Multiorgan Development of Oxidative and Nitrosative Stress in LPS-Induced Endotoxemia in C57Bl/6 Mice: DHE-Based In Vivo Approach

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.

Figure 1

Monomeric DHE oxidation products formed in vivo in the liver (a, d, and g), lung (b, e, and h), and kidney (c, f, and i) tissues in a murine model of endotoxemia, 6 and 12 hours after LPS challenge. Using HPLC-Fl detection, the superoxide-specific 2-OH-E+ and nonspecific E+ monomeric products of DHE oxidation were measured according to the details presented in Materials and Methods. Additionally, the ratio of 2-OH-E+/E+ was calculated for each sample. Data are presented as mean ± SD or median ± IQR, and statistical significance was tested using one-way ANOVA or the nonparametric Kruskal-Wallis ANOVA, depending on the distribution and homoscedasticity of data. p values <0.05 were considered significant, with ∗ <0.05 and ∗∗ <0.01 versus the control group and ○○ <0.01 versus the LPS 6 h group.

Figure 2

Superoxide-specific and unspecific oxidation of DHE accumulated in the liver and lung tissue in a murine model of endotoxemia, 6 and 12 hours after LPS challenge. Ratios of 2-OH-E⁺/DHE accumulated in the liver and lung tissues suggest that the liver is under increased superoxide-specific oxidant stress (a), as opposed to the lung (b). Moreover, the ratio of E⁺/DHE also suggests increased superoxide-independent oxidative stress in the liver (c), which is not the case in the lung (d). Data are presented as median ± IQR, and statistical significance was tested using nonparametric Kruskal-Wallis ANOVA. p values <0.05 were considered significant, with ^∗<0.05 and ^∗∗<0.01 versus the control group, and <0.01 versus the LPS 6 h group.

Figure 3

Nitric oxide production ex vivo in tissues and nitrite/nitrate burst in plasma in a murine model of endotoxemia, 6 and 12 hours after LPS challenge. EPR spin trapping as described in Materials and Methods was used to detect nitric oxide ex vivo in the aorta, lung, and liver tissues (a, b, and c, respectively), whereas nitrite (d) and nitrate (e) were measured in the plasma using the ENO-20 apparatus. Gradually increased NO production was seen in the lungs at 6 and 12 hours post-LPS injection (b), while the liver showed a much higher NO overproduction (c, 6-7-fold change). Data are presented as mean ± SD or median ± IQR (in c), and statistical significance was tested using one-way ANOVA post hoc LSD's test or Kruskal-Wallis's, post hoc Dunn's (in c), depending on the distribution and homoscedasticity of data. p values < 0.05 were considered significant, with ∗ <0.05, ∗∗ <0.01, ∗∗∗ <0.005, and ∗∗∗∗ <0.001 versus the control group.

Figure 4

Markers of the organ injury and systemic inflammation in a murine model of endotoxemia, 6 and 12 hours after LPS challenge. Liver damage—ALT (a) and AST (b), kidney damage—creatinine (c), and systemic inflammation—SAA (d). Data are presented as mean ± SD or median ± IQR, and statistical significance was tested using one-way ANOVA post hoc LSD's test or Kruskal-Wallis, post hoc Dunn's, depending on the distribution and homoscedasticity of data. p values <0.05 were considered significant, with ∗ <0.05, ∗∗ <0.01, and ∗∗∗∗ <0.001 versus the control group and ○ <0.05 and ○○ <0.01 versus the LPS 6 h group.

Figure 5

Eicosanoid concentration in plasma in a murine model of endotoxemia, 6 and 12 hours after LPS challenge. PGE₂ (a), TXB₂ (b), and 12-HETE (c). Data are presented as mean ± SD or median ± IQR, and statistical significance was tested using one-way ANOVA post hoc LSD's test or Kruskal-Wallis, post hoc Dunn's (for b, c), depending on the distribution and homoscedasticity of data. p values <0.05 were considered significant, with ^∗ <0.05, ^∗∗ <0.01, and ^∗∗∗∗ <0.001 versus the control group, and ○ <0.05 and ○○ <0.01 versus the LPS 6 h group.