Long-term administration of a small molecular weight catalytic metalloporphyrin antioxidant, AEOL 10150, protects lungs from radiation-induced injury

Abstract

Purpose: To determine whether administration of a catalytic antioxidant, Mn(III) tetrakis(N,N'-diethylimidazolium-2-yl) porphyrin, AEOL 10150, with superoxide dismutase (SOD) mimetic properties, reduces the severity of radiation-induced injury to the lung from single-dose irradiation (RT) of 28 Gy.

Methods and materials: Rats were randomly divided into four different dose groups (0, 1, 10, and 30 mg/kg/day of AEOL 10150), receiving either short-term (1 week) or long-term (10 weeks) drug administration via osmotic pumps. Rats received single-dose irradiation (RT) of 28 Gy to the right hemithorax. Breathing rates, body weights, blood samples, histopathology, and immunohistochemistry were used to assess lung damage.

Results: There was no significant difference in any of the study endpoints between the irradiated controls and the three groups receiving RT and short-term administration of AEOL 10150. For the long-term administration, functional determinants of lung damage 20 weeks postradiation were significantly worse for RT + phosphate-buffered saline (PBS) and RT + 1 mg/kg/day of AEOL 10150 as compared with the irradiated groups treated with higher doses of AEOL 10150 (10 or 30 mg/kg/day). Lung histology at 20 weeks revealed a significant decrease in structural damage and collagen deposition in rats receiving 10 or 30 mg/kg/day after radiation in comparison to the RT + PBS and 1 mg/kg/day groups. Immunohistochemistry demonstrated a significant reduction in macrophage accumulation, oxidative stress, and hypoxia in rats receiving AEOL 10150 (10 or 30 mg/kg/day) after lung irradiation compared with the RT + PBS and 1 mg/kg/day groups.

Conclusions: The chronic administration of a novel catalytic antioxidant, AEOL 10150, demonstrates a significant protective effect from radiation-induced lung injury. AEOL 10150 has its primary impact on the cascade of events after irradiation, and adding the drug before irradiation and its short-term administration have no significant additional benefits.

Figure 1

Figure 1(A) Experimental Design: Animals were randomly divided into four duplicate groups for short (one day before through one week following irradiation) and long-term (from one day after through 10 weeks post-radiation) drug administration experiments. AEOL 10150 was delivered as a continuous infusion with an osmotic pump implanted subcutaneously, at three different concentrations of 1, 10 and 30 mg/kg/day. Figure 1(B) Plasma concentrations of AEOL 10150 at 7^th week of infusion. AEOL 10150 was delivered as a continuous infusion with an osmotic pump implanted subcutaneously, at three different concentrations of 1, 10 and 30mg/kg/day. Significant steady state plasma levels of AEOL 10150 were achieved at both the 10 and 30mg/kg/day treatments. Error bars represent ± SEM.

Figure 2

All groups exhibited steady gain in body weight throughout the treatment period. Both the RT+PBS and RT+ 1mg/kg/dayAEOL 10150 animals, however, exhibited significantly less weight gain beginning at week 14 compared with RT+10 or 30 mg/kg/day AEOL 10150 groups (RT+PBS and RT+1mg/kg/day vs. RT+10mg/kg/day at 14-20 weeks, # p<0.05, RT+PBS and RT+1mg/kg/day vs. RT+30mg/kg/day at 16-20 weeks, * p<0.05, respectively). Error bars represent ± SEM.

Figure 3

Time course of breathing rates after 28 Gy of single dose irradiation to the right hemithorax with or without AEOL 10150 treatment at dose of 1, 10 and 30 mg/kg. Breathing rates increased beginning at 8 weeks and reached a peak 18 weeks after irradiation in animals treated with PBS and 1mg AEOL 10150. The average breathing rates in irradiated groups treated with AEOL 10150 (10 or 30mg/kg/day) were significantly lower than the group receiving RT+PBS and RT+1mg/kg/day (RT+PBS and RT+1mg/kg/day vs. RT+10mg/kg/day at 12-20 weeks, # p<0.05, RT+1mg and RT+10mg/kg/day vs. RT+30mg/kg/day at 12-20 weeks, * p<0.05, respectively). Error bars represent ± SEM.

Figure 4

Figure. 4(A) Histologic comparison by hematoxylin and eosin staining among RT+PBS, RT+1mg mg/kg/day and RT+10 or 30mg/kg/day of AEOL 10150 at 20 weeks after 28 Gy of irradiation. Rats treated with 10 or 30 mg/kg/day AEOL 10150 after single dose irradiation showed less pulmonary damage than rats received irradiation plus PBS or AEOL 10150 1mg/kg/day. Figure. 4 (B) Semiquantitative analyses of lung histology at 20 weeks revealed a significant decrease in structural damage and its severity in animals receiving 10 and 30 mg/kg/day after radiation in comparison to RT+PBS group or RT+1mg/kg/day (RT+10 and 30mg/kg/day vs. RT+PBS and RT+1mg/kg/day AEOL 10150, p=0.01). Figure. 4(C) RT+PBS and RT+1mg/kg/day AEOL 10150 groups had greater number of focal areas with increased collagen deposition than did the RT+10 or 30 mg/kg/day AEOL 10150 groups. This translated into a higher lung fibrosis score for the RT+PBS and RT+1mg AEOL 10150 animals than the other groups, although no animals were found to have strong or intense staining for collagen (RT+PBS vs. RT+10 and 30mg/kg/day, * p=0.003, 0.005 respectively: RT+1mg/kg/day vs. RT+10 and 30mg/kg/day, # p=0.002, 0.003 respectively ). Error bars represent ± SEM.

Figure 4

Figure. 4(A) Histologic comparison by hematoxylin and eosin staining among RT+PBS, RT+1mg mg/kg/day and RT+10 or 30mg/kg/day of AEOL 10150 at 20 weeks after 28 Gy of irradiation. Rats treated with 10 or 30 mg/kg/day AEOL 10150 after single dose irradiation showed less pulmonary damage than rats received irradiation plus PBS or AEOL 10150 1mg/kg/day. Figure. 4 (B) Semiquantitative analyses of lung histology at 20 weeks revealed a significant decrease in structural damage and its severity in animals receiving 10 and 30 mg/kg/day after radiation in comparison to RT+PBS group or RT+1mg/kg/day (RT+10 and 30mg/kg/day vs. RT+PBS and RT+1mg/kg/day AEOL 10150, p=0.01). Figure. 4(C) RT+PBS and RT+1mg/kg/day AEOL 10150 groups had greater number of focal areas with increased collagen deposition than did the RT+10 or 30 mg/kg/day AEOL 10150 groups. This translated into a higher lung fibrosis score for the RT+PBS and RT+1mg AEOL 10150 animals than the other groups, although no animals were found to have strong or intense staining for collagen (RT+PBS vs. RT+10 and 30mg/kg/day, * p=0.003, 0.005 respectively: RT+1mg/kg/day vs. RT+10 and 30mg/kg/day, # p=0.002, 0.003 respectively ). Error bars represent ± SEM.

Figure 4

Figure. 4(A) Histologic comparison by hematoxylin and eosin staining among RT+PBS, RT+1mg mg/kg/day and RT+10 or 30mg/kg/day of AEOL 10150 at 20 weeks after 28 Gy of irradiation. Rats treated with 10 or 30 mg/kg/day AEOL 10150 after single dose irradiation showed less pulmonary damage than rats received irradiation plus PBS or AEOL 10150 1mg/kg/day. Figure. 4 (B) Semiquantitative analyses of lung histology at 20 weeks revealed a significant decrease in structural damage and its severity in animals receiving 10 and 30 mg/kg/day after radiation in comparison to RT+PBS group or RT+1mg/kg/day (RT+10 and 30mg/kg/day vs. RT+PBS and RT+1mg/kg/day AEOL 10150, p=0.01). Figure. 4(C) RT+PBS and RT+1mg/kg/day AEOL 10150 groups had greater number of focal areas with increased collagen deposition than did the RT+10 or 30 mg/kg/day AEOL 10150 groups. This translated into a higher lung fibrosis score for the RT+PBS and RT+1mg AEOL 10150 animals than the other groups, although no animals were found to have strong or intense staining for collagen (RT+PBS vs. RT+10 and 30mg/kg/day, * p=0.003, 0.005 respectively: RT+1mg/kg/day vs. RT+10 and 30mg/kg/day, # p=0.002, 0.003 respectively ). Error bars represent ± SEM.

Figure 5

Figure. 5(A) Panel of ED-1, 8-OHdG and CA9 immunohistochemistry: Lung tissues were immunostained for macrophage activation (ED1), oxidative stress (8OHdG) and tissue hypoxia (CA 9) (brown staining), in RT+PBS, RT+1mg AEOL 10150, RT+10or 30 mg/kg/day AEOL 10150 groups. The lungs of rats treated with high-doses of AEOL 10150 (10 or 30 mg/kg/day) exhibited much fewer activated macrophages, less oxidative stress and less hypoxia than rats treated with RT+PBS and RT+1 mg/kg/day AEOL 10150. These photomicrographs are representative of results obtained from 6-8 animals in each group. For ED1 and 8OHdG, each image is 400× magnification and CA9 100x. Figure. 5(B) Semi quantitative analysis of activated macrophages in the lungs of rats 20 weeks after 28 Gy of single dose irradiation with or without AEOL 10150 treatment. RT+PBS and RT+1 mg/kg/day AEOL 10150 groups had significantly more activated macrophages per field of lung tissue than high-doses (RT+PBS vs. RT+10 and 30mg/kg/day, * p=0.0006, 0.016 respectively: RT+1mg/kg/day vs. RT+10mg/kg/day, # p=0.02). There was no significant difference between group receiving RT+1mg/kg/day AEOL 10150 compared with RT+PBS and RT+30mg/kg/day (p > 0.05). Error bars represent ± SEM. Figure. 5(C) Semi quantitative analysis for the expression of 8-OHdG marker of DNA oxidation and oxidative stress, after 28 Gy of single dose irradiation. A significant reduction in the expression of DNA oxidation was seen in radiation plus high-dose AEOL 10150 treatment groups (RT+PBS vs. RT+10 and 30mg/kg/day, * p=0.006, 0.014 respectively: RT+1mg/kg/day vs. RT+10mg/kg/day and 30mg/kg/day, # p=0.004, 0.009 respectively). Error bars represent ± SEM. Figure. 5(D) RT+PBS and RT+1mg/kg/day AEOL 10150 animals expressed strong CA 9 immunoreactivity, which was seen mainly in the irradiated, damaged tissue and inflammatory cell The tissue protein expression of CA 9 was markedly reduced in the groups that received radiation plus high doses of AEOL 10150 (10 and 30 mg/kg/day) (RT+PBS vs. RT+10 and 30mg/kg/day, * p=0.001, 0.0009 respectively: RT+1mg/kg/day vs. RT+10mg/kg/day and 30mg/kg/day, # p=0.00001, 0.00005 respectively). Error bars represent ± SEM.

Figure 5

Figure. 5(A) Panel of ED-1, 8-OHdG and CA9 immunohistochemistry: Lung tissues were immunostained for macrophage activation (ED1), oxidative stress (8OHdG) and tissue hypoxia (CA 9) (brown staining), in RT+PBS, RT+1mg AEOL 10150, RT+10or 30 mg/kg/day AEOL 10150 groups. The lungs of rats treated with high-doses of AEOL 10150 (10 or 30 mg/kg/day) exhibited much fewer activated macrophages, less oxidative stress and less hypoxia than rats treated with RT+PBS and RT+1 mg/kg/day AEOL 10150. These photomicrographs are representative of results obtained from 6-8 animals in each group. For ED1 and 8OHdG, each image is 400× magnification and CA9 100x. Figure. 5(B) Semi quantitative analysis of activated macrophages in the lungs of rats 20 weeks after 28 Gy of single dose irradiation with or without AEOL 10150 treatment. RT+PBS and RT+1 mg/kg/day AEOL 10150 groups had significantly more activated macrophages per field of lung tissue than high-doses (RT+PBS vs. RT+10 and 30mg/kg/day, * p=0.0006, 0.016 respectively: RT+1mg/kg/day vs. RT+10mg/kg/day, # p=0.02). There was no significant difference between group receiving RT+1mg/kg/day AEOL 10150 compared with RT+PBS and RT+30mg/kg/day (p > 0.05). Error bars represent ± SEM. Figure. 5(C) Semi quantitative analysis for the expression of 8-OHdG marker of DNA oxidation and oxidative stress, after 28 Gy of single dose irradiation. A significant reduction in the expression of DNA oxidation was seen in radiation plus high-dose AEOL 10150 treatment groups (RT+PBS vs. RT+10 and 30mg/kg/day, * p=0.006, 0.014 respectively: RT+1mg/kg/day vs. RT+10mg/kg/day and 30mg/kg/day, # p=0.004, 0.009 respectively). Error bars represent ± SEM. Figure. 5(D) RT+PBS and RT+1mg/kg/day AEOL 10150 animals expressed strong CA 9 immunoreactivity, which was seen mainly in the irradiated, damaged tissue and inflammatory cell The tissue protein expression of CA 9 was markedly reduced in the groups that received radiation plus high doses of AEOL 10150 (10 and 30 mg/kg/day) (RT+PBS vs. RT+10 and 30mg/kg/day, * p=0.001, 0.0009 respectively: RT+1mg/kg/day vs. RT+10mg/kg/day and 30mg/kg/day, # p=0.00001, 0.00005 respectively). Error bars represent ± SEM.