Involvement Of Superoxide Radicals Research Articles

Demethylation and denitrosation of nitrosamines by cytochrome P-450 isozymes

Metabolism of nitrosamines was studied in a reconstituted monooxygenase system composed of cytochrome P-450 isozymes purified from liver microsomes of ethanol- and phenobarbital-treated rats. The ethanol-induced isozyme ( P-450 et) was efficient in catalyzing the demethylation of N-nitrosodimethylamine (NDMA), with a K m of 2.4 m m and V max of 7.2 nmol min −1 nmol P-450 −1, but less active with N-nitrosomethylbenzylamine and N-nitrosomethylaniline. The phenobarbital-induced form ( P-450 b) was ineffective in NDMA metabolism but was active in catalyzing the demethylation of N-nitrosomethylaniline, with an estimated K m of 0.08 m m and a V max of 7.2 nmol min −1 nmol −1. P-450 et also catalyzed the denitrosation of NDMA with a K m of 13.6 m m and a V max of 1.36 nmol min −1 nmol −1. With control liver microsomes, multiple K m values were observed for the demethylation and denitrosation of NDMA. Involvement of superoxide radicals in the metabolism of NDMA was suggested by the action of superoxide dismutase, which inhibited the denitrosation by 43 to 73% and the demethylation by 13 to 22% in different monooxygenase systems. The P-450 et-dependent NDMA demethylation was strongly inhibited by 2-phenylethylamine and 3-amino-1,2,4-triazole; these compounds were previously believed not to be inhibitors of P-450-dependent reactions but were found to inhibit microsomal NDMA demethylase. The present results establish the role of P-450 in nitrosamine metabolism and help to clarify some of the previous confusion in this area of research.

Involvement of superoxide radicals on adrenochrome formation stimulated by arachidonic acid in bovine heart sarcolemmal vesicles

Highly purified sarcolemmal membranes prepared from bovine heart muscle produced superoxide radicals, especially when incubated with NADPH or NADH, as revealed by the oxidation of adrenaline to adrenochrome. The reaction was inhibited by superoxide dismutase or by heat denaturation of the sarcolemmal vesicles. Less evident was the inhibitory effect shown by catalase, while mannitol, deferoxamine or dicumarol were uneffective. The formation of adrenochrome was an oxygen-dependent reaction with a K m for adrenaline of 8–10 μM. Moreover, the reaction was inhibited by preincubating the sarcolemmal membranes with propranolol, while the α-antagonist phentolamine was without effect. Adrenaline oxidation was unaffected by the presence of exogenous linolenic acid or methylarachidonic acid, while arachidonic acid, with a K m for this reaction of 175 μM, showed a marked stimulatory effect. This activation was suppressed by superoxide dismutase, catalase and NaCN, while mannitol was without effect. Moreover, the reaction was blocked by the cyclooxygenase inhibitor indomethacin, differently from the lipooxygenase inhibitor nordihydroguaiaretic acid. Also, the incubation of the sarcolemmal vesicles with phospholipase A 2 and calcium produced a stimulation of adrenochrome formation which was partially suppressed by albumin. In the experiments using arachidonic acid or phospholipase A 2, the addition of indomethacin blocked the adrenaline oxidation. These results indicate that arachidonic acid accentuated the heart sarcolemmal adrenochrome formation presumably by participating in the cyclooxygenase reaction.

The production of free radicals during the autoxidation of cysteine and their effect on isolated rat hepatocytes

Autoxidizing cysteine has been shown to produce thiyl and hydroxyl radicals. Hydrogen peroxide increased the yield of both radicals which was inhibited by catalase but stimulated by copper / zinc superoxide dismutase. This effect is due to increased bydrogen peroxide production by copper / zinc superoxide dismutase as a result of superoxide dismutation. The production of superoxide radicals could not be detected probably because of its low reactivity, however, measurement of oxygen uptake and reduction of ferricytochrome c by autoxidizing cysteine clearly implicate the involvement of super oxide radicals. The production of hydroxyl radicals is postulated to proceed through a Fenton reaction, however, this may not necessarily be metal ion controlled. Autoxidizing cysteine disrupts the integrity of heptocytes causing release of glutathione, adenosine triphosphate and lactate dehydrogenase indicating that it is of little use as a therapeutic agent.

Interconversion of nitrate reductase from Ankistrodesmus braunii related to redox changes

Reversible inactivation of homogeneous nitrate reductase (NAD(P)H: nitrate oxidoreductase, EC 1.6.6.2) from the green alga Ankistrodesmus braunii has been carried out by aerobic incubation of the enzyme with reduced pyridine nucleotide. The involvement of superoxide radicals in the inactivation process is inferred from the fact that it does not take place in the absence of oxygen or in the presence of superoxide dismutase. On the other hand, cyanide also causes the inactivation of the enzyme under reducing conditions. The inactivation of A. braunii nitrate reductase takes place in two steps; the first is the one-electron reduction of the enzyme probably involving the molybdenum centers, and the second, and rate-limiting step, results from the interaction of the reduced enzyme with a nucleophylic agent such as superoxide or cyanide. The mean potential value, at pH 7.5, of the inactivation process, measured by reductive titration with dithionite in the presence of cyanide, was −50 mV. Inactive nitrate reductase, previously dialyzed to remove the inactivating agents, can be immediately reactivated by treatment with ferricyanide in a process requiring the removal of only one electron. This process showed a mean potential value, measured by oxidative titration with ferricyanide, of +230 mV at pH 7.5, independent of the system used to inactivate the enzyme.

Involvement of superoxide radicals in the formation of methemoglobin from oxyhemoglobin: Inhibition of superoxide dismutase by diethyldithiocarbamate.

Diethyldithiocarbamate (DDC), a copper chelate agent, was used to examine a possible participation of superoxide radicals (O2-) in the formation of methemoglobin (metHb) under the O2- generating system of photoactivated riboflavin. Superoxide dismutase in the lysate from DDC-injected rats was inhibited to 93% and catalase remained unchange, however, the formation of metHb was scarcely observed. In the experiments in vitro, superoxide dismutase was completely inhibited by the pretreatment with DDC above 3 mM whereas catalase was partially inhibited. In this case, the formation of metHb was accelerated with increase of DDC concentration. Addition of catalase to the dialyzed lysate resulted in the inhibition of formation of metHb to almost the same level as that in the normal lysate. Superoxide dismutase alone, however, caused a slight increase in the rate of formation of metHb, indicating that catalase but not superoxide dismutase has protective ability to the formation of metHb by O2-. From these results, O2-appears to be the source of hydrogen peroxide which accounts for most the observed metHb formation.

Free radical and ionic reaction of bisulfite with reduced nicotinamide adenine dinucleotide and its analogs

1,4-Dihydronicotinamide adenine dinucleotide (NADH) and its analogues undergo two reactions in sulfite buffers in the pH range 5.5-7.1: (1) an oxygen-mediated free-radical chain reaction which results in the oxidation of the dihydropyridine to the pyridinium salt, and (2) an ionic reaction which results in the hydration of the 5,6 double bond of the dihydropyridine. The free-radical reaction is inhibited by superoxide dismutase (indicating the involvement of superoxide radicals) and by free-radical inhibitors. The ionic reaction is not affected by free-radical inhibitors and follows the rate law: rate = [substrate] [HSO3-] (K + SIGMAK' [HA]), where HA is a general acid of hydronium ion. The occurrence of third-order terms of the type [substrate] X [HSO3-] [HA] is consistent with the formation of a reactive bisulfite-substrate complex, which undergoes general acid catalyzed hydration.