Comparison of two Mn porphyrin-based mimics of superoxide dismutase (SOD) in pulmonary radioprotection

Abstract

Development of radiation (RT) induced lung injury is associated with chronic production of reactive oxygen and nitrogen species (ROS/RNS). MnTE-2-PyP⁵⁺ is a catalytic Mn porphyrin (MnP) mimic of SOD, already shown to protect lungs from RT induced injury by scavenging ROS/RNS. The purpose of this study was to compare MnTE-2-PyP⁵⁺ with the newly synthesized MnTnHex-2-PyP⁵⁺ which is expected to be a more effective radioprotector due to its lipophilic properties. This study shows that Fischer rats which were irradiated to their right hemithorax (28 Gy) have less pulmonary injury as measured with breathing frequencies when treated with daily subcutaneous injections of MnTE-2-PyP⁵⁺ (3 and 6 mg/kg) or MnTnHex-2-PyP⁵⁺ (0.3 – 0.6 – 1.0 mg/kg) for 2 weeks after RT. However, at 16 weeks post RT, only MnTE-2-PyP⁵⁺ at a dose of 6 mg/kg is able to ameliorate oxidative damage, block activation of HIF-1α and TGF-β and impair upregulation of CA-IX and VEGF. MnTnHex-2-PyP⁵⁺ at a dose of 0.3 mg/kg is effective only in reducing RT-induced TGF-β and CA-IX expression. Significant loss in bodyweight was observed in animals receiving MnTnHex-2-PyP⁵⁺ (0.3 and 0.6 mg/kg). MnTnHex-2-PyP⁵⁺ has the ability to dissolve lipid membranes causing local irritations/necrosis at injection sites if given at doses of 1 mg/kg or higher. In conclusion, both compounds show ability to ameliorate lung damage as measured with breathing frequencies and histopathologic evaluation. However, MnTE-2-PyP⁵⁺ at 6 mg/kg proved to be more effective in reducing expression of key molecular factors known to play an important role in radiation-induced lung injury.

Introduction

Radiation-induced lung injury can develop after therapeutic as well as accidental exposure to ionizing radiation. Experience with nuclear accident victims suggests that when gastrointestinal and bone marrow syndromes are successfully treated, respiratory failure due to radiation pneumonitis and lung fibrosis later became the major cause of death [1-4]. Furthermore, in clinical settings, approximately 20% of patients receiving thoracic radiation therapy (RT) go on to develop injury, thereby limiting the delivery of higher and thus more effective radiation doses. Therefore, strategies to prevent and/or ameliorate radiation-induced lung injury could be beneficial in both the clinical setting as well as in the event of accidental radiation exposure.

Recent insights into the mechanism of radiation-induced lung injury [5-7] have opened new opportunities for therapeutic intervention. A new therapeutic approach is targeted against the continuous production of reactive oxygen/reactive nitrogen species (ROS/RNS) in an ongoing process that perpetuates lung injury [5]. Superoxide dismutase (SOD) is an endogenous enzyme capable of neutralizing superoxide (O₂^•-), and subsequently reducing the production of other highly damaging species, particularly hydroxyl radical (OH^•), peroxynitrite (ONOO^-), and its decomposition products carboxyl radical (CO₃^•-) and nitric dioxide (NO₂^•). Previous studies have shown that chronic overproduction of ROS/RNS and subsequent pathological disease can be alleviated in a rodent model of radiation injury through overexpression of SOD [8, 9]. Furthermore, it has been shown that manganese (III) tetrakis (N-ethylpyridinium-2-yl) porphyrin (MnTE- 2-PYP⁵⁺, MnP) and other derivatives of SOD are effective radioprotectors [10-14].

Nevertheless, a limiting factor for biological efficacy of MnTE-2-PYP⁵⁺ is its hydrophilicity, which prevents the complete translocation of this compound through lipid membranes. Addition of lengthened alkyl groups to the pyridyl rings results in manganese porphyrins with higher lipophilicity and preserved charges and thus maintained antioxidant potency (MnTnHex-2-PyP⁵⁺ and MnTnOct-2-PyP⁵⁺) [15]. The herein tested MnTnHex-2-PyP⁵⁺ is a hexyl analogue of MnTE-2-PyP⁵⁺ with hexylpyridyl groups instead of ethyl groups on ortho quaternized nitrogens. In vitro data, generated in an E.coli model of oxidative stress, suggest that MnTnHex-2-PyP⁵⁺ has at least 10-fold higher efficacy in protecting from oxidative stress than MnTE-2-PyP⁵⁺ [16] therefore allowing smaller doses limiting costs and potential side effects.

The purpose of this study is to compare the radioprotective effect of MnTE-2-PyP⁵⁺ with a compound showing higher lipophilicity, MnTnHex-2-PyP⁵⁺, in an established rat model of radiation-induced lung injury.

Materials and Methods

Animals

Forty-eight female Fischer-344 rats weighing between 125 and 140 g were used in this study with prior approval from the Duke University Institutional Animal Care and Use Committee. Three animals were housed per cage and maintained under identical conditions with food and water provided ad libitum. All rats were sacrificed at a predetermined time of sixteen weeks post-radiation by pentobarbital overdose.

Drugs

MnTE-2-PyP⁵⁺ and MnTnHex-2-PyP⁵⁺ were synthesized and characterized as previously described [15]. MnTnHex-2-PyP⁵⁺ was additionally purified by ultrafiltration through 500-cutoff filter to remove excess of free Manganese and then analyzed for the purity. Solutions of both Manganese porphyrins used in this study were dissolved in PBS.

Irradiation and SOD mimetics

At the time of irradiation all rats weighed between 140 and 160 g to minimize possible variations in lung size. The 48 animals were divided equally into the following groups to receive: 1) right hemithoracic irradiation (RT) and PBS injection (2 ml), 2) RT and MnTE-2-PyP⁵⁺ (1 mg/kg), 3) RT and MnTE-2-PyP⁵⁺ (3 mg/kg), 4) RT and MnTE-2-PyP⁵⁺ (6 mg/kg), 5) RT and MnTnHex-2-PyP⁵⁺ (0.3 mg/kg), 6) RT and MnTnHex-2-PyP⁵⁺ (0.6 mg/kg), 7) RT and MnTnHex-2-PyP⁵⁺ (1 mg/kg), and 8) Control (sham-RT and PBS injection). The animals were anesthetized before RT with intraperitoneal injection of ketamine (65 mg/kg) and xylazine (4.5 mg/kg) and placed in a prone position. Hemithoracic radiation was delivered to the right lung with a single dose of 28 Gy using 150 kV x-rays with a dose rate of 0.71 Gy/min (Therapax 320, Pantak Inc., East Haven, CT). 12 mm lead blocks were used to protect the left thorax and the rest of the body. This is a prerequisite for studying long-term changes (months, [5, 6, 11, 13]) after RT which might not be tolerated by the animals if both lungs were irradiated with 28 Gy, a dose necessary to induce a robust fibrotic damage in the lung.

For the irradiated rats in the treatment groups, subcutaneous (s.c.) injection of MnTE-2-PyP⁵⁺ or MnTnHex-2-PyP⁵⁺ were administered daily for 14 days beginning 2 hours after RT. Irradiated and non-irradiated control animals received an s.c. injection of equivalent volume of PBS.

Follow-up and functional assessment of lung injury

All animals were followed-up for sixteen weeks after RT. Bodyweight was measured bi-weekly. Breathing frequency using whole-body plethysmography (Model RM-80, Columbus Instruments, Columbus, OH, USA) was measured as an indicator of pulmonary injury. Measurements were taken every 2 weeks for 16 weeks, starting 4 weeks after RT. The mean values of five measurements were recorded for 5 animals of each group.

Histology

At the time of sacrifice, the right upper lobes of all animals were obtained for immunohistochemistry and histopathology studies. Animals were euthanized with pentobarbital overdose. Both lungs were infused by tracheal instillation of a solution containing 2% glutaraldehyde and 0.085 M sodium cacodylate buffer for 25 minutes for fixation prior to removal of the lung. After removal the four lobes of the irradiated right lung were separated and embedded in paraffin. The tissue was then cut into 5 μm thick sections at a microtome and stored on slides.

Histopathology

Five-micrometer thick sections of the lung tissue embedded in paraffin were stained with hematoxylin and eosin (H&E) and Masson’s trichrome to visualize the extent of fibrosis and collagen deposition in the lung. Slides were systematically scanned under a microscope using a 10x objective and eight to ten fields containing the highest degree of fibrosis were selected. The extent of radiation induced fibrosis for each field was graded on a scale from 0 (normal lung) to 8 (total fibrous obliteration of the field), as described by Ashcroft et al. [17]. Average scoring below grade 4 (moderate thickening of walls without obvious damage to lung architecture) resulted in the animal’s placement in the “no damage” group while average scoring above grade 4 (increased fibrosis with definite damage to lung architecture) led to the respective animal’s placement in the “damage” group.

Immunohistochemistry

Lungs were assessed for the number and activity of macrophages with ED-1 staining, the level of expression of 8-OHdG (8-hydroxy-2’-deoxyguanosine) as a marker for oxidative stress/ ROS production, levels of TGF-β (transforming growth factor-beta), a profibrogenic cytokine, HIF-1α (hypoxia inducible factor 1α), a pro-angiogenic transcription factor and its products VEGF (A) (vascular endothelial growth factor A), a pro-angiogenic cytokine, and CA-IX (carbonic anhydrase IX), a marker associated with cellular hypoxia.

Immunohistochemistry was performed as described by Hsu et al. [18] Briefly, the tissue sections were deparaffinized and rehydrated with xylene and decreasing alcohol concentrations from100% to 80%. Endogenous peroxidase activity was blocked with 3% hydrogen peroxide for 15 minutes. The slides were then placed in a citrate buffer (Biogenex, San Ramon, CA) and heated in a microwave for 2 × 5 minutes for antigen retrieval. The tissue sections were rinsed with phosphate-buffered saline and incubated with primary antibodies to activated macrophage marker ED1 (MCA341, 1:100, Serotec, Oxford, UK), 8-OHdG (MOG-020P, 1:1000, JaICA, Shizuoka, Japan), VEGF (A) (Sc-152, 1:100, Santa Cruz Biotechnology Inc., Santa Cruz, CA), active TGF-β1 (Sc-146, 1:200, Santa Cruz Biotechnology Inc., Santa Cruz, CA), CA-IX (NB 100-417, 1:1000, Novus Biologicals, Littleton, CO), and HIF-1α (NB 100-105, 1:100, Novus Biologicals, Littleton, CO) overnight at 4°C. Slides were then washed three times in phosphate-buffered saline for 5 minutes followed by the incubation with the appropriate secondary antibody (1:1000, Jackson ImmunoResearch, West Grove, PA) for 30 minutes at room temperature. Again slides were washed three times in phosphate-buffered saline for 5 minutes followed by incubation with ABC-Elite (Vector Laboratories, Burlingame, CA) for 30 minutes at room temperature and developed using DAB working solution (Laboratory Vision, Fremont, CA). Finally, the slides were counterstained with Harris hematoxylin (Fisher Scientific, Pittsburgh, PA) and mounted with coverslips.

Image Analysis

For image analysis, tissue sections from the upper right lobe from each animal were examined. Image analysis was carried out as previously described [13]. Eight digital images were acquired from each slide using a 20x (VEGF, TGF-β, CA-IX, ED-1) or 40x (HIF-1, 8-OHDG) objective. Activated macrophage marker ED1, 8-OHdG and HIF-1α staining were quantified by manual counting per image. Results were expressed as the number of positively stained macrophages or percent of 8-OHdG/HIF-1α positive nuclei per digital image (average of eight digital images per animal, average of five to six animals per group).

Active TGF-β, VEGF, and CA-IX were analyzed in Adobe Photoshop (Version 9.0.2; Adobe Systems, San Jose, CA). After quantifying the positive expression per image, the total tissue area regardless of expression was quantified. Results represent the average percentage of positive staining as the ratio of positive staining over total tissue area per digital image.

Statistical Analysis

The discrepancies between binomial proportions were tested using Fisher’s exact test [19]. The distributions of the biomarkers with respect to drug (or dose) were compared in a pairwise fashion using the exact two-sample Wilcoxon test [20]. For breathing frequencies and body weight, an aggregate measure was computed as the empirical area under the curve. The distributions of these curves were compared in a pairwise fashion using the exact two-sample Wilcoxon test [20]. The R [21] environment was used for all statistical calculations.

Results

In preliminary toxicity experiments, female Fischer 344 rats were injected daily for 14 days subcutaneously with MnTnHex-2-PyP⁵⁺, doses ranging from 0.5 to 3 mg/kg. Local alopecia at injection sites turning into necrosis was observed at doses ≥ 1.0 mg/kg. Therefore, a dose range from 0.3 to 1 mg/kg for MnTnHex-2-PyP⁵⁺ was defined for the radiation experiment while a dose range from 1 to 6 mg/kg was used for MnTE-2-PyP⁵⁺ which proved to be effective in earlier studies [10].

Body Weight and Breathing Frequencies

Body weight was measured on the day of RT, one week after RT, and biweekly starting from week two until week 16, the time point of sacrifice. Average starting bodyweight over all groups was 152.5 ± 1.4 g. The control group gained weight throughout the process until 16 weeks after sham-RT. In comparison, all irradiated animals showed a significant delay in weight gain (p < 0.001 vs. control), irrespective of MnP injections. This difference sustained until the end of experiment. Injection of MnTnHex-2-PyP⁵⁺ (0.3 mg/kg (p = 0.05 vs. RT) and 0.6 mg/kg (p < 0.001 vs. RT) resulted in a greater weight delay in comparison to all irradiated and MnP injected animals after 2 weeks of MnP injection. This delay in gain of bodyweight was not more apparent after 16 weeks (see Figure 1).

Figure 1.

Course of bodyweight, as measured from week zero (pre-RT) to week sixteen (time-point of sacrifice).

All irradiated groups showed significant delay in rise of weight throughout the whole course. MnP injection resulted in greater loss in comparson to RT +PBS. This effect was not more seen after MnP injections were stopped. MnTnHex-2-PyP⁵⁺ 0.3 mg/kg (p = 0.05 vs. RT group) and 0.6 mg/kg (p < 0.001 vs. RT group) resulted in significantly more weight loss than all other tested MnP.

Breathing frequencies were measured in a representative group (n = 5) before RT and in 5 animals per group biweekly, starting 4 weeks after RT until 16 weeks post RT. Significant lower breathing frequencies were seen in animals receiving RT + MnTE-2-PyP⁵⁺ (6 mg/kg, p = 0.008 vs. RT and 3 mg/kg, p = 0.016 vs. RT) and RT + MnTnHex-2-PyP⁵⁺ (all groups, p = 0.008 vs. RT) in comparison with the RT + PBS group (see Figure 2).

Figure 2.

Breathing frequencies, as recorded bi-weekly from week zero (pre-RT) to week sixteen (time-point of sacrifice). Starting approx. four weeks after RT, animals which received no treatment presented significantly elevated breathing frequencies. No significant difference was seen between normal control and animals which received for two weeks s.c. injections of MnTE-2-PYP⁵⁺ (3 [p = 0.016 vs. RT] and 6 mg/kg [p = 0.008 vs. RT] or MnTnHex-2-PyP⁵⁺ [all tested dosages p = 0.008 vs. RT]) after RT.

Histopathology

H&E staining revealed no damage in the control group (average scoring grade = 0). In comparison, all animals in the RT + PBS group showed an average damage score above 4, composed of severe distortion of lung structure, large fibrous masses, accumulation of alveolar macrophages, and interstitial/alveolar edema. In contrast, only animals which received RT + the highest dose of MnTE-2-PyP⁵⁺ (6 mg/kg, p = 0.015 vs. RT) and RT + the lowest dose of MnTnHex-2-PyP⁵⁺ (0.3 mg, p = 0.015 vs. RT) displayed an average damage grading below 4, reflecting mostly thickening of the alveolar wall, interstitial/alveolar edema and, sporadic accumulation of macrophages but no definitive damage to lung structures (see Table 1 and Figure 3).

Table 1.

P-values of tested parameters in histopathology (HP) and immunhistochemistry (IHC) studies.

	Compound	MnTE-2-PyP5+			MnTnHex-2-PyP5+
	Groups	1 mg/kg	3 mg/kg	6 mg/kg	0.3 mg/kg	0.6 mg/kg	1 mg/kg
HP	H & E	0.182	0.061	0.015	0.015	0.182	0.455
	MT	0.242	0.545	0.242	1.000	1.000	0.545
IHC	8-OHDG	1.000	1.000	0.041	0.429	1.000	0.818
	TGF-β	1.000	0.041	0.002	0.004	0.177	0.937
	HIF-1 α	1.000	0.931	0.009	0.151	0.082	0.429
	VEGF (A)	1.000	1.000	0.016	0.841	1.000	0.931
	CA-IX	0.429	0.222	0.004	0.016	0.030	0.247
	ED-1	1.000	1.000	0.310	0.537	0.792	0.699

Group comparison vs. RT + PBS injection group. P-values were determined by Wilcoxon rank sum test; p –value < 0.05 is considered significant.

HP: Hematoxylin & Eosin (H&E; structural damage), Masson’s Trichrome (MT; fibrous structural damage). IHC: 8-hydroxydiguanosine (8-OHDG; DNA-oxidation), transforming growth factor-beta (TGF-β; key factor in development of lung fibrosis), hypoxia inducible factor 1 alpha (HIF-1α; alpha subunit of the transcription factor responsible for VEGF and CA-IX), vascular endothelial growth factor (A) (VEGF (A); growth factor responsible for vasculogenesis and endothelial leakage, regulated by HIF-1), carbonic anhydrase IX (CA-IX; strongly expressed in hypoxic cells and regulated by HIF-1), ED-1 (ED-1 antibody for CD 68 antigen in activated rat macrophages).

Figure 3.

Representative images of histotpathology (H&E, Masson’s Trichrome) and Immunohistchemistry (8-OHDG, TGF-β, HIF-1α, VEGF (A), CA-IX, ED-1) studies. Magnification 10X. Groups: Control, RT + PBS injection (8-OHDG and HIF-1α images with 40X insert), RT + MnTE-2-PyP⁵⁺ injection (6 mg/kg), RT + MnTnHex-2-PyP⁵⁺ injection (0.3 mg/kg). Negative control shows normal lung structure, no positive (brown) immunostaining. RT + PBS shows large area of fibrous damage (honey comb lung) and prominent immunostaining as well as activated macrophages (brown, localized interstitial and intra-alveolar). RT + MnTE-2-PyP⁵⁺ and RT + MnTnHex-2-PyP⁵⁺ depict focal localized damage with thickening of alveolar wall, interstitial edema, diminished immunostaining and localized activated macrophages.

Masson’s trichrome staining showed no damage in the control group and 83 % fibrous damage with grading above 4 in the RT + PBS group (p = 0.015 vs. control). Statistical analysis revealed no significant difference between irradiated animals which received treatment with MnP in comparison to the RT only group (see Table 1 and Figure 3).

Immunohistochemistry

8-OHdG is a biomarker for DNA-oxidation [22]. After 16 weeks, no DNA-oxidation could be detected in the control group. In contrast, a distinct staining pattern positive for 8-OHdG was seen in the RT + PBS group but also in animals which were treated with MnTE-2-PyP⁵⁺ (1 and 3 mg/kg) as well as all dosages of MnTnHex-2-PyP⁵⁺. A significant reduction in DNA-oxidation was seen only in animals which received RT + MnTE-2-PyP⁵⁺ (6 mg/kg, p = 0.041 vs. RT; see Table 1 and Figure 3).

Active TGF-β is a key factor in the development of lung fibrosis after RT. In our study, the control group showed no explicit staining for TGF-β. In comparison, TGF-β positive staining was found in the animals which received PBS, MnTE-2-PyP⁵⁺ (1 mg/kg) or MnTnHex-2-PyP⁵⁺ (0.6 and 1 mg/kg) after RT. Animals which received RT + MnTE-2-PyP⁵⁺ (3 mg/kg, p = 0.041 vs. RT and 6 mg/kg, p = 0.002 vs. RT) or RT + MnTnHex-2-PyP⁵⁺ (0.3 mg/kg, p = 0.004 vs. RT) showed significantly reduced staining for active TGF-β at 16 weeks after RT (see Table 1 and Figure 3)

No positive staining for the transcription factor HIF-1α was seen in the control group. Animals in the RT + PBS, RT + MnTE-2-PyP⁵⁺ (1 mg/kg and 3 mg/kg), and all RT + MnTnHex-2-PyP⁵⁺ groups showed positive staining for HIF-1α. Only animals which received RT + MnTE-2-PyP⁵⁺ (6 mg/kg) showed significantly decreased staining for HIF-1α (p = 0.009 vs. RT; see Table 1 and Figure 3).

VEGF promotes vascular genesis and endothelial leakage and has been linked to the development of lung fibrosis. No positive staining for VEGF (A) was seen in the control group. 16 weeks after RT, significant positive staining for VEGF (A) could be detected in all groups apart from the animals which received RT + MnTE-2-PyP⁵⁺ (6 mg/kg, p = 0.016 vs. RT; see Table 1 and Figure 3).

CA-IX is related to local cellular hypoxia. No CA-IX staining was seen in the control group. Positive staining for CA-IX was observed in the RT + PBS group and in animals receiving MnTE-2-PyP⁵⁺ (1 and 3 mg/kg) or MnTnHex-2-PyP⁵⁺ (1 mg/kg) after RT. Significantly reduced positive CA-IX staining was seen in the RT + MnTE-2-PyP⁵⁺ (6 mg/kg, p = 0.004 vs. RT) and in MnTnHex-2-PyP⁵⁺ (0.3 mg/kg, p = 0.016 vs. RT) and 0.6 mg/kg, p = 0.030 vs. RT) groups (see Table 1 and Figure 3).

Activated macrophages stain positive for ED-1. Activation and recruitment of alveolar macrophages is a hallmark of radiation induced lung injury. No activation of macrophages was seen in the control animals. Positive staining for ED-1 was observed in all irradiated animals. No statistical evidence was found, suggesting reduced activation of macrophages by MnP (see Table 1 and Figure 3).

Discussion

This is the first study in a rodent model of radiation induced lung injury where two super oxide (SOD) mimics of identical ROS/RNS scavenging abilities, yet different lipophilicities, MnTE-2-PyP⁵⁺ and MnTnHex-2-PyP⁵⁺ were compared.

Herein, we found the more hydrophilic SOD mimic, MnTE-2-PyP⁵⁺ (6 mg/kg) and the more lipophilic MnTnHex-2-PyP⁵⁺ at a 20-fold lower dose (0.3 mg/kg) can prevent higher degree of histological lung structure damage with direct effect on vital parameters like breathing frequency if administered directly after RT and if injections are continued for 14 days. However, at 16 weeks post RT, only MnTE-2-PyP⁵⁺ at a dose of 6 mg/kg is able to ameliorate oxidative damage, block activation of HIF-1α and TGF-β and impair upregulation of CA-IX and VEGF. MnTnHex-2-PyP⁵⁺ at a dose of 0.3 mg/kg is effective only in reducing RT-induced TGF-β and CA-IX expression. MnTnHex-2-PyP⁵⁺ appears to be more toxic as seen in local effects at injection sites and body weight measurements.

So far, three groups of SOD-mimics have been developed and tested in different models of oxidative stress: manganese porphyrins (MnP) [23, 24], manganese (II) penta-azamacrocylclic complexes [25], and manganese (III) salen complexes [26]. MnP and manganese (III) salen complexes have been tested as radioprotectors in models of radiation-induced lung damage [10, 13, 14]. Recently, it has been shown, that EUK-189, a representative of the manganese (III) salen complexes is able to prevent irradiation-induced DNA-double strand breaks in partial lung irradiation if given immediately after RT. EUK-189 failed to enhance survival of rats 16 weeks post RT if given once daily i.p. for three days starting 5 min after RT in a model of whole lung irradiation (15.5 – 18 – 20.5 Gy) [14].

In our study, at 16 weeks after RT, only MnTE-2-PyP⁵⁺ at a dose of 6 mg/kg significantly reduced DNA-oxidation. Furthermore only in animals treated with MnTE-2-PyP⁵⁺ at 6 mg/kg, robust effects were seen on all investigated gene products regulated by transcription factors with redox sensitive active centers [27-31]. This seems to be in discrepancy with the observation that almost all animals treated with MnTE-2-PyP⁵⁺ or MnTnHex-2-PyP⁵⁺ displayed normalizing breathing frequencies after RT. We hypothesize that both MnP exert some degree of functional radioprotection. Right hemithoracic irradiation spares the left lung which is able to compensate deteriorating lung function of the right side within limits. This adds to the weak protective effects of the less potent MnP, therefore leading to the false conclusion that both MnP have equal radioprotective properties. Immunohistochemistry studies on the other hand are undertaken in tissue from the irradiated right side and take into account the typical focal damage induced by ionizing radiation. Therefore, in the model of hemithoracic irradiation, qualitative tests can display even small differences in efficacy of the tested compounds.

Interestingly, activation of macrophages was not significantly decreased by any tested MnP. These cells are the main producers of ROS in the model of chronic lung injury after RT. We hypothesize that MnP act as direct scavengers of ROS/RNS or modulators of redox-regulated transcription factors. We therefore do not expect necessarily the overall production of ROS/RNS or activation of macrophages to be diminished due to treatment with MnP in a state of inflammatory injury. This might explain the elevated breathing frequencies in all groups despite treatment with MnP at 8 weeks after RT, a time point at which the RT-induced alveolitis has completely developed (see Figure 3).

MnP scavenge reactive oxygen and nitrogen species, such as O₂^•-, ONOO^-, NO₂^•, and CO₃^•- [32]. Furthermore, evidence suggests that MnP have a pivotal role in activation /deactivation of redox sensitive transcription factors. The p50 subunit of NFκB is oxidized by MnTE-2-PyP⁵⁺ once p50 enters the nucleus therefore preventing DNA-binding of NFκB [33]. Also, HIF-1α, activated by RT, has been shown to be blocked by MnTE-2-PyP⁵⁺ [30]. This direct action of MnP on relevant biological factors is due to the metal-centered redox potential (+228 mV vs. NHE) [15]. According to the redox-state of the environment, MnP can therefore act as anti- or pro-oxidants and deactivate transcription factors once they are activated [33]. Consequently, MnP proved to be effective in various animal models of oxidative stress [10-13, 34-36].

The investigated compounds in this study (MnTE-2-PyP⁵⁺ and MnTnHex-2-PyP⁵⁺) differ in their lipophilicity due to the difference in the length of alkyl chains. The higher lipophilicity of MnTnHex-2-PyP⁵⁺ facilitates progression of the compound through lipid membranes into the cell and therefore has direct consequence for bioactivity as has been seen in an E.coli model of oxidative stress and in a model of renal ischemia/reperfusion [16, 37]. Nevertheless, the hydrophilic center and the lipophilic alkyl chains of MnTnHex-2-PyP⁵⁺ create a micelle-like structure which can lead to disruption of lipid membranes [16]. We have observed local irritations turning into necrosis after continuous s.c. injections of MnTnHex-2-PyP⁵⁺ into rat skin at dosages ranging upwards from 1 mg/kg (data not shown). Due to the higher lipophilicity, efficacy as seen in other models, and the local toxicity at doses higher than 1 mg/kg, we have chosen to test MnTnHex-2-PyP⁵⁺ at dosages of 0.3, 0.6, and 1.0 mg/kg. We observed an inverse dose-effectiveness relationship for MnTnHex-2-PyP⁵⁺. While the lowest dose of MnTnHex-2-PyP⁵⁺ (0.3 mg/kg) proved to have limited effect as radioprotector, higher dosages (0.6 and 1 mg/kg) did not show that effect to the same extent. Currently, there is no sufficient explanation why MnTnHex-2-PyP⁵⁺ at the higher tested dosages failed to be as effective. In sight of the observed toxicity of dosages above 1 mg/kg which we believe is due to disruption of lipid cell membranes, it might be plausible to consider toxic effects at cellular level even in dosages which did not reveal macroscopic injury at the injection sites. This might be substantiated by our finding that animals which were treated after RT with MnTnHex-2-PyP⁵⁺ at its most effective dose (0.3 mg/kg), showed significantly reduced bodyweight during the period of daily s.c. injections. Although these animals recovered in bodyweight once the injections were stopped, the question arises whether this loss in bodyweight was due to systemic toxic effects of MnTnHex-2-PyP⁵⁺.

Recently, MnTnHex-2-PyP⁵⁺ at a 6-fold smaller dose than the herein tested lowest dose has been shown to reduce renal injury and mitochondrial damage in a model of renal ischemia/reperfusion [37]. We are currently testing whether such a reduced dose of MnTnHex-2-PyP⁵⁺ (50 μg/kg) is effective in our model.

In conclusion, MnTnHex-2-PyP⁵⁺ did not prove to be more effective than MnTE-2-PyP⁵⁺ in this study of pulmonary radioprotection. At tested doses, MnTE-2-PyP⁵⁺ appears less toxic and more effective.

List of Abbreviations

RT

Radiotherapy

MnTE-2-PyP⁵⁺

Manganese (III) tetrakis (N-ethylpyridinium-2-yl) porphyrin

MnTnHex-2-PyP⁵⁺

Manganese (III) tetrakis (N-hexylpyridinium-2-yl) porphyrin

NO₂^•

Nitric dioxide

CO₃^•-

Carboxyl radical

OH^•

Hydroxyl radical

O₂^•-

Superoxide

ONOO^-

Peroxynitrite

MT

Masson’s trichrome

H&E

Hematoxylin and eosin

8-OHdG

8-hydroxy-2’-deoxyguanosine

TGF-β

Transforming growth factor-beta

HIF-1α

Hypoxia inducible factor 1α

VEGF (A)

Vascular endothelial growth factor A

CA-IX

Carbonic anhydrase IX