Non-enzymatic Oxidative Stress Research Articles

Endogenous Glutathione Adducts

This review provides an overview of the formation, pharmacology, and toxicology of endogenous glutathione (GSH)-adducts with particular emphasis on GSH-adducts that arise from lipid peroxidation. GSH is the major low-molecular-weight thiol in mammalian cells. It is involved in the formation of endogenous bioactive eicosanoids and is a source of reducing equivalents in a number of biosynthetic reactions. GSH has long been recognized to act as a co-factor in the reduction of reactive oxygen species and lipid hydroperoxides by glutathione peroxidases and glutathione-S-transferases (GSTs). It also plays an important role in the reduction of reactive intermediates derived from arylamines and in the conjugation of reactive intermediates to form S-substituted endogenous GSH-adducts through its nucleophilic cysteine sulfhydryl group. Although some reactive intermediates can form adducts directly, GST-mediated reactions generally predominate. This results in the formation of bioactive endogenous GSH-adducts derived from eicosanoids, isoprostanes, estrogens, catecholamines, and 4-hydroxy-2(E)-nonenal (HNE). Cellular oxidative stress causes increased lipid peroxidation with the concomitant formation of DNA- and protein-reactive bifunctional electrophiles. It has generally been considered that HNE is the most abundant bifunctional electrophile that is formed. Several years ago we discovered that 4-oxo-2(E)-nonenal (ONE) was also a major lipid hydroperoxide-derived bifunctional electrophile. From in vitro studies, we showed that ONE and HNE arose from the common intermediate, 4-hydroperoxy-2(E)-nonenal and also showed that ONE was formed in greater amounts than HNE. We have recently made the unexpected discovery that GSH addition to ONE leads to the formation of an unusual thiadiazabicyclo-ONE-GSH-adduct (TOG), which was characterized as (2S,7R) - 7 - [N - (carboxymethyl)carbamoyl] - 5 - oxo - 12 - pentyl - 9 - thia - 1,6 - diazabicyclo[8.2.1]trideca - 10(13), 11-diene-2-carboxylic acid. TOG is one of the most abundant GSH-adducts formed during peroxide/Fe(II)- or Fe(II)-mediated oxidative stress in EA.hy 926 endothelial cells. As TOG is formed from ONE, these experiments have confirmed that ONE is a major lipid hydroperoxide-derived bifunctional electrophile formed during intracellular oxidative stress. TOG represents the first member of a new class of endogenous GSH-adduct biomarkers that can be used to quantify intracellular oxidative stress. Two other members of the TOG family arise from GST-mediated GSH-adduct formation with dioxododecenoic acid and dioxooctenoic acid, bifunctional electrophiles derived from the carboxy terminus of lipid hydroperoxides. The formation of TOG and TOG-related endogenous GSH-adducts can result from free radical- as well as cyclooxygenase- and lipoxygenase-mediated pathways. Analysis of the GSH-adducts by stable isotope dilution mass spectrometry-based methodology will provide a quantitative measure of enzymatic and non-enzymatic cellular oxidative stress to complement isoprostane measurements. In future studies, it will also be important to establish the biological activity of TOG and its analogs in view of the potent activity of many other endogenous GSH-adducts such as the leukotrienes.

The Unfolded Protein Response in Lens Epithelial Cells from Galactosemic Rat Lenses

Diabetic complications are associated with hypoglycemia and hyperglycemia. The purpose of this study was to investigate the effect of both glucose deprivation and hyperglycemia on the induction of endoplasmic reticulum (ER) stress and the subsequent activation of the unfolded protein response (UPR) that results in apoptosis in in vitro cultured lens epithelial cells (LECs) and in vivo cataract formation in galactose-fed rats. Lenses from rats fed a standard diet containing 50% galactose with or without an aldose reductase inhibitor (ARI) were investigated. Transformed human LECs were cultured in standard 10% FCS-DMEM containing various concentrations of sugar. UPR-specific proteins from both the rat lenses and lens cultures were quantified by protein blot analysis. Cell death was evaluated with TUNEL staining and ethidium homodimer-1 (EthD) dyes. Reactive oxygen species (ROS) were quantified with H2-DCF, and free glutathione (GSH) levels were measured with a commercial GSH quantification kit. Increased apoptosis of the LECs was observed in the lenses of rats fed the galactose diet for 5 to 9 days, and nuclear cataracts subsequently developed in these lenses after 13 to 15 days. Protein blot analysis of the LECs from these galactose-fed rats showed higher levels of the UPR-specific proteins Bip/GRP78, ATF4, and CHOP. These LECs also demonstrated activation of the UPR-specific procaspase-12 and the increased presence of ROS, whereas GSH was reduced. Because these results indicate that the UPR is activated in LECs along with the production of ROS and apoptosis during cataract formation in the galactose-fed rats, subsequent studies were conducted to determine the role of nonenzymatic glycation, osmotic stress, and oxidative stress on these biochemical processes. In vitro cultures of human LECs showed that the UPR was induced by osmotic and oxidative stress, but not by glycation. In addition, the UPR and apoptosis in LECs was induced by glucose deprivation. The ARI blocked the induction of the UPR, cell death, and cataract formation. The UPR that is induced by abnormally high or low concentrations of sugar is linked to the production of ROS, increased apoptosis in LECs, and cataract formation. The inhibition of the UPR induction by ARI suggests that osmotic stress may be the primary inducer of the UPR. Modulation of the UPR pathways may offer novel methods for the development of therapeutic tools to delay cataracts.

Non-enzymatic glycation and oxidative stress in chronic illnesses and diabetes mellitus

New approaches in biochemistry and molecular biology have increased the knowledge on the pathophysiology of chronic diseases as late diabetic complications, Alzheimer's disease, arteriosclerosis and vascular disease by defining the concept of "AGE-formation and oxidative stress." Nonenzymatic glycation, in which reducing sugars are covalently bound to free aminogroups of macromolecules, results in the formation of Advanced Glycation End products (AGEs) which accumulate during aging and at accelerated rate during the course of diabetes. Glycation accompanying oxidation processes support AGE-formation. AGE-formation changes the physicochemical properties of proteins, lipids and nucleic acids. In addition, binding of AGEs to specific surface receptors induces cellular signalling and cell activation. Interaction of AGEs with one of the receptors, RAGE, generates intracellular oxidative stress, which results in activation of the transcription factor NF-kappa B and subsequent gene expression, which might be relevant in late diabetic complications. Knowledge of the basis molecular mechanisms allows to understand the interplay of different inducers such as redicals, cytokines, AGE-proteins and amyloid-beta-peptids and to define oxidative stress as a "common endpoint" of cell dysfunction. With respect to therapeutic options it is now possible not only to optimize blood glycemic control, but also to design drugs such as AGE-inhibitors and AGE-"cross-link" breakers. In addition patients with chronic disease associated with increased oxidative stress ay benefit from an antioxidant rich (and AGE protein poor?) nutrition.

Cardiovascular complications of diabetes.

Cardiovascular complications are the leading causesof mortality in both insulin-dependent (IDDM) andnon-insulin-dependent diabetes mellitus (NIDDM).The severity of this problem is clearly illustrated bythe finding that IDDM patients suffer from mortalityrates many times higher than of the general popula-tion [1]. In spite of the gravity of this complication,there is a relative paucity of data specifically on thepathogenesis, prevention and the treatment of thecardiovascular lesions in diabetes. The goal of this ar-ticle is to highlight certain areas which are pertinentto diabetic cardiovascular complications and to sug-gest research goals which will bring understanding ofthe pathogenesis of this grave complication and itspotential treatments.George L. King: Hyperglycaemia and insulin resistance:how do they increase cardiovascular risk in diabeticpatients?A major cause of morbidity and mortality in diabeticpatients is macrovascular disease affecting the heartand large vessels. In diabetes there is an accelerationof atherosclerosis with excessive extracellular matrixthickening. In the heart, the major pathology is athe-rosclerosis of the coronary arteries, and the fibrosis,and hypertrophy of the myocardium. Hyperglycae-mia, hyperlipidaemia and insulin resistance are someof the recognized risk factors [2, 3]. The basic ab-normalities are in glucose metabolism and insulinaction which can lead to a whole range of vascularchanges in the areas of coagulation, contractility, leu-kocyte adhesions, and smooth muscle cell prolifera-tion. The mechanisms by which hyperglycaemia cau-ses vascular dysfunction are probably multiple. Thesemechanisms include non-enzymatic glycation, oxida-tive stress, polyol-myoinositol alteration, and activa-tion of diacylglycerol (DAG) and protein kinase C(PKC) pathways. The role of the DAG and PKCpathways which regulate many vascular functionswere discussed since this is the newest of the fourmain theories.Studies over the last 7 years have shown that thereis an increase in DAG levels and activation of PKCactivities in vascular cells from the retina, glomeruli,aorta and the heart when exposedto high glucose lev-els in culture or in the diabetic state in vivo [4]. In-creased glycolysis can elevate DAG which in turn ac-tivates protein kinase C in the vascular cells. The

Diabetes mellitus, hypertension, and cardiovascular disease: Which role for oxidative stress?

Accelerated atherosclerotic vascular disease is the leading cause of mortality in patients with diabetes mellitus. Endothelium-derived nitric oxide (NO) is a potent endogenous nitrovasodilator and plays a major role in modulation of vascular tone. Selective impairment of endothelium-dependent relaxation has been demonstrated in aortas of both nondiabetic animals exposed to elevated concentrations of glucose in vitro and insulin-dependent diabetic animals. The impaired NO release in experimentally induced diabetes may be prevented by a number of antioxidants. It has been hypothesized that oxygen-derived free radicals (OFR) generated during both glucose autoxidation and formation of advanced glycosylation end products may interfere with NO action and attenuate its vasodilatory activity. The oxidative injury may also be increased in diabetes mellitus because of a weakened defense due to reduced endogenous antioxidants (vitamin E, reduced glutathione [GSH]). A defective endothelium-dependent vascular relaxation has been found in animal models of hypertension and in hypertensive patients. An imbalance due to reduced production of NO or increased production of free radicals, mainly superoxide anion, may facilitate the development of an arterial functional spasm. Treatment with different antioxidants increases blood flow in the forearm and decreases blood pressure and viscosity in normal humans; vitamin E inhibits nonenzymatic glycosylation, oxidative stress, and red blood cell microviscosity in diabetic patients. Long-term randomized clinical trials of adequate size in secondary and primary prevention could support the free-radical hypothesis for diabetic vascular complications and the use of antioxidants to reduce the risk of coronary heart disease.

Suppression of the Hydrazine-Induced Formation of Megamitochondria in the Rat Liver by α-Tocopherol

The effects of α-tocopherol on the hydrazine-induced changes in the structure of mitochondria and those in the enzymatic and nonenzymatic antioxidant systems of the liver were investigated using rats as experimental animals. Animals were divided into four groups: animals of the first group were placed on a powdered diet containing 1.0% hydrazine for 7 days; those of the second and third groups received a control diet and α-tocopherol (ip, daily, 700 mg/kg body wt). On the fourth day and thereafter for up to 7 days, the control diet was replaced by a 1.0% hydrazine diet for the animals of the second group; those of the fourth group served as the control. After 10 days (in the case of animals of the first group, 7 days), animals were sacrificed. Results obtained were as follows: (1) Treatment of animals with α-tocopherol partly prevented the hydrazine-induced formation of megamitochondria in the liver. (2) Treatment of animals with hydrazine induced remarkable increases in the contents of α-tocopherol in mitochondria (4.8 times), microsome (1.4 times), and homogenate (2.9 times) of the liver compared with those of the control. Treatment of animals with α-tocopherol did not raise the concentration of α-tocopherol in mitochondria of the liver. The highest concentration of α-tocopherol in motochondria of the liver was obtained in animals given hydrazine plus α-tocopherol (7.2 times higher than the control). (3) The amount of lipid-soluble fluorophores as an indicator of nonenzymatic oxidative stress was remarkably increased in mitochondria, microsome, and homogenate of the liver of hydrazine-treated animals. (4) Among enzymes protecting the cell from the oxidative stress activities of superoxide dismutase and glutathione peroxidase were almost the same among four groups of animals, while the activity of catalase was decreased distinctly in hydrazine-treated animals. Administration of α-tocopherol to these animals did not improve its activity. The present study has clearly demonstrated that α-tocopherol, a typical scavenger for free radicals, prevents the hydrazine-induced formation of megamitochondria in the liver. However, we failed to correlate free radicals to biochemical and physicochemical changes of mitochondrial membranes induced by hydrazine. Previously, we have demonstrated that a ratio of unsaturated to saturated fatty acids in phospholipid domains of mitochondrial membranes increases with hydrazine treatment. Since increases in the ratio specified above are a key event in the membrane fusion process we are now studying how the ratio is modified by hydrazine focussing on desaturase activity in the liver, and results will be reported soon.

A comparative study of free radicals in vertebrates—II. Non-enzymatic antioxidants and oxidative stress

1. 1. The three main non-enzymatic endogenous soluble antioxidants and three estimators of oxidative stress were measured in the liver, lung and brain of seven animal species of different vertebrate classes. 2. 2. The more concentrated antioxidant was GSH, followed by ascorbate and finally by uric acid. Liver showed higher levels of GSH and uric acid than the other two organs in the majority of the species. 3. 3. GSSG/GSH ratio was highest in lung, probably due to the high pO 2 prevalent in the tissue. Nevertheless, this did not result in higher tissue peroxidation, suggesting that the lung antioxidants are capable of coping with a high tissue pO 2. 4. 4. Tissue peroxidation was maximal in the brain when assayed by the TBA test, but this was not confirmed by HPLC of malondialdehyde (MDA). HPLC resulted in much lower MDA values than TBA.