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Biomedical subjects

A Sevanian

Publications and source records attributed to A Sevanian.

At least 91 records · Page 5Linked to original sources
PubMed1991

Histochemical localization of superoxide production in experimental autoimmune uveitis.

Although the presence and role of oxygen reactive species in uveal inflammation is the subject of intense investigation, there is little direct evidence that oxygen metabolites are present at the site of inflammation. We used the nitroblue tetrazolium test for superoxide to determine production of this oxygen reactive species in experimental autoimmune uveitis (EAU). The choroidal tissues of animals with this disease contained intracellular, blue-staining granules. Most of the positive staining cells appeared to be polymorphonuclear leukocytes. This localization of superoxide in EAU is further evidence of the generation of oxygen reactive species in uveal inflammation.

Animals↗
PubMed1991

Uptake, metabolism, and cytotoxicity of isomeric cholesterol-5,6-epoxides in rabbit aortic endothelial cells.

The isomeric cholesterol-5,6-epoxides represent two common cholesterol autoxidation products and along with their principal metabolic product, 3 beta,5 alpha,6 beta-cholestane triol, are purportedly angiotoxic. The uptake and cytotoxic action of these compounds was examined in cultured rabbit aortic endothelial cells emphasizing mechanisms of uptake and metabolic fate. The isomeric cholesterol epoxides are incorporated with equal facility and in a dose-dependent manner. The pattern of uptake, which is markedly influenced by media serum concentration and by temperature, suggests that these compounds are partly incorporated through association with serum lipoproteins. After incorporation, both epoxide isomers are rapidly converted to cholestane triol which readily exits the cells. Cholestane triol is further metabolized to an ester-type product representing up to 10% of the added cholesterol epoxides by 24 h of incubation. The order of cytotoxic potency of these cholesterol oxides is: cholestane triol greater than cholesterol-beta-epoxide greater than cholesterol-alpha-epoxide, with LD50 concentrations ranging from 23 to greater than 150 microM in confluent cells. Cholestane triol and cholesterol-beta-epoxide are twice as cytotoxic to subconfluent cells as compared to confluent cells, whereas cholesterol-alpha-epoxide is essentially equitoxic to confluent and subconfluent cells. Cholesterol epoxide cytotoxicity is significantly reduced by treatments in the absence of serum in accord with substantial reduction in uptake when incubations are performed in serum-free media. Our findings show that these cytotoxic cholesterol oxides are incorporated by endothelial cells through a combination of receptor-mediated and nonspecific or passive mechanisms; however, the efficacy of uptake and resulting toxicity is substantially influenced by serum lipoproteins.

Animals↗
PubMed1990

Ferrylmyoglobin-catalyzed linoleic acid peroxidation.

The addition of linoleic acid (18:2) to a solution containing oxymyoglobin (MbIIO2), metmyoglobin (MbIII), or metmyoglobin-azide complex (MbIII-N3-) resulted in the formation of a common complex with identical absorption spectral properties. The addition of H2O2 to a MbIII/linoleic acid mixture revealed a spectral profile with lambda max at 530 nm and different from that observed in the reaction of MbIII with H2O2 and identical to that of ferrylmyoglobin. This was accompanied by a progressive decrease in the absorption in the visible region, indicating heme degradation during the lipid peroxidation process. The oxidation products of linoleic acid during the MbIII/18:2/H2O2 interaction were assessed by HPLC under anaerobic and aerobic conditions. In both instances, the chromatograms at lambda 234 nm revealed the formation of a main peak with a retention time of 11.1 min, which cochromatographed with a standard of 9-hydroperoxide of linoleic acid. The latter adduct was not degraded by the oxoferryl complex of myoglobin. The conclusions originating from this research are two-fold. On the one hand, the identical spectral properties exhibited by the product originating from the reaction of either MbIIO2 or MbIII with linoleic acid bridge the apparent discrepancy between the different reactivities of MbIIO2 and MbIII toward H2O2 and their ability to promote lipid peroxidation. On the other hand, the pattern of oxidation products of linoleic acid observed during the MbIII/H2O2 interaction, i.e., the formation of a 9-hydroperoxide adduct as a major product, points to a specific binding character and a regioselectivity of the oxoferryl complex in the oxidation of unsaturated fatty acids or a catalytic preference for decomposition of the various isomeric hydroperoxides over that of the 9-hydroperoxide.

Animals↗
PubMed1990

Inhibition of arachidonic acid release by nordihydroguaiaretic acid and its antioxidant action in rat alveolar macrophages and Chinese hamster lung fibroblasts.

The ability of nordihydroguaiaretic acid (NDGA) to inhibit arachidonic acid (AA) release from rat alveolar macrophages treated with t-butyl hydroperoxide (tBOOH) or from Chinese hamster lung fibroblasts (V79 cells) treated with linoleic acid hydroperoxide (LOOH) was examined. Treatment of alveolar macrophages with 100 microM tBOOH significantly increased arachidonic acid release and its conversion to metabolites. Pretreatment of macrophages with NDGA (greater than or equal to 2.5 microM) inhibited the release of AA and its subsequent metabolism following addition of tBOOH. Treatment of V79 cells with 1 microM LOOH stimulated the release of AA. Pretreatment with either 1 or 10 microM NDGA prior to the addition of LOOH inhibited the release of AA. A23187 (2 microM)-stimulated release of AA from V79 cells was less sensitive to NDGA inhibition. Pretreatment with 10 microM NDGA, but not with 1 microM NDGA, inhibited A23187-stimulated release of AA. PLA2-dependent hydrolysis of micelle preparations of disaturated phosphatidylcholine was not inhibited by NDGA. Previous studies have suggested that the addition of peroxides alters cells by inducing lipid peroxidation so that the action of phospholipases upon their membranes is enhanced. The results suggest that NDGA, a lipid-soluble antioxidant which traps free radicals, indirectly blocked the action of phospholipases upon cell membranes by inhibiting lipid peroxidation.

Animals↗
PubMed1990

Microsomal lipid peroxidation: the role of NADPH--cytochrome P450 reductase and cytochrome P450.

The role of NADPH--cytochrome P450 reductase and cytochrome P450 in NADPH- and ADP--Fe3(+)-dependent lipid peroxidation was investigated by using the purified enzymes and liposomes prepared from either total rat-liver phospholipids or a mixture of bovine phosphatidyl choline and phosphatidyl ethanolamine (PC/PE liposomes). The results suggest that NADPH- and ADP--Fe3(+)-dependent lipid peroxidation involves both NADPH--cytochrome P450 reductase and cytochrome P450. Just as in the case of cytochrome P450-linked monooxygenations, the role of these enzymes in lipid peroxidation may be to provide two electrons for O2 reduction. The first electron is used for reduction of ADP--Fe3+ and subsequent addition of O2 to the perferryl radical (ADP--Fe3(+)-O2-), which then extracts an H atom from a polyunsaturated lipid (LH) giving rise to a free radical (LH.) that reacts with O2 yielding a peroxide free radical (LOO.). The second electron is then used to reduce LOO. to the lipid hydroperoxide (LOOH). In the latter capacity, reduced cytochrome P450 can be replaced by EDTA--Fe2+ or by the superoxide radical as generated through redox cycling of a quinone such as menadione.

Adenosine Diphosphate↗
PubMed1990

Minimally modified low density lipoprotein stimulates monocyte endothelial interactions.

The effect of minimally modified LDL (MM-LDL) on the ability of large vessel endothelial cells (EC) to interact with monocytes and neutrophils was examined. These LDL preparations, obtained by storage or by mild iron oxidation, were indistinguishable from native LDL to the LDL receptor and were not recognized by the scavenger receptor. Treatment of EC with as little as 0.12 micrograms/ml MM-LDL caused a significant increase in the production of chemotactic factor for monocytes (sevenfold) and increased monocyte binding (three- to fivefold). Monocyte binding was maximal after 4 h of EC exposure to MM-LDL, persisted for 48 h, and was inhibited by cycloheximide. In contrast, neutrophil binding was not increased after 1-24 h of exposure. Activity in the MM-LDL preparations was found primarily in the polar lipid fraction. MM-LDL was toxic for EC from one rabbit but not toxic for the cells from another rabbit or any human umbilical vein EC. The resistant cells became sensitive when incubated with lipoprotein in the presence of cycloheximide, whereas the sensitive strain became resistant when preincubated with sublethal concentrations of MM-LDL. We conclude that exposure of EC to sublethal levels of MM-LDL enhances monocyte endothelial interactions and induces resistance to the toxic effects of MM-LDL.

Animals↗
PubMed1989

DPH lifetime distributions in vesicles containing phospholipid hydroperoxides.

Multifrequency phase fluorometry was used to determine the lifetime distributions of 1,6 diphenyl-1,3,5-hexatriene in 1-palmitoyl-2 linoleoyl phosphatidylcholine small unilamellar vesicles containing 2% incorporation of phospholipid hydroperoxides. A biexponential decay was observed in both vesicle preparations over a temperature range of 5 to 35 degrees C. Vesicles containing phospholipid hydroperoxides showed an overall longer lifetime as well as a greater distribution in width. These findings suggest that phospholipid hydroperoxides create structural heterogeneity in membrane structure.

Chromatography, High Pressure Liquid↗
PubMed1989

Incorporation of arachidonic, dihomogamma linolenic and eicosapentaenoic acids into cultured V79 cells.

The uptake and distribution of three common dietary polyunsaturated fatty acids was studied using Chinese hamster lung fibroblasts (V79 cells). Treatment of V79 cells with arachidonic (20:4), eicosapentaenoic (20:5) and dihomogammalinolenic (20.3) acids for 24 hr produced a marked uptake of 20:3 and 20:4, both of which were assimilated to a considerably greater degree than 20:5. All polyunsaturated fatty acids were incorporated primarily into phospholipids; however, there were considerable differences in their distribution into individual phospholipid species. Although 20:4 was incorporated primarily into phosphatidylcholine, 20:3 entered largely into phosphatidylethanolamine and phosphatidylglycerol, and 20:5 was distributed about equally between phosphatidylcholine, phosphatidylethanolamine and phosphatidylinositol. A marked conversion of 20:3 to 20:4 was found after 24 hr and, in several phospholipids, there was as much derived-radiolabeled 20:4 as there was radiolabeled 20:3. There was little evidence of 20:4 and 20:5 metabolism. V79 cells undergo substantial changes in phospholipid fatty acid composition following supplementation with these polyunsaturated fatty acids; however, these fatty acids are assimilated to different degrees and their distribution among cellular phospholipids is distinct, suggesting incorporation via independent mechanisms.

8,11,14-Eicosatrienoic Acid↗
PubMed1989

Induction of cytotoxicity and mutagenesis is facilitated by fatty acid hydroperoxidase activity in Chinese hamster lung fibroblasts (V79 cells).

The metabolic activation of benzo[a]pyrene and 7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene was studied in V79 Chinese hamster fibroblasts after supplementations with arachidonic acid or treatments with linoleic acid hydroperoxide. The extent of metabolic activation was estimated using cytotoxicity and mutagenesis as endpoints. Pretreatment of cells with arachidonic acid for 24 h resulted in significant elevations in the content of this fatty acid in cell phospholipids and increased prostaglandin synthesis. Arachidonic acid and linoleic acid hydroperoxide facilitated 7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene cytotoxicity and mutagenesis, and to a lesser extent increased the cytotoxicity and mutagenicity of benzo[a]pyrene. No other compounds tested were mutagenic under these conditions, however, linoleic acid hydroperoxide markedly increased their cytotoxicity. Arachidonic acid-facilitated toxicity and mutagenesis was inhibited by indomethacin, whereas no inhibition was seen when linoleic acid hydroperoxide was used. Nordihyroquairaretic acid abolished the cytotoxicity and mutagenesis facilitated by arachidonic acid and linoleic acid hydroperoxide. Our findings demonstrate that induction of cytotoxicity and mutagenesis following treatment of V79 cells with carcinogens may be limited by low levels of arachidonic acid in these cells. A peroxidatic mechanism is proposed, with limited substrate specificity, for the metabolic activation of chemicals in V79 cells.

Animals↗
PubMed1988

Lipid peroxidation and phospholipase A2 activity in liposomes composed of unsaturated phospholipids: a structural basis for enzyme activation.

The effect of lipid peroxidation on membrane structure and phospholipase A2 activity was studied using liposomes composed of bovine liver phosphatidylcholine (PC) and phosphatidylethanolamine (PE). The phospholipids were mixed at set ratios and sonicated to yield small unilamellar vesicles. The liposome preparations were subjected to lipid peroxidation as induced by cumene hydroperoxide and hematin. Under these conditions, a sharp increase in lipid peroxidation was noted over a 30 min incubation period and was accompanied by loss of polyunsaturated fatty acids (PUFA). Liposomes enriched in PE were most extensively peroxidized with a preferred oxidation of this phospholipid. The extent of PC oxidation was also greater in liposomes containing the largest proportions of PE. Analysis of liposome anisotropy, via steady-state fluorescence polarization of diphenylhexatriene indicated that progressive increases in either PE content or the level of lipid peroxidation increased the apparent microviscosity of the vesicles. Moreover, lipid peroxidation increased anisotropy more effectively than variations in the ratios of PE vs. PC. Thus, peroxidation of 5-10% of the phospholipids produced the same anisotropy increase as a 20% increase in the ratio of PE vs. PC. Analysis of vesicle turbidity suggested that fusion was also more readily achieved through lipid peroxidation. When liposomes were incubated with 0.4 U/ml of snake venom phospholipase A2, a direct correlation was found between the degree of lipid peroxidation and the extent of phospholipid hydrolysis. The more unsaturated phospholipid, PE, was most extensively hydrolyzed following peroxidation. Increasing the proportion of PE also resulted in more extensive phospholipid hydrolysis. These findings indicate that lipid peroxidation produces a general increase in membrane viscosity which is associated with vesicle instability and enhanced phospholipase A2 attack. A structural basis for membrane phospholipase A2 activation as a consequence of lipid peroxidation is discussed in light of these findings.

Animals↗
PubMed1988

4-Hydroxynonenal: a specific indicator for canine neuronal-retinal ceroidosis.

Previous attempts to demonstrate abnormalities in lipid peroxidation in various forms of the neuronal ceroid-lipofuscinoses (NCL) have been unrewarding up to and including the peroxide level (peroxidase). In this experiment a survey was made in a canine model of NCL to study the relative concentration of 4-hydroxynonenal (HNE), a fragment derived from an acute oxidation product of unsaturated fatty acids. Peripheral blood cells and various tissues from an affected and a normal control dog were surveyed. HNE was assayed after reacting with O-(2,3,4,5,6-pentafluorobenzyl) hydroxylamine to form the 4-hydroxynonenal (O-pentafluorobenzyl) oxime. This reaction product was then separated by capillary gas liquid chromatography (g/c) and quantitated by flame ionization. The survey showed that neutrophils isolated from affected dogs and carriers contained abnormal amounts of HNE when compared with normal control animals. Two carriers had mean values of +3,289% above normal, and neutrophils from two affected animals were +4,873% above normal. In addition, an examination of the relative HNE levels in brain, retina, retinal pigment epithelium (RPE), and kidney of an affected dog compared with a control animal also showed abnormal levels of HNE, particularly in brain (+168%) and in the RPE (+135%), the two organs exhibiting the most severe pathologic damage unique to these disorders. These findings, although preliminary, clearly document a role for HNE in this canine form of human NCLs. The well-known cytotoxic properties of HNE and other alpha,beta unsaturated aldehydes suggest a primary role in the pathogenetic events of this disorder.

Aldehydes↗
PubMed1988

Co-oxidation of salicylate and cholesterol during the oxidation of metmyoglobin by H2O2.

The reaction between metmyoglobin and H2O2 proceeds with oxidation of the hemo-protein iron to a higher valence state and consumption of the peroxide. This reaction is further associated with (a) O2 evolution; (b) hydroxylation of the aromatic compound salicylate to yield a set of dihydroxybenzoic acid derivatives (analyzed by HPLC with electrochemical detection); (c) autoxidation of cholesterol with formation of 3 beta-hydroxy-5-alpha-cholest-6-ene-5-hydroperoxide; and (d) formation of electronically excited states detected by low-level chemiluminescence. The heterolytic scission of the O-O bond of hydroperoxides by metmyoglobin causes the formation of an oxidizing equivalent capable of promoting peroxidation of linoleate and arachidonate (as indicated by the parallel formation of thiobarbituric acid-reactive material and an enhancement of chemiluminescence intensity). The identity of the oxidizing equivalent(s) is discussed in terms of the formation of a relatively stable higher state of oxidation of heme Fe (FeIV-OH or FeV = O) as well as on possible intermediate species derived during the decomposition of H2O2 by metmyoglobin, such as HO.and 1O2. These species might be involved either simultaneously or sequentially in the peroxidation of fatty acids as well as in the tissue damage associated with the formation of H2O2 in ischemic-reperfusion states.

Cholesterol↗
PubMed1988

Mutagenic characterization of cholesterol epoxides in Chinese hamster V79 cells.

The uptake, metabolism and alkylating properties of the diastereomeric cholesterol epoxides were studied using Chinese hamster lung fibroblasts (V79 cells). Specific emphasis is given to the comparative cyto- and geno-toxic effects of cholesterol 5 beta,6 beta-epoxide (beta CE) and cholesterol 5 alpha,6 alpha-epoxide (alpha CE) and data are provided for the first time indicating that beta CE can induce more 6-thioguanine-resistant cells than alpha CE. Cholesterol 5 beta,6 beta-epoxide induced colonies of cells resistant to 6-thioguanine at 2-3-fold the frequencies observed with the alpha-isomer, but neither compound produced ouabain-resistant colonies. The cytotoxicity (LD50) of alpha CE was estimated to be 45-50 microM whereas beta CE displayed an LD50 of 25-29 microM. Inhibition of DNA synthesis (IC50) was observed over the same dose ranges as the LD50 for each epoxide isomer. The epoxides were assimilated by cells to an equal extent, however, beta CE was metabolized to cholestane 3 beta,5 alpha-6 beta-triol twice as rapidly as the alpha-isomer. Both epoxides reacted with 4-(4'-nitrobenzyl)-pyridine to a similar extent, and with identical nucleophilic selectivity at pH 7.4, but their alkylating activity was estimated on this basis to be two orders of magnitude less than methyl methanesulfonate. Binding experiments with the DNA or cultured V79 cells or with calf-thymus DNA indicated that interactions were noncovalent and DNA binding did not correlate with the potency of the epoxides to induce the 6-thioguanine-resistant phenotype. Our results could be interpreted as indicating that both cholesterol epoxide isomers are weak mutagens or that they might induce some epigenetic event repressing the hypoxanthine guanine-phosphoribosyltransferase gene. The similarity of the epoxides' alkylating activity and their DNA-binding properties are inconsistent with their different potencies in inducing the 6-thioguanine-resistant phenotype, suggesting that the mechanism leading to this phenotype is not necessarily the result of DNA alkylation.

Alkylating Agents↗
PubMed1988

Effect of cholesterol epoxides on the inhibition of intercellular communication and on mutation induction in Chinese hamster V79 cells.

Cholesterol, cholesterol-5 alpha, 6 alpha-epoxide, cholesterol-5 beta, 6 beta-epoxide and cholestane-3 beta,5 alpha,6 beta-triol were tested for their ability to induce mutations at the Na+/K+-ATPase loci of the Chinese hamster V79 cells. None of these compounds induced ouabain-resistant mutations compared to the background mutation frequency in the control cells. These compounds were further tested for their ability to inhibit intercellular communication, using the Chinese hamster V79 cell metabolic cooperation assay. The diastereomeric epoxides and cholestane-triol, but not cholesterol, were found to be inhibitors of intercellular communication in a manner similar to other known tumor promoters.

Animals↗
PubMed1988

Treatment of experimental lens-induced uveitis by dimethyl thiourea.

Dimethyl thiourea is a highly specific hydroxyl radical scavenger. The antiinflammatory potential of this hydroxyl radical scavenger was studied in Lewis rats sensitized with soluble lens protein. Intraperitoneal injection of dimethyl thiourea into the experimental animals at the onset of the disease resulted in marked reduction of intraocular inflammation, hemorrhage and edema, and eliminated the retinal and choroidal necrotizing vasculitis that was characteristic in untreated animals. These findings indicate that dimethyl thiourea may act as a potent antiphlogistic agent in the treatment of experimental lens-induced uveitis.

Animals↗
PubMed1988

Anti-inflammatory effect of glutathione peroxidase on experimental lens-induced uveitis.

An acute lens-induced necrotizing intraocular inflammation was produced in Lewis rats. Treatment of these animals by intraperitoneal injection of glutathione peroxidase resulted in marked decrease in uveal inflammation, retinal edema, hemorrhage and vasculitis. Intraperitoneal injection of glutathione peroxidase resulted in increased levels of this enzyme in ocular tissue. These findings suggest that glutathione peroxidase can modulate acute lens-induced ocular inflammation by serving as a potent anti-inflammatory agent.

Animals↗