Abstract
Detection of free radicals in tissues is challenging. Most approaches rely on incubating excised sections or homogenates with reagents, typically at supraphysiologic oxygen tensions, to finally detect surrogate, nonspecific end products. In the present work, we explored the potential of using intravenously (i.v.) injected dihydroethidine (DHE) to detect superoxide radical (O2∙-) abundance in vivo by quantification of the superoxide-specific DHE oxidation product, 2-hydroxyethidium (2-OH-E+), as well as ethidium (E+) and DHE in multiple tissues in a murine model of endotoxemia induced by lipopolysaccharide (LPS). LPS was injected intraperitoneally (i.p.), while DHE was delivered via the tail vein one hour before sacrifice. Tissues (kidney, lung, liver, and brain) were harvested and subjected to HPLC/fluorescent analysis of DHE and its monomeric oxidation products. In parallel, electron spin resonance (EPR) spin trapping was used to measure nitric oxide (∙NO) production in the aorta, lung, and liver isolated from the same mice. Endotoxemic inflammation was validated by analysis of plasma biomarkers. The concentration of 2-OH-E+ varied in the liver, lung, and kidney; however, the ratios of 2-OH-E+/E+ and 2-OH-E+/DHE were increased in the liver and kidney but not in the lung or the brain. An LPS-induced robust level of ∙NO burst was observed in the liver, whereas the lung demonstrated a moderate yet progressive increase in the rate of ∙NO production. Interestingly, endothelial dysfunction was observed in the aorta, as evidenced by decreased ∙NO production 6 hours post-LPS injection that coincided with the inflammatory burden of endotoxemia (e.g. elevated serum amyloid A and prostaglandin E2). Combined, these data demonstrate that systemic delivery of DHE affords the capacity to specifically detect O2∙- production in vivo. Furthermore, the ratio of 2-OH-E+/E+ oxidation products in tissues provides a tool for comparative insight into the oxidative environments in various organs. Based on our findings, we demonstrate that the endotoxemic liver is susceptible to both O2∙--mediated and nonspecific oxidant stress as well as nitrosative stress. Oxidant stress in the lung was detected to a lesser extent, thus underscoring a differential response of liver and lung to endotoxemic injury induced by intraperitoneal LPS injection.
Highlights
Reactive oxygen species (ROS) are critical components of various disease processes with superoxide anion radical (O2∙-) often assuming the center of attention
There have been studies describing the topical administration of DHE to murine carotid arteries [4], where DHE was applied in vivo intraperitoneally [5,6,7,8,9,10,11] or intravascularly [12, 13] to study oxidative stress ex vivo in the brain using DHE-derived red fluorescence cianisfijaeccmtmiaoerntkheoorfdoDfoHOfE2O∙h-2pa∙-srodbdeetueenccttuiioosnend; u[t1on4fs]ot.urMtduyonOraet2oe∙lv-yei,nrt,hvsiausrbiisocnuuostattniasessoupueesswith fluorimetric detection [15, 16]; the authors did not take into account that the major fluorescent product of DHE oxidation is ethidium (E+) nor did they elucidate the abundance of the parent compound in analyzed tissues
The LPS insult is known to increase the level of proinflammatory cytokines [18, 19], which leads to excessive production of nitric oxide (∙NO) via NOS-2 and increased production of prostanoids due to cyclooxygenase2 (COX-2) induction as well as concomitant alternative mechanisms leading to endotoxemic multiorgan failure [20]
Summary
Reactive oxygen species (ROS) are critical components of various disease processes with superoxide anion radical (O2∙-) often assuming the center of attention. The aim of this study was to assess the feasibility of multiorgan in vivo oxidative stress detection using intravenously injected DHE, with subsequent quantitative HPLC-based analysis of the tissue accumulation of DHE, 2-OH-E+, and E+ and their ratios. To evaluate this methodology, we used mice with lipopolysaccharide- (LPS-) induced endotoxemia, as this model is well characterized by an inflammatory reaction with an NADPH-oxidase-dependent oxidant burst and elevation in RNS through the inducible nitric oxide synthase (NOS-2) pathway [17]. Additional analysis from the blood was used to validate endotoxemia severity for reliable interpretation
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