Stobadine inhibits doxorubicin-induced apoptosis through a caspase-9 dependent pathway in P815 mastocytoma cells

Doxorubicin (DOXO), a widely used chemotherapeutic agent, induces apoptosis in transformed and non-transformed cells. The apoptotic effect of DOXO has been linked to the generation of reactive oxygen species (ROS). Antioxidants may be effective in the prevention of DOX-induced apoptosis. In the present study we investigated the effects of stobadine, a pyridoindole antioxidant in a DOXO-induced apoptosis model of P815 cells by flow cytometric analyses and by measuring caspase-3 and caspase-9 activities. Pretreating cells with stobadine significantly increased cell viability and decreased apoptosis rate. Inhibition in apoptosis was observed at maximum levels following treatment of cells with 10(-7)M stobadine as evident from flow cytometric analyses. The antiapoptotic effect of stobadine was further confirmed by inhibition of caspase-3 and caspase-9 activities. We found that the antioxidative effects of stobadine were comparable to the effects of a well known antioxidant, N-acetyl l-cysteine (NAC).     

The effect of stobadine on the copper-induced peroxidation of egg yolk phosphatidylcholine in multilamellar liposomes

Stobadine, (-)- cis-2,8-dimethyl-2 ,3,4,4a,5,9b-hexahydro-1H-pyrido-[4,3b]-indole, is a pyridoindole derivative with antioxidant, antiarrhytmic, neuroprotective, local anesthetic, alpha-adrenolytic, antihistaminic, myorelaxant and other pharmacodynamic effects. The antioxidant properties were tested in a model system of egg yolk phosphatidylcholine (EYPC) multilamellar liposomes. The lipoperoxidation was induced by adding Cu2+ ions and tert-butylhydroperoxide. The course of EYPC peroxidation was monitored spectrophotometrically for conjugated diene formation. We found that stobadine prolonged the lag phase and decreased the rate of peroxidation during the lag phase in a dose-dependent manner. Surprisingly, an increase in the rate of peroxidation was observed at low stobadine concentration in the propagation phase. The possible cause of prooxidative action of stobadine is discussed.     

Development of the New Group of Indole-Derived Neuroprotective Drugs Affecting Oxidative Stress

Summary 1. The role of oxidative stress, and accordingly uncontrolled reactive oxygen species generation/action, have been widely documented in a number of different neuronal pathologies. However, the concept of pharmacological interventions in prevention and therapy of oxidative stress-related diseases has not found adequate application in clinical practice. This may be due to the insufficient efficacy of drugs available, their unsuitable pharmacokinetics, side effects, toxicity, etc.2. Based on stobadine, (−)-cis-2,8-dimethyl-2,3,4,4a,5,9b-hexahydro-1H-pyrido[4,3-b]indole, a well-known antioxidant, free radical scavenger, and neuroprotectant, it was attempted to develop new stobadine derivatives with improved pharmacodynamic and toxicity profiles, on applying molecular design, synthesis and adequate tests. Stobadine molecule was modified mostly by electron donating substitution on the benzene ring and by alkoxycarbonyl substitution at N-2 position. A total of >70 derivatives were prepared.3. In a mice model of head trauma, some of the new stobadine derivatives administered i.v. immediately after the trauma, significantly improved sensomotoric outcome in the animals assessed 1 h later. Accordingly, decrease in brain edema was proved histologically as well as by brain wet weight assessment.4. Putative neuroprotective action of the compounds was confirmed on rat hippocampal slices exposed to reversible 6 min hypoxia/low glucose by analysis of synaptic transmission in CA1 region neurons. Irreversible impairment of neurotransmission resulting from the hypoxia was significantly reduced by the presence of SMe1EC2, one of the new compounds, in concentration range 0.03−10.0×10⁻⁶ mol l⁻¹. Both the neuroprotective and antioxidant effect of the compound closely resembled those of stobadine, melatonin, 21-aminosteroids, alpha-phenyl-tert-butylnitrone and others, all well-established antioxidants, except the range of effective concentrations was by 1–2 orders lower in SMe1EC2.5. A remarkable antioxidant efficacy was observed in the new compounds in rat brain homogenates exposed to iron/ascorbate system by protection of lipids and creatine kinase against the oxidative impairment. A link between the neuroprotective and antioxidant/ scavenger properties in the compounds can be assumed.6. Acute toxicity of some of the new pyridoindoles was diminished compared to stobadine. That might be due to the virtually full elimination of stobadine's undesired alpha ₁-adrenolytic activity attained by appropriate modifications of its molecule.7. The new pyridoindoles extend the range of available neuroprotectants interfering with oxidative stress in neuronal tissue.     

Structural aspects of antioxidant activity of substituted pyridoindoles

Stobadine and its two structural analogues, dehydrostobadine and N-acetylated stobadine were used to examine how structural alteration in the close proximity of the indolic nitrogen would influence the antioxidant activity of the substituted pyridoindoles. The compounds were tested for their efficiency to scavenge stable free radicals of alpha,alpha'-diphenyl-beta-picrylhydrazyl as well as for their ability to prevent 2,2'-azobis-(2-amidinopropane)hydrochloride induced peroxidation of dioleoyl phosphatidylcholine liposomes. The results proved that the substituted pyridoindoles can act as potent scavengers of peroxyl radicals both in aqueous and lipid phases, the antioxidant activity being comparable with that of Trolox. Structural changes in the proximity of the indolic nitrogen were found crucial for the radical scavenging efficiency: aromatisation of the pyridoindole skeleton in dehydrostobadine lowered the antioxidant activity, while acetylation of the indolic nitrogen completely abolished the ability to scavenge peroxyl radicals. The results are in agreement with the notion that the antioxidant activity of stobadine and of the related pyridoindoles may be mediated via the indolic nitrogen centre. When stobadine and Trolox were present simultaneously in liposomal incubations, Trolox spared stobadine in a dose-dependent manner; a direct interaction of Trolox with stobadinyl radical appears to be a plausible explanation with possible consequences for the antioxidant capacity of stobadine under in vivo conditions, where re-cycling of stobadine by vitamin E might occur.     

Antioxidant Activity of Diethyldithiocarbamate

Diethyldithiocarbamate (DDC), a potent copper chelating agent, has long been used for the treatment of oxygen toxicity to the central nervous system, as an immunomodulator to treat cancer, and in HIV-infected patients. We evaluated the antioxidant properties of DDC, including its scavenging of reactive oxygen species, its reducing properties, its iron-chelating properties, and its protective effects on oxidant-induced damage to brain tissue, protein, human LDL, and DNA. It is found that DDC is a powerful reductant and antioxidant since it scavenges hypochlorous acid, hydroxyl radical and peroxynitrite; it chelates, then oxidizes ferrous ions; it blocks the generation of hydroxyl radicals and inhibits oxidative damage to deoxyribose, protein, DNA, and human LDL. These findings may provide an explanation for the apparent beneficial effects of DDC against oxidative stress-related diseases that have been observed in experimental and clinical studies.     

Effects of antioxidant stobadine on protein carbonylation, advanced oxidation protein products and reductive capacity of liver in streptozotocin-diabetic rats: role of oxidative/nitrosative stress

BACKGROUND: Increased oxidative/nitrosative stress is important in the pathogenesis of diabetic complications, and the protective effects of antioxidants are a topic of intense research. The purpose of this study was to investigate whether a pyridoindole antioxidant stobadine (STB) have a protective effect on tissue oxidative protein damage represented by the parameters such as protein carbonylation (PC), protein thiol (P-SH), total thiol (T-SH) and non-protein thiol (Np-SH), nitrotyrosine (3-NT), and advanced oxidation protein products (AOPP) in streptozotocin-diabetic rats. METHODS: Diabetes was induced in male Wistar rats by intraperitonal injection of streptozotocin (55 mg/kg). Some of the non-diabetic (control) and diabetic rats treated with STB (24.7 mg/kg/day) during 16 weeks, and the effects on blood glucose, PC, AOPP, 3-NT, P-SH, T-SH and Np-SH were studied. Biomarkers were assayed by enzyme-linked immunosorbent assay (ELISA) or by colorimetric methods. RESULTS: Administration of stobadine to diabetic animals lowered elevated blood glucose levels by approximately 16% relative to untreated diabetic rats. Although stobadine decreased blood glucose, poor glycemic control was maintained in stobadine treated diabetic rats during the treatment period. Biochemical analyses of liver proteins showed significant diminution of sulfhydryl groups, P-SH, T-SH, Np-SH, and elevation of carbonyl groups in diabetic animals in comparison to healthy controls. As a biomarker of nitrosative stress, 3-NT levels did not significantly change by diabetes induction or by stobadine treatment when compared to control animals. However, the treatment with stobadine resulted in a significant decrease in PC, AOPP levels and normalized P-SH, T-SH, Np-SH groups in liver of diabetic animals. CONCLUSIONS: The results are in accordance with the pro-oxidant role of chronic hyperglycemia, and the ability of stobadine to attenuate protein oxidation and improving tissue reductive capacity may account, at least partly for its observed beneficial effects on tissue function in diabetes.     

Antioxidants and vision health: facts and fiction

A number of nutritional supplements containing antioxidants are advertised for better vision health. Do they benefit the average consumer? The literature was examined for the effectiveness of antioxidants for human eye health, and for the intricacies in collection of such evidence. The following diseases were considered: cataract, glaucoma, age-related macular degeneration (AMD), retinopathy, retinitis pigmentosa, eye infections, and uveitis. The literature indicates that antioxidant supplements plus lutein have a reasonable probability of retarding AMD. For glaucoma, such supplements were ineffectual in some studies but useful in others. In some studies, antioxidant rich fruits and vegetables were also useful for protection against glaucoma. For diabetic retinopathy, antioxidant supplements may have a small benefit, if any, but only as an adjunct to glycemic control. In very high-risk premature retinopathy and retinitis pigmentosa, antioxidant supplements may be beneficial but those with excess Vitamin E should be avoided. For cataract, there is no evidence for an advantage of such nutritional supplements. However, lubricant drops containing N-acetylcarnosine may be helpful in initial stages of the disease. For eye infections and other causes of uveitis, antioxidants have not been found useful. We recommend that a diet high in antioxidant rich foods should be developed as a habit from an early age. However, when initial signs of vision health deterioration are observed, the appropriate nutritional supplement products may be recommended but only to augment the primary medical treatments.     

Lipid peroxidation of phosphatidylcholine liposomes depressed by the calcium channel blockers nifedipine and verapamil and by the antiarrhythmic-antihypoxic drug stobadine

Nifedipine, verapamil and stobadine were tested and compared with butylated hydroxytoluene (BHT) as possible free radical scavengers inhibiting lipid peroxidation in phosphatidylcholine liposomes. Liposomes were peroxidized by incubation in air at 50 degrees C. Verapamil less than nifedipine less than BHT less than stobadine depressed the lipid peroxidation as detected spectroscopically for conjugate diene and thiobarbituric acid product formation. Verapamil and stobadine were tested as OH radical scavengers in a Fenton-type reaction against spin trap 5,5'-dimethyl-1-pyrroline-N-oxide (DMPO), as detected by ESR spectroscopy. The tested drugs competed with DMPO in trapping OH radicals, with stobadine being more effective than verapamil. ESR spectra of nifedipine in the incubated liposomes revealed that nifedipine could be involved in free radical reactions in the liposomes leading to nifedipine-stable radical(s) which were immobilized in the membrane. The obtained results suggest that some of the beneficial effects of the studied drugs can be mediated in disease by their ability to scavenge free radicals and by their protective effect on lipid peroxidation.     

Aggregation of human blood platelets in the presence of the pyridoindole stobadine

The antiarrhythmic and cardioprotective drug stobadine, possessing antioxidant and neuroprotective properties, was studied as to its in vitro effect on aggregation of human blood platelets. Pretreatment of platelets with stobadine for 30 s inhibited stimulated platelet aggregation in a dose-dependent way. Depending on the aggregation stimulus used, the minimal effective concentrations of the drug were 1 micromol/l (adrenaline), 200 micromol/l (ADP), and 1,000 micromol/l (PMA). Aggregation induced with thrombin or Ca2+-ionophore A23187 was not changed in the presence of stobadine even in the concentration of 1,000 micromol/l. Addition of stobadine 30 s after adrenaline was also effective and terminated aggregation (100 and 1,000 micromol/l) or prolonged onset of its second phase (10 micromol/l). The presented experiments showed stobadine as a potent inhibitor of adrenaline-induced aggregation, indicating its involvement in the observed antithrombotic and cytoprotective activity.     

Antioxidant drug mechanisms: transition metal-binding and vasodilation

In our work evaluating the antioxidant properties of a number of cardiovascular drugs, we have emphasized the importance of lipophilicity as a property contributing to antioxidant potency. Thus, the dihydropyridine calcium channel blockers and propranolol, one of the most lipophilic beta-blockers, were found to exhibit the greatest potency in membrane and cellular models. Both beta-blockers and calcium channel blockers are classified as antihypertensive agents. We found that the specific chemical moieties of various drugs may participate in the antioxidant mechanism of action. While reviewing relevant work from the past literature, it became apparent that some of the chemical moieties of antihypertensive and vasodilator drugs may bind transition metals. Thus, this present review focuses on common properties of transition metal-interaction that are shared, to a greater or lesser degree, by a number of vasoactive drugs and chemical agents. Although this observation has been pursued by other investigators in the past, we submit that the potential relevance to the newer pharmacological agents needs to be explored further. In addition, new information regarding the role of transition metals and free radicals involving vascular cells focuses greater importance on transition metal-interaction as a potential mechanism in vasodilation. This review does not intend to be inclusive of all chemical structures capable of binding transition metals; only those that are clinically relevant will be considered in some detail. Potential mechanisms of metal-chelating actions leading to vasodilation are also discussed.     

Effect of stobadine on cardiac injury induced by ischemia and reperfusion

The aim of this work was to evaluate the effect of the antioxidant stobadine on ischemia/reperfusion-induced injury of the isolated rat heart. Experiments were performed according to Langendorff. Ischemia was induced by stop-flow lasting 30 minutes and the duration of repefusion was 30 minutes. Reperfusion of the ischemic heart induced dysrhythmias, with the most severe ones occurring in the first minutes of reperfusion. A significant increase in coronary perfusion pressure was observed starting after 15 min of reperfusion. Stobadine (10(-6) M applied 3 minutes before onset of ischemia and during reperfusion) prevented the deleterious effects to develop fully. The protective effect of stobadine observed in our experiments seems to be a consequence of its antioxidant properties.     

EPR spectroscopy of free radical intermediates of antiarrhythmic-antihypoxic drug stobadine, a pyridoindole derivative

Mechanisms of antioxidant action of stobadine, a pyridoindole derivative with cardioprotective and antihypoxic properties, has been probed using EPR spectroscopy. Oxidation of stobadine by PbO2/tBuOOH in benzene results in the formation of nitroxide radical observable directly by EPR spectroscopy at room temperature, indicating conversion of indolic amino group to the corresponding nitroxide.     

Effect of stobadine on opsonized zymosan stimulated generation of reactive oxygen species in human blood cells

To predict more precisely the effect of stobadine, a pyridoindole antioxidant agent, in the whole organism, we studied its effect on opsonized zymosan-stimulated free radical generation in whole blood, on superoxide generation in the mixture of PMNL : platelets (1:50), as well as on superoxide generation and myeloperoxidase release in isolated PMNL. Without stimulation, stobadine had no effect on reactive oxygen species (ROS) generation and myeloperoxidase release. Stobadine in a concentration of 10 or 100 micromol/l significantly decreased luminol-enhanced chemiluminescence in opsonized zymosan-stimulated whole blood. In concentrations of 10 and 100 micromol/l, it reduced myeloperoxidase release from isolated neutrophils. Stobadine significantly decreased superoxide generation in isolated neutrophils in 100 micromol/l concentration. Its effect was much less pronounced in the mixture of neutrophils and platelets in the ratio close to physiological conditions (1:50). Our results suggest that stobadine might exert a beneficial effect in diseases or states where superfluous ROS generation could be deleterious.     

Antioxidant activity of β-blockers: An effect mediated by scavenging reactive oxygen and nitrogen species?

The therapeutic effects of beta-blockers are normally explained by their capacity to block the beta-adrenoceptors, however, some of the beneficial cardiovascular effects shown by this group of compounds have already been associated with the antioxidant properties that some of them seem to possess. The beta-blockers atenolol, labetalol, metoprolol, pindolol, propranolol, sotalol, timolol, and carvedilol were tested for their putative scavenging activity for ROS (O(2)(-), H(2)O(2), HO(.), HOCl, and ROO(.)) and RNS ((.)NO and ONOO(-)). Some of the studied compounds are effective ROS and/or RNS scavengers, these effects being possibly useful in preventing oxidative damage verified in hypertension as well as in other cardiovascular diseases that frequently emerge in association with oxidative stress.     

Non-antioxidant properties of α-tocopherol reduce the anticancer activity of several protein kinase inhibitors in vitro

The antioxidant properties of α-tocopherol have been proposed to play a beneficial chemopreventive role against cancer. However, emerging data also indicate that it may exert contrasting effects on the efficacy of chemotherapeutic treatments when given as dietary supplement, being in that case harmful for patients. This dual role of α-tocopherol and, in particular, its effects on the efficacy of anticancer drugs remains poorly documented. For this purpose, we studied here, using high throughput flow cytometry, the direct impact of α-tocopherol on apoptosis and cell cycle arrest induced by different cytotoxic agents on various models of cancer cell lines in vitro. Our results indicate that physiologically relevant concentrations of α-tocopherol strongly compromise the cytotoxic and cytostatic action of various protein kinase inhibitors (KI), while other classes of chemotherapeutic agents or apoptosis inducers are unaffected by this vitamin. Interestingly, these anti-chemotherapeutic effects of α-tocopherol appear to be unrelated to its antioxidant properties since a variety of other antioxidants were completely neutral toward KI-induced cell cycle arrest and cell death. In conclusion, our data suggest that dietary α-tocopherol could limit KI effects on tumour cells, and, by extent, that this could result in a reduction of the clinical efficacy of anti-cancer treatments based on KI molecules.     

Bilirubin and oxidative stress in term and preterm infants

Hyperbilirubinemia is the most frequent clinical problem neonatologists must deal with during the newborn period. It has been suggested that bilirubin is involved in the balance between antioxidant and pro-oxidant agents due to its antioxidant properties. However, the relevance of these effects in vivo in term and preterm infants is still debated. We performed a literature review of studies that investigated the association between total serum bilirubin (TSB) and oxidative stress in newborn infants. We found that studies in term infants give contradictory results, while studies in preterm infants suggest that the TSB increase is associated with an oxidative stress increase due to concurrent factors other than bilirubin level, such as heme oxygenase (HO) activity. Moreover, it could be speculated that low physiologic TSB values are associated with antioxidant effects, while high pathologic TSB values are associated with pro-oxidant effects. Literature data do not allow the establishment of whether if the antioxidant properties of bilirubin are important from a clinical point of view and can affect the outcome in ill infants.     

Antimicrobial activity and effects of resveratrol on human pathogenic bacteria

Resveratrol (3,4′,5-trihydroxistilbene) is a phytoalexin commonly found in food and drinks, which is thought to possess antimicrobial activity. These effects together with its well known antioxidant properties are beneficial for the prevention of some diseases, e.g. cancer. In this study we have verified that resveratrol has antibacterial activity against all tested Gram-positive bacteria using both the disk diffusion and broth microdilution methods. Time kill assays of this compound against Gram-positive bacteria showed that its effects on the growth of bacterial cells were due to bacteriostatic action. The addition of resveratrol has allowed the identification of changes in cell morphology and DNA contents, which have been assessed through microscopic analysis and flow cytometry; this suggests that the cell cycle is affected by resveratrol. This study indicates that this compound may have potential as a natural antibacterial agent for both food preservation and medicinal use.     

Selenium as an adjuvant for modification of radiation response

Ionizing radiation plays a central role in several medical and industrial purposes. In spite of the beneficial effects of ionizing radiation, there are some concerns related to accidental exposure that could pose a threat to the lives of exposed people. This issue is also very critical for triage of injured people in a possible terror event or nuclear disaster. The most common side effects of ionizing radiation are experienced in cancer patients who had undergone radiotherapy. For complete eradication of tumors, there is a need for high doses of ionizing radiation. However, these high doses lead to severe toxicities in adjacent organs. Management of normal tissue toxicity may be achieved via modulation of radiation responses in both normal and malignant cells. It has been suggested that treatment of patients with some adjuvant agents may be useful for amelioration of radiation toxicity or sensitization of tumor cells. However, there are always some concerns for possible severe toxicities and protection of tumor cells, which in turn affect radiotherapy outcomes. Selenium is a trace element in the body that has shown potent antioxidant and radioprotective effects for many years. Selenium can potently stimulate antioxidant defense of cells, especially via upregulation of glutathione (GSH) level and glutathione peroxidase activity. Some studies in recent years have shown that selenium is able to mitigate radiation toxicity when administered after exposure. These studies suggest that selenium may be a useful radiomitigator for an accidental radiation event. Molecular and cellular studies have revealed that selenium protects different normal cells against radiation, while it may sensitize tumor cells. These differential effects of selenium have also been revealed in some clinical studies. In the present study, we aimed to review the radiomitigative and radioprotective effects of selenium on normal cells/tissues, as well as its radiosensitive effect on cancer cells.     

Flavonoids-potent and versatile biologically active compounds interacting with cytochromes P450.

Flavonoids represent a group of phytochemicals exhibiting a wide range of biological activities arising mainly from their antioxidant properties and ability to modulate several enzymes or cell receptors. Flavonoids have been recognized to exert anti-bacterial and anti-viral activity, anti-inflammatory, anti-angionic, analgesic, anti-allergic effects, hepatoprotective, cytostatic, apoptotic, estrogenic and anti-estrogenic properties. However, not all flavonoids and their actions are necessarily beneficial. Some flavonoids have mutagenic and/or prooxidant effects and can also interfere with essential biochemical pathways. Among the proteins that interact with flavonoids, cytochromes P450 (CYPs), monooxygenases metabolizing xenobiotics (e.g. drugs, carcinogens) and endogenous substrates (e.g. steroids), play a prominent role. Flavonoid compounds influence these enzymes in several ways: flavonoids induce the expression of several CYPs and modulate (inhibit or stimulate) their metabolic activity. In addition, some CYPs participate in metabolism of flavonoids. Flavonoids enhance activation of carcinogens and/or influence the metabolism of drugs via induction of specific CYPs. On the other hand, inhibition of CYPs involved in carcinogen activation and scavenging reactive species formed from carcinogens by CYP-mediated reactions can be beneficial properties of various flavonoids. Flavonoids show an estrogenic or anti-estrogenic activity owing to the structural similarity with the estrogen skeleton. Mimicking natural estrogens, they bind to estrogen receptor and modulate its activity. They also block CYP19, a crucial enzyme involved in estrogen biosynthesis. Flavonoids in human diet may reduce the risk of various cancers, especially hormone-dependent breast and prostate cancers, as well preventing menopausal symptoms. For these reasons the structure-function relationship of flavonoids is extensively studied to provide an inspiration for a rational drug and/or chemopreventive agent design of future pharmaceuticals.     

Evaluation of the antioxidant properties of the angiotensin-converting enzyme inhibitor,captopril and the nucleotide enhancing agent,acadesine

The angiotensin-converting enzyme inhibitor, captopril and the nucleotide enhancing agent, acadesine, protect myocardial tissue from ischaemia/reperfusion-induced injury. Although both drugs have well established, independent mechanisms of cardiac protection, they may also have antioxidant activity which could contribute to their beneficial action. In this study we have examined the antioxidant activity of captopril and acadesine by examining their ability to scavenge ABTS radicals, formed from the interaction of ferryl metmyoglobin with phenothiazine in the presence of hydrogen peroxide. For comparison, we compared these results to those obtained for a range of other drugs commonly used for the treatment of cardiovascular disorders. These included verapamil (arrhythmia), isosorbide dinitrate (angina), atenolol (hypertension) and enalapril (congestive heart failure). The antioxidant properties of these drugs were then compared to the well characterised antioxidants, Trolox (a water soluble vitamin E analogue), ascorbate and glutathione. Captopril and acadesine were both shown to be efficient scavengers of ABTS radicals, importantly at drug concentrations expected to be found in vivo. These data confirm that the antioxidant potential of captopril and acadesine may be an important component of their mechanism of action, with both drugs probably protecting the myocardium against oxygen derived free radicals during ischaemia/reperfusion.