Suppression of tumor promoter phorbolmyristate acetate-induced chromosome breakage by antioxidants and inhibitors of arachidonic acid metabolism

To gain insight into the mechanism of formation of chromosomal aberrations by the tumor promoter phorbolmyristate acetate (PMA) in human lymphocytes, we investigated the effect of antioxidants and inhibitors of arachidonic acid metabolism. Among the antioxidants bovine erythrocyte CuZn superoxide dismutase, glutathione peroxidase, mannitol (a scavenger of hydroxyl radicals), butylated hydroxytoluene and butylated hydroxyanisole were anticlastogenic while catalase and dimethylfuran (a scavenger of singlet oxygen) were inactive. These results show that the induction of aberrations by PMA occurs via indirect action, i.e. the intermediacy of superoxide and hydroxyl radicals. The following inhibitors of arachidonic acid metabolism were strongly anticlastogenic: the cyclo-oxygenase inhibitors indomethacin and flufenamic acid and the lipoxygenase inhibitor BN1015. Imidazole, nordihydroguaiaretic acid BN 1048 and 5,8,11,14-eicosatetraynoic acid were moderately active. The inhibitor of phospholipase A₂, fluocinolone acetonide, was also anticlastogenic.

We conclude that the oxidative metabolism of arachidonic acid is involved in the induction of chromosomal aberrations by PMA in human lymphocytes. However, because of the limited selectivity of these drugs, it is not yet possible to identify unambiguously the step(s) in the arachidonic acid cascade responsible for PMA clastogenicity.     

The effects of arachidonic and other fatty acids on canine neutrophil metabolism

The effect of arachidonic acid on the metabolic activity and chemiluminesence of canine neutrophils was investigated to gain further insight into its role in the neutrophil metabolic burst. Arachidonic acid was found to stimulate metabolic activity and luminol-augmented chemiluminescence. The increased metabolic activity was detected by both oxygen uptake measurements and assays of hexose monophosphate shunt activity. An inhibitor of lipoxygenase and cyclooxygenase,5, 8, 11, 14-eicosatetraynoic acid prevented the hexose monophosphate shunt response to arachidonic acid. Aspirin or indomethacin, blockers of cyclooxygenase, inhibited chemiluminescence but failed to block the metabolic response to arachidonic acid. Since superoxide dismutase and 2-deoxyglucose, a blocker of glucose metabolism, inhibited the chemiluminescent response of neutrophils to arachidonic acid, it is likely that oxygen radicals produced via the hexose monophosphate shunt are required for the chemiluminescent reaction. In addition it was found that inhibition of cyclooxygenase activity blocked chemiluminescence but not the metabolic stimulation induced by sodium fluoride, suggesting that the chemiluminescence stimulated by sodium fluoride is associated with endogenous fatty acid stores. From these studies it can be concluded that arachidonic acid products of the cyclooxygenase pathway do not play a significant role in the metabolic response of neutrophils when arachidonic acid or sodium fluoride is the stimulant while the lipoxygenase pathway appears to be involved. The metabolic response is not linked to the chemical reaction that causes neutrophil, chemiluminesence, although the chemiluminescent response depends on hexose monophosphate shunt activity and presumably the oxygen radicals that ultimately result from that process.     

Fatty acid changes during the induction of differentiation of human promyelocytic leukemia (HL-60) cells by phorbolmyristate acetate.

We studied fatty acid changes that are likely to occur during phorbolmyristate acetate (PMA)-induced differentiation of HL-60 cells. It was observed that PMA-induced differentiation is associated with increased uptake, but not synthesis, of fatty acids. Fatty acid analysis revealed that arachidonic acid (AA), 20:5 n-3 and 22:6 n-3 levels are reduced with a concomitant increase in 22:5 n-6 in the phospholipid fraction. In the FFA fraction there are increases in free AA, free 20:5 n-3, 22:5 n-3 and 22:6 n-3, and a fall in free 22:5 n-6 in PMA-treated cells. PMA-induced differentiation and nitroblue tetrazolium reduction by PMA-treated cells was only partially inhibited (about 20-30%) by indomethacin and nordihydroguiaretic acid (cyclooxygenase and lipoxygenase inhibitors respectively), but not by superoxide dismutase, catalase or mannitol. These results indicate that PMA-induced differentiation of HL-60 cells is accompanied by specific changes in the fatty acid composition of the cells.     

A novel approach to study the reaction of adrenaline autooxidation: A possibility for polarographic determination of superoxide dismutase activity and antioxidant properties of various preparations

The reaction of adrenaline autooxidation in an alkaline carbonate buffer followed by formation of superoxide radicals and the product of its oxidation, adrenochrome, which models the quinone pathway of adrenaline metabolism in the body, is accompanied by oxygen consumption. A study of this process by the polarographic method enabled one to apply this reaction to determine the activity of superoxide dismutase and antioxidant properties of biological and chemical compounds; it is based on evaluation of a latent period and the rate of oxygen consumption, which are measured in the presence of examined compounds. It was suggested that known neuroand cardiotoxicity of the quinone products of adrenaline oxidation may be associated not only to their intrinsic properties and reactive oxygen species formed but also local hypoxia of those regions of the cell and tissue where the quinone oxidation of adrenaline occurs.     

NAD(P)H oxidation elicits anion superoxide formation in radish plasmalemma vesicles

Radish plasmalemma-enriched fractions show an NAD(P)H-ferricyanide or NAD(P)H-cytochrome c oxidoreductase activity which is not influenced by pH in the 4.5-7.5 range. In addition, at pH 4.5-5.0, NAD(P)H elicits an oxygen consumption (NAD(P)H oxidation) inhibited by catalase or superoxide dismutase (SOD), added either before or after NAD(P)H addition. Ferrous ions stimulate NAD(P)H oxidation, which is again inhibited by SOD and catalase. Hydrogen peroxide does not stimulate NADH oxidation, while it does stimulate Fe2+-induced NADH oxidation. NADH oxidation is unaffected by salicylhydroxamic acid and Mn2+, is stimulated by ferulic acid, and inhibited by KCN, EDTA and ascorbic acid. Moreover, NADH induces the conversion of epinephrine to adrenochrome, indicating that anion superoxide is formed during its oxidation. These results provide evidence that radish plasma membranes contain an NAD(P)H-ferricyanide or cytochrome c oxidoreductase and an NAD(P)H oxidase, active only at pH 4.5-5.0, able to induce the formation of anion superoxide, that is then converted to hydrogen peroxide. Ferrous ions, sparking a Fenton reaction, would stimulate NAD(P)H oxidation.     

Standardization and regulation of the rate of the superoxide-generating reaction of adrenaline autoxidation used for evaluation of pro/antioxidant properties of various materials

The superoxide-generating reaction of adrenaline autoxidation is widely used for determination of superoxide dismutase activity and pro/antioxidant properties of various materials. There are two variants of the spectrophotometric registration of the products of this reaction. The first one is based on registration of adrenochrome (a product of adrenaline autoxidation) at 347 nm; the second approach employs nitro blue tetrazolium (NBT) and registration of diformazan (a product of NBT reduction) at 560 nm. In the present work, recommendations for the standardization of the reaction rate in both variants have been given. The main approach consists in the use of a pharmaceutical form of 0.1% adrenaline hydrochloride solution. Although each of two adrenaline preparations available in the Russian market has some individual features in kinetic behavior of adrenaline autoxidation, they are applicable for the superoxide generating system. Performing measurements at 560 nm, the reaction rate can be regulated by lowering concentration of added adrenaline, whereas during spectrophotometric registration at 347 nm, this is not applicable. These features of the adrenaline autoxidation reaction may be attributed to the multistage process of adrenaline conversion to adrenochrome and also to coupled electron transfer from adrenaline and intermediate products of its oxidation to oxygen, carbon dioxide, and carbonate bicarbonate ions. This results in formation of corresponding radicals detectable by adding NBT.     

Superoxide dismutase triggers activation of "primed" platelets

Superoxide dismutase (SOD) triggers activation of human platelets exposed to subthreshold concentrations of arachidonic acid and collagen. The subthreshold concentrations used are not able to activate platelets but "prime" platelets to be activated by SOD. The addition of SOD to arachidonic acid-or collagen-primed platelets induced aggregation, thromboxane A2 production, and release of [3H]serotonin. Superoxide dismutase does not have any effect on resting platelets and ADP-, thrombin-, calcium ionophore A23187-, PAF-, or U46619-stimulated platelets. Furthermore, superoxide dismutase-dependent platelet activation is fully prevented by catalase and/or aspirin, suggesting a role for H2O2 and the involvement of the cyclooxygenase pathway of arachidonic acid in such activation.     

Relationship between the reduction of oxygen, artificial acceptors and cytochrome P-450 by NADPH--cytochrome c reductase.

The interaction of NADPH--cytochrome c reductase with oxygen, artificial acceptors and cytochrome P-450 was studied. The generation of superoxide anion radicals (O2-.) from the oxidation of adrenaline to adrenochrome catalysed by NADPH--cytochrome c reductase proceeds independently of the interaction of the enzyme with the artificial anaerobic acceptors cytochrome c or 2,6-dichlorophenol-indophenol. Propyl 3,4,5-trihydroxybenzoate inhibited competitively the adrenaline oxidation by isolated NADPH--cytochrome c reductase (Ki 3.2--4.7 micrometer) and inhibited non-competitively the cytochrome c reduction (Ki 92--109 micrometer). In contrast with the process of electron transfer to cytochrome c, the rate of reduction of cytochrome P-450 and the rate of oxidation of adrenaline in liver microsomal fraction are correlated. Hexobarbital increases the Vmax. of adrenaline oxidation without affecting the Km value, whereas metyrapone, a metabolic inhibitor decreases Vmax. without affecting the Km. From the results obtained, some conclusions about NADPH--cytochrome c reductase function were made.     

Interrelationship between the generation of oxygen anion-radicals and the reduction of artificial acceptors and cytochrome P-450 by NADPH-cytochrome c reductase]

The interaction of NADPH-cytochrome c reductase with oxygen, artificial acceptors and cytochrome P-450 is investigated. It is found that generation of oxygen anion-radicals (O2-), determined from the reaction of adrenaline oxidation into adrenochrome, proceeds independently on the reactions of interaction with artificial "anaerobic" acceptors-cytochrome c, dichlorophenolindophenol. Propylgallate competitively inhibits the reaction of adrenaline oxidation by isolated DADPH-cytochrome c reductase and non-competitively suppress the reaction of cytochrome c reduction. In contrast to the process of electron transfer on cytochrome c, there is a direct correlation between the rate of cytochrome P-450 reduction and the rate of adrenaline oxidation in liver microsomes. Hexobarbital increases V of the adrenaline oxidation reaction and does not affect the Km value, while metirapon, a metabolic inhibitor, decreases the Vmax and does not change Km. On the basis of the data obtained it is suggested that the reactions of NADPH-cytochrome c reductase interaction with oxygen and artificial "anaerobic" acceptors are connected with different redox-states of flavoprotein or with different flavine coenzymes, and that the electron transport on cytochrome P-450 and directly on oxygen takes place in interrelated redox-states of flavoprotein.     

Inhibition of O2-- and HO - Mediated Processes by a New Class of Free Radical Scavengers: The N-ACYL Dehydroalanines

N-phenylacetyl dehydroalanines are captodative olefins. They inhibit two processes mediated by superoxide anion (O₂-) in a concentration dependent manner: reduction of NBT to blue formazan and oxidation of epinephrine to adrenochrome. They also inhibit in a dose related way the degradation of deoxyribose produced during either the Fenton reaction or the radiolysis of water, which are the two experimental sources of hydroxyl radical (HO^-) production. Based on the results obtained with superoxide dismutase, mannitol, thiourea, and uric acid, we postulate that these competitive inhibitory effects suggest a reaction between the dehydroalanine derivatives and the two oxygen derived radicals. Hydroxyl free radical is scavenged more efficiently than superoxide anion. Substitution of the phenyl ring by methoxy groups does not modify significantly the activity. These molecules possess three target active sites which can react with free radicals.     

Inhibitory effect of superoxide radicals on cardiac myofibrillar ATPase activity

The superoxide radicals generated by the xanthine oxidase reaction reduced the myofibrillar Ca2+-ATPase activity. This negative effect was prevented by superoxide dismutase or by dithiothreitol, a protective thiol compound. Partial protection was achieved by catalase, while mannitol was ineffective. The myofibrillar Ca2+-ATPase exposed to O2-. radicals did not modify the affinity for Ca2+ while it showed a remarkable reduction of Vmax measured at the saturating level of Ca2+. The O2-. inhibited myofibrillar ATPase showed a higher value of Km for the cofactor associated to a reduced value of Vmax when studied in the presence of increasing concentration of ATP. Thus, circumstances that enhance the production of cardiac O2- radicals can be considered a negative metabolic event capable of depressing the myofibrillar Ca2+-ATPase activity.     

Effect of drugs on oxidation and precipitation of the isolated chains of human hemoglobin

Summary The paper deals with the action of: primaquine, epinephrine, adrenochrome, acetylphenylhydrazine and sulphanilamide on the autoxidation of the isolated chains from human hemoglobin and on the precipitation which follows. The effect of superoxide dismutase and catalase on the drug induced autoxidation allows the assessment of the possible role of 0₂ derivatives (notably superoxide or peroxide) in the overall reaction mechanism. It is also shown that primaquine and acetylphenylhydrazine enhance precipitation of the isolated oxidized chains, while epinephrine and adrenochrome display a small inhibitory effect on precipitation. These effects do not involve O₂ radicals, but have presumably to be related to a destabilizing (or stabilizing) action of the drugs on the structure of the protein.     

Inhibition of O2−- and HO - Mediated Processes by a New Class of Free Radical Scavengers: The N-ACYL Dehydroalanines

N-phenylacetyl dehydroalanines are captodative olefins. They inhibit two processes mediated by superoxide anion (O₂-) in a concentration dependent manner: reduction of NBT to blue formazan and oxidation of epinephrine to adrenochrome. They also inhibit in a dose related way the degradation of deoxyribose produced during either the Fenton reaction or the radiolysis of water, which are the two experimental sources of hydroxyl radical (HO^?) production. Based on the results obtained with superoxide dismutase, mannitol, thiourea, and uric acid, we postulate that these competitive inhibitory effects suggest a reaction between the dehydroalanine derivatives and the two oxygen derived radicals. Hydroxyl free radical is scavenged more efficiently than superoxide anion. Substitution of the phenyl ring by methoxy groups does not modify significantly the activity. These molecules possess three target active sites which can react with free radicals.     

NADH- and NADPH-dependent formation of superoxide anions by bovine heart submitochondrial particles and NADH–ubiquinone reductase preparation

1. Both NADH and NADPH supported the oxidation of adrenaline to adrenochrome in bovine heart submitochondrial particles. The reaction was completely inhibited in the presence of superoxide dismutase, suggesting that superoxide anions (O(2) (-)) are responsible for the oxidation. The optimal pH of the reaction with NADPH was at pH7.5, whereas that with NADH was at pH9.0. The reaction was inhibited by treatment of the preparation with p-hydroxymercuribenzoate and stimulated by treatment with rotenone. Antimycin A and cyanide stimulated the reaction to the same extent as rotenone. The NADPH-dependent reaction was inhibited by inorganic salts at high concentrations, whereas the NADH-dependent reaction was stimulated. 2. Production of O(2) (-) by NADH-ubiquinone reductase preparation (Complex I) with NADH or NADPH as an electron donor was assayed by measuring the formation of adrenochrome or the reduction of acetylated cytochrome c which does not react with the respiratory-chain components. p-Hydroxymercuribenzoate inhibited the reaction and rotenone stimulated the reaction. The effects of pH and inorganic salts at high concentrations on the NADH- and NADPH-dependent reactions of Complex I were essentially similar to those on the reactions of submitochondrial particles. 3. These findings suggest that a region between a mercurialsensitive site and the rotenone-sensitive site of the respiratory-chain NADH dehydrogenase is largely responsible for the NADH- and NADPH-dependent O(2) (-) production by the mitochondrial inner membranes.     

NADPH2 and organic hydroperoxide-dependent oxidation of adrenaline to adrenochromes in liver and brain microsomes

It was shown that oxidation of adrenaline to adrenochrome in microsomal membranes of the brain and liver in the presence of NADP . H2 or NAD . H2 is mainly accounted for by the formation of a superoxide anion radical. The formation of adrenochrome from adrenalin was found to depend on organic hydroperoxides (natural and synthetic). The organic hydroperoxide-dependent oxidation of adrenochrome involves singlet oxygen. In microsomal fractions of the liver the organic peroxide-dependent oxidation of adrenalin was catalyzed by cytochrome P-450.     

Adriamycin stimulated superoxide formation in submitochondrial particles.

Adriamycin (doxorubicin), an anticancer agent, stimulated the formation of superoxide in submitochondrial particles isolated from bovine heart. Superoxide formation was detected by oxygen uptake, by the cooxidation of epinephrine to adrenochrome and by the reduction of acetylated cytochrome c. These processes were sensitive to superoxide dismutase (SOD). Rotenone-insensitive oxidation of NADH by the mitochondrial respiratory chain in the presence of oxygen caused the formation of approx 4 nmol of superoxide per min/mg of protein. Adriamycin at a concentration of 400 micron stimulated the rate of superoxide formation 6-fold to 25 nmol.min-1.mg-1, but this was not a maximum rate. Approximately 50 micron adriamycin was estimated to be sufficient for obtaining one-half maximal stimulation. Hydrogen peroxide accumulated as a final reaction product. Measurements of the relative catalase activity of blood-free tissues of rabbits and rats indicated that heart contained 2 to 4% of the catalase activity of liver or kidney. An enhanced production of superoxide and hydrogen peroxide and the relatively low catalase content of heart tissue may be factors in the cardiotoxicity induced by adriamycin chemotherapy if a similar reaction occurs in vivo.     

Superoxide and hydrogen peroxide production and NADPH oxidation stimulated by nitrofurantoin in lung microsomes: possible implications for toxicity.

The addition of nitrofurantoin to aerobic incubation mixtures containing rat lung microsomes strongly enhanced the generation of adrenochrome from epinephrine. Adrenochrome formation in this system was blocked by superoxide dismutase, but not by catalase. Hydrogen peroxide production was also strongly enhanced by nitrofurantoin in these preparations; superoxide dismutase did not significantly alter the amount of H₂O₂ measured, but no H₂O₂ was detected in incubation mixtures in the presence of catalase. Nitrofurantoin enhanced the oxidation of NADPH in lung microsomal suspensions under aerobic conditions; the enhancement was unaffected by catalase but was partially prevented by superoxide dismutase. Neither adrenochrome formation nor H₂O₂ production were enhanced by nitrofurantoin under anaerobic (N₂) conditions, but NADPH oxidation in the presence of nitrofurantoin was greater under anaerobic conditions than under aerobic conditions. These results are consistent with the view that the redox cycling of nitrofurantoin in lung microsomes in the presence of oxygen results in the consumption of NADPH and the production of activated oxygen species, emphasizing some $\text{in vitro}$ metabolic similarities with the lung-toxic herbicide, paraquat.     

Direct and respiratory chain-mediated redox cycling of adrenochrome

Adrenochrome is reduced by ascorbate in a reaction accompanied by a large and rapid oxygen uptake. The rates of adrenochrome reduction and the concomitant oxygen uptake are decreased in the presence of superoxide dismutase or catalase. The species formed on the one-electron reduction of adrenochrome (i.e., the semiquinone) was shown by pulse radiolysis to rapidly react with oxygen (9.10(8) M-1.s-1), indicating the occurrence of a redox cycling in a system formed by adrenochrome, a reducing agent, and oxygen. Adrenochrome is also reduced to the corresponding semiquinone by complex I of beef heart submitochondrial particles supplemented with NADH, while succinate is unable to support this reduction. The o-semiquinone is the intermediate species in the superoxide-generating cycle resulting from both non-enzymatic and enzymatic reduction. The toxic effects of adrenochrome and its pathophysiological role can be explained, at least in part, on the basis of the demonstrated cycle.     

Superoxide dismutase and catalase protect cultured hepatocytes from the cytotoxicity of acetaminophen

Superoxide dismutase, catalase and mannitol prevent the killing of cultured hepatocytes by acetaminophen in the presence of an inhibitor of glutathione reductase, BCNU. Under these conditions, the cytotoxicity of acetaminophen depends upon its metabolism, since beta-naphthoflavone, an inhibitor of mixed function oxidation, prevents the cell killing. In hepatocytes made resistant to acetaminophen by pretreatment with the ferric iron chelator, deferoxamine, addition of ferric or ferrous iron restores the sensitivity to acetaminophen. In such a situation, both superoxide dismutase and catalase prevent the killing by acetaminophen in the presence of ferric iron. By contrast, catalase, but not superoxide dismutase, prevents the cell killing dependent upon addition of ferrous iron. These results document the participation of both superoxide anion and hydrogen peroxide in the killing of cultured hepatocytes by acetaminophen and suggest that hydroxyl radicals generated by an iron catalyzed Haber-Weiss reaction mediate the cell injury.     

Prostaglandin H synthase: distinct binding sites for cyclooxygenase and peroxidase substrates

Prostaglandin H synthase has two distinct catalytic activities: a cyclooxygenase activity that forms prostaglandin G2 from arachidonic acid; and a peroxidase activity that reduces prostaglandin G2 to prostaglandin H2. Lipid hydroperoxides, such as prostaglandin G2, also initiate the cyclooxygenase reaction, probably via peroxidase reaction cycle enzyme intermediates. The relation between the binding sites for lipid substrates of the two activities was investigated with an analysis of the effects of arachidonic and docosahexaenoic acids on the reaction kinetics of the peroxidase activity, and their effects on the ability of a lipid hydroperoxide to initiate the cyclooxygenase reaction. The cyclooxygenase activity of pure ovine synthase was found to have an apparent Km value for arachidonate of 5.3 microM and a Ki value (competetive inhibitor) for docosahexaenoate of 5.2 microM. When present at 20 microM neither fatty acid had a significant effect on the apparent Km value of the peroxidase for 15-hydroperoxyeicosatetraenoic acid: the values were 7.6 microM in the absence of docosahexaenoic acid and 5.9 microM in its presence, and (using aspirin-treated synthase) 13.7 microM in the absence of arachidonic acid and 15.7 microM in its presence. Over a range of 1 to 110 microM the level of arachidonate had no significant effect on the initiation of the cyclooxygenase reaction by 15-hydroperoxyeicosatetraenoic acid. The inability of either arachidonic acid or docosahexaenoic acid to interfere with the interaction between the peroxidase and lipid hydroperoxides indicates that the cyclooxygenase and peroxidase activities of prostaglandin H synthase have distinct binding sites for their lipid substrates.