Daila S. Gridley1, Michael J. Pecaut1, Lora M. Green1,
Daniela Salvemini2, and Gregory A. Nelson1
1Radiobiology Program, Department of Radiation Medicine, Loma
Linda University and Medical Center Loma Linda, CA and 2MetaPhore
Pharmaceuticals, Inc., St. Louis, MO
Introduction. Immune system dysfunction has been identified as a major risk of long duration space flight, since it can lead to infection, reactivation of endogenous viruses, serious performance degradation, and permanent disability. Furthermore, in addition to the well established role of radiation as a carcinogen, numerous reports indicate a strong relationship between immunodepression and the development of cancer. The generation of reactive oxygen radicals by ionizing radiation is responsible for many of the damaging effects observed. Free radicals are also formed as an indirect consequence of irradiation by phagocytic cells that become activated during removal of radiation-injured tissues. Studies strongly suggest that oxidative stress from radiation exposure can trigger a cascade of events, including altered immune function, transformation of damaged cells, and late effects in tissues. Superoxide dismutases (SOD)s are oxidoreductases that contain Cu, Fe, or Mn at the active site and catalyze the dismutation of O2·- to O2 and H2O2. Along with catalase and glutathione peroxidase, they are the major intracellular enzymes that protect against oxygen toxicity. We hypothesized that early intervention by M40403 [1,4,7,10,13,-pentaazacyclopentadecane containing added bis(cyclohexylpyridine)], a low MW SODm with access to all tissue and cell compartments may arrest such a cascade. The present study represents preliminary data obtained with M40403 and radiation-induced damage in a mouse model.
Materials and Methods. C57BL/6 female mice (n = 32), 8-9 wk old at arrival, were acclimatized for 1 week prior to assignment into the following groups: 1) control (0 Gy); 2) 3 Gy; 3) M40403 + radiation (3 Gy); and 4) radiation (3 Gy) + M40403. Whole-body irradiation (g-rays, 60Co) was administered in a single fraction at a dose rate of ~0.7 Gy/min. M40403, an SODm developed by MetaPhore, Inc., was dissolved in 26 mM Na bicarbonate buffer to a concentration of 2 mg/ml immediately prior to use and injected subcutaneously (s.c.) according to two different time schedules: 1) 30 min before irradiation and at 1, 2, and 3 days post-irradiation and 2) 15 min and 1, 2, and 3 days post-irradiation. The amount of M40403 injected each time was 10 mg/kg body weight. The mice were euthanized at 4 days post-irradiation, close to the nadir for immune system damage, for a series of assays on blood, spleen, and bone marrow. Assays included: body, spleen, thymus, and liver mass; white blood cell (WBC), red blood cell (RBC), and thrombocyte counts; 3-part differentials of major leukocyte populations; hemoglobin; hematocrit; flow cytometry analysis of lymphocyte subsets (CD3+ T cells, CD3+/CD4+ T helper cells, CD3+/CD8+ T cytotoxic cells, CD19+ B cells, and NK1.1+ natural killer or NK cells); basal DNA synthesis; response to T cell (phytohemagglutinin, PHA; concanavalin A, ConA) and B cell (lipopolysaccharide, LPS) mitogens; and micronucleus formation and apoptosis induction in bone marrow.
Results. Most of the measured parameters were depressed in all three irradiated groups at the 4-day post-irradiation time point. However, based on spleen cell response to both T cell mitogens (PHA and ConA) and the B cell mitogen (LPS), the more effective regimen in counteracting the immunodepressive effect of radiation was the administration of M40403 beginning at 30 min before irradiation. Furthermore, higher stimulation indices (SI) and raw cpm values were obtained for this group compared to radiation alone. In addition, initiation of M40403 treatment before irradiation, compared to after irradiation, resulted in consistently (although not always significantly) higher mean values in the following assays: a) spleen and thymus weight; b) spleen and thymus weight relative to body weight; c) spleen cell responsiveness to T and B cell mitogens; d) numbers of WBC, lymphocytes, granulocytes, CD3+ T cells, and CD3+/CD4+ T helpers in the blood; and e) CD4:CD8 ratios in the blood. A similar pattern was noted in the spleens from these mice. The fact that this trend was observed employing a variety of different assays supports the premise that the administration of M40403 before irradiation was more beneficial than giving the drug after exposure.
Furthermore, the micronuclei assay, showed that bone marrow cells from unirradiated mice had a spontaneous rate of micronuclei formation of 6%; in the 3 Gy group, micronuclei were present in 8% of the cells scored (25% increase above basal levels). The groups treated with M40403, irrespective of whether administration was initiated before or after irradiation, had a rate of 6% (equivalent to non-irradiated levels). Additionally, there was the dramatic reduction of apoptosis in the groups treated with M40403. In non-irradiated mice there was an 11% incidence of apoptotic bone marrow cells, whereas a 50% rate of apoptosis was found in the irradiated group. Treatment with M40403 beginning prior to irradiation reduced the rate to 29%; when M40403 was given only after radiation it was 21%. Thus, M40403 prevented chromosome damage and dramatically reduced radiation-induced apoptosis.
Conclusions. The data show that M40403 preserved the ability of splenic lymphocytes to respond to a stimulating agent after whole-body irradiation of the intact animal. Initiation of drug treatment prior to irradiation generally appeared to be more beneficial than when treatment was initiated post-irradiation. Furthermore, micronucleus formation is a sensitive measure of radiation-induced damage and the reductions in micronuclei formation and apoptosis introduced by treatment with M40403 are highly significant. Collectively, these findings suggest that the immune system was left more intact with M40403 treatment and that subsequent recovery may be more rapid in these mice. Taken a step further, one could speculate that immunological defenses against infectious agents, and possibly also immune surveillance against neoplastically transformed cells, may be more vigorous with M40403 treatment. Longer term follow-up and immune system challenge are obviously essential in order to prove (or disprove) these possibilities.
Acknowledgements: This study was supported by the National Aeronautics and Space Administration (NASA NCC5-236), MetaPhore Pharmaceuticals, Inc., and the Chan Shun International Foundation.