Elizabeth L. Travis

Models for evaluating agents intended for the prophylaxis, mitigation and treatment of radiation injuries. Report of an NCI Workshop, December 3-4, 2003

Abstract Stone, H. B., Moulder, J. E., Coleman, C. N., Ang, K. K., Anscher, M. S., Barcellos-Hoff, M. H., Dynan, W. S., Fike, J. R., Grdina, D. J., Greenberger, J. S., Hauer-Jensen, M., Hill, R. P., Kolesnick, R. N., MacVittie, T. J., Marks, C., McBride, W. H., Metting, N., Pellmar, T., Purucker, M., Robbins, M. E., Schiestl, R. H., Seed, T. M., Tomaszewski, J., Travis, E. L., Wallner, P. E., Wolpert, M. and Zaharevitz, D. Models for Evaluating Agents Intended for the Prophylaxis, Mitigation and Treatment of Radiation Injuries. Report of an NCI Workshop, December 3–4, 2003. Radiat. Res. 162, 711–728 (2004). To develop approaches to prophylaxis/protection, mitigation and treatment of radiation injuries, appropriate models are needed that integrate the complex events that occur in the radiation-exposed organism. While the spectrum of agents in clinical use or preclinical development is limited, new research findings promise improvements in survival after whole-body irradiation and reductions in the risk of adverse effects of radiotherapy. Approaches include agents that act on the initial radiochemical events, agents that prevent or reduce progression of radiation damage, and agents that facilitate recovery from radiation injuries. While the mechanisms of action for most of the agents with known efficacy are yet to be fully determined, many seem to be operating at the tissue, organ or whole animal level as well as the cellular level. Thus research on prophylaxis/protection, mitigation and treatment of radiation injuries will require studies in whole animal models. Discovery, development and delivery of effective radiation modulators will also require collaboration among researchers in diverse fields such as radiation biology, inflammation, physiology, toxicology, immunology, tissue injury, drug development and radiation oncology. Additional investment in training more scientists in radiation biology and in the research portfolio addressing radiological and nuclear terrorism would benefit the general population in case of a radiological terrorism event or a large-scale accidental event as well as benefit patients treated with radiation.

Prevention of late effects of irradiation lung damage by manganese superoxide dismutase gene therapy

Organ and tissue damage caused by ionizing irradiation is directly related to volume irradiated, total dose and dose rate. The acute effects are in part mediated by cellular activation of early response genes, including those for transcriptional activators of genes for humoral cytokines. In the lung, as in other organs, recovery from the acute effects of ionizing irradiation does not always correlate with prevention of the critical late effects, including fibrosis, which contribute to organ failure. An interventional technique by which to protect normal organs from the late effects of irradiation has remained elusive. We now demonstrate that overexpression of a transgene for human manganese superoxide dismutase (MnSOD) delivered by plasmid–liposome, or adenovirus to the lungs of C57BL/6J or Nu/J mice, respectively, before irradiation exposure, decreases the late effects of whole lung irradiation (organizing alveolitis/fibrosis). These data provide a rational basis for the design of gene therapy approaches to organ protection from irradiation damage.

Radiation pneumonitis and fibrosis in mouse lung assayed by respiratory frequency and histology

TRAVIS, E. L., DOWN, J. D., HOLMES, S. J., AND HOBSON, B. Radiation Pneumonitis and Fibrosis in Mouse Lung Assayed by Respiratory Frequency and Histology. Radiat. Res. 84, 133-143 (1980). The response of mouse lung to radiation was assessed by measuring breathing frequency up to 52 weeks after graded single doses of X rays to both lungs. Breathing frequency was increased after both lethal and sublethal doses, with damage appearing sooner after higher doses. Dose-response curves were obtained after 14 weeks with a threshold of 12 Gy. All mice given doses greater than 15 Gy died by 22 weeks. Some return of function was observed in the lower dose groups by 36 weeks, but breathing frequency remained elevated for 52 weeks after a nonlethal dose. The changes in breathing frequency were compared with histological changes in the same mice at sacrifice. The increased breathing frequency between 14 and 24 weeks was associated with radiation pneumonitis seen histologically. After 36 weeks, the elevation in breathing frequency above the control value was associated with fibrosis. Comparison of dose-response curves for the pathological changes and for breathing frequency 16, 36, and 52 weeks after irradiation indicated that increases in breathing frequency were associated with increases in the severity of the histological changes scored.

ISOEFFECT MODELS AND FRACTIONATED RADIATION THERAPY

Since the advent of the Ellis formula, a number of isoeffect formulae have been proposed for estimating the total dose required in different fractionation schedules to produce equivalent biological effects.‘*3*” Most often these formulae are applied retrospectively to clinical data thus making it impossible to control factors such as the endpoint for assessing damage, non-biased scoring of the damage by (preferably) more than one independent observer, the criteria for patient eligibility, and adequate follow-up records for assessment. Thus, these formulae often are viewed skeptically by clinicians. However, there is an urgent need to accurately and concisely define isoeffect relationships for human normal tissues in vivo because of the implied steepness of dose-response curves as derived from animal data. Also, the initiation of new fractionation schedules, for example, hyperfractionation, are all based on experimental data and, although a comparison of mouse and human data indicates that the shape of the dose-response curves for the two species is similar, the absolute dose level for response varies, generally in the direction of a lower isoeffect dose in humans. The lung is one dose-limiting normal tissue for which there are some dose-response data available for humans. The lung is dose-limiting not only in the treatment of malignant diseases of the thorax but in the use of total body irradiation as a preparative regimen for bone marrow transplantation. This latter treatment and the use of upper half body irradiation have resulted in published dose-response curves for the incidence of radiation pneumonitis in humans, at least after large single doses of radiation. However, limited data are available after fractionated doses and these are, again, from retrospective studies.

Intratracheal injection of adenovirus containing the human MnSOD transgene protects athymic nude mice from irradiation-induced organizing alveolitis

PURPOSE A dose and volume limiting factor in radiation treatment of thoracic cancer is the development of fibrosis in normal lung. The goal of the present study was to determine whether expression prior to irradiation of a transgene for human manganese superoxide dismutase (MnSOD) or human copper/zinc superoxide dismutase (Cu/ZnSOD) protects against irradiation-induced lung damage in mice. METHODS AND MATERIALS Athymic Nude (Nu/J) mice were intratracheally injected with 10(9) plaque-forming units (PFU) of a replication-incompetent mutant adenovirus construct containing the gene for either human MnSOD, human copper/zinc superoxide dismutase (Cu/ZnSOD) or LacZ. Four days later the mice were irradiated to the pulmonary cavity to doses of 850, 900, or 950 cGy. To demonstrate adenoviral infection, nested reverse transcriptase-polymerase chain reaction (RT-PCR) was carried out with primers specific for either human MnSOD or Cu/ZnSOD transgene on freshly explanted lung, trachea, or alveolar type II cells, and immunohistochemistry was used to measure LacZ expression. RNA was extracted on day 0, 1, 4, or 7 after 850 cGy of irradiation from lungs of mice that had previously received adenovirus or had no treatment. Slot blot analysis was performed to quantitate RNA expression for IL-1, tumor necrosis factor (TNF)-alpha, TGF-beta, MnSOD, or Cu/ZnSOD. Lung tissue was explanted and tested for biochemical activity of MnSOD or Cu/ZnSOD after adenovirus injection. Other mice were sacrificed 132 days after irradiation, lungs excised, frozen in OCT, (polyvinyl alcohol, polyethylene glycol mixture) sectioned, H&E stained, and evaluated for percent of the lung demonstrating organizing alveolitis. RESULTS Mice injected intratracheally with adenovirus containing the gene for human MnSOD had significantly reduced chronic lung irradiation damage following 950 cGy, compared to control mice or mice injected with adenovirus containing the gene for human Cu/ZnSOD or LacZ. Immunohistochemistry for LacZ protein in adenovirus LacZ (Ad-LacZ)-injected mice demonstrated expression of LacZ in both the upper and lower airway. Nested RT-PCR showed lung expression of MnSOD and Cu/ZnSOD for at least 11 days following infection with each respective adenovirus construct. Nested RT-PCR using primers specific for human MnSOD demonstrated increased expression of the human MnSOD transgene in the trachea and alveolar type II cells 4 days after virus injection on the day of irradiation. At this time point, increased biochemical activity of MnSOD and Cu/ZnSOD respectively, was detected in lungs from these two adenovirus groups, compared to each other or to control or adenovirus LacZ mice. Slot blot analysis of RNA from lungs of mice in each group following 850 cGy irradiation demonstrated decreased expression of mRNA for interleukin-I (IL-1), TNF-alpha, and transforming growth factor-beta (TGF-beta) in the MnSOD adenovirus-injected mice, compared to irradiated control, LacZ, or Cu/ZnSOD adenovirus-injected, irradiated mice. Mice receiving adenovirus MnSOD showed decreased organizing alveolitis at 132 days in all three dose groups, compared to irradiated control or Ad-LacZ, or Ad-Cu/ZnSOD mice. CONCLUSIONS Overexpression of MnSOD in the lungs of mice prior to irradiation prevents irradiation-induced acute and chronic damage quantitated as decreased levels of mRNA for IL-1, TNF-alpha, and TGF-beta in the days immediately following irradiation, and decrease in the percent of lung demonstrating fibrosis or organizing alveolitis at 132 days. These data provide a rational basis for development of gene therapy as a method of protection of the normal lung from acute and chronic sequelae of ionizing irradiation.

In Vivo Respiratory-Gated Micro-CT Imaging in Small-Animal Oncology Models

Micro-computed tomography(micro-CT) is becoming an accepted research tool for the noninvasive examination of laboratory animals such as mice and rats, but to date, in vivo scanning has largely been limited to the evaluation of skeletal tissues. We use a commercially available micro-CT device to perform respiratory gated in vivo acquisitions suitable for thoracic imaging. The instrument is described, along with the scan protocol and animal preparation techniques. Preliminary results confirm that lung tumors as small as 1 mm in diameter are visible in vivo with these methods. Radiation dose was evaluated using several approaches, and was found to be approximately 0.15 Gy for this respiratory-gated micro-CT imaging protocol. The combination of high-resolution CT imaging and respiratory-gated acquisitions appears well-suited to serial in vivo scanning.

Effect of dose-rate on total body irradiation: Lethality and pathologic findings

The effect of low dose-rate total body irradation (TBI) on hemopoietic and nonhemopoietic lethality has been studied in BALB/c mice using dose-rates ranging from 25 to 1 cGy/min. Deaths were scored at 10 days, 30 days, and one year after irradiation, and dose-response curves were constructed to determine the dose-rate dependence of deaths from the gastrointestinal syndrome, hemopoietic syndrome, and late lethal syndrome(s), respectively. A plot of the LD50S for each of these lethal syndromes versus dose-rate showed the dose-rate dependence for late lethality to be somewhat greater than that for gut death, but both of these endpoints were markedly more dose-rate dependent than was hemopoietic lethality, particularly at dose rates less than 5 cGy/min. To determine which late responding normal tissues might be critical for low dose-rate TBI, complete necropsies were performed on all mice dying later than 60 days after irradiation and on all mice surviving at one year; all tissues were examined histologically. Morphologic evidence of radiation injury was present in only three tissues, lung (fibrosis and scarring) kidney (tubule depletion), and liver (presence of mitoses). Subjectively, the lung changes were most severe up to 9 months while kidney changes became more prominent after this time, suggesting that late death after low dose-rate TBI may not be entirely attributable to lung injury. However, regardless of which late responding normal tissue is dose-limiting, it is clear that low dose-rate TBI preferentially spares these tissues compared with hemopoietic stem cells.

Damage and morbidity from pneumonitis after irradiation of partial volumes of mouse lung

PURPOSE The aims of this study were to: (a) define the relationship of dose and volume irradiated to damage and morbidity in mouse lung, (b) determine the threshold volume for morbidity after partial lung irradiation; and (c) determine whether the response to radiation of mouse lung is independent of the region irradiated. METHODS AND MATERIALS C3Hf/Kam female mice were used in this study. The fractional volume of the lung to be irradiated was determined by two methods, weights and computed tomography (CT) scanning. Two experiments were performed to define the volume effect and to determine whether the response of the mouse lung to radiation was homogeneous. In the first experiment, single doses of x-rays ranging from 12 to 20 Gy were given to partial volumes of 84%, 70%, and 40% including the base, 50%, 33%, and 17% including the apex, to 43% in the middle, and to the sum of 57% as 17% in the apex and 40% in the base. In the second experiment, the same volumes of 50% and 70-75% in the apex and base of the lung were irradiated with single doses ranging from 12-19.25 Gy. Morbidity from radiation pneumonitis was quantitated by two end points, breathing rate and lethality between 12 and 32 weeks after irradiation. Damage was assessed by histopathological evidence of pneumonitis. RESULTS Clear well-defined dose-response curves were obtained for both breathing rate and lethality after all volumes irradiated. There was a clear volume-dependent shift of the dose-response curves for breathing rate and lethality at 28 weeks after irradiation, the end of the pneumonitis phase of damage, to higher doses compared with these data after whole-lung irradiation. In addition, the slopes of the dose-response curves for irradiation of partial lung volumes were more shallow compared to those after whole-lung irradiation. Increases in breathing rate correlated with lethality when the volume irradiated was equal to or greater than 50% of the reference volume. However, after irradiation of volumes smaller than 40%, breathing rate increases were not accompanied by death. A heterogeneous response of the mouse lung to radiation was observed in the first experiment and confirmed by the second experiment. For a given volume irradiated, the isoeffect dose was always less for the base than for the apex of the lung. The threshold volume for breathing rate changes was less than 17 and 40% when the irradiated volumes involved the apex and base, respectively. For lethality, the threshold volume was between 40 and 70% for the base and greater than 50% for the apex of the lung. Finally, damage as assessed by histological evidence of pneumonitis was observed in the irradiated area only. CONCLUSIONS (a) The volume effect was resolvable in mice, (b) the volume effect in mouse lung exhibits a clear threshold for morbidity, (c) the threshold volume for morbidity is dependent on the end point, (d) the response of mouse lung is heterogeneous, dependent on the site irradiated, and is always greater for the same volumes irradiated in the base than the apex, and, (e) histopathological damage does not always produce observable morbidity.

Decreased pulmonary radiation resistance of manganese superoxide dismutase (MnSOD)-deficient mice is corrected by human manganese superoxide dismutase-plasmid/liposome (SOD2-PL) intratracheal gene therapy

Abstract Epperly, M. W., Epstein, C. J., Travis, E. L. and Greenberger, J. S. Decreased Pulmonary Radiation Resistance of Manganese Superoxide Dismutase (MnSOD)-Deficient Mice is Corrected by Human Manganese Superoxide Dismutase-Plasmid/Liposome (SOD2-PL) Intratracheal Gene Therapy. The pulmonary ionizing radiation sensitivity of C57BL/6 Sod2+/– mice heterozygous for MnSOD deficiency was compared to that Sod2+/+ control littermates. Embryo fibroblast cell lines from Sod2–/– (neonatal lethal) or Sod2+/– mice produced less biochemically active MnSOD and demonstrated a significantly greater in vitro radiosensitivity. No G2/M-phase cell cycle arrest after 5 Gy was observed in Sod2–/– cells compared to the Sod2+/– or Sod2+/+ lines. Subclonal Sod2–/– or Sod2+/– embryo fibroblast lines expressing the human SOD2 transgene showed increased biochemical activity of MnSOD and radioresistance. Sod2+/– mice receiving 18 Gy whole-lung irradiation died sooner and had an increased percentage of lung with organizing alveolitis between 100 and 160 days compared to Sod2+/+ wild-type littermates. Both Sod2+/– and Sod2+/+ littermates injected intratracheally with human manganese superoxide dismutase-plasmid/liposome (SOD2-PL) complex 24 h prior to whole-lung irradiation showed decreased DNA strand breaks and improved survival with decreased organizing alveolitis. Thus underexpression of MnSOD in the lungs of heterozygous Sod2+/– knockout mice is associated with increased pulmonary radiation sensitivity and parallels increased radiation sensitivity of embryo fibroblast cell lines in vitro. The restoration of cellular radioresistance in vitro and in lungs in vivo by SOD2-PL transgene expression supports a potential role for SOD2-PL gene therapy in organ-specific radioprotection.

Direct Estimation of Latent Time for Radiation Injury in Late-responding Normal Tissues: Gut, Lung, and Spinal Cord

Mixture models are proposed for simultaneous analysis of the latency and fractionation characteristics of radiation injury in late-responding normal tissues. The method is an extension of the direct analysis for quantal response data. Conceptually, the application of the mixture model is based on the biological observation that over a wide range of doses a proportion of the irradiated subjects will never express damage. Mixture models allow the time of occurrence to be utilized in the analysis. Furthermore, this type of model takes time-censored observations into account in a natural way and provides an adequate framework for modelling and analysis of effect-dependent latency. Mixture models with complete and incomplete repair are applied to dose-incidence data for four late endpoints in rodents: death from radiation-induced pneumonitis, leg paralysis after spinal-cord irradiation, and radiation-induced rectal stenosis and anal discharge. Radiation-induced pneumonitis had an effect-dependent latency. The modelling of this phenomenon correlates well with the results of histologic studies. Interestingly, the ratio of hazard rates was not constant for this endpoint. The dominating feature in the latency of radiation injury to the spinal cord was a strong dependency on dose per fraction. After correction for this effect a tendency towards a longer latent time for lower effect levels was observed. For the rectal complications, there was no difference between latency with radiation only vs. radiation combined with cis-platin.