Lipid-soluble Free Radical Research Articles

Comparative evaluation of dose dependent efficacy of vitamin C, in relation to oxidative stress level in subjects with Oral Submucus Fibrosis

Background Oral submucus fibrosis (OSMF) has a 0.2-0.5 prevalence rate in India. The malignancy rate of OSMF was found to be 7.6%. The focus of pathogenesis is currently on reactive oxygen species levels which can rise to extremely high levels, exceeding the human body’s antioxidant defense system and causing oxidative stress that goes beyond physiological limitations. Since oxidative stress plays such a significant part in carcinogenesis and other degenerative diseases, it is fair to believe that antioxidants will help to reduce or even stop these processes. Vitamin C is known for its antioxidant properties which act as a lipid-soluble free radical scavenger in cell membranes. But in some of studies a paradoxical effect of antioxidant doses was found. Thus, the present study will be conducted to evaluate the dose dependent efficacy of Vitamin C level in relation to oxidative stress level in subjects of OSMF. Methods After informed consent, the patient’s examination will be conducted. For each individual, 5ml of the blood sample will be withdrawn under all the aseptic precautions and oxidative stress level will be assessed depending on MDA & SOD level in a central research laboratory. Depending on oxidative stress level patients will be divided into two groups. Group I - Moderate oxidative stress in OSMF SI less than 240 but more than 192 Group II - High Oxidative Stress in OSMF SI more than 240 Single and double doses of Vitamin C will be given to patients accordingly and the MDA & SOD levels will be calculated on day 30,90 days and on 180th day Conclusions Positive results of the study will give direction for choosing the correct dose of the antioxidant Vitamin C for OSMF depending on oxidative stress level.

Plant Nitric Oxide Signaling under Drought Stress.

Water deficit caused by drought is a significant threat to crop growth and production. Nitric oxide (NO), a water- and lipid-soluble free radical, plays an important role in cytoprotection. Apart from a few studies supporting the role of NO in drought responses, little is known about this pivotal molecular amendment in the regulation of abiotic stress signaling. In this review, we highlight the knowledge gaps in NO roles under drought stress and the technical challenges underlying NO detection and measurements, and we provide recommendations regarding potential avenues for future investigation. The modulation of NO production to alleviate abiotic stress disturbances in higher plants highlights the potential of genetic manipulation to influence NO metabolism as a tool with which plant fitness can be improved under adverse growth conditions.

Concentration and time dependent cytoprotective effects of anthocyanins against oxidative hemolysis induced by water and lipid soluble free radical initiators: an <i>in vitro</i> study

The non-enzymatic, free radical-mediated peroxidation of erythrocyte membranes is now correlated with a variety of pathological conditions, and considered as a primary event associated with hematotoxicity of variety of drugs and chemicals that induce erythrocyte hemolysis. The plant derived polyphenols including anthocyanins have been suggested to enhance red blood cell resistance to oxidative stress both in vitro and in vivo. The present study was designed to investigate the concentration and time dependent anti-hemolytic activity of anthocyanins using in vitro models of azo compounds-induced erythrocyte membrane peroxidative damage. Three different concentrations (1, 0.1 and 0.01 mg/ml) of anthocyanins solution were incubated with erythrocytes suspension 30 min before addition of either water soluble (AAPH) or lipid soluble (AMVN) free radical initiators. The level of hemoglobin released into the solution was determined spectrophotometrically, both in presence and absence of anthocyanins. Moreover, the most effective concentration was selected to be added at different time intervals (zero time, 2 and 3 hours) post challenge with either AAPH or AMVN and the extent of hemolysis was determined as mentioned before. The results showed that anthocyanins, concentration dependently attenuated hemolysis induced by azo compounds, and substantial cytoprotective effects of anthocyanins were observed when they added simultaneously with azo compounds, while delayed addition of anthocyanins for 2 and 3 h diminished but not abolished the protective effect of anthocyanins. In conclusion, anthocyanins can effectively display concentration and time dependent cytoprotective properties against azo compounds-induced peroxidative hemolytic damage.

UBIQUINONE-10 AS AN ANTIOXIDANT

ABSTRACT Ubiquinol, the reduced form of coenzyme Q, is known as a lipid antioxidant. Its fully oxidized form, ubiquinone, would theoretically not have this activity. However, we report that ubiquinone-10, the oxidized form of coenzyme Q, demonstrated antioxidant activity in model studies against a lipid-soluble free radical generator, 2,2′-azobis(2,4-dimethylvaleronitrile). This was demonstrated at both 1:100 and 1:1,000 ratios of ubiquinone-10 to lipid (for both methyl linoleate and methyl linolenate). Ubiquinone-10 should therefore not be discounted as a possible antioxidant in more complex systems such as food. PRACTICAL APPLICATIONS Coenzyme Q (ubiquinol/ubiquinone) is an important component of electron transport in biological tissues and is also classified as a potent antioxidant. It is generally believed that the form that is active as an antioxidant is the reduced ubiquinol. In evaluating the loss of antioxidants in mackerel light and dark muscle tissues, it was observed that the oxidized form, ubiquinone, comprised over 87% of the total coenzyme Q in light muscle and over 93% in dark muscle and this observation did not change under anaerobic conditions. This study was done to determine if the oxidized form of coenzyme Q, ubiquinone, was capable of acting as an antioxidant. In this study, it is shown that it can function in this manner.

Differential antioxidant properties of red wine in water soluble and lipid soluble peroxyl radical generating systems.

Red wine and its components have been shown to possess cardioprotective and anti-atherogenic effects. Additionally, red wine and many of its components like catechin, epicatechin, rutin, transresveratrol and quercetin possess antioxidant properties. Oxidized low density lipoprotein (LDL) is involved in the development of an atherosclerotic lesion. Red wine, therefore, may be anti-atherogenic because of its antioxidant effects on LDL modification. This study examined the antioxidant effects of catechin, epicatechin, rutin, transresveratrol, quercetin and Merlot wines on LDL oxidation. Merlot was chosen because although other red wines have been tested, limited information exists for this variety. Oxidation was carried out with AAPH (2,2'-Azo-bis(2-amidinopropane) dihydrochloride) and AMVN (2,2'-Azo-bis(2,4-dimethylvaleronitrile)), as water and lipid soluble peroxyl radical generating systems (FRGS), respectively. This allowed us to determine the lipophilic antioxidant characteristics of the wine and its components. Conjugated diene assays were used to measure LDL oxidation over 6 hrs. In an AAPH system, all polyphenolic compounds except transresveratrol displayed an antioxidant effect. LDL oxidation by AAPH was also inhibited by aliquots of Merlot wine. No antioxidant effects were observed in an AMVN environment except for a mild antioxidant effect by quercetin. Surprisingly, incubation of LDL with Merlot wine strongly protected against oxidation by AMVN. In summary, the five phenolic compounds displayed antioxidant effects in a water soluble free radical generating system, but only quercetin showed this in a lipid soluble one. However, red wine inhibited LDL oxidation by both water and lipid soluble free radical generating systems. Our data suggest, therefore, that red wines contain unidentified antioxidants that provide protection against LDL oxidation within a lipid soluble environment. (Mol Cell Biochem 263: 211-215, 2004).

Nitric Oxide Regulation of Maspin Expression in Normal Mammary Epithelial and Breast Cancer Cells

In this study, we examined the unique relationship of maspin, a serine protease inhibitor (serpin), that plays a critical role in mammary gland development and is silenced during breast cancer progression, and nitric oxide (NO), a multifaceted water and lipid soluble free radical. The hypothesis tested was that there is a correlation between endothelial nitric oxide synthase and maspin in MCF-7 cells and that NO is capable of regulating maspin expression. An experimental system was developed in which cellular levels of NO in normal human mammary epithelial cells and the breast cancer cell line MCF-7 could be altered using NO modulators. The effect(s) of NO modulators on maspin was measured using reverse transcriptase-polymerase chain reaction and Western blot analysis of subcellular fractions of both cell types. The data revealed that NO induced maspin expression in MCF-7 cells, and the induced maspin resulted in diminished cell motility and invasion, concomitant with an increase in the apoptotic index. This novel finding provides new information regarding the molecular role of maspin in regulating mammary epithelial growth, remodeling, tumor progression, and the metastatic process. More significantly, these findings could have a potential impact on future therapeutic intervention strategies for breast cancer. Targeted delivery of NO within the tumor microenvironment could provide a feasible noninvasive approach for effective treatment.

A Review of Polymorphisms in the Human Gene for Inducible Nitric Oxide Synthase (NOS2) in Patients with Malaria

Significant variability exists in host responses to malaria in different human populations. These epidemiological data have prompted a search for genetic variations that determine host responses to malaria. This review focuses on polymorphisms in the promoter region of inducible nitric oxide synthase (NOS2) as a source of genetic differences in the response of patients to infection with malaria. Nitric oxide (NO), a lipid soluble free radical, mediates host resistance to infectious organisms including parasites. NO is produced by three different NO synthases (NOS) in humans. Research on NO and malaria has focused on the cytokine inducible isoform of NOS (NOS2). The role of NO in host responses to infection and inflammation is likely to be both detrimental and beneficial to the host; this is reflected by the effects of NO on different host tissues and on parasite viability. Therefore, studies of the regulation of NOS2 transcription by different human cell types are important to gain a better understanding of the effects of malaria infection on different host tissues. Several important transcription factor binding sites in the NOS2 promoter region have been identified and represent potential sites of genetic heterogeneity in NOS2 transcription during malaria infection. Furthermore, a variety of studies have been performed to determine the role of NO in the pathogenesis of malaria in humans. Taken together, these studies suggest that high systemic NO production is associated with milder disease and increased parasite clearance while impaired systemic NO production (but increased central nervous system NOS2 expression) is associated with cerebral malaria. There are several possible mechanisms that might explain differences in NO production in malaria patients including functional polymorphisms in the promoter region of NOS2. To date, two NOS2 promoter polymorphisms, G-954C and a CCTTT microsatellite repeat, have been identified, and are associated with disease severity in populations of African patients with malaria. The relationship of these polymorphisms to malaria disease severity in other populations remains controversial and is the subject of ongoing studies. Likewise, the functional significance (relative to NO production) of these polymorphisms in unknown. Therefore, future studies will be necessary to determine whether other polymorphisms in the NOS2 gene are associated with malaria disease severity and to determine the functional effects of these polymorphisms on malaria disease severity and NOS2 transcription.

Antioxidant properties of merlot wines in water soluble and lipid soluble free radical generating systems

Antioxidant properties of merlot wines in water soluble and lipid soluble free radical generating systems

Oxidative Stress in Brain Ischemia

Brain ischemia initiates a complex cascade of metabolic events, several of which involve the generation of nitrogen and oxygen free radicals. These free radicals and related reactive chemical species mediate much of damage that occurs after transient brain ischemia, and in the penumbral region of infarcts caused by permanent ischemia. Nitric oxide, a water- and lipid-soluble free radical, is generated by the action of nitric oxide synthases. Ischemia causes a surge in nitric oxide synthase 1 (NOS 1) activity in neurons and, possibly, glia, increased NOS 3 activity in vascular endothelium, and later an increase in NOS 2 activity in a range of cells including infiltrating neutrophils and macrophages, activated microglia and astrocytes. The effects of ischemia on the activity of NOS 1, a Ca2+-dependent enzyme, are thought to be secondary to reversal of glutamate reuptake at synapses, activation of NMDA receptors, and resulting elevation of intracellular Ca2+. The up-regulation of NOS 2 activity is mediated by transcriptional inducers. In the context of brain ischemia, the activity of NOS 1 and NOS 2 is broadly deleterious, and their inhibition or inactivation is neuroprotective. However, the production of nitric oxide in blood vessels by NOS 3, which, like NOS 1, is Ca2+-dependent, causes vasodilatation and improves blood flow in the penumbral region of brain infarcts. In addition to causing the synthesis of nitric oxide, brain ischemia leads to the generation of superoxide, through the action of nitric oxide synthases, xanthine oxidase, leakage from the mitochondrial electron transport chain, and other mechanisms. Nitric oxide and superoxide are themselves highly reactive but can also combine to form a highly toxic anion, peroxynitrite. The toxicity of the free radicals and peroxynitrite results from their modification of macromolecules, especially DNA, and from the resulting induction of apoptotic and necrotic pathways. The mode of cell death that prevails probably depends on the severity and precise nature of the ischemic injury. Recent studies have emphasized the role of peroxynitrite in causing single-strand breaks in DNA, which activate the DNA repair protein poly(ADP-ribose) polymerase (PARP). This catalyzes the cleavage and thereby the consumption of NAD+, the source of energy for many vital cellular processes. Over-activation of PARP, with resulting depletion of NAD+, has been shown to make a major contribution to brain damage after transient focal ischemia in experimental animals. Neuronal accumulation of poly(ADP-ribose), the end-product of PARP activity has been demonstrated after brain ischemia in man. Several therapeutic strategies have been used to try to prevent oxidative damage and its consequences after brain ischemia in man. Although some of the drugs used in early studies were ineffective or had unacceptable side effects, other trials with antioxidant drugs have proven highly encouraging. The findings in recent animal studies are likely to lead to a range of further pharmacological strategies to limit brain injury in stroke patients.

Licorice extract and its major polyphenol glabridin protect low-density lipoprotein against lipid peroxidation: in vitro and ex vivo studies in humans and in atherosclerotic apolipoprotein E-deficient mice

Polyphenolic flavonoids are powerful antioxidants. In the present study we investigated the antioxidative activity against low-density-lipoprotein (LDL) oxidation of a not yet studied subclass of polyphenols, the isoflavans, which are present in licorice alcoholic extract. The study was performed in humans as well as in atherosclerotic apolipoprotein E-deficient mice (E zero), because their LDL is highly susceptible to oxidation. LDL oxidation was induced by incubating it with copper ions as well as with the aqueous or lipid-soluble free radical generators 2,2'-azobis'2-amidino propane hydrochloride (AAPH) and 2,2'-azobis 2,4-dimethylvaleronitrile (AMVN), respectively. The extent of LDL oxidation was determined by measuring the formation of conjugated dienes, thiobarbituric acid reactive-substances (TBARS), and lipid peroxides. By all methods in human studies, licorice ethanolic extract as well as a pure material, which was identified by gas chromatography-mass spectroscopy as the isoflavan glabridin, were shown to inhibit LDL oxidation by a mechanism involving scavenging of free radicals. In an ex vivo study, LDL isolated from the plasma of 10 normolipidemic subjects who were orally supplemented for 2 wk with 100 mg licorice/d was more resistant to oxidation than was LDL isolated before licorice supplementation. Dietary supplementation of each E zero mouse with licorice (200 micrograms/d) or pure glabridin (20 micrograms/d) for 6 wk resulted in a substantial reduction in the susceptibility of their LDL to oxidation along with a reduction in the atherosclerotic lesion area. These results could be related to the absorption and binding of glabridin to the LDL particle and subsequent protection of the LDL from oxidation by multiple modes as shown in humans and in E zero mice.

Effect of sofalcone on gastric mucosal injury induced by ischemia-reperfusion and its antioxidant properties.

The effect of sofalcone on gastric mucosal injury induced by ischemia-reperfusion (I-R) injury was studied in rats. I-R injury was produced in rat stomach by applying a small clamp to the celiac artery for 30 min and by removal of the clamp for 60 min. The increase in total area of erosions in the stomach after I-R and the increase in lipid peroxides in the gastric mucosa were significantly inhibited by intragastric administration of sofalcone. In addition, sofalcone significantly inhibited the lipid-soluble free radical initiator-induced increase in lipid peroxides of the gastric mucosal homogenate, and could show scavenging action of superoxide radicals in aprotic solvent. These results showed that the protective effect of sofalcone against I-R-induced gastric mucosal injury is attributable to its antioxidant activities in the lipophilic phase.

ANTIOXIDANT STATUS AND ALCOHOL-RELATED DISEASES

Chronic ethanol consumption is associated with increased incidence of a variety of illnesses, including cancer. Studies have shown that ethanol consumption may result in increased oxidative stress with formation of lipid peroxides and free radicals. The susceptibility of a given tissue to peroxidation is, however, a function of the overall balance between prooxidants and antioxidant defence systems. The latter involve both intracellular and extracellular protective factors were nutrients play an important role. Impaired nutritional status of different vitamins and trace elements have been reported in alcoholics. Reduced levels of vitamin E have been found in serum of alcoholics with and without liver disease and in liver biopsies from alcoholics with cirrhosis. These findings may be due to the increased oxidative stress as reported in experimental animals, and may be of importance since vitamin E is the major, if not the only, lipid-soluble free radical scavenger in some tissues. Reduced antioxidant capacity has been found in several tissues and may promote the generation of free radicals and lipid peroxides which may damage cells directly, induce inflammation and accelerate collagen synthesis. These events may progress to tissue damage and disease. The importance of radicals in cancer initiation and promotion is presently of great interest. The role of lipid peroxides and free radicals in alcohol-related disease and cancer remains unresolved. Further research is required to establish the role of these reactive species in the pathogenesis of alcohol-related disease, and to evaluate the role of nutrition in favour of the antioxidant defence mechanisms.

Antioxidant activity of carnosine in cooked ground pork

Carnosine (0·5–1·5%) reduced ( P < 0·05) the formation of thiobarbituric acid reactive substances (TBARS) in cooked unsalted ground pork after 7 days of storage at 4°C. The antioxidant activity of carnosine was less in cooked salted ground pork, with only 1·5% carnosine inhibiting (P < 0·05) TBARS formation during refrigerated storage. The antioxidant activity of carnosine in cooked salted and unsalted ground pork was greater than those of the lipid-soluble free radical scavengers, butylated hydroxytoluene and α-tocopherol ( P < 0·05) but less than that of sodium tripolyphosphate ( P < 0·05). These data suggest that carnosine could be used to reduce the oxidative deterioration of cooked salted and unsalted ground pork.

Synergistic interaction of ozone and respirable aerosols on rat lungs. IV. Protection by quenchers of reactive oxygen species

In previous studies we have shown interactions between ozone and acidic respirable aerosols as evaluated by sensitive biochemical and quantitative morphological endpoints. In the present paper we have attempted to test the hypothesis that active oxygen specis might play a role in lung damage caused by ozone ± sulfuric acid aerosol. Rats were administered various scavengers of active oxygen species via the intraperitoneal (i.p.) route before and during exposure to 0.12, 0.20 or 0.64 ppm of ozone ± 0.04, 0.1, 0.2, 0.5, or 1.0 mg/m 3 of sulfuric acid aerosol. Vitamin E, a lipid-soluble free radical scavenger, β-carotene (the precursor of vitamin A), a singlet oxygen scavenger, and dimethylthiourea, a hydrogen peroxide scavenger, were tested. Dimethylthiourea at doses of 50 – 500 mg/kg per day was able to protect rats from the effets of ozone, suggesting a role for hydrogen peroxide in ozone-induced lung damage in vivo. Neither vitamin E nor β-carotene at 100 mg/kg doses had any protective activity in our experiments, despite documentation of increased lung content of the scavengers in animals receiving these agents. These results suggest that water solubility may play a role in the efficacy of the scavenging agents under our exposure protocols. We conclude that: (1) hydrogen peroxide may be involved in ozone-induced lung damage; and (2) that lung injury by ozone and by ozone + sulfuric acid aerosol may share common pathways.

Effect of antioxidants on primary alloantigen-induced T cell activation and proliferation.

Because oxygen-centered free radicals are involved in cell-mediated immune responses, we examined the possibility that these reactive species could also have a role in the lymphoproliferative response to alloantigens in the mixed lymphocyte culture (MLC). Three classes of agents that prevent the formation, or damaging effects, of oxygen radicals were tested: the non-permeant electron acceptor ferricyanide; the iron chelators desferrioxamine, desferrithiocin, octanohydroxamic acid, and pyridoxal isonicotinoyl hydrazone, which are thought to be capable of preventing the iron-catalyzed reduction of H2O2 to more reactive species; and the lipid-soluble free radical scavenger butylated hydroxyanisole. These compounds inhibited the proliferation of all potential responder cells in the MLC in a dose-dependent manner. Inhibition was observed only if these agents were present early (less than 40 hr) in the MLC; if added later, their effects were significantly reduced. In contrast, the interleukin 2 (IL 2)-dependent proliferation of CTLL-2 cells or Con A blasts was not affected by these compounds at concentrations that inhibited proliferation in MLC by greater than 90%. Interleukin 1 (IL 1) production by peritoneal exudate cells and IL 2 and IL 1 levels in MLC were not affected by any of the agents tested. By flow microfluorometry, the expression of IL 2 receptors on stimulated T cells was found to be inhibited in the presence of these drugs. Taken together, the data point to an important role for free radical-mediated processes during the early stages of T lymphocyte activation, before IL 2 receptor expression.

Alpha tocopherol decreases lipid peroxidation, neuronal necrosis, and reactive gliosis in reaggregate cultures of fetal rat brain.

This study explored the effects of the lipid-soluble free radical scavenger, alpha tocopherol (vitamin E), on neuronal injury and glial protein accumulation in a well-characterized, three-dimensional, mixed neuronal and glial culture system derived from fetal rat prosencephalon. As these reaggregated spheroidal cultures grew and enlarged, they developed small central areas of necrosis (presumably due to nutritional compromise) which we used as a model for central nervous system injury. Treatment with vitamin E (delivered in egg phosphatidylcholine liposomes beginning on day 8 in vitro) did not alter the appearance of the central necrotic areas, but it strongly suppressed the reaction of adjacent astroglia and microglia. Histological examination showed that by day 35 in vitro control cultures which received liposomes without tocopherol were nearly devoid of neurons and contained many glial and microglial cells. In contrast, tocopherol-treated cultures contained many viable-appearing neurons and did not exhibit an overgrowth of glia. Both glial fibrillary acidic protein (as measured by immunoassay) and lipid peroxidation (as estimated by malondialdehyde levels) were markedly reduced in the tocopherol-treated cultures. We speculate that the vitamin exerts its protective effect on injured nervous tissues by scavenging free radicals, stabilizing cellular membranes, and quenching the cascade of biochemical events that follows necrosis in brain. This work suggests that the signal(s) to initiate gliosis are mediated, at least indirectly, by free radical formation.