Measurement and Characterization of Superoxide Generation from Xanthine Dehydrogenase: A Redox-Regulated Pathway of Radical Generation in Ischemic Tissues

The enzyme xanthine oxidoreductase (XOR) is an important source of oxygen free radicals and related postischemic injury. Xanthine dehydrogenase (XDH), the major form of XOR in tissues, can be converted to xanthine oxidase (XO) by oxidation of sulfhydryl residues or by proteolysis. The conversion of XDH to XO has been assumed to be required for radical generation and tissue injury. It is also possible that XDH could generate significant quantities of superoxide, ^•O₂^–, for cellular signaling or injury; however, this possibility and its potential ramifications have not been previously considered. To unambiguously determine if XDH can be a significant source of ^•O₂^–, experiments were performed to measure and characterize ^•O₂^– generation using XDH from chicken liver that is locked in the dehydrogenase conformation. Electron paramagnetic resonance spin trapping experiments with 5-(diethoxyphosphoryl)-5-methyl-1-pyrroline-<i>N</i>-oxide demonstrated that XDH in the presence of xanthine produces significant amounts of ^•O₂^–. NAD⁺ and NADH inhibited the generation of ^•O₂^– from XDH in a dose-dependent manner, with NAD⁺ exhibiting stronger inhibition than NADH at low physiological concentrations. Decreased amounts of NAD⁺ and NADH, which occur during and following tissue ischemia, enhanced the generation of ^•O₂^– from XDH in the presence of xanthine. It was observed that XDH-mediated oxygen radical generation markedly depressed Ca²⁺-ATPase activity of isolated sarcoplasmic reticulum vesicles from cardiac muscle, and this was modulated by NAD⁺ and NADH. Thus, XDH can be an important redox-regulated source of ^•O₂^– generation in ischemic tissue, and conversion to XO is not required to activate radical formation and subsequent tissue injury

Effects of adeno-SF1 on responsiveness of KGN cells (mean ± SD) to 10 mol/L atrazine or 10 mol/L simazine

<p><b>Copyright information:</b></p><p>Taken from "Atrazine-Induced Aromatase Expression Is SF-1 Dependent: Implications for Endocrine Disruption in Wildlife and Reproductive Cancers in Humans"</p><p></p><p>Environmental Health Perspectives 2007;115(5):720-727.</p><p>Published online 5 Feb 2007</p><p>PMCID:PMC1867956.</p><p>This is an Open Access article: verbatim copying and redistribution of this article are permitted in all media for any purpose, provided this notice is preserved along with the article's original DOI</p> () Basal aromatase mRNA (; relative copies) was significantly increased in cells transfected with adeno-SF-1 relative to controls infected with adeno-LacZ. () Aromatase enzymatic activity (fold change) also increased in response to atrazine or simazine in adeno-SF-1 infected KGN cells, but not in the control adeno-LacZ infected cells. Letters above bars indicate statistical groups (ANOVA, < 0.05)

Effects of atrazine and simazine (10 mol/L each for and as marked for ) of three cell types, measured by RLA

<p><b>Copyright information:</b></p><p>Taken from "Atrazine-Induced Aromatase Expression Is SF-1 Dependent: Implications for Endocrine Disruption in Wildlife and Reproductive Cancers in Humans"</p><p></p><p>Environmental Health Perspectives 2007;115(5):720-727.</p><p>Published online 5 Feb 2007</p><p>PMCID:PMC1867956.</p><p>This is an Open Access article: verbatim copying and redistribution of this article are permitted in all media for any purpose, provided this notice is preserved along with the article's original DOI</p> () Atrazine and simazine stimulated ArPII in H295R cells without exogenous SF-1 supplementation. () ArPII response to atrazine and simazine in NIH-3T3 cells required coexpression of SF1. () Atrazine and simazine stimulation of SF-1–mediated ArPII in SF-1–co-transfected NIH-3T3 cells was dose dependent. Both triazines were effective at concentrations as low as 10 mol/L (ANOVA, < 0.05). Bars show mean ± SD; letters above bars indicate statistical groups (ANOVA, < 0.05)