Activity of the glutathione antioxidant system and cytochrome P-450 in rat liver under induction and inhibition of xanthine oxidase

The effects of specific xanthine oxidase induction and inhibition on glutathione antioxidant system activity, lipid peroxidation, cytochrome P-450 quantity and corticosteroids concentration in the rat liver were studied. It was dependence established that there was a straight between xanthine oxidase activity and the activity of glutathione antioxidant system, lipid peroxidation and the ascorbic acid formation. The reciprocal dependence was established between xanthine oxidase activity and the concentrations of cytochrome P-450 and corticosteroids.     

The effect of CoCl2 on xanthine oxidase,nitric oxide synthase,and protein kinase C activity as well as cytochrome P450 1A1, 1A2 and 1B1 quantities in rat liver

Established that CoCl2 induced oxidative stress activates xanthine oxidase, inhibit nitric oxide synthase and cytochrome P450 in the rat liver in vivo. The concentration of S-nitrosothiols was respectively decreased and PKC was activated. The quantities of general cytochrome P450 as well as its 1A1, 1A2 and 1B1 isoforms were decreased.     

Comparative study of chicken liver xanthine dehydrogenase and bovine liver xanthine oxidase. dehydrogenase activity of xanthine oxidase (author's transl)]

A method to purify bovine liver xanthine oxidase in described, with which samples of 256-fold specific activity with respect to the initial homogenate are obtained. Bovine liver xanthine oxidase and chicken liver xanthine dehydrogenase with oxygen as electron acceptor exhibit similar profile in pKM and log V versus pH plots. With NAD+ as electron acceptor a different profile in the pKM xanthine plot is obtained for chicken liver xanthine dehydrogenase. However three inflection points at the same pH values appear in all plots. Both enzymes are irreversibly inhibited by pCMB and reversibly by N-ethylmaleimide and by iodoacetamide, with competitive and uncompetitive type inhibitions respectively. These results suggest that NAD+ alters the enzymatic action since its binding to the enzyme antecedes the binding of xanthine to the xanthine oxidase molecule, without undergoing itself any modification. 0.15 M DDT of DTE treatment of bovine liver xanthine oxidase gives to the enzyme a permanent activity with NAD+ without modifying its activity with oxygen. The enzyme thus treated produces parallel straight lines in Lineweaver-Burk plots.     

In vitro study of regulation of rat liver xanthine oxidase activity by antioxidant reducing agents]

It was learned the regulation of xanthine oxidase activity from rat liver in the partly purified prepared by ascorbic acid, glutathione-SH, dithiothreitol, cysteine++ and hydrocortisone++. It was shown that ascorbic acid glutathione-SH, dithiothreitol, and cysteine++ can be activators and uncompetitor inhibitors of xanthine oxidase in dependence from concentration. As far as hydrocortisone is concerned, it is a powerful uncompetitor inhibitor of xanthine oxidase, that is bind with it. It was considered the mechanism of activation and inhibition of xanthine oxidase by these reductors-antioxidants.     

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.     

A role for xanthine oxidase in the loss of cytochrome P-450 evoked by interferon.

It has been suggested that the loss of cytochrome P-450, which is mediated by interferon and its inducers, can result from the generation of free radical species by the enzyme xanthine oxidase. Cytochrome P-450, aminopyrine N-demethylase, and ethoxyresorufin deethylase were depressed by 35, 36, 38%, respectively, in the livers of hamsters 24 h following the administration of a synthetic interferon (IFN-alpha-Con1) which contains the most frequent amino acid sequences of the human subtypes. Interferon increased the activities of the D and O forms of xanthine oxidase by 65 and 74%, respectively, in the same animals. The induction of the D form of xanthine oxidase, which is the precursor of the O form, preceded the loss in cytochrome P-450. The protein synthesis inhibitor, actinomycin D, prevented the interferon-induced loss of drug biotransformation and the increase in xanthine oxidase. The free radical scavenger, alpha-tocopherol, and the xanthine oxidase inhibitor, allopurinol, also prevented the loss of cytochrome P-450 mediated by the interferon inducer poly rI.rC. In chickens in which xanthine oxidase cannot be formed, poly rI.rC had no effect on cytochrome P-450 levels. These results suggest that xanthine oxidase induction may play some role in the interferon-mediated loss of cytochrome P-450.     

A molybdopterin-free form of xanthine oxidase

A previously unidentified fraction lacking xanthine:O2 activity has been isolated during affinity chromatography of bovine milk xanthine oxidase preparations on Sepharose 4B/folate gel. Unlike active, desulfo, or demolybdo forms of xanthine oxidase, this form, which typically comprises about 5% of an unfractionated enzyme solution, passes through the affinity column without binding to it, and is thus easily separated from the other species. The absorption spectrum of this fraction is very similar to that of the active form, but has a 7% lower extinction at 450 nm. Analysis of the fraction has shown that it is a dimer of normal size, but that it does not contain molybdenum or molybdopterin (MPT). The "MPT-free" xanthine oxidase contains 90-96% of the Fe found in active xanthine oxidase, and 100% of the expected sulfide. EPR and absorption difference spectroscopy indicate that the MPT-free fraction is missing approximately half of its Fe/S I centers. The presence of a new EPR signal suggests that an altered Fe/S center may account for the nearly normal Fe and sulfide content. Microwave power saturation parameters for the Fe/S II and Fe/S I centers in the MPT-free fraction are normal, with P1/2 equal to 1000 and 60 mW, respectively. The new EPR signal shows intermediate saturation behavior with a P1/2 = 200 mW. The circular dichroism spectrum of the MPT-free fraction shows distinct differences from that of active enzyme. The NADH:methylene blue activity of the MPT-free fraction is the same as that of active xanthine oxidase which exhibits xanthine:O2 activity, but NADH:cytochrome c and NADH:DCIP activities are diminished by 54 and 37%, respectively.     

Application of electron-donor properties of glucose oxidase and xanthine oxidase for reduction of microsomal NAD(P)H-dependent electron-transport chains

The reduction of cytochromes b5 and P-450 in mammalian hepatic microsomes by glucose oxidase and xanthine oxidase has been investigated. Under anaerobic conditions cytochrome b5 is reduced by glucose oxidase to the "dithionite" level, while cytochrome P-450 remains oxidized. Under the same conditions xanthine oxidase completely reduces both hemoproteins. Besides, neither glucose oxidase nor xanthine oxidase reduces isolated cytochromes. They can be reduced only after addition of microsomes to incubation media. Only in this case are the cytochromes, both isolated and included in microsomal membranes, reduced. The participation of microsomal flavoproteins in the reduction reaction is discussed. The method suggested makes it possible to substantially decrease the rates of reduction of microsomal hemoproteins, thus permitting the investigation of interactions between microsomal NADH- and NADPH-dependent electron-transport chains and electron carriers.     

NADH oxidase activity of rat liver xanthine dehydrogenase and xanthine oxidase-contribution for damage mechanisms

The involvement of xanthine oxidase (XO) in some reactive oxygen species (ROS) -mediated diseases has been proposed as a result of the generation of O*- and H2O2 during hypoxanthine and xanthine oxidation. In this study, it was shown that purified rat liver XO and xanthine dehydrogenase (XD) catalyse the NADH oxidation, generating O*- and inducing the peroxidation of liposomes, in a NADH and enzyme concentration-dependent manner. Comparatively to equimolar concentrations of xanthine, a higher peroxidation extent is observed in the presence of NADH. In addition, the peroxidation extent induced by XD is higher than that observed with XO. The in vivo-predominant dehydrogenase is, therefore, intrinsically efficient at generating ROS, without requiring the conversion to XO. Our results suggest that, in those pathological conditions where an increase on NADH concentration occurs, the NADH oxidation catalysed by XD may constitute an important pathway for ROS-mediated tissue injuries.     

The role of iron chelates in hydroxyl radical production by rat liver microsomes,NADPH-cytochrome P-450 reductase and xanthine oxidase

Uninduced rat liver microsomes and NADPH-Cytochrome P-450 reductase, purified from phenobarbital-treated rats, catalyzed an NADPH-dependent oxidation of hydroxyl radical scavenging agents. This oxidation was not stimulated by the addition of ferric ammonium sulfate, ferric citrate, or ferric-adenine nucleotide (AMP, ADP, ATP) chelates. Striking stimulation was observed when ferric-EDTA or ferric-diethylenetriamine pentaacetic acid (DTPA) was added. The iron-EDTA and iron-DTPA chelates, but not unchelated iron, iron-citrate or iron-nucleotide chelates, stimulated the oxidation of NADPH by the reductase in the absence as well as in the presence of phenobarbital-inducible cytochrome P-450. Thus, the iron chelates which promoted NADPH oxidation by the reductase were the only chelates which stimulated oxidation of hydroxyl radical scavengers by reductase and microsomes. The oxidation of aminopyrine, a typical drug substrate, was slightly stimulated by the addition of iron-EDTA or iron-DTPA to the microsomes. Catalase inhibited potently the oxidation of scavengers under all conditions, suggesting that H₂O₂ was the precursor of the hydroxyl radical in these systems. Very high amounts of superoxide dismutase had little effect on the iron-EDTA-stimulated rate of scavenger oxidation, whereas the iron-DTPA-stimulated rate was inhibited by 30 or 50% in microsomes or reductase, respectively. This suggests that the iron-EDTA and iron-DTPA chelates can be reduced directly by the reductase to the ferrous chelates, which subsequently interact with H₂O₂ in a Fenton-type reaction. Results with the reductase and microsomal systems should be contrasted with results found when the oxidation of hypoxanthine by xanthine oxidase was utilized to catalyze the production of hydroxyl radicals. In the xanthine oxidase system, ferric-ATP and -DTPA stimulated oxidation of scavengers by six- to eightfold, while ferric-EDTA stimulated 25-fold. Ferric-desferrioxamine consistently was inhibitory. Superoxide dismutase produced 79 to 86% inhibition in the absence or presence of iron, indicating an iron-catalyzed Haber-Weiss-type of reaction was responsible for oxidation of scavengers by the xanthine oxidase system. These results indicate that the ability of iron to promote hydroxyl radical production and the role that superoxide plays as a reductant of iron depends on the nature of the system as well as the chelating agent employed.     

Immunohistochemical localization of aldehyde and xanthine oxidase in rat tissues using polyclonal antibodies

Tissues from male Wistar rats, fixed with 4% paraformaldehyde and embedded in paraffin, were studied with immunoperoxidase techniques using polyclonal antibodies raised against aldehyde oxidase or xanthine oxidase purified from rat liver. Immunohistochemical studies demonstrated that aldehyde oxidase-bearing cells were strongly stained in renal tubules, esophageal, gastric, intestinal and bronchial epithelium as well as liver cytoplasm. Weak but positive immunoreactivity was observed on the pulmonary alveolar epithelial cells, gastric glands and intestinal goblet cells. In contrast, it was demonstrated that cells with xanthine oxidase were strongly stained in renal tubules, esophageal, gastric, and small and large intestinal and bronchial epithelia etc. Positive immunostaining was also found in adrenal gland, skeletal muscle, spleen and cerebral hippocampus. Immunoreactivity againt aldehyde oxidase was not found in adrenal gland, spleen, mesentery or aorta, while immunoreactivity against xanthine oxidase was not found in mesentery or aorta. Although the significance of this ubiquitous and similar localization of aldehyde and xanthine oxidase seems unclear at present, these results may provide a clue as to the full understanding of the pathophysiological role of these oxidases in tissues.     

The role of xanthine oxidase in paraquat intoxication.

The role of xanthine oxidase in the mechanism of paraquat toxicity was assessed by in vitro and in vivo experiments. Paraquat stimulated the reduction of cytochrome c by xanthine-xanthine oxidase system in vitro. Paraquat, when added in vitro, stimulated hypoxanthine-dependent superoxide production in the cytosol of rat lung. Tungsten-feeding inhibits xanthine oxidase activity in a variety of tissues in experimental animals. Its therapeutic effect on paraquat intoxication was studied in this paper. In rats fed a tungsten-enriched diet for 5 weeks prior to intraperitoneal injection of 50 mg/kg paraquat dichloride, the mortality decreased significantly compared with rats fed a standard diet. Pretreatment with oxypurinol (1000 mg/kg, s.c.) also ameliorated the paraquat toxicity in rats. We conclude that xanthine oxidase plays an important role in paraquat toxicity and that xanthine oxidase inhibitors may become antidotes for paraquat intoxication.     

Lysosomal enzyme leakage during the hypoxanthine/xanthine oxidase reaction

Impairment of lysosomal stability due to reactive oxygen species generated during the oxidation of hypoxanthine by xanthine oxidase was studied in rat liver lysosomes isolated in a discontinuous Nycodenz gradient. Production of O2.- and H2O2 during the hypoxanthine/xanthine oxidase reaction occurred for at least 5 min, while lysosomal damage, indicated by the release of N-acetyl-beta-glucosaminidase, occurred within 30 s, there being no further damage to these organelles thereafter. The extent of lysosomal enzyme release increased with increasing xanthine oxidase concentration. Superoxide dismutase and catalase did not prevent lysosomal damage during the hypoxanthine/xanthine oxidase reaction. Lysosomes reduced xanthine oxidase activity, as assessed in terms of O2 consumption, only slightly but substantially inhibited in a competitive manner the O2.- -mediated reduction of cytochrome c. This inhibition was almost completely reversed by potassium cyanide, thus pointing to the presence of a cyanide-sensitive superoxide dismutase in the lysosomal fraction. However, potassium cyanide did not affect the hypoxanthine/xanthine oxidase-mediated lysosomal damage, thus suggesting an inability of the lysosomal superoxide dismutase to protect the organelles. Negligible malondialdehyde formation was observed in the lysosomes either during the hypoxanthine/xanthine oxidase reaction or with different selective experimental approaches known to produce lipid peroxidation in other organelles such as microsomes and mitochondria.(ABSTRACT TRUNCATED AT 250 WORDS)     

Age-related increase in xanthine oxidase activity in human plasma and rat tissues

This study assessed the role of xanthine oxidase in vascular ageing. A positive correlation between xanthine oxidase activity and age was found in human plasma. Similar results were found in rat plasma. Xanthine oxidase expression and activity in homogenates from the aortic wall were significantly higher in samples from old rats than in their young counterparts (p < 0.01). In rat skeletal muscle homogenates both xanthine oxidase expression and activity showed a similar age-related profile. Superoxide production by xanthine oxidase in aortic rings was higher in aged rats. Uric acid, the final product of xanthine oxidase has been proposed as a risk factor for coronary heart disease and an independent marker of worse prognosis in patients with moderate-to-severe chronic heart failure. These results give a possible explanation for this correlation and underscore the role of xanthine oxidase in ageing.     

NADH oxidase activity of rat and human liver xanthine oxidoreductase: potential role in superoxide production

To characterise the NADH oxidase activity of both xanthine dehydrogenase (XD) and xanthine oxidase (XO) forms of rat liver xanthine oxidoreductase (XOR) and to evaluate the potential role of this mammalian enzyme as an O₂ ^•− source, kinetics and electron paramagnetic resonance (EPR) spectroscopic studies were performed. A steady-state kinetics study of XD showed that it catalyses NADH oxidation, leading to the formation of one O₂ ^•− molecule and half a H₂O₂ molecule per NADH molecule, at rates 3 times those observed for XO (29.2 ± 1.6 and 9.38 ± 0.31 min⁻¹, respectively). EPR spectra of NADH-reduced XD and XO were qualitatively similar, but they were quantitatively quite different. While NADH efficiently reduced XD, only a great excess of NADH reduced XO. In agreement with reductive titration data, the XD specificity constant for NADH (8.73 ± 1.36 μM⁻¹ min⁻¹) was found to be higher than that of the XO specificity constant (1.07 ± 0.09 μM⁻¹ min⁻¹). It was confirmed that, for the reducing substrate xanthine, rat liver XD is also a better O₂ ^•− source than XO. These data show that the dehydrogenase form of liver XOR is, thus, intrinsically more efficient at generating O₂ ^•− than the oxidase form, independently of the reducing substrate. Most importantly, for comparative purposes, human liver XO activity towards NADH oxidation was also studied, and the kinetics parameters obtained were found to be very similar to those of the XO form of rat liver XOR, foreseeing potential applications of rat liver XOR as a model of the human liver enzyme.     

Genistein effect on xanthine oxidase activity

Genistein was defined to be an allosteric xanthine oxidase inhibitor in the concentrations 0.1-4.0 microM and xanthine oxidase activator with superoxide scavenging activity in the concentrations 5.0 microM and higher. But the most effective allosteric binding with the highest affinity was observed in the genistein concentrations 0.1-1.0 microM. Intraperitoneum injections of genistein (500 micrograms/kg) during three days with the interval 24 hours decrease xanthine oxidase activity in the liver, lung and brain of the Vistar rats.     

Age-related increase in xanthine oxidase activity in human plasma and rat tissues

This study assessed the role of xanthine oxidase in vascular ageing. A positive correlation between xanthine oxidase activity and age was found in human plasma. Similar results were found in rat plasma. Xanthine oxidase expression and activity in homogenates from the aortic wall were significantly higher in samples from old rats than in their young counterparts (p<0.01). In rat skeletal muscle homogenates both xanthine oxidase expression and activity showed a similar age-related profile. Superoxide production by xanthine oxidase in aortic rings was higher in aged rats. Uric acid, the final product of xanthine oxidase has been proposed as a risk factor for coronary heart disease and an independent marker of worse prognosis in patients with moderate-to-severe chronic heart failure. These results give a possible explanation for this correlation and underscore the role of xanthine oxidase in ageing.     

Purification and characterization of mouse liver xanthine oxidase

Xanthine oxidase (EC 1.1.3.22) is purified to homogeneity from mouse liver after induction with bacterial lipopolysaccharide. The enzyme has an apparent molecular weight of 300,000 in its native state and it is suggested to be constituted of two identical subunits of Mr 150,000 each. The isoelectric point is 6.7 and the apparent Km value for xanthine is 3.4 microM. The amino acid composition of mouse xanthine oxidase is quite similar to that of Drosophila xanthine dehydrogenase.     

Quantitation of mRNAs specific for the mixed-function oxidase system in rat liver and extrahepatic tissues during development

Evaluation of ontogenetic expression of the cytochrome P450PCN and cytochrome P450b gene families as well as the NADPH-cytochrome P450 oxidoreductase and epoxide hydrolase genes in Holtzmann rats showed that basal levels of mRNAs encoding these enzymes could be detected in most tissues. Distinct developmental patterns of mRNA expression are evident for these four proteins in liver and extrahepatic tissues. Levels of cytochrome P450b-like mRNA were comparable in adult lung and liver, while cytochrome P450PCN-homologous mRNA exhibited low levels in lung and approximately 100-fold higher levels in liver. Cytochrome P450PCN-homologous mRNA also reached substantial levels in adult intestine, and was also present in placenta, where it increased approximately 4-fold 24 h before birth. Epoxide hydrolase mRNA was demonstrated to be highest in liver followed by kidney, lung, and intestine but was extremely low in brain. NADPH-cytochrome P450 oxidoreductase mRNA in kidney, lung, prostate, adrenal, and intestine exhibited levels comparable to that found in liver; however, the pattern of expression for oxidoreductase mRNA was unique in that levels declined at maturity in liver, kidney, and intestine but not in lung and brain. Development of mixed-function oxidase and epoxide hydrolase activities in liver was distinct from that in other tissues in that mRNAs for all four proteins rose dramatically after parturition. Testis from immature males demonstrated low levels of all the mRNAs assayed, which ranged from 20% (oxidoreductase) to less than 1% (cytochrome P450PCN and epoxide hydrolase) of the levels found in liver.     

Absence of xanthine oxidase or xanthine dehydrogenase in the rabbit myocardium

We directly measured the activity of the enzymes xanthine oxidase and xanthine dehydrogenase in rabbit and rat hearts, using a sensitive radiochemical assay. Neither xanthine oxidase activity nor xanthine dehydrogenase activity was detected in the rabbit heart. In the rat heart, xanthine oxidase activity was 9.1 +/- 0.5 mIU per gram wet weight and xanthine dehydrogenase activity was 53.0 +/- 1.9 mIU per gram wet weight. These results argue against the involvement of the xanthine oxidase/xanthine dehydrogenase system as a mechanism of tissue injury in the rabbit heart, and suggest that the ability of allopurinol to protect the rabbit heart against hypoxic or ischemic damage must be due to a mechanism other than inhibition of these enzymes.