The free radical scavenging activity of four flavonoids determined by the comet assay

Flavonoids are reported to exhibit a wide variety of biological effects, including antioxidant and free radical scavenging activities. The aim of our study was to determine the cytotoxicity of flavonoids quercetin, rutin, apigenin and luteolin and their ability to protect DNA molecules against H2O2-induced damage. Cytotoxicity of studied flavonoids was tested in murine leukemia L1210 cells by the trypan blue exclusion technique. DNA strand breaks were determined using the alkaline single-cell gel electrophoresis (comet assay). Quercetin was found to possess the highest protective effect among the flavonoids studied (45%).The protective activity determined was lower for luteolin (40%). Protective effect of apigenin (600 microM/L) was only marginal (2%). However, at the higher concentration of apigenin (1200 microM/L), this flavonoid induced DNA single strand breaks. This indicates the ability of apigenin to serve as a pro-oxidant. Rutin had no protective effect on DNA single strand breaks induced by H2O2.     

Determination of free radical scavenging activity of quercetin, rutin, luteolin and apigenin in H2O2-treated human ML cells K562

We investigated protective effects of four flavonoids against H2O2- induced DNA damage in human myelogenous leukemia cells (K562) using the comet assay. The structural difference of studied flavonoids -- quercetin, rutin, luteolin and apigenin -- are characterized by the number of hydroxyl groups on the B ring. The presence of an o-dihydroxy structure on the B-ring confers a higher degree of stability to the flavonoid phenoxyl radicals by participating in electron delocalization and is, therefore, an important determinant for antioxidative potential. The results correlate with earlier published data obtained in murine leukemia cell line L1210. Hydrogen peroxide induced in human K562 cells a concentration-dependent increase of single cell DNA strand breaks. The strongest inhibition against H2O2-induced DNA damage (44%, 42%) was found in a range of luteolin and quercetin concentrations of 20-100 micromol/l. Protective effect of rutin (100 and 1000 micromol/l) was only marginal (8-10%). Apigenin had no protective effect on DNA single strand breaks induced by H2O2. Luteolin and quercetin are therefore effective in the protection of human single cell DNA from oxidative attack.     

The pineal secretory product melatonin reduces hydrogen peroxide-induced DNA damage in U-937 cells

Melatonin, the chief secretory product of the pineal gland, is a potent and efficient endogenous radical scavenger. Thus, melatonin was shown to protect different biomolecules, such as DNA, membrane lipids, and cytosolic proteins, from oxidative damage induced by oxygen-derived free radicals. In order to study the protective role of melatonin in hydrogen peroxide (H2O2)-induced DNA damage, U-937 cells were treated with different concentrations of H2O2, either in the presence or absence of melatonin, and DNA damage was assessed using the cytokinesis-block micronucleus technique. Melatonin diminished H2O2-induced micronuclei production both in short and long treatments. Additionally, melatonin concentrations higher than 1 microM were capable of protecting cells from spontaneous micronuclei production. These data suggest that melatonin, an endogenous antioxidant and nontoxic compound, may have an important role in protecting cells from genetic damage due to free radicals, supporting the idea of this hormone as a possible therapeutic agent in preventing aging and age-related diseases.     

Preventive effect of melatonin against DNA damage induced by cyanide, kainate, glutathione/Fe3+/O2, or H2O2/Fe2+

We have investigated hydroxyl free radical (.OH)-mediated damage to calf thymus DNA produced by potassium cyanide, kainate, H(2)O(2)/Fe(2+), or glutathione/Fe(3+) by in vitro method. When calf thymus DNA was exposed to potassium cyanide (0.5, 0.75, 1.0, 1.5, or 2.0 mM) or kainate (0.25, 0.5, or 1.0 mM) for 90 min at 37 degrees C in homogenate or the 9000g supernatant of mice brain, the quantity of DNA damage was observed to be concentration-dependent. Similarly, glutathione (1.0, 2.0, 4.0, 5.0, or 6.0 mM) inflicted damage on calf thymus DNA in the presence of Fe(3+) (3.0 microM). In addition, hydrogen peroxide (0.15, 0.30, 0.75, 1.50, or 3.0 mM) also caused damage to calf thymus DNA in the presence of Fe(2+) (3.0 microM) in the same manner. Furthermore, it was observed that the DNA damage induced by potassium cyanide (2.0 mM), kainate (0.5 mM), glutathione (4.0 mM)/Fe(3+), and H(2)O(2) (1.5 mM)/Fe(2+) was prevented by the treatment with melatonin (1.0 or 1.5 mM), a potent .OH scavenger. These results suggest that cyanide, kainate, glutathione/Fe(3+), and H(2)O(2)/Fe(2+)-mediated .OH may play a cardinal role for DNA damage induced by these chemicals. Hence the conclusion of the present study is that melatonin protects against DNA damage induced by the .OH produced by these chemicals.     

Adenosine A1 receptor activation attenuates cardiac injury produced by hydrogen peroxide.

Adenosine has been shown to protect the ischemic and reperfused myocardium. To examine whether the protective effect of the nucleoside is mediated by modulation of oxidative stress, isolated rat hearts were perfused for 30 minutes with 100 microM H2O2 or an exogenous free radical-generating system consisting of purine (3.06 mM) and xanthine oxidase (10 units/l) in the presence or absence of drugs acting on adenosine A1 or A2 receptors. H2O2 alone produced a greater than 90% loss in contractility concomitant with a threefold elevation in resting tension, although these effects occurred in the absence of ultrastructural damage. Two A1 receptor agonists N6-cyclopentyladenosine (CPA, 1 microM) and R(-)-N6-(2-phenylisopropyl)adenosine (R-PIA, 1 microM) significantly attenuated the cardiodepressant effects of H2O2 and depressed the elevation in resting tension; however, only the effect of CPA was found to be significant with regard to the latter parameter. A similar concentration of S(+)-N6-(2-phenylisopropyl)adenosine (S-PIA), a markedly less potent A1 receptor agonist, was found to be without beneficial effect. However, a significant protective effect against both the reduction in contractility and the elevation in resting tension was seen with a 10-fold elevation in the concentration of S-PIA (10 microM). The protective effects on functional parameters were associated with preservation of high-energy phosphate and adenine nucleotide contents after 30 minutes of H2O2 treatment. The salutary effects of all drugs were reversed in the presence of the A1 receptor antagonist 1,3-dipropyl-8-cyclopentylxanthine (0.5 microM). An A2 receptor agonist 2-[p-(carboxyethyl)phenethylamino]-5'-N-ethylcarboxamidoadenosine, termed CGS 21680 (1 microM), failed to alter the cardiac response to H2O2 with regard to all parameters studied. Neither a 50% reduction in external CaCl2 concentration nor treatment with 10 microM DL-propranolol exerted salutary effects against H2O2-induced dysfunction. None of the A1 receptor agonists modulated the response to purine plus xanthine oxidase. Our results demonstrate a selective protective effect of adenosine A1 receptor activation against the cardiac toxicity of H2O2 and provide, at least in part, a basis for the cardioprotective actions of adenosine and its analogues.     

Comparison of the effect of hydroxyurea and methotrexate on DNA fragmentation at various reaction conditions.

Using the changes in DNA breakage as a marker of DNA damage, the direct action of hydroxyurea (HU) and methotrexate (MTX) on DNA was examined. The experimental design was to expose isolated DNA to HU and MTX alone or HU and MTX with accelerators of free radical reaction (H2O2, Fe..) and to determine DNA fragmentation assessed by electrophoresis. The results indicated that HU can damage DNA, but to demonstrate this ability it needs H2O2, Fe.. or prolonged incubation in solution. Unlike HU, MTX with H2O2 was ineffective; MTX with Fe.. at certain degree protected DNA against lesions induced by Fe.. alone. It is concluded that despite several common features of HU- and MTX-induced toxic side-effects in the cells suggesting interference of these drugs with free radical reactions, their direct effect on DNA under oxidizing conditions is quite different at least at the concentrations used by us.     

Reduction of calcium channel antagonist binding sites by oxygen free radicals in rat heart

In view of the importance of Ca2+-channels in controlling the entry of Ca2+ into the myocardium, this study was undertaken to examine the effects of oxygen free radicals on the binding of Ca2+-channel antagonists in rat heart by employing [3H]-nitrendipine as a ligand. Isolated heart membranes were incubated with xanthine + xanthine oxidase (a superoxide anion radicals generating system), hydrogen peroxide (an activated species of oxygen), or hydrogen peroxide + Fe2+ (a hydroxyl radicals generating system). The assay of the [3H]-nitrendipine binding activity revealed that the maximal number of binding sites (Bmax) were reduced in a time-dependent manner by superoxide radicals without any changes in the binding constant (Kd); a significant reduction of Bmax was seen after incubating membranes with xanthine + xanthine oxidase for a 10-min-period. Superoxide dismutase showed a protective effect on the superoxide radicals induced reduction in Bmax. Both hydrogen peroxide and hydroxyl radicals also depressed the Bmax for [3H]-nitrendipine binding without any significant change in Kd; catalase and mannitol showed protective effects on hydrogen peroxide or hydroxyl radicals induced depression in Bmax, respectively. These results indicate that oxygen free radicals may reduce the number of Ca2+-channels in the cell membrane and this change may contribute towards decreasing the voltage-dependent Ca2+ influx in the cardiac cell.     

In vitro studies on the OH* and O2(-*) free radical scavenger properties of idebenone in chemical systems

OH(*) free radicals were generated by Fenton reaction in the presence of bovine serum albumin (BSA). The decreasing water-solubility of BSA with increasing Fe(2+) concentrations of the system is a sensitive indicator of the cross-linking effects of the OH(*) free radicals. Idebenone (oxidized form) was solubilized for this experiment in DMSO and added to the system in final concentrations of 0.01 or 0.1%. Neither of these concentrations displayed any protective effect against the insolubilization of BSA. Therefore, oxidized idebenone has to be considered as a substance which reacts with OH(*) free radicals slower than the BSA itself, i.e., its oxidized form is not an efficient scavenger of this type of free radicals under the given circumstances. The ability of idebenone to scavenge superoxide radicals was tested in ( [Formula: see text] ) the pyrogallol system; and (ii) the xanthine-xanthine oxidase-nitro blue tetrazolium (XXO-NBT) system. Idebenone did not show any O(2)(-*) radical scavenging ability as revealed by these two in vitro methods, in the concentration ranges studied (up to 75 or 220 microg/ml, respectively). On the contrary, an increasing O(2)(-*) radical generation was observed with increasing concentrations of the drug in both test systems used. The possible biological significance of these observations is discussed in the light of other results like ESR spin trapping and measurements of superoxide dismutase (SOD) activity in various tissues.     

Protection of cardiomyocyte function by propofol during simulated ischemia is associated with a direct action to reduce pro-oxidant activity

To demonstrate a direct protective effect of propofol on myocardial contractile performance during an ischemic episode and investigate underlying mechanisms, isolated adult rat ventricular cardiomyocytes were subjected for 2 h to (i) ischemic medium containing 2-deoxyglucose (20 mM), gassed with 100% N(2) at pH 6.4, (ii) normal medium with 95% O(2)/5% CO(2) at pH 7.4 or (iii) normal medium with addition of H(2)O(2) (50 microM). Propofol under normal conditions decreased the peak amplitude of electrically stimulated contraction of cardiomyocytes from a basal value of 6.5+/-0.37 microm to a maximum attenuation ( approximately 37%) at 0.44 to 56 microM. Under ischemic conditions, the contraction amplitude at baseline was 2.8+/-0.34 microm, but propofol, despite having a cardiodepressant effect per se, stimulated contraction, such that at >or=0.44 microM, normal and ischemic values in the presence of propofol were similar. Comparably, pro-oxidant (H(2)O(2))-induced attenuation of cell shortening was reversed by propofol (0.5 microM) to the level of contractile activity produced by the anaesthetic alone. The protective effect against ischemia-induced injury was not reflected in an improved ATP/ADP ratio nor was it mediated through diltiazem-sensitive L-type Ca(2+) channels. Propofol (0.5 microM) did, however, attenuate the ischemia- and H(2)O(2)-induced increases in the membrane lipid hydroperoxides, MDA (by 83% and 30%) and 4-HNE (by 47% and 69%). It is concluded that propofol, at clinically relevant concentrations, can counteract the effects of increased production of free radical compounds by cardiomyocytes subjected to oxidant stress and improve contractile performance.     

Serotonin potentiates angiotensin II--induced vascular smooth muscle cell proliferation.

Vascular smooth muscle cell (VSMC) proliferation is a key feature in the development of atherosclerosis and restenosis after angioplasty, which can occur in response to many different humoral and mechanical stimuli. We investigated the growth promoting activities of two potent vasoactive substances, angiotensin II (Ang II) and serotonin (5-HT), on cultured rabbit VSMCs. Growth-arrested VSMCs were incubated with serum-free medium containing different concentrations of Ang II in the presence or absence of 5-HT. [3H]thymidine incorporation into VSMC DNA was measured as an index of cell proliferation. Ang II and 5-HT stimulated DNA synthesis in a dose-dependent manner with a maximal effect at 1.75 microM for Ang II (202%) and 50 microM for 5-HT (205%). When added together, low concentrations of Ang II (1 microM) and 5-HT (5 microM) synergistically induced DNA synthesis (363%). Candesartan (1 microM), an AT(1) receptor antagonist, but not PD 123319 (1 microM), an AT(2) receptor antagonist, inhibited the mitogenic effect on Ang II and its interaction with 5-HT. Sarpogrelate (10 microM), a 5-HT(2A) receptor antagonist, and pertussis toxin (10 ng/ml) inhibited the mitogenic effect of 5-HT and its interaction with Ang II. The protein kinase C inhibitor Ro 31-8220 (0.1 microM), the Raf-1 inhibitor radicicol (10 microM), and the MAPK kinase inhibitor PD 098059 (10 microM) abolished mitogenic effects of Ang II and 5-HT, and also their synergistic interaction. The JAK2 inhibitor AG 490 (10 microM) had only a minimal inhibitory effect of Ang II-induced DNA synthesis but significantly inhibited the interaction of Ang II with 5-HT. The synergistic effect on Ang II (1 microM) with 5-HT (5 microM) on DNA synthesis was completely reversed by the combined use of both candesartan (1 microM) and sarpogrelate (10 microM). Our results suggest that Ang II and 5-HT exert a synergistic interaction on VSMC proliferation via AT(1) and 5-HT(2A) receptors. The activation of MAPK and JAK/STAT pathways may explain the synergistic interaction between Ang II and 5-HT.     

Prevention of postischemic cardiac injury by the orally active iron chelator 1,2-dimethyl-3-hydroxy-4-pyridone (L1) and the antioxidant (+)-cyanidanol-3

In this study, we investigated the role of oxygen-derived free radicals and iron in mediating myocardial injury during ischemia and reperfusion. Iron is of special interest because it may enhance tissue injury during ischemia and reperfusion by catalyzing the formation of highly reactive hydroxyl radicals (by modified Haber-Weiss or Fenton reactions). Rat hearts, perfused by the Langendorff method, were subjected to global ischemia (15 minutes at 37 degrees C) and reperfusion. The effects of two iron chelators, 1,2-dimethyl-3-hydroxy-4-pyridone (L1) and 5-hydroxy-2-hydroxymethyl-4-pyrone (kojic acid), and one antioxidant, (+)-cyanidanol-3, on contractile function, coronary flow, lactate dehydrogenase release, and lactate production were studied. The combination of these iron chelators is of special importance because L1 is known to prevent lipid peroxidation, induced by ADP/Fe3+ and NADPH in microsomes, in contrast to kojic acid. We found significant protection of contractile function (apex displacement) during reperfusion with 50 microM L1 and 20 microM (+)-cyanidanol-3 (p less than 0.01, n = 6), whereas no protection was found with 50 microM kojic acid (n = 6). Measurements of lactate dehydrogenase release during reperfusion showed a protective pattern similar to that found for heart contractile function, although 50 microM kojic acid also showed a significantly lower lactate dehydrogenase release during the first 10 minutes of reperfusion. No differences in coronary resistance or lactate release were found between the various groups. Our findings indicate that iron and oxygen-derived free radicals are important in the pathogenesis of postischemic reperfusion injury probably because of the formation of hydroxyl radicals.(ABSTRACT TRUNCATED AT 250 WORDS)     

Transition metals in legume root nodules: iron-dependent free radical production increases during nodule senescence

The cytosol from root nodules of soybean, bean, and cowpea contained Fe and Cu capable of catalyzing the formation of highly reactive free radicals. Specific and sensitive assays based on free radical-mediated DNA degradation revealed that most catalytic Fe and Cu were present as small chelates (300-600 Da). The involvement of catalytic Fe in free radical production during nodule senescence, which was induced by exposure of plants to continuous darkness for 2-4 days, was investigated. (i) Free heme remained at a constant and low concentration (1-4% of total nodule heme) during senescence, indicating that it is not an important constituent of the catalytic Fe pool of nodules. (ii) Catalytic Fe of nodule cytosol promoted deoxyribose degradation and linolenic acid peroxidation in reaction mixtures containing physiological concentrations of ascorbate and H2O2. Deoxyribose degradation but not lipid peroxidation required hydroxyl radicals to proceed. (iii) The cytosol from senescent nodules, particularly of bean and cowpea, sustained in vitro higher rates of deoxyribose degradation and lipid peroxidation than the cytosol from unstressed nodules. Both degradative processes were inhibited by the Fe chelator desferrioxamine and were correlated with the content of catalytic Fe in the nodule cytosol. (iv) Although other transition metals (Cu, Mn, Mo, and Ni) were present in significant amounts in the low molecular mass fraction (<3 kDa) of the nodule cytosol, Fe is most likely the only metal involved in free radical generation in vivo. (v) By using dimethyl sulfoxide as a molecular probe, formation of significant amounts of hydroxyl radical was observed in vivo during senescence of bean and cowpea nodules.     

Pyridoindole antioxidant stobadine protected bovine serum albumin against the hydroxyl radical mediated cross-linking in vitro

On exposure to free radicals generated by the Fenton reaction system of Fe(2+)/EDTA/H(2)O(2)/ascorbate, bovine serum albumin (BSA), used as a model of water-soluble protein, was losing its water solubility depending on the concentration of the chelated iron. The precipitate was found irreversibly insoluble even in concentrated urea. In the soluble fraction, SDS-PAGE analysis proved the presence of dimers and trimers of BSA, accompanied by enhanced bityrosine fluorescence. The pyridoindole antioxidant stobadine inhibited the process of albumin insolubilization in a concentration-dependent manner, the protective effect being more efficient than that of 2-keto-4-methiolbutyric acid (KMBA). Stobadine was, however, less effective than trolox. The inhibitory effect of the antioxidants, expressed as IC(50), correlated well with the reciprocal values of corresponding second order rate constants for scavenging OHz.rad; radicals. The results indicated that the insolubilization of BSA induced by the Fenton system of Fe(2+)/EDTA/H(2)O(2)/ascorbate was caused by OHz.rad; radical mediated cross-linking of the albumin. The model system proved to be suitable for convenient testing of OHz.rad; radical scavenging ability of new antioxidants in a non-lipid environment.     

Inhibition of sarcolemmal carbon-centered free radical formation by propranolol

The mechanism of propranolol-inhibited sarcolemmal membrane lipid peroxidation was investigated by electron spin resonance spin-trapping technique using 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) and 2-methyl-2-nitrosopropane (MNP). Highly purified canine myocytic sarcolemma were peroxidized by a superoxide-driven (from dihydroxyfumarate) and Fe3+-catalyzed free radical-generating system. Hydroxyl radicals (.OH), identified by electron spin resonance signals as DMPO-OH adducts, were generated in the aqueous phase. Propranolol up to 500 microM did not effectively reduce the intensity of the DMPO-OH adducts. When the sarcolemma were incubated with MNP before the addition of free radicals, MNP adducts characteristic of carbon-centered radicals were produced. Pretreatment of the membranes with propranolol (3-100 microM) decreased the intensity of the MNP adducts in a log concentration-dependent manner; the EC50 is about 7 microM. D- and L-propranolol were found equally effective. When protein-depleted sarcolemmal lipids were similarly incubated with MNP and the free radical system, identical MNP adducts were observed; this finding suggests that the adducts were lipid-derived products arising from lipid peroxidation. Furthermore, their formation was also inhibited by propranolol pretreatment. Since propranolol is not an effective scavenger of oxygen radicals in the aqueous phase, the data suggest that the antiperoxidative effect of propranolol is due to its lipophilic interaction with the membrane and thus subsequent interruption of the free radical chain reactions.     

Electron spin resonance spectroscopic demonstration of the hydroxyl free radical scavenger properties of dimethylaminoethanol in spin trapping experiments confirming the molecular basis for the biological effects of centrophenoxine

The ADP-Fe(II)-H2O2 system generates OH free radicals which can be trapped by 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) thus yielding a measurable signal by electron spin resonance spectroscopy. The amount of DMPO-OH spin adduct formed under certain conditions decreased considerably, if dimethylaminoethanol (DMAE), p-chlorophenoxyacetic acid (PCPA) or centrophenoxine (CPH) were present in comparable concentrations to that of DMPO. It has been demonstrated that such an effect cannot be attributed to any interference of the tested compounds with the Fe(II) and its oxidability by H2O2. The reaction of DMAE with OH free radicals was demonstrated also by using other spin traps. These spin traps reacted with OH free radicals either not at all (phenyl-tert-butylnitrone, PBN) or only to a slight extent (alfa-pyridyl-l-oxide-N-tert-butylnitrone, 4-POBN). DMAE was also a competitive OH free radical scavenger with proline and hydroxyproline, both of which have recently been shown to react with OH free radicals to form nitroxyl free radicals. On the basis of the experimental results, the OH free radical scavenger property of DMAE can be regarded as firmly established. This result supports the molecular mechanism proposed for the explanation of the anti-aging effects of CPH in terms of the membrane hypothesis of aging.     

Free radical-mediated tolbutamide desensitization of K+ATP channels in rat pancreatic beta-cells

To study the effects of hydroxyl radicals on the sensitivity of the ATP-sensitive K+ (K+ ATP) channel to tolbutamide, we used patch clamp and microfluorometric techniques in pancreatic beta-cells isolated from rats. cell-attached membrane patches, exposure of the cells to 0.3 mM H2O2 increased the probability of opening of K+ATP channels in the presence of 2.8 mM glucose. Tolbutamide dose-dependently inhibited the K+ATP channel with half-maximal inhibition (IC50) at 0.8 microM before and immediately after exposure to H2O2. After prolonged exposure (>20 min) to H2O2, the IC50 was increased to 15 microM. The presence of both ATP and ADP at concentrations ranging from 0.01 to 0.1 mM in the inside-out bath solution significantly enhanced the inhibition of the channels by 10 microM tolbutamide. Addition of 0.3 mM H2O2 induced a transient minute increase in the cytoplasmic Ca2+ concentration ([Ca2+]i) within 10 min, followed by a sustained pronounced increase in [Ca2]i. After more than 20 min of exposure of cells to 0.3mM H2O2, [Ca2]i was increased to above 2 microM. Treatment of the cytoplasmic face of inside-out membrane patches with 1 microM Ca2+ attenuated the tolbutamide-sensitivity of the K+ATP channel, but not the ATP-sensitivity of the channel. These findings indicate that H2O2 reduces tolbutamide sensitivity by inducing a sustained increase in [Ca2+]i.     

Lipid peroxidation product 4-hydroxy-2-nonenal acts synergistically with serotonin in inducing vascular smooth muscle cell proliferation

Formation of an atherosclerotic lesion is in part mediated by inflammatory and oxidative mechanisms including lipid peroxidation. To characterize the potential role of lipid peroxidation products in atherogenesis, we assessed the effect of 4-hydroxy-2-nonenal (HNE), a component of oxidatively modified lipids on vascular smooth muscle cells (VSMCs) proliferation, and its interaction with serotonin (5-hydroxytryptamine, 5-HT), a known mitogen for VSMCs. Growth-arrested rabbit VSMCs were incubated with different concentrations of HNE in the absence or presence of 5-HT. VSMCs proliferation was examined by increases in [3H]thymidine incorporation into DNA and cell number. HNE and 5-HT stimulated DNA synthesis in a dose-dependent manner. HNE had a maximal proliferative effect at a concentration of 1 microM (143% of the control) and 5-HT at 50 microM (211%). When added together, low concentrations of HNE (0.1 microM) and 5-HT (5 microM) synergistically induced DNA synthesis (273%). These effects on DNA synthesis were paralleled by an increase in cell number. A 5-HT2 receptor antagonist LY 281067 (10 microg/ml) and pertussis toxin (10 ng/ml) inhibited the mitogenic effect of 5-HT only. Protein tyrosine kinase inhibitor erbstatin A (10 microM) completely inhibited the mitogenic effect of HNE and partially that of 5-HT and the combined effect of HNE+5-HT. Protein kinase C inhibitor Ro 31-8220 (0.1 microM) completely inhibited mitogenic effects of both HNE and 5-HT, and also the combined effect of HNE+5-HT. The synergistic effect of HNE+5-HT on DNA synthesis was completely reversed by the combined use of LY 281067 (10 microg/ml) and antioxidants N-acetylcysteine (400 microM), vitamin C (200 microM), or vitamin E (20 microM). Our results suggest that HNE acts synergistically with 5-HT in inducing VSMCs proliferation. Combined use of both antiplatelet and antioxidant therapies may be useful for the prevention of VSMCs proliferative disorders associated with atherosclerosis and restenosis after angioplasty.     

Halothane modifies oxygen free radical activity on the voltage-sensitive calcium channels in canine myocardial membranes

Background: The functional derangements in the myocyte cell membrane, the sarcolemma, during short myocardial ischaemia and reperfusion are attributed to excessive influx of Ca2+ ions via the voltage-sensitive calcium channels (VSCC) and to the free radical-related injury. However, it is unclear whether the primary changes in the VSCC should be attributed to the ischaemic effect or to free radical action on channel constituents. Under these circumstances of ischaemia and reperfusion, volatile anaesthetics have exhibited protective properties on the myocardium. The present study is aimed at characterizing the effect of artificially-generated oxygen free radicals on the VSCC in canine sarcolemma, independently of the effect of ischaemia, and the effect of halothane on the membranes during the surge of the free radicals. Methods: Selective production of free radicals (O2-, CO2-) was made by gamma irradiation of isolated sarcolemma membranes with 137 Cesium (Cs), in the presence of 20 mM sodium formate. Control studies were performed without formate in the aqueous solution. In an additional group, liquid halothane (3 microl. 1.9 vol%) was added to the sarcolemma / formate preparation immediately prior to irradiation. The effects of free radicals on the VSCC was evaluated by redioligand binding studies of the calcium channel blocker [3H] isradipine to the sarcolemma. Results: In six control studies, the rediolytic aqueous species produced by 137 Cs irradiation resulted in unchanged [3 H] isradipine binding. In the presence of formate [n=9], the free radicals have caused a 23% to 25% decrease, both, in density and dissociation constant (P=0.05) of [3 H]isradipine to the VSCC binding sites. When superoxide radicals were generated in the presence of 1.9% halothane and formate (n=6), a significant increase in maximal binding capacity (by 55% +/- 2; P<0.01) and in the dissociation constant (by 209% +/- 35, P<0.01) occurred. Conclusion: Oxidative free radicals which are generated by gamma irradiation exerted minimal changes on the normal function of the VSCC as reflected by the non-significant changes in [3 H] isradipine specific binding. Introduction of halothane into free radical generating system causes acute perturbations to the VSCC kinetics, and does not provide protection to the cardiac membranes.     

Mutagenesis by the autoxidation of iron with isolated DNA.

Oxygen free radicals are highly reactive species generated by many cellular oxidation-reduction processes. These radicals damage cellular constituents and have been causally implicated in the pathogenesis of many human diseases. We report here that oxygen free radicals generated by Fe2+ in aqueous solution are mutagenic. Aerobic incubation of luminal diameter X174 am3 (amber 3 mutation) DNA with Fe2+ results in decreased phage survival when the treated DNA is transfected into Escherichia coli spheroplasts. Transfection of the treated DNA into SOS-induced spheroplasts results in an increase in mutagenesis as great as 50-fold. Both killing and mutagenesis can be prevented by binding of Fe2+ with deferoxamine or by the addition of catalase or mannitol. These results suggest that DNA damage and mutagenesis brought about by Fe2+ are likely to occur by a Fenton-type mechanism that involves the generation of (i) hydrogen peroxide by the autoxidation of iron and (ii) hydroxyl radicals by the interaction of the hydrogen peroxide with Fe2+. DNA sequence analysis of the Fe2+-induced mutants indicates that reversion of the phage phenotype to wild type occurs largely by a transversion type of mutation involving substitution of deoxyadenosine for thymidine opposite a template deoxyadenosine. Mutagenesis is not abolished by incubation of Fe2+-treated luminal diameter X174 am3 DNA with an apurinic endonuclease and only partially abolished by incubation with alkali, suggesting that a large fraction of the mutagenesis by oxygen free radicals is not caused by formation of apurinic sites but instead involves an as-yet-to-be-defined alteration in deoxyadenosine. These findings raise the possibility that free iron localized in cellular DNA may cause mutations by the generation of oxygen free radicals.     

Effects of phorbol ester treatment on dibutyryl cyclic adenosine-5' monophosphate- and carbachol-stimulated aminopyrine accumulation in isolated rat parietal cells

BACKGROUND: The functional role of the intracellular diacylglycerol/protein kinase C second-messenger pathway in the regulation of gastric acid secretion and the effects on the involved inositotrisphosphate/Ca2+/calmodulin system are not well understood, and contradictory data have been reported. We therefore evaluated the effects of phorbol ester treatment (tetradecanoylphorbol-12,13-acetate (TPA)) on dibutyryl cyclic adenosine-5' monophosphate (dBcAMP)- and carbachol-stimulated aminopyrine (AP) accumulation in comparison with intracellular alterations of the phospholipase C/inostol phosphate signal transduction pathway in isolated rat gastric parietal cells. METHODS: [14C]AP accumulation was determined as an indirect measure of gastric acid secretion. Inositolphosphate second-messenger activation was investigated with [3H]inositolmonophosphate release in [3H]-myoinositol prelabeled rat gastric parietal cells. RESULTS: TPA at a low concentration of 5 nM caused a small (45%) but significant increase in carbachol (0.1 mM)-stimulated AP accumulation, which was dose-dependently inhibited by higher concentrations of TPA with corresponding shifts in the dose-response curve for carbachol-stimulated AP accumulation. AP uptake stimulated by dBcAMP (0.1 mM) and the synergistic stimulatory effect induced by carbachol together with dBcAMP were inhibited by TPA at all concentrations investigated. In the presence of increasing concentrations of the calcium ionophore ionomycin (10(-8)-10(-5) M) TPA at 5 nM increased AP accumulation (AP ratio was 4.02 with 5 nM TPA versus 1.23 in the absence of TPA; P < 0.05), indicating that phorbol ester stimulates AP uptake in rat parietal cells. Simultaneous investigation of [14C]AP accumulation and [3H]inositol monophosphate release showed that inhibitory effects of TPA on carbachol- and carbachol plus dBcAMP-stimulated cells are mediated by an inhibition of the receptor/G-protein/phospholipase C interaction, leading to a reduction of inositolphosphate release. The costimulation of rat parietal cells with dBcAMP, ionomycin, and TPA (5 nM) did not reproduce the synergistic effects of carbachol together with dBcAMP on AP accumulation, suggesting that carbachol-stimulated AP uptake seems to be additionally mediated by a still unknown pathway independent of intracellular calcium release or protein kinase C activation.