Mary A. Cotter, Alan Keegan, and Norman E. Cameron
Biomedical Sciences, Aberdeen University, Scotland, United Kingdom
Diabetes is associated with impaired erectile performance and impotence. Using an in-vitro rat corpus cavernosum (CC) preparation, we examined the effects of streptozotocin-induced diabetes and antioxidant treatment with a-lipoic acid (LA; 300 mg/kg dietary supplement). For phenylephrine (1 µM) precontracted CC, endothelium- dependent relaxation to acetylcholine (ACh) was abolished by the nitric oxide (NO) synthase inhibitor (NOSI), NG-nitro-l-arginine (10 µM); indicating a major contribution by a NO-mediated endothelial mechanism. Two months of diabetes reduced maximum ACh relaxation from 55.8 ± 2.9% to 35.8 ± 2.8% (SEM; p < 0.001. This deficit did not develop with LA treatment (52.8 ± 3.9%; p < 0.001 vs untreated diabetes). Diabetes did not alter endothelium-independent relaxation to sodium nitroprusside, or contractile responses to phenylephrine or electrical field stimulation (EFS) of noradrenergic fibers. For phenylephrine precontracted CC in the presence of 1 µM atropine and 10 µM guanethidine, EFS caused non-adrenergic non-cholinergic (NANC) relaxation that was abolished by NOSI and tetrodotoxin (1 µM). Nondiabetic rat CC had a maximum NANC relaxation of 41.2 ± 2.6%, which was reduced to 23.9 ± 2.0% (P<0.001) by diabetes. In contrast, with LA treatment relaxation was within the nondiabetic range (41.7 ± 3.1%; p < 0.001). Thus, diabetes compromised the NO- mediated systems responsible for endothelial and NANC CC vasodilation in rats. Defects in these systems are likely to cause erectile dysfunction. Prevention by LA treatment in rats suggests a potential therapeutic approach to diabetic impotence.
Thomas G. Cotter and Adrian J. McGowan
Tumour Biology Laboratory, Department of Biochemistry, University College Cork, Ireland
Apoptosis is a genetically regulated process by which cells die under a variety of pathological and physiological circumstances. A number of reports have suggested a role for reactive oxygen intermediates (ROI) as either inducers of apoptosis or as part of the signal transduction process. Here we report the protective effects of two anti-oxidant compounds 3 ß-doxyl a cholestane and 2,2,6,6-tetra methyl-piperdinoxyl, in HL-60 and U937 leukemic cells, subjected to a number of cytotoxic insults. In addition, the rapid production of peroxide is demonstrated as a general response to cytotoxic agents in these leukemic cell lines, indicating that changes in the redox status of the cells may contribute to their ultimate death. Closer examination of this peroxide production has identified enzymic production and/or disruption of resident anti-oxidants as possible sources. However, in contrast to recent reports mitochondrial depolarisation did not appear to be required for the production of peroxides in these cells. Finally, we demonstrated the activation of the redox sensitive transcription factor NF-kB in response to these cytotoxic insults.
In press Exp Cell Res 1998
Felice D¹Agnillo, Dan Goldman and Abdu I. Alayash
Center for Biologics Evaluation and Research, FDA, Bethesda, MD, 20892
We tested the hypothesis that hemoglobin (Hb) and lipopolysaccharide (LPS) may alter the oxidative status of endothelial cells, and lead to cellular toxicity. Confluent bovine aortic endothelial cells (BAECs) were incubated in media with or without fetal bovine serum (control), Hb, LPS, or Hb/LPS. In the presence of serum, LPS induced a dose-dependent increase in BAEC apoptosis as measured by increased DNA fragmentation. In the absence of serum, LPS concentrations up to 1mg/ml did not induce apoptosis. Hb (50 mM) alone induced a small, but non-significant increase in apoptosis with or without serum. In the presence of serum, Hb slightly inhibited LPS-induced apoptosis at £10 ng/ml LPS, but increased apoptosis at higher LPS concentrations. In the absence of serum, the combination of Hb and LPS (1 mg/ml) significantly increased apoptosis compared to Hb alone. This enhanced apoptotic effect was inhibited by the water-soluble vitamin E analogue, Trolox, but not by superoxide dismutase or catalase. A reduction in total soluble thiols was measured in the presence of LPS and Hb/LPS, whereas a slight increase occurred with Hb alone. The addition of Trolox inhibited the decrease in total thiols. Under these conditions, LPS induced only minor increases in nitrite accumulation and Hb oxidation suggesting that nitric oxide (NO) overproduction may not be an important factor. These results suggest that Hb and LPS may affect the oxidative status of endothelial cells via a cellular thiol-mediated process. These Hb/LPS interactions may play a role at infected injury sites or during septic shock, and may be especially relevant when modified hemoglobin-based blood substitutes are administered during septic shock or when used as an NO scavenger.
F. Dreher1, B. Gabard2, D.A. Schwindt1, H.I. Maibach1
1University of California, School of Medicine, Department of Dermatology, San Francisco, U.S.A., 2Spirig Ltd., Department of Biopharmacy, Pharmaceuticals, Egerkingen, Switzerland
In a randomized, double-blinded human study, the short-term photoprotective effects of different antioxidants and of their combinations were evaluated in vivo. Vitamin C (ascorbic acid), vitamin E (a tocopherol) and melatonin (N-acetyl-5-methoxytryptamine) were topically applied alone or in combination 30 min before UV-irradiation of the skin. The erythemal reaction was evaluated visually and non-invasively with different bioengineering methods (skin color and skin blood flow). The results showed a modest protective effect of the vitamins when applied alone and a dose-dependent photoprotective effect of melatonin. Topical application of combinations of both vitamins, or of melatonin with vitamins enhanced the photoprotective response. Better protection was obtained by using the combination of melatonin with both vitamins. The role of reactive oxygen species and of oxygen-derived free radicals is discussed in view of a possible mechanism explaining this elevated photoprotective effect.
Rat retina light degeneration: Protective effects of EGb 761 and DMTU on the retinal function
Marie-Thérèse Droy-Lefaix*, Isabelle Ranchon**, Jacques Cluzel**, Michel Doly**
*IPSEN Institute, Paris & ** Biophysics Lab., INSERM U. 71, Clermont Ferrand, France
In the retina, constant illumination can lead to structural damage due to lipoperoxidation. To prevent these disorders, we tested two antioxidants, the Ginkgo biloba extract (EGb 761, IPSEN France), and the dimethylthiurea (DMTU).Wistar rats were divided into 4 groups, a control group and 3 groups submitted to fluorescent light, treated or not with EGb 761 (100 mg/kg/po) or DMTU (500 mg/ kg/ip). Light damage were induced using a continuous fluorescent light set at 1700 lux during 24h.To evaluate the retinal degeneration, electroretinograms (ERGs) were recorded on exposed and non exposed rats. The present analysis showed that b and PIII amplitudes from the control group (unexposed, untreated) were reproducible all over the experimental period. A strong light exposure was responsible of severe functional damage of the retina, with a 60 % reduction of the b wave and a 40 % reduction of the PIII wave amplitudes. If the rats were pretreated with EGb 761 or DMTU, the ERG responses were not affected. Histological analysis confirmed these results. On unexposed-untreated rat, the photoreceptor layer and the outer nuclear layer thickness were reduced.With EGb 761 or DMTU treatment, no morphometric repercussion was noted. In conclusion, as oxygen radical scavengers, such as EGb 761 and DMTU were effective in limiting the photoreceptor retinal degeneration, there is evidence that prolonged light stimulus can induce oxidative reactions in the retina, linked to functional disorders. These two antioxidants can preserve the morphology and the function of the retina after light exposure.
Are antioxidants health effective or preventing disease progeny. The task of validity
Stellan Eriksson
Occupational Health Center, S-613 30 Oxelosund, Health Center Järna, S-153 00 Järna, Sweden
Attempts to evaluate if antioxidants are health effective are many and some with doubtful interpretation. One seeks for an answer whether they also could prevent disease progeny. In these evaluations there are many potential pitfalls, some of which will be emphasized.
One first order criteria is of course to know whether a study is validated or not. It means that you should know if the substance in question really functions as an antioxidant in vivo in contrast to a prooxidant. Before starting a large investigation, pilot studies should be done to establish whether there is functional variable in the study.
It is generally observed that antioxidants in low amount can act instead of ameliorating as deteriorating in metabolic processes. Therefore, validity aspects have to be judged upon from the very beginning in planning of studies and studies of studies. Ethics committees will also have to burden a greater responsibility in study planning. The form of the supplements is of great importance as for selenium and b-carotene. The Swedish Council on Technology Assessment in Health Care has made an evaluation on prevention of diseases with antioxidants with validation of non functional parameters of forms and concentrations with the negative results of antioxidants as preventive in diseases.
Regular training improves plasma antioxidant status in young soccer players
Pablo A. Evelson1, Fernando D. Brites2, Marina García Christiansen2, María F. Nicol1, Regina W. Wikinski2, and Susana F. Llesuy1
1Cátedra de Química General e Inorgánica y 2Departamento de Bioquímica Clínica, Facultad de Farmacia y Bioquímica, Universidad de Buenos Aires, Argentina
Physical activity is known to induce oxidative stress in individuals subjected to intense exercise. In this study, we investigated the lipid and lipoprotein profile and the plasma antioxidant status (measure ments of total plasma antioxidant capacity, hydrosoluble antioxidants (ascorbic acid, uric acid and bilirrubin levels), liposoluble antioxidants (a-tocopherol levels) and superoxide dismutase activity) in a group of soccer players engaged in a regular training program. As it was expected for aerobic exercise, high density lipoprotein-cholesterol (HDL-C) and HDL3-C levels were significantly increased in the sportsmen group (P < 0.05). Total plasma antioxidant capacity was 25 % higher in sportsmen than in controls (P < 0.005). Accordingly, plasma hydrosoluble antioxidant levels were found significantly elevated in the soccer player group (P < 0.005). In addition, these subjects showed high vitamin E concentration in plasma as compared to controls (P < 0.005). Furthermore, an increase in plasma superoxide dismutase activity was also observed in relation to exercise (P < 0.01). The elevation in plasma activities of antioxidant enzymes and the higher levels of free radicals scavengers of low molecular weight may compensate the oxidative stress caused by physical activity. High HDL plasma levels may offer additional protection by inhibiting LDL oxidation and liposoluble antioxidant consumption. Therefore, regular training may be improving the antioxidant overall conditions in soccer players.
Antioxidant activity of different phenolic fractions separated from an Italian red wine
Andrea Ghiselli*, Mirella Nardini*, Alessandro Baldi**, and Cristina Scaccini*
*Istituto Nazionale della Nutrizione, Via Ardeatina 543 00178 Roma and **Dipartimento di Scienze Farmaceutiche, University of Florence, Via Gino Capponi 9 50121 Firenze - Italy
To discriminate the antioxidant action of the various phenols in wine, different classes of polyphenols were extracted from an Italian red wine (Chianti -produced using Sangiovese R 10 clone), using liquid/liquid extraction. Three fractions were obtained, containing single polyphenolic sub fractions: (1) phenolic acids and quercetin- 3-glucuronide, (2) catechins and quercetin-3-glucoside, and (3) anthocyanins. After qualitative and quantitative analyses, the antioxidant capacity of the different fractions was tested: beside the scavenging capacity against hydroxyl and peroxyl radicals, the in vitro inhibition of low density lipoprotein oxidation (both catalyzed by Cu(II) and initiated by AAPH) and of platelet aggregation was tested. In each phase of the study, the quantitative relation among the different classes of polyphenols was maintained constant (1:500 with respect to the original wine) to differentiate the relative contribution of each fraction to the total antioxidant activity of the original wine. The antioxidant activity of the fractions has been compared with that of the original red wine before and after dealcoholization.
In spite of a loss of antioxidant capacity after dealcoholization, wine and its fractions still maintained an appreciable free radical scavenging activity, inhibiting LDL oxidative modification and platelet aggregation. Among the wine fractions, anthocyanins were always the most effective.
The high inhibitory effect exhibited by the anthocyanins was not only explained by their relative abundance in red wine, but also by their antioxidant efficiency. In fact, for example, when the scavenging of peroxyl radical was expressed as molar efficiency (mM trolox equivalents/mM phenolic concentration) anthocyanins had an antioxidant efficiency (0.60) higher than those of the dealcoholized wine (0.34), phenolic acid (0.22) and catechin (0.12) fractions.
Our results suggest that anthocyanins (~ 70% w/w of the total phenols in red wine) could be a key component in red wine in light of its supposed protection against diseases in which the involvement of oxygen radicals has been suggested.
Bound malondialdehyde: Bioavailability of the N-2-propenals of lysine
Julio Giron-Calle1,2, Valentina Ruiz-Gutierrez1, Manuel Alaiz1, and Eduardo Vioque1
1Instituto de la Grasa y sus Derivados (Consejo Superior de Investigaciones Cientificas), Seville, Spain; 2 University of Southern California, Los Angeles, CA
Malondialdehyde is a very reactive product of the peroxidative decomposition of polyunsaturated fatty acids, and it may be involved in the detrimental effects of oxidative stress. Diverse evidence indicates that the N-2-propenals of lysine (R-NH-CH=CH-CH=O), especially N-e-(2-propenal) lysyl residues in proteins, are a major bound form of this aldehyde both in living tissues and in foods. The bioavailability in vivo and in vitro of the lysine contained in the N-2 propenals of this amino acid has been studied in order to find out how stable the endogenously formed N-2-propenals of lysine are in tissues, and whether foods containing these adducts may be a source of free malondialdehyde. For the in vivo studies, radiolabeled lysine was used to synthesize a mixture of the two monopropenals N-a- and N-e-(2 propenal)lysine, and N,N'-di-(2-propenal) lysine, which were administered to rats. Radioactivity distribution after oral administration indicated that although both preparations were well absorbed from the digestive tract, only minor incorporation into tissues occurred. After intraperitoneal injection of the same mono- and dipropenal preparations, no radioactivity was found in hepatic microsomes, showing that regeneration of free lysine suitable for protein synthesis did not occur. For the in vitro studies, N-e-(2-propenal)lysine and N,N'-di-(2 propenal)-lysine were incubated with whole tissue homogenates obtained from rat liver, kidney, and intestinal mucosa, which again did not produce any release of free lysine. These results indicate that the N-2-propenals of lysine are very stable once they are endogenously formed in tissues or absorbed from the diet.
Regulation of focal adhesion kinase (p125FAK) phosphorylation in ROS-stimulated endothelial cells
Alexia Gozin, Lydie Da Costa, Sophie Faure, Valérie Andrieu, and Catherine Pasquier
INSERM U. 479, CHU Bichat, BP16 75870 Paris cedex 18, France
Focal adhesion kinase (p125FAK) is a 125-kDa cytoplasmic tyrosine kinase associated with focal adhesions in several different cells. It associates with many proteins, via their SH2 or SH3 domains, including paxillin and p130cas. p125FAK is phosphorylated on tyrosine in response to stimulation of various G protein-coupled receptors. The first step appears to be the autophosphorylation of Tyr-397. We have previously shown that reactive oxygen species (ROS) activated tyrosine phosphorylation of p125FAK and cytoskeleton proteins, paxillin and p130cas in endothelial cells, as well as adhesion of PMN to these cells. Its role in adhesion in endothelial cells as well as the mechanisms of its activation by ROS is not clear. As it has been shown that cAMP regulated p125FAK phosphorylation in neuronal cells, we investigated the effect of cAMP on PMN adhesion to ROS-stimulated HUVEC and on tyrosine phosphorylation of p125FAK, paxillin and p130cas. HUVEC were previously treated with intracellular cyclic AMP (cAMP) inducers such as inhibitors of phosphodiesterases or activator of adenylate cyclase (isobutylmethylxanthine, pentoxifylline, isoproterenol and forskoline) as well as cell-permeant analogs of cAMP. We show here that cAMP inhibited the adhesion of PMN to ROS-stimulated- HUVEC and also inhibited phosphorylation of p125FAK paxillin and p130cas. When HUVEC were stimulated with PMA, adhesion of PMN was increased but was not inhibited by cAMP; p125FAK paxillin and p130cas tyrosine phosphorylation was not modified with increased intracellular cAMP, suggesting that cAMP regulates specifically ROS-induced tyrosine phosphorylation and adhesion.
cAMP stimulates protein kinase A (PKA). Mechanism by which PKA regulates tyrosine phosphorylation is not known. It may involve inhibition of a tyrosine kinase or stimulation of a tyrosine phosphatase itself known to be inhibited by ROS. The understanding of this mechanism will allow to correlate adhesion of PMN to ROS-stimulated HUVEC and tyrosine phosphorylation of HUVEC p125FAK.
Poly-ADP-ribose mediated regulation of the 20S proteasome
Tilman Grune, Nicolle Sitte, and Oliver Ullrich
Humboldt University Berlin
Oxygen radicals cause various modifications to structure of proteins, often resulting in a loss of protein function. Whereas the repair and removal of oxidized proteins within the cytosol was studied to a certain level, nothing is known about the fate of oxidized proteins within the nucleus. The removement of oxidized proteins from the chromatin structure and further the degradation of damaged proteins seems to be of great importance in the genomic integrity because of the requirement of non-protein coated DNA for DNA-repair after oxidative stress. Therefore our aim was to investigate the fate of oxidatively modified histones, the main DNA-binding proteins, and the mechanism of their selective degradation by nuclear proteolytic systems. The 20S proteasome is able to degrade oxidized nuclear proteins preferentially, but unlike the cytosolic 20S proteasome the nuclear pool of the protease undergoes an activation during oxidative stress. We could demonstrate, that the activation of the 20S proteasome is due to poly-ADP-ribosylation. After this modification the nuclear 20S proteasome is responsible for the selective degradation of oxidative damaged histones. As well soluble as DNA-bound oxidized nuclear proteins are selectively recognized and degraded by the poly-ADP-ribosylated 20S proteasome.
This is a new mechanism of regulation of the proteolytic degradation of oxidized proteins within mammalian cells.