Rafael Medina-Navarro

Hyperglycemia induces apoptosis and p53 mobilization to mitochondria in RINm5F cells.

The mechanisms related to hyperglycemia-induced pancreatic β-cell apoptosis are poorly defined. Rat insulin-producing cells (RINm5F) cultured in high glucose concentrations (30 mM) showed increased apoptosis and protein p53 translocation to mitochondria. In addition, hyperglycemia induced both the disruption of mitochondrial membrane potential (Δ < eqid1 > m), and an increase in reactive oxygen species (ROS), as shown by fluorescence changes of JC-1 and dichlorodihydrofluorescein-diacetate (DCDHF-DA), respectively. The increased intracellular ROS by high glucose exposure was blunted by mitochondrial-function and NADPH-oxidase inhibitors. We postulate that the concomitant mobilization of p53 protein to the mitochondria and the subsequent changes on the Δ < eqid2 > m, lead to an important pancreatic β-cell apoptosis mechanism induced by oxidative stress caused by hyperglycemia.

Pro-oxidating properties of melatonin in the in vitro interaction with the singlet oxygen.

In an aqueous system, the oxidation of the erythrocyte membrane by the singlet oxygen formed during the photoactivation of the rose bengal coloring was examined. The effects of the singlet oxygen on lipids and proteins were studied through the simultaneous quantification of peroxidation products, lipoperoxides and carbonyl groups, the oxidation of protein SH groups and the activity of the glyceraldehyde 3-phosphate dehydrogenase (G3PD) associated with the erythrocyte membrane. The antioxidant activity of melatonin was tested and compared to that of two antioxidants in extreme cases of hydrosolubility, ascorbate and beta-carotene, with the purpose of comparing the protective ability of melatonin against singlet oxygen. The results show the expected effect even at low (0.125-0.75 mM; 0.015-0.90 mM, respectively) for ascorbate and beta-carotene, antioxidants known to possess important antioxidant qualities against singlet oxygen. It is shown that melatonin, under the conditions described, and at the concentrations at which the other two compounds are efficacious, not only confers little antioxidant protection, but that a pro-oxidant tendency was proven both on lipids and proteins, as well as on G3PD enzymatic activity. The results show that the antioxidant protective effect that melatonin can exert on biological systems is probably not by a direct interaction with oxidant species, but probably, as has been previously proposed, through the regulation of antioxidant defense systems. The formation of secondary oxidation products, such as melatonin-derived endoperoxides, may explain the evidence found on pro-oxidant qualities of this molecule.

Effect of an aqueous extract of Cucurbita ficifolia Bouché on the glutathione redox cycle in mice with STZ-induced diabetes

ETHNOPHARMACOLOGICAL IMPORTANCE Cucurbita ficifolia is used in Mexican traditional medicine as an anti-diabetic and anti-inflammatory agent and its actions can be mediated by antioxidant mechanisms. Disturbance in the homeostasis of glutathione has been implicated in the etiology and progression of diabetes mellitus and its complications. MATERIAL AND METHODS It was evaluated, the effect of an aqueous extract of Cucurbita ficifolia on glycemia, plasma lipid peroxidation; as well as levels of reduced (GSH) and oxidized (GSSG) glutathione and activities of enzymes involved in glutathione redox cycle: glutathione peroxidase (GPx) and glutathione reductase (GR) in liver, pancreas, kidney and heart homogenates of streptozotocin-induced diabetic mice. RESULTS Increased blood glucose and lipid peroxidation, together with decreased of GSH concentration, GSH/GSSG ratio and its redox potential (E(h)), and enhanced activity of GPx and GR in liver, pancreas and kidney were the salient features observed in diabetic mice. Administration of the aqueous extract of Cucurbita ficifolia to diabetic mice for 30 days, used at a dose of 200 mg/kg, resulted in a significant reduction in glycemia, polydipsia, hyperphagia and plasma lipid peroxidation. Moreover, GSH was increased in liver, pancreas and kidney, and GSSG was reduced in liver, pancreas and heart, therefore GSH/GSSG ratio and its E(h) were restored. Also, the activities involved in the glutathione cycle were decreased, reaching similar values to controls. CONCLUSIONS An aqueous extract of Cucurbita ficifolia with hypoglycemic action, improve GSH redox state, increasing glutathione pool, GSH, GSH/GSSG ratio and its E(h), mechanism that can explain, at least in part, its antioxidant properties, supporting its use as an alternative treatment for the control of diabetes mellitus, and prevent the induction of complications by oxidative stress.

Glucose-stimulated acrolein production from unsaturated fatty acids

Glucose auto-oxidation may be a significant source of reactive oxygen species (ROS), and also be important in the lipid peroxidation process, accompanied by the release of toxic reactive products. We wanted to demonstrate that acrolein can be formed directly and actively from free fatty acids in a hyperglycemic environment. A suspension of linoleic and arachidonic acids (2.5 mM) was exposed to different glucose concentrations (5, 10 and 15 mmol/L) in vitro. The samples were extracted with organic solvents, partitioned, followed at 255 / 267 nm, and analysed using capillary electrophoresis and mass spectroscopy. The total release of aldehydes significantly (P ≤ 0.01) increased from 1.0 to 5.1, 8.3 and 13.1 μmol/L after 6 hours of incubation, proportional to glucose concentrations. It was possible to verify a correlate hydroperoxide formation as well. Among the lipid peroxidation products, acrolein (5% of total) and its condensing product, 4-hydroxy-hexenal, were identified. From the results presented here, it was possible to demonstrate the production of acrolein, probably as a fatty acid product, due to free radicals generated from the glucose auto-oxidation process. The results led us to propose that acrolein, which is one of the most toxic aldehydes, is produced during hyperglycemic states, and may lead to tissue injury, as one of the initial problems to be linked to high levels of glucose in vivo.

Nitric oxide synthesis inhibition suppresses implantation and decreases cGMP concentration and protein peroxidation

The effect of Nw-nitro-L-arginine on embryonic implantation and cGMP carbonyl group concentration was assessed at the rat implantation site (IS) and non-implantation site (NIS). The intraluminal administration of 25 microg (2.3 mM) of Nw-nitro-L-arginine inhibited implantation in 34.7% and embryo survival (100%), while in addition, decreasing cGMP concentration both at the site (1664.2 +/- 333.8 pmoles/mg of protein for the control and 1321 +/- 384.3 for those treated), as well as at the NIS (1203.7 +/- 200 to 780.2 +/- 168.5). Carbonyl group concentration was considerably less at the implantation site treated with Nw-nitro-L-arginine than in the control (0.062 +/- 0.012 nmoles/mg of protein and 0.45 +/- 0.1, respectively). Nonetheless, the NIS was not significantly different (0.12 +/- 0.04 and 0.15 +/- 0.05). Our results show that a nitric oxide (NO) dependent system parallel to the formation of cGMP and protein peroxidation products is important at the blastocyst implantation site in order for the endometrium to acquire the necessary properties for an adequate receptivity.

Oxidation by reactive oxygen species (ROS) alters the structure of human insulin and decreases the insulin-dependent D-glucose-C14 utilization by human adipose tissue.

The formation of dityrosine of human insulin oxidized by metal-catalyzed oxidation system (H2O2/Cu) was estimated by fluorescent methods. The oxidation of tyrosine and phenylalanine residues present on the insulin molecule was evident after 2 minutes of in vitro oxidation due to the formation of protein-bound dityrosine. The success of oxidative protein modification was followed until available aromatic residues were consumed (60 minutes), measured by their emission at 405 nm. The structural and chemical changes on insulin molecule are related to the loss of biological activity as assessed by measuring the increase of U-14C-glucose utilization by human adipose tissue in a radiorespirometry system. The oxidation of glucose (14CO2 production) of the adipose cells was increased 35 % (301 +/- 119 to 407 +/- 182 cpm/mg in dry weight. P < 0.05) in presence of 0.1 IU and 69 % (301 +/- 119 to 510 +/- 266 cpm/dry weight. P < 0.05) for 1.0 IU of insulin. The recombinant human insulin oxidized for 5 minutes only increased the glucose oxidation by 25 %. In conclusion, these observations show that dityrosine formation and other oxidative chemical changes of insulin due to its in vitro oxidation decrease and can abolish its biological activity.

Chemical and functional changes of human insulin by in vitro incubation with blood from diabetic patients in oxidative stress

Oxidative stress damage to biomolecules has been implicated in several diseases including diabetes mellitus. In the present study, we investigated the effect of oxidative stress in whole blood (WB) from diabetic patients (n = 60) on recombinant human insulin. Insulin was incubated with WB obtained from diabetic patients (DP) who had hyperglycemia (>300 mg/dL) or from 41 healthy volunteers (HV). Whole blood of DP, unlike WB of HV, induced higher values of formazan (142%), dityrosines (279%), and carbonyls (58%) in the insulin residues. Interestingly, the insulin modified by WB of DP showed less hypoglycemic activity in rat (30%) in comparison with insulin incubated with WB of HV. The incubation of insulin in WB from DP induces chemical changes in insulin and a decrease in its biological activity, events that might be associated with the high levels of oxidative stress markers found in the plasma of these patients.

Identification of acrolein from the ozone oxidation of unsaturated fatty acids

By-products of lipoperoxidation reactions may be associated with the genesis or the progression of several diseases as arteriosclerosis, diabetes and cancer, among many others. Acrolein, at first a widely distributed environmental pollutant, is currently known as a compound capable of being generated as a result of metabolic reactions within biological systems, highly toxic and the most electrophilic of the a, b-unsaturated aldehydes formed during lipoperoxidation. In the present study: 1 The separation of acrolein and malondialdehyde was achieved at alkaline pH with the use of high voltage capillary electrophoresis in uncoated fused-silica capillaries. 2 It was demonstrated how the oxidation of fatty acids (arachidonic/linoleic) with ozone generates, in dose-dependent form, acrolein as one of the by-products of the lipoperoxidation process. The oxidation of open human erythrocyte membranes with ozone also generated acrolein. 3 After aldolic condensation, aldol-acrolein derivative has a positive reaction with 2-thiobarbituric acid (TBA) and shows a maximum absorption at 498 nm. This novel characteristic is used in its identification after the separation of the by-products. 4 It is possible to suggest that in the classic reaction of the denominated thiobarbituric acid reactive substances (TBARS), when used as an indicator of the degree of peroxidation in biological systems, a portion of acrolein could be present but dwarfed by the TBAMDA adduct.

Diabetogenic Effect of STZ Diminishes with the Loss of Nitric Oxide: Role of Ultraviolet Light and Carboxy-PTIO

Nitric oxide has been demonstrated to participate in β-cell damage during streptozotocin (STZ)-induced diabetes. STZ consists of 2-deoxy-D-glucose substituted by N-methyl-N-nitrosourea at C-2 and therefore can liberate ·NO. However, it has not been proven whether ·NO generation from STZ is responsible for the disease. We found that STZ treated in vitro with ultraviolet (UV) light liberated significantly more ·NO than non-irradiated STZ (1,134.4 ± 104 vs. 256.9 ± 240 nmol). Moreover, the diabetogenic effect of STZ was abolished by UV irradiation before its administration to experimental animals. In these animals the glucose and insulin values were significantly different from those of the diabetic group (151.3 ± 16.6 vs. 364.6 ± 63.4 mg/dl and 36.3 ± 17.9 vs. 0.08 ± 5.5 µIU/ml, respectively) and similar to those of the non-diabetic group (127.2 ± 34.1 mg/dl and 41.7 ± 13.9 µIU/ml, respectively). Carboxy-PTIO treatment returned glycemia to nearly normal levels in 60% of STZ-induced diabetic rats (157.5 ± 11.8 vs. 364.6 ± 63.6 mg/dl of the diabetic group). L-NAME and dexamethasone cannot return either glucose or insulin to normal levels. In conclusion, UV light increased ·NO liberation from STZ and suppressed its diabetogenic activity. It is possible that the diabetogenic activity of STZ is related to the liberation of nitric oxide from STZ, since carboxy-PTIO scavenger had a protective effect, while L-NAME and dexamethasone did not. It is possible that an increase in ·NO concentration into cell, independently of its endogenous or exogenous origin, can induce β-cell damage and diabetes.

Protein conjugated with aldehydes derived from lipid peroxidation as an independent parameter of the carbonyl stress in the kidney damage

BackgroundOne of the well-defined and characterized protein modifications usually produced by oxidation is carbonylation, an irreversible non-enzymatic modification of proteins. However, carbonyl groups can be introduced into proteins by non-oxidative mechanisms. Reactive carbonyl compounds have been observed to have increased in patients with renal failure. In the present work we have described a procedure designed as aldehyde capture to calculate the protein carbonyl stress derived solely from lipid peroxidation.MethodsAcrolein-albumin adduct was prepared as standard at alkaline pH. Rat liver microsomal membranes and serum samples from patients with diabetic nephropathy were subjected to the aldehyde capture procedure and aldol-protein formation. Before alkalinization and incubation, samples were precipitated and redisolved in 6M guanidine. The absorbances of the samples were read with a spectrophotometer at 266 nm against a blank of guanidine.ResultsEvidence showed abundance of unsaturated aldehydes derived from lipid peroxidation in rat liver microsomal membranes and in the serum of diabetic patients with advanced chronic kidney disease. Carbonyl protein and aldol-proteins resulted higher in the diabetic nephropathy patients (p < 0.004 and p < 0.0001 respectively).ConclusionThe aldehyde-protein adduct represents a non oxidative component of carbonyl stress, independent of the direct amino acid oxidation and could constitute a practical and novelty strategy to measure the carbonyl stress derived solely from lipid peroxidation and particularly in diabetic nephropathy patients. In addition, we are in a position to propose an alternative explanation of why alkalinization of urine attenuates rhabdomyolysis-induced renal dysfunction.