γ-GT) activity of the different tissues. In vitro and ex vivo experiments with tubular renal cells and isolated perfused rat kidney showed that the cellular uptake of intact GSH was correlated with the extracellular concentrations of GSH. CONCLUSION: s. The prominent in vivopharmacological effect of ADT was a marked increase of GSH concentration in the kidney and a decrease of some systemic and renal biomarkers of oxidative stress. In particular, by inhibition of γ-GT activity, it decreased the production cysteinylglycine, a thiol that has prooxidant effects as the consequence of its autooxidation. The activity of ADT as GSH enhancer in both the circulation and the kidney was long-lasting. All these characteristics make ADT a promising drug to protect the kidney, and in particular proximal tubule cells, from xenobiotic-induced damage.


Assuntos
Anetol Tritiona/administração & dosagem , Glutationa/metabolismo , gama-Glutamiltransferase/metabolismo , Anetol Tritiona/farmacologia , Animais , Linhagem Celular , Cisteína/sangue , Cisteína/metabolismo , Dipeptídeos/sangue , Dipeptídeos/metabolismo , Dissulfetos/sangue , Glutationa/sangue , Humanos , Rim/efeitos dos fármacos , Rim/enzimologia , Rim/metabolismo , Túbulos Renais Proximais/citologia , Túbulos Renais Proximais/metabolismo , Masculino , Malondialdeído/sangue , Estresse Oxidativo/efeitos dos fármacos , Ratos , Ratos Sprague-Dawley , gama-Glutamiltransferase/antagonistas & inibidores
18.
Apoptosis ; 25(9-10): 674-685, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-32638182

RESUMO

Costunolide, a natural sesquiterpene lactone, has multiple pharmacological activities such as neuroprotection or induction of apoptosis and eryptosis. However, the effects of costunolide on pro-survival factors and enzymes in human erythrocytes, e.g. glutathione and glucose-6-phosphate dehydrogenase (G6PDH) respectively, have not been studied yet. Our aim was to determine the mechanisms underlying costunolide-induced eryptosis and to reverse this process. Phosphatidylserine exposure was estimated from annexin-V-binding, cell volume from forward scatter in flow cytometry, and intracellular glutathione [GSH]i from high performance liquid chromatography. The oxidized status of intracellular glutathione and enzyme activities were measured by spectrophotometry. Treatment of erythrocytes with costunolide dose-dependently enhanced the percentage of annexin-V-binding cells, decreased the cell volume, depleted [GSH]i and completely inhibited G6PDH activity. The effects of costunolide on annexin-V-binding and cell volume were significantly reversed by pre-treatment of erythrocytes with the specific PKC-α inhibitor chelerythrine. The latter, however, had no effect on costunolide-induced GSH depletion. Costunolide induces eryptosis, depletes [GSH]i and inactivates G6PDH activity. Furthermore, our study reveals an inhibitory effect of chelerythrine on costunolide-induced eryptosis, indicating a relationship between costunolide and PKC-α. In addition, chelerythrine acts independently of the GSH depletion. Understanding the mechanisms of G6PDH inhibition accompanied by GSH depletion should be useful for development of anti-malarial therapeutic strategies or for synthetic lethality-based approaches to escalate oxidative stress in cancer cells for their sensitization to chemotherapy and radiotherapy.


Assuntos
Benzofenantridinas/farmacologia , Inibidores Enzimáticos/farmacologia , Eriptose/genética , Glucosefosfato Desidrogenase/genética , Proteína Quinase C-alfa/genética , Apoptose/efeitos dos fármacos , Cálcio/metabolismo , Eriptose/efeitos dos fármacos , Eritrócitos/efeitos dos fármacos , Eritrócitos/patologia , Glucosefosfato Desidrogenase/antagonistas & inibidores , Glutationa/genética , Humanos , Estresse Oxidativo/efeitos dos fármacos , Proteína Quinase C-alfa/antagonistas & inibidores , Espécies Reativas de Oxigênio , Sesquiterpenos/farmacologia
19.
PLoS One ; 15(5): e0233788, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32470081

RESUMO

In pre-hypertension, moderate control of blood pressure (BP) can be obtained by a nutritional approach. The effects of a diet enriched with defatted larvae of the mealworm Tenebrio molitor (Coleoptera: Tenebrionidae) (TM) endowed with ACE inhibitory activity was studied in both spontaneously hypertensive rats (SHR) and in the age-matched normotensive Wistar Kyoto strain. These were fed for 4 weeks with standard laboratory rodent chow supplemented with or without TM or captopril. In SHR, the TM diet caused a significant reduction in BP, heart rate and coronary perfusion pressure, as well as an increase in red blood cell glutathione/glutathione disulphide ratio. Rat brain slices of SHR were more resistant to oxidative stress and contained lower levels of inflammatory cytokines, while vascular and liver enzyme-activities were not affected. These results suggest that TM can be considered a new functional food that can lower BP in vivo and thus control cardiovascular-associated risk factors such as hypertension.


Assuntos
Pressão Sanguínea , Suplementos Nutricionais , Frequência Cardíaca , Hipertensão/dietoterapia , Animais , Anti-Hipertensivos/farmacologia , Captopril/farmacologia , Hipertensão/tratamento farmacológico , Larva , Masculino , Ratos , Ratos Endogâmicos SHR , Ratos Endogâmicos WKY , Tenebrio
20.
Toxicol In Vitro ; 65: 104818, 2020 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-32135238

RESUMO

Cigarette smoke (CS) is one of the most important preventable risk factors for the development of respiratory diseases, cardiovascular diseases, stroke, and various types of cancer. Due to its high intracellular concentration and central role in maintaining the cellular redox state, glutathione (GSH) is one of the key players in several enzymatic and non-enzymatic reactions necessary for protecting cells against CS-induced oxidative stress. A plethora of in vitro cell models have been used over the years to assess the effects of CS on intracellular GSH and its disulphide forms, i.e. glutathione disulphide (GSSG) and S-glutathionylated proteins. In this review, we described the effects of cell exposure to CS on cellular GSH and formation of its oxidized forms and adducts (GSH-conjugates). We also discussed the limitations and relevance of in vitro cell models of exposure to CS and critically assessed the congruence between smokers and in vitro cell models. What emerges clearly is that results obtained in vitro should be interpreted with extreme caution, bearing in mind the limitations of the specific cell model used. Despite this, in vitro cell models remain important tools in the assessment of CS-induced oxidative damage.


Assuntos
Glutationa/metabolismo , Modelos Biológicos , Nicotiana , Fumaça/efeitos adversos , Animais , Humanos
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