Radioprotection of normal tissues against gamma rays and cyclotron neutrons with WR-2721: LD50 studies and 35S-WR-2721 biodistribution

The ability of WR-2721 to protect mice against two modes of death following whole-body radiation with 137Cs gamma rays or d(22)+Be neutrons was examined. For single fractions, 400 mg/kg WR-2721 was administered prior to irradiation. In two-fraction exposures, the dose was 275 mg/kg given prior to each fraction. Dose modification factors (DMFs) were calculated as ratios of LD50 values. For single fractions of gamma rays, the DMF was 1.74 for the LD50/7 end point and for LD50/30, the DMF for single fractions was 2.25. For two fractions 3 hr apart, it was 1.88. For single fractions of cyclotron neutrons, the DMF was 1.32 for LD50/7. Measured with the LD50/30 end point, the DMF for single neutron doses was 1.41 and for two fractions, 1.19. Substantial radioprotection of bone marrow and intestinal epithelium against cyclotron neutrons was seen in these investigations. Biodistribution studies were done following ip injection of 35S-labeled WR-2721 into C3H mice bearing RIF-1 tumors. Blood levels peaked at 10 min after injection and declined thereafter. Most normal tissues achieved maximum levels of 35S at 30 to 60 min postinjection and high concentrations were retained in most tissues for up to 2 hr. Assuming that all 35S is in parent compound or dephosphorylated radioprotective metabolites, the concentration of protector (milligram per gram tissue) in various organs at 30 min postinjection ranked as follows: kidney greater than submandibular gland much greater than liver = lung greater than gut greater than heart much greater than blood greater than skin greater than tumor greater than brain. High levels of 35S were achieved and retention times were long in certain normal tissues which respond at early or late times postradiation and may be dose limiting in radiotherapy: kidney, liver, salivary gland, and lung. These combined observations suggest that there is potential for protecting dose-limiting, late-responding normal tissue in the radiotherapy of human cancer with both neutrons and conventional radiotherapy.     

A comparison of radioprotection from three neutron sources and 60Co by WR-2721 and WR-151327

Two thiophosphoroate radiation protectors (WR-2721 and WR-151327) were assessed for their ability to modify the effects of neutron or gamma irradiation on the gastrointestinal tract. Three neutron sources (DOSAR, JANUS, and FERMILAB) were compared to the response obtained after 60Co irradiation. The end points studied were intestinal stem cell survival and LD50(6). DOSAR and JANUS, both fission-spectrum neutrons, showed somewhat different gut sensitivities [LD50(6)] of about 240 and 400 cGy respectively. The intestinal LD50 obtained with FERMILAB neutrons (25 meV) was closer (875 cGy) to that obtained after 60Co (1068 cGy) irradiation. WR-151327 protected against the lethal effects of fission neutron (DOSAR and JANUS) to a greater degree (DMF = 2.2) than with lower LET sources such as FERMILAB neutrons (DMF = 1.7) or 60Co (DMF = 1.7). The results did not correlate with the intestinal stem cell assays where WR-2721 when compared to WR-151327 showed either similar (DOSAR; fission spectrum neutrons) or somewhat better (60Co and FERMILAB neutrons) protection. Possible explanations for the differing results are discussed.     

Postirradiation glucan administration enhances the radioprotective effects of WR-2721

Based on murine survival studies, endogenous hemopoietic spleen colony formation (E-CFU), and recovery of bone marrow and splenic granulocyte-macrophage colony-forming cells (GM-CFC), it was demonstrated that the postirradiation administration of glucan, an immunomodulator and hemopoietic stimulant, enhances the radioprotective effects of WR-2721. LD50/30 dose reduction factors for mice treated with WR-2721 (200 mg/kg approximately 30 min before irradiation), glucan (250 mg/kg approximately 1 h after irradiation), or both agents were 1.37, 1.08, and 1.52, respectively. Enhanced survival in mice treated with both agents appeared to be due in part to glucan's ability to accelerate hemopoietic regeneration from stem cells initially protected from radiation-induced lethality by WR-2721. Following a 10-Gy radiation exposure, E-CFU numbers in mice treated with saline, WR-2721, glucan, or both WR-2721 and glucan were 0.05 +/- 0.03, 6.70 +/- 1.05, 0.95 +/- 0.24, and 33.90 +/- 2.96, respectively. Similarly, bone marrow and splenic GM-CFC numbers were greater in mice treated with both WR-2721 and glucan than in mice treated with either agent alone. These results demonstrated at least additive radioprotective effects when mice were given WR-2721 prior to irradiation and glucan following irradiation. These effects appeared to depend on the sequential cell protection mediated by WR-2721 and hemopoietic repopulation mediated by glucan.     

Radioprotective effects of the thiols GSH and WR-2721 against X-ray-induction of micronuclei in erythroblasts

The frequency of micronucleated polychromatic erythrocytes (MNPCEs) was assessed in the bone marrow and peripheral blood of adult male Swiss mice treated with reduced glutathione (GSH) and S-2-/3-aminopropylamino/ethyl phosphorothioic acid (WR-2721), at a dose of 400 mg/kg body weight, and exposed to 6 Gy X-rays. GSH or WR-2721 was applied alone, or 60 and 30 min, respectively, prior to X-ray-exposure. The number of MNPCEs was determined at 24 h after the thiol treatment and X-irradiation. The radioprotection and toxicity caused in the mouse erythroblasts by GSH and WR-2721, as indicated by the number of MNPCEs were dependent on the thiol applied. The stronger radioprotective effect is obtained following WR-2721 administration than after GSH application. WR-2721 showed greater toxicity than GSH. The combination of GSH and WR-2721 given before X-ray-exposure resulted in the most radioprotective effect as compared to the respective single-drug treatment of mice. Application of the both thiols, without subsequent X-irradiation appeared to be the most toxic, compared with administration of WR-2721 or GSH alone. The effective radioprotection by the combined action of GSH and WR-2721 against genomic instability induced in the mouse erythroblasts by X-rays was shown.     

WR-2721, its derivatives and their radioprotective effects on mammalian cells in culture

Although it is well known that WR-2721 is very efficient in protecting mice against lethal irradiation, we could not find any radioprotective effect of WR-2721 on mouse L cells in culture. But WR-1065 alone (free SH form of WR-2721), and WR-2721 pre-incubated with mouse liver homogenate, showed radioprotective ability. It was found that mouse liver homogenate dephosphorylated WR-2721 to WR-1065. The highest WR-2721 metabolizing activity was found in mouse liver homogenate and Chang liver cell homogenate. Homogenates of human liver and kidney were also shown to possess moderate activity for metabolizing WR-2721. These results suggest to us that WR-2721 must be dephosphorylated before exerting its radioprotective effect and that this dephosphorylating activity varies with tissues. It is demonstrated therefore that mouse L cell in culture is a novel system to assess the extent of dephosphorylation of WR-2721 in various tissues.     

Induction of micronucleated erythrocytes by MEA,AET, WR-2721 and X-rays

The induction of micronucleated polychromatic erythrocytes (MNPCEs) was assessed in the bone marrow of adult male Swiss mice treated with MEA (cysteamine HCl), AET (2-aminoethylisothiouronium Br.HBr), or WR-2721 (S-2-(3-aminopropylamino)ethyl phosphorothioic acid), at a dose of 200 mg/kg body weight, and/or exposed to 6 Gy X-rays. MEA, AET, or WR-2721 was given alone or 15 min prior to X-ray exposure, and the frequency of MNPCEs was determined 24 h after the aminothiol treatment and X-irradiation of mice. A genotoxic effect was shown for MEA, AET, WR-2721, and X-rays, as well as a protective effect of the aminothiols against X-ray-induced genotoxicity in the mouse erythropoietic system. The aminothiol drugs given alone, without subsequent X-irradiation, elevated the frequency of MNPCEs, and WR-2721 appeared to be less toxic than AET and MEA. After exposure of mice to X-rays, the number of MNPCEs was distinctly increased. MEA, AET, or WR-2721 administration prior to X-irradiation resulted in a reduction of the X-ray-induced elevation of the frequency of micronuclei, but a stronger radioprotective effect was obtained following WR-2721 and AET treatment than after MEA application. So, the genotoxic and radioprotective effect of the aminothiols was dependent on the compound applied.     

Protection of mice against mixed fission neutron-gamma (n: gamma = 1:1) irradiation by WR-2721, 16,16-dimethyl PGE2, and the combination of both agents

The survival of mice after whole-body exposure to a modified fission neutron-gamma field (n: gamma = 1:1) was used to examine radiation protection by WR-2721, 16,16-dimethyl PGE2(DiPGE2), and the combination of both agents. Administration of WR-2721 (453 mg/kg) increased the LD50/30 from 5.24 to 7.17 Gy (DMF = 1.37), whereas pretreatment with DiPGE2 (1.6 mg/kg) increased the LD50/30 to 5.77 Gy (dose modification factor (DMF) = 1.10). The combination of 453 mg/kg WR-2721 and 0.4 mg/kg DiPGE2 resulted in an LD50/30 of 7.33 Gy, yielding a DMF of 1.39. However, no significant difference in protection was obtained with the combination of the two agents compared to that seen with WR-2721 alone.     

Phosphatidylserine externalization during bone marrow cell death after treatment of mice with WR-2721 and gamma-rays

The cell surface exposure of phosphatidylserine (PS) and the plasma membrane impairment were assessed in the bone marrow of adult male Swiss mice exposed to a single 6 Gy dose of 60 Co gamma-rays, and treated intraperitoneally with the aminothiol WR-2721 (Amifostine, S-2-/3-aminopropylamino/ethyl phosphorothioic acid), at a dose of 400 mg/kg body weight, 30 min prior to gamma-irradiation. The bone marrow cells were stained with a combination of fluoresceinated annexin V (annexin V--FITC) and propidium iodide (PI) at 3 h, 7 h, and 24 h after treatment of mice with WR-2721 and 60Co gamma-irradiation. The number of early apoptotic cells (annexin V--FITC positive/PI negative), and late apoptotic and necrotic cells (annexin V--FITC positive/PI positive), was increased at 3 h after exposure of mice to 60Co gamma-rays and thereafter declined with the frequency of apoptotic and necrotic cells remaining lower in WR-2721 pre-treated mice. Using the annexin V--FITC flow cytometric assay, the radioprotective effect of WR-2721 against induction of apoptosis and necrosis in normal cells of the haematopoietic system was shown.     

Effect of WR-2721 on fetal development in the rat

The radioprotector WR-2721 has been shown to radioprotect all tissues studied except the central nervous system. However, it has not yet been used to radioprotect the fetus. In this study we determined that [14C]WR-2721 injected into pregnant rats quickly passed the placenta and was concentrated by the fetus. In addition, we evaluated the toxicity of WR-2721 (2.5-600 mg/kg) to pregnant rats and their fetuses during the period of major organogenesis at Days 9, 11, and 14 postconception. Pregnant animals were only slightly more (10%) sensitive (LD50, 580 mg/kg) to WR-2721 than nonpregnant cohort animals (LD50, 640 mg/kg). At concentrations of 50 mg/kg or less there was a small but statistically significant increase in fetal deaths, while at doses greater than 300 mg/kg a larger degree of fetal mortality occurred. Maximal fetal weight loss, to about 84% of control, was found at 500 mg/kg. No changes in head dimensions or gross malformations of the surviving fetuses were detected at any time or concentration. Of all the parameters measured in this study none demonstrated a predilection for any specific period of major organogenesis. The results of this study indicate that while WR-2721 demonstrates a dose-related embryotoxicity it is not teratogenic.     

Effect of 60Co-Gamma whole body irradiation and treatment with radioprotector on the retention of 144Ce, in mice

In animal experiments the effects of 7.5 Gy whole-body irradiation (LD50/30) and/or of radioprotecting compound (WR-2721) on the retention and elimination of 144Ce were investigated. It was stated that in the first three days there was an increase in the deposition of 144Ce after whole-body irradiation and upon treatment with radioprotective compound WR-2721.     

Radiation protection of murine intestine by WR-2721, 16,16-dimethyl prostaglandin E2, and the combination of both agents

The survival of murine intestinal clonogenic cells (ICC) and the survival of mice after whole-body exposure to 137Cs irradiation were used to measure radiation protection by ethiophos (WR-2721), 16,16-dimethyl prostaglandin E2, and the combination of the two. Doses from 2 to 12.5 mg/mouse of WR-2721 increased cell survival linearly from 3.2 +/- 0.3 in controls given 15.0 Gy to 93.1 +/- 5.2 per jejunal circumference. In contrast, 16,16-dm PGE2 increased ICC survival at 15.0 Gy rapidly from 1 to 10 micrograms/mouse, followed by a plateau up to 100 micrograms/mouse. Animal survival at 6 days (LD50/6) increased from 16.3 +/- 0.4 Gy (95% confidence limits) in controls to 20.3 +/- 0.6 Gy in the PG-treated animals. WR-2721 increased the LD50/6 to 26.1 +/- 1.4 Gy. The dose modification factors were 1.25 and 1.60, respectively. The combination of agents increased ICC survival above that seen with each agent alone up to 8 mg WR-2721, above which no additional protection was seen. Animals given 10 micrograms PG plus 10 mg WR-2721 survived longer than with either agent given alone. The LD50/6 was 36.3 +/- 1.8 Gy for a dose modification factor (DMF) of 2.23. In addition, the slope of the probit curve was reduced from those of each agent alone. PG-induced changes in villus epithelial cell morphology and survival may account, in part, for these observations. The results suggest that either the mechanisms for these two types of radiation protectors are different or they act on separate subcellular targets which are critical to survival from radiation injury.     

Radioprotective effect of combinations of WR-2721 and mercaptopropionylglycine on mouse bone marrow chromosomes

The radioprotective and toxic effects of low to moderate doses of S-2-(3-aminopropylamino)ethyl phosphorothioic acid (WR-2721) and its combination with mercaptopropionylglycine (MPG, 20 mg/kg body wt) on the chromosomes of the bone marrow cells of Swiss albino mice were studied at 24 h and 14 days postirradiation. Significant protection against radiation-induced chromosome aberrations was observed with 50 mg/kg WR-2721. The protection increased with the dose of the drug administered, and the degree of protection per unit dose increment was more pronounced at lower than at higher doses. A combination of WR-2721 and MPG given before exposure resulted in a significantly greater number of normal metaphases at 24 h postirradiation compared to the respective single-drug treatment groups. On Day 14 postirradiation, when the presence of WR-2721 resulted in an increase in the frequency of aberrant cells, combination with MPG helped to reduce this value markedly, especially at WR-2721 doses below 200 mg/kg. On the basis of these results it is suggested that 150 mg/kg WR-2721 may be considered an optimum dose for combination with MPG for protection of chromosomes of bone marrow cells when repeated drug administrations are not needed. Changes in the level of glutathione (GSH) in the blood were studied at different times following the administration of 150 mg/kg WR-2721 and its combination with MPG (20 mg/kg) before sham irradiation or exposure to 4.5 Gy 60Co gamma rays. The results showed that WR-2721 elevated blood GSH levels significantly above normal values by the time radiation was delivered, while MPG did not. Glutathione appears to have an important role in the action of WR-2721, while protection by MPG may not be mediated through GSH. Injection of MPG after WR-2721 helps to maintain the higher GSH level for a longer duration compared to treatment with WR-2721 alone. It is possible that MPG delays the metabolism of GSH.     

Selenium Pretreatment Enhances the Radioprotective Effect and Reduces the Lethal Toxicity of Wr-2721

Although WR-2721, S-2-(3-aminopropylamino)ethylphosphorothioic acid, is an effective radioprotector, its use is limited by its toxicity. Combining WR-2721 with other agents might decrease its toxicity and/or increase its effectiveness. The effect of selenium (Se) pretreatment on the acute toxicity and radioprotective effect of WR-2721 was studied in male CD2F1 mice. Injection of 1.6mg/kg Se 24 hr before WR-2721 (800-1200 mg/kg, IP) decreased the lethality of WR-2721 significantly. Lower doses of Se were also effective, but simultaneous administration was not effective. Se injection alone (1.6 mg/kg) 24 hr before cobalt-60 irradiation increased the survival (dose reduction factor, DRF = 1.1) significantly. A synergistic effect on post-irradiation survival was observed when Se was injected 24 hr before WR-2721 (200-600 mg/ kg IP before irradiation). For example, after exposure to 22 Gy (1 Gy/min), 30-day survival was 100% when mice were treated with both Se and 600mg/kg WR-2721, and was 13% with WR-2721 alone. The DRF after 400 mg/kg WR-2721 was 2.6 with Se compared to 2.2 without Se pretreatment. Alkaline phosphatase activity in bone marrow cells and serum was significantly depressed after treatment with 1.6 mg/kg Se, suggesting that a retardation of conversion of WR-2721 to its active free sulfhydryl form through the action of alkaline phosphatase might be partly responsible for the effects of Se. Other possible mechanisms related to the antioxidant properties of Se are under investigation.     

Selenium Pretreatment Enhances the Radioprotective Effect and Reduces the Lethal Toxicity of Wr-2721

Although WR-2721, S-2-(3-aminopropylamino)ethylphosphorothioic acid, is an effective radioprotector, its use is limited by its toxicity. Combining WR-2721 with other agents might decrease its toxicity and/or increase its effectiveness. The effect of selenium (Se) pretreatment on the acute toxicity and radioprotective effect of WR-2721 was studied in male CD2F1 mice. Injection of 1.6mg/kg Se 24 hr before WR-2721 (800-1200 mg/kg, IP) decreased the lethality of WR-2721 significantly. Lower doses of Se were also effective, but simultaneous administration was not effective. Se injection alone (1.6 mg/kg) 24 hr before cobalt-60 irradiation increased the survival (dose reduction factor, DRF = 1.1) significantly. A synergistic effect on post-irradiation survival was observed when Se was injected 24 hr before WR-2721 (200-600 mg/ kg IP before irradiation). For example, after exposure to 22 Gy (1 Gy/min), 30-day survival was 100% when mice were treated with both Se and 600mg/kg WR-2721, and was 13% with WR-2721 alone. The DRF after 400 mg/kg WR-2721 was 2.6 with Se compared to 2.2 without Se pretreatment. Alkaline phosphatase activity in bone marrow cells and serum was significantly depressed after treatment with 1.6 mg/kg Se, suggesting that a retardation of conversion of WR-2721 to its active free sulfhydryl form through the action of alkaline phosphatase might be partly responsible for the effects of Se. Other possible mechanisms related to the antioxidant properties of Se are under investigation.     

Histopathologic effects of soluble glucan and WR-2721, independently and combined in C3H/HeN mice.

Soluble glucan, an immunomodulator, and Walter Reed (WR)-2721, a radioprotectant, increase postirradiation survival when administered before and after exposure, respectively. Combined, these agents act synergistically through WR-2721's ability to spare hematopoietic stem/progenitor cells from radiation injury and glucan's ability to subsequently stimulate spared cells to proliferate. In this study, the histopathologic effects of WR-2721 (200 mg/kg, ip) and glucan (250 mg/kg, iv), at doses capable of increasing survival in lethally irradiated mice, were evaluated in unirradiated and irradiated female C3H/HeN mice. After treatment, whole body weights and wet organ weights of liver, spleen, and kidney, as well as gross and histologic changes in these and other tissues, were monitored on Days 1, 4, 7, 11, 15, 21, and 28. Morphometric studies of splenic white and red pulps were also performed. Soluble glucan, with or without WR-2721, in unirradiated groups, was associated with splenomegaly, transient morphometrically determined perturbations of white and red pulp areas, and histologic alterations of white pulp. In irradiated mice, splenic weight loss was initially dampened in glucan groups and accompanied by morphologic and histologic changes similar to those seen in unirradiated counterparts. The subsequent rebound of splenic parameters in irradiated mice was limited to WR-2721-treated mice and was associated with hematopoietic reconstitution. Glucan, with or without WR-2721, in unirradiated groups was associated with transient hepatomegaly and associated histologic changes. Similar changes in irradiated animals were seen only in the combined treatment group.     

WR-2721 inhibition of radiation-induced prostaglandin excretion in rats

Pre-irradiation administration of the radioprotectant drug WR-2721 to rats resulted in a significant reduction in radiation-induced increases in excretion rates of prostaglandins (PGE and PGF2 alpha) and thromboxane (TxB2). In animals not irradiated. WR-2721 did not significantly alter these excretion rates. Dramatic reductions in the levels of urinary PGE and TxB2 were observed following exposure to 9.0 Gy of whole-body, unilateral gamma-radiation in WR-2721-treated animals, whereas changes in PGF2 alpha levels were less pronounced. Radiation-induced diuresis was also significantly depressed in animals given WR-2721 before irradiation. Reduced prostaglandin excretion rates may reflect the general radioprotective capacity of the chemoprotector WR-2721 on the release of prostaglandins from radiation-damaged tissue. The decrease in diuresis may be related to the observed prostaglandin decreases.     

WR-2721 entry into the brain across a modified blood-brain barrier

Radioprotection of the CNS by WR-2721 has not been possible because of its inability to cross the blood-brain barrier (BBB) and so gain access to the neural tissue. Modification of the BBB using hypertonic arabinose (1.8 m), injected via the internal carotid artery (ica), permitted entry of ip-injected [14C]WR-2721 into the ipsilateral cerebral hemisphere. The BBB-modified hemisphere had a 5.34-fold increased uptake compared to nonmodified controls. Delivery as a bolus via the ica further enhanced uptake after BBB opening; WR-2721 was 3.73 times greater than by ip injection. A 20-fold increase of WR-2721 brain uptake has been calculated for ica administration with the BBB opened as compared to the ip route without BBB modification. Toxicity of ip-administered WR-2721 with the BBB open was only 1.4 times greater than non-modified controls and 1.96 times more toxic when delivered via the ica. These data demonstrate significant uptake of WR-2721 into the CNS, a previously unprotected organ, and provide a model for future radioprotective studies.     

Induction of apoptosis in bone marrow cells after treatment of mice with WR-2721 and gamma-rays: relationship to the cell cycle

Apoptosis and cell proliferation are accepted to be responsible for the maintenance of homeostasis in the hematopoietic system. Understanding of the mechanisms of action of the aminothiols and ionizing radiation on normal hematopoietic cells requires determination of the correlation between apoptotic cell death and cell cycle distribution. The effects of WR-2721 ((S)-2-/3-aminopropylamino/ethylphosphorothioic acid; Amifostine) and ⁶⁰Co gamma-rays on apoptosis and cell cycle progression in the mouse bone marrow were determined. Adult male Swiss mice were exposed to 6 Gy gamma-rays only, or pretreated with WR-2721, at a dose of 400 mg/kg body weight, 30 min before gamma-irradiation. The laser scanning cytometry APO-BRDU^TM assay based on simultaneous analysis of cellular DNA content and the in situ detection of DNA strand breaks was used to identify apoptotic cells and to reveal the cell cycle position of apoptotic and nonapoptotic cells. Temporary changes in the frequency of apoptotic cells with fluorescein isothiocyanate (FITC) labeling of DNA strand breaks, and all bone marrow cells including apoptotic and nonapoptotic ones, whose DNA stained with propidium iodide, were observed in the particular phases of the cell cycle throughout the 96-h period after WR-2721 application and gamma-irradiation. The cell cycle phase specificity of WR-2721 and ⁶⁰Co gamma-irradiation was shown in terms of induction of apoptosis in bone marrow cells. The patterns of alterations in the frequency of apoptotic cells and all bone marrow cells with respect to their cell cycle position were dependent on the agent(s) applied and the time interval after treatment of mice with WR-2721 and/or gamma-rays. A modulatory, suppressive action of WR-2721 on apoptosis induction and the cell cycle perturbation caused in normal cells of the mouse bone marrow by gamma-rays was found.     

Pathological effects of the radiation protector WR-151327 in mice

The systemic effects of the radiation protective agent, S-3-(3-methylaminopropylamino) propylphosphorothioic acid (WR-151327), were studied in unirradiated B6CF1 male mice. Fifty mice were injected intraperitoneally with 540 mg/kg WR-151327, and groups of five mice were sacrificed at 14-day intervals up to and including 140 days post-treatment. Ten mice served as sham-injected controls. A necropsy was performed and gross morphological abnormalities were noted. Tissues (brain, eyes, harderian gland, salivary glands, sternal bone marrow, thyroid, lung, thymus, esophagus, trachea, skeletal muscle, heart, liver, kidney, adrenal gland, spleen, small intestine, pancreas, and testes) were fixed in 10% formalin, embedded in paraffin, and sectioned. Slides were routinely stained with hematoxylin and eosin while Alizarin red stain was used to test specifically for the presence of calcium salts. Histopathological effects of WR-151327 were restricted to the testes, salivary gland, and pancreas. The caudal pole of the testes was observed to undergo progressive changes from coagulation necrosis to dystrophic calcification. The cells of the submandibular salivary gland showed mainly hyperchromatic nuclei while the pancreas showed enlarged islets of Langerhans.     

Late functional and biochemical changes in mouse lung after irradiation: differential effects of WR-2721

The radioprotective effect of WR-2721 on late damage after whole thorax irradiation has been studied after split doses of radiation using the standard death and breathing rate assays at monthly intervals between 3 and 15 months after irradiation, as well as two biochemical measurements of injury at 15 months, hydroxyproline (HP), an indicator of tissue fibrosis, and DNA content, an indicator of tissue cellularity. A comparison of HP/lung and breaths per minute (BPM) in each dose group in the WR-2721 and non-WR-2721-treated mice 15 months after irradiation showed that the relationship between these two assays of late lung injury was not the same. There were large dose-related increases in breathing rate corresponding to relatively small changes in HP in the lungs of mice given radiation alone. In contrast, the mice given WR-2721 before irradiation showed large dose-related increases in HP/lung, but BPM remained relatively constant independent of dose. These data suggest then that changes in breathing rate and deaths later than 9 months after whole lung irradiation may not be due to collagen accumulation in the lung. WR-2721 did protect better against late lung functional changes (protection factors (PF) = 1.6) and late deaths (PF = 1.51) than against earlier changes in these same assays (PF = 1.4 and 1.28, respectively). Although the earlier-appearing injury after whole thoracic irradiation is most likely related to lung damage with deaths and increases in breathing rate resulting from pneumonitis, the cause of the late-appearing functional injury in the lung after radiation is not clear. Thus protection of late lung damage measured from either lethality or breathing rate is not related to the prevention of lung fibrosis.