Targeting the Renin-angiotensin system combined with an antioxidant is highly effective in mitigating radiation-induced lung damage

Abstract

Purpose: To investigate the outcome of suppression of the renin angiotensin system using captopril combined with an antioxidant (Eukarion [EUK]-207) for mitigation of radiation-induced lung damage in rats.

Methods and materials: The thoracic cavity of female Sprague-Dawley rats was irradiated with a single dose of 11 Gy. Treatment with captopril at a dose of 40 mg/kg/d in drinking water and EUK-207 given by subcutaneous injection (8 mg/kg daily) was started 1 week after irradiation (PI) and continuing until 14 weeks PI. Breathing rate was monitored until the rats were killed at 32 weeks PI, when lung fibrosis was assessed by lung hydroxyproline content. Lung levels of the cytokine transforming growth factor-β1 and macrophage activation were analyzed by immunohistochemistry. Oxidative DNA damage was assessed by 8-hydroxy-2-deoxyguanosine levels, and lipid peroxidation was measured by a T-BARS assay.

Results: The increase in breathing rate in the irradiated rats was significantly reduced by the drug treatments. The drug treatment also significantly decreased the hydroxyproline content, 8-hydroxy-2-deoxyguanosine and malondialdehyde levels, and levels of activated macrophages and the cytokine transforming growth factor-β1 at 32 weeks. Almost complete mitigation of these radiation effects was observed by combining captopril and EUK-207.

Conclusion: Captopril and EUK-207 can provide mitigation of radiation-induced lung damage out to at least 32 weeks PI after treatment given 1-14 weeks PI. Overall the combination of captopril and EUK-207 was more effective than the individual drugs used alone.

Figure 1

Breathing rate (mean breaths per minute) as a function of time after irradiation. Control = No irradiation; WTR = whole thorax irradiation (11Gy); WTR+Euk = whole thorax irradiation + Euk-207; WTR+Capt = whole thorax irradiation + Captopril; WTR+Euk+Capt = whole thorax irradiation + Euk-207 + Captopril; Euk+Capt = Euk-207 + Captopril. The drug treatments were started 1 week post-irradiation (PI) and terminated at 14 weeks PI. The rats were euthanized at 32 weeks PI. Each point represents the mean (±SEM) for all rats available for analysis at the different times. All groups contained 8 rats at the start of the experiment. At 8 weeks this was reduced to 6 rats for the WTR group and to 7 rats for the WTR + Euk-207 group and the WTR + Capt group due to morbidity requiring euthanasia. These numbers remained constant out to sacrifice at 32 weeks.

Figure 2

Body weight (mean in grams) as a function of time after irradiation. Labeling of the treatment groups (and number of rats per group) as indicated in the legend to Figure 1. Each point represents the mean (±SEM) for all rats available for analysis at the different times.

Figure 3

A) Hydroxyproline (µg of hydroxyproline/100mg of wet lung tissue) content of the lung tissue at 32 weeks after irradiation; B) Aperio analysis of TGF-beta staining (percent positivity) at 32 weeks after irradiation. C) Definiens analysis of TGF-beta staining (percent of cells stained). Each bar represents the mean (±SEM) for all rats available for analysis. Labeling of the treatment groups (and number of rats per group) as indicated in the legend to Figure 1. The asterisks indicate groups that are significantly different from the WTR group in each panel.

Figure 4

A) Analysis of 8-OHdG staining (percent positivity assessed using Tissue Scope) at 32 weeks after irradiation; B) Analysis of MDA levels (mM) in the lung tissue at 32 weeks after irradiation. Each bar represents the mean (±SEM) for all rats available for analysis. Labeling of the treatment groups (and number of rats per group) as indicated in the legend to Figure 1. The asterisks indicate groups that are significantly different from the WTR group in each panel.

Figure 5

Analysis of macrophage activation (ED-1 staining) at 32 weeks after irradiation as percent positivity (assessed using Tissue Scope). Each bar represents the mean (±SEM). Labeling of the treatment groups (and number of rats per group) as indicated in the legend to Figure 1. The asterisks indicate groups that are significantly different from the WTR group.