Ron Kohen

OXIDATION OF BIOLOGICAL SYSTEMS: OXIDATIVE STRESS PHENOMENA, ANTIOXIDANTS, REDOX REACTIONS, AND METHODS FOR THEIR QUANTIFICATION

Reactive oxygen species (ROS) and other radicals are involved in a variety of biological phenomena, such as mutation, carcinogenesis, degenerative and other diseases, inflammation, aging, and development. ROS are well recognized for playing a dual role as deleterious and beneficial species. The objectives of this review are to describe oxidative stress phenomena, terminology, definitions, and basic chemical characteristics of the species involved; examine the biological targets susceptible to oxidation and the defense mechanisms of the organism against these reactive metabolites; and analyze methodologies, including immunohistochemical markers, used in toxicological pathology in the visualization of oxidative stress phenomena. Direct detection of ROS and other free radicals is difficult, because these molecules are short-lived and highly reactive in a nonspecific manner. Ongoing oxidative damage is, thus, generally analyzed by measurement of secondary products including derivatives of amino acids, nuclei acids, and lipid peroxidation. Attention has been focused on electrochemical methods based on voltammetry measurements for evaluating the total reducing power of biological fluids and tissues. This approach can function as a tool to assess the antioxidant-reducing profile of a biological site and follow changes in pathological situations. This review thus includes different topics essential for understanding oxidative stress phenomena and provides tools for those intending to conduct study and research in this field.

Oxidative Stress in Closed-Head Injury: Brain Antioxidant Capacity as an Indicator of Functional Outcome

It has been suggested that reactive oxygen species (ROS) play a role in the pathophysiology of brain damage. A number of therapeutic approaches, based on scavenging these radicals, have been attempted both in experimental models and in the clinical setting. In an experimental rat and mouse model of closed-head injury (CHI), we have studied the total tissue nonenzymatic antioxidant capacity to combat ROS. A major mechanism for neutralizing ROS uses endogenous low-molecular weight antioxidants (LMWA). This review deals with the source and nature of ROS in the brain, along with the endogenous defense mechanisms that fight ROS. Special emphasis is placed on LMWA such as ascorbate, urate, tocopherol, lipoic acid, and histidine-related compounds. A novel electrochemical method, using cyclic voltammetry for the determination of total tissue LMWA, is described. The temporal changes in brain LMWA after CHI, as part of the response of the tissue to high ROS levels, and the correlation between the ability of the brain to elevate LMWA and clinical outcome are addressed. We relate to the beneficial effects observed in heat-acclimated rats and the detrimental effects of injury found in apolipoprotein E-deficient mice. Finally, we summarize the effects of cerebroprotective pharmacological agents including the iron chelator desferal, superoxide dismutase, a stable radical from the nitroxide family, and HU-211, a nonpsychotoropic cannabinoid with antioxidant properties. We conclude that ROS play a key role in the pathophysiology of brain injury, and that their neutralization by endogenous or exogenous antioxidants has a protective effect. It is suggested, therefore, that the brain responds to ROS by increasing LMWA, and that the degree of this response is correlated with clinical recovery. The greater the response, the more favorable the outcome.

Skin antioxidants: Their role in aging and in oxidative stress — New approaches for their evaluation

Skin is a highly metabolic tissue which possesses the largest surface area in the body and serves as the protective layer for internal organs [1]. Skin is also a major candidate and target of oxidative stress. It is designed to give both physical and biochemical protection, and is equipped with a large number of defense mechanisms. The skin tissue is exposed to a variety of damaging species which originate in the outer environment, in the skin itself, and in various endogenous sources [2, 3]. The structure of skin is quite complex being composed of several layers, each of which plays a specific role and carries out different functions [4]. Each layer is equipped with its own arsenal of defense molecules, and the various systems differ from each other based on the layers susceptibility to oxidative stress and its function. It is generally agreed that one of the major and important contributions to skin aging, skin disorders and skin diseases results from reactive oxygen species (ROS) [1, 5]. Due to the high occurrence of potential biological targets for oxidative damage, skin is very susceptible to such reactions. For example, skin is rich in lipids, proteins, and DNA, all of which are extremely sensitive to the oxidation process [6-8]. Elucidation of the mechanisms involved in skin oxidation and the examination of the defense systems may contribute to the understanding of skin aging and of the mechanisms involved in the various pathological processes of skin. This review addresses the antioxidant defense mechanism of the skin, the role it plays during the aging process, and the role skin has following exposure to oxidative stresses.

Skin low molecular weight antioxidants and their role in aging and in oxidative stress

The overall skin low molecular weight antioxidant (LMWA) capacity was evaluated during the aging process and following exposure to oxidative stress. Several invasive and non-invasive techniques were developed for evaluating total antioxidant activity. It was found that the skin possesses an extremely efficient and unique antioxidant activity that is better than other tissues. During the aging process a significant decrease in the levels and activity of the water-soluble LMWA was detected while no change and even a slight increase was recorded for the lipophilic LMWA. Similar results were obtained following exposure to oxidative stress. A significant decrease in the water soluble LMWA was recorded in all the stress induced procedures indicating a common mechanism of response. It has also been shown that along with the reduction in total water soluble antioxidant activity there is an accumulation of oxidized adducts. This was observed both on the surface of the skin and in deeper layers. It has been found that skin releases LMWA from its surface. This secretion phenomenon was found to be age dependent. Following exposure to oxidative stress of various kinds, the release of LMWA from the skin was significantly enhanced. This may suggest a physiological mechanism of the skin to cope with oxidative stress, which would open new possibilities for intervention.

A novel function of red wine polyphenols in humans: prevention of absorption of cytotoxic lipid peroxidation products

Current evidence supports a contribution of polyphenols to the prevention of cardiovascular disease, but their mechanisms of action are not understood. We investigated the impact of red wine polyphenols on postprandial cytotoxic lipid peroxidation products (MDA) levels in humans. In a randomized, crossover study, the effect of red wine polyphenols on postprandial levels of plasma and urine MDA was investigated. Three meals of 250 g turkey cutlets supplemented by water (A); soaked in red wine after heating plus 200 ml of red wine (B); or soaked in red wine prior to heating plus 200 ml of red wine (C) were administered to 10 healthy volunteers. Subject baseline plasma levels of MDA were 50 ± 20 nM. After a meal of turkey meat cutlets, plasma MDA levels increased by 160 nM (P<0.0001);after (B) there was a 75% reduction in the absorption of MDA (P<0.0001). However, after (C), the elevation of plasma MDA was completely prevented (P<0.0001). Similar results were obtained for MDA accumulation in urine. Our study suggests that red wine polyphenols exert a beneficial effect by the novel new function, absorption inhibition of the lipotoxin MDA. These findings explain the potentially harmful effects of oxidized fats found in foods and the important benefit of dietary polyphenols in the meal.— Gorelik, S., Ligumsky, M., Kohen, R., Kanner, J. A novel function of red wine polyphenols in humans: prevention of absorption of cytotoxic lipid peroxidation products. FASEB J. 22, 41–46 (2008)

Melatonin-induced neuroprotection after closed head injury is associated with increased brain antioxidants and attenuated late-phase activation of NF-kappaB and AP-1.

Traumatic brain injury (TBI) is followed by massive production of reactive oxygen species (ROS), which mediate secondary cellular damage. Low molecular weight antioxidants (LMWA) constitute one of the defense mechanisms of the brain, and their levels correlate with post‐TBI outcome. Melatonin, the main pineal hormone, possesses antioxidant properties. We investigated the effects of melatonin on neurobehavioral recovery, brain LMWA, and activation of the redox‐sensitive transcription factors nuclear factor‐kappaB (NF‐κB) and AP‐1 in mice subjected to closed head injury (CHI). Given 1 h after CHI, melatonin facilitated recovery during at least 1 wk (P<0.05) and decreased lesion size by ∼ twofold (P<0.01). The dose response displayed a bell‐shape, i.e., neuroprotection was achieved with 5 but not 1 or 10 mg/kg. At the neuroprotective dose, melatonin treatment was associated with sustained (4 days) elevation of brain LMWA, including ascorbic acid (P<0.05). In contrast, LMWA were unaffected by the administration of the neuroprotective endocannabinoid 2‐arachidonoyl glycerol. Furthermore, melatonin did not alter early phase (24 h) CHI‐induced activation of NF‐κB and AP‐1; however, it blocked the robust late‐phase (8 days) activation of NF‐κB and decreased that of AP‐1 to below basal levels. Our results demonstrate that melatonin induces neuroprotection, presumably via potentiation of brain antioxidants and attenuation of NF‐κB and AP‐1 activation.

EVALUATION OF PLASMA LOW MOLECULAR WEIGHT ANTIOXIDANT CAPACITY BY CYCLIC VOLTAMMETRY

The low molecular weight antioxidants (LMWA) of biological samples include many compounds and contribute to the total antioxidant capacity of the system. They act as direct chemical scavengers neutralizing, reactive oxygen-derived species (ROS), and contribute to the ability of the sample to cope with oxidative stress. We propose cyclic voltammetry (CV) as a new method for evaluating the antioxidant capacity of plasma-LMWA and the severity of oxidative stress exerted on the plasma. It is based on the reducing properties of these molecules. CV has been proven to be a simple, sensitive and reliable method. Its tracing does not change during storage of frozen plasma for up to six months. We analyzed the CV tracings by the oxidation potential E1/2, and the current heights Ia of its anodic wave(s). E1/2 indicates the specific component of the LMWA and its ability to donate electron(s); Ia indicates the concentration of this component. Two anodic waves have been identified in human plasma, at E1/2 = 420 +/- 25 and 920 +/- 25 mV. Ascorbate (AA) and urate (UA) were identified as major LMWA components of the first anodic wave, and were confirmed by HPLC-electrochemical detection. Ia was shown to depend linearly on the concentration of either of these LMWA, both in buffer and in plasma. Oxidative stress exerted by exposure to peroxyl radicals, copper ions and ionizing irradiation caused marked changes in the CV tracing. These changes represent corresponding alterations particularly in la, rather than in E1/2. The Ia and E1/2 values reflect the antioxidant capacity of the plasma, while the change of Ia value represents the severity of the oxidative stress induced.

The Stomach as a “Bioreactor”: When Red Meat Meets Red Wine

To determine the stomach bioreactor capability for food oxidation or antioxidation, rats were fed red turkey meat cutlets (meal A) or red turkey meat cutlets and red wine concentrate (meal B). The hydroperoxides (LOOH) and malondialdehyde (MDA) levels of the stomach contents were evaluated during and after digestion; the postprandial plasma MDA level was also evaluated. In independently fed rats, the stomach LOOH concentration fell substantially 90 min following the meal, and the addition of red wine polyphenols enhanced LOOH reduction 3-fold. A similar trend was obtained for MDA. After pyloric ligation, the stomach contents of rats fed red meat homogenate showed >2-fold increases in LOOH and MDA accumulation. The postprandial plasma MDA level increased significantly by 50% following meal A and was maintained or even fell by 34% below basal level following meal B. The findings show that consumption of partially oxidized food could increase lipid peroxidation in the stomach and the absorption of cytotoxic lipid peroxidation products into the body. The addition of antioxidants such as red wine polyphenols to the meal may alter these outcomes. These findings explain the potentially harmful effects of oxidized fats in foods and the important benefit of consuming dietary polyphenols during the meal.

Quantification of the overall REACTIVE OXYGEN SPECIES scavenging capacity of biological fluids and tissues

A method has been developed for measuring and evaluating the overall antioxidant activity derived from the low-molecular weight antioxidants (scavengers). The principle governing this method is based on a common chemical characteristic of the scavengers, their reducing properties. It was hypothesized and then demonstrated that an evaluation of the overall reducing power of a biological sample correlates with the overall scavenging activity of the sample. In order to quantify the total reducing power, the cyclic voltammetry methodology was applied. The resulting measurements correlated with the antioxidant activity of both hydrophilic and lipophilic scavengers. The method is suitable for use in biological fluids and in tissue homogenates, and can supply information concerning the type of antioxidants and their total concentration without having to determine specific compounds. A noninvasive procedure for determining skin overall scavenging activity is also described. This method is based on a well containing an extraction solution that is attached to the skins surface. Following incubation time the extraction solution is analyzed using the cyclic voltammeter instrument and other methods. We have found these methods suitable for evaluating the reducing capacity status in various clinical conditions such as diabetes, ionizing and nonionizing irradiation, brain degenerative diseases, head trauma, and inflammatory bowel diseases. This method is also an efficient tool for evaluating the overall antioxidant capacity of mixtures of antioxidant preparations in vitro. The measurements themselves are simple and rapid. Furthermore, they do not require manipulation of the samples.

Neuroprotective and antioxidant activities of HU-211, a novel NMDA receptor antagonist

This study examines the ability of (+)-(3S,4S)-7-hydroxy-delta 6-tetrahydrocannabinol-1,1-dimethylheptyl (HU-211), a non-competitive NMDA receptor antagonist to: (1) rescue neurons in culture from injury evoked by sodium nitroprusside, hydrogen peroxide (H2O2) and oxygen glucose deprivation; and (2) scavenge reactive oxygen species in vitro. Qualitative and quantitative assessments of cell survival have indicated that: (1) Neuronal cell injury produced following deprivation of oxygen and glucose was significantly attenuated by 5 microM HU-211. (2) Glial and neuronal cell damage induced by sodium nitroprusside was markedly ameliorated by 10 microM HU-211. (3) HU-211 reduced protein oxidation initiated by gamma irradiation, and scavenged peroxyl radicals. (4) HU-211 carries an oxidation potential of 550 mV. These findings suggest that HU-211 holds a unique position among putative neuroprotectant agents in that it combines NMDA receptor antagonistic activity and free radical scavenging abilities in a single molecule.