Bacq and Alexander Award lecture--chemical radioprotection: past, present, and future prospects.

PURPOSE: To review and evaluate the development of effective radiation protectors. OUTLINE: Sulphydryl radioprotectors are the best radioprotectors known today. Their use encounters two great difficulties: their toxicity and the short period during which they are active. The biological response modifiers (BRM), developed mainly in the 1990s, demonstrated some protective effects. They can also modulate radiation injury when given after irradiation. In parallel with the use of single radioprotectors, observations have been made in mice using combined treatments with BRM and other radioprotectors. Low-to-moderate doses of several radioprotective agents acting via different mechanisms markedly improved the degree of protection in rodents while maintaining toxicity within acceptable limits, but applications in man remain doubtful. CONCLUSIONS: No radioprotective drug available today has all the requisite qualities to be an ideal radioprotector. Although combinations of radioprotective drugs acting via different mechanisms markedly improve the degree of protection and keep toxicity to acceptable levels in small rodents, attempts to use such treatments in large mammals have been less successful. It is thus questionable whether chemical protection has any prospects for the future.

Survival and diseases in C57BL mice exposed to X rays or 3.1 MeV neutrons at an age of 7 or 21 days.

Survival and causes of mortality were studied in 7- or 21-day-old male C57BL/Cnb mice exposed to 0.5, 1 or 3 Gy of 250 kVp X rays or 0.125, 0.25, 0.5 or 1 Gy of accelerator neutrons (modal energy 3.1 MeV). A total of 1287 animals were used in the experiments. Survival of irradiated animals was reduced significantly only in the mice receiving the highest doses (1 Gy neutrons, 3 Gy X rays ). Mice exposed to the lowest doses (0.125 Gy neutrons, 0.5 Gy X rays) lived significantly longer than controls, apparently reflecting a reduction in non-neoplastic lung and liver diseases. All malignant tumors increased significantly from (and including) doses of 0.5 Gy neutrons and 1 Gy X rays. Hepatocellular carcinoma was the principal contributor to the increase in tumor incidence, at least after exposure to neutrons. No significant increase in hepatocellular carcinoma was seen in 21-day-old mice exposed to X rays. An increase, especially after 3 Gy X rays, was also observed for all leukemias. Controls in the present study lived significantly longer than those in our earlier studies of irradiated adult mice, making a direct comparison of the radiation-induced effects in adult and infant mice difficult. Based on percentage life shortening, it appears that exposure during infancy does not shorten total survival or survival from cancer much more than exposure of adults, although such exposure, especially to neutrons, causes more hepatocellular carcinoma. Due to the nonlinearity of the dose-effect relationships, it is difficult to calculate the RBE of neutrons. For survival time at higher doses an RBE of about 3 is obtained. When the incidence of all malignant tumors and of hepatocellular cancer is fitted to a linear or a linear-quadratic function, an RBE from 5 to 8 is obtained. No RBE can be estimated for hepatocellular carcinoma in mice of an age of 21 days because exposure to X rays does not seem to cause this tumor at that age.

Effect of neutrons alone or combined with diethylnitrosamine on tumor induction in the livers of infant C57BL mice.

The possible combined effects of the initiator diethylnitrosamine (DEN)+neutrons on the induction of foci, adenomas and carcinomas in the livers of C57BL/Cnb mice were evaluated. Four groups of infant mice were treated as follows: DEN alone, neutrons alone, DEN followed by neutrons and neutrons followed by DEN. Ten mice in each group were killed at 10-week intervals over 70 weeks. The following parameters were measured: body weight, liver weight, number and size of superficial macroscopic liver lesions, and number and total surface area of the different types of microscopic liver lesions. The rate of appearance of foci increased significantly at different times when a dose of 0.125 Gy of neutrons was administered 7 days before or after a dose of 1.25 micrograms of DEN. No significant differences were observed in the total surface area of foci and/or adenomas and carcinomas when increasing doses of neutrons were given 7 days before or after the administration of 1.25 and 2.5 micrograms of DEN.

Reduction of short-term radiation lethality by biological response modifiers given alone or in association with other chemical protectors.

The advantages gained by a combined treatment of different chemical protectors on short-term lethality of X-irradiated adult male mice have been studied. The following compounds were given alone or in a mixture of two or three compounds: 16,16-dimethyl PGE2 (PGE2), cysteine (Cys), glucan, glutathione (GSH), 5-hydroxytryptamine (5-HT), mercaptoproprionylglycine (MPG), or WR-2721. The survival of mice treated before X irradiation with the optimal dose of each radioprotector given separately shows that WR-2721 and 5-HT yield the best protection with dose reduction factors (DRFs) of 2.2 and 1.7, respectively. Cysteine, glucan, PGE2, MPG, and GSH, with DRFs of 1.4, 1.4, 1.2, 1.1, and 1.1, respectively, are less efficient radioprotectors. When PGE2 was combined with a low dose of WR-2721 (200 mg/kg), the protection increased in a synergistic way. The increase in protection offered by a combination of PGE2 with Cys, glucan, GSH, or 5-HT is less marked and the effect obtained is only additive. A synergistic action is also obtained with a combination of WR-2721 (200 mg/kg) and 5-HT (8 mg/kg) (DRF 2.7).

Effect of X rays alone or combined with diethylnitrosamine on tumor induction in infant mouse liver.

The possible combined effects of the initiator diethylnitrosamine (DEN) with X rays on cancer induction in C57BL/Cnb mouse liver were evaluated. Four groups of infant mice were treated as follows: with DEN alone, with X rays alone, with DEN + X rays, and with X rays + DEN. Mice in each group were killed at 10-week intervals over 70 weeks. The following parameters were measured: body weight, liver weight, number and size of macroscopic liver lesions, and number and total surface of the different types of microscopic liver lesions. The number of induced liver foci and carcinomas was found to depend essentially on the dose of DEN. X irradiation did not produce any combined effect on the induction of foci and carcinomas when given 7 days before or after DEN administration.

Brain atrophy after foetal exposure to very low doses of ionizing radiation.

Acute, high dose-rate, exposure of the rat embryo on day 15 post-conception (PC) causes a reduction of brain weight in adult life that is proportional to the dose received. Doses as low as 10 mGy of 600 keV neutrons, from a Van de Graaff accelerator, or 100 mGy of 250 kV X-rays are capable of eliciting a significant effect. The relative biological effectiveness for acute neutron exposure compared with 250 kV X-rays was 3.5. A brain weight reduction was also observed after gamma-ray exposures protracted over 4 or 6 days, during cerebral corticogenesis. The dose-rate reduction factor was only 1.5 for exposure from days 12 to 16 PC and 3.3 for exposure from days 14 to 20 PC. In relation with the decrease in brain weight, the cingulum bundle, a myelinated structure associated with the corpus callosum, displayed a significant reduction in size. The implications of these observations for human exposures are discussed.

Life-shortening and disease incidence in mice after exposure to gamma rays or high-energy neutrons.

Male C57Bl/Cnb and BALB/c mice were exposed to single and fractionated d(50) + Be neutrons or 137Cs gamma rays at 12 weeks of age and were followed for life-shortening and disease incidence as ascertained by autopsy and histological examinations at the time of spontaneous death. Fractionation schedules used were 10 exposures at 24-h intervals and 8 exposures at 3-h intervals for gamma rays, and 8 exposures at 3-h intervals for neutrons. The data were analyzed by the Kaplan-Meier procedure using as criteria causes of death and possible causes of death. Individual groups were compared by a modified Wilcoxon test according to Hoel and Walburg (J. Natl. Cancer Inst. 49, 361-372 (1972)). No significant difference was found in C57Bl/Cnb and BALB/c male mice between a single gamma-ray exposure and a single neutron exposure. Gamma-ray fractionation was clearly less effective in reducing survival time than a single exposure. In contrast, fractionation of neutrons was slightly, although not significantly, more effective in reducing survival time than a single exposure. The relative biological effectiveness (RBE) for life-shortening for d(50)-Be neutrons compared to gamma rays is of the order of 1 to 2 for a single exposure to neutrons and between 2 and 3 for fractionated neutrons compared to a single exposure to gamma rays. Neutron irradiation caused somewhat more cancer than gamma irradiation, and the RBE for cancer induction may be higher, probably between 2 and 3 in the range of 1 to 3 Gy, although the present data do not allow a more precise assessment.

Toxicity of 241Am in male C57BL mice: relative risk versus 226Ra.

A life-span study on male C57BL mice after injection of various doses of 241Am was conducted. The effects on life span were evaluated and the incidence of tumors was determined by procedures that take competing risks into account. Bone tumors were induced in the mice by injections of 22 and 58 Bq 241Am per g. The mice died early from nonneoplastic diseases at the higher dose levels (190, 373, and 1197 Bq 241Am/g). Additionally, spontaneously occurring tumors such as liver carcinomas, lymphosarcomas, and lymphoreticulosarcomas occurred at an enhanced rate with increasing dose level. The data for survival time after 241Am injection and death with bone tumor were compared to data collected previously for 226Ra-injected mice of the same C57BL strain. This enabled direct comparison in the same strain of the effects of the bone-surface seeker 241Am to the effects of the bone-volume seeker 226Ra. The proportional hazards model was applied and the rate of death with bone tumor was 12.9 +/- 5.2 times higher after 241Am injection than after 226Ra injection if the regression covariate was the average dose to the skeleton. The relative risk was 3.5 +/- 1.7 if regressed on the injected radioactivity. The mortality rate after 241Am injection was 20.4 +/- 3.6 times higher than after 226Ra injection if regressed on average dose to the skeleton.

Protracted treatment of C57B1 mice with Zn-DTPA after 241Am injection reduces the long-term radiation effects.

Repeated intraperitoneal injections of ZnNa3-diethylenetriaminepenta-acetate (ZnDTPA), once a week, during 8 successive weeks, and starting 4 days after injection of 58 and 373 kBq 241Am/kg to C57B1 mice, were an effective protection against long-term radiation damage. At both dose levels of 241Am, Zn-DTPA reduced the 241Am concentration in bones by between 33% and 45%, and in the liver by 97%. Mean survival with 241Am was shortened in Zn-DTPA-treated mice, by 17% at the lower dose level and by 70% at the higher dose level. After treatment with DTPA at the lower 241Am level, survival became equal to that of control mice without 241Am, while at the higher level life span was still shortened. After the lower 241Am dose the incidence of bone tumours, liver carcinomas and the total number of all malignant tumours were significantly reduced by chelation therapy. The decrease in bone tumour incidence was proportional to the decrease in 241Am concentration and reduction in cumulative radiation dose in bone after chelation therapy. The incidence of liver carcinomas was reduced to that in non-241Am-injected mice and the reduction was thus proportional to the 97% reduction in 241Am concentration of the liver at the end of chelation therapy. After the higher 241Am dose no tumours showed up in sham-treated mice, probably due to the overkill effect on the cells at risk. In the corresponding Zn-DTPA-treated mice, bone tumours and a few other malignant tumours were observed.

Toxicity of 99Tc: can it represent a risk to man?

The movement through the environment and the toxicity to plants of 99Tc have been studied. However, information is scanty on 99Tc toxicity to mammals even though this is the decisive criterion for assessing the consequences of releases of Tc. A critical tissue could be the thyroid because of its preferential accumulation of Tc and the developing organism because of its greater radiosensitivity; moreover, this toxicity might be enhanced under conditions of a low iodine (I) diet. These questions were studied in rats given large amounts of 99Tc in either a normal or an I-deficient diet for several months starting 2 wk before mating. Newborns were continued on these diets after weaning. The parameters determined were: occurrence of pregnancy and litter size, triiodothyronine and thyroxin in serum, uptake of 131I by the thyroid 24 h after injection, histology of the thyroid and concentration of Tc in tissues of the mothers and their decendants. Thyroid damage, as well as the effects on pregnancy, could be observed after amounts of 10 micrograms Tc g-1 food. Iodine deficiency only slightly influences the 99Tc toxicity. The chemical and radiological toxicity of 99Tc to rats is small. Consequently, it seems unlikely that contamination levels in the environment would ever reach levels that could lead to serious non-stochastic effects, even in the developing organism.

Chemical protection against ionizing radiation.

Some of the problems related to chemical protection against ionizing radiation are discussed with emphasis on: definition, classification, degree of protection, mechanisms of action and toxicity. Results on the biological response modifyers (BRMs) and on the combination of nontoxic (i.e. low) doses of sulphydryl radioprotectors and BRMs are presented.

Response of mouse lung air-blood barrier to X-irradiation: ultrastructural and stereological analysis.

Male mice of the Balb/c strain were exposed, at an age of three months, to a single dose of 10 or 20 Gy on the right hemithorax. At 3, 4, 6, 9 and 12 months after exposure, lungs were processed for electron microscopy following a standardized procedure in order to allow stereological analysis. By this method, the arithmetical mean thickness and, the air-blood barrier mean thickness in the lung parenchyma was shown to increase quickly with time by oedemization and fibrinization of the septal space. The ratio endothelium/epithelium surfaces (SI/SE) gradually decreased by reduction of both surfaces but this was more marked for Si. The endothelium and epithelium were both highly damaged. Quantitative results indicate that damage to the epithelial cells and mainly to type II, appear at the same time as damage to the endothelium. From the time lapse quantitation it is not possible to determine which one plays the predominant role in the radiation pneumonitis. The strong reaction of the basement membrane and mainly of the interstitial cells could play a decisive role in the evolution of the illness.

Provirus integration and myc amplification in 90Sr induced osteosarcomas of CF1 mice.

In mice, endogenous retroviruses are known to be activated during the course of radiation osteosarcomagenesis. Using the Southern blotting procedure, we have studied the presence of somatically acquired proviruses in genomic DNA isolated from seven primary 90Sr induced osteosarcomas and one osteosarcoma cell line, 0-127a1, of the CF1 mouse strain. Specific hybridization probes demonstrated the presence of newly integrated ecotropic proviruses in four primary tumors. Probably, clonally integrated proviruses were present at distinct locations in different subpopulations of tumor cells, reflecting tumor heterogeneity. Genomic DNA isolated from cultured osteosarcoma cells contained different additional MCF-related proviruses. No proviruses were found integrated in the vicinity of c-myc, but a large domain containing the complete c-myc gene was found amplified in one primary tumor (greater than 22 kbp) and in 0-127a1 cells (greater than 39 kbp). Our data suggest that activated retroviruses are not essential for the development of radiogenic osteosarcomas in CF1 mice, but they might be responsible for the deregulated expression of a growth promoting gene in some bone tumor cells.

Life-shortening and disease incidence in C57Bl mice after single and fractionated gamma and high-energy neutron exposure.

C57Bl Cnb mice were exposed to single or fractionated d(50)+Be neutrons or 137Cs gamma-ray exposure at 12 weeks of age and were followed for life-shortening and disease incidence. The data were analyzed by the Kaplan-Meier procedure using as criteria cause of death and possible cause of death. Individual groups were compared by a modified Wilcoxon test according to Hoel and Walburg, and entire sets of different doses from one radiation schedule were evaluated by the procedure of Peto and by the Cox proportional hazard model. No significant difference was found in life-shortening of C57Bl mice between a single gamma and neutron exposure. Gamma fractionation was clearly less effective in reducing survival time than a single exposure. On the contrary, fractionation of neutrons was slightly although not significantly more effective in reducing life span than a single exposure. Life-shortening appeared to be a linear function of dose in all groups studied. The data on causes of death show that malignant tumors, particularly leukemias including thymic lymphoma, and noncancerous late degenerative changes in lung were the principal cause of life-shortening after a high single gamma exposure. Exposure delivered in 8 fractions 3 h apart was more effective in causing leukemias and all carcinomas and sarcomas than one delivered in 10 fractions 24 h apart or in a single session. Following a single neutron exposure, leukemias and all carcinomas and sarcomas appeared to increase somewhat more rapidly with dose than after gamma irradiation. No significant difference in the incidence of leukemias and all carcinomas and sarcomas was noted between a single and a fractionated neutron exposure.

The effect of prenatal or early postnatal irradiation on the production of anti-arsonate antibodies and cross-reactive idiotypes.

Preliminary studies on the long-term effects of prenatal and early postnatal irradiation on the immune response to arsonate were performed using A/J mice. Pregnant mice were irradiated (0.5 Gy, X-rays) or sham-irradiated on a single occasion during gestation (between day 5 and 18 post-conception). Alternatively, newborn mice received the same treatment between day 2 and 7 after birth. Mice were immunized with keyhole limpet haemocyanin-arsonate (KLH-Ars) in adjuvant from 2 months after birth. The levels of specific antibodies to arsonate (anti-Ars) were measured by radioimmunoassay. In addition, the Ars-related cross-reactive idiotype (CRIA) was measured by the haemagglutination technique. In the primary response the titre of anti-Ars was reduced in animals that had been irradiated between day 12 and 15 of gestation. In the second response, in contrast, they had increased levels of anti-Ars. After immunization with KLH-Ars, high levels of CRIA were observed in all groups. However, in mice irradiated 18-20 days after conception the level of CRIA was often much higher than the level of anti-Ars, indicating that a large proportion of the CRIA-positive molecules were not specific for Ars. Thus, in this particular case, some specificity of the immune response was lost after irradiation. The expression of recurrent idiotypes may be a sensitive indicator of immunological perturbations after irradiation.

Effects of radioprotective glycans on the arrest of blood-borne lymphoma cells.

The mode of action of radioprotective glycans is not understood. In view of the known importance of cell migration in haematology, on the one hand, and of carbohydrates in homing processes, on the other, we have investigated the effect of several glycans on blood-borne arrest of lymphoma cells. Radioprotective glycans (but not heparin) modified the arrest of injected cells in the spleen. Altered recirculation and homing processes may play a role in the radioprotective properties of glycans.

Effect of X-rays alone or combined with diethylnitrosamine on cancer induction in mouse liver.

The possible combined effects of the initiator diethylnitrosamine (DEN) with X-rays on cancer induction in infant C57BL/Cnb mice were evaluated. Preliminary data show that a dose of X-rays administered 7 days before a single injection of DEN was more effective in inducing liver nodules than when administered 7 days after DEN administration.