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.     

Oxidation of 4-methylcatechol: implications for the oxidation of catecholamines

At alkaline pH, 4-methylcatechol oxidizes more rapidly than the related catecholamines: dopamine, norepinephrine, and epinephrine. This oxidation is not inhibited by superoxide dismutase or catalase, indicating that O2 itself is the oxidant, but the reduction potential of O2/O2-* is too low for it to oxidize 4-methylcatechol directly. Instead, O2 oxidizes the 4-methylcatechol semiquinone, which is formed by comproportionation of 4-methylcatechol and its o-quinone. Aniline reacts very quickly with the o-quinone and thus stops the comproportionation reaction that oxidizes 4-methylcatechol to the semiquinone. Oxidation of 4-methylcatechol then requires superoxide, and in the presence of aniline, oxidation of 4-methylcatechol by O2 is inhibited by superoxide dismutase. When catecholamines oxidize, the side chain amine inserts into the catechol o-quinone, forming a bicyclic compound. By eliminating the quinone, this ring closure prevents comproportionation and the consequent oxidation of catecholamines by O2. It also prevents reaction of the quinone with other compounds and the formation of potentially toxic products.     

The Antioxidant properties of <Emphasis Type="Italic">para</Emphasis>-Aminobenzoic acid and its sodium salt

The antioxidant properties of para-aminobenzoic acid, a substance from the group of vitamins, and its sodium salt has been found using the reaction of adrenaline autoxidation in an alkaline medium as a superoxide-generating model system. These compounds inhibited the accumulation of adrenochrome, which is a product of adrenaline oxidation, and the formation of superoxide anions, detected by their reaction with nitro blue tetrazolium. Approaches have been developed to produce a true solution of para-aminobenzoic acid and conditions were established to measure the antioxidant activity of para-aminobenzoic acid and its sodium salt. The antioxidant properties of these compounds indicate their possible participation in the redox reactions of the cell and can also be one reason that they are essential.     

On the mechanism of the Mn3(+)-induced neurotoxicity of dopamine:prevention of quinone-derived oxygen toxicity by DT diaphorase and superoxide dismutase

Dopamine (DA) is rapidly oxidized by Mn3(+)-pyrophosphate to its cyclized o-quinone (cDAoQ), a reaction which can be prevented by NADH, reduced glutathione (GSH) or ascorbic acid. The oxidation of DA by Mn3+, which appears to be irreversible, results in a decrease in the level of DA, but not in a formation of reactive oxygen species, since oxygen is neither consumed nor required in this reaction. The formation of cDAoQ can initiate the generation of superoxide radicals (O2-.) by reduction-oxidation cycling, i.e. one-electron reduction of the quinone by various NADH- or NADPH-dependent flavoproteins to the semiquinone (QH.), which is readily reoxidized by O2 with the concomitant formation of O2-.. This mechanism is believed to underly the cytotoxicity of many quinones. Two-electron reduction of cDAoQ to the hydroquinone can be catalyzed by the flavoprotein DT diaphorase (NAD(P)H:quinone oxidoreductase). This enzyme efficiently maintains DA quinone in its fully reduced state, although some reoxidation of the hydroquinone (QH2) is observed (QH2 + O2----QH. + O2-. + H+; QH. + O2----Q + O2-.). In the presence of Mn3+, generated from Mn2+ by O2-. (Mn2+ + 2H+ + O2-.----Mn3+ + H2O2) formed during the autoxidation of DA hydroquinone, the rate of autoxidation is increased dramatically as is the formation of H2O2. Furthermore, cDAoQ is no longer fully reduced and the steady-state ratio between the hydroquinone and the quinone is dependent on the amount of DT diaphorase present. The generation of Mn3+ is inhibited by superoxide dismutase (SOD), which catalyzes the disproportionation of O2-. to H2O2 and O2. It is noteworthy that addition of SOD does not only result in a decrease in the amount of H2O2 formed during the regeneration of Mn3+, but, in fact, prevents H2O2 formation. Furthermore, in the presence of this enzyme the consumption of O2 is low, as is the oxidation of NADH, due to autoxidation of the hydroquinone, and the cyclized DA o-quinone is found to be fully reduced. These observations can be explained by the newly-discovered role of SOD as a superoxide:semiquinone (QH.) oxidoreductase catalyzing the following reaction: O2-. + QH. + 2H+----QH2 + O2. Thus, the combination of DT diaphorase and SOD is an efficient system for maintaining cDAoQ in its fully reduced state, a prerequisite for detoxication of the quinone by conjugation with sulfate or glucuronic acid. In addition, only minute amounts of reactive oxygen species will be formed, i.e. by the generation of O2-., which through disproportionation to H2O2 and further reduction by ferrous ions can be converted to the hydroxyl radical (OH.). Absence or low levels of these enzymes may create an oxidative stress on the cell and thereby initiate events leading to cell death.     

Redox cycling of 2-(x'-mono, -di, -trichlorophenyl)- 1, 4-benzoquinones, oxidation products of polychlorinated biphenyls

Polychlorinated biphenyl (PCB) preparations are complete liver carcinogens in rodents and efficacious promoters in two-stage hepatocarcinogenesis. Cytochrome P450 isozymes catalyze the oxidation of PCBs to mono- and dihydroxy metabolites. The potential for further enzymatic or nonenzymatic oxidation of ortho- and para-dihydroxy PCB metabolites to (semi)quinones raises the possibility that redox cycling involving reactive oxygen species may be involved in PCB toxicity. Seven synthetic 2-(x'-chlorophenyl)-1, 4-benzoquinones (containing one to three chlorines) were investigated for their participation in oxidation-reduction reactions by following the oxidation of NADPH. These observations were made: (i) NADPH alone directly reduced all quinones but only 2-(2'-chlorophenyl)- and 2-(4'-chlorophenyl)-1,4-benzoquinone supported NADPH consumption beyond that required to quantitatively reduce the quinone. (ii) For all quinones, superoxide dismutase increased NADPH oxidation in excess of the amount of quinone, demonstrating the participation of the superoxide radical. (iii) The presence of microsomal enzymes from rat liver increased the rate of NADPH consumption, but only 2-(2'-chlorophenyl)- and 2-(4'-chlorophenyl)-1,4-benzoquinone autoxidized. (iv) The combination of superoxide dismutase with microsomal enzymes accelerated autoxidation from 1.6- to 6.8-fold higher than that found in the absence of microsomal protein. These data support the concept that in the absence of microsomal protein, there occurs a two-electron reduction of the quinone by NADPH to the corresponding hydroquinone that comproportionates with the large reservoir of quinone to initiate autoxidation. In the presence of microsomes, enzymatic one-electron reduction generates a semiquinone radical whose autoxidation with oxygen propagates the redox cycle. These results show the potential of some 2-(x'-chlorophenyl)-1, 4-benzoquinones to initiate the wasteful loss of NADPH.     

The enzymic oxidation of chlorogenic acid and some reactions of the quinone produced

1. Partially purified preparations of tobacco-leaf o-diphenol oxidase (o-quinol-oxygen oxidoreductase; EC 1.10.3.1) oxidize chlorogenic acid to brown products, absorbing, on average, 1.6atoms of oxygen/mol. oxidized, and evolving a little carbon dioxide. 2. The effect of benzenesulphinic acid on the oxidation suggests that the first stage is the formation of a quinone; the solution does not go brown, oxygen uptake is restricted to 1 atom/mol. oxidized, and a compound is produced whose composition corresponds to that of a sulphone of the quinone derived from chlorogenic acid. 3. Several other compounds that react with quinones affect the oxidation of chlorogenic acid. The colour of the products formed and the oxygen absorbed in their formation suggest that the quinone formed in the oxidation reacts with these compounds in the same way as do simpler quinones. 4. Some compounds that are often used to prevent the oxidation of polyphenols were tested to see if they act by inhibiting o-diphenol oxidase, by reacting with quinone intermediates, or both. 5. Ascorbate inhibits the enzyme and also reduces the quinone. 6. Potassium ethyl xanthate, diethyldithiocarbamate and cysteine inhibit the enzyme to different extents, and also react with the quinone. The nature of the reaction depends on the relative concentrations of inhibitor and chlorogenic acid. Excess of inhibitor prevents the solution from turning brown and restricts oxygen uptake to 1 atom/mol. of chlorogenic acid oxidized; smaller amounts do not prevent browning and slightly increase oxygen uptake. 7. 2-Mercaptobenzothiazole inhibits the enzyme, and also probably reacts with the quinone; inhibited enzyme is reactivated as if the inhibitor is removed as traces of quinone are produced. 8. Thioglycollate and polyvinylpyrrolidone inhibit the enzyme. Thioglycollate probably reduces the quinone to a small extent.     

Use of nitro blue tetrazolium in the reaction of adrenaline autooxidation for the determination of superoxide dismutase activity

The addition of nitro blue tetrazolium (NBT) into the reaction of adrenaline autooxidation allows direct identification of superoxide anion formation (O₂^−⊙) as well as demonstration of kinetics of their accumulation in this superoxide-generating system. The kinetics of adrenochrome and O₂^−⊙ formation has been compared under the same conditions. Three possible approaches to the use of the adrenaline autooxidation reaction for the determination of superoxide dismutase activity (SOD) and revealing antioxidant properties of various compounds are discussed. Two of these approaches have been described previously: the spectro-photometric method of registration of adrenochrome, an end product of adrenaline autooxidation, at 347 nm (Sirota, 1999) and the polarographic method, which measures oxygen consumption used for O₂^−⊙ formation (Sirota, 2011). Here, a novel approach to this problem is presented; it is based on spectrophotometric determination of O₂^−⊙ using NBT. The employment of this approach results in a significant decrease of the pH value of carbonate buffer from 10.5 to 9.7 and a 4-fold decrease in the amounts of added adrenaline, thus creating milder conditions for the revealing and investigation of antioxidant properties of materials being examined.     

Reaction of hydrogen peroxide with ferrylhemoglobin: superoxide production and heme degradation

The reaction of Fe(II) hemoglobin (Hb) but not Fe(III) hemoglobin (metHb) with hydrogen peroxide results in degradation of the heme moiety. The observation that heme degradation was inhibited by compounds, which react with ferrylHb such as sodium sulfide, and peroxidase substrates (ABTS and o-dianisidine), demonstrates that ferrylHb formation is required for heme degradation. A reaction involving hydrogen peroxide and ferrylHb was demonstrated by the finding that heme degradation was inihibited by the addition of catalase which removed hydrogen peroxide even after the maximal level of ferrylHb was reached. The reaction of hydrogen peroxide with ferrylHb to produce heme degradation products was shown by electron paramagnetic resonance to involve the one-electron oxidation of hydrogen peroxide to the oxygen free radical, superoxide. The inhibition by sodium sulfide of both superoxide production and the formation of fluorescent heme degradation products links superoxide production with heme degradation. The inability to produce heme degradation products by the reaction of metHb with hydrogen peroxide was explained by the fact that hydrogen peroxide reacting with oxoferrylHb undergoes a two-electron oxidation, producing oxygen instead of superoxide. This reaction does not produce heme degradation, but is responsible for the catalytic removal of hydrogen peroxide. The rapid consumption of hydrogen peroxide as a result of the metHb formed as an intermediate during the reaction of reduced hemoglobin with hydrogen peroxide was shown to limit the extent of heme degradation.     

Dual effects of superoxide dismutase on the autoxidation of 1,4-naphthohydroquinone

The autoxidation of 1,4-naphthohydroquinone, in a phosphate, EDTA buffer at pH 7.4, exhibits an autocatalysis whose lag phase becomes more pronounced in the presence of either the Cu,Zn- or the Mn-containing superoxide dismutases. In contrast, the autoxidation of a second aliquot of the hydroquinone, added after complete oxidation of the first, is linear and is accelerated by superoxide dismutase. Catalase or inactive superoxide dismutase were without effect in either situation. These results are explicable in terms of a free radical chain reaction which is initially propagated by O2- and then, as the quinone accumulates, by univalent reduction of the quinone by the hydroquinone. Reduction of the quinone by O2- diminishes the overall rate of oxidation. It is not necessary to postulate catalysis by superoxide dismutase of the reduction of the semiquinone by O2-.     

Evidence that a quinone may be required for the production of superoxide and hydrogen peroxide in neutrophils

Neutrophils contain a quinone that may function as an electron carrier during production of superoxide and hydrogen peroxide. First, addition of exogenous coenzyme Q-10, coenzyme Q-6, vitamin K₁, benzoquinone or duroquinone to rat peritoneal neutrophils resulted in increased rates of oxygen consumption and increased rates of hydrogen peroxide and superoxide production. Duroquinone titration studies showed saturation kinetics at submillimolar concentrations for oxygen consumption and for hydrogen peroxide and superoxide production. Second, tropolone, 2-hydroxy-2,4,6-cycloheptatrienone, effectively inhibited oxygen metabolism in neutrophils perhaps because of its structural similarity to quinone. Dibromothymoquinone, a known inhibitor at the quinone level in chloroplasts and mitochondria, was also inhibitory in neutrophils.     

The catalytic effect of the carcinogen "4-nitroquinoline-N-oxide" on the oxidation of vitamin C.

The carcinogen 4-nitroquinoline-N-oxide was found to mediate the reaction between ascorbate and oxygen. The oxidation of ascorbate was initiated by the production of the nitro radical anion which reacted with oxygen to produce the oxygen superoxide radical anion, peroxide and hydroxyl radical. The production of partially reduced oxygen intermediates resulted in additional reactions with ascorbate. The consumption of oxygen could be either completely blocked by reacting the nitro radical with ferricytochrome c or partially blocked by the combined effects of superoxide dismutase and catalase. The consumption of oxygen could be enhanced by reducing the hydroxyl radicals with dimethylsulfoxide.     

The oxidation of phenylhydrazine: superoxide and mechanism.

The oxidation of phenylhydrazine in buffered aqueous solutions is a complex process involving several intermediates. It can be initiated by metal cations, such as Cu2+; in which case EDTA acts as an inhibitor. It can also be intiated by oxyhemoglobin; in which case chelating agents do not interfere. Superoxide radical is both a product of this reaction and a chain propagator. The formation of O2- could be demonstrated in terms of a reduction of nitroblue tetrazolium, which was prevented by superoxide dismutase. The importance of O2- in carrying the reaction chains was shown by the inhibition of phenylhydrazine oxidation by superoxide dismutase. Hydrogen peroxide accumulated during the reaction and could be detected with catalase. The progress of this oxidation could be monitored in terms of oxygen consumption and by following increases in absorbance at 280 or 320 nm. The oxidation was markedly autocatalytic and superoxide dismutase had the effect of extending the lag period. The absorbance at 280 nm was due to an intermediate which first accumulated and was then consumed. This intermediate appears to be benzendiazonium ion. The absorbance at 320 nm was due to a stable product, which was not identified. The time course of oxygen consumption paralleled the increase in absorbance at 320 nm and lagged behind the changes at 280 nm. Exogenous benzenediazonium ion accelerated the oxidation of phenylhydrazine and eliminated the lag phase. Benzenediazonium ion must therefore react with phenylhydrazine to produce a very reactive intermediate, possibly phenyldiazene. A mechanism was proposed which is consistent with the data. The intermediates and products of the oxidation of phenylhydrazine include superoxide radical, hydrogen peroxide, phenylhydrazyl radical, phenyldiazene, and benzenediazonium ion. This is a minimal list: others remain to be detected and identified. It appears likely that the diverse biological effects of phenylhydrazine are largely due to the reactivities of these intermediates and products.     

Effect of superoxide dismutase on the autoxidation of various hydroquinones--a possible role of superoxide dismutase as a superoxide:semiquinone oxidoreductase

The autoxidation of DT-diaphorase-reduced 1,4-naphthoquinone, 2-OH-1,4-naphthoquinone, and 2-OH-p-benzoquinone is efficiently prevented by superoxide dismutase. This effect was assessed in terms of an inhibition of NADPH oxidation (over the amount required to reduce the available quinone), O2 consumption, and H2O2 formation. Superoxide dismutase also affects the distribution of molecular products -hydroquinone/quinone-involved in autoxidation, by favoring the accumulation of the reduced form of the above quinones. In contrast, the rate of autoxidation of DT-diaphorase-reduced 1,2-naphthoquinone is enhanced by superoxide dismutase, as shown by increased rates of NADPH oxidation, O2 consumption, and H2O2 formation and by an enhanced accumulation of the oxidized product, 1,2-naphthoquinone. These findings suggest that superoxide dismutase can either prevent or enhance hydroquinone autoxidation. The former process would imply a possible new activity displayed by superoxide dismutase involving the reduction of a semiquinone by O2-.. This activity is probably restricted to the redox properties of the semiquinones under study, as indicated by the failure of superoxide dismutase to prevent autoxidation of 1,2-naphthohydroquinone.     

The role of catalytic superoxide formation in the O2 inhibition of nitroreductase.

Many nitroreductases are strongly inhibited by oxygen. The first intermediate of nitroreductase activity, the nitroaromatic anion free radical, cannot be detected in aerobic microsomal incubations. Even though the nitro compounds are unchanged, both nitrofurantoin and $\text{p}$-nitrobenzoate profoundly increase the NADPH-supported oxygen uptake. This catalytic oxygen consumption is partially reversed by superoxide dismutase, suggesting that superoxide anion free radical is being formed by the rapid air oxidation of the nitroaromatic anion radical.     

Studies on the NADPH oxidation by subcellular particles from phagocytosing polymorphonuclear leucocytes: evidence for the involvement of three mechanisms

1. The NADPH-oxidizing activity of a 100 000 X g particulate fraction of the postnuclear supernatant obtained frm guinea-pig phagocytosing poymorphonuclear leucocytes has been assayed by simultaneous determination of oxygen consumption, NADPH oxidation and O2- generation at pH 5.5 and 7.0 and with 0.15 mM and 1 mM NADPH. 2. The measurements of oxygen consumption and NADPH oxidation gave comparable results. The stoichiometry between the oxygen consumed and the NADPH oxidized was 1:1. 3. A markedly lower enzymatic activity was observed, under all the experimental conditions used, when the O2- generation assay was employed as compared to the assays of oxygen uptake and NADPH oxidation. 4. The explanation of this difference came from the analysis of the effect of superoxide dismutase and of cytochrome c which removes O2- formed during the oxidation of NADPH. 5. Both superoxide dismutase and cytochrome c inhibited the NADPH-oxidizing reactin at pH 5.5. The inhibition was higher with 1 mM NADPH than with 0.15 mM NADPH. 6. Both superoxide dismutase and cytochrome c inhibited the NADPH-oxidizing reaction at pH 7.0 with 1 mM NADPH but less than at pH 5.5 with 1 mM NADPH. 7. The effect of superoxide dismutase at pH 7.0 with 0.15 mM NADPH was negligible. 8. In all instances the inhibitory effect of cytochrome c was greater than that of superoxide dismutase. 9. It was concluded that the NADPH-oxidizing reaction studied here is made up of three components: an enzymatic univalent reduction of O2; an enzymatic, apparently non-univalent, O2 reduction and a non-enzymatic chain reaction. 10. These three components are variably and independently affected by the experimental conditions used. For example, the chain reaction is freely operative at pH 5.5 with 1 mM NADPH but is almost absent at pH 7.0 with 0.15 mM NADPH, whereas the univalent reduction of O2 is optimal at pH 7.0 with 1 mM NADPH.     

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.     

Discrimination between two possible reaction sequences that create potential risk of generation of deleterious radicals by cytochrome bc1: Implications for the mechanism of superoxide production

In addition to its bioenergetic function of building up proton motive force, cytochrome bc₁ can be a source of superoxide. One-electron reduction of oxygen is believed to occur from semiquinone (SQ_o) formed at the quinone oxidation/reduction Q_o site (Q_o) as a result of single-electron oxidation of quinol by the iron-sulfur cluster (FeS) (semiforward mechanism) or single-electron reduction of quinone by heme b_L (semireverse mechanism). It is hotly debated which mechanism plays a major role in the overall production of superoxide as experimental data supporting either reaction exist. To evaluate a contribution of each of the mechanisms we first measured superoxide production under a broad range of conditions using the mutants of cytochrome bc₁ that severely impeded the oxidation of FeS by cytochrome c₁, changed density of FeS around Q_o by interfering with its movement, or combined these two effects together. We then compared the amount of generated superoxide with mathematical models describing either semiforward or semireverse mechanism framed within a scheme assuming competition between the internal reactions at Q_o and the leakage of electrons on oxygen. We found that only the model of semireverse mechanism correctly reproduced the experimentally measured decrease in ROS for the FeS motion mutants and increase in ROS for the mutants with oxidation of FeS impaired. This strongly suggests that this mechanism dominates in setting steady-state levels of SQ_o that present a risk of generation of superoxide by cytochrome bc₁. Isolation of this reaction sequence from multiplicity of possible reactions at Q_o helps to better understand conditions under which complex III might contribute to ROS generation in vivo.     

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 Km for adrenaline of 8-10 microM. Moreover, the reaction was inhibited by preincubating the sarcolemmal membranes with propranolol, while the alpha-antagonist phentolamine was without effect. Adrenaline oxidation was unaffected by the presence of exogenous linolenic acid or methylarachidonic acid, while arachidonic acid, with a Km for this reaction of 175 microM, 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 A2 and calcium produced a stimulation of adrenochrome formation which was partially suppressed by albumin. In the experiments using arachidonic acid or phospholipase A2, 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.     

Formation of superoxide radicals in the mitochondria of skeletal muscle

The superoxide-dependent oxidation of adrenaline by skeletal muscle mitochondria (maximal inhibition by superoxide dismutase from 50 to 80%) is described. The oxidation reaction is initiated by antimycin A but not by rotenone. It was assumed that the main source of superoxide radicals in skeletal muscle mitochondria is the site of the respiratory chain between the rotenone and antimycin block. It was found that skeletal muscle mitochondria are characterized by a higher rate of superoxide anion formation and by a lower activity of superoxide dismutase as compared to heart mitochondria.     

A convenient approach for evaluating the toxicity profiles of in vitro neuroprotective alkylaminophenol derivatives

The cytotoxicity profiles of a series of quinol-type derivatives were examined through simple Escherichia coli plate assays discriminating the two main cytotoxicity mechanisms associated with polyphenol oxidation to quinone. Toxicity mediated by reactive oxygen species (ROS-TOX) was detected in the OxyR(-) assay using cells sensitive to oxidative stress due to a deficiency in the OxyR function. Toxicity arising from the high susceptibility of quinone toward endogenous nucleophiles (Q-TOX) was detected using OxyR(+) cells, in the presence of a nitric oxide donor to promote the quinol oxidation to the corresponding quinone. The toxicity profile markedly depended on structural features. Strong ROS-TOX required a pyrogallol arrangement (exifone; 2,3,4-trihydroxybenzophenone, 1; baicalein) or a 2-aminoresorcinol sequence (3-amino-2,4-dihydroxybenzophenone, 4). The pyrogallol moiety determined a low Q-TOX, suggesting the conversion of quinones into oxidation products of low toxicity. Compounds lacking a 2-hydroxyl substituent (derivatives 2 and 5, related to 1 and 4, respectively) induced a weak ROS-TOX, but a significant Q-TOX. The electrochemical oxidation of the studied compounds corroborated the crucial role of the 2-hydroxyl group, which had two effects: to protect the quinonoid species from Michael addition, the reaction at the origin of Q-TOX, and, due to the contraction of hydrogen bonding, to stabilize every intermediary oxidation product, very likely involved in ROS-TOX.