The mechanism and significance of the conversion of xanthine dehydrogenase to xanthine oxidase in mammalian secretory gland cells.

The conversion of xanthine dehydrogenase (XDH) to xanthine oxidase (XO) occurs only in mammalian species. In fresh bovine milk, the enzyme exists predominantly as the oxidase form, in contrast to various normal organs where it is found primarily as the dehydrogenase: the mechanism of conversion to the oxidase in milk remains obscure. A systematic search for the essential factors for conversion from XDH to XO has been performed within fresh bovine milk using the highly purified dehydrogenase form after removal endogenous oxidase form by fractionation analysis. We find that conversion to the oxidase form requires four components under air: lactoperoxidase (LPO), XDH, SCN-, and substrate hypoxanthine or xanthine; the contribution of sulfhydryl oxidase appears to be minor. Disulfide bond formation between Cys-535 and Cys-995 is principally involved in the conversion, consistent with the result obtained from previous work with transgenic mice. In vitro reconstitution of LPO and SCN- results in synergistic conversion of the dehydrogenase to the oxidase the presence of xanthine, indicating the conversion is autocatalytic. Milk from an LPO knockout mouse contains a significantly greater proportion of the dehydrogenase form of the enzyme, although some oxidase form is also present, indicating that LPO contributes principally to the conversion, but that sulfhydryl oxidase (SO) may also be involved to a minor extent. All the components XDH/LPO/SCN- are necessary to inhibit bacterial growth in the presence of xanthine through disulfide bond formation in bacterial protein(s) required for replication, as part of an innate immunity system in mammals. Human GTEx Data suggest that mRNA of XDH and LPO are highly co-expressed in the salivary gland, mammary gland, mucosa of the airway and lung alveoli, and we have confirmed these human GTEx Data experimentally in mice. We discuss the possible roles of these components in the propagation of SARS-CoV-2 in these human cell types.

Xanthine Oxidoreductase Is Involved in Chondrocyte Mineralization and Expressed in Osteoarthritic Damaged Cartilage.

Pathologic calcification of cartilage consists of the formation of basic calcium phosphate (BCP) and/or calcium pyrophosphate dihydrate (CPPD) containing calcium crystals in mature hyaline or articular cartilage and is associated with aging, cartilage injury and likely plays a role in accelerating the pathology of osteoarthritis (OA). The pathways regulating joint calcification, in particular cartilage calcification, are not completely understood, but inflammation and the formation of reactive oxygen species (ROS) are contributory factors. The xanthine oxidase (XO) form of xanthine oxidoreductase (XOR), the key enzyme in xanthine and uric acid metabolism, is a major cellular source of superoxide. We hypothesized that XOR could be implicated in chondrocyte mineralization and cartilage calcification and degradation in OA. We showed both in murine primary chondrocyte and chondrogenic ATDC5 cells, that mineralization was inhibited by two different XOR inhibitors, febuxostat and allopurinol. In addition, XOR inhibition reduced the expression of the pro-mineralizing cytokine interleukin-6 (IL-6). We next generated XOR knock-out chondrocyte cell lines with undetectable XOR expression and XO activity. XOR knock-out chondrocyte cells showed decreased mineralization and reduced alkaline phosphatase (Alp) activity. To assess the precise form of XOR involved, primary chondrocytes of XOR mutant mice expressing either the XDH form (XDH ki) or the XO form (XO ki) were studied. We found that XO ki chondrocytes exhibited increased mineralization compared to XDH ki chondrocytes, and this was associated with enhanced Alp activity, ROS generation and IL-6 secretion. Finally, we found increased XOR expression in damaged vs. undamaged cartilage obtained from OA patients and XOR expression partially co-localized with areas showing pathologic calcification. Altogether, our results suggest that XOR, via its XO form, contribute to chondrocyte mineralization and pathological calcification in OA cartilage.

Targeted knock-in mice expressing the oxidase-fixed form of xanthine oxidoreductase favor tumor growth.

Xanthine oxidoreductase has been implicated in cancer. Nonetheless, the role played by its two convertible forms, xanthine dehydrogenase (XDH) and oxidase (XO) during tumorigenesis is not understood. Here we produce XDH-stable and XO-locked knock-in (ki) mice to address this question. After tumor transfer, XO ki mice show strongly increased tumor growth compared to wild type (WT) and XDH ki mice. Hematopoietic XO expression is responsible for this effect. After macrophage depletion, tumor growth is reduced. Adoptive transfer of XO-ki macrophages in WT mice increases tumor growth. In vitro, XO ki macrophages produce higher levels of reactive oxygen species (ROS) responsible for the increased Tregs observed in the tumors. Blocking ROS in vivo slows down tumor growth. Collectively, these results indicate that the balance of XO/XDH plays an important role in immune surveillance of tumor development. Strategies that inhibit the XO form specifically may be valuable in controlling cancer growth.

New Strategy That Delays Progression of Amyotrophic Lateral Sclerosis in G1H-G93A Transgenic Mice: Oral Administration of Xanthine Oxidoreductase Inhibitors That Are Not Substrates for the Purine Salvage Pathway.

Amyotrophic lateral sclerosis (ALS), Lou Gehrig's disease, is a progressive fatal neurodegenerative disease that involves both upper and lower motor neurons. We orally administered 4 xanthine oxidoreductase (XOR) inhibitors to G1H-G93A mice carrying 25 transgene copy numbers of human mutant G93A superoxide dismutase 1, from 80 days of age. Three nonpurine-analogue inhibitors (TEI-6720: Febuxostat, Y-700 and FYX-051), but not allopurinol with a purine analogue ring (pyrazolo pyrimidine ring), significantly delayed disease onset, prolonged survival and the duration of disease stages, improved clinical signs, and alleviated weight loss. Exercise testing (extension reflex, inclined plane, footprint, rotarod, and beam balance tests) showed significantly improved motor function in the G1H-G93A mice treated with these 3 inhibitors. Significant amelioration of disease was seen even when TEI-6720 or Y-700 was administered after the appearance of early signs. Histopathological evaluation in the late stage revealed that G1H-G93A mice treated with TEI-6720 had well-preserved motor neurons and fewer inclusion bodies, compared with mice treated with placebo or with allopurinol. Our results indicate that these 3 nonpurine-analogue XOR inhibitors might increase the supply of high-energy compounds via the purine salvage pathway, thereby protecting motor neurons against death. This strategy may be applicable for oral therapy of ALS patients.

The Effects of Xanthine Oxidoreductase Inhibitors on Oxidative Stress Markers following Global Brain Ischemia Reperfusion Injury in C57BL/6 Mice.

We demonstrated that 3-nitrotyrosine and 4-hydroxy-2-nonenal levels in mouse brain were elevated from 1 h until 8 h after global brain ischemia for 14 min induced with the 3-vessel occlusion model; this result indicates that ischemia reperfusion injury generated oxidative stress. Reactive oxygen species production was observed not only in the hippocampal region, but also in the cortical region. We further evaluated the neuroprotective effect of xanthine oxidoreductase inhibitors in the mouse 3-vessel occlusion model by analyzing changes in the expression of genes regulated by the transcription factor nuclear factor-kappa B (including pro-inflammatory cytokines interleukin-1ß (IL-1ß) and tumor necrosis factor-α (TNF-α), matrix metalloproteinase-9 and intercellular adhesion molecules-1). Administration of allopurinol resulted in a statistically significant decrease in IL-1ß and TNF-α mRNA expression, whereas febuxostat had no significant effect on expression of these genes; nevertheless, both inhibitors effectively reduced serum uric acid concentration. It is suggested that the neuroprotective effect of allopurinol is derived not from inhibition of reactive oxygen species production by xanthine oxidoreductase, but rather from a direct free-radical-scavenging effect.

Evaluation of neuronal protective effects of xanthine oxidoreductase inhibitors on severe whole-brain ischemia in mouse model and analysis of xanthine oxidoreductase activity in the mouse brain.

Global cerebral ischemia and reperfusion (I/R) often result in high mortality. Free radicals play an important role in global cerebral I/R. Xanthine oxidoreductase (XOR) inhibitors, such as allopurinol, have been reported to protect tissues from damage caused by reactive oxygen species (ROS) by inhibiting its production through XOR inhibition. The recently introduced XOR inhibitor febuxostat, which is a more potent inhibitor than allopurinol, is expected to decrease free radical production more effectively. Here, we analyzed the effects of allopurinol and febuxostat in decreasing global severe cerebral I/R damage in mice. Mice were divided into three groups: a placebo group, an allopurinol group, and a febuxostat group. Pathological examinations, which were performed in each group in the CA1 and CA2 regions of the hippocampus 4 days after I/R surgery, revealed that there was a decrease in the number of neuronal cells in the 14-min occlusion model in both regions and that drugs that were administered to prevent this damage were not effective. The enzymatic activity was extremely low in the mouse brain, and XOR could not be detected in the nonischemic and ischemic mice brains with western blot analyses. Thus, one of the reasons for the decreased effectiveness of XOR inhibitors in controlling severe whole-brain ischemia in a mouse model was the low levels of expression of XOR in the mouse brain.

Phosphorylation of Thr(1495) of nestin in a mouse model of cerebral ischemia and reperfusion damage.

Nestin, a class VI intermediate filament protein, is expressed by neuronal progenitor cells in the subventricular zone (SVZ). In the present study, we analyzed the nestin expression and phosphorylation levels in nerve cells in a mouse model of cerebral ischemia and reperfusion. C57BL/6 mice were subjected to three-vessel occlusion for 14 min, and were killed either 1 or 4 days after the procedure. The percentages of cells in the SVZ that were positive for nestin, Thr(1495)-phosphorylated nestin or Ki67 did not significantly differ between the ischemic reperfusion and sham groups. Conversely, in the striatum and cornu ammonis 2 (CA2) regions, the mice at 4 days after ischemic reperfusion showed significantly higher numbers and percentages of nerve cells that were positive for nestin, Thr(1495)-phosphorylated nestin and Ki67 compared to results from the other groups. To our knowledge, this is the first description of phosphorylated nestin expression in neural progenitor cells in the SVZ of adult mice. In this cerebral ischemia and reperfusion mouse model, cells positive for Thr(1495)-phosphorylated nestin were increased in the striatum and CA2 field of the hippocampus; suggesting that nestin phosphorylation may play an important role in mitotically active neuronal progenitor cells.

Chemical nature and reaction mechanisms of the molybdenum cofactor of xanthine oxidoreductase.

Xanthine oxidoreductase (XOR), a complex flavoprotein, catalyzes the metabolic reactions leading from hypoxanthine to xanthine and from xanthine to urate, and both reactions take place at the molybdenum cofactor. The enzyme is a target of drugs for therapy of gout or hyperuricemia. We review the chemical nature and reaction mechanisms of the molybdenum cofactor of XOR, focusing on molybdenum-dependent reactions of actual or potential medical importance, including nitric oxide (NO) synthesis. It is now generally accepted that XOR transfers the water-exchangeable -OH ligand of the molybdenum atom to the substrate. The hydroxyl group at OH-Mo(IV) can be replaced by urate, oxipurinol and FYX-051 derivatives and the structures of these complexes have been determined by xray crystallography under anaerobic conditions. Although formation of NO from nitrite or formation of xanthine from urate by XOR ischemically feasible, it is not yet clear whether these reactions have any physiological significance since the reactions are catalyzed at a slow rate even under anaerobic conditions.