Early and late administration of MnTE-2-PyP5+ in mitigation and treatment of radiation-induced lung damage

Abstract

Chronic production of reactive oxygen and nitrogen species is an underlying mechanism of irradiation (IR)-induced lung injury. The purpose of this study was to determine the optimum time of delivery of an antioxidant and redox-modulating Mn porphyrin, MnTE-2-PyP(5+), to mitigate and/or treat IR-induced lung damage. Female Fischer-344 rats were irradiated to their right hemithorax (28 Gy). Irradiated animals were treated with PBS or MnTE-2-PyP(5+) (6 mg /kg/24 h) delivered for 2 weeks by sc-implanted osmotic pumps (beginning after 2, 6, 12, 24, or 72 h or 8 weeks). Animals were sacrificed 10 weeks post-IR. Endpoints were body weight, breathing frequency, histopathology, and immunohistochemistry (8-OHdG, ED-1, TGF-beta, HIF-1alpha, VEGF A). A significant radioprotective effect on functional injury, measured by breathing frequency, was observed for all animals treated with MnTE-2-PyP(5+). Treatment with MnTE-2-PyP(5+) starting 2, 6, and 12 h but not after 24 or 72 h resulted in a significant decrease in immunostaining for 8-OHdG, HIF-1alpha, TGF-beta, and VEGF A. A significant decrease in HIF-1alpha, TGF-beta, and VEGF A, as well as an overall reduction in lung damage (histopathology), was observed in animals beginning treatment at the time of fully developed lung injury (8 weeks post-IR). The catalytic manganese porphyrin antioxidant and modulator of redox-based signaling pathways MnTE-2-PyP(5+) mitigates radiation-induced lung injury when given within the first 12 h after IR. More importantly, this is the first study to demonstrate that MnTE-2-PyP(5+) can reverse overall lung damage when started at the time of established lung injury 8 weeks post-IR. The radioprotective effects are presumably mediated through its ability both to suppress oxidative stress and to decrease activation of key transcription factors and proangiogenic and profibrogenic cytokines.

Figure 1

Breathing frequencies, as recorded bi-weekly from week zero (pre-IR) to week ten (time-point of sacrifice). In comparison to nonirradiated control animals, significantly higher breathing rates were measured in animals which received no treatment after IR (P = 0.0079). Animals, which were irradiated and received MnTE-2-PyP⁵⁺ post-IR displayed significantly lower breathing rates in comparison to the IR-only group. Animals, which received late treatment starting 8 weeks post-IR displayed a trend towards elevated breathing frequencies which was not more seen 5 days after starting of treatment (implantation of pumps at day 5 of week 7; measurement of breathing frequencies five days later at day 3 of week 8).

Figure 1

Breathing frequencies, as recorded bi-weekly from week zero (pre-IR) to week ten (time-point of sacrifice). In comparison to nonirradiated control animals, significantly higher breathing rates were measured in animals which received no treatment after IR (P = 0.0079). Animals, which were irradiated and received MnTE-2-PyP⁵⁺ post-IR displayed significantly lower breathing rates in comparison to the IR-only group. Animals, which received late treatment starting 8 weeks post-IR displayed a trend towards elevated breathing frequencies which was not more seen 5 days after starting of treatment (implantation of pumps at day 5 of week 7; measurement of breathing frequencies five days later at day 3 of week 8).

Figure 2

Comparison of experimental groups on histopathological damage, macrophage activation (ED-1), oxidative stress (8-OHdG), transforming growth factor-β (TGF-β), hypoxia inducible factor-1α (HIF-1α), and vascular endothelial growth factor (A) (VEGF(A)). Results are displayed as vertical bar plots with standard deviation. Asterixes indicate statistical significant differences between IR-only group and IR + MnP treatment groups (p < 0.05).

Figure 3

Representative images of histotpathology (H&E staining) and Immunohistchemistry (8-OhDG, HIF-1α, VEGF (A), TGF-β, ED-1) studies. Magnification 100× for H&E, TGF-ß, VEGF(A), ED-1, Magnification 400× for 8-OHdG and HIF-1α. Groups: Control (no IR + PBS), IR + PBS (TGF-ß and HIF-1α images with 400× insert), IR + MnTE-2-PyP⁵⁺ (6 mg/kg) 2h group, IR + MnTE-2-PyP⁵⁺ 8 weeks group. Negative control shows normal lung structure, no positive (brown) immunostaining. IR + PBS shows large area of alveolar edema and cell infiltrates with beginning formation of fibrous masses and prominent immunostaining as well as activated macrophages (brown, localized interstitial and intra-alveolar). IR + MnTE-2-PyP⁵⁺ (2 h and 8 weeks groups) depict focal localized damage with thickening of alveolar wall, interstitial edema, diminished immunostaining and localized activated macrophages.