Aminoglutethimide-induced protein free radical formation on myeloperoxidase: a potential mechanism of agranulocytosis

Aminoglutethimide (AG) is a first-generation aromatase inhibitor used for estrogen-dependent breast cancer. Unfortunately, its use has also been associated with agranulocytosis. We have investigated the metabolism of AG by myeloperoxidase (MPO) and the formation of an MPO protein free radical. We hypothesized that AG oxidation by MPO/H2O2 would produce an AG cation radical that, in the absence of a biochemical reductant, would lead to the oxidation of MPO. We utilized a novel anti-DMPO antibody to detect DMPO (5,5-dimethyl-1-pyrroline N-oxide) covalently bound to protein, which forms only by the reaction of DMPO with a protein free radical. We found that AG metabolism by MPO/H2O2 induced the formation of DMPO-MPO, which was inhibited by MPO inhibitors and ascorbate. Glutethimide, a congener of AG that lacks the aromatic amine, did not cause DMPO-MPO formation, indicating the necessity of oxidation of the aniline moiety in AG. When analyzed by electron spin resonance spectroscopy, we detected a phenyl radical adduct, derived from AG, which may be involved in the free radical formation on MPO. Furthermore, we also found protein-DMPO adducts in MPO-containing, intact human promyelocytic leukemia cells (HL-60). MPO was affinity-purified from HL-60 cells treated with AG/H2O2 and was found to contain DMPO. These findings were also supported by the detection of protein free radicals with electron spin resonance in the cellular cytosolic lysate. The formation of an MPO protein free radical is believed to be mediated by one of two free radical drug metabolites of AG, one of which was characterized by spin trapping with 2-methyl-2-nitrosopropane. These results are the first demonstration of MPO free-radical detection by the anti-DMPO antibody that results from drug oxidation. We propose that drug-dependent free radical formation on MPO may play a role in the origin of agranulocytosis.     

Possible role of free radical formation in clozapine (clozaril)-induced agranulocytosis.

The use of clozapine, a unique antipsychotic drug, has been restricted due to a 1-2% incidence of drug-induced agranulocytosis. Metabolic activation of clozapine in neutrophils or stem cells could be the molecular mechanism underlying this side effect. Clozapine oxidation by human myeloperoxidase and horseradish peroxidase was evident from the disappearance of the UV absorbance at 290 nm. High performance liquid chromatography analysis revealed the formation of at least four radioactive peaks as a result of clozapine metabolism, including radioactivity coeluting with the protein. The tight association of radioactivity with the enzymatic protein was metabolism-dependent. This protein binding, which correlates with the total metabolism of clozapine, was reduced in the presence of glutathione and was absent in the presence of ascorbate. Similarly, in the presence of both reducing agents, the metabolite peaks in the high performance liquid chromatography radiogram, which are not associated with protein, disappeared. In contrast, in the presence of glutathione, two additional metabolites were found that could be isolated and identified by NMR and mass spectroscopy as clozapine glutathionyl adducts. Evidence for one-electron transfer reactions or the intermediate formation of a clozapine radical during the peroxidase-mediated metabolism of clozapine stems from the observation of thiyl and ascorbyl radicals in the presence of glutathione and ascorbate, respectively. The ascorbyl radical was detected by direct ESR spectroscopy in a peroxidase system. Its steady state concentration was significantly increased in the presence of clozapine. Glutathionyl radical formation was demonstrated by radical trapping with 5,5-dimethyl-1-pyrroline N-oxide in a peroxidase system. Again, the radical adduct concentration was significantly increased in the presence of clozapine. Similarly, when oxygen consumption was measured in peroxidase systems in the presence of glutathione or NADPH, the rate of oxygen uptake was markedly enhanced upon addition of clozapine. Thus, the data support the possibility of clozapine activation to free radical metabolites, which may cause oxidative stress or lead to adduct formation. Further, it can be concluded from these data that radical scavengers such as ascorbic acid, when coadministered with clozapine to patients, may reduce oxidative stress and protein adduct formation.     

Evidence for hydroxyl free radical formation during paraquat but not for nifurtimox liver microsomal biotransformation. A dimethyl-sulfoxide scavenging study

The effect of several experimental conditions on methane (CH₄) production from dimethylsulfoxide (DMSO) in incubation mixtures containing liver microsomes and NADPH generating systems was studied. The process was heat sensitive in part but a significant fraction was non-enzymatic in nature. CH₄ formation from DMSO was not significantly modified by 2-diethylaminoethyl-2,2-diphenylvalerate. HC1 (SKF 525 A) or EDTA 1 mM and significantly enhanced under an atmosphere of (CO 80%+O₂ 20%) rather than under air. A marked increase in CH₄ production was observed when paraquat (PQ) was included in incubation mixtures but not when nifurtimox (Nfx) was added. Results support the hypothesis of hydroxyl free radical (·OH) formation during PQ biotransformation but cast doubts about its production for the case of Nfx. The low temperature gas chromatographic separation of d₃-CH₄ from CH₄ described opens the future possibility for detecting trace formation of ·OH in vivo, without interference from fecal CH₄ formation by administering d₆-DMSO to animals and collecting exhaled gases produced, in chambers containing the entire animal.This work was supported by Grant AM 13 195-18 from the National Institutes of Health (USA)     

In vivo formation of a free radical metabolite of ethanol

Free radical metabolism of ethanol has been suggested as a factor in its hepatotoxicity. Although evidence of lipid radical formation due to ethanol treatment in vivo has been reported, free radicals from ethanol itself have not been detected in living animals. However, by applying the EPR spectroscopy technique of spin trapping to the study of ethanol-treated alcohol dehydrogenase-deficient deermice (Peromyscus maniculatus), we have detected the alpha-(4-pyridyl-1-oxide)-N-t-butylnitrone (POBN)/alpha-hydroxyethyl radical adduct in bile from animals administered [1-13C]ethanol and the spin trap POBN. Hyperfine coupling constants were aN = 15.48, a beta H = 2.02, and a beta 13C = 4.61 G. In addition, an ethanol-dependent but 13C-invariant radical adduct, presumably lipid derived, was detected. Hyperfine coupling constants were aN = 15.38 and a beta H = 2.5 G. This report demonstrates, for the first time, the in vivo formation of the alpha-hydroxyethyl free radical metabolite of ethanol.     

Procainamide, a drug causing lupus, induces prostaglandin H synthase-2 and formation of T cell-sensitizing drug metabolites in mouse macrophages.

Procainamide (PA) may cause drug-induced lupus, and its reactive metabolites, hydroxylamine-PA (HAPA) and nitroso-PA, are held responsible for this. Here, we show that N-oxidation of PA to these metabolites can take place in macrophages and lead to formation of neoantigens that sensitize T cells. Murine peritoneal macrophages (PMvarphi), exposed to PA in vitro, generated neoantigens related to HAPA as indicated by (1) their capacity to elicit a specific recall response of HAPA-primed T cells in the adoptive transfer popliteal lymph node (PLN) assay and (2) the appearance of metabolite-bound protein in PA-pulsed PMvarphi, as determined by Western blot. Analysis of five phase I enzymes that might be responsible for HAPA formation by PMvarphi pointed to prostaglandin H synthase-2 (PGHS-2) as a likely candidate. Experimental evidence that PA can be oxidized to HAPA by PGHS was obtained by exposing PA to PGHS in vitro. The resulting metabolites were identified by mass spectral analysis and covalent protein binding in ELISA. In vitro, PA exposure of PMvarphi of slow acetylator A/J and fast acetylator C57BL/6 mice failed to show significant strain differences in enzyme mRNA expression, enzyme activities, or formation of HAPA-related neoantigens. By contrast, after long-term PA treatment in vivo only in slow acetylators the PMvarphi harbored HAPA-related neoantigens and T cells were sensitized to them. PMvarphi of fast acetylator C57BL/6 mice only contained HAPA-related neoantigens, and their T cells were only sensitized to them if, in addition to long-term PA treatment, their donors had received injections of phorbol myristate acetate (PMA), a known enhancer of oxidative enzymes in phagocytes. In conclusion, PA treatment leads to N-oxidation of PA by enzymes, in particular PGHS-2, present in antigen-presenting cells (APC) and, hence, to generation of neoantigens which sensitize T cells. The enhanced neoantigen formation and T cell sensitization seen in slow acetylators might be explained by their higher concentration of PA substrate that is available for extrahepatic N-oxidation in APC.     

Clozapine bioactivation induces dose-dependent, drug-specific toxicity of human bone marrow stromal cells: a potential in vitro system for the study of agranulocytosis

Clozapine, an atypical antipsychotic drug effective in treatment of refractory schizophrenia causes potentially life-threatening agranulocytosis. The drug undergoes bioactivation to a toxic, chemically reactive intermediate with capacity to target stromal cells, central components of the bone marrow microenvironment implicated in neutrophil development. To identify possible mechanisms underpinning disruption of stroma as a site of drug bioactivation, toxicity was induced in vitro. Therefore metabolite generation procedures utilizing HOCl or HRP-H(2)O(2) as primary components involved in clozapine metabolism were adapted for stromal culture and coupled with viability determinations. Drug oxidation by HOCl was less toxic to stromal cells than HRP-H(2)O(2) based methods. More specifically, clozapine bioactivation by HRP-H(2)O(2) caused dose-dependent inhibition of stromal viability at therapeutically relevant concentrations. Differences in susceptibility of HAS303 and LP101 cells to the clozapine nitrenium ion were also evident. Stromal cell death was attributed to clozapine in the presence of a complete metabolising system comprising HRP and H(2)O(2). In the absence of a complete metabolising system clozapine was not cytotoxic. For LP101 cells, drug plus HRP (minus H(2)O(2)) also induced toxicity. Importantly, other antipsychotic drugs including risperidone, olanzapine and haloperidol when bioactivated, were not cytotoxic, indicating system specificity for clozapine. Exogenous GSH, N-acetylcysteine, l-ascorbic acid, catalase, and sodium azide afforded protection to cells whereas S-methylGSH, GSSG, ketoprofen and proadifen did not. Thus functional data derived from the in vitro stromal system defined in these studies may enable further investigation of the mechanisms subserving stromal impairment in clozapine-induced agranulocytosis and direct attention to improved methods for its prevention.     

Pretreatment with ramiprilat induces cardioprotection against free radical injury in guinea-pig isolated heart: involvement of bradykinin, protein kinase C and prostaglandins

1. Pretreatment with ramiprilat, an angiotensin-converting enzyme (ACE) inhibitor, induced cardioprotection and its possible mechanism of action was investigated in guinea-pig Langendorff perfused heart. 2. Superoxide anion (*O2-), produced by hypoxanthine and xanthine oxidase, and the 1,1-diphenyl-2-picryl-hydrazyl (DPPH) free radical were used for triggering free radical injury in cardiac tissue. 3. 1,1-Diphenyl-2-picryl-hydrazyl and *O2- significantly reduced left ventricular developed pressure (LVDP), +/-dP/dt(max), heart rate and coronary flow. Left ventricular end-diastolic pressure (LVEDP) was elevated and lactate dehydrogenase (LDH) leakage and the formation of thiobarbituric acid-reactive substances (TBARS) formation were significantly increased. 4. Pretreatment with ramiprilat induced cardioprotection against DPPH and *O2- free radical injury. Cardiac functions (LVDP, LVEDP and +/-dP/dt(max)) were significantly improved. Both LDH and TBARS were reduced. 5. HOE 140 (a selective bradykinin B2 receptor antagonist), calphostin C (a protein kinase C (PKC) inhibitor) and indomethacin (a cyclo-oxygenase inhibitor) all abolished the cardiac protective effect of ramiprilat. However, N(G)-nitro-L-arginine methyl ester, a nitric oxide synthase inhibitor, had no effect. 6. In conclusion, ramiprilat pretreatment induces cardioprotection against either DPPH or *O2- free radical injury. The protective effect depends on activation of B2 receptors and PKC. Prostaglandin synthesis is also involved.     

Diphenylene iodonium, an inhibitor of free radical formation, inhibits platelet aggregation.

Diphenylene iodonium is an inhibitor of the enzyme NADPH-oxidase and prevents the generation of oxygen-derived free radicals in neutrophils (Cross and Jones, 1986). Here we show that diphenylene iodonium (0.25-2 microM) inhibited, according to the dose, thrombin-induced platelet-aggregation in human washed platelets and ADP-induced platelet aggregation in platelet-rich plasma. At the concentrations which inhibited platelet aggregation diphenylene iodonium did not alter platelet concentrations of cAMP or cGMP but enhanced the anti-platelet activity of iloprost, sodium nitroprusside or cultured endothelial cells. These findings highlight the importance of free radicals as platelet pro-aggregatory agents.     

The effect of a brominated flame retardant, tetrabromobisphenol-A, on free radical formation in human neutrophil granulocytes: the involvement of the MAP kinase pathway and protein kinase C.

This study investigates the effects of one of the most frequently used brominated flame-retardants (BFR), tetrabromobisphenol-A (TBBPA), on formation of reactive oxygen species (ROS) and calcium levels in human neutrophil granulocytes. TBBPA enhanced ROS production in a concentration-depended manner (1-12 microM), measured as 2,7-dichlorofluorescein diacetate amplified (DCF) fluorescence. The results on ROS production by TBBPA was confirmed by lucigenin-amplified chemiluminescence. The TBBPA induced formation of ROS was due to activation of respiratory burst, as shown by the NADPH oxidase inhibitor DPI (10 microM). TBBPA induced activation of respiratory burst was also inhibited by the MEK 1/2 inhibitor U0126 (10 microM), the PKC inhibitor BIM (0.25 microM), and the tyrosine kinase inhibitor erbstatin-A (25 microM). We also found a small reduction in ROS formation in the absence of extracellular calcium and when verapamil was added. The phosphorylation of ERK 1/2 was confirmed by Western blotting. TBBPA also induced a concentration dependent increase in intracellular free calcium measured with Fura-2/AM. We suggest that exposure of human neutrophil granulocytes to the brominated flame retardant TBBPA leads to an activation of the NADPH oxidase primarily by an ERK 1/2 stimulated pathway. The data also show that PKC, calcium, and tyrosine kinases may be involved in the activation.     

Ukrain modulates glial fibrillary acidic protein, but not connexin 43 expression, and induces apoptosis in human cultured glioblastoma cells

Glioblastoma is a highly malignant tumor, characterized by an unfavorable prognosis even in response to multidisciplinary treatment strategies, owing to its high-invasive phenotype. Ukrain, a semisynthetic thiophosphoric acid derivative of the purified alkaloid chelidonine, has been used in the therapy of several solid tumors, but little is known about its effect on glioblastoma and, in general, about the molecular mechanisms responsible for its effects. In particular, we previously demonstrated that Ukrain modulates the expression of genes and proteins involved in tumor invasion, and here we investigate some unreported effects of Ukrain on human cultured glioblastoma cells. We used morphological and molecular biology methods to analyze the expression and the intracellular distribution pattern of glial fibrillary acidic protein, the expression of the gap junction protein connexin 43 and the apoptotic effect in human glioblastoma cells treated with 0.1, 1 and 10 micromol/l Ukrain for 72 h. After treatment with 10 micromol/l Ukrain, glial fibrillary acidic protein fluorescence increased and a higher number of cells displayed glial fibrillary acidic protein organized into a filamentous state. Western blot analysis of glial fibrillary acidic protein confirmed that Ukrain tended to upregulate the protein. Connexin 43 was not modulated by Ukrain both at the mRNA and at the protein level. Ukrain-induced apoptotic rate was 4.63, 10.9 and 28.9% after 0.1, 1 and 10 micromol/l Ukrain, respectively, likely mediated by cytochrome c release in the cytoplasm. Considered as a whole, these findings provide new information to complete the understanding of the mechanisms of Ukrain antitumor and chemopreventive effect, and support the possible potential of Ukrain for the therapy of brain tumors.     

Preferential efflux by P-glycoprotein, but not MRP1, of compounds containing a free electron donor amine

Multidrug resistance (MDR) in model systems is known to be conferred by two different integral proteins, the 170-kDa P-glycoprotein (P-gp) and the 190-kDa multidrug resistance-associated protein (MRP1), both of which pump drugs out of MDR cells. The presence of a nitrogen atom, charged at physiological pH, has frequently been considered to be a hallmark of P-gp substrates and inhibitors. The present study was aimed at investigating the role of nitrogen in the ability of the pump to recognise substrate. We measured the kinetics of active efflux of seven new anthracycline derivatives in P-gp-expressing K562/ADR cells and in MRP1-expressing GLC4/ADR cells. Six of these compounds represent analogues of daunorubicin in which the amino sugar nitrogen is bound to an amino- or a nitro-substituted benzyl moiety, the seventh is a doxorubicin derivative in which benzyl group is bound with 4'-oxygen. We found that the compounds with a nitro group on the benzyl ring were poor substrates for P-gp despite the presence of a secondary amine that can be protonated. In contrast, compounds that have a free amino group were very good substrates even though this amine is not protonated in the pH range studied (pK approximately 3). These results show that the nitrogen atom does not interact with P-gp in a charged form but rather as an electron donating group.     

Silica nanoparticle uptake induces survival mechanism in A549 cells by the activation of autophagy but not apoptosis

We report here an in vitro evaluation of silica nanoparticle uptake by lung epithelial cells (A549), the cytotoxic effect of the particles and we propose autophagy as possible survival strategy. The effect of surface charge, serum proteins and the influence of inhibitors on the uptake of 20 nm monodispersed nanoparticles with various functional groups are discussed. Uptake rate of the particles with various functional groups is demonstrated to be similar in the presence of serum proteins, while the uptake rate ranking is COOH > NH₂ > OH under serum free conditions. Our results suggest an actin-dependent, macropinocytotic uptake process that was also confirmed by scanning and transmission electron microscopy. In spite of the intensive active uptake, significant cytotoxic effect is detected only at relatively high concentrations (above 250 μg/mL). Blebbing of the cell surface is observed already at 5 h of exposure and is shown to be related to autophagy rather than apoptotic cell death. The A549 cells display elevated levels of autophagosomes, however they do not express typical apoptosis markers such as increased amount of active caspase-3 and release of mitochondrial cytochrome C. Based on these results, we propose here an autophagic activity and cross-talk between autophagic and apoptotic pathways as a mechanism allowing the survival of A549 cells under exposure to silica nanoparticles.     

Intra-striatal infusion of D-amphetamine induces hydroxyl radical formation: inhibition by MK-801 pretreatment

Recent evidence suggests that free radicals can be produced in the brain following systemic administration of repeated or high doses of D-amphetamine (AMPH). However, it has been proposed that the toxic effects of AMPH are mostly secondary to AMPH-induced hyperthermia, and agents that protect against AMPH neurotoxicity do so by blocking AMPH-induced hyperthermia or causing hypothermia. In this study, we examined the effects of AMPH on the formation of hydroxyl radicals (*OH) following its infusion into the rat striatum via a microdialysis probe. We found that intra-striatal perfusion of AMPH (10 microM) caused an increased formation of hydroxyl radicals but did not raise the core temperatures of the rats. Pretreatment with the NMDA antagonist MK-801 (0.5 mg/kg) attenuated hydroxyl radical production elicited by AMPH infusion, although core body temperatures in AMPH-treated rats were not significantly altered. Additionally, infusion of AMPH in the striatum increased extracellular dopamine concentration and this effect was potentiated by MK-801 pretreatment. Thus, these results demonstrate that direct infusion of AMPH in the striatum induces hydroxyl radical production without causing hyperthermia, and also imply that activation of glutamate NMDA receptors mediates, at least in part, AMPH-induced hydroxyl radical formation in the rat striatum.     

Free radicals as potential mediators of metal-allergy: Ni- and Co-mediated free radical generation

The generation of free radicals by Ni²⁺ and Co²⁺ was studied at physiological pH in H₂O₂-containing solutions in the absence and presence of various radical-mediating ligands and in human peripheral blood mononuclear cell (PBMC) cultures. With ESR spectroscopy, free radical species were identified and quantitated by spin trapping with 5,5-dimethyl-1-pyrroline-N-oxide (DMPO). Co²⁺ generated hydroxyl radicals from H₂O₂ in PBS solutions containing glutathione (GSH) or histidine (His). Omission of GSH or His from the reaction mixture significantly reduced the ESR-signal, indicating the importance of metal-chelation in free radical generation. Carnosine did not significantly enhance the reactivity of Co²⁺ toward H₂O₂, whereas cysteine (Cys) and N-acetylcysteine (NAC) suppressed free radical generation. Under identical reaction conditions, Ni²⁺ was markedly less reactive toward H₂O₂ in comparison with Co²⁺. GSH, His, Cys and NAC did not enhance free radical generation of Ni²⁺ from H₂O₂. However, in the presence of carnosine weak but significantly enhanced ESR intensities were found. Incubation of PBMC cultures from healthy subjects with Co²⁺ (10–50 μM) yielded the DMPO-OH adduct, suggesting Co²⁺-mediated hydroxyl radical generation. In contrast, incubation of PBMC cultures with Ni²⁺ (10–50 μM) did not produce a detectable ESR-signal. Ascorbic acid efficiently inhibited Co²⁺-mediated free radical generation in PBS solutions and PBMC cultures. The observed difference in free radical generating capacity between Ni²⁺ and Co²⁺ is of interest with respect to the absence of cross-reactivity between the two metal-ions in experimental allergic contact dermatitis.     

Raclopride,but not SCH 23390, induces maldirected jumping in rats trained to perform a run-climb-run behavioral task

In order to explore further the putative differential behavioral consequences of D₁ dopamine and D₂ dopamine receptor antagonism, SCH 23 390 (0.01–0.12 mg/kg) and raclopride (0.12–1.0 mg/kg) were administered to two separate groups of rats that had been trained in an eight-trial-per-day format to run down an alleyway, climb a vertical rope, and run across a horizontal board to access sweetened milk. Although both drugs dose-dependently reduced the speed of task completion, only raclopride produced vigorous, maldirected jumping behavior in the floor segment of the apparatus. The number of such jumps increased with dose. This raclopride-specific jumping phenomenon may provide a new behavioral arena for investigating the functional differences between D₁ and D₂ receptor antagonism.Supported by MH43429. The data were gathered during the course of doctoral dissertation research by L.S. at the University of Mississippi     

Immunosuppressive effect of emodin, a free radical generator.

The possible mechanism of immunosuppressive effect of emodin (1,3,8-trihydroxy-6-methylanthraquinone) was investigated in this study. Human mononuclear cells (10(6) cells/ml) were stimulated with 0.25% phytohemagglutinin for 24, 48 and 72 h, and the proliferative response was determined by the uptake of tritiated thymidine. In the presence of emodin (10(-6) to 3 x 10(-5) M), the proliferative response was reduced in a dose-dependent manner. Emodin (3 x 10(-7) to 3 x 10(-5) M) also dose dependently reduced the proliferative response to mixed lymphocyte reaction. After 72 h exposure to emodin (10 microM), interleukin-1 (IL-1), interleukin-2 (IL-2) production and IL-2 receptor expression were all reduced. The structure-activity relationship of emodin and 10 other anthraquione derivatives indicates that the free hydroxyl group at the beta-position of the anthraquinone nucleus plays an important role in the immunosuppressive effect. The suppressive activity of emodin was significantly inhibited by catalase (a scavenger of hydrogen peroxide), but little affected by superoxide dismutase (a scavenger of superoxide radical) and mannitol (a scavenger of hydroxyl radical). Methylene blue and hemoglobin, guanylate cyclase inhibitors, did not significantly affect the suppressive activity of emodin. Nordihydroguaiaretic acid (a lipoxygenase inhibitor) significantly potentiated the suppressive activity whereas quinacrine (a phospholipase A2 inhibitor) and indomethacin (a cyclooxygenase inhibitor) did not significantly affect it. The results suggest that the immunosuppressive effect of emodin may be partly mediated through hydrogen peroxide generated from semiquinone and regulated by arachidonic acid metabolites or byproducts.     

Inhibitory activity of kinetin on free radical formation of activated platelets in vitro and on thrombus formation in vivo

Kinetin has been shown to have anti-aging effects on several different systems, including plants and human cells. Recently, we demonstrated that kinetin markedly inhibited platelet aggregation in washed human platelets. In the present study, an electron spin resonance (ESR) method was used to further evaluate the scavenging activity of kinetin on the free radicals formed. Kinetin (70 and 150 microM) concentration dependently reduced the ESR signal intensity of hydroxyl radicals in collagen (1 microg/ml)-activated platelets. Furthermore, kinetin was effective in reducing the mortality of ADP-induced acute pulmonary thromboembolism in mice when administered intravenously at doses of 4 and 6 mg/kg. In addition, intravenous injection of kinetin (4 and 6 mg/kg) significantly prolonged the bleeding time by approximately 1.9- and 2.1-fold as compared with normal saline in severed mesenteric arteries of rats. A continuous infusion of kinetin (0.6 mg/kg/min) for 10 min also significantly increased the bleeding time by about 2.3-fold, and the bleeding time returned to baseline within 120 min after cessation of kinetin infusion. Platelet thrombi formation was induced by irradiation of mesenteric venules with filtered light in mice pretreated intravenously with fluorescein sodium. When kinetin was administered at 13 and 14 mg/kg in mice pretreated with fluorescein sodium (5 mg/kg), the occlusion time was significantly prolonged. In conclusion, these results suggest that kinetin has effective free radical-scavenging activity in vitro and antithrombotic activity in vivo. Treatment with kinetin may lower the risk of thromboembolic-related disorders. Therefore, kinetin may be a potential therapeutic agent for arterial thrombosis, but its toxicity must be further assessed.