2[125I]iodomelatonin binding sites in spleens of guinea pigs.

2-[125I]Iodomelatonin was found to bind specifically to the membrane preparations of the spleens of guinea pigs with high affinity. The binding was rapid, stable, saturable and reversible. Scatchard analysis of the binding assays revealed an equilibrium dissociation constant (Kd) of 49.8 +/- 4.12 pmol/l and binding site density (Bmax) of 0.69 +/- 0.082 fmol/mg protein at mid-light (n = 10). There was no significant change in the Kd (41.8 +/- 3.16 pmol/l) or the Bmax (0.58 +/- 0.070 fmol/mg protein) at mid-dark (n = 10). Kinetic analysis showed a Kd of 23.13 +/- 4.81 pmol/l (mean +/- SE, n = 4), in agreement to that derived from the saturation studies. The 2-[125I]iodomelatonin binding sites have the following order of potency: 2-iodomelatonin greater than melatonin greater than 6-chloromelatonin much greater than N-acetylserotonin, 6-hydroxymelatonin greater than 5-methoxytryptamine, 5 methoxytryptophol greater than serotonin, 5-methoxyindole-3-acetic acid greater than 5-hydroxytryptophol, 3-acetylindole, 1-acetylindole-3-carboxyaldehyde, L-tryptophan greater than tryptamine, 5-hydroxyindole-3-acetic acid. Differential centrifugation studies showed that the binding sites are localized mainly in the nuclear fraction (65.5%), the rest are distributed in the microsomal fraction (17.4%), mitochondrial fraction (14.7%) and cytosolic fraction (0.3%). The demonstration of 2-[125I]iodomelatonin binding sites in the spleen suggests the presence of melatonin receptors and a direct mechanism of action of melatonin on the immune system.     

Examination of pineal indoles and 6-methoxy-2-benzoxazolinone for antioxidant and antimicrobial effects

Oxidative modification of low density lipoprotein (LDL) induced by free radicals is implicated in the development of atherosclerosis. The aim of the present study was to examine the ability of various pineal indoles in inhibiting LDL oxidation which is accompanied by an increase in mobility in agarose gel electrophoresis and by an augmented generation of thiobarbituric acid-reactive substance induced by Cu²⁺. It was found that the order of potencies in inhibiting malondialdehyde formation was 5-hydroxytryptamine (serotonin)>5-hydroxytryptophol and 5-hydroxyindole-3-acetic acid when tested at 4 mM. 5-Methoxytryptamine was as effective as 5-hydroxytryptophol and 5-hydroxyindole-3-acetic acid when tested at 4 mM but was inactive at 1 mM. 5-Methoxytryptophol was marginally active at 4 mM. Melatonin, 5-methoxyindole-3-acetic acid and 6-methoxy-2-benzoxazolinone were inactive even at 4 mM. The ranking of antioxidative potencies as reflected in the shift of mobility in agar gel electrophoresis was 5-hydroxytryptamine>5-methoxytryptamine>5-hydroxyindole-3-acetic acid and 5-methoxytryptophol>5-hydroxytryptophol and melatonin. Another aim of this investigation was to ascertain the action of the aforementioned pineal indoles on the enhanced lipid peroxidation brought about in the mouse kidney and liver by intraperitoneal administrations of carbon tetrachloride. It was found that all pineal indoles tested demonstrated an inhibitory effect in the kidney but not in the liver. 6-Methox-2-benzoxazolinone and 5-methoxyindole-3-acetic acid exerted antifungal activity against Mycosphaerella arachidicola, Botrytis cinerea and Physalospora piricola. 6-Methoxy-2-benzoxazolinone exhibited antibacterial activity against Proteus vulgaris and 5-methoxytryptamine against Staphylocccus aureus and Bacillus subtilis. Other pineal indoles did not possess antifungal or antibacterial action.     

Transplacental carcinogenic effect of the serotonin derivative 5-methoxyindoleacetic acid

Transplacental administration of serotonin metabolite 5-methoxyindole-3-acetic acid possessing blastomogenic activity induced the development of malignant and benign neoplasms (lymphosarcomas, adenomas, hepatomas and other tumours) in 84% of C57BL/6 mouse progeny. The number of neoplasms was significantly higher, than in the control, they appeared earlier and were more malignant.     

Indole-3-acetic acid catabolism in Zea mays seedlings. Metabolic conversion of oxindole-3-acetic acid to 7-hydroxy-2-oxindole-3-acetic acid 7'-O-beta-D-glucopyranoside

A new metabolite of the plant growth substance indole-3-acetic acid has been extracted from Zea mays seedlings and characterized as the 7'-O-beta-D-glucopyranoside of 7-hydroxy-2-oxindole-3-acetic acid. This compound was the major product formed from [5-3H] 2-oxindole-3-acetic acid, incubated with intact plants or root and coleoptile sections. Identification was by gas chromatography-mass spectrometry of the trimethylsilyl derivative and by analysis of the hydrolysis products. A synthesis is reported for 7-hydroxy-2-oxindole-3-acetic acid. These results and prior work demonstrate the following catabolic route for indole-3-acetic acid in Zea: indole-3-acetic acid----2-oxindole-3-acetic acid----7-hydroxy-2-oxindole-3-acetic acid----7-hydroxy-2-oxindole-3-acetic acid glucoside.     

Metabolism of indole-3-acetic acid by orange (Citrus sinensis) flavedo tissue during fruit development

[5-3H, 1'-14C, 13C6, 12C] Indole-3-acetic acid (IAA), was applied to the flavedo (epicarp) of intact orange fruits at different stages of development. After incubation in the dark, at 25 degrees C, the tissue was extracted with MeOH and the partially purified extracts were analyzed by reversed phase HPLC-RC. Six major metabolite peaks were detected and subsequently analyzed by combined HPLC-frit-FAB MS. The metabolite peak 6 contained oxindole-3-acetic acid (OxIAA), indole-3-acetyl-N-aspartic acid (IAAsp) and also indole-3-acetyl-N-glutamic acid (IAGlu). The nature of metabolite 5 remains unknown. Metabolites 3 and 4 were diastereomers of oxindole-3-acetyl-N-aspartic acid (OxIAAsp). Metabolite 2 was identified as dioxindole-3-acetic acid and metabolite 1 as a DiOx-IAA linked in position three to a hexose, which is suggested to be 3-(-O-beta-glucosyl) dioxindole-3-acetic acid (DiOxIAGlc). Identification work as well as feeding experiments with the [5-3H]IAA labeled metabolites suggest that IAA is metabolized in flavedo tissue mainly through two pathways, namely IAA-OxIAA-DiOxIAA-DiOxIAGlc and IAA-IAAsp-OxIAAsp. The flavedo of citrus fruit has a high capacity for IAA catabolism until the beginning of fruit senescence, with the major route having DiOxIAGlc as end product. This capacity is operative even at high IAA concentrations and is accelerated by pretreatment with the synthetic auxins 2,4-D, NAA and the gibberellin GA3.     

Formation of Thiazolidine-4-Carboxylic Acid Represents a Main Metabolic Pathway of 5-Hydroxytryptamine in Rat Brain

Incubation of 5-hydroxytryptamine (5-HT) with rat brain homogenate resulted in the formation of (4R)-2-[3'-(5'-hydroxyindolyl)-methyl]-1,3-thiazolidine-4-carboxyl ic acid (5'-HITCA) as the major metabolite. The substance represents the condensation product of 5-hydroxyindole-3-acetaldehyde with L-cysteine. The chemical structure was confirmed by chromatographic and chemical methods as well as by fast atom bombardment mass spectrometry. Incubation of 5-HT in the presence of L-cysteine yielded the thiazolidine as the main metabolite up to 4 h. Under these conditions, the concentration of 5-hydroxyindole-3-acetic acid (5-HIAA) amounted to about 20% and 57% of 5'-HITCA (0.5 h and 4 h, respectively). In contrast to these findings, indole-3-acetic acid (IAA) was identified as the major metabolite when tryptamine was incubated under similar conditions. (4R)-2-(3'-Indolylmethyl)-1,3-thiazolidine-4-carboxylic acid (ITCA) was found to be the main conversion product of tryptamine only during the first 30 min. To investigate the fate of the thiazolidines, radiolabelled and unlabelled ITCA was incubated with rat brain homogenate. The compound was degraded enzymatically and rapidly. Subcellular fractionation revealed that the enzyme activity was present mainly in the cytosolic fraction whereas the preparation of mitochondria showed less activity. The responsible enzyme is presumably a carbon-sulfur lyase (EC 4.4.1.-). The major metabolite was isolated by HPLC and identified by mass spectrometry as well as by comparison with reference compounds to be IAA.(ABSTRACT TRUNCATED AT 250 WORDS)     

Thromboxane (TX)A3 and prostaglandin (PG)I3 are formed in man after dietary eicosapentaenoic acid: identification and quantification by capillary gas chromatography-electron impact mass spectrometry

The low incidence of myocardial infarction in Greenland Eskimos has been related to their traditional marine diet rich in eicosapentaenoic acid. However, whether dietary eicosapentaenoic acid is indeed transformed in man to antiaggregatory PGI3 and weakly proaggregatory TXA3 has not been clarified. In our studies we ingested either cod liver oil or mackerel both rich in eicosapentaenoic acid. Formation of TXB3, the hydrolysis product of TXA3, in platelet-rich plasma stimulated ex vivo with collagen was traced by capillary GC/EIMS. Via external standard, TXB3 formation in platelets was estimated to be 5-15% of TXB2 formation. From urine we extracted dinor metabolites of PGI according to a selective method. We utilized delta 17-2,3-dinor-6-keto-PGF1 alpha (PGI3-M) as an index of total body production of PGI3 in analogy to 2,3-dinor-6-keto-PGF1 alpha (PGI2-M), the major urinary metabolite of PGI2. We separated PGI2-M and PGI3-M as the Me, MO, Me3Si derivatives by capillary gas chromatography and identified PGI3-M by EI mass spectrometry. Excretion of PGI3-M, which was not detectable under control conditions, was 83 +/- 25 ng/24 h (SD) after ingestion of cod liver oil and 134 +/- 38 ng/24 h after mackerel ingestion, while excretion of PGI2-M was 162 +/- 52 ng/24 h and 236 +/- 32 ng/24 h, respectively. Our findings with diets rich in EPA show that it is possible in man to change in vivo the spectrum of biologically active prostanoids by nutritional means and alter it in a favourable direction.     

Metabolism of Indole-3-Acetic Acid by Pericarp Discs from Immature and Mature Tomato (Lycopersicon esculentum Mill)

[1′-¹⁴C, ¹³C₆]Indole-3-acetic acid was infiltrated into immature pericarp discs from fruits of tomato (Lycopersicon esculentum Mill., cv Moneymaker). After a 24-h incubation period the discs were extracted with methanol and the partially purified extract was analyzed by reversed-phase high-performance liquid chromatography-radiocounting. Five metabolite peaks (1-5) were detected and subsequently analyzed by combined high-performance liquid chromatography-frit-fast atom bombardment-mass spectrometry. The metabolite 4 fraction was found to contain [¹³C₆]-indole-3-acetylaspartic acid, and analysis of metabolite 5 identified [¹³C₆]indole-3-acetyl-β-d-glucose. The other metabolites could not be identified, but alkaline hydrolysis studies and gel permeation chromatography indicated that metabolites 1 and 3 were both amide conjugates with a molecular weight of approximately 600. Studies with radiolabeled indole-3-acetic acid, indole-3-acetylaspartic acid, and indole-3-acetyl-β-d-glucose demonstrated that in immature pericarp indole-3-acetic acid is deactivated primarily via metabolism to indole-3-acetylaspartic acid, which is further converted to metabolites 1, 2, and 3. In mature, pink pericarp discs, indole-3-acetic acid is converted more extensively to its glucosyl conjugate. Conjugation of indole-3-acetic acid to indole-3-acetylaspartic acid appears to be dependent upon protein synthesis because it is inhibited by cycloheximide. In contrast, cycloheximide has little effect on the further conversion of indole-3-acetylaspartic acid to metabolites 1, 2, and 3.     

Identification of 3-(O-beta-Glucosyl)-2-Indolone-3-Acetylaspartic Acid as a New Indole-3-Acetic Acid Metabolite in Vicia Seedlings

A new indole-3-acetic acid metabolite was isolated from broad bean (Vicia faba L. cv Chukyo) seedlings. It was a conjugate of dioxindole-3-acetic acid, aspartic acid, and glucose and was identified as 3-(O-β-glucosyl)-2-indolone-3-acetylaspartic acid (molecular weight 484) from ultraviolet, infrared, nuclear magnetic resonance, and mass spectra. Its natural content in 4-day-old Vicia seedlings was estimated to be 8.6 nanomoles per gram fresh weight. It was suggested that oxidation of indole-3-acetic acid not accompanied by decarboxylation might regulate endogenous level of the hormone.     

The biosynthesis and conjugation of indole-3-acetic acid in germinating seed and seedlings ofDalbergia dolichopetala

Germinating seed ofDalbergia dolichopetala converted both [²H₅]l-tryptophan and [²H₅]indole-3-ethanol to [²H₅]indole-3-acetic acid (IAA). Metabolism of [2-¹⁴C]IAA resulted in the production of indole-3-acetylaspartic acid (IAAsp), as well as several unidentified components, referred to as metabolites I, II, IV and V. Re-application of [¹⁴C]IAAsp to the germinating seed led to the accumulation of the polar, water-soluble compound, metabolite V, as the major metabolite, together with a small amount of IAA. Metabolites I, II and IV were not detected, nor were these compounds associated with the metabolism of [2-¹⁴C]IAA by shoots and excised cotyledons and roots from 26-d-oldD. dolichopetala seedlings. Both shoots and cotyledons converted IAA to IAAsp and metabolite V, while IAAsp was the only metabolite detected in extracts from excised roots. The available evidence indicates that inDalbergia, and other species, IAAsp may not act as a storage product that can be hydrolysed to provide the plant with a ready supply of IAA.Abbreviations HPLC-RC high-performance liquid chromatography-radiocounting - IAA indole-3-acetic acid - IAAsp indole-3-acetylaspartic acid - IAlnos 2-O-indole-3-acetyl-myo-inositol - IEt indole-3-ethanol     

An electron spin resonance study of free radical intermediates in the oxidation of indole acetic acid by horseradish peroxidase

The oxidation of indole-3-acetic acid by horseradish peroxidase was studied using the spin traps t-nitrosobutane and 5,5-dimethyl-1-pyrroline N-oxide to trap free radical intermediates. The major free radical metabolite of indole acetic acid was unambiguously determined by the use of indole-3-[2,2-2H2]acetic acid to be the skatole carbon-centered free radical. In the presence of oxygen, superoxide was also trapped.     

Indole-3-methanol—a metabolite of indole-3-acetic acid in pea seedlings

Summary Degradation of indole-3-acetic acid was investigated in etiolated pea shoots; the study was limited to indolic metabolites. The products formed were fractionated by column chromatography and identified by thin-layer chromatography and chemical methods. The pathway of indole-3-acetic acid degradation involving indole-3-aldehyde was found to be more significant than stated in literature, and indole-3-methanol was established as the major indolic metabolite.The following abbreviations will be used: IAA: indole-3-acetic acid; IM: indole-3-methanol; IAld: indole-3-aldehyde; ICA: indole-3-carboxylic acid.     

2,3,7,8-Tetrachlorodibenzo-p-dioxin alters melatonin metabolism in fish hepatocytes

Pineal hormone melatonin is an important regulator of endocrine and circadian rhythms in vertebrates. Since liver is assumed to be the major organ in the metabolism of this indole hormone, we investigated the effect of the known Ah-receptor agonist, 2,3,7, 8-tetrachlorodibenzo-p-dioxin (TCDD) on melatonin metabolism in fish hepatocytes as well as the in vitro effect of melatonin on trout hepatic microsomal cytochrome P4501A (CYP1A) catalyst. Primary cell cultures of rainbow trout hepatocytes were exposed to [3H]melatonin (1 nM to 1 microM) alone and in combination with TCDD (50 pM) at 15 degrees C for 24 or 48 h. Analysis of melatonin and its metabolites in the culture medium and hepatocytes by HPLC revealed that about 96% of the added [3H]melatonin was metabolised after 24 h in both control and TCDD treated cultures. 3H-radioactivity was found mainly in the culture medium and less than 5% of the total 3H-radioactivity retained inside hepatocytes. Of the HPLC separated metabolites, one coeluted with 6-hydroxymelatonin and one unknown metabolite eluted after 6-hydroxymelatonin. In addition, two other metabolites were more water-soluble than 6-hydroxymelatonin and were considered to be conjugated products. Treatment of the hepatocytes with TCDD increased the amount of the major oxidated product, 6-hydroxymelatonin, about 2.5-fold after 24 h and 1.2-fold after 48 h exposure, respectively when compared with the control cultures. Whereas the amount of the unknown metabolite eluting after 6-hydroxymelatonin decreased about 1.3-fold after 24 h and 1.2-fold after 48 h exposure, respectively. Melatonin alone did not affect P4501A associated EROD-activity or CYP1AmRNA levels in the primary hepatocyte cultures. TCDD-treatment increased EROD-activity 3 to 5-fold and respective CYP1AmRNA content 6 to 14-fold, when compared with the control or melatonin-treated cultures. Furthermore, melatonin competitively inhibited EROD-activity in liver microsomes with a Ki value of 62.06+/-3.78 microM. The results show that TCDD alters metabolic degradation of melatonin in hepatocytes and suggest that P4501A may be an important P450 isoenzyme involved in oxidative metabolism of melatonin in fish liver.     

Translocation of Radiolabeled Indole-3-Acetic Acid and Indole-3-Acetyl-myo-Inositol from Kernel to Shoot of Zea mays L.

Either 5-[³H]indole-3-acetic acid (IAA) or 5-[³H]indole-3-acetyl-myo-inositol was applied to the endosperm of kernels of dark-grown Zea mays seedlings. The distribution of total radioactivity, radiolabeled indole-3-acetic acid, and radiolabeled ester conjugated indole-3-acetic acid, in the shoots was then determined. Differences were found in the distribution and chemical form of the radiolabeled indole-3-acetic acid in the shoot depending upon whether 5-[³H]indole-3-acetic acid or 5-[³H]indole-3-acetyl-myo-inositol was applied to the endosperm. We demonstrated that indole-3-acetyl-myo-inositol applied to the endosperm provides both free and ester conjugated indole-3-acetic acid to the mesocotyl and coleoptile. Free indole-3-acetic acid applied to the endosperm supplies some of the indole-3-acetic acid in the mesocotyl but essentially no indole-3-acetic acid to the coleoptile or primary leaves. It is concluded that free IAA from the endosperm is not a source of IAA for the coleoptile. Neither radioactive indole-3-acetyl-myo-inositol nor IAA accumulates in the tip of the coleoptile or the mesocotyl node and thus these studies do not explain how the coleoptile tip controls the amount of IAA in the shoot.     

Aminoethyl-substituted indole-3-acetic acids for the preparation of tagged and carrier-linked auxin

Indole-3-acetic acid is an indispensable hormone (auxin) in plants and an important metabolite in humans, animals, and microorganisms. Here we introduce its 5- and 6-(2-aminoethyl)-derivatives for use in the design of novel research tools, such as immobilized and carrier-linked forms of indole-3-acetic acid and its conjugates with biochemical tags or biocompatible molecular probes. The aliphatic nitrogens of 5- and 6-(2-aminoethyl)indole were acetylated and the products were converted to the corresponding 3-(N,N-dimethylamino)methyl derivatives (gramines). These were reacted with cyanide. Saponification of the resulting acetonitriles was accompanied by N-deprotection to yield 5- and 6-(2-aminoethyl)indole-3-acetic acids. The latter were chemically stable and could be linked, via their amino groups, and without prior protection of their carboxyl moieties, to bovine serum albumin and to biotin, including appropriate spacer modules. One of the protein conjugates was used to elicit the formation of monoclonal antibodies, which were evaluated using the biotin conjugates in an enzyme-linked immunosorbent assay employing streptavidin-coupled alkaline phosphatase, and thus shown to recognize predominantly the indole-3-acetic acid moiety.     

Inhibition of small conductance K+ -channels attenuated melatonin-induced relaxation of serotonin-contracted rat gastric fundus

The aim of this study was to investigate the effects of melatonin on rat gastric fundus smooth muscle. Melatonin (10(-4) to 10(-3) M) had no effect on the basal tone of gastric smooth muscle. After precontraction with carbachol (10(-6) M) or serotonin (10(-7) M), melatonin caused a concentration dependent inhibitory action. The half maximal effect on serotonin-induced contraction was found with 1.12 +/- 0.86 x 10(-5) M of melatonin. Increasing concentrations of melatonin (10(-5) to 10(-3) M) resulted in a right shift of the serotonin concentration response curve (10(-10) to 10(-5) M). This inhibitory effect of melatonin was partially blocked in the presence of apamin (10(-10) to 10(-7) M), a specific blocker of the small conductance calcium-dependent potassium channel, but not in the presence of other potassium channel blockers like charybdotoxin (10(-8) M), glibenclamide (l0(-5) M), or tetraethylammonium (ODQ, 10(-4) M). The inhibitory effect was not changed in the presence of the neuronal blocker tetrodotoxin (10(-6) M), the selective P2-receptor antagonist pyridoxalphosphate-6-azophenyl-2',4'-disulphonic acid (3 x 10(-5) M), the nitric-oxide synthase inhibitor N-nitro-L-arginine (3 x 10(-4) M), or the guanylate cyclase inhibitor 1H-[1,2,4]oxadiazolo[4,3-a]-quinoxalin-1-one (10(-4) M), suggesting that neither the purinergic, nitrergic, nor guanylate cyclase pathways were involved. We further investigated inhibitory responses to electrical field stimulation (EFS) at different frequencies under non-adrenergic, non-cholinergic (NANC) conditions on a serotonin-induced contraction in the presence of melatonin (10)-5 to 10(-4) M). Melatonin significantly reduced these inhibitory NANC responses in higher (8-32 Hz), but not lower (05-4 Hz), frequencies (16 Hz without melatonin, 103 +/- 6.3%; melatonin 10(-5) M, 80.4 +/- 7.5%; melatonin 10(-4) M, 39.1 +/- 17.1%). Melatonin had no effect on contractile responses induced by EFS under basal tone. These results demonstrate that the inhibitory effect of melatonin in rat gastric fundus smooth muscle is apamin sensitive, but is not affected by other potassium channel blockers. This suggests that melatonin may be another transmitter candidate for the apamin sensitive responses within the gastrointestinal tract.     

Intestinal and hepatic microsomal metabolism of testosterone and progesterone by a 3 alpha-hydroxysteroid dehydrogenase to the 3 alpha-hydroxy derivatives in the channel catfish,Ictalurus punctatus

Intestinal or hepatic microsomes from channel catfish converted [4-14C]-testosterone to three major metabolites: 6 beta-hydroxytestosterone, androstenedione and a third metabolite. Formation of the unknown metabolite required NADPH as cofactor. When incubated with 200 microM testosterone, the rate of formation of the unknown metabolite was 265+/-158 pmol/(min mg) protein (mean+/-S.D.) in microsomes from the proximal intestine, 515+/-93 pmol/(min mg) protein in distal intestine and 226+/-42 pmol/(min mg) protein in hepatic microsomes. Comparison of the chromatographic and spectral properties of the unknown metabolite with those of authentic testosterone derivatives showed that this metabolite corresponded to 4-androstene-3 alpha,17 beta-diol. No 3 alpha-reduced metabolite was formed in incubations of testosterone with catfish intestinal cytosol. Testosterone was reduced to 5 alpha-dihydrotestosterone primarily in the cytosolic fraction and not in microsomes. Incubation of progesterone with intestinal microsomes resulted in the formation of a metabolite with properties similar to that of the 3 alpha-reduced testosterone, and this metabolite was identified by co-chromatography with authentic standard as 3 alpha-reduced progesterone. Thus, 3 alpha-hydroxysteroid dehydrogenase is an important pathway in intestinal microsomes of the channel catfish.     

Effect of the carcinogenic metabolites of aromatic amino acids on oxidative processes in lipids in vitro and in vivo

A comparative study of the effect produced by endogenous carcinogens (p-hydroxyphenyllactic and 5-methoxyindole-3-acetic acids) and their non-cancerogenic analogues on lipid peroxidation in vitro and in vivo was performed. It has been found that cancerogenic tyrosine and serotonin metabolites, unlike their non-cancerogenic analogues, increase lipid peroxidation in vitro. In vivo, cancerogenic tyrosine metabolite--p-hydroxyphenyllactic acid--is capable both of increasing and decreasing antioxidative lipid activity in the animal liver.     

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.