Melatonin produced metabolic changes in testis and did not prevent indomethacin-induced testicular lipid peroxidation in adult rat

Melatonin was orally given to rats at the dosage of 0.75 mg/rat/day for 7 days and challenged on the day 7 with a single toxic dose of indomethacin (20 mg/kg, intramuscularly) to test either protection afforded by melatonin against indomethacin-induced oxidative tissue damage or effects of repeated administration of this hormone on some testicular metabolic parameters. The results showed increased lipid peroxidation, as evidenced by the formation of thiobarbituric acid reactive substances, accompanied by non-significantly decreased glutathione content in the testis of rats treated with indomethacin. However, prior administration of melatonin failed to prevent indomethacin-induced testicular lipid peroxidation. No change in the production of lipid peroxidation and glutathione was observed as well after treatment with melatonin alone. Meanwhile, exogenous melatonin inhibited testicular levels of total lipid, total protein, and activity of aspartate aminotransferase, alanine aminotransferase, and alkaline phosphatase. All treated rats exhibited unchanged activity of both acid phosphatase and lactate dehydrogenase. The results indicated inability of oral administration of melatonin to prevent some of the oxidative damaging effects of indomethacin in the rat testis. In addition, the study provided an evidence that melatonin has an inhibitory action on the testicular metabolism in adult rats and thereby suggests a possible role of this hormone in modulating functions of rat testis.     

Degradation of tryptamine in pig brain: identification of a new condensation product

Incubation of tryptamine with pig brain homogenate led to the formation of a product which is not identical with other known tryptamine metabolites. The same results were observed with rat brain tissue and bovine brain tissue. The compound has been isolated and identified by NMR spectroscopy, fast atom bombardment mass spectroscopy, and by chemical synthesis as a thiazolidine derivative, (4R)-2-(3-indolylmethyl)-1,3-thiazolidine-4-carboxylic acid. It is formed by a condensation reaction of indole-3-acetaldehyde generated enzymatically from tryptamine and of free L-cysteine present in the tissue. The compound inhibited monoamine oxidase (preferentially type A) and the neuronal gamma-aminobutyric acid uptake.     

Comparison of potential protective effects of melatonin, indole-3-propionic acid, and propylthiouracil against lipid peroxidation caused by potassium bromate in the thyroid gland

Potassium bromate (KBrO3) is a prooxidant and carcinogen, inducing thyroid tumors. Melatonin and indole-3-propionic acid (IPA) are effective antioxidants. Some antioxidative effects of propylthiouracil (PTU)--a thyrostatic drug--have been found. The aim of the study was to compare protective effects of melatonin, IPA, and PTU against lipid peroxidation in the thyroids, collected from rats treated with KBrO3, and in homogenates of porcine thyroids, incubated in the presence of KBrO3. Wistar rats were administered KBrO3 (110 mg/kg b.w., i.p., on the 10th day of the experiment) and/or melatonin, or IPA (0.0645 mmol/kg b.w., i.p., twice daily, for 10 days), or PTU (0.025% solution in drinking water, for 10 days). Homogenates of porcine thyroids were incubated for 30 min in the presence of KBrO3 (5 mM) plus one of the antioxidants: melatonin (0.01, 0.1, 0.5, 1.0, 5.0, 7.5 mM), or IPA (0.01, 0.1, 0.5, 1.0, 5.0, 7.5, 10.0 mM), or PTU (0.01, 0.1, 0.5, 1.0, 5.0, 7.5, 10.0 mM). The level of lipid peroxidation products (MDA + 4-HDA) was measured spectrophotometrically in thyroid homogenates. In vivo pretreatment with either melatonin or with IPA or with PTU decreased lipid peroxidation caused by KBrO3--injections in rat thyroid gland. Under in vitro conditions, PTU (5.0, 7.5, and 10.0 mM), but neither melatonin nor IPA, reduced KBrO3-related lipid peroxidation in the homogenates of porcine thyroids. In conclusion, melatonin and IPA may be of great value as protective agents under conditions of exposure to KBrO3.     

Partial suppressive effect of melatonin on indomethacin-induced renal injury in rat

Intramuscular injection of a single high dose of indomethacin (20 mg/kg) in fasted rats produced renal injury. The results showed increases in the level of lipid peroxidation and cholesterol, and activity of acid phosphatase and alkaline phosphatase in the kidney. Also, the renal contents of both reduced glutathione and activity of total adenosine triphosphatase were decreased by the toxicant. In serum, indomethacin increased activity of lactate dehydrogenase and acid phosphatase, and levels of creatinine and inorganic phosphorus. Paradoxically, administration of melatonin (0.75 mg/rat/day) alone for 7 days decreased significantly the activity of lipid peroxidation and acid phosphatase, and increased, but not significantly, the level of reduced glutathione in the kidney. Also, serum level of creatinine tended to decrease, but not significantly. Pretreatment with melatonin prevented the increase by subsequently administered indomethacin in the renal activity of lipid peroxidation and acid phosphatase. However, this pretreatment regimen partially suppressed the adverse changes in the remaining analyzed cytotoxic parameters induced by indomethacin in both serum and kidney. These results indicate that oral administration of melatonin at a low dose level exerted moderate antioxidant action, thereby it protected against some of the renal detrimental effects produced by indomethacin.     

Relative efficacies of indole antioxidants in reducing autoxidation and iron-induced lipid peroxidation in hamster testes

Increased iron stores are associated with free radical generation and carcinogenesis. Lipid peroxidation is involved in DNA damage, thus indirectly participating in the early steps of tumor initiation. Melatonin and structurally related indoles are effective in protecting against oxidative stress. The aim of the study was to compare the relative efficacies of melatonin, N-acetylserotonin (NAS), indole-3-propionic acid (IPA), and 5-hydroxy-indole-3-acetic acid (5HIAA) in altering basal and iron-induced lipid peroxidation in homogenates of hamster testes. To determine the effect of the indoles on the autoxidation of lipids, homogenates were incubated in the presence of each agent in concentrations of 0.0, 0.01, 0.05, 0.1, 0.25, 0.5, 0.75, 1.0, 2.0, 2.5, or 5.0 mM. To study their effects on induced lipid peroxidation, homogenates were incubated with FeSO(4) (30 microM + H(2)O(2) (0.1 mM) + each of the indoles in the same concentrations as above. The degree of lipid peroxidation was expressed as concentrations of malondialdehyde + 4-hydroxyalkenals (MDA + 4-HDA) per mg protein. The indoles decreased both basal and iron-related lipid peroxidation in a concentration-dependent manner. Melatonin reduced basal MDA + 4-HDA levels when used at the concentrations of 0.25 mM or higher, and prevented iron-induced lipid peroxidation at concentrations of 1.0, 2.0, 2.5, or 5.0 mM. The lowest effective concentrations of NAS required to lower basal and iron-related lipid peroxidation were 0.05 mM and 0.25 mM, respectively. IPA, only when used in the highest concentrations of 2.5 mM or 5 mM inhibited basal lipid peroxidation levels and it was ineffective on the levels of MDA + 4-HDA due to iron damage. 5HIAA reduced basal lipid peroxidation when used at concentrations of 0.25 mM or higher, and it prevented iron-induced lipid peroxidation only at the highest applied concentration (5 mM). In conclusion, melatonin and related indoles at pharmacological concentrations protect against both the autoxidation of lipids as well as induced peroxidation of lipids in testes. In doing so, these agents would be expected to reduce testicular cancer that is initiated by products of lipid peroxidation.     

Melatonin preserves arachidonic and docosapentaenoic acids during ascorbate-Fe2+ peroxidation of rat testis microsomes and mitochondria

The pineal hormone melatonin (N-acetyl, 5-methoxytryptamine) was recently accepted to act as an antioxidant under both in vivo and in vitro conditions. In this study, we examined the possible preventive effect of melatonin on ascorbate-Fe(2+) lipid peroxidation of rat testis microsomes and mitochondria. Special attention was paid to the changes produced on the highly polyunsaturated fatty acids C20:4 n6 and C22:5 n6. The lipid peroxidation of testis microsomes or mitochondria produced a significant decrease of C20:4 n6 and C22:5 n6. The light emission (chemiluminescence) used as a marker of lipid peroxidation was similar in both kinds of organelles when the control and peroxidized groups were compared. Both long chain polyunsaturated fatty acids were protected when melatonin was incorporated either in microsomes or mitochondria. The melatonin concentration required to inhibit by 100% the lipid peroxidation process was 5.0 and 1.0mM in rat testis microsomes and mitochondria, respectively. IC 50 values calculated from the inhibition curve of melatonin on the chemiluminescence rates were higher in microsomes (4.98 mM) than in mitochondria (0.67 mM). The protective effect observed by melatonin in rat testis mitochondria was higher than that observed in microsomes which could be explained if we consider that the sum of C20:4 n6+C22:5 n6 in testis microsomes is two-fold greater than present in mitochondria.     

Metabolism of Tryptophan, Indole-3-acetic Acid, and Related Compounds in Parasitic Plants from the Genus Orobanche

Metabolic reactions involving the aliphatic side chain of tryptophan were studied in the holoparasitic dicotyledonous plants Orobanche gracilis Sm., O. lutea Baumg., and O. ramosa L. Unlike known autotrophic plants, the parasite metabolized l-tryptophan directly to indole-3-carboxaldehyde, which was further converted to indole-3-methanol and indole-3-carboxylic acid. Independently, these metabolites were also formed from d-tryptophan, tryptamine, indole-3-lactic acid, and indole-3-acetic acid. As in autotrophic plants, tryptophan and tryptamine were also converted, via indole-3-acetaldehyde, to indole-3-acetic acid, indole-3-ethanol, and its glucoside. The branch of tryptophan metabolism relevant to auxin biogenesis and catabolism is, therefore, not rudimentary in Orobanche but even more complex than in autotrophic higher plants.     

Hepatic oxidative stress and related defenses during treatment of mice with acetylsalicylic acid and other peroxisome proliferators

The peroxisome proliferators perfluorooctanoic acid (PFOA; 0.02% w/w), perfluorodecanoic acid (PFDA; 0.02%, w/w), nafenopin (0.125%, w/w), clofibrate (0.5%, w/w), and acetylsalicylic acid (ASA; 1%, w/w) were administered to male C57 BL/6 mice in their diet for two weeks. Parameters for Fe³⁺ ADP, NADPH or ascorbic acid-initiated lipid peroxidation in vitro were measured. Approximately a twofold increase in susceptibility to lipid peroxidation was obtained for all the peroxisome proliferators tested. Cotreatment of mice with the peroxisome proliferator ASA (1%, w/w) and a catalase inhibitor, 3-amino-1,2,4-triazole (AT; 0.4%, w/w) for 7 days resulted in little inhibition of peroxisome proliferation, an elevated level of H₂O₂ in vivo, and total inhibition of the increased susceptibility to lipid peroxidation in vitro. No increase in lipid peroxidation in vivo was observed. Certain antioxidant enzymes (DT-diaphorase, superoxide dismutase, glutathione transferase, glutathione peroxidase, and glutathione reductase) and components (ubiquinone and α-tocopherol) were also measured. The results showed that there was some induction of these antioxidant enzymes and components by ASA or aminotriazole, except for glutathione peroxidase and superoxide dismutase, which were inhibited. The possible involvement of oxidative stress in the carcinogenicity of peroxisome proliferators is discussed.     

Synthesis and evaluation of N-substituted indole-3-carboxamide derivatives as inhibitors of lipid peroxidation and superoxide anion formation

The in vitro antioxidant effects of novel N-substituted indole-3-carboxamides (I3CDs) 1-10 on rat liver microsomal NADPH-dependent lipid peroxidation (LP) levels and their free radicals scavenging properties were determined by the inhibition of superoxide anion formation (SOD). Among the synthesized compounds, 4, 5, 8 and 9 significantly inhibited SOD with an inhibition range at 84-100% at 10(-3) M concentration. The presence of halo substituents both ortho- and para- positions of these compounds resulted 100% inhibition of SOD. Comparison the activity results of halogenated and non-halogenated derivatives suggested that the halogenated compounds are more active than the non-halogenated compounds. On the other hand, the introduction of a para fluoro benzyl in the 1-position of indole (compounds 7, 8) has more impact on the SOD inhibition when the benzamide ring was mono halogenated. However, none of other compounds had a significant inhibitory effects on the level of lipid peroxidation.     

Melatonin directly scavenges free radicals generated in red blood cells and a cell-free system: chemiluminescence measurements and theoretical calculations

Melatonin, a pineal secretory product, has properties of both direct and indirect powerful antioxidant. The aim of the present study was to compare the radical-scavenging, structural and electronic properties of melatonin and tryptophan, precursor of melatonin. Using the alkoxyl- and peroxyl radical-generating systems [the organic peroxide-treated human erythrocytes and a cell-free system containing the azo-initiator 2,2'-azobis(2-amidinopropane)dihydrochloride], we evaluated the radical-scavenging effects of melatonin and tryptophan. Melatonin rather than tryptophan at concentrations of 100-2000 microM markedly inhibited membrane lipid peroxidation in human erythrocytes treated with organic hydroperoxide as well as radical-induced generation of luminol-dependent chemiluminescence. The apparent Stern-Volmer constants for inhibition of membrane lipid peroxidation by melatonin and tryptophan were estimated to be (0.23+/-0.05) x 10(4) M(-1) and (0.02+/-0.005) x 10(4) M(-1), respectively. The apparent Stern-Volmer constants for inhibition of azo-initiator-derived peroxyl radical generation by melatonin and tryptophan were determined to be (0.42+/-0.05) x 10(4) M(-1) and (0.04+/-0.01) x 10(4) M(-1), respectively. The structural and electronic properties of melatonin and its precursor, tryptophan, were determined theoretically by performing semi-empirical and ab initio calculations. The high radical-scavenging properties of melatonin may be explained by the high surface area value and high dipole moment value. From the thermodynamic standpoint, based on our calculations, N(1)-acetyl-N(2)-formyl-5-methoxykynuramine (AFMK), was the most stable end oxidative product of melatonin.     

Role of Pinoline and Melatonin in Stabilizing Hepatic Microsomal Membranes against Oxidative Stress

We investigated the influence of pinoline (0.01–1.5 mM) on microsomal membrane fluiditybefore and after rigidity was induced by oxidative stress. In addition, we tested the effect ofpinoline in the presence of 1 mM melatonin. The fluidity in rat hepatic microsomes wasmonitored using fluorescence spectroscopy and it was compared to the inhibition ofmalonaldehyde (MDA) plus 4-hydroxyalkenals (4-HDA) production as a reflection of lipid peroxidation.Below 0.6 mM, pinoline inhibited membrane rigidity in a manner parallel to its inhibitoryeffect on MDA + 4–HDA formation. At concentrations between 1–1.5 mM, pinoline wasless effective in stabilizing microsomal membranes than was predicted from its inhibition oflipid peroxidation. The addition of 1 mM melatonin enhanced the membrane-stabilizing activityof pinoline (0.01–0.6 mM). This cooperative effect was not observed for concentrations ofpinoline between 1–1.5 mM. When pinoline was tested without induced oxidative damage,1–1.5 mM pinoline maintained membrane fluidity at the same level as that recorded afterinduced lipid peroxidation. The results suggest that pinoline may be another pineal moleculethat prevents membrane rigidity mediated by lipid peroxidation and this ability is enhancedby melatonin.     

Endogenous and dietary indoles: a class of antioxidants and radical scavengers in the ABTS assay

Indoles are very common in the body and diet and participate in many biochemical processes. A total of twenty-nine indoles and analogs were examined for their properties as antioxidants and radical scavengers against 2,2'-Azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) ABTS*+ radical cation. With only a few exceptions, indoles reacted nonspecifically and quenched this radical at physiological pH affording ABTS. Indoleamines like tryptamine, serotonin and methoxytryptamine, neurohormones (melatonin), phytohormones (indoleacetic acid and indolepropionic acid), indoleamino acids like L-tryptophan and derivatives (N-acetyltryptophan, L-abrine, tryptophan ethyl ester), indolealcohols (tryptophol and indole-3-carbinol), short peptides containing tryptophan, and tetrahydro-beta-carboline (pyridoindole) alkaloids like the pineal gland compound pinoline, acted as radical scavengers and antioxidants in an ABTS assay-measuring total antioxidant activity. Their trolox equivalent antioxidant capacity (TEAC) values ranged from 0.66 to 3.9 mM, usually higher than that for Trolox and ascorbic acid (1 mM). The highest antioxidant values were determined for melatonin, 5-hydroxytryptophan, trp-trp and 5-methoxytryptamine. Active indole compounds were consumed during the reaction with ABTS*+ and some tetrahydropyrido indoles (e.g. harmaline and 1-methyl-1,2,3,4-tetrahydro-beta-carboline-3-carboxylic acid ethyl ester) afforded the corresponding fully aromatic beta-carbolines (pyridoindoles), that did not scavenge ABTS*+. Radical scavenger activity of indoles against ABTS*+ was higher at physiological pH than at low pH. These results point out to structural compounds with an indole moiety as a class of radical scavengers and antioxidants. This activity could be of biological significance given the physiological concentrations and body distribution of some indoles.     

Protection against oxidative damage in cold-stored rabbit kidneys by desferrioxamine and indomethacin

The storage of rabbit kidneys in hypertonic citrate solution at 0 degree C for 48-72 hr of cold ischemia resulted in oxidative damage to membranes as measured by the in vitro formation of two markers of lipid peroxidation (Schiff's base and thiobarbituric acid (TBA)-reactive material). This damage was further increased when the organs were autografted and reperfused for 60 min. The intravenous (iv) administration of desferrioxamine (a powerful iron-chelating agent) prior to the removal of the kidneys reduced the production of Schiff's bases and TBA-reactive material to low levels in the cortex of stored kidneys and decreased these measures of lipid peroxidation in the medulla by approximately 50%. Intravenous administration of indomethacin (a cyclooxygenase inhibitor) had no effect on the rate of lipid peroxidation in the renal cortex, but significantly reduced the formation of TBA-reactive material and Schiff's bases in the medulla of kidneys following storage for 72 hr. The existence of two separate pathways of lipid peroxidation (one iron-catalyzed and the other cyclooxygenase-catalyzed) in the medulla of stored kidneys was further confirmed when administration of desferrioxamine and indomethacin together resulted in significantly greater protection against lipid peroxidation than when these compounds were administered singly. The value of this combination of agents for protecting kidneys against the damage due to cold ischemia followed by reperfusion was further suggested by a trend toward improved long-term survival of the animals following replantation of the stored kidneys.     

Liposomal Drug with Carnosine and Lipoic Acid: Preparation,Antioxidant and Antiplatelet Properties

The conditions for producing phosphatidylcholine liposomes containing lipoic acid and carnosine together were determined. The obtained liposomes are 180–250-nm spherical particles with an efficiency of lipoic acid inclusion of 50–70% (for carnosine, 17–33%). Based on the model of the oxidation of phosphatidylcholine by hydrogen peroxide, an antioxidant effect of carnosine, lipoic acid or lipoic acid with carnosine together was demonstrated; it consisted in inhibition of lipid peroxidation process, which was manifested in a decrease in the formation of lipid peroxidation products that react with thiobarbituric acid. It was established that lipoic acid (5 mM) and carnosine (0.1–10 mM) in liposomes exhibit an antioxidant effect. At the same time, it was demonstrated that the content of the appropriate lipid peroxidation products in liposomes with antioxidants (lipoic acid + carnosine) was 15 times lower than in control liposomes (without antioxidants). The effect of the obtained liposomal drugs on the platelet aggregation induced by arachidonic acid was evaluated. It was found that the liposomal drug containing lipoic acid (1.5 mM) and carnosine (2.1 mM) inhibited platelet aggregation by 50–55% relative to the control (platelets and arachidonic acid), while liposomes without antioxidants and water-soluble forms of carnosine and lipoic acid had almost no effect on platelet aggregation caused by arachidonic acid.

     

In vivo regulation of intermediate reactions in the pathway of tryptophan biosynthesis in Neurospora crassa

The in vivo regulation of intermediate reactions in the pathway of tryptophan synthesis in Neurospora crassa was examined in a double mutant (tr-2, tr-3) which lacks the functions of the first and last enzymes in the pathway from chorismic acid to tryptophan. The double mutant can convert anthranilic acid to indole and indole-3-glycerol, and the production of these indolyl compounds by germinated conidia was used to estimate the activity of the intermediate enzymes in the pathway. Indole-synthesizing activity was maximal in germinated conidia obtained from cultures in which the levels of l-tryptophan were growth-limiting; the formation of this activity was markedly repressed when the levels of l-tryptophan exceeded those required for maximal growth. d-, 5-methyl-dl-, and 6-methyl-dl-tryptophan were less effective than l-tryptophan, and 4-methyl-dl-tryptophan, tryptamine, and indole-3-acetic acid were ineffective in repressing the formation of indole-synthesizing activity; anthranilic acid stimulated the formation of indole-synthesizing activity. Preformed indole-synthesizing activity was strongly and specifically inhibited by low levels of l-tryptophan; several related compounds were ineffective as inhibitors. These results suggest that, in addition to repression, an end product feedback inhibition mechanism is operative on an intermediate enzyme(s) in tryptophan biosynthesis. The relation of these results to other in vivo and in vitro studies and to general aspects of the regulation of tryptophan biosynthesis in N. crassa are discussed.     

Synthesis of N-substituted indole-2-carboxamides and investigation of their biochemical responses against free radicals

The antioxidant role of novel N-substituted indole-2-carboxamides (I2CDs) was investigated for their inhibitory effects on superoxide anion (O2-) and lipid peroxidation (LP). Among the synthesized I2CDs, 3, 4, 6, 8 and 9 significantly inhibited O2*- with an inhibition range at 70-98%. Examination of substituent effects on activity showed that both the ortho- and para- positions of the benzamide residue needs to be dichlorinated in order to get a maximum inhibitory effect on superoxide anion. In general, halogenated derivatives were found more active then the non-halogenated ones. However, none of the I2CDs had a significant inhibitory effects on the level of lipid peroxidation; only compounds 7 and 10 moderately decreased LP levels by over 50% at 10(-3) M concentrations.     

Melatonin reduces phenylhydrazine-induced oxidative damage to cellular membranes: evidence for the involvement of iron

Phenylhydrazine and iron overload result in augmented oxidative damage and an increased likelihood of cancer. Melatonin is a well known antioxidant and free radical scavenger. The aim of this study was to determine whether melatonin would protect against phenylhydrazine-induced oxidative damage to cellular membranes and to evaluate the possible role of iron in this process. Changes in lipid peroxidation and microsomal membrane fluidity were estimated after the treatment of rats with phenylhydrazine (15 mg/kg body weight, daily, 7 days) alone and melatonin or ascorbic acid (15 mg/kg body weight, two times daily, 8 days), or their combination. Additionally, lipid peroxidation was measured in liver homogenates from untreated and melatonin or ascorbic acid-treated rats in vivo and exposed to iron in vitro. Melatonin, but not ascorbic acid, reduced phenylhydrazine-induced lipid peroxidation in vivo in spleen (3.16+/-0.06 vs. 3.83+/-0.12 nmol/mg protein, P<0.05) and plasma (7. 73+/-0.52 vs. 9.96+/-0.71 nmol/ml, P<0.05) and attenuated the decrease in hepatic microsomal membrane fluidity (1/polarization, 3. 068+/-0.007 vs. 3.027+/-0.008, P<0.05). In vitro exposure to iron significantly enhanced the lipid peroxidation in liver homogenates from untreated (3.34+/-0.75 vs. 1.25+/-0.28, P<0.05) or ascorbic acid-treated rats (2.72+/-0.39 vs. 0.88+/-0.06, P<0.05) but not from melatonin-treated rats (1.49+/-0.55 vs. 0.68+/-0.20, NS). It is concluded that free radical mechanisms are involved in the toxicity of phenylhydrazine and that the antioxidant melatonin, but not ascorbic acid, reduces the toxic affects of phenylhydrazine in vivo and of iron in vitro in cell membranes. Therefore, melatonin co-treatment in conditions of iron overload may prove beneficial.     

Arachidonic acid-containing phosphatidylcholine inhibits lymphocyte proliferation and decreases interleukin-2 and interferon-gamma production from concanavalin A-stimulated rat lymphocytes

The proliferation of concanavalin A (Con A)-stimulated rat lymphocytes was markedly inhibited by phosphatidylcholine containing arachidonic and stearic acids (PC(A-S)), but not by phosphatidylcholine containing oleic and stearic acids or phosphatidylinositol containing arachidonic and stearic acids. The concentration of PC(A-S) which inhibited Con A-stimulated proliferation by 50% was 31 microM and near total inhibition was observed at 154 microM . Phosphatidylserine containing only oleic acid enhanced proliferation by 37% at a concentration of 31 microM , but phosphatidylethanolamine and phosphatidylcholine containing only oleic acid did not affect proliferation at this concentration. It is concluded that both the head group and the fatty acid composition contribute to the influence of phospholipids on lymphocyte proliferation. The effects of PC(A-S) on T-lymphocyte responses were investigated further. In parallel with the inhibition of proliferation PC(A-S) caused a concentration-dependent decrease in the production of the Th1-type cytokines interleukin (IL)-2 and interferon (IFN)-gamma; inhibition of cytokine production was >85% at the highest concentration of PC(A-S) used (154 microM ). Production of the Th2-type cytokines IL-4 and IL-10 was not affected. The possible role of prostaglandins in mediating the effects of PC(A-S) was examined by adding indomethacin into the medium and the participation of lipid peroxidation was examined by adding vitamin E and vitamin C. Indomethacin and vitamin E did not affect the inhibition caused by PC(A-S) but vitamin C caused a partial reversal. It is concluded that inhibition of T-lymphocyte proliferation by phospholipids involves both the head group and the fatty acyl chains, that this inhibition is not mediated by prostaglandins but may involve some form of oxidant stress and that some phospholipids (e.g., PC(A-S)) can markedly influence cytokine profiles.     

Inhibition of neuronal nitric oxide synthase activity by N1-acetyl-5-methoxykynuramine, a brain metabolite of melatonin

We assessed the effects of melatonin, N(1)-acetyl-N (2)-formyl-5-methoxykynuramine (AFMK) and N(1)-acetyl-5-methoxykynuramine (AMK) on neuronal nitric oxide synthase (nNOS) activity in vitro and in rat striatum in vivo. Melatonin and AMK (10(-11)-10(-3) m), but not AFMK, inhibited nNOS activity in vitro in a dose-response manner. The IC(50) value for AMK (70 microm) was significantly lower than for melatonin (>1 mm). A 20% nNOS inhibition was reached with either 10(-9) m melatonin or 10(-11) m AMK. AMK inhibits nNOS by a non-competitive mechanism through its binding to Ca(2+)-calmodulin (CaCaM). The inhibition of nNOS elicited by melatonin, but not by AMK, was blocked with 0.05 mm norharmane, an indoleamine-2,3-dioxygenase inhibitor. In vivo, the potency of AMK to inhibit nNOS activity was higher than that of melatonin, as a 25% reduction in rat striatal nNOS activity was found after the administration of either 10 mg/kg of AMK or 20 mg/kg of melatonin. Also, in vivo, the administration of norharmane blocked the inhibition of nNOS produced by melatonin administration, but not the inhibition produced by AMK. These data reveal that AMK rather than melatonin is the active metabolite against nNOS, which may be inhibited by physiological levels of AMK in the rat striatum.     

Biomarkers of oxidative stress study III. Effects of the nonsteroidal anti-inflammatory agents indomethacin and meclofenamic acid on measurements of oxidative products of lipids in CCl4 poisoning

Plasma and urinary levels of malondialdehyde-like products (MDA) and isoprostanes were identified as markers of in vivo lipid peroxidation in an animal model of CCl4 poisoning. We sought to determine the extent to which the formation of these oxidation products is influenced by inhibition of the cyclooxygenase enzymes which catalytically generate proinflammatory lipid peroxidation products known as prostaglandins and thromboxane. In the present studies, after induction of oxidant stress in rats with CCl4, lipid peroxidation products measured in plasma and urine demonstrate that isoprostanes and MDA can be partially inhibited by cyclooxygenase inhibitors, albeit to different extents. The lowering of isoprostane and MDA formation, however, may not to due primarily to the diminution of catalytic generation of isoprostanes or MDA by the cyclooxygenases but, rather, may be the result of the suppression of nonenzymatic lipid peroxidation. This is suggested since 8,12-iso-iPF2alpha-VI is also reduced by indomethacin, yet, unlike other isoprostanes and MDA, it is not generated catalytically by the cyclooxygenase. Thus, although the two cyclooxygenase inhibitors we tested have statistically significant effects on the measurements of both isoprostanes and MDA in this study, the results provide evidence that these lipid-degradation products primarily constitute markers of oxidative stress.