Inhibition of in vitro lipid peroxidation by stable steroidic nitroxyl radicals

4',4'-dimethylspiro (5 alpha-cholestane-3,2'-oxazolidin)-3'-yloxy (IK-1) and 7 alpha,12 alpha-dihydroxy-4',-4'-dimethylspiro (5 beta-cholan-24-oic-3,2'-oxazolidin)-3'-yloxy acid (IK-2), two stable steroidic nitroxyl radicals, were newly synthesized and tested as possible inhibitors of lipid peroxidation, induced by Fenton's reagent in both rat liver microsomes and egg phosphatidylcholine liposomes. The inhibitory activity, evaluated through the formation of thiobarbituric acid reactive substances (TBARS) and the conjugated diene, was compared with that of alpha-tocopherol and 2,2,6,6-tetramethylpiperidine-1-yloxy (TEMPO). In each model system IK-1 and IK-2 exhibited an IC50 of 8 microM and reduced the formation of TBARS and conjugated diene, showing IK-1 a potency comparable to alpha-tocopherol and higher than TEMPO. Moreover IK-1 and, to a lesser extent IK-2, reduced the lipid peroxidation induced in the microsomes by the water-soluble azo-initiator 2,2'-Azobis (2-methylpropionamidine) dihydrochloride (AMPH), indicating the IK-1 and IK-2 ability as chain-breaking antioxidants. The hydroxylamine 4',4'-dimethylspiro (5 alpha-cholestane-3,2'-oxazolidin)-3'-hydroxide (IK-3), obtained by chemical reduction of IK-1, was completely inactive as an inhibitor of lipid peroxidation in heat pre-treated microsomes and in liposomes. However in microsomes it was active since it was oxidized to the corresponding nitroxyl radical IK-1.     

Inhibition of lipid peroxidation mediated by indolizines

Esters, ethers, carbonates and carbamates of 1-indolizinols and azaindolizinols exhibit a profound inhibition of lipid peroxidation in vitro. The antioxidants were prepared by cyclization of pyridines and diazines with diphenylcyclopropenone followed by introduction of the O-substituent.     

In vitro desaturation or elongation of monotrans isomers of linoleic acid by rat liver microsomes

The mammalian rasGAPs constitute a group of widely expressed proteins involved in the negative regulation of ras-mediated signaling. In this study we have isolated a novel human gene, RASAL (Ras GTPase-activating-like) and its murine ortholog, MRASAL which are most similar to the GAP1 family of rasGAP proteins, based upon the presence and organization of specific conserved domains. Full-length human and murine mRNA sequences are predicted to encode 804 and 799 amino acid polypeptides, respectively. Sequence analysis of these two proteins revealed the presence of two N-terminal calcium-dependent phospholipid binding C2 domains, a conserved GAP related domain (GRD) and a C-terminal pleckstrin homology (PH) domain. Northern blot and mRNA in situ hybridization analyses indicate that RASAL, in contrast to other mammalian rasGAP proteins, has a limited expression pattern; RASAL is highly expressed in the follicular cells of the thyroid and the adrenal medulla and expressed at lower levels in brain, spinal cord and trachea. Human RASAL has been localized by radiation hybrid mapping to chromosome 12q23-24.     

Inhibition of microsomal lipid peroxidation and monooxygenase activities by eugenol

Previously we reported that eugenol (4-allyl-2-methoxyphenol) inhibits non-enzymatic peroxidation in liver mitochondria (E. Nagababu and N. Lakshmaiah, 1992, Biochemical Pharmacology. 43, 2393-2400). In the present study, we examined the effect of eugenol on microsomal mixed function oxidase mediated peroxidation using Fe+3-ADP-NADPH, carbon tetrachloride (CCL4)-NADPH and cumene hydroperoxide (CumOOH) systems. In the presence of eugenol the formation of thiobarbituric acid reactive substances (TBARS) was decreased in all the systems (IC50 values: 14 microM for Fe+3-ADP-NADPH, 4.0 microM for CCl4-NADPH and 15 microM for CumOOH). Oxygen uptake was also inhibited to a similar extent with Fe+3-ADP-NADPH and CumOOH systems. A comparative evaluation with other antioxidants showed that in Fe+3-ADP-NADPH and CumOOH systems, the antioxidant efficacy was in the order: butylated hydroxytoluene (BHT) > eugenol > alpha-tocopherol, while in CCl4-NADPH system the order was alpha-tocopherol > BHT > eugenol. Time course of inhibition by eugenol indicated interference in initiation as well as propagation of peroxidation. Eugenol did not inhibit cytochrome P-450 reductase activity but it inhibited P-450 - linked monooxygenase activities such as aminopyrine-N-demethylase, N-nitrosodimethylamine demethylase, benzo(a)pyrene hydroxylase and ethoxyresorufin-O-deethylase to different extents. However, CumOOH supported monooxygenases (aminopyrine-N-demethylase and benzo(a)pyrene hydroxylase) required much higher concentrations of eugenol for inhibition. The concentration of eugenol required to inhibit monooxygenase activities was more than that required to inhibit peroxidation in all the systems. Eugenol elicited type 1 changes in the spectrum of microsomal cytochrome P-450. These results suggest that the inhibitory effect of eugenol on lipid peroxidation is predominantly due to its free radical quenching ability. Eugenol significantly protected against the degradation of cytochrome P-450 during lipid peroxidation with all the systems tested. These findings suggest that eugenol has the potential to be used as a therapeutic antioxidant. Further evaluation may throw more light on this aspect.     

Inhibition of embryonic retinoic acid synthesis by aldehydes of lipid peroxidation and prevention of inhibition by reduced glutathione and glutathione S-transferases

Inhibition of conceptal biosynthesis of all-trans-retinoic acid (t-RA) by aldehydes generated from lipid peroxidation was investigated. Oxidative conversion of all-trans-retinal (t-RAL, 18 microM) to t-RA catalyzed by rat conceptal cytosol (RCC) was sensitive to inhibition by trans-2-nonenal (tNE), nonyl aldehyde (NA), 4-hydroxy-2-nonenal (4HNE), and hexanal. With an initial molar ratio of aldehyde/t-RAL of 2:1, tNE, NA, and 4HNE caused 70, 65, and 40% reductions of t-RA synthesis, respectively. Hexanal reduced generation of t-RA by approximately 50% as the ratio of aldehyde/t-RAL was raised to 20:1. tNE significantly increased the Km of the reaction and kinetic analyses indicated a mixed competitive/noncompetitive inhibition. By contrast, analogous reactions catalyzed by adult rat hepatic cytosol (ARHC) were highly resistant to inhibition by the same aldehydes. Significant inhibition (> 40% reduction of t-RA generation) by 4HNE, NA, and tNE were achieved at high molar ratios of aldehyde/t-RAL (> 175:1). Hexanal did not inhibit the reaction significantly even at very high ratios of aldehyde/t-RAL (> 2,000:1). Interestingly, when reduced glutathione (GSH, 10 mM) alone or GSH plus glutathione S-transferase (GST) were added to RCC-catalyzed reactions, additions of tNE or 4HNE showed either no significant inhibition or a partial lack of inhibition. Results suggested that GSH-dependent conjugation with 4HNE proceeded slowly compared to conjugation with tNE. To test the hypothesis that GST-catalyzed GSH conjugation can effectively prevent inhibition of t-RA synthesis by aldehydic products of lipid peroxidation, triethyltin bromide (TEB, a potent inhibitor of GST, 20 microM) was added to ARHC-catalyzed reactions when hexanal or tNE were present in the incubations. Eighty and 60% of hexanal and tNE inhibition, respectively, were observed. This was apparently due to TEB blockage of GST-catalyzed GSH conjugation reactions and thus strongly supported the stated hypothesis.     

Inhibition of fos-jun-DNA complex formation by dihydroguaiaretic acid and in vitro cytotoxic effects on cancer cells

The main cause of acquired inguinal hernia is weakness of Fruchaud's deep muscolofascial floor, following metabolically-determined collagen disorders. A technique for the anterior reinforcement of this structure with polypropylene mesh is described here. Following intermuscular decollement, the mesh is placed in direct contact with the surface formed by the transversalis fascia and the transversus abdominis muscle and stretched as extensively as possible. Because the posterior aspect of the inguinal canal is the true barrier to abdominal pressure, the author believe that its direct reinforcement, without interposition of the internal oblique muscle, constitutes the most correct anatomo-surgical approach to hernia repair. This is the case for both indirect hernias, in which the internal ring is reconstructed at a deeper level, and for direct hernias, in which the "tent effect" of the prosthesis is prevented. Ninety-two primary inguinal hernias (56 indirect, 29 direct and 7 direct and indirect) in 87 patients were repaired with this technique. Seventy-nine patients were followed up from 2 to 24 months. Early complications included: 7 ecchymosis, 3 seromas, 2 subcutaneous infections, 3 testicular swellings. Incision and testicular pain for longer than 6 months occurred in 2 cases. No prosthetic infections or recurrences have been detected up to the present.     

Inhibition of cholesteatoma migration in vitro with all-trans retinoic acid

Retinoids have recently become of interest to clinicians because of their ability to inhibit migration and proliferation of premalignant squamous cells while enhancing growth and proliferation of normal cells. An in vitro investigation was undertaken to determine whether retinoic acid exhibits similar inhibitory effects on cholesteatoma cells. Cholesteatoma specimens were obtained intraoperatively from 10 patients undergoing mastoidectomy or revision mastoidectomy for chronic ear disease. Cholesteatoma explant growth and en mass migration were observed daily, and topographic maps were constructed at various time intervals to quantity rate and direction of explant migration in the presence or absence of all-trans retinoic acid. Before all-trans retinoic acid administration, explants migrated very rapidly (1 to 2 mm/day). A maximum threefold inhibition of migratory rate occurred, with explants exposed to 0.1 micromol/L retinoic acid when compared with controls. A sixfold maximum inhibition was observed at higher retinoic acid concentrations (5 micromol/L). On removal of all-trans retinoic acid, twofold and fourfold increases in migratory rates were observed. The direction of explant migration varied significantly for long periods of time and appeared not to be affected by retinoic acid. This investigation suggests that all-trans retinoic acid has an inhibitory effect on cholesteatoma cell migration. Retinoids may have a role in controlling cholesteatoma disease in the future.     

Modification of red cell membrane lipids by hypochlorous acid and haemolysis by preformed lipid chlorohydrins

Hypochlorous acid (HOCl), a strong oxidant generated by the myeloperoxidase system of neutrophils and monocytes, has been implicated in inflammatory tissue damage by these cells. Reaction of HOCl with the double bonds of unsaturated lipids produces alpha, beta-chlorohydrin isomers. We have exposed red cell membranes to HOCl and used thin layer chromatography (TLC) of the extracted lipids and enzyme-linked immunosorbent assay (ELISA), using an antichlorohydrin monoclonal antibody, to show that fatty acyl chlorohydrins are formed. The ELISA was approximately 25 fold more sensitive than TLC, and chlorohydrins were detected when membranes from 10(6) cells were treated with > or = 0.16 nmoles HOCl. Lipid chlorohydrins are more polar and bulky than their parent lipids and as such could affect membrane stability and function. To determine the effect of incorporation of lipid chlorohydrins into cell membranes, preformed fatty acid and cholesterol chlorohydrins were incubated with red cells. Lysis was measured as release of haemoglobin and incorporation of lipids was determined by 14C scintillation counting. Addition of HOCl-treated oleic acid to red cells resulted in rapid lysis of a fraction of the cells in a concentration dependent manner. HOCl-treated cholesterol also caused a small amount of cell lysis that was predominantly due to chlorohydrin 3, one of the three major cholesterol chlorohydrin products. Chlorohydrin 3, which has a decreased planarity and polarity, was also primarily responsible for altering the critical micelle concentration of HOCl-treated cholesterol-containing liposomes.     

Inhibition of hemopoiesis in vitro by neuroblastoma-derived gangliosides

Hemopoiesis is disturbed in bone marrow-involving cancers like leukemia and neuroblastoma. Shedding of gangliosides by tumor cells may contribute to this tumor-induced bone marrow suppression. We studied in vitro the inhibitory effects of murine neuroblastoma cells (Neuro-2a and C1300) and their gangliosides on hemopoiesis using normal murine hemopoietic progenitor colony-forming assays. Transwell cultured neuroblastoma cells showed a dose-dependent inhibition on hemopoiesis, indicating that a soluble factor was responsible for this effect. Furthermore, the supernatant of Neuro-2a cultured cells inhibited hemopoietic proliferation and differentiation. To determine whether the inhibitory effect was indeed due to shed gangliosides and not, for instance, caused by cytokines, the effect of DL-threo-1 -phenyl-2-decanoylamino-3-morpholino-1-propanol (DL-PDMP) on Neuro-2a cells was studied. DL-PDMP is a potent inhibitor of glucosylceramide synthase, resulting in inhibition of the synthesis and shedding of gangliosides. The initially observed inhibitory effect of supernatant of Neuro-2a cells was abrogated by culturing these cells for 3 days in the presence of 10 microM DL-PDMP. Moreover, gangliosides isolated from Neuro-2a cell membranes inhibited hemopoietic growth. To determine whether the described phenomena in vitro are a reflection of bone marrow suppression occurring in vivo, gangliosides isolated from plasma of neuroblastoma patients were tested for their effects on human hemopoietic progenitor colony-forming assays. These human neuroblastoma-derived gangliosides inhibited normal erythropoiesis (colony-forming unit-erythroid/burst-forming unit-erythroid) and myelopoiesis (colony-forming unit-granulocyte/macrophage) to a higher extent compared with gangliosides isolated from control plasma. Altogether these results suggest that gangliosides shed by neuroblastoma cells inhibit hemopoiesis and may contribute to the observed bone marrow depression in neuroblastoma patients.     

Changes in lipid peroxide level in retinal pigment epithelial cells in vitro upon addition of linoleic acids or linoleic acid hydroperoxides under varying concentrations of oxygen

BACKGROUND: Plasma prolactin response to fenfluramine, a serotonergic agent, is typically blunted in moderately to severely depressed adults when compared to healthy controls. It is not clear, however, whether this dysregulation represents an acute change during symptomatic depression or a chronic disturbance. METHODS: In the current study, the prolactin responses to D,L-fenfluramine (weight-adjusted oral dose) of 29 adults who had a history of at least one major depressive episode (DSM-III-R criteria), but not during the past year, were compared to the prolactin responses of 58 age-, sex-, and socioeconomic status-matched adults without a lifetime history of major depression. RESULTS: Individuals with a positive history of major depression had significantly lower peak prolactin responses than controls. This finding was not attributable to weight, fenfluramine bioavailability, or baseline prolactin levels. CONCLUSIONS: This is the first investigation to compare men and women with a history of depression but not depressed at the time of the fenfluramine challenge to a similar group of healthy controls. The results are consistent with the hypothesis that central serotonergic activity is persistently disturbed in adults who experience depressive episodes.     

Inhibition by dexamethasone of human neutrophil apoptosis in vitro

The efficacy of collagen-sponge to reduce postoperative scar formation was investigated in 65 Japanese white rabbits that received laminectomy in the 7th and 8th thoracic vertebra. The defect after laminectomy was filled by collagen-sponge in 25 rabbits, by free fat in 20 rabbits, and by nothing in 20 rabbits as controls. The animals were sacrificed 2, 4, 8 and 12 weeks and additional 5 rabbits of which defects were filled with collagen-sponge were sacrificed after 24 weeks. All the defects were examined histologically. At 4 weeks after laminectomy, the defects filled by collagen-sponge showed that fibrous tissue had invaded into the sponge, but there was no remarkable adhesion to the dura mater. At 8 weeks, the defect with collagen sponge showed foaming cells, and no thickening of the dura mater was observed. At 12 weeks, the grouping of foaming cells was partially replaced by fat cells. At 24 weeks, most of the foaming cells were replaced by fat cells, and the defect was then similar to that filled by free fat at 12 weeks. In contrast, the defect with no interposed membrane was already filled with fibrous tissue at 4 weeks, and adhesion to the dura mater was observed. Although the free-fat graft at 12 weeks postoperatively showed no remarkable adhesion around the dura mater, infiltration of fat tissue into the spinal canal was observed in 2 of 5 rabbits. These results indicated that collagen-sponge can be utilized as a new biomaterial to effectively prevent scar formation after laminectomy.     

Inhibition of pancreatic lipase by phenolic acids--examination in vitro

The influence of addition 2,4,6,8, and 10 microM of benzoic and cinnamic acids and selected phenolic acids (salicylic, p-hydroxybenzoic, gentisic, protocatechuic, vanillic, syringic, o-coumaric, p-coumaric, caffeic, ferulic, sinapic) on the activity of pancreatic lipase was examined in vitro. The strongest inhibition activities were observed with caffeic, ferulic and benzoic acid, while sinapic and gentisic acids produced the lowest inhibition.     

Inhibition of cytokine production and arachidonic acid metabolism by eucalyptol (1.8-cineole) in human blood monocytes in vitro

The authors implanted to a group of 10 patients incontinent after prostate surgery (on account of BPH and adenocarcinoma) an artificial AMS 800 sphincter. After a mean follow-up period of 29 months they evaluate based on a questionnaire the therapeutic effect and its influence on the patients quality of life as well as the adequacy of the approval procedure of indication on the part of the insurance company as it influences the quality of life. The effect of treatment and influence on quality of life is evaluated without exception very highly while the approval process is evaluated negatively. The authors draw attention to the risk of suicide in mentally otherwise sound subjects due to unsatisfactory solution of urinary incontinence. Correctly indicated treatment by an artificial sphincter can achieve very satisfactory results. The approval procedure must combine medical and rational aspects, it must be however revised, incl. the economic aspects of the system of health services.     

Inhibition of 2,2'-azobis(2,4-dimethylvaleronitrile)-induced lipid peroxidation by sesaminols

We found that sesaminols, a mixture of sesaminol and its stereoisomers, are potent inhibitors of the oxidation of low-density lipoprotein induced by 2,2'-azobis(2,4-dimethylvaleronitrile). Although sesaminols strongly inhibit lipid peroxidation related to their ability to scavenge free radicals, their antioxidant effects have not been investigated. To confirm the involvement of the phenolic moiety in the sesaminol structure in antioxidant activity, sesaminols were reacted with 2,2'-azobis(2,4-dimethylvaleronitrile). The reaction products were isolated by high-performance liquid chromatography and found to have a 1-cyano-1,3-dimethyl-butyl-peroxyl group in their structures. These chemical structures suggest that the sesaminols reacted with the alkylperoxyl radicals to form four major reaction products that are stereoisomers of each other, although the stereochemistry of each isomer has not yet been confirmed. Further instrumental analyses of the reaction products may increase our understanding of the antioxidant activity of sesaminols.     

Conjugated linoleic acid reduces arachidonic acid content and PGE2 synthesis in murine keratinocytes

Using a T-maze, the influence of transient global cerebral ischemia on working memory in gerbils was investigated. Furthermore, it was examined whether a correlation exists between impairment in choice accuracy in the T-maze and neuron loss in the hippocampus. In two experiments, male Mongolian gerbils were tested in a previously learned delayed alternation T-maze task 1 week after a 4 min occlusion of the common carotid arteries. In both experiments memory was significantly impaired and in the second experiment, where the design allowed a separation between working and reference memory deficits, a selective impairment in working memory was seen. The results suggest that ischemia-induced disruption of delayed alternation in the T-maze in gerbils is a model which is relevant to the clinical manifestations of vascular dementia.