Amifostine

Low-level radiation exposures are expected to have long-term health implications but few near-term effects that would impair function. These assumptions are based on extrapolation from acute exposure responses, not on studies of a larger array of exposure scenarios (e.g., protracted exposures) that might present as operational threats. Protracted exposure is one scenario that needs to be better defined in terms of both the initial effect and the long-term health consequences. Reported here are the near-term effects of chronic, low daily-dose gamma-irradiation (3-128 mGy per day) on the blood-forming system of canines. Change in hematopoietic capacity was monitored along with time of exposure and cumulative radiation dose. The rate, magnitude, and timing of suppression and accommodation were determined. The ability of periodic treatment with a lipopolysaccharide immunomodulator to alleviate the suppressive hematopoietic effects of chronic exposure was tested. The effects of other pharmacologics (amifostine, granulocyte colony-stimulating factor, cytokine) are projected on the basis of current research using rodent models. Results indicated that low but significant suppression of blood leukocyte and platelet levels occurred at 3 mGy per day. As the dose rate increased from 3 mGy to 128 mGy per day, the rate of suppression increased approximately eightfold, whereas the time to accommodate declined from 2,000 days to approximately 150 days. Within the time frame required to reach the upper limit of 700 mGy, none of the dose rates examined elicited blood cell decrements large enough to severely compromise near-term immune function. Pharmacological intervention with lipopolysaccharide minimized hematopoietic suppression in only a small fraction of the treated animals that displayed distinctive long-term survival and pathology patterns. Comparable shortterm benefits of treatment with hematopoietic cytokines or chemoprotectants are predicted on the basis of responses noted in rodent models. Long-term benefits of such treatments remain to be determined. Future work will require the application of advanced molecular tools to more fully assess potential pathophysiological responses and their modulation after low-level radiation exposure.

Introduction North Atlantic Treaty Organization Allied Command Europe directive 80-63 defines low-level radiation (LLR) and addresses the issue of defensive measures against low-level radiological hazards during military operations. Health risks can result from exposure to radiation levels ranging from background levels to as much as 70 cGy.1 Unfortunately, the health risks associated with such LLR exposures remain ill defined. The health implications of LLR exposure, especially chronic exposure, need to be better understood. To ensure greater operational flexibility and minimize operational casualties, LLR health risks need to be clearly identified and preventive methods developed and applied.

Under extremely low rates of exposure to ionizing radiation, sensitive organ systems of the body (e.g., the blood-forming system) have the capacity to adapt and repair injury.2,3 The radiological and temporal parameters under which these adaptive responses are elicited remain ill defined, as do the long-term health consequences of these naturally occurring, short-term protective responses. The precise mechanism or mechanisms by which these adaptive or accommodative processes occur are largely unknown, although repair, cell cycle, and cell selection are all thought to play a role.3,4

The intent of this article is to (1) evaluate the effects of chronic, low daily-dose gamma-irradiation on the blood-forming system, (2) focus on near-term effects of LLR exposure, and (3) examine the possible benefits of pharmacological interventions.

Materials and Methods

General

Two animal models were used in these radiation studies: a

canine (beagle) model and a mouse (C3H/NIHHe) model. The canine model was developed and extensively used at the Argonne National Laboratory for assessing general survival patterns and health consequences of chronic exposure to low daily doses of cobalt-60 gamma-rays.2-5 The blood work described here was based on the Argonne studies database. The mouse model is an acute, whole-body radiation injury and hematopoietic repair assay system developed at our laboratory and modified to test the efficacy of new cytokine therapies.6

Animals

The canine model used beagles that were approximately 400

days old at the start of the exposure. For survival responses and for the analyses of induced pathologies, a total of 739 dogs were used, of which 143 served as nonirradiated controls and 596 served as irradiated test animals. Specifically, for the analysis of blood, responses of 19 nonirradiated controls and 78 irradiated test animals were used. Additional dogs and their recorded bioresponses were used for other measured endpoints of interest, including 75 (49 test animals and 26 controls) for radiosensitivity and repair measurements of marrow progenitors and 57 (28 lipopolysaccharide [LPS]-treated, irradiated test animals and 29 non-LPS-treated, irradiated controls) for the LPS-induced survival responses.

The mouse model used male C3H/NIHHe mice that were approximately 10 weeks old at the start of the protocol. Approximately 240 mice (120 irradiated and 120 nonirradiated mice) were used to assess the hematopoietic recovery following cytokine (granulocyte colony-stimulating factor [G-CSF] and interleukin 11) treatments given after radiation exposure. Cytokine-induced enhancement of blood levels of leukocytes (and granulocyte, monocyte, and lymphocyte subsets), erythrocytes, and platelets, along with changes in bone marrow cellularity and selected progenitorial cell compartments, were measured and used as principal endpoints.

Chronic Irradiation

Canines were chronically exposed to gamma-rays using a specifically designed, live-in exposure facility equipped with a cobalt-60 gamma irradiator. Exposure was carried out in a near continuous mode for 22 hours each day for the duration of life. Daily rates of exposure ranged from 3 to 128 mGy. Mouse irradiations were carried out in a high-level cobalt-60 gamma irradiator at 0.6 Gy per minute for total doses up to 7 Gy.

Physical dosimetry was conducted for both model systems. The determination of details was carried out as previously deSCribed.7,8

Treatments

Canines under chronic irradiation (75 mGy per day) were treated periodically at 100-day intervals with 5 (mu)g/kg of purified bacterial cell wall LPS from Salmonella typhimurium.

Acutely irradiated mice were given a series of five cytokine injections (G-CSF or interleukin 11 at 2.5 and 10 (mu)g per mouse per day, respectively) every other day for 10 days.

Assessments and Endpoints

Clinical, hematological, and histopathological evaluations of irradiated experimental animals or nonirradiated controls were conducted using standard, previously described methods.2-5 Marrow cellularity was determined by direct microscopic evaluation and by electronic cell counting. Marrow progenitor levels were quantitated by in vitro cloning as previously described.2-6

Results

Blood Responses under Chronic Irradiation

Characteristic dose-rate-dependent patterns of suppression and accommodation of blood leukocyte levels were noted in chronically irradiated dogs (Fig. 1). As the irradiation rate increased from 3 mGy to 75 mGy per day, the duration of the initial suppressive phase increased, as did the magnitude of blood-cell loss. Similar response patterns were noted for blood platelets and for many of the other leukocyte subsets, including granulocytes, monocytes, and lymphocytes (but not erythrocytes). These patterns reflect the pronounced differential responses of the proliferative myeloid and erythroid compartments within the bone marrow of dogs under these very low daily rates of gamma-irradiation.

Radiological and Temporal Parameters Affecting Accommodation

Time and radiation dose parameters that constrained the transition from the initial suppressive phase to an accommodative phase were extracted from the blood response data and analyzed (Fig. 2). The leukocyte blood level at which the accommodative hematopoietic state was achieved declined as the dose rate rose from 3 to 75 mGy per day (Fig. 3a). Similarly, the time required to achieve accommodation decreased as the daily dose rate increased (Fig. 3b). In addition, the cumulative radiation dose at which accommodation occurred increased as the daily rate of irradiation increased (Fig. 3c).

Initial Responses to LLR Exposure

Relative to the amount of time under chronic irradiation, the initial rates of blood leukocyte cell loss increased as the daily rate of irradiation increased (Fig. 3d). Relative to absorbed dose, the rate of cell loss during the initial phase of irradiation was low (~6% per Gy) and largely independent of dose rate (3-75 mGy per day) (Fig. 4). Only at the higher dose rate tested (128 mGy per day) was there substantially elevated rates of cell loss (~25% per Gy) (Fig. 4). Even at these higher rates of cell loss, however, there would be little impact on blood capacity and function and near-term health if the exposures were limited to total doses of 700 mGy or less.

Responses of Hematopoietic Progenitors

Accommodation of the blood-forming system under chronic irradiation is associated with an inherent shift in radiosensitivity from a sensitive to a resistant state. This shift in hematopoietic tissue sensitivity is mediated in part by a change in the resistance, repair, and recovery potential of rare but vital progenitorial compartments within the bone marrow.3-5 Direct proof of this concept comes from the observed change in radiosensitivity of marrow progenitors committed to granulocyte and monocyte differentiation (granulocyte-macrophage colonyforming unit) as a function of the accommodative state of the animal undergoing various rates of irradiation5 (Fig. 5). These data demonstrate that prior to accommodation (labeled prerecovery), the repair capacity of these vital progenitorial cells (as assessed by measures of sublethal and potentially lethal damage capacities) was either unchanged or diminished under a wide range of radiation dose rates. With onset of accommodation (labeled postrecovery), there was a significant rise in these damage and repair capacities with increasing daily rates of irradiation (Fig. 5).

Discussion

Effects of Pharmacologics

Select types of radioprotectants and immunomodulators can elicit shifts in radiosensitivity (comparable with those noted previously) in target cells in vital tissues of the body, and this induced shift is augmented by chronic irradiation.9 In this regard, we tested the effects of periodic administration (approximately every 100 days) of low doses (5 (mu)g/ko of LPS on the hematopoietic responses of dogs under chronic, low daily-dose gamma-irradiation. As anticipated, we observed an enhanced but short-lived mobilization and increased blood levels of leukocytes (in particular, granulocytes) for approximately 12 to 24 hours after LPS injection.10 The magnitude of these LPS-induced responses was strictly dependent on the functional status of the hematopoietic system (i.e., cellular reserves of the marrow) at the time of injection. In comparing the blood responses of LPS-- treated dogs vs. those of control dogs not receiving LPS, the LPS treatments seemingly promoted earlier, more robust accommodative hematopoietic responses. Further, the LPS-treated dogs exhibited increased survival under chronic irradiation compared with irradiated dogs not treated with LPS. This LPS-induced increase in survival was particularly marked in irradiated animals that had accommodative capacity (Fig. 6).

We modeled the expected reparative effects of G-CSF under chronic irradiation on the noted mobilizing effects of periodic LPS administration and the degree to which cytokine (G-CSF) treatments enhance repair and recovery in hematopoietic tissues. The model, a proportional target cell response model, suggests that a given dose of cytokine (G-CSF) will elicit a proportional rise in blood granulocytes at the start of chronic exposure. This initial cytokine dose-dependent rise in blood granulocyte levels and its time-dependent decline serve to minimize both the magnitude and the period of acute myelosuppression while fostering recovery and maintenance of critical blood-cell levels (Fig. 7). Although the model predicts substantial nearterm reparative responses of G-CSF cytokine treatments under chronic irradiation, it does not provide information about longterm consequences of such treatments. Regardless, the model needs to be thoroughly tested and its predictions challenged before any advanced preclinical development is undertaken.

New Tools for Analysis of LLR Effects

New molecular tools are being applied to the assessment of LLR-related injuries.11 One of the more promising of these tools is the cDNA expression arrays, in which the responses of thousands of genes from LLR-exposed cells and tissues can be simultaneously examined.12 This technology provides the opportunity to examine the ability of a given pharmacologic to modify these LLR-induced genetic responses, either globally or relative to a class of genes (stress genes, cell cycle genes, etc.) or an individual. We have had promising initial results when using this technology for this purpose. For example, in one experiment, we compared the early (24 hours) transcriptional responses of a 588-gene array (Clontech Atlas cDNA array; Clontech Laboratories, Palo Alto, CA) in hematopoietic tissues of mice that had been treated with the radioprotective drug amifostine (WR2721) and irradiated with 3 Gy gamma-rays. Preliminary results indicate that amifostine has a global effect of downregulating a large number of radiation-responsive genes; for example, in the absence of amifostine protection, approximately 32% of the responsive genes were up-regulated (~68% downregulated), whereas with amifostine treatment, only 13% were up-regulated (~87% down-regulated). Of the six classes of genes analyzed, genes tied to stress response and to signal transduction were most affected by the amifostine treatment; for example, amifostine treatment exerted a seven- and twofold reduction in HSP84 and HSP86 heat shock genes, respectively, which exhibited 12- and 20-fold inducible increases (3 Gy induction) in the absence of amifostine treatment.

It is highly probable that this cDNA expression array technology will prove to be a powerful tool in determining the modes of radioprotective action of given pharmacologics.

In addition to molecular tools, exposure facilities designed for low-dose and low-dose-rate irradiation are critical to the advancement of our understanding of the pathological potential of LLR exposure. The development of safe and effective preventive treatments and therapies is dependent on a full understanding of the nature of LLR-associated injuries and the relationship to dose and dose rate. The need for LLR exposure capability is particularly critical in light of the closure of older exposure facilities at various national laboratories within the U.S. Department of Energy. A new LLR facility that at press time is online at the Armed Forces Radiobiology Research Institute is expected to play an important role in furthering our ability to assess, prevent, and treat LLR-associated injuries.

Conclusion

Under protracted gamma-ray exposure at very low dose rates, the normally highly radiosensitive hematopoietic system adapts and becomes radioresistant. This adaptive process is constrained by both time and radiological exposure parameters (dose and dose rate) and is mediated, at least in part, by damage capacity acquired by vital hematopoietic progenitors found in the bone marrow. This naturally occurring adaptive process can seemingly be elicited and augmented by periodic administration of strong immunodulating pharmacologics such as bacterial LPS or by hematopoietic cytokines.

Theoretical modeling of the bioresponses of periodic hematopoietic cytokine (G-CSF) treatments under chronic exposure suggests possible short-term therapeutic gain but does not address long-term health consequences. Testing of the model relative to both short- and long-term hematopoietic effects is essential before doing advanced preclinical testing in higher animal models.

Finally, molecular assays and new exposure facilities that should aid in the development of countermeasures for LLR-- associated injuries are becoming available.

Acknowledgments

The authors wish to acknowledge Dr. Ken Anderson (Rush St. Luke's Presbyterian Medical Center, Chicago, IL) for his initial advice and guidance on using the cDNA arrays. We would also like to thank Susan Myers, Lillian Kaspar, and Donald Doyle for their clinical and hematological assessments.

References

1. North Atlantic Treaty Organization Allied Command Europe Directive Number 80-63: In Potential Radiation Exposures in Military Operations: Protecting the Soldier Before, During and After, pp 119-128. Edited by Thaul S, O'Maonaigh H. Washington, DC, National Academy Press, 1999.

2. Fritz TE, Seed TM, Tolle DV, Lombard LS: Late effects of protracted whole-body irradiation of beagles by cobalt-60 gamma rays. In Life-Span Radiation Effects

Studies in Animals: What Can They Tell Us? pp 116-141. Edited by Thompson RC, Mahaffey JA. CONF-830951, Office of Scientific and Technical Information. Washington, DC, U.S. Department of Energy, 1986.

3. Seed TM, Kaspar LV, Fritz TE, Tolle DV: Cellular responses in chronic radiation leukemogenesis. In Carcinogenesis. Vol 10, pp 363-79. Edited by Huberman E, Barr SH. New York, Raven Press, 1985.

4. Seed TM. Kaspar LV, Tolle DV, Fritz TE, Frazier ME: Analyses of critical target cell responses during preclinical phases of evolving chronic radiation-induced myeloproliferative disease: exploitation of a unique canine model. In Multilevel Health Effects Research: From Molecules to Man, pp 245-55. Edited by Park JF, Pelroy RA. Columbus, Ohio/Richland, Washington, Battelle Press. 1988.

5. Seed TM, Kaspar LV: Acquired radioresistance of hematopoietic progenitors (granulocyte/monocyte colony-forming units) during chronic radiation leukemogenesis. Cancer Res 1992; 52: 1469-76.

6. Patchen ML: Single and combination cytokine therapies for the treatment of radiation-induced hemopoietic injury. In Advances in the Treatment of Radiation Injuries/Advances in the Biosciences, Vol 94, pp 21-36. Edited by MacVittie TJ, Weiss JF, Browne D. Oxford, UK, Pergamon/Elsevier Science, Ltd., 1996.

7. Schulz J, Almond PR, Cunningham JR, et al: A protocol for the determination of absorbed dose for high energy photon and electron beams. Med Phys 1983; 10: 741-71.

8. Williamson FS, Hubbard LB, Jordan DL: The response of beagle dogs to protracted exposure to Co gamma-rays at 5-35 R/day: I. Dosimetry. In Annual Report, pp 153-6. Argonne, IL, Division of Biological and Medical Research, Argonne National Laboratory, 1968.

9. FitzGerald TJ, Henault S, Santucci MA, et al: Recombinant murine GM-CSF increases resistance of some factor dependent hematopoietic progenitor cells to low-dose-rate gamma irradiation. Int J Radiat Oncol Biol Phys 1989; 17: 323-35.

10. Seed TM, Cullen SM, Kaspar LV, Tolle DV, Fritz TE: Hemopathological consequences of protracted gamma irradiation: alterations in granulocyte reserves and granulocyte mobilization. Blood 1980: 56: 42-51.

11. Blakely WF, Miller AC, Pogozelski WK, et al: Nucleic acid molecular biomarkers for diagnostic biodosimetry applications: use of the fluorogenic 5'-nuclease polymerase chain reaction assay. Milit Med 2002; 167: 16-19.

12. Amundson SA, Do KT, Fornace AJ Jr: Induction of stress genes by low doses of gamma rays. Radiat Res 1999; 152: 225-31.

Guarantor: Thomas M. Seed, PhD

Contributors: Thomas M. Seed, PhD*; Cyndi Inal, BS*; Michael E. Dobson, CDR USN*; Shameek Ghose, BS*; Edward Hilyard, LCDR USN*; David Tolle, MSc^; Thomas E. Fritz, DVM^

*Radiation Casualty Research Team, Armed Forces Radiobiology Research Institute, 8901 Wisconsin Avenue, Bethesda, MD 20889-5603.

^Center for Mechanistic Biology, Argonne National Laboratory, 9700 South Cass Avenue, Argonne, IL 60437.

This manuscript was received for review in February 2001. The revised manuscript was accepted for publication in November 2001.

Copyright Association of Military Surgeons of the United States Feb 2002
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