Internal mammary artery smooth muscle cells resist migration and possess high antioxidant capacity

Objective- This study investigated whether differences exist in atherogen-induced migratory behaviors and basal antioxidant enzyme capacity of vascular smooth muscle cells (VSMC) from human coronary (CA) and internal mammary (IMA) arteries. Methods- Migration experiments were performed using the Dunn chemotaxis chamber. The prooxidant [NAD(P)H oxidase] and antioxidant [NOS, superoxide dismutase, catalase and glutathione peroxidase] enzyme activities were determined by specific assays. Results- Chemotaxis experiments revealed that while both sets of VSMC migrated towards platelet-derived growth factor-BB (1-50 ng/ml) and angiotensin II (1-50 nM), neither oxidized-LDL (ox-LDL, 25-100 �g/ml) nor native LDL (100 �g/ml) affected chemotaxis in IMA VSMC. However, high dose ox-LDL produced significant chemotaxis in CA VSMC that was inhibited by pravastatin (100 nM), mevastatin (10 nM), losartan (10 nM), enalapril (1 �M), and MnTBAP (a free radical scavenger, 50��M). Microinjection experiments with isoprenoids i.e. geranylgeranylpyrophosphate (GGPP) and farnesylpyrophosphate (FPP) showed distinct involvement of small GTPases in atherogen-induced VSMC migration. Significant increases in antioxidant enzyme activities and nitrite production along with marked decreases in NAD(P)H oxidase activity and O2 .- levels were determined in IMA versus CA VSMC. Conclusions- Enhanced intrinsic antioxidant capacity may confer on IMA VSMC resistance to migration against atherogenic agents. Drugs that regulate ox-LDL or angiotensin II levels also exert antimigratory effects.

Impact of weight loss on oxidative stress and inflammatory cytokines in obese type 2 diabetic patients.

Background: Type 2 diabetes mellitus is associated with abnormal markers of inflammatory cytokines and oxidative stress markers. Although, these abnormalities could be modulated with weight reduction; there is limitation in clinical studies that have addressed the beneficial effects of weight reduction in modulating biomarkers of inflammatory cytokines and oxidative stress for obesity associated with type 2 diabetes mellitus. Objective: This study was designed to detect the effects of weight loss on the inflammatory cytokines, oxidative stress markers in obese type 2 diabetic patients. Material and Methods: Eighty obese patients with type 2 diabetes mellitus, their age ranged from 35-57 years and their body mass index ranged from 31-35 kg/m2 were equally assigned into 2 groups: the weight reduction group received aerobic exercises, diet regimen, where as the control group received medical treatment only for 12 weeks. Results: The mean values of body mass index (BMI), tumor necrosis factor–alpha (TNF-α), interleukin-6 (IL-6), C-reactive protein (sCRP), conjugated dienes (CD) and malondialdehyde (MDA) were significantly decreased, while the mean values of glutathione peroxidase (GPx), superoxide dismutase (SOD) and glutathione (GSH) were significantly increased in patients of group (A), while changes were not significant in group (B). Also, there were significant differences between mean levels of the investigated parameters in group (A) and group (B) at the end of the study. Conclusion: Weight loss ameliorates inflammatory cytokines and oxidative stress markers in obese type 2 diabetic patients.

Differential mechanisms of angiotensin II and PDGF-BB on migration and proliferation of coronary artery smooth muscle cells

Angiotensin II (Ang II) and platelet-derived growth factor-BB (PDGF-BB) are associated with excessive cell migration, proliferation and many growth-related diseases. However, whether these agents utilise similar mechanisms to trigger vascular pathologies remains to be explored. The effects of Ang II and PDGF-BB on coronary artery smooth muscle cell (CASMC) migration and proliferation were investigated via Dunn chemotaxis assay and the measurement of [3H]thymidine incorporation rates, respectively. Both atherogens produced similar degrees of cell migration which were dramatically inhibited by mevastatin (10 nM). However, the inhibitory effects of losartan (10 nM) and MnTBAP (a free radical scavenger; 50 μM) were found to be unique to Ang II-mediated chemotaxis. In contrast, MnTBAP, apocynin (an antioxidant and phagocytic NADPH oxidase inhibitor; 500 μM), mevastatin and pravastatin (100 nM) equally suppressed both Ang II and PDGF-BB-induced cellular growth. Although atherogens produced similar changes in NADPH oxidase, NOS and superoxide dismutase activities, they differentially regulated antioxidant glutathione peroxidase activity which was diminished by Ang II and unaffected by PDGF-BB. Studies with signal transduction pathway inhibitors revealed the involvement of multiple pathways i.e. protein kinase C, tyrosine kinase and MAPK in Ang II- and/or PDGF-BB-induced aforementioned enzyme activity changes. In conclusion, Ang II and PDGF-BB may induce coronary atherosclerotic disease formation by stimulating CASMC migration and proliferation through agent-specific regulation of oxidative status and utilisation of different signal transduction pathways.

Régulation épigénétique de la défense antioxydante et de l'Angiopoietin-like 2 dans le contexte du vieillissement et des maladies cardiovasculaires

Suite à l’exposition à des facteurs de risque incluant la malnutrition, la dyslipidémie, la sédentarité et les désordres métaboliques, les maladies cardiovasculaires (MCV) sont caractérisées par un état pro-oxydant et pro-inflammatoire, et une dérégulation de l’expression de divers facteurs responsables de l’homéostasie de l’environnement rédox et inflammatoire. L’implication d’enzymes antioxydantes telles que les superoxyde dismutases (SOD) et les glutathion peroxydases (Gpx), ainsi que la contribution de médiateurs pro-inflammatoires tels que l’angiopoietin-like 2 (Angptl2) ont été rapportées dans le cadre des MCV. Toutefois, les mécanismes moléculaires sensibles aux facteurs de risque et menant au développement des MCV sont peu connus. L’épigénétique est un mécanisme de régulation de l’expression génique sensible aux stimuli extracellulaires et pourrait donc contribuer au développement des MCV. La méthylation de l’ADN est un des mécanismes épigénétiques pouvant varier tant de manière gène-spécifique qu’à l’échelle génomique, et la conséquence de tels changements sur l’expression des gènes ciblés dépend du site de méthylation. Puisqu’il a été démontré que des variations au niveau de la méthylation de l’ADN peuvent être associées à divers contextes pathologiques incluant les MCV, le but de nos travaux était d’étudier le lien entre la méthylation de gènes antioxydants et pro-inflammatoires avec leurs répercussions fonctionnelles biologiques en présence de facteurs de risques associés aux MCV, tels que le vieillissement, la dyslipidémie et la sédentarité. Dans la première étude, nous avons observé que dans l’artère fémorale de souris vieillissantes, la méthylation au niveau du promoteur du gène Sod2, codant pour l’enzyme antioxydante superoxyde dismutase de type 2 (SOD2 ou MnSOD), diminue avec l’âge. Ceci serait associé à l’induction de l’expression de MnSOD, renforçant ainsi la défense antioxydante endogène. Le vieillissement étant associé à une accumulation de la production de radicaux libres, nous avons étudié la vasodilatation dépendante de l’endothélium qui est sensible au stress oxydant. Nous avons observé que la capacité vasodilatatrice globale a été maintenue chez les souris âgées, aux dépens d’une diminution des facteurs hyperpolarisants dérivés de l’endothélium (EDHF) et d’une contribution accentuée de la voie du monoxyde d’azote (NO). Nous avons ensuite utilisé deux approches visant à réduire les niveaux de stress oxydant in vivo, soit la supplémentation avec un antioxydant, la catéchine, et l’exposition chronique à de l’exercice physique volontaire. Ces interventions ont permis de prévenir à la fois les changements au niveau de la fonction endothéliale et de l’hypométhylation de Sod2. Cette première étude démontre donc la sensibilité de la méthylation de l’ADN à l’environnement rédox. Dans la deuxième étude, nous avons démontré une régulation de l’expression de l’enzyme antioxydante glutathion peroxydase 1 (Gpx1) en lien avec la méthylation de son gène codant, Gpx1, dans un contexte de dyslipidémie sévère. Nos résultats démontrent que dans le muscle squelettique de souris transgéniques sévèrement dyslipidémiques (LDLr-/-; hApoB+/+), Gpx1 est hyperméthylé, ce qui diminue l’expression de Gpx1 et affaiblit la défense antioxydante endogène. Chez ces souris, l’exercice physique chronique a permis d’augmenter l’expression de Gpx1 en lien avec une hypométhylation transitoire de son gène. Cette étude démontre que le stress oxydant associé à la dyslipidémie sévère altère les mécanismes de défense antioxydante, en partie via un mécanisme épigénétique. De plus, on observe également que l’exercice physique permet de renverser ces effets et peut induire des changements épigénétiques, mais de manière transitoire. La troisième étude avait pour but d’étudier la régulation de l’Angptl2, une protéine circulante pro-inflammatoire, dans le contexte des MCV. Nous avons observé que chez des patients coronariens, la concentration circulante d’Angptl2 est significativement plus élevée que chez des sujets sains et ce, en lien avec une hypométhylation de son gène, ANGPTL2, mesurée dans les leucocytes circulants. Nous sommes les premiers à démontrer qu’en réponse à l’environnement pro-inflammatoire associé à une MCV, l’expression de l’Angptl2 est stimulée par un mécanisme épigénétique. Nos études ont permis d’identifier des nouvelles régions régulatrices différentiellement méthylées situées dans les gènes impliqués dans la défense antioxydante, soit Sod2 en lien avec le vieillissement et Gpx1 en lien avec la dyslipidémie et l’exercice. Nous avons également démontré un mécanisme de régulation de l’Angptl2 dépendant de la méthylation d’ANGPTL2 et ce, pour la première fois dans un contexte de MCV. Ces observations illustrent la nature dynamique de la régulation épigénétique par la méthylation de l’ADN en réponse aux stimuli environnementaux. Nos études contribuent ainsi à la compréhension et l’identification de mécanismes moléculaires impliqués dans le développement du phénotype pathologique suite à l’exposition aux facteurs de risque, ce qui ouvre la voie à de nouvelles approches thérapeutiques.

Chronic contamination assessment integrating biomarkers' responses in transplanted mussels-A seasonal monitoring

This study aimed to provide the first biomonitoring integrating biomarkers and bioaccumulation data in São Paulo coast, Brazil and, for this purpose, a battery of biomarkers of defense mechanisms was analyzed and linked to contaminants' body burden in a weigh-of-evidence approach. The brown mussel Perna perna was selected to be transplanted from a farming area (Caraguatatuba) to four possibly polluted sites: Engenho D'Agua, DTCS (Dutos e Terminais do Centro-Oeste de São Paulo) oil terminal (Sao Sebastiao zone), Palmas Island, and Itaipu (It; Santos Bay zone). After 3 months of exposure in each season, mussels were recollected and the cytochrome P4501A (CYP1A)- and CYP3A-like activities, glutathione-S-transferase and antioxidants enzymes (catalase, glutathione peroxidase, and glutathione reductase) were analyzed in gills. The concentrations of polycyclic aromatic hydrocarbons, linear alkylbenzenes, and nonessential metals (Cr, Cd, Pb, and Hg) in whole tissue were also analyzed and data were linked to biomarkers' responses by multivariate analysis (principal component analysisfactor analysis). A representation of estimated factor scores was performed to confirm the factor descriptions and to characterize the studied stations. Biomarkers exhibited most significant alterations all year long in mussels transplanted to It, located at Santos Bay zone, where bioaccumulation of organic and inorganic compounds was detected. This integrated approach using transplanted mussels showed satisfactory results, pointing out differences between sites, seasons, and critical areas, which could be related to land-based contaminants' sources. The influence of natural factors and other contaminants (e.g., pharmaceuticals) on biomarkers' responses are also discussed. (C) 2010 Wiley Periodicals, Inc. Environ Toxicol, 2012.

Potential protective role of reactive astrocytes in the periventricular parenchyma in congenital hydrocephalus

Background Cerebrospinal fluid accumulation in hydrocephalus produces an elevation of intraventricular pressure with pathological consequences on the periventricular brain parenchyma including ischemia, oedema, oxidative stress, and accumulation of metabolic waste products. Here we studied in the hyh mouse, an animal model of congenital hydrocephalus, the role of reactive astrocytes in this clinical degenerative condition. Materials and Methods Wild type and hydrocephalic hyh mice at 30 days of postnatal age were used. Three metabolites related to the oxidative and neurotoxic conditions were analysed in ex vivo samples (glutathione, glutamine and taurine) using High Resolution Magic Angle Spinning (HR-MAS). Glutathione synthetase and peroxidase, glutamine synthetase, kidney-type glutaminase (KGA), and taurine/taurine transporter were immunolocated in brain sections. Results Levels of the metabolites were remarkably higher in hydrocephalic conditions. Glutathione peroxidase and synthetase were both detected in the periventricular reactive astrocytes and neurons. Taurine was mostly found free in the periventricular parenchyma and in the reactive astrocytes, and the taurine transporter was mainly present in the neurons located in such regions. Glutamine synthetase was found in reactive astrocytes. Glutaminase was also detected in the reactive astrocytes and in periventricular neurons. These results suggest a possible protective response of reactive astrocytes against oxidative stress and neurotoxic conditions. Conclusions Astrocyte reaction seems to trigger an anti-oxidative and anti-neurotoxic response in order to ameliorate pathological damage in periventricular areas of the hydrocephalic mice.

Multibiomarker assessment of cadmium-based quantum dots effects in the marine mussel Mytilus galloprovincialis

Tese de Doutoramento, Ciências do Mar, da Terra e do Ambiente, Ramo: Ciências e Tecnologias do Ambiente, Especialização em Ecotoxicologia, Faculdade de Ciências e Tecnologia, Universidade do Algarve, 2016

Effect of Astragalus membranaceus (Fisch) Bunge extract on streptozocin-induced diabetic in rats

Purpose: To investigate the effect of Astragalus membranaceus (Fisch.) Bunge. extract (AMBE) on streptozotocin-induced diabetic rats. Methods: The aqueous extract of AMB was obtained by steeping the dried Astragalus membranaceus (Fisch.) Bunge. in water at 60 oC three times, each for 1 h, before first drying in an oven at 100 oC and then freeze-drying the last extract thus obtained. Diabete model rats was induced by a single intraperitoneal injection of a freshly prepared solution of streptozotocin (50 mg/kg). The rats were randomly divided into 6 groups of ten rats each: negative control group, normal control group, reference group (glibenclamide1 mg/kgbody weight) as well as AMB extract groups, namely, 40, 80 and 160 mg/kg body weight. Antihyperglycemic effect was measured by blood glucose and plasma insulin levels. Oxidative stress was evaluated in liver and kidney by antioxidant markers, viz, lipidperoxidation (LPO), superoxide dismutase (SOD), reduced glutathione (GSH), glutathione peroxidase (GPx) and catalase (CAT), while blood serum levels of creatinine and urea were also determined in both diabetic control and treated rats. Results: Compared with diabetic rats, oral administration of AMBE at a concentration of 160 mg/kg daily for 30 days showed a significant decrease in fasting blood glucose (109.438 ± 3.52, p < 0.05) and increased insulin level (13.96 ± 0.74, p < 0.05). Furthermore, it significantly reduced biochemical parameters (serum creatinine, 0.86 ± 0.29, p < 0.05) and serum urea (45.14 ± 1.79, p < 0.05). The treatment also resulted in significant increase in GSH (49.21 ± 2.59, p < 0.05), GPx (11.96 ± 1.16, p < 0.05), SOD (14.13 ± 0.49, p < 0.05), CAT (83.25 ± 3.14, p < 0.05) level in the liver and kidney of diabetic rats. Conclusion: The results suggest that AMBE may effectively normalize impaired antioxidant status in streptozotocin-induced diabetes in a dose-dependent manner. AMBE has a protective effect against lipid peroxidation by scavenging free radicals and is thus capable of reducing the risk of diabetic complications.

Protective effect of rhamnopyranosyl vanilloyl isolated from Scrophularia ningpoensis Hemsl (Scrophulariaceae) root against acute liver injury in mice

Purpose: To investigate the protective effect of rhamnopyranosyl vanilloyl (RV) from Scrophularia ningpoensis root against tetrachloromethane (CCl4)-induced acute liver injury (ALI) in mice. Methods: RV was isolated from S. ningpoensis by column chromatography. ALI model of mice was established by intraperitoneal injection of CCl4. Liver index, liver function indices, as well as serum alanine transaminase (ALT), aspartate aminotransferase (AST) and total bilirubin (TBIL) were evaluated. Lipid peroxidation (LPO)-related indices, including malonaldehyde (MDA), glutathione (GSH), superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px). Apoptotic proteins (Bcl-2, Bax and caspase-3) in liver tissue were determined by enzyme-linked immunosorbent assay (ELISA) and Western blot. Results: After treatment with RV (10, 20 or 40 mg/kg), liver index (5.65 - 5.21 vs. 6.68 %), ALT (90.18 - 79.68 vs. 112.47 U/L), AST (64.44 - 57.63 vs. 75.41 U/L) and TBIL (2.68 - 1.95 vs. 3.21 U/L) activities, as well as MDA (3.58 - 2.88 vs. 4.13 μmol/g), Bax and caspase-3 levels significantly (p < 0.05 or 0.01) decreased, compared with those in control group. After treatment with RV (10, 20 or 40 mg/kg), GSH (16.58 - 22.14 vs. 12.34 μmol/g), Bcl-2, SOD (86.45 - 107.61 vs. 68.43 U/mg) and GSH-Px (295.64 - 329.47 vs. 268.49 U/mg) levels or activities significantly (p < 0.05 or 0.01) increased, compared with those in control group. Conclusion: RV has protective effect against CCl4-induced ALI in mice, and the mechanisms involve the inhibition of LPO and apoptosis in liver cells. Thus, RV is a potential drug for the treatment of liver injury

Influence of levels and forms of selenium associated with levels of vitamins C and E on the performance, yield and composition of tilapia fillet

Objetivou-se avaliar a influência da vitamina C (VC), vitamina E (VE) e de duas formas de selênio sobre o desempenho, rendimento e qualidade do filé de tilápia Nilótica. O experimento foi realizado num delineamento inteiramente casualizado composto por seis tratamentos, os quais foram constituídos pela adição de 100, 200 e 400 mg kg-1 das vitaminas C e E, associados com 0,1, 0,2 e 0,4 mg kg-1 de duas formas de selênio. Cada tratamento foi constituído por cinco repetições de 30 peixes cada. A dieta com 200 mg kg-1 de VC e VE + 0,2 mg kg-1 de selênio orgânico proporcionou ganho de peso, comprimento corporal e fator de conversão alimentar semelhantes aos tratamentos com 400 mg kg-1 de VC e VE + 0,4 mg kg-1 de selênio orgânico ou inorgânico. A adição de selênio orgânico à dieta melhorou o ganho de peso e o fator de conversão alimentar em comparação com o selênio inorgânico. A dieta com 0,2 mg kg-1 de selênio orgânico apresentou nível de glutationa peroxidase igual ao da dieta com 0,4 mg kg-1 de selênio inorgânico. Os rendimentos de carcaça e filé não foram influenciados pelos tratamentos, entretanto houve efeito dos tratamentos na composição química do filé.

Development of an ecotoxicological protocol for the deep-sea fauna using the hydrothermal vent shrimp Rimicaris exoculata

In light of deep-sea mining industry development, particularly interested in massive-sulphide deposits enriched in metals with high commercial value, efforts are increasing to better understand potential environmental impacts to local fauna. The aim of this study was to assess the natural background levels of biomarkers in the hydrothermal vent shrimp Rimicaris exoculata and their responses to copper exposure at in situ pressure (30MPa) as well as the effects of depressurization and pressurization of the high-pressure aquarium IPOCAMP. R. exoculata were collected from the chimney walls of the hydrothermal vent site TAG (Mid Atlantic Ridge) at 3630m depth during the BICOSE cruise in 2014. Tissue metal accumulation was quantified in different tissues (gills, hepatopancreas and muscle) and a battery of biomarkers was measured: metal exposure (metallothioneins), oxidative stress (catalase, superoxide dismutase, glutathione-S-transferase and glutathione peroxidase) and oxidative damage (lipid peroxidation). Data show a higher concentration of Cu in the hepatopancreas and a slight increase in the gills after incubations (for both exposed groups). Significant induction of metallothioneins was observed in the gills of shrimps exposed to 4μM of Cu compared to the control group. Moreover, activities of enzymes were detected for the in situ group, showing a background protection against metal toxicity. Results suggest that the proposed method, including a physiologically critical step of pressurizing and depressurizing the test chamber to enable the seawater exchange during exposure to contaminants, is not affecting metal accumulation and biomarkers response and may prove a useful method to assess toxicity of contaminants in deep-sea species.

Ohr (Organic Hydroperoxide Resistance Protein) Possesses a Previously Undescribed Activity, Lipoyl-dependent Peroxidase

The Ohr (organic hydroperoxide resistance) family of 15-kDa Cys-based, thiol-dependent peroxidases is central to the bacterial response to stress induced by organic hydroperoxides but not by hydrogen peroxide. Ohr has a unique three-dimensional structure and requires dithiols, but not monothiols, to support its activity. However, the physiological reducing system of Ohr has not yet been identified. Here we show that lipoylated enzymes present in the bacterial extracts of Xylella fastidiosa interacted physically and functionally with this Cys-based peroxidase, whereas thioredoxin and glutathione systems failed to support Ohr peroxidase activity. Furthermore, we could reconstitute in vitro three lipoyl-dependent systems as the Ohr physiological reducing systems. We also showed that OsmC from Escherichia coli, an orthologue of Ohr from Xylella fastidiosa, is specifically reduced by lipoyl-dependent systems. These results represent the first description of a Cys-based peroxidase that is directly reduced by lipoylated enzymes.

Inhibitory Effect of Gallic Acid and Its Esters on 2,2 '-Azobis(2-amidinopropane)hydrochloride (AAPH)-Induced Hemolysis and Depletion of Intracellular Glutathione in Erythrocytes

The protective effect of gallic acid and its esters, methyl, propyl, and lauryl gallate, against 2,2'-azobis(2-amidinopropane)hydrochloride (AAPH)-induced hemolysis and depletion of intracellular glutathione (GSH) in erythrocytes was studied. The inhibition of hemolysis was dose-dependent, and the esters were significantly more effective than gallic acid. Gallic acid and its esters were compared with regard to their reactivity to free radicals, using the DPPH and AAPH/pyranine free-cell assays, and no significant difference was obtained. Gallic acid and its esters not only failed to inhibit the depletion of intracellular GSH in erythrocytes induced by AAPH but exacerbated it. Similarly, the oxidation of GSH by AAPH or horseradish peroxidase/H(2)O(2) in cell-free systems was exacerbated by gallic acid or gallates. This property could be involved in the recent findings on proapoptotic and pro-oxidant activities of gallates in tumor cells. We provide evidence that lipophilicity and not only radical scavenger potency is an important factor regarding the efficiency of antihemolytic substances.

Inhibitory effect of gallic acid and its esters on 2,2'-azobis(2-amidinopropane)hodrpchloride (AAPH)-induced hemolysis and depletion of intracellular glutathione in erythrocytes

The protective effect of gallic acid and its esters, methyl, propyl, and lauryl gallate, against 2,2'-azobis(2-amidinopropane)hydrochloride (AAPH)-induced hemolysis and depletion of intracellular glutathione (GSH) in erythrocytes was studied. The inhibition of hemolysis was dose-dependent, and the esters were significantly more effective than gallic acid. Gallic acid and its esters were compared with regard to their reactivity to free radicals, using the DPPH and AAPH/pyranine free-cell assays, and no significant difference was obtained. Gallic acid and its esters not only failed to inhibit the depletion of intracellular GSH in erythrocytes induced by AAPH but exacerbated it. Similarly, the oxidation of GSH by AAPH or horseradish peroxidase/H(2)O(2) in cell-free systems was exacerbated by gallic acid or gallates. This property could be involved in the recent findings on pro-apoptotic and pro-oxidant activities of gallates in tumor cells. We provide evidence that lipophilicity and not only radical scavenger potency is an important factor regarding the efficiency of antihemolytic substances.

Role of the Ascorbate-Glutathione Cycle of Mitochondria and Peroxisomes in the Senescence of Pea Leaves1

We investigated the relationship between H2O2 metabolism and the senescence process using soluble fractions, mitochondria, and peroxisomes from senescent pea (Pisum sativum L.) leaves. After 11 d of senescence the activities of Mn-superoxide dismutase, dehydroascorbate reductase (DHAR), and glutathione reductase (GR) present in the matrix, and ascorbate peroxidase (APX) and monodehydroascorbate reductase (MDHAR) activities localized in the mitochondrial membrane, were all substantially decreased in mitochondria. The mitochondrial ascorbate and dehydroascorbate pools were reduced, whereas the oxidized glutathione levels were maintained. In senescent leaves the H2O2 content in isolated mitochondria and the NADH- and succinate-dependent production of superoxide (O2·−) radicals by submitochondrial particles increased significantly. However, in peroxisomes from senescent leaves both membrane-bound APX and MDHAR activities were reduced. In the matrix the DHAR activity was enhanced and the GR activity remained unchanged. As a result of senescence, the reduced and the oxidized glutathione pools were considerably increased in peroxisomes. A large increase in the glutathione pool and DHAR activity were also found in soluble fractions of senescent pea leaves, together with a decrease in GR, APX, and MDHAR activities. The differential response to senescence of the mitochondrial and peroxisomal ascorbate-glutathione cycle suggests that mitochondria could be affected by oxidative damage earlier than peroxisomes, which may participate in the cellular oxidative mechanism of leaf senescence longer than mitochondria.