Down-regulated xanthine oxidoreductase is a feature of aggressive breast cancer.

PURPOSE: Xanthine oxidoreductase (XOR) is a key enzyme in the degradation of DNA, RNA, and high-energy phosphates and also plays a role in milk lipid globule secretion. Given the strong and regulated expression of XOR in normal breast epithelium, and the previously shown alterations of its expression in experimental tumorigenesis, we hypothesized that XOR may be differentially expressed in breast cancer. EXPERIMENTAL DESIGN: XOR expression was analyzed by immunohistochemistry in tissue microarray specimens of 1,262 breast cancer patients with a median follow-up of 9.5 years. RESULTS: Expression of XOR was moderately decreased in 50% and undetectable in another 7% of the tumors. Decreased XOR expression was associated with poor histologic grade of differentiation, ductal and lobular histologic types, large tumor size, high number of positive axillary lymph nodes, and high cyclooxygenase-2 expression, but not with estrogen or progesterone receptor status, Ki-67, p53, or ERBB2 amplification. Absence of XOR expression was associated with unfavorable outcome, and patients with no XOR expression had more than twice the risk of distant recurrence as compared with those with a moderately decreased or normal expression (hazard ratio, 2.21; P < 0.0001). This was also true in patients with node-negative disease (hazard ratio, 2.75; P < 0.0001) as well as in patients with small (< or = 1 cm) tumors (hazard ratio, 3.09; P = 0.027). In a multivariate survival analysis, negative XOR emerged as an independent prognostic factor both in the entire series (P = 0.01) and among patients with node-negative disease (P = 0.0009). CONCLUSION: Loss of XOR identifies breast cancer patients with unfavorable prognosis.

Increased sensitivity of homozygous Sod2 mutant mice to oxygen toxicity.

Induction or overexpression of pulmonary manganese superoxide dismutase (MnSOD) has been shown to protect against oxygen (O2) toxicity. Genetic inactivation of MnSOD (Sod2) results in multiple organ failure and early neonatal death. However, lungs or O2-tolerance of Sod2 knockout mice have not been investigated. We evaluated survival, lung histopathology, and other pulmonary antioxidants (glutathione cycle) of homozygous (-/-) and heterozygous (+/-) Sod2 mutant mice compared with wild-type controls (Sod2+/+) following 48 h exposure to either room air or to O2. The ability of antioxidant N-acetylcysteine to compensate for the loss of MnSOD was explored. Mortality of Sod2-/- mice increased from 0% in room air to 18 and 83% in 50 and 80% O2, respectively. N-acetylcysteine did not alter mortality of Sod2-/- mice. Histopathological analysis revealed abnormalities in saccules of Sod2-/- mice exposed either to room air or to 50% O2 suggestive of delayed postnatal lung development. In 50% O2, activities of glutamate-cysteine ligase (GCL) (previously known as gamma-glutamylcysteine synthetase, gamma-GCS) and glutathione peroxidase increased in Sod2-/- (35 and 70%, respectively) and Sod2+/- (12 and 70%, respectively) mice, but glutathione levels remained unaltered. We conclude that MnSOD is required for normal O2 tolerance and that in the absence of MnSOD there is a compensatory increase in pulmonary glutathione-dependent antioxidant defense in hyperoxia.

Adenine nucleotide metabolism and cell fate after oxidant exposure of rat cortical neurons: effects of inhibition of poly(ADP-ribose) polymerase.

We exposed cultured neurons prelabeled with 14C-adenine to H2O2 with or without the poly(ADP-ribose) polymerase (PARP) inhibitor 3,4-Dihydro-5-[4-(1-piperidinyl)butoxy]-1(2H)-isoquinolinone (DPQ) to quantify its effects on acute ATP depletion, later ATP synthesis, cellular and nuclear morphology, extent of DNA fragmentation, and PARP cleavage. According to the extent of the acute ATP depletion, the exposures were classified as 'mild' (50 microM H2O2), 'moderate' (100-250 microM H2O2), or 'severe' (500 microM-1 mM H2O2) insults. Mild exposure had no significant effects on the parameters studied. In the 'moderately' exposed neurons, ATP depletion to 59+/-6% of control was associated with a decrease in the cell counts, apoptotic morphology, and cleavage of PARP. In this group, DPQ prevented the acute ATP (to 95+/-15% of control), preserved cell morphology, and improved cell survival. In the 'severe' group, ATP depletion to 18+/-4% was associated with necrosis and intact PARP. DPQ elevated ATP levels (to 44+/-12% of control) and post-insult ATP synthesis, improved cell counts, and altered cell morphology towards apoptosis rather than necrosis. Post-insult application of DPQ was less effective. Our results show that the extent of oxidant-induced ATP depletion and cell fate can be modified by PARP inhibition, to some extent also after the insult.

Biphasic ATP depletion caused by transient oxidative exposure is associated with apoptotic cell death in rat embryonal cortical neurons.

Hypoxia-ischemia leads to an acute depletion of high-energy phosphates in neonatal brain. After reperfusion, energy status is restored, but may show progressive secondary failure, associated with neuronal loss, brain damage, or death. Oxidants are produced on reperfusion. We investigated whether a biphasic energy failure develops in cultured neurons after oxidant exposure, and whether the degree of primary disturbance correlates with later ATP synthesis and mode of cell death. Embryonic rat cortical neurons were exposed to varying doses of hydrogen peroxide for 60 min and incubated for 12, 24, or 48 h. Adenine nucleotides and the incorporation of [(14)C]adenine into adenine nucleotides were quantified. Apoptosis was evaluated by DNA electrophoresis and in situ end-labeling. A mild insult (10-50 microM) caused no ATP depletion or change in subsequent growth or energy metabolism, whereas an intermediate insult (100 microM) caused acute ATP depletion (49 +/- 12% of control). This recovered to 91 +/- 28% by 12 h, but then declined to 61 +/- 18% at 24 h. A severe insult (1 mM) depleted ATP to 15 +/- 3% of control, with no recovery. Moderate ATP depletion was associated with apoptotic cell death, whereas a severe insult caused acute necrosis. Transient oxidant exposure of embryonal cortical neurons causes a biphasic energy depletion followed by apoptosis in analogy with asphyxiated brains. This model may prove useful for the study of pathogenesis and treatment of hypoxic-ischemic encephalopathy.

Regulation of xanthine oxidoreductase by intracellular iron.

Xanthine oxidoreductase (XOR) may produce reactive oxygen species and play a role in ischemia-reperfusion injury. Because tissue iron levels increase after ischemia, and because XOR contains functionally critical iron-sulfur clusters, we studied the effects of intracellular iron on XOR expression. Ferric ammonium citrate and FeSO(4) elevated intracellular iron levels and increased XOR activity up to twofold in mouse fibroblast and human bronchial epithelial cells. Iron increased XOR protein and mRNA levels, whereas protein and RNA synthesis inhibitors abolished the induction of XOR activity. A human XOR promoter construct (nucleotides +42 to -1937) was not induced by iron in human embryonic kidney cells. Hydroxyl radical scavengers did not block induction of XOR activity by iron. Iron chelation by deferoxamine (DFO) decreased XOR activity but did not lower endogenous XOR protein or mRNA levels. Furthermore, DFO reduced the activity of overexpressed human XOR but not the amount of immunoreactive protein. Our data show that XOR activity is transcriptionally induced by iron but posttranslationally inactivated by iron chelation.

Posttranslational inactivation of human xanthine oxidoreductase by oxygen under standard cell culture conditions.

Xanthine oxidoreductase (XOR) catalyzes the final reactions of purine catabolism and may account for cell damage by producing reactive oxygen metabolites in cells reoxygenated after hypoxia. We found a three- to eightfold higher XOR activity in cultured human bronchial epithelial cells exposed to hypoxia (0.5-3% O2) compared with cells grown in normoxia (21% O2) but no difference in XOR protein or mRNA. XOR promoter constructs failed to respond to hypoxia. The cellular XOR activity at 3% O2 returned to basal levels when the cells were returned to 21% O2, and hyperoxia (95% O2) abolished enzyme activity with no change in XOR protein. Our data suggest reversible enzyme inactivation by oxygen or its metabolites. NADH was normally oxidized by the oxygen-inactivated enzyme, which rules out damage to the flavin adenine dinucleotide cofactor. Hydrogen peroxide partially inactivated the molybdenum center of XOR, as shown by a parallel decrease in XOR-catalyzed xanthine oxidation and dichlorophenolindophenol reduction. We conclude that the transcription or translation of XOR is not influenced by hypoxia or hyperoxia. Instead, the molybdenum center of XOR is posttranslationally inactivated by oxygen metabolites in "normal" (21% O2) cell culture atmosphere. This inactivation is reversed in hypoxia and accounts for the apparent induction.

Intrauterine growth restriction and postnatal steroid treatment effects on insulin sensitivity in preterm neonates.

OBJECTIVES: To study whether intrauterine growth restriction (IUGR) is associated with decreased sensitivity to the main fetal growth factor, insulin, and the effect of glucocorticoid therapy on insulin sensitivity in preterm infants. STUDY DESIGN: Newborn infants with a birth weight (BW) of< 1500 g were classified as appropriate for gestational age ([AGA], BW within +/- 1 SD, n = 10), or small for gestational age ([SGA], BW <-2 SD, n = 13); 5 AGA infants and 8 SGA infants received systemic steroids. An abbreviated modified minimal model test was performed, consisting of sequential blood samples for glucose and insulin assays, and intravenous infusions of 0.3 g/kg glucose and 0.02 U/kg regular human insulin. The insulin sensitivity index (S(I)) was calculated using a computer program. RESULTS: The basal insulin/glucose ratio (I/G) and S(I) did not differ between the AGA and SGA groups. Steroids did not influence the I/G nor the S(I) of AGA infants (10.2 +/- 6.7 vs 8.2 +/- 2.3), but decreased the S(I) in the SGA group (12.2 +/- 5.1 vs 5.3 +/- 2.7, P <.05). CONCLUSIONS: Insulin sensitivity of neonates can be measured by the modified minimal model. IUGR is not associated with impaired fetal glucose tolerance. Early neonatal steroid treatment decreases insulin sensitivity in SGA infants, which may contribute to their risk of having hyperglycemia.

Plasma 8-isoprostane is increased in preterm infants who develop bronchopulmonary dysplasia or periventricular leukomalacia.

Our aim was to assess the plasma free 8-epi-prostaglandin F(2alpha) (8-isoprostane) and ascorbyl radical as risk indicators for oxidative damage in extremely low birth weight infants (ELBWIs) and the effect of N-acetylcysteine (NAC) on these markers. Plasma samples were collected on days 3 and 7 of life from infants who were enrolled in a randomized, controlled trial in which i.v. NAC or placebo was administered to ELBWIs during the first week of life, with the aim of preventing bronchopulmonary dysplasia (BPD). Plasma 8-isoprostane was analyzed in 83 infants using an enzyme immunoassay kit. Ascorbyl radical concentration was measured in 61 infants with electron spin resonance spectroscopy. The 8-isoprostane concentrations were similar in the NAC and placebo groups. In infants who later developed BPD or died (n = 29), the median (range) 8-isoprostane concentration was significantly higher (p = 0.001) on day 3 and day 7 [50.0 pg/mL (19-360) and 57.0 pg/mL (14-460), respectively] than in survivors without BPD [n = 54; 34.5 pg/mL (5-240) and 39.5 pg/mL (7-400), respectively]. The 8-isoprostane levels increased significantly more (p < 0.05) in infants who later developed periventricular leukomalacia. NAC treatment or the later development of BPD was not related to the ascorbyl radical levels. The ascorbyl radical level decreased significantly in all groups from day 3 to day 7, but the difference between the groups was not significant. The mean (SD) ascorbyl radical level on day 3 was significantly higher (p < 0.01) in infants who later developed periventricular leukomalacia [287 (124) versus 194 (90)]. These data suggest that plasma 8-isoprostane could serve as a marker in assessing the risk for BPD development in ELBWIs.

N-acetylcysteine does not prevent bronchopulmonary dysplasia in immature infants: a randomized controlled trial.

OBJECTIVE: To evaluate whether N-acetylcysteine (NAC) infusion during the first week of life reduces the risk of death or bronchopulmonary dysplasia (BPD) in infants with extremely low birth weight. Study design In a Nordic multicenter, double-blind trial, infants (n=391) weighing 500 to 999 g and on ventilator or nasal continuous positive airway pressure were randomized before the age of 36 hours to receive NAC 16 to 32 mg/kg/d (n=194) or placebo (n=197) intravenously for 6 days. Primary end points were death or BPD, defined as supplementary oxygen requirement at 36 weeks' gestational age. RESULTS: There was no difference in the combined incidence of the primary end points death or BPD, 51% vs. 49%, between the NAC group and control group. Also similar was the incidence of BPD in survivors at 36 weeks' gestational age, 40% vs. 40%, and the mean oxygen requirement at the age of 28 days, 31.2% vs. 30.7%, respectively. The severity of BPD was similar in both groups. CONCLUSIONS: A 6-day course of intravenous N-acetylcysteine at the dosage used does not prevent BPD or death in infants with extremely low birth weight.