Histochemistry of reactive oxygen-species (ROS)-generating oxidases in cutaneous and mucous epithelia of laboratory rodents with special reference to xanthine oxidase

Summary Cutaneous and mucous epithelia of various organs of laboratory rodents were analysed histochemically for reactive oxygen species (ROS)-generating oxidases using cerium methods. High activities of xanthine oxidase and also superoxide dismutase were present in orthokeratotic stratified squamous epithelia of skin, lips, esophagus and forestomach and parakeratotic keratinizing stratified epithelia of vagina, tongue and penis. Moreover, activity was found in simple epithelium of the uterus and intestine of rats, mice and guinea-pigs. Moderate activities of monoamine oxidase and d-amino acid oxidase were only seen in enterocytes of large and small intestine, whereas -hydroxy acid oxidase could not be detected at all. With the use of specific inhibitors for superoxide anions-producing xanthine oxidase and H₂O₂-generating superoxide dismutase it was shown that epithelial cells of all studied external and internal surface epithelia contain a highly effective xanthine oxidase-superoxide dismutase system. It is hypothesized that this system might have a general microbicidal function and might play a special role in tumor promotion of the skin.Dedicated to Prof. Dr. T. Günther on the occasion of his 60th birthday     

Reactive oxygen species (ROS)-generating oxidases in the normal rabbit cornea and their involvement in the corneal damage evoked by UVB rays

The corneas of albino rabbits were irradiated (5 min exposure once a day) with UVB rays (312 nm) for 4 days (shorter procedure) or 8 days (longer procedure). The eyes were examined microbiologically and only the corneas of sterile eyes or eyes with non-pathogenic microbes were employed. Histochemically, the activities of reactive oxygen species (ROS)-generating oxidases (xanthine oxidase, D-amino acid oxidase and alpha-hydroxy acid oxidase) were examined in cryostat sections of the whole corneas. Biochemically, the activity of xanthine oxidoreductase/xanthine oxidase was investigated in the scraped corneal epithelium. UVB rays significantly changed enzyme activities in the corneas. In comparison to the normal cornea, where of ROS-generating oxidases only xanthine oxidase showed significant activity in the corneal epithelium and endothelium, D-amino acid oxidase was very low and alpha-hydroxy acid oxidase could not be detected at all, in the cornea repeatedly irradiated with UVB rays, increased activities of xanthine oxidase and D-amino acid oxidase were observed in all corneal layers. Only after the longer procedure the xanthine oxidase and D-amino acid oxidase activities were decreased in the thinned epithelium in parallel with its morphological disturbances. Further results show that the xanthine oxidase/xanthine oxidoreductase ratio increased in the epithelium together with the repeated irradiation with UVB rays. This might suggest that xanthine dehydrogenase is converted to xanthine oxidase. However, in comparison to the normal corneal epithelium, the total amount of xanthine oxidoredutase was decreased in the irradiated epithelium. It is presumed that xanthine oxidoreductase might be released extracellularly (into tears) or the enzyme molecules were denatured due to UVB rays (particulary after the longer procedure). Comparative histochemical and biochemical findings suggest that reactive oxygen species-generating oxidases (xanthine oxidase, D-amino acid oxidase) contribute to the corneal damage evoked by UVB rays.     

Effect of chelating agents and superoxide on human neutrophil NAD(P)H oxidation

NAD(P)H oxidation is frequently measured to assay the activity of the neutrophil O-2-generating oxidase. It was found that 10(-4) M ethylene glycol bis (beta-aminoethyl ether)-N-N'-tetraacetic acid (EGTA) increased NAD(P)H oxidation by the 27,000 g granule fraction of resting and stimulated human neutrophils without altering net O-2 production. The commonly used chelating agents EDTA and diethylene triamine pentaacetic acid had similar effects. The addition of superoxide dismutase eliminated the effect of the chelating agents and thus demonstrated that the stimulated reaction was dependent upon O-2. KCN and bathophenanthroline disulfonate, an iron-chelating agent, prevented O-2-dependent NADPH oxidation by neutrophil granule fractions in the presence of EGTA. In contrast, bathocuproine disulfonate, a copper-chelating agent, mimicked the EGTA effect. The effects of both bathophenanthroline disulfonate and bathocuproine disulfonate were completely abolished when the agents were saturated with iron and copper, respectively. All the chelating agents studied, except bathophenonthroline disulfonate, also promoted O-2-dependent NADPH oxidation in a system wherein O-2 was generated by xanthine oxidase. Thus, commonly used chelating agents, by interacting with available iron and copper, may alter the apparent stoichiometry of the neutrophil O-2-generating oxidase and artifactually increase NADPH oxidation in other systems where O-2 is present.     

Intracellular utilization of superoxide anion by indoleamine 2,3-dioxygenase of rabbit enterocytes.

The participation of superoxide anion (O2-) in the intracellular indoleamine 2,3-dioxygenase activity was studied using the dispersed cell suspension of the rabbit small intestine. The dioxygenase activity was assayed by measuring [14C]formate released from DL-[ring-2-14C]tryptophan. The addition of diethyldiethiocarbamate, a superoxide dismutase inhibitor, markedly accelerated the intracellular dioxygenase activity while the superoxide dismutase activity decreased concomitantly. Furthermore, substrates of xanthine oxidase such as inosine, adenosine, and hypoxanthine also increased the dioxygenase activity in the cells, particularly in the presence of methylene blue. This increase was completely abolished by the addition of allopurinol, a specific inhibitor of xanthine oxidase. These results, taken together, indicate that the intracellular accumulation of O2- results in acceleration of the in situ dioxygenase activity, and that indoleamine 2,3-dioxygenase utilizes O2- in the isolated intestinal cells.     

Studies on indoleamine 2,3-dioxygenase. I. Superoxide anion as substrate.

Indoleamine 2,3-dioxygenase purified to apparent homogeneity from rabbit intestine was inhibited by scavengers for superoxide anion such as superoxide dismutase and 1,2-dihydroxybenzene-3,5-disulfonic acid (Tiron). On the other hand, beta-carotene and 1,4-diazobicyclo-(2,2,2)-octane, scavengers for singlet oxygen, did not affect the enzyme activity significantly. The degree of inhibition of the dioxygenase by superoxide dismutase preparations from bovine erythrocytes, green peas, spinach leaves, and Escherichia coli paralleled that observed with these dismutase preparations on the aerobic reduction of cytochrome c by xanthine oxidase and its substrate. The pH profiles of the inhibition by dismutase of the dioxygenase and cytochrome c reduction were also similar and the maximal inhibition was observed around pH 10 in both cases. The degree of inhibition was not affected by the concentration of substrate but was a function of the concentration of dismutase. It was inversely related to the concentrations of the dioxygenase and its cofactors, ascorbic acid and methylene blue, both of which were required for maximum activity. Ascorbic acid could be replaced either by xanthine oxidase and its substrate, or by tetrabutylammonium superoxide prepared by electrolytic reduction of molecular oxygen, or by potassium superoxide. When limited amounts of superoxide anion were added to the reaction mixture containing a substrate amount of the dioxygenase, the ratio of the amount of superoxide anion added to that of the product formed was approximately unity both under aerobic and anaerobic conditions. Taken together, these findings indicate that superoxide anion, rather than molecular oxygen, is utilized as substrate by indoleamine 2,3-dioxygenase.     

Differences in activities of antioxidant superoxide dismutase, glutathione peroxidase and prooxidant xanthine oxidoreductase/xanthine oxidase in the normal corneal epithelium of various mammals

Under normal conditions, antioxidants at the corneal surface are balanced with the production of reactive oxygen species without any toxic effects. Danger from oxidative stress appears when natural antioxidants are overwhelmed leading to antioxidant/prooxidant imbalance. The aim of the present study was to examine the activities of enzymes contributing to the antioxidant/prooxidant balance in normal corneal epithelium of various mammals. The enzyme activities of antioxidant superoxide dismutase and glutathione peroxidase, as well as prooxidant xanthine oxidoreductase/xanthine oxidase were examined using biochemical methods. Results show that superoxide dismutase activity is high in rabbits and guinea pigs, whereas in pigs the activity is low and in cows it is nearly absent. In contrast, glutathione peroxidase activity is high in cows, pigs and rabbits, whereas in guinea pigs the activity is low. As far as prooxidant enzymes are concerned, elevated xanthine oxidoreductase/xanthine oxidase activities were found in rabbits, lower activities in guinea pigs, very low activity in cows and no activity in pigs. In conclusion, the above results demonstrate inter-species variations in activities of enzymes participating in antioxidant/prooxidant balance in the corneal epithelium. It is suggested that the levels of antioxidant and prooxidant enzymes studied in the corneal epithelium might be associated with the diurnal or nocturnal activity of animals. UV rays decompose hydrogen peroxide to damaging hydroxyl radicals and perhaps for this reason large animals with diurnal activity (cow, pig) require more effective peroxide removal (high glutathione peroxidase activity) together with the suppression of peroxide production (low superoxide dismutase activity, low xanthine oxidoreductase activity).     

Monooxygenase activities of dioxygenases. Benzphetamine demethylation and aniline hydroxylation reactions catalyzed by indoleamine 2,3-dioxygenase

Benzphetamine demethylase and aniline hydroxylase activities were determined with various hemoproteins including indoleamine 2,3-dioxygenase in a cytochrome P-450-like reconstituted system containing NADPH, NADPH-cytochrome P-450 reductase, and O2. The highest specific activities, almost comparable to those of liver microsomal cytochrome P-450, were detected with indoleamine 2,3-dioxygenase from the rabbit intestine. The indoleamine 2,3-dioxygenase-catalyzed benzphetamine demethylation reaction was inhibited by catalase but not by superoxide dismutase. Exogenous H2O2 or organic hydroperoxides was able to replace the reducing system and O2. The stoichiometry of H2O2 added to the product formed was essentially unity. These results indicate that the dioxygenase catalyzes the demethylation reaction by the so-called "peroxygenation" mechanism using H2O2 generated in the reconstituted system. On the other hand, the dioxygenase-catalyzed aniline hydroxylation reaction was not only completely inhibited by catalase but also suppressed by superoxide dismutase by about 60%. Although the O2- and H2O2-generating system (e.g. hypoxanthine-xanthine oxidase) was also active as the reducing system, neither exogenous H2O2 nor the generation of O2- in the presence of catalase supported the hydroxylation reaction, indicating that both H2O2 and O2- were essential for the hydroxylation reaction. However, typical scavengers for hydroxyl radical and singlet oxygen were not inhibitory. These results suggest that a unique, as yet unidentified active oxygen species generated by H2O2 and O2- participates in the dioxygenase-mediated aniline hydroxylation reaction.     

Superoxide anion increases intracellular free calcium in human myometrial cells

We investigated the effects of superoxide anion on the intracellular free calcium concentration ([Ca2+]i) in human cultured myometrial cells using a calcium-sensitive fluorescent dye, indo-1, and a digital imaging fluorescence microscopic system. Hypoxanthine (HX) plus xanthine oxidase induced a rise in [Ca2+]i in a manner dose-dependent on xanthine oxidase. The increase in [Ca2+]i in the absence of extracellular calcium ([Ca2+]ex) was 10% of that in the presence of [Ca2+]ex. Nifedipine, which blocks voltage-sensitive calcium channels, also reduced the increase in [Ca2+]i induced by HX-xanthine oxidase. Superoxide dismutase or superoxide dismutase plus catalase, which metabolizes superoxide anion, inhibited the effect of HX-xanthine oxidase on [Ca2+]i. The desensitization of the effect of superoxide anion on [Ca2+]i was investigated by pulsatile administration of HX and xanthine oxidase. Desensitization was observed on pulsatile administration of HX-xanthine oxidase at 2-min intervals. These data suggest that superoxide production may participate in uterine contraction via [Ca2+]i increase.     

Superoxide-dependent and -independent mechanisms of iron mobilization from ferritin by xanthine oxidase. Implications for oxygen-free-radical-induced tissue destruction during ischaemia and inflammation.

Xanthine oxidase is able to mobilize iron from ferritin. This mobilization can be blocked by 70% by superoxide dismutase, indicating that part of its action is mediated by superoxide (O2-). Uric acid induced the release of ferritin iron at concentrations normally found in serum. The O2(-)-independent mobilization of ferritin iron by xanthine oxidase cannot be attributed to uric acid, because uricase did not influence the O2(-)-independent part and acetaldehyde, a substrate for xanthine oxidase, also revealed an O2(-)-independent part, although no uric acid was produced. Presumably the amount of uric acid produced by xanthine oxidase and xanthine is insufficient to release a measurable amount of iron from ferritin. The liberation of iron from ferritin by xanthine oxidase has important consequences in ischaemia and inflammation. In these circumstances xanthine oxidase, formed from xanthine dehydrogenase, will stimulate the formation of a non-protein-bound iron pool, and the O2(-)-produced by xanthine oxidase, or granulocytes, will be converted by 'free' iron into much more highly toxic oxygen species such as hydroxyl radicals (OH.), exacerbating the tissue damage.     

Detergent-amplified chemiluminescence of lucigenin for determination of superoxide anion production by NADPH oxidase and xanthine oxidase

The detergent-induced amplification of lucigenin-dependent chemiluminescence of O2-, generated by xanthine oxidase or microsomal NADPH oxidase was studied. An assay system is described which is at least 10 times more sensitive than normal lucigenin-dependent chemiluminescence due to the amplification by high concentrations of octylphenylpolyethylene glycol (Triton X-100). Compared to the superoxide dismutase-sensitive reduction of acetylated cytochrome c, a 3750-fold lower amount of microsomal protein was necessary to produce an O2- signal 10-fold above the background. In contrast to cytochrome c reduction, detergent-amplified chemiluminescence of lucigenin was completely inhibited by superoxide dismutase and therefore more selective for O2-. The membrane-bound and Triton X-100-solubilized NADPH oxidase from microsomes of macrophages was activated by ethylene glycol bis(beta-aminoethyl ether)-N,N'-tetraacetic acid and inhibited by Ca2+ and sodium dodecyl sulfate. The membrane-bound enzyme showed a Km value of 1.35 microM, which decreased to 0.95 microM after the addition of 12% (g/g) Triton X-100. The Km and Vmax values of soluble xanthine oxidase were not influenced by Triton X-100, indicating that the enzyme activities were not impaired by the high concentrations of detergent.     

Biosensors for determination of total and natural antioxidant capacity of red and white wines: comparison with other spectrophotometric and fluorimetric methods

Research was carried out to experimentally evaluate the antioxidant capacity of several red and white wines using a superoxide dismutase (SOD) biosensor recently developed by the present authors. Measurements were performed by comparing the biosensor response to increasing concentration of the superoxide radical produced in solution by the xanthine/xanthine oxidase system, both in the presence and absence of the test sample.The results were compared with those of two traditional spectrophotometric methods and of a spectrofluorimetric method described in literature.Lastly, also the polyphenol, sulfite and ascorbic acid contents of the different wine samples examined were measured using a tyrosinase biosensor, a sulfite oxidase biosensor and an ascorbate oxidase biosensor, respectively.     

Reduction of iodonitrotetrazolium violet by superoxide radicals

p-Iodonitrotetrazolium (2-(4-iodophenyl-3-(4-nitrophenyl)- 5-phenyltetrazolium; INT) was reduced to a water-soluble product with an absorbance maxima at about 505 nm (reddish pink) by superoxide anion (O2-.) generated by xanthine/xanthine oxidase. The rate of INT reduction was linearly related to the xanthine oxidase activities, and was inhibited by superoxide dismutase. The soluble product may further be converted to an insoluble product, presumably nonionic formazan, with an absorbance maxima of 490 nm (purplish), under certain conditions, and the rate of the formazan formation depended on pH and protein concentration.     

Allopurinol-insensitive oxygen radical formation by milk xanthine oxidase systems.

Oxygen radical generation in the xanthine- and NADH-oxygen reductase reactions by xanthine oxidase, was demonstrated using the ESR spin trap 5,5'-dimethyl-1- pyrroline-N-oxide. No xanthine-dependent oxygen radical formation was observed when allopurinol-treated xanthine oxidase was used. The significant superoxide generation in the NADH-oxygen reductase reaction by the enzyme was increased by the addition of menadione and adriamycin. The NADH-menadione and -adriamycin reductase activities of xanthine oxidase were assessed in terms of NADH oxidation. From Lineweaver-Burk plots, the Km and Vmax of xanthine oxidase were estimated to be respectively 51 microM and 5.5 s-1 for menadione and 12 microM and 0.4 s-1 for adriamycin. Allopurinol-inactivated xanthine oxidase generates superoxide and OH.radicals in the presence of NADH and menadione or adriamycin to the same extent as the native enzyme. Adriamycin radicals were observed when the reactions were carried out under an atmosphere of argon. The effects of superoxide dismutase and catalase revealed that OH.radicals were mainly generated through the direct reaction of H2O2 with semiquinoid forms of menadione and adriamycin.     

Characterization of free radical generation by xanthine oxidase. Evidence for hydroxyl radical generation

Xanthine oxidase has been hypothesized to be an important source of biological free radical generation. The enzyme generates the superoxide radical, .O2- and has been widely applied as a .O2- generating system; however, the enzyme may also generate other forms of reduced oxygen. We have applied electron paramagnetic resonance (EPR) spectroscopy using the spin trap 5,5'-dimethyl-1-pyrroline-N-oxide (DMPO) to characterize the different radical species generated by xanthine oxidase along with the mechanisms of their generation. Upon reaction of xanthine with xanthine oxidase equilibrated with air, both DMPO-OOH and DMPO-OH radicals are observed. In the presence of ethanol or dimethyl sulfoxide, alpha-hydroxyethyl or methyl radicals are generated, respectively, indicating that significant DMPO-OH generation occurred directly from OH rather than simply from the breakdown of DMPO-OOH. Superoxide dismutase totally scavenged the DMPO-OOH signal but not the DMPO-OH signal suggesting that .O2- was not required for .OH generation. Catalase markedly decreased the DMPO-OH signal, while superoxide dismutase + catalase totally scavenged all radical generation. Thus, xanthine oxidase generates .OH via the reduction of O2 to H2O2, which in turn is reduced to .OH. In anaerobic preparations, the enzyme reduces H2O2 to .OH as evidenced by the appearance of a pure DMPO-OH signal. The presence of the flavin in the enzyme is required for both .O2- and .OH generation confirming that the flavin is the site of O2 reduction. The ratio of .O2- and .OH generation was affected by the relative concentrations of dissolved O2 and H2O2. Thus, xanthine oxidase can generate the highly reactive .OH radical as well as the less reactive .O2- radical. The direct production of .OH by xanthine oxidase in cells and tissues containing this enzyme could explain the presence of oxidative cellular damage which is not prevented by superoxide dismutase.     

Relationship between mechanical dysfunction and depression of sarcolemmal Ca2+-pump activity in hearts perfused with oxygen free radicals

Although in vitro studies have shown that oxygen free radicals depress the sarcolemmal Ca²⁺-pump activity and thereby may cause the occurrence of intracellular Ca²⁺ overload for the genesis of contractile failure, the exact relationship between changes in sarcolemmal Ca²⁺-pump activity and cardiac function due to these radicals is not clear. In this study we examined the effects of oxygen radicals on sarcolemmal Ca²⁺ uptake and Ca²⁺-stimulated ATPase activities as well as contractile force development by employing isolated rat heart preparations. When hearts were perfused with medium containing xanthine plus xanthine oxidase, the sarcolemmal Ca²⁺-stimulated ATPase activity and ATP-dependent Ca²⁺ accumulation were depressed within 1 min whereas the developed contractile force, rate of contraction and rate of relaxation were increased at 1 min and decreased over 3–20 min of perfusion. The resting tension started increasing at 2 min of perfusion with xanthine plus xanthine oxidase. Catalase showed protective effects against these alterations in heart function and sarcolemmal Ca²⁺-pump activities upon perfusion with xanthine plus xanthine oxidase whereas superoxide dismutase did not exert such effects. The combination of catalase and superoxide dismutase did not produce greater effects in comparison to catalase alone. These results are consistent with the view that the depression of heart sarcolemmal Ca²⁺ pump activities may result in myocardial dysfunction due to the formation of hydrogen peroxide and/or hydroxyl radicals upon perfusing the hearts with xanthine plus xanthine oxidase.     

EPR studies on the superoxide-scavenging capacity of the nutraceutical resveratrol

Resveratrol (3,4',5-trihydroxystilbene), a polyphenolic compound found in mulberries, grapes, and red wine, has received considerable attention because of its apparent protective effects against various degenerative diseases due to its potential antioxidant activities. However, direct evidence for the superoxide-scavenging capacity of resveratrol is lacking in literature. In this study, electron paramagnetic resonance spectroscopy in combination with 5-(diethoxyphosphoryl)-5-methylpyrroline-N-oxide (DEPMPO)-spin trapping technique was utilized to determine the ability of resveratrol in scavenging superoxide anions generated from both potassium superoxide and the xanthine oxidase/xanthine system. We have demonstrated here for the first time that the presence of resveratrol resulted in decreased formation of DEPMPO-superoxide adduct (DEPMPO-OOH) in both the potassium superoxide and xanthine oxidase/xanthine systems, indicating that resveratrol could directly scavenge superoxide anions. The inhibition of DEPMPO-OOH in the xanthine oxidase/xanthine system, however, was found to be much potent as compared to that observed in potassium superoxide system. It was further shown that resveratrol could also directly inhibit xanthine oxidase activity as assessed by oxygen consumption and formation of uric acid. Taken together, the dual role of resveratrol in directly scavenging superoxide and inhibiting its generation via xanthine oxidase reported in this study may explain, at least in part, the protective role of this compound against oxidative injury in various disease processes.     

Effects of reactive oxygen and nitrogen species on cyclooxygenase-1 and -2 activities

The effects of reactive oxygen species (superoxide anion radical--O(2)*-, hydrogen peroxide--H(2)O(2) and hydroxyl radical--*OH; the reaction products of xanthine plus xanthine oxidase system) and reactive nitrogen species [nitric oxide--NO*; from 1-hydroxyl-2-oxo-3-(N-methyl-3-aminopropyl)-3-methyl-1-triazene--NOC7 and peroxynitrite--ONOO(-)] on the activities of purified cyclooxygenase (COX)-1 and -2 were studied. Xanthine plus xanthine oxidase suppressed the COX-1 and -2 activities in a xanthine oxidase concentration-dependent fashion. This effect was reversed by addition of catalase to the reactive oxygen species-generating system but not by superoxide dismutase or mannitol, indicating that H(2)O(2) is the responsible metabolite. NOC7 activated the COX-1 activity but inhibited the COX-2 activity at concentrations ranging from 1 to 50 microM. Experiments utilizing a NO* antidote, carboxy-2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl 3-oxide revealed that the observed effects of NOC7 are caused by NO*.ONOO(-), a product of NO* and O(2)*-, both activated and inhibited the COX-1 and -2 activities, depending on ONOO(-) concentration. At a low concentration of ONOO(-) (5 microM) there was enhancement of the COX-1 and -2 activities, but with higher concentrations there was suppression of these two enzyme activities (COX-1, at 200 microM; COX-2, >50 microM). These results suggest that H(2)O(2), NO* and ONOO(-) can have different modulatory effects on the COX-1 and -2 activities.     

Identification of superoxide dismutase in rat liver peroxisomes.

In this paper we have investigated whether or not superoxide dismutase is localized in peroxisomes from rat liver. Using an improved method to prepare peroxisomes from clofibrate induced rat livers, we identified superoxide dismutase activity in peroxisomes. This activity was found to be predominantly of the copper-zinc type. The finding of superoxide dismutase activity in peroxisomes makes sense since peroxisomes also contain superoxide generating enzyme activities such as xanthine oxidase.     

Role of cellular superoxide dismutase against reactive oxygen metabolite injury in cultured bovine aortic endothelial cells.

We examined the protective effect of cellular superoxide dismutase against extracellular hydrogen peroxide in cultured bovine aortic endothelial cells. 51Cr-labeled cells were exposed to hydrogen peroxide generated by glucose oxidase/glucose. Glucose oxidase caused a dose-dependent increase of 51Cr release. Pretreatment with diethyldithiocarbamate enhanced injury induced by glucose oxidase, corresponding with the degree of inhibition of endogenous superoxide dismutase activity. Inhibition of cellular superoxide dismutase by diethyldithiocarbamate was not associated either with alteration of other antioxidant defenses or with potentiation of nonoxidant injury. Enhanced glucose oxidase damage by diethyldithiocarbamate was prevented by chelating cellular iron. Inhibition of cellular xanthine oxidase neither prevented lysis by hydrogen peroxide nor diminished enhanced susceptibility by diethyldithiocarbamate. These results suggest that, in cultured endothelial cells: 1) cellular superoxide is involved in mediating hydrogen peroxide-induced damage; 2) superoxide, which would be generated upon exposure to excess hydrogen peroxide independently of cellular xanthine oxidase, promotes the Haber-Weiss reaction by initiating reduction of stored iron (Fe3+) to Fe2+; 3) cellular iron catalyzes the production of a more toxic species from these two oxygen metabolites; 4) cellular superoxide dismutase plays a critical role in preventing hydrogen peroxide damage by scavenging superoxide and consequently by inhibiting the generation of the toxic species.     

Role of superoxide in the N-oxidation of N-(2-methyl-1-phenyl-2-propyl)hydroxylamine by the rat liver cytochrome P-450 system

The N-oxidation of N-(2-methyl-1-phenyl-2-propyl)hydroxylamine (N-hydroxyphentermine, MPPNHOH) and the N-hydroxylation of 2-methyl-1-phenyl-2-propylamine (phentermine) by reconstituted systems that contained cytochromes P-450 purified from rat liver microsomes were demonstrated. The oxidation of MPPNHOH, but not of phentermine, could also be mediated by a superoxide and hydrogen peroxide generating system that contained xanthine and xanthine oxidase. Superoxide dismutase completely inhibited the oxidation of MPPNHOH by the xanthine/xanthine oxidase system and inhibited by 70% the oxidation mediated by a reconstituted cytochrome P-450 oxidase system. The majority of the microsomal oxidation was inhibited by an antibody raised against the major isozyme of cytochrome P-450 purified from livers of phenobarbital-pretreated rats. 2-Methyl-2-nitroso-1-phenylpropane (MPPNO) was found to be an intermediate in the overall oxidation of MPPNHOH to 2-methyl-2-nitro-1-phenylpropane (MPPNO2). Superoxide dismutase appeared to inhibit the first step, the conversion of MPPNHOH to MPPNO. These observations are accounted for by a sequence of two mechanistically distinct P-450-mediated oxidations. In the first reaction, N-hydroxylation of phentermine occurs by a normal cytochrome P-450 pathway. The formed hydroxylamine then uncouples the cytochrome P-450 system to generate superoxide and hydrogen peroxide. The superoxide oxidizes MPPNHOH to MPPNO which is then oxidized to MPPNO2, the ultimate product. This superoxide-mediated oxidation represents another pathway for N-oxidation by cytochrome P-450.