José Augusto Nogueira-Machado

The Role of Oxidative Stress in the Aging Process

The aging of organisms is characterized by a gradual functional decline of all organ systems. An appropriate theory must explain four main characteristics of aging: it is progressive, endogenous, irreversible, and deleterious for the individual. The aging of the immune system, or immunosenescence, is manifested by an increased susceptibility to infections with increased morbidity and mortality. Phagocytic capacity, synthesis of reactive oxygen intermediaries, and the intracellular killing efficiency of neutrophils are impaired in the elderly. Among all aging theories, the most updated one describes the free radicals. It implies that progressive aging is associated with higher levels of oxidative biomolecules reacted with free radicals. Although reactive oxygen species (ROS) are predominantly implicated in causing cell damage, they also play a major physiological role in several aspects of intracellular signaling and regulation. ROS include a number of chemically reactive molecules derived from oxygen. Not only oxygen, but also nitrogen can be deleterious species. The overproduction of reactive nitrogen species (RNS) is called nitrosative stress. ROS/RNS are known to play a dual role in biological systems since they can be either harmful or beneficial to living systems.

Increase of reactive oxygen (ROS) and nitrogen (RNS) species generated by phagocyting granulocytes related to age.

In this present paper the age-induced effect on reactive oxidizing species generated by oxygen (ROS) and nitrogen (RNS) was studied using human phagocyting granulocytes. The ROS and RNS were quantified, respectively, in a chemiluminescence assay and by the measurement of nitrite production. The age-induced reactive oxidizing species generation was studied in healthy subjects ranging from 20 to 80 years old, divided into six age groups: group I, 20-29 years old; group II, 30-39 years old; group III, 40-49 years old; group IV, 50-59 years old; group V, 60-69 years old; and group VI, 70-80 years old. Our results demonstrate a parallelism between generation of the ROS and RNS induced by the age. A significant increase of ROS production was observed from 40 years old (age groups III, IV, V and VI while for RNS this increase was observed only from 50 years old (groups IV, V and VI). These data suggest an increase of oxidizing species generation (ROS/RNS) related to age. The increased generation of ROS (40-49 years old) was induced before the increasing of RNS (50-59 years old) and it may have consequences on inflammation and host defences.

Oxidative stress: a review on metabolic signaling in type 1 diabetes

Diabetic complications appear to be multifactorial process. The biochemical and pathological mechanisms are associated with chronic hyperglycemia of diabetes and the increased oxidative stress which has been postulated to play a central role in these disorders. Accumulating evidence suggests that oxidative cell injury caused by free radicals contributes to the development of type 1 diabetes (DM1) complications and decreased efficiency of antioxidant defenses (both enzymatic and nonenzymatic) seems to correlate with the severity of pathological tissue changes in DM1. In this review, we report as oxidative stress may exert deleterious effects in diabetes, as well as address current strategies in study to down-regulating vascular injury.

Modulation of the Reactive Oxygen Species (ROS) generation mediated by cyclic AMP-elevating agents or Interleukin 10 in granulocytes from type 2 diabetic patients (NIDDM): a PKA-independent phenomenon

UNLABELLED SUMMARY-BACKGROUND: The present study investigates the hypothesis that cells from ill patients and from healthy subjects may have different reactivity under metabolic stimulation as a consequence of an disease-induced metabolic adaptation. METHODS Granulocytes either from healthy subjects or from type II-Non Insulin Dependent Diabetes Mellitus (NIDDM) patients were compared in their capacities to generate Reactive Oxygen Species (ROS). The ROS generation was comparatively determined in a chemiluminescence assay, luminol-dependent, after cell incubation in the presence of either cyclic AMP - elevating agents or Interleukin 10. In some experiments the cells were pretreated with H89 compound (a PKA inhibitor) or with diphenylene iodonium (DPI), a NADPH-oxidase inhibitor. RESULTS Our results showed an increased ROS generation in granulocytes from diabetic patients in absence of cyclic AMP-elevating agents or IL-10. In the presence of cyclic AMP-elevating agents was observed an inverse metabolic response in granulocytes from diabetic patients in comparison to cells from healthy subjects. The granulocytes were pre-incubated in the presence of cyclic AMP-elevating agents--amminophylline (AMF) or dibutyryl cyclic AMP (dbcAMP)--or interleukin 10 (IL-10). The AMF, dbcAMP and IL-10 inhibited ROS production by granulocytes from healthy subjects. By contrast, AMF and dbcAMP activated cells from diabetic patients while IL-10 had no effect. The inhibition of ROS induced by AMF, dbcAMP or IL-10 was promptly abolished by the pretreatment of the cells with either PKA H89 inhibitor or NADPH-oxidase inhibitor (DPI) in granulocytes from healthy subjects. In relation to the granulocytes from type 2 diabetics patients, the activation of ROS generation mediated by AMF and dbcAMP was fully abolished by NADPH-oxidase DPI-inhibitor, but not by PKA H89 inhibitor. CONCLUSIONS Our present results reinforce the hypothesis that cells from ill patients (type II diabetic) when compared to cells from healthy subjects have different reactivity under metabolic stimulation. ROS production by human granulocytes was modulated by cyclic AMP elevating agents and IL-10. The inhibition of the ROS production in cells from healthy subjects was PKA-dependent while the activation in granulocytes from patients was PKA-independent. This inverse metabolic response, in cells from patients, suggests the use of an alternative metabolic pathway PKA-independent, possible cAMP/Epac/PKB-dependent. The correlation between activation of ROS production in granulocytes from diabetic patients and pathogenesis of diabetes can be suggested, however, further and extensive studies are needed for demonstrating this suggestion.

Ascorbic acid, alpha-tocopherol, and beta-carotene reduce oxidative stress and proinflammatory cytokines in mononuclear cells of Alzheimer's disease patients.

Abstract Objectives The in vitro effect of a vitamin complex in generating and reducing oxidative species in peripheral blood mononuclear cells (PBMNC) and plasma of patients with Alzheimers disease (AD) and healthy subjects (HS) was evaluated. Methods Two concentrations of a vitamin complex ([A] and [20A]) with ascorbic acid, alpha-tocopherol, and beta-carotene were incubated with either mononuclear cells or plasma. The generation of oxidizing species was measured in a luminol-dependent chemiluminescence assay and the reducing response by the MTT dye reduction assay. The levels of cytokines (interleukin [IL]-1β, IL-6, and IL-4) were measured by sandwich enzyme-linked immunosorbent assay. Results Our results demonstrated that the increase in the vitamin complex concentration reduced the reactive oxygen species (ROS) production and enhanced cellular reduction capacity in cells of AD patients in concentration [20A]. Plasma reduction capacity rose significantly for both groups (AD and HS). Concentration [A] did not alter the IL-1β production, increased IL-4 production in both groups and lowered IL-6 production in AD cells. Concentration [20A] increased pro-inflammatory cytokines (IL-1β and IL-6) and decreased IL-4 production by PBMNC of HS leading to a pro-inflammatory status. Discussion The antioxidant vitamin complex was effective in reducing oxidative stress in PBMNC of AD patients by lowering ROS production, improving cellular antioxidant capacities and modifying cytokine induced inflammation.

Resistance to oxamniquine of a Schistosoma mansoni strain isolated from patient submitted to repeated treatments.

A strain of Schistosoma mansoni (R1) was isolated from patient previously submitted to four treatments with oxamniquine, and to another one with praziquantel. The results obtained with chemotherapeutic test, by using oxamniquine in mice infected with the strains R1 and LE (standard), showed an evident resistance to the drug in worms of the strain R1. Thus, at the dose of 250 mg/kg oxamniquine, all mice (17) infected with the LE strain did not show surviving worms, whereas 12 out of 17 mice infected with the R1 strain presented surviving worms. At the dose of 200 mg/kg, the LE strain showed recovery rates of 1.06% and 20.58%, whereas the R1 strain presented 18.57% and 61.14%, for male and female worms, respectively. At the dose of 100 mg/kg, the recovery of male worms was 2.6% for the LE strain, and 29.9% for the R1 strain. At the same dose, the recovery of females did not show statistically significant differences between the two strains (LE = 76.38%, R1 = 79.12%). Praziquantel showed similar antischistosomal activity against both studied strains, when administered at the dose of 500 mg/kg.

The Production of Nitric Oxide, IL-6, and TNF-Alpha in Palmitate-Stimulated PBMNCs Is Enhanced through Hyperglycemia in Diabetes

We examined nitric oxide (NO), IL-6, and TNF-α secretion from cultured palmitate-stimulated PBMNCs or in the plasma from type 2 diabetes mellitus (T2MD) patients or nondiabetic (ND) controls. Free fatty acids (FFA) have been suggested to induce chronic low-grade inflammation, activate the innate immune system, and cause deleterious effects on vascular cells and other tissues through inflammatory processes. The levels of NO, IL-6, TNF-α, and MDA were higher in supernatant of palmitate stimulated blood cells (PBMNC) or from plasma from patients. The results obtained in the present study demonstrated that hyperglycemia in diabetes exacerbates in vitro inflammatory responses in PBMNCs stimulated with high levels of SFA (palmitate). These results suggest that hyperglycemia primes PBMNCs for NO, IL-6, and TNF-alpha secretion under in vitro FFA stimulation are associated with the secretion of inflammatory biomarkers in diabetes. A combined therapy targeting signaling pathways activated by hyperglycemia in conjunction with simultaneous control of hyperglycemia and hypertriglyceridemia would be suggested for controlling the progress of diabetic complications.

Correlation between NADPH Oxidase and Protein Kinase C in the ROS Production by Human Granulocytes Related to Age

Background: Aging may be defined as gradual and progressive changes in an organism that increase the probability of death. Accumulating evidence now indicates that the sum of deleterious free radical reactions going on continuously throughout cells and tissues constitutes the aging process or is a major contributor to it. Objective: The aim of this paper was to study the correlation between NADPH oxidase and protein kinase C (PKC) in the reactive oxygen species (ROS) production related to age. Methods: The age-induced ROS generation was studied in healthy subjects ranging in age from 20 to 80 years, divided into six age groups: (1) 20–29, (2) 30–39, (3) 40–49, (4) 50–59, (5) 60–69, and (6) 70–80 years. The ROS were quantified using a chemiluminescence assay (luminol dependent) and the results expressed as RLU/s at maximum peak and total chemiluminescence (integral under the curve RLU/s). Results: Our results demonstrate a significant increase of the ROS production from 40 years of age (age groups 3–6). In the age groups 1 and 2, we did not observe a significant difference (p > 0.05). These data suggest an increase of the ROS production from 40 to 49 years of age which may be induced by the PKC activity. The selective PKC inhibitor (calphostin C) abrogated the stimulatory effect of phorbol-12,13-dibutyrate on the ROS production. However, the NADPH oxidase inhibitor diphenylene iodonium did not inhibit the total ROS production by granulocytes in relation to age. Conclusions: These data suggest a correlation between age-related PKC activity, NADPH oxidase phosphorylation, and ROS production. The above correlations between unspecific and inflammatory responses related to age are discussed.

Cellular death, reactive oxygen species (ROS) and diabetic complications

Chronic or intermittent hyperglycemia is associated with the development of diabetic complications. Several signaling pathways can be altered by having hyperglycemia in different tissues, producing oxidative stress, the formation of advanced glycation end products (AGEs), as well as the secretion of the pro-inflammatory cytokines and cellular death (pathological autophagy and/or apoptosis). However, the signaling pathways that are directly triggered by hyperglycemia appear to have a pivotal role in diabetic complications due to the production of reactive oxygen species (ROS), oxidative stress, and cellular death. The present review will discuss the role of cellular death in diabetic complications, and it will suggest the cause and the consequences between the hyperglycemia-induced signaling pathways and cell death. The signaling pathways discussed in this review are to be described step-by-step, together with their respective inhibitors. They involve diacylglycerol, the activation of protein kinase C (PKC) and NADPH-oxidase system, and the consequent production of ROS. This was initially entitled the “dangerous metabolic route in diabetes”. The historical usages and the recent advancement of new drugs in controlling possible therapeutical targets have been highlighted, in order to evaluate the evolution of knowledge in this sensitive area. It has recently been shown that the metabolic responses to stimuli (i.e., hyperglycemia) involve an integrated network of signaling pathways, in order to define the exact responses. Certain new drugs have been experimentally tested—or suggested and proposed—for their ability to modulate the possible biochemical therapeutical targets for the downregulation of retinopathy, nephropathy, neuropathy, heart disease, angiogenesis, oxidative stress, and cellular death. The aim of this study was to critically and didactically evaluate the exact steps of these signaling pathways and hence mark the indicated sites for the actions of such drugs and their possible consequences. This review will emphasize, besides others, the therapeutical targets for controlling the signaling pathways, when aimed at the downregulation of ROS generation, oxidative stress, and, consequently, cellular death—with all of these conditions being a problem in diabetes.

The dual role of free fatty acid signaling in inflammation and therapeutics.

Obesity, type 2 diabetes, insulin resistance, dyslipidemia, cardiovascular diseases and atherosclerosis have all been associated with high levels of free fatty acid (FFA). In the present review, we suggest that FFA may act as either pro- or anti-inflammatory agents depending on the chemical structure. Saturated fatty acids (SFA) and polyunsaturated fatty acids (PUFA) significantly differ in their contributions to inflammation. While SFAs have been shown to induce inflammation, PUFAs have anti-inflammatory effects by downregulating NF-kappaB, IL-1β, TNF-α and IL-6 despite upregulating of IL-10. It is suggested that FFA may activate Toll Like Receptor-4 (TLR4) and G protein-coupled receptors (GPCR) activating signaling pathways that promote production and release of inflammatory cytokines (IL-6 and TFN-α). Fatty acid action on TLR4, peroxisome proliferator-activated receptors (PPARs) and GPCRs are potential therapeutic targets for controlling FFA-induced inflammation. Approaches that downregulate the inflammatory properties of free fatty acid are discussed in this manuscript. In this review, some patents associated with controlling FFA effects are also reported.