Joaquín Romá

β-Amyloid-induced activation of Caspase-3 in primary cultures of rat neurons

It is known that beta-amyloid peptide (Abeta) contributes to the neurodegeneration in Alzheimers disease (AD) and operates through activation of an apoptotic pathway. Apoptotic signal is driven by a family of cysteine proteases called caspases. The beta-amyloid precursor protein (APP) is directly and efficiently cleaved by caspases during apoptosis, resulting in elevated beta-amyloid peptide formation. Cerebellar neurons from rat pups were treated with the aged Abeta(25-35) at 1 and 5 microM and fluorescence assays of caspase activity performed over 4 days. We observed an increase in caspase activity after 48 h treatment in both 1 and 5 microM treated cells, then (72-96 h) caspase activity decreased to control values. The data presented support the hypothesis that Abeta(25-35)-induced apoptosis is mediated by the activation of Caspase-3 and that this is a transient effect.

Interferon decreases serum lipid peroxidation products of hepatitis C patients

Thiobarbituric acid reactive substances (TBARS) concentration in serum has been determined in healthy subjects and in patients suffering acute hepatitis and chronic cases of hepatitis C. Treatment with interferon of the chronic active hepatitis C patients, 5 x 10(6) U three times a week during 2 months, led in those patients whose SGPT activity normalized in serum, to a concomitant decrease in serum TBARS content. The possible theoretical involvement of peroxidation and antioxidants in this beneficial effect of interferon in hepatitis C patients is discussed. The results presented confirm the value of TBARS as laboratory test in the management of liver diseases and as a useful tool for the study of pathogenic and/or therapeutic mechanisms of this viral infection.

Oxygen Toxicity in the Nervous Tissue: Comparison of the Antioxidant Defense of Rat Brain and Sciatic Nerve

Nervous tissue, central and peripheral, is, as any other, subject to variations in oxygen tension, and to the attack of different xenobiotics; these situations may promote the generation of activated oxygen species of free radical character. Results are presented showing that the content of total glutathione (GSH) in brain is 10-fold that found in the sciatic nerve of the rat (2620 vs. 261 nmol/g wet weight, respectively). The existence of a relatively high superoxide dismutase activity in peripheral nervous tissue, when compared with brain or liver, in combination with the DT-diaphorase activity detected in the sciatic nerve might represent an effective defense mechanism against quinone toxicity, as is also discussed. Nervous tissue, both central and peripheral lack Se-independent GSH peroxidase activity. Finally, the activities of other glutathione-related enzymes studied in the sciatic nerve are very low, when compared with the central nervous tissue, thus suggesting a higher susceptibility of peripheral tissue to oxidative stress damage, since GSH concentration and/or any GSH-related enzymatic activities, e.g. GSH peroxidase or glutathione disulfide reductase, might become limiting.

Decreased glutathione peroxidase activity in sciatic nerve of alloxan-induced diabetic mice and its correlation with blood glucose levels

The effect of alloxan-induced diabetes on glutathione peroxidase (GSH-Px) activity in sciatic nerve of mice has been studied. We have found, 7 days after alloxan treatment, a significant decrease in this enzymatic activity in the cytosol of sciatic nerve of diabetic mice, and moreover, that these changes remained unaltered up to 21 days after alloxan injection. No modification in the glutathione content of sciatic nerve of diabetic mice was observed throughout the experiment when compared with controls. The decrease in GSH-Px activity in this tissue shows a good correlation with the increase of blood glucose levels throughout the experiment. It is hypothesized whether a combination of mechanisms could be involved in this decrease of GSH-Px activity and if oxygen radicals might be the common mediators of these processes.

Role of oxygen and nitrogen species in experimental uveitis: Anti-inflammatory activity of the synthetic antioxidant ebselen

This study was aimed at examining the role of oxygen and nitrogen reactive species in a model of experimental uveitis upon intravitreal injection of bacterial endotoxin to albino New Zealand rabbits. The inflammatory response was evaluated in terms of: (i) the integrity of the blood aqueous barrier (protein and cell content in samples of aqueous humor), (ii) histopathological changes of the eyes, (iii) clinical evaluation (with a score index based on clinical symptoms), and (iv) the concentration of malondialdehyde (MDA), in aqueous humor, as a marker of oxidative stress. Betamethasone was used as reference treatment, superoxide dismutase as quencher of superoxide anion, L-N(G)-nitro-L-arginine-methyl-esther (L-NAME) and chlorpromazine as nitric oxide synthase inhibitors, and ebselen, a glutathione peroxidase mimic, as peroxynitrite reductant. All the substances were injected subconjunctivally to the rabbits immediately after the intravitreal endotoxin injection. Ebselen was the only treatment able to decrease MDA concentration to control values, exerting an effect similar to that elicited by L-NAME on the rest of the parameters tested. The data presented render ebselen a notable choice for the treatment of uveitis, with implications for clinical trials.

Efficacy of the antioxidant ebselen in experimental uveitis

Inflammation results in the production of free radicals. In a model of experimental uveitis upon subcutaneous injection of endotoxin to Lewis rats, i.e., endotoxin-induced experimental uveitis (EIU), we have evaluated the status of the antioxidant capacity of ocular tissues. EIU results in a decrease of glutathione (GSH) content and glutathione peroxidase (GPx) activity in whole eye homogenates 24-h after endotoxin administration. Furthermore, an increase in malondialdehyde (MDA) content was observed in these same samples, thus confirming the involvement of oxidative stress in the pathophysiology of the process. In view of the ability of the antioxidant ebselen as GPx enzyme mimic, we tested the effect of the oral treatment with two doses of 100 mg/kg body weight of ebselen (first dose administered at the same time of endotoxin, and the second after 12 h). Ebselen administration normalized the GSH and MDA contents and protected the GPx activity of the EIU rat eyes. The GPx activity in the eye homogenate of the treated rats could be completely acounted for by the ebselen-dependent GPx-like activity, i.e., GPx activity measured in the acidic supernatant of the homogenate after neutralization. Unmodified ebselen was detected in whole eye homogenates, thus it shows for the first time the penetration of ebselen through the blood-aqueous and blood-retina barrier. The results herein may allow the proposal of ebselen as a suitable antiinflammatory agent in ocular tissues.

Glutathiione system of human retina: Enzymatic conjugation of lipid peroxidation products

The major aspects of the glutathione (GSH)-related antioxidant defense of human retina are presented. These include concentration of GSH and activities of some GSH-dependent enzymes: glutathione peroxidase, glutathione disulfide reductase, and glutathione S-transferase toward a broad spectrum substrate 1-chlor-2,4-dinitrobenzene and a toxic product of lipid peroxidation (4-hydroxynonenal). The presence of a relatively high GSH concentration, GSH peroxidase activity, and GSH S-transferase specific activity toward 4-hydroxynonenal in human retina might constitute a central defense mechanism in inflammation-promoted oxidative stress and subsequent lipid peroxidation. The use of different substrates for the determination of the GSH peroxidase activity showed no statistically significant difference, thus suggesting the lack of Se-independent GSH peroxidase in human retina. Large individual variations were obtained for GSH concentration and the different activities tested; the apparent correlation with age of these findings is currently under investigation.

4-Hydroxynonenal, a Lipid Peroxidation Product, Induces Relaxation of Human Cerebral Arteries

The relaxant effect of 4-hydroxynonenal (4-HNE), a lipid peroxidation product, on human cerebral arteries was studied. Addition of 4-HNE to artery rings promoted no contraction, and after stimulation with prostaglandin F2α (PFG2α; 10−7-3 × 10−6 M), 100% relaxation was obtained with 3 × 10−5 M 4-HNE. Inhibition of nitric oxide formation with NG-nitro-l-arginine methyl ester hydrochloride (l-NAME; (10−4 M), as well as prostaglandin synthesis with indomethacin (3 × 10−6 M), partially prevented 4-HNE-induced relaxation, but each of these substances separately failed to inhibit complete relaxation. Addition of both inhibitors together reduced 4-HNE-induced relaxation to ≈50%, but relaxation could not be abolished. When the endothelium was removed, 4-HNE did not promote relaxation after PGF2α stimulation. The possible roles of different intracellular signaling systems in the vascular effect of 4-HNE are discussed.

CHRONIC ALCOHOL FEEDING INDUCES BIOCHEMICAL, HISTOLOGICAL, AND FUNCTIONAL ALTERATIONS IN RAT RETINA

AIMS Ethanol consumption originates a wide spectrum of disorders, including alteration of visual function. Oxidative stress is included among the mechanisms by which alcohol predisposes nervous tissue to injury. Retina, which is the neurosensorial eye tissue, is particularly sensitive to oxidative stress. METHODS In this study we analyze the effect of long-term alcohol consumption on oxidative stress parameters of the rat retina, and its correlation to retinal function, as well as to the expression of the antiapoptotic protein Bcl-2. We also study the protective effect of ebselen, a synthetic selenoorganic antioxidant. RESULTS Herein we show that ethanol has a toxic effect on rat retina associated with oxidative stress. Decreases in retina glutathione concentration and increases in malondialdehyde content in whole eye homogenate significantly correlate with ERG b-wave decrease and Bcl-2 overexpression. We also show how ebselen is able to prevent all the alterations observed. CONCLUSION Chronic ethanol consumption induces oxidative stress in rat retina associated with an impairment of ERG and Bcl-2 overexpression, suggesting a role for glial cells. All these alterations in the rat allow the proposal of an alcoholic retinopathy in this species.

Antioxidant and glutathione-related enzymatic activities in rat sciatic nerve

The present work tries to establish the antioxidant capacity of the peripheral nervous tissue of the rat, in terms of the enzymatic activities present in this tissue that either prevent the formation of activated species as the semiquinone radical (DT-diaphorase), protect against activated oxygen species (superoxide dismutase, glutathione peroxidase), conjugate natural toxic products or xenobiotics (glutathione S-transferase, especially the activity conjugating 4-hydroxy-nonenal), or complete the glutathione system metabolism (glutathione disulfide reductase, gamma-glutamyl transpeptidase). All the activities studied are lower in this tissue than they are in liver, except for gamma-glutamyl transpeptidase. The relevance of the results obtained and its possible relationship with different neuropathies is discussed. It is concluded that the peripheral nervous tissue is by far less protected than the liver against oxidative damage.