Seasonal variation of xanthine oxidoreductase activity in the digestive gland cells of the mussel Mytilus galloprovincialis: a biochemical, histochemical and immunochemical study

The activity and the tissue distribution of the oxygen radical producing enzyme xanthine oxidoreductase (XOR) were measured in the digestive gland of the common marine mussel Mytilus galloprovincialis Lmk along an annual cycle. No xanthine oxidase (XOX) activity could be measured, the enzyme only displaying xanthine dehydrogenase (XDH) activity in all the cases. This is interpreted as a mechanism to avoid the harmful effects of the oxygen radicals that would be produced by XOX during periods following anoxic conditions at low tide. The highest XDH activities coincided with the late spring/early summer months, the activity maxima being recorded from May to July. Histochemically XOR activity was very pronounced in duct and stomach epithelial cells as well as in the surrounding connective tissue and hemolymph vessels, the activity increasing towards the summer months. These seasonal variations in XDH or XOR activities are possibly linked to hormonal changes governing the reproductive cycle and to changes in food availability. The localization of the protein in the connective tissue lining the hemolymph vessels was confirmed immunohistochemically using a polyclonal antibody against rat liver protein that cross-reacted specifically with a polypeptide of 150 kDa of molecular mass in homogenates of the digestive gland. This polypeptide was linked to cytosolic fractions isolated by differential centrifugation from mussel digestive glands. In paraffin sections the antibody labeled the digestive cells of digestive tubules, as well as the connective tissue surrounding the hemolymph vessels, gonadal follicles, digestive epithelia and certain protozoan parasites. Taken together our results suggest that in the digestive gland of bivalve molluscs XOR is involved in the metabolism of purines and in the scavenging of oxygen free radicals.     

Oxidative stress to human lymphocytes by xanthine oxidoreductase activity

The in vitro toxicity of the reactive oxygen species generating enzyme xanthine oxidoreductase (XOR) to human peripheral blood lymphocytes was studied after stimulation with phytohaemoagglutinin or anti-CD3/CD28 antibodies. Apoptosis and necrosis were induced by the XOR/hypoxanthine system in a time- and concentration-dependent manner. CD8+ lymphocytes showed a higher sensitivity than CD4+ cells to the XOR/hypoxanthine system. The occurrence of apoptosis was demonstrated by annexin-V binding to injured cell membrane, which was the most precocious alteration observed, followed by the increment of transglutaminase activity, which was significant at the lowest XOR concentration used. Nuclear damage was assessed by the increased hypodiploid nuclei and by DNA migration on gel electrophoresis, which turned to an apoptotic pattern before the occurrence of cell membrane necrotic lesions. Apoptosis was induced by XOR activity proportionally to substrate concentration and was prevented by the competitive enzyme inhibitor, allopurinol. The hydrogen peroxide scavenging enzyme, catalase, gave a higher protection than superoxide dismutase from the toxicity caused by the XOR/hypoxanthine system. Necrosis occurs in a variable percentage indicating that reactive oxygen species may trigger both apoptosis and necrosis in proliferating human lymphocytes, mostly depending on XOR concentration.     

Oxidative stress to human lymphocytes by xanthine oxidoreductase activity

The in vitro toxicity of the reactive oxygen species generating enzyme xanthine oxidoreductase (XOR) to human peripheral blood lymphocytes was studied after stimulation with phytohaemoagglutinin or anti-CD3/CD28 antibodies. Apoptosis and necrosis were induced by the XOR/hypoxanthine system in a time- and concentration-dependent manner. CD8+ lymphocytes showed a higher sensitivity than CD4+ cells to the XOR/hypoxanthine system. The occurrence of apoptosis was demonstrated by annexin-V binding to injured cell membrane, which was the most precocious alteration observed, followed by the increment of transglutaminase activity, which was significant at the lowest XOR concentration used. Nuclear damage was assessed by the increased hypodiploid nuclei and by DNA migration on gel electrophoresis, which turned to an apoptotic pattern before the occurrence of cell membrane necrotic lesions. Apoptosis was induced by XOR activity proportionally to substrate concentration and was prevented by the competitive enzyme inhibitor, allopurinol. The hydrogen peroxide scavenging enzyme, catalase, gave a higher protection than superoxide dismutase from the toxicity caused by the XOR/hypoxanthine system. Necrosis occurs in a variable percentage indicating that reactive oxygen species may trigger both apoptosis and necrosis in proliferating human lymphocytes, mostly depending on XOR concentration.     

Improved method for measurement of human plasma xanthine oxidoreductase activity

The XOR activity in human plasma was measured by quantifying the XOR-derived uric acid (UA) in plasma using the high-performance liquid chromatography (HPLC) equipped with a UV detector. Chromatographic separation consisted of the mobile phase (a mixture of 0.1% trifluoroacetic acid in Milli-Q water and 0.085% trifluoroacetic acid in acetonitrile in a mix ratio of 99:1) running through a Zorbax StableBond SB-C(18) column at a flow-rate of 1 ml/min. Deproteinization with heat-treatment of plasma samples after the reaction was used in the assay to avoid splitting of the UA and xanthine peaks caused by acid deproteinization that could interfere the accurate determination of human plasma XOR activity in our case. Based on the examination of the dependence of XOR activity on added amounts of xanthine and reaction times, the amount of xanthine and reaction time for XOR activity assay were determined to prevent the errors caused by the limiting effect of substrates and plateau phase of the reaction. Using this method, human plasma XOR activities of 25 healthy people were measured. The average human plasma XOR activity was 2.1+/-0.8 (x10(-3) U/ml).     

Xanthine oxidoreductase and xanthine oxidase in human cornea

Xanthine oxidoreductase (xanthine dehydrogenase + xanthine oxidase) is a complex enzyme that catalyzes the oxidation of hypoxanthine to xanthine, subsequently producing uric acid. The enzyme complex exists in separate but interconvertible forms, xanthine dehydrogenase and xanthine oxidase, which generate reactive oxygen species (ROS), a well known causative factor in ischemia/reperfusion injury and also in some other pathological states and diseases. Because the enzymes had not been localized in human corneas until now, the aim of this study was to detect xanthine oxidoreductase and xanthine oxidase in the corneas of normal post-mortem human eyes using histochemical and immunohistochemical methods. Xanthine oxidoreductase activity was demonstrated by the tetrazolium salt reduction method and xanthine oxidase activity was detected by methods based on cerium ion capture of hydrogen peroxide. For immunohistochemical studies. we used rabbit antibovine xanthine oxidase antibody, rabbit antihuman xanthine oxidase antibody and monoclonal mouse antihuman xanthine oxidase/xanthine dehydrogenase/aldehyde oxidase antibody. The results show that the enzymes are present in the corneal epithelium and endothelium. The activity of xanthine oxidoreductase is higher than that of xanthine oxidase, as clearly seen in the epithelium. Further studies are necessary to elucidate the role of these enzymes in the diseased human cornea. Based on the findings obtained in this study (xanthine oxidoreductase/xanthine oxidase activities are present in normal human corneas), we hypothesize that during various pathological states, xanthine oxidase-generated ROS might be involved in oxidative eye injury.     

Studies of the ferroxidase activity of native and chemically modified xanthine oxidoreductase.

The O2-utilizing (type O, oxidase) form of xanthine oxidoreductase is primarily responsible for its ferroxidase activity. This form of xanthine oxidoreductase has 1000 times the ferroxidase activity of the serum ferroxidase caeruloplasmin. It has the ability to catalyse the oxidative incorporation of iron into transferrin at very low Fe2+ and O2 concentrations. Furthermore, the pH optimum of the ferroxidase activity of the enzyme is compatible with the conditions of pH that normally exist in the intestinal mucosa, where it has been proposed that xanthine oxidoreductase may facilitate the absorption of ionic iron. Modification of the molybdenum (Mb) centres of the enzyme in vitro by treatment with cyanide, methanol or allopurinol completely abolishes its ferroxidase activity. The feeding of dietary tungsten to rats, which prevents the incorporation of molybdenum into newly synthesized intestinal xanthine oxidoreductase, results in the progressive loss of the ferroxidase activity of intestinal-mucosa homogenates. Removal of the flavin centres from the enzyme also results in the complete loss of ferroxidase activity; however, the ferroxidase activity of the flavin-free form of the enzyme can be restored with artificial electron acceptors that interact with the molybdenum or non-haem iron centres. The presence of superoxide dismutase or catalase in the assay system results in little inhibition of the ferroxidase activity of xanthine oxidoreductase.     

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.     

Comparative localization of aldehyde oxidase and xanthine oxidoreductase activity in rat tissues

The distribution of aldehyde oxidase activity was evaluated in unfixed cryostat sections from tissues of male Wistar rats using a tissue protectant, polyvinyl alcohol, with Tetranitro BT as a final electron acceptor. The distribution of aldehyde oxidase activity was compared with that of xanthine oxidoreductase. The enzyme histochemical method demonstrated aldehyde oxidase activity in the epithelium of the tongue, renal tubules and bronchioles, as well as in the cytoplasm of liver cells. Such activity was not detected in oesophagus, stomach, spleen, adrenal glands, small or large intestine or skeletal and heart muscle fibres. In contrast, xanthine oxidoreductase activity was demonstrated in the tongue, renal tubules, bronchioles, oesophageal, gastric, small and large intestinal epithelial cells, adrenal glands, spleen and liver cytoplasm but not in skeletal and heart muscle fibres. The significance of the ubiquitous distribution of aldehyde oxidase activity, especially in surface epithelial cells from various tissues, except for the gastrointestinal tract, is unclear. However, aldehyde oxidase may possess some physiological activity other than in the metabolism of N-heterocyclics or of certain drugs. © 1998 Chapman & Hall     

High inter-individual variability of serum xanthine oxidoreductase activity in IBD patients

Objectives: Thiopurines play an essential role in the management of inflammatory bowel diseases (IBD, i.e. Crohn's disease and ulcerative colitis). Over the past decade, several strategies to optimize treatment with thiopurines have been evaluated, including co-administration of allopurinol, a xanthine-oxidoreductase (XO) inhibitor, to low-dose thiopurine therapy. We aimed to assess the inter-individual variability of XO-activity between IBD-patients.

Methods: We assessed XO activity in serum of IBD-patients of two medical centers in The Netherlands using the Amplex® Red Xanthine/Xanthine Oxidase Assay Kit, which measures the superoxide formation in a coupled reaction to the red-fluorescent oxidation product, resofurine.

Results: We observed a high inter-individual variability of XO-activity in 119 patients, with a median activity of 16 µU/ml/hour (range 1–85 µU/ml/hour). The XO-activity was influenced by gender (male 19.5 vs. female 14.0 µU/ml/hour, p < 0.01), patient's age (Pearson's correlation r = 0.21, p = 0.02) and duration of IBD (r = 0.23, p = 0.01). The XO activity was not affected by the type of IBD, smoking status, body mass index or (type of) thiopurine use (p > 0.05).

Conclusions: There is a high inter-individual variability of XO-activity in IBD-patients; XO-activity is positively associated with male gender and patient's age.     

Migratory activity of human breast cancer cells is modulated by differential expression of xanthine oxidoreductase

Xanthine oxidoreductase (XOR) may exert an important, but poorly defined, role in the pathogenesis of breast cancer (BC). Loss of XOR expression was linked to aggressive BC, and recent clinical observations have suggested that decreasing XOR may be functionally linked to BC aggressiveness. The goal of the present investigation was to determine whether the decreased XOR observed in clinically aggressive BC was an intrinsic property of highly invasive mammary epithelial cells (MEC). Expression of XOR was investigated using HC11 mouse MEC, HB4a and MCF-10A normal human MEC, and several human mammary tumor cells including MCF-7 and MDA-MB-231. Consistent with clinical observations, data shown here revealed high levels of XOR in normal HC11 and MCF-10A cells that was markedly reduced in highly invasive mammary tumor cells. The contribution of XOR to tumor cell migration in vitro was investigated using MDA-MB-231 and MCF-7 cells and clonally selected derivatives of HC11 that exhibit either weak or strong migration in vitro. We observed that over-expression of an XOR cDNA in MDA-MB-231 and in HC11-C24, both possessing weak XOR expression and high migratory capacity, inhibited their migration in vitro. Conversely, pharmacological inhibition of XOR in MCF-7 and HC11-C4, both possessing high XOR expression and weak migratory capacity, stimulated their migration in vitro. Further experiments suggested that XOR derived ROS mediated this effect and also modulated COX-2 and MMP levels and function. These data demonstrate a functional link between XOR expression and MEC migration and suggest a potential role for XOR in suppressing BC pathogenesis.     

Intermediate dehydrogenase-oxidase form of xanthine oxidoreductase in rat liver.

A spectrophotometric method for the determination of three forms of xanthine oxidoreductase, namely dehydrogenase (D), dehydrogenase-oxidase (D/O) and oxidase (O), is described. Enzymic fractions obtained from rat liver were found to contain either all three forms, or (under special conditions of preparation) only two forms, D and D/O. The conversion of form D leads to form D/O leads to form O in the presence of Cu2+ ions was shown. Form D/O acted with NAD+ as well as with O2 as electron acceptors, it exhibited greater affinity to NAD+ than to O2, and NAD+ abolished the oxidase activity of this form. Moreover, oxidase activity of form D/O was inhibited by NADH. These facts indicate that NAD+ and O2 compete for the same active site on the enzyme molecule.     

A histochemical method for detecting xanthine oxidase activity in the liver

A histochemical method is described for localizing xanthine oxidase--the key enzyme in the purine catabolic pathway. The above method is based on the reduction of p-Nitroblue tetrazolium during hypoxanthine enzymatic oxidation, phenazine methosulfate being an intermediate electron acceptor. The patterns of the reaction product distribution suggest that the Kupffer cells and the sinusoidal endothelium possess the highest xanthine oxidase activity.     

Mammalian xanthine oxidoreductase - mechanism of transition from xanthine dehydrogenase to xanthine oxidase

Reactive oxygen species are generated by various biological systems, including NADPH oxidases, xanthine oxidoreductase, and mitochondrial respiratory enzymes, and contribute to many physiological and pathological phenomena. Mammalian xanthine dehydrogenase (XDH) can be converted to xanthine oxidase (XO), which produces both superoxide anion and hydrogen peroxide. Recent X-ray crystallographic and site-directed mutagenesis studies have revealed a highly sophisticated mechanism of conversion from XDH to XO, suggesting that the conversion is not a simple artefact, but rather has a function in mammalian organisms. Furthermore, this transition seems to involve a thermodynamic equilibrium between XDH and XO; disulfide bond formation or proteolysis can then lock the enzyme in the XO form. In this review, we focus on recent advances in our understanding of the mechanism of conversion from XDH to XO.