Circaannual changes in antioxidants and oxidative stress in the heart and liver in rats

Reactive oxygen species are formed in physiological and pathological conditions in mammalian tissues. Because of their high reactivity, they may interact with biomolecules, inducing oxidative injury. Increases in lipid peroxidation can result in oxidative damage to cellular membranes. Protection against oxidative damage is provided by enzymatic and non-enzymatic antioxidant defenses. Antioxidant enzyme activities and lipid peroxidation, as an index of oxidative stress injury, were evaluated in different seasons over one year in the heart and liver of rats, maintained on a 12 h light and dark cycle. Glutathione peroxidase and catalase activities, in both tissues, were maximal in the summer season. Lipid peroxidation in the heart was maximal in the spring as compared to the other seasons and it did not vary in the liver during the year. These findings suggest that any study of antioxidants or oxidative stress must take into account such seasonal variations for a more precise analysis of changes due to any pathological condition.     

4-Hydroxy-2-nonenal: a product and mediator of oxidative stress

The onset of lipid peroxidation within cellular membranes is associated with changes in their physiochemical properties and with the impairment of enzymatic functions located in the membrane environment. There is increasing evidence that aldehydic molecules generated endogenously during the process of lipid peroidation are causally involved in most of the pathophysiological effects associated with oxidative stress in cells and tissues. 4-Hydroxy-2-nonenal (HNE), among them, is believed to be largely responsible for cytopathological effects observed during oxidative stree in vivo and has achieved the status of one of the best recognized and most studied of the cytotoxic products of lipid peroxidation. In the present review, I provide a comprehensive summary of HNE, as the product and mediator or oxidative stress.     

Effect of splenosid on lipid peroxidation process and glutathione antioxidant system in rats exposed to fractionated radiation

The dynamics of lipid peroxidation, antioxidant glutathione system condition in blood and viscerals (brain, heart, liver, spleen) of rats which were fractionally irradiated (10 fractions) in the total dose 1.0 Gy and oxidative homeostasis increase of primary and secondary lipid peroxidation products and glutathione system disturbances were established in the irradiated rats. The administration of splenosid diminished the disturbances of oxidative homeostasis but does not completely normalize the latter. The administration of splenosid during the irradiation course and after its finishing is more effective than only during the irradiation course.     

Glycolaldehyde induces growth inhibition and oxidative stress in human breast cancer cells

Glycolaldehyde (GA) is formed by oxidative degradation of glucose, from glycated proteins, lipid peroxidation, and oxidation of amino acids, and by human neutrophils during phagocytosis. The exact purpose of GA production by phagocytes is unclear, but it is tempting to speculate that it is part of the defense against invading bacteria and tumor cells. We have already reported that GA induces apoptosis in breast cancer cells. Because the GA carbonyl group cannot be blocked by cyclization, it is prone to enolization followed by air oxidation with concomitant production of glyoxal and superoxide. Since both these products can induce oxidative stress, in this work we focused on the ability of GA to cause oxidative cell damage. MCF7 human breast cancer cells were incubated with different GA concentrations and O2*- production, lipid peroxidation, and carbonylated protein were assessed. GA was cytotoxic at 20 microM, inhibiting cell proliferation, and at 100 microM, induced p53 expression and caused apoptosis. These events were accompanied by increases of O2*- production, lipid peroxidation, and accumulation of protein carbonyl. It thus appears that alpha-hydroxy aldehydes can induce oxidative stress. Prevention of oxidative stress, however, did not abolish the effects of GA on cell growth and viability, which appeared to be a direct consequence of glyoxal toxicity.     

Synergistic interactions of Ferulic acid with Ascorbic acid: Its cardioprotective role during isoproterenol induced myocardial infarction in rats

Studies on the lipid peroxidation and antioxidant changes and their significance during myocardial injury have provided a new insight into the pathogenesis of heart disease. The heart failure subsequent to myocardial infarction may be associated with an antioxidant deficit as well as increased myocardial oxidative stress. The present study was designed to evaluate the effect of the combination of ferulic acid and ascorbic acid on antioxidant defense system and lipid peroxidation against isoproterenol (ISO)-induced myocardial infarction in rats. Induction of rats with isoproterenol (150 mg/kg body weight daily, i.p.) for 2 days resulted in a marked elevation in lipid peroxidation, serum marker enzymes (LDH, CPK, GOT, and GPT), and a significant decrease in activities of endogenous antioxidants (SOD, GPx, GST, CAT, and GSH). Pre-co-treatment with the combination of ferulic acid (20 mg/kg body weight/day) and ascorbic acid (80 mg/kg body weight/day) orally for 6 days, significantly attenuated these changes when compared to the individual treatment groups. Histopathological observations were also in correlation with the biochemical parameters. Thus, ferulic acid and ascorbic acid significantly counteracted the pronounced oxidative stress effect of ISO by the inhibition of lipid peroxidation, restoration of antioxidant status, and myocardial marker enzymes levels. In conclusion, these findings indicate the synergistic protective effect of ferulic acid and ascorbic acid on lipid peroxidation and antioxidant defense system during ISO-induced myocardial infarction and associated oxidative stress in rats.     

Effect of docosahexaenoic acid and eicosapentaenoic acid supplementation on oxidative stress levels during pregnancy

Docosahexaenoic acid (DHA) is an indispensable component of cell membranes with high requirements during pregnancy. DHA supplementation is thought to enhance oxidative stress because of increased likelihood of lipid peroxidation. We estimated the oxidative stress levels in two groups of pregnant women who received daily supply of required vitamins with (n = 23) or without (n = 23) 500 mg of DHA and 150 mg of eicosapentaenoic acid (EPA) from 20 weeks of gestation to the time of delivery. Urinary excretions of 8-hydroxy-2'-deoxyguanosine (8-OHdG), a marker of oxidative DNA damage and of malondialdehyde (MDA), a marker of lipid peroxidation, were measured at 20, 30 weeks and at the time of delivery. Urinary MDA excretion remained unchanged throughout the study period in both groups. Urinary 8-OHdG excretion at delivery was significantly higher than at 20 and 30 weeks (p < 0.05), but there were no group differences at the three time points. There were no differences between the two groups in plasma a-tocopherol levels. We conclude that under the conditions studied, a daily supplementation of 500 mg DHA and 150 mg EPA with vitamins to pregnant women did not enhance lipid peroxidation or oxidative DNA damage.     

The LEC rat: a useful model for studying liver carcinogenesis related to oxidative stress and inflammation

Growing evidence indicates oxidative stress as a mechanism of several diseases including cancer. Oxidative stress can be defined as the imbalance between cellular oxidant species production and antioxidant capability shifted towards the former. Lipid peroxidation is one of the processes that takes place during oxidative stress. Lipid peroxidation products, such as malondialdehyde (MDA) and 4-hydroxy-2-nonenal (HNE), are closely related to carcinogenesis as they are potent mutagens and they have been suggested as modulators of signal pathways related to proliferation and apoptosis, two processes implicated in cancer development. Mechanisms by which oxidative stress leads to tumor formation are still under investigation. The need of suitable in vivo models that could reflect that inflammation-related human carcinogenesis is evident. In this regard, the mutant strain Long Evans Cinnamon-like (LEC) rat provides a promising model for investigation of the relationship between hepatitis induced by oxidative stress and hepatocarcinogenesis because it has been demonstrated to develop spontaneous liver tumor formation related to copper accumulation and oxidative stress. In this review, the findings regarding oxidative stress and its relation with liver pathologies in LEC rats are discussed; we focus on the mechanisms proposed for HNE carcinogenesis.     

The relationship between cell membrane damage and lipid peroxidation under the condition of hypoxia‐reoxygenation: analysis of the mechanism using antioxidants and electron transport inhibitors

We consecutively observed lipid peroxidation and cell membrane damage under the condition of hypoxia‐reoxygenation (H/R) in cells and analyzed their mechanisms by using electron transport inhibitors and an antioxidant. In H/R experiments, lipid peroxidation and cell membrane damage were observed during the hypoxia phase. In the reoxygenation phase, lipid peroxidation stopped, while cell membrane damage did not. An antioxidant, n‐acetylcystein (NAC), and potassium cyanide (KCN) inhibited lipid peroxidation and cell membrane damage, while rotenone did not inhibit either of them. Although antimycin A did not inhibit lipid peroxidation, it inhibited cell membrane damage during the hypoxia phase but not during the reoxygenation phase. These results suggested that lipid peroxidation can affect cell membrane damage as a trigger during the hypoxia phase and the generation of oxidative stress can vary depending on the inhibition locations in the electron transport system. Copyright © 2009 John Wiley & Sons, Ltd.     

Accumulation of Nepsilon-(Hexanoyl)lysine, an oxidative stress biomarker, in rice seeds during storage

Nepsilon-(hexanoyl)lysine (HEL) is a potentially useful marker of oxidative stress in animals. We investigated whether HEL might be useful as a marker in rice seeds damaged by oxidative stress during storage, as well as in animals. The germination ability of rice decreased with lipid peroxidation during storage at 40 degrees C for three months. Moreover, we observed accumulation of HEL in the damaged rice. In addition, the activities of antioxidative enzymes, catalase and superoxide dismutase, significantly decreased in the rice seeds during storage at 40 degrees C. These results suggest that HEL might be a useful marker of oxidative stress in rice.     

Alpha-tocopherol improves biochemical and dynamic parameters in cryopreserved boar semen

Cryopreservation is associated with the production of reactive oxygen species which lead to lipid peroxidation of sperm membranes. The objective was to determine an alpha-tocopherol concentration capable of improving the quality of cryopreserved porcine semen. Boar spermatozoa frozen with 200, 500 or 1000 microg/mL alpha-tocopherol were thawed and incubated at 37 degrees C for 4 h. Routine parameters of semen quality, susceptibility to lipid peroxidation 2-thiobarbituric acid (TBARS) and oxygen uptake were evaluated. Motility was higher (P<0.05) in samples treated with different concentrations of alpha-tocopherol up to 2 h of incubation. Viability and acrosome integrity significantly decreased during incubation (no significant differences between treatments). Two hundred micrograms per milliliter alpha-tocopherol protected spermatozoa against lipid peroxidation during incubation, but 1000 microg/mL failed to protect after 2 h of incubation. There was a negative association between TBARS and motility, suggesting that lipid peroxidation affected sperm motility. Both control and 200 microg/mL alpha-tocopherol samples preserved the capacity to generate oxidative energy up to 1 h of incubation. The addition of 200 microg/mL alpha-tocopherol in the semen extender could be useful to preserve boar spermatozoa against the oxidative stress generated by cryopreservation.     

Hydroxynonenal and uncoupling proteins: a model for protection against oxidative damage

In this mini review we summarize recent studies from our laboratory that show the involvement of superoxide and the lipid peroxidation product 4-hydroxynonenal in the regulation of mitochondrial uncoupling. Superoxide produced during mitochondrial respiration is a major cause of the cellular oxidative damage that may underlie degenerative diseases and ageing. Superoxide production is very sensitive to the magnitude of the mitochondrial protonmotive force, so can be strongly decreased by mild uncoupling. Superoxide is able to give rise to other reactive oxygen species, which elicit deleterious effects primarily by oxidizing intracellular components, including lipids, DNA and proteins. Superoxide-induced lipid peroxidation leads to the production of reactive aldehydes, including 4-hydroxynonenal. These aldehydic lipid peroxidation products are in turn able to modify proteins such as mitochondrial uncoupling proteins and the adenine nucleotide translocase, converting them into active proton transporters. This activation induces mild uncoupling and so diminishes mitochondrial superoxide production, hence protecting against disease and oxidative damage at the expense of energy production.     

Oxidative stress causes a decline in lysosomal integrity during hypothermic incubation of rat hepatocytes

Oxidative stress during cold preservation has been identified as a significant cause of cell injury but the process by which injury occurs is poorly understood. We examined loss of lysosomal integrity as a possible cause of cell injury during extended cold storage of isolated rat hepatocytes. After 21 h of hypothermia, there was a marked decline in lysosomal integrity, which was correlated with an increase in lipid peroxidation. When lipid peroxidation was prevented with the antioxidant Trolox (a vitamin E analog) or the iron chelator desferrioxamine, lysosomal integrity was preserved. In contrast, increasing lysosomal iron with ferric chloride caused an increase in lipid peroxidation and decreased lysosomal integrity. Loss of lysosomal integrity during cold preservation in this experimental model was consistent with iron-initiated oxidative stress. The progressive loss of lysosomal integrity during hypothermic incubation has the potential to affect liver function after transplantation.     

Correlation of serum toll like receptor 9 and trace elements with lipid peroxidation in the patients of breast diseases

Toll-like receptors are recognized as redox sensitive receptor proteins and have been implicated in cellular response to oxidative stress. Altered pro-oxidant-antioxidant balance leads to an increased oxidative damage and consequently play an important role in breast diseases. The study was designed to access the oxidative stress status by quantification of byproducts generated during lipid peroxidation and inadequate trace elements during oxidative damage and its effects on the toll like receptor (TLR) activity in patients of breast diseases. Decreased levels of selenium, copper, zinc, magnesium and iron with elevated levels of malondialdehyde (marker of lipid peroxidation) were accompanied by decreased TLR activity in patients of benign breast diseases as well as breast carcinoma. A similar pattern was observed with the advancement of disease and its subsequent progression in breast carcinoma patients. Results of multinomial regression analysis suggest benign breast disease patients are at higher risk of developing breast cancer with high odds ratio of lipid damage.     

Lipid peroxidation during ischemia depends on ischemia time in warm ischemia and reperfusion of rat liver

Prolonged hepatic warm ischemia has been incriminated in oxidative stress after reperfusion. However, the magnitude of oxidative stress during ischemia has been controversial. The aims of the present study were to elucidate whether lipid peroxidation progressed during ischemia and to clarify whether oxidative stress during ischemia aggravated the oxidative damage after reperfusion. Rats were subjected to 30 to 120 min of 70% warm ischemia alone or followed by reperfusion for 60 min. Lipid peroxidation (LPO) was evaluated by amounts of phosphatidylcholine hydroperoxide (PC-OOH) and phosphatidylethanolamine hydroperoxide (PE-OOH) as primary LPO products. Total amounts of malondialdehyde and 4-hydroxy-2-nonenal (MDA + 4-HNE), degraded from hydroperoxides, were also determined. PC-OOH and PE-OOH significantly increased at 60 and 120 min ischemia with concomitant increase of oxidized glutathione. These hydroperoxides did not increase at 60 min reperfusion after 60 min ischemia, whereas they did increase at 60 min reperfusion after 120 min ischemia with deactivation of phospholipid hydroperoxide glutathione peroxidase and superoxide dismutase. The amount of MDA + 4-HNE exhibited similar changes, but the velocity of production dropped with ischemic time longer than 60 min. In conclusion, oxidative stress progressed during ischemia and triggered the oxidative injury after reperfusion. Secondary LPO products are less sensitive, especially during ischemia, which may cause possible underestimation and discrepancy.     

Protective effect of exogenous coenzyme Q in rats subjected to partial hepatic ischemia and reperfusion.

In a surgical model of liver ischemia lipid peroxidation occurs, as shown by increase of lipid peroxidation end products, endogenous CoQ9 is oxidized and mitochondrial respiration is lowered; however, pre-treatment of the rats by i.p. injection of CoQ10 for 14 days normalizes the above parameters, presumably by way of the observed high extent of reduction of the incorporated quinone; moreover, liver homogenates of the CoQ10-treated rats are more resistant than those of non-treated rats to oxidative stress induced by an azido free radical initiator. This preliminary study suggests that CoQ10 pre-treatment can be of beneficial effect against oxidative damage during liver surgery transplantation.     

Lipid peroxidation induces cholesterol domain formation in model membranes

Numerous reports have established that lipid peroxidation contributes to cell injury by altering the basic physical properties and structural organization of membrane components. Oxidative modification of polyunsaturated phospholipids has been shown, in particular, to alter the intermolecular packing, thermodynamic, and phase parameters of the membrane bilayer. In this study, the effects of oxidative stress on membrane phospholipid and sterol organization were measured using small angle x-ray diffraction approaches. Model membranes enriched in dilinoleoylphosphatidylcholine were prepared at various concentrations of cholesterol and subjected to lipid peroxidation at physiologic conditions. At cholesterol-to-phospholipid mole ratios (C/P) as low as 0.4, lipid peroxidation induced the formation of discrete, membrane-restricted cholesterol domains having a unit cell periodicity or d-space value of 34 A. The formation of cholesterol domains correlated directly with lipid hydroperoxide levels and was inhibited by treatment with vitamin E. In the absence of oxidative stress, similar cholesterol domains were observed only at C/P ratios of 1.0 or higher. In addition to changes in sterol organization, lipid peroxidation also caused reproducible changes in overall membrane structure, including a 10 A reduction in the width of the surrounding, sterol-poor membrane bilayer. These data provided direct evidence that lipid peroxidation alters the essential organization and structure of membrane lipids in a manner that may contribute to changes in membrane function during aging and oxidative stress-related disorders.     

Comparison of protein oxidation and aldehyde formation during oxidative stress in isolated mitochondria

Oxidative stress is known to cause oxidative protein modification and the generation of reactive aldehydes derived from lipid peroxidation. Extent and kinetics of both processes were investigated during oxidative damage of isolated rat liver mitochondria treated with iron/ascorbate. The monofunctional aldehydes 4-hydroxynonenal (4-HNE), n-hexanal, n-pentanal, n-nonanal, n-heptanal, 2-octenal, 4-hydroxydecenal as well as thiobarbituric acid reactive substances (TBARS) were detected. The kinetics of aldehyde generation showed a lag-phase preceding an exponential increase. In contrast, oxidative protein modification, assessed as 2,4-dinitrophenylhydrazine (DNPH) reactive protein-bound carbonyls, continuously increased without detectable lag-phase. Western blot analysis confirmed these findings but did not allow the identification of individual proteins preferentially oxidized. Protein modification by 4-HNE, determined by immunoblotting, was in parallel to the formation of this aldehyde determined by HPLC. These results suggest that protein oxidation occurs during the time of functional decline of mitochondria, i.e. in the lagphase of lipid peroxidation. This protein modification seems not to be caused by 4-HNE.     

Purified human chondroitin-4-sulfate reduced MMP/TIMP imbalance induced by iron plus ascorbate in human fibroblast cultures

Imbalance between matrix metalloproteinases (MMPs) and tissue inhibitor of matrix metalloproteinases (TIMPs) is an important control point in tissue remodelling. Several findings have reported a marked MMP/TIMP imbalance in a variety of in vitro models in which oxidative stress was induced. Since previous studies showed that commercial hyaluronan and chondroitin-4-sulphate are able to limit lipid peroxidation during oxidative stress, we investigated the antioxidant capacity of purified human plasma chondroitin-4-sulfate in reducing MMP and TIMP imbalance in a model of ROS-induced oxidative injury in fibroblast cultures. Purified human plasma chondroitin-4-sulfate was added to the fibroblast cultures exposed to FeSO4 plus ascorbate. We assayed cell death, MMP and TIMP mRNA expression and protein activities, DNA damage, membrane lipid peroxidation, and aconitase depletion. FeSO4 plus ascorbate produced severe death of cells and increased MMP-1, MMP-2 and MMP-9 expression and protein activities. It also caused DNA strand breaks, enhanced lipid peroxidation and decreased aconitase. TIMP-1 and TIMP-2 protein levels and mRNA expression remain unaltered. Purified human plasma C4S, at three different doses, restored the MMP/TIMP homeostasis, increased cell survival, reduced DNA damage, inhibited lipid peroxidation and limited impairment of aconitase. These results further support the hypothesis that these biomolecules possess antioxidant activity and by reducing ROS production C4S may limit cell injury produced by MMP/TIMP imbalance.     

Aging-related oxidative stress depends on dietary lipid source in rat postmitotic tissues

We investigate mitochondrial-lipid peroxidation of mitotic (liver) and postmitotic (heart and skeletal muscle) tissues of rats fed lifelong on two different lipid sources: virgin olive oil (monounsaturated fatty acids) and sunflower oil (n–6 polyunsaturated fatty acids). Two groups of 80 rats each were fed over 24 months on a diet differing in the lipid source (virgin olive oil or sunflower oil). Twenty rats per group were killed at 6, 12, 18, and 24 months; liver, heart, and skeletal muscle mitochondria were isolated and the lipid profile, hydroperoxides, vitamin E, and ubiquinone as well as catalase activity measured. Lipid peroxidation was higher in postmitotic tissues, and sunflower oil led to a higher degree of polyunsaturation and peroxidation. The levels of -tocopherol adapted to oxidative stress and preferentially accumulated during aging in heart and skeletal muscle. In conclusion, the type of dietary fat should be considered in studies on aging, since oxidative stress is directly modulated by this factor. This study confirms that postmitotic tissues are more prone to oxidative stress during aging and proposes a hypothesis to explain this phenomenon.     

Migrant blackbirds,Turdus merula,have higher plasma levels of polyunsaturated fatty acids compared to residents,but not enhanced fatty acid unsaturation index

Birds have been observed to have dietary preferences for unsaturated fatty acids during migration. Polyunsaturated fatty acids (PUFAs) may increase the exercise performance of migrant birds; however, PUFAs are also peroxidation prone and might therefore incur increased costs in terms of enhanced oxidative damage in migratory individuals. To shed light on this potential constraint, we analyzed plasma fatty acid (FA) composition and estimated the unsaturation index as a proxy for susceptibility to lipid peroxidation of migrants and residents of the partially migratory common blackbird (Turdus merula) at a stopover site during autumn migration. As predicted, migrant birds had higher relative and absolute levels of PUFAs compared to resident birds. This included the strictly dietary ω‐3 PUFA α‐linolenic acid, suggesting a dietary and/or storage preference for these FAs in migrants. Interestingly, the FA unsaturation index did not differ between migrants and residents. These findings suggest a mechanism where birds alter their levels of metabolic substrate without simultaneously increasing the susceptibility of the substrate to lipid peroxidation. In summary, our results are in line with the hypothesis that increased exercise performance during migration might be constrained by oxidative stress, which is manifested in changes in the composition of key FAs to retain the unsaturation index constant despite the increased levels of peroxidizable PUFAs.