Murine intestinal crypt survival after combined taxol plus radiation exposure.

Taxol is active against a number of cancers, some of which are also radiosensitive. The role of combined Taxol and radiotherapy has not been established. A theoretical benefit exists, however, due to the G2/M phase specificity of Taxol, and radiation-sensitizing effects have been observed in vitro. This study focused on the combined Taxol/radiation effect on murine intestinal epithelium. HA/ICR random-bred male mice were used to examine the temporal effect of injecting Taxol (40 mg/kg) before and after irradiation (12.5 Gy). The microcolony assay was used to determine the effect of single and combined modalities. Control groups consisted of no treatment, drug solvent only (Cremophor/ethanol/saline), radiation only, and Taxol only. A complete radiation dose-response study of treatment with and without Taxol was performed 12 hr after injection. Preirradiation Taxol appears not to enhance intestinal cell killing, and may even offer some protection when injected at 4 hr prior to irradiation. Postirradiation Taxol was found to enhance radiation response most dramatically at times greater than 12 hr. Dose-response data appeared to confirm the increased radiation effect of combined treatment. In conclusion, Taxol administered prior to irradiation results in either no sensitization or possible protection of the normal tissue response. Postirradiation Taxol appears to sensitize intestinal crypts when injected at least 12 hr after gamma irradiation. These data suggest that care should be exercised when using Taxol after irradiation.

Chemical modification of normal tissue damage induced by photodynamic therapy.

One of the limitations of successful use of photodynamic therapy (PDT) employing porphyrins is the acute and long-term cutaneous photosensitivity. This paper describes results of experiments designed to test the effects of two radiation protective agents (WR-2721, 500 mg kg-1 or WR-3689, 700 mg kg-1) on murine skin damage induced by PDT. C3H mice were shaved and depilated three days prior to injection with the photosensitiser, Photofrin (5 or 10 mg kg-1). Twenty-four hours later, the mice were injected intraperitoneally with a protector 30 min prior to Argon dye laser (630 nm) exposure. The skin response was followed for two weeks post irradiation using an arbitrary response scale. A light dose response as well as a drug dose response was obtained. The results indicate that both protectors reduced the skin response to PDT, however WR-2721 was demonstrated to be the most effective. The effect of the protectors on vascular stasis after PDT was determined using a fluorescein dye exclusion assay. In mice treated with Photofrin (5 mg kg-1), and 630 nm light (180 J cm-2) pretreatment with either WR-2721 or WR-3689 resulted in significant protection of the vascular effects of PDT. These studies document the ability of the phosphorothioate class of radiation protective agents to reduce the effects of light on photosensitized skin. They do so in a drug dose-dependent fashion with maximum protection at the highest drug doses.

Modification of neutron-induced hematopoietic effects by chemical radioprotectors.

Nine drugs, WR-347, WR-1065, WR-2529, WR-2721, WR-3689, WR-44923, WR-151327, WR-109342, and WR-168643, were assayed for intraperitoneal pharmacological toxicity, hematopoietic lethality, and spleen stem cell survival in C57/Bl mice in conjunction with whole-body fission neutron irradiation. In addition, WR-3689, WR-109342, and WR-168643 were used with per os administration to determine hematopoietic lethality. Drug toxicities after intraperitoneal administration, as determined by LD50's, ranged from 37 mg/Kg for WR-109342 to 2901 mg/Kg for WR-2529. Following oral administration drug toxicities ranged from 58 mg/Kg (WR-109342) to 1816 mg/Kg (WR-3689). Dose modification factors as determined by LD50(30)'s following intraperitoneal administration ranged from 1.08 for WR-347 to 1.49 for WR-2529. Of the drugs orally administered, WR-168643 was the best protector with a DMF of 1.51. Spleen stem cell survival, which was performed only after intraperitoneal drug administration, provided no significant differences in dose modification factors.

Chemical protection and cell-cycle effects on radiation-induced mutagenesis.

Chinese hamster ovary cells in the exponential phase of growth were harvested and separated by the method of centrifugal elutriation into subpopulations enriched with up to 95% G1 phase, 70% S phase and 65% G2 + M phase cells. Cell cycle distributions were routinely monitored by flow cytometry. Following elutriation, aliquots of cells from each of the enriched cell fractions were incubated in the presence or absence of 4 mM of 2-[(aminopropyl)amino] ethanethiol (WR-1065) for 30 min at 37 degrees C. The cells were then irradiated with 60Co gamma-rays or fission-spectrum neutrons from the JANUS research reactor. Both cell killing and mutagenesis were determined. Regardless of the radiation quality used, cells enriched in G1 phase were the most sensitive to radiation-induced mutagenesis at the hypoxanthine-guanine phosphoribosyl transferase locus. The relative magnitude of protection exerted by WR-1065 differed for each of the elutriator separated cell populations. The greatest magnitude of protection, however, was observed for G1-enriched populations, regardless of the radiation quality used or the biological end-point tested.

Protection against late effects of radiation by S-2-(3-aminopropylamino)-ethylphosphorothioic acid.

We have demonstrated that S-2-(3-aminopropylamino)ethylphosphorothioic acid (WR2721) administered to mice 30 min prior to a relatively low dose of ionizing radiation is effective in protecting against radiation-induced carcinogenesis and subsequent life shortening. Female C57BL/6JANL x BALB/cJANL F1 mice, 200 per group, were exposed to gamma radiation at a dose of 206 cGy. Additional groups of 200 animals were sham treated, given injections of 400 mg/kg of WR2721, or administered WR2721 and the irradiated with 60Co photons at doses of 206 cGy or 417 cGy. Mice were treated at 110 days of age. They were housed five to a cage and were checked daily throughout life. All deceased animals were necropsied, and tissues were removed and fixed for histopathological analysis. Over 90% of the animal deaths were due to tumor involvement. WR2721 afforded significant protection (P = 0.0016) against radiation-induced malignancies (i.e., a total of 164 tumor codes were used) following a dose of 206 cGy. Protection against lymphoreticular tumors in particular was significant (P = 0.0165). Subsequent survival time in WR2721-protected animals (compared with matched irradiated controls) was extended by 65 days. Mice irradiated with 417 cGy following administration of WR2721 exhibited a response similar to those irradiated without the protector at a dose of 206 cGy (P = 0.26). Cumulative survival curves for unirradiated mice were unaffected by a single dose of WR2721. These data indicate a potential novel benefit for radioprotectors in cancer therapy. WR2721 and similar aminothiols may be effective adjuvants for reducing the risk of therapy-induced secondary cancers in patients who have an excellent prognosis for cure and long-term survival.

The effect of chemical radiation protectors on cell cycle progression after gamma or neutron irradiation.

The effects of two chemical radiation protectors, WR-1065 and WR-151326, were characterized in V79 Chinese hamster cells after either cobalt-60 (60Co) gamma or fission spectrum neutron irradiation. Each protector was administered at a concentration of 4 mM to exponentially growing cultures for 30 min prior to and during irradiation with either 60Co gamma or JANUS fission spectrum neutrons. After irradiation the cells were either plated immediately for survival or returned to the incubator and assayed for cell progression. Aliquots of cells were removed at selected times, counted, fixed and stained with 4'6-diamidino-2-phenylindole (DAPI). Analysis of DNA histograms indicate that the presence of the protector during irradiation reduced the division delay experienced at the G2-M interface. Implications of these effects are discussed.

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.

Protection by WR1065 and WR151326 against fission-neutron-induced mutations at the HGPRT locus in V79 cells.

The radioprotectors WR1065 and WR151326, each at a concentration of 4 mM, protect against cell killing and mutagenesis at the hypoxanthine-guanine phosphoribosyl transferase (HGPRT) locus in V79 Chinese hamster fibroblast cells exposed to fission-spectrum neutrons (mean energy of 0.85 MeV) from the JANUS reactor. Significant protection against neutron-induced cell lethality occurred only when the radioprotectors were present during irradiation; e.g., D0's and n's were 82 Gy, 1.27 for control cells; 97 Gy, 1.51 for WR1065-protected cells; and 120 Gy, 1.00 for WR151326-protected cells, respectively. Mutation induction by JANUS fission-spectrum neutrons was linear over the dose range tested giving rise to a mutation frequency of 109.3 x 10(-6)/Gy. In comparison with 60Co gamma rays (mutation frequency 8.7 X 10(-6)/Gy), JANUS neutrons, at a dose rate of 24 cGy/min, were over 12 times more effective in inducing HGPRT mutations. Both WR1065 and WR151326 afforded protection against the induction of mutants by neutrons, even when they were administered up to 3 h after irradiation; i.e., mutation frequencies were 40.9, 48.8 and 68.6 X 10(-6)/Gy for WR1065 present during, present immediately after, or added 3 h after irradiation, respectively; and 61.7, 47.8, and 68.5 X 10(-6)/Gy for WR151326 present at the same times.

Protection against radiation-induced mutagenesis in V79 cells by 2-[(aminopropyl)amino] ethanethiol under conditions of acute hypoxia.

The effects of the radioprotector 2-[(aminopropyl)amino] ethanethiol (WR-1065) on radiation-induced cell killing and mutagenesis at the hypoxanthine-guanine phosphoribosyl transferase (HGPRT) locus in V79 Chinese hamster cells under hypoxic or aerobic conditions were examined. Conditions of acute hypoxia were attained by gassing 10(6) cells in 1-ml volumes in individual glass ampoules for 2 min with nitrogen. Ampoules were then sealed and incubated at 37 degrees C for 60 min. Following this treatment, cell survival after irradiation as expected was significantly enhanced. The effect of acute hypoxia on the formation of HGPRT mutants by irradiation was also investigated. Mutation frequencies were determined with a 6-day expression time and corrected for the number of spontaneous background mutants. Although mutation induction was approximately linear as a function of radiation dose under most conditions tested, it was significantly reduced in cell populations made acutely hypoxic prior to irradiation. Protection against mutation induction was apparent and similar when cells were irradiated in the presence of the radioprotector, regardless of whether they were also hypoxic or aerated. If cells were irradiated in air and then made hypoxic, no significant protection was still observed. These results suggest that the antimutagenic effect of WR-1065 is not due solely to its ability to scavenge radiation-induced oxygen-free radicals, but rather that it may also modulate these effects through the scavenging of metabolically induced free radicals and/or the chemical repair of radiation-induced DNA lesions.

The effect of 2-[(aminopropyl)amino] ethanethiol on fission-neutron-induced DNA damage and repair.

The effect(s) of the radioprotector 2-[(aminopropyl)amino] ethanethiol (WR 1065) on fission-neutron-induced DNA damage and repair in V79 Chinese hamster cells was determined by using a neutral filter elution procedure (pH 7.2). When required, WR1065, at a final working concentration of 4 mM, was added to the culture medium, either 30 min before and during irradiation with fission spectrum neutrons (beam energy of 0.85 MeV) from the JANUS research reactor, or for selected intervals of time following exposure. The frequency of neutron-induced DNA strand breaks as measured by neutral elution as a function of dose equalled that observed for 60Co gamma-ray-induced damage (relative biological effectiveness of one). In contrast to the protective effect exhibited by WR1065 in reducing 60Co-induced DNA damage, WR1065 was ineffective in reducing or protecting against induction of DNA strand breaks by JANUS neutrons. The kinetics of DNA double-strand rejoining were measured following neutron irradiation. In the absence of WR1065, considerable DNA degradation by cellular enzymes was observed. This process was inhibited when WR1065 was present. These results indicate that, under the conditions used, the quality (i.e. nature), rather than quantity, of DNA lesions (measured by neutral elution) formed by neutrons was significantly different from that formed by gamma-rays.

Differential protection of radiation-induced DNA single-strand breaks and cell survival by solcoseryl.

V79 Chinese hamster cells were studied in vitro for modification of cobalt-60 gamma radiation effects by solcoseryl. This treatment did not modify cell survival but did protect against DNA single-strand breaks.

Cell cycle redistribution of cultured cells after treatment with chemical radiation protectors.

The effect of two radioprotective agents (WR-1065 and WR-151326) was tested for their ability to modify cell cycle progression. Each protector was administered at a concentration of 4 mmol to exponentially growing cultures of V79 cells for periods of time up to 3 h. Under these conditions no cell toxicity was observed. At selected times up to and after removal of the protector, aliquots of cells were removed, counted and fixed in cold 70% ethanol. The cells were stained with DAPI in a 0.1% citrate solution and DNA histograms were obtained using a PARTEC PAS-II flow cytometer. The coefficient of variation of the G1 peaks obtained for unperturbed cell samples routinely ranged from 1.5 to 2.5%. During exposure, both radioprotectors effectively perturbed cell cycle progression, as characterized by a build-up of cells in S and G2 phases. After the protectors were removed, cells began to redistribute throughout the cell cycle. Twelve hours were required before cells exposed to WR-1065 approached levels commensurable with controls. In contrast, cells treated with WR-151236 required about 24 h to redistribute to control levels. These data demonstrate that different thiol-containing radioprotective compounds can differentially affect the progression and redistribution of exposed cells.

Modification of radiation response in mice by fractionated extracts of Panax ginseng.

A water-soluble extract of the root of Panax ginseng, a plant native to northeastern China, was fractionated into three components: carbohydrate, protein, and saponin fractions. The fractions obtained were tested for their ability to protect against the lethal effects of 60Co gamma irradiation in C3H mice. The results were compared to the protective ability of the water-soluble fraction of whole ginseng. An experiment designed to test the optimum time of injection of whole ginseng showed that administration 24 h prior to irradiation was optimal. Ginseng extract or one of its three fractions was dose adjusted and injected intraperitoneally into mice that 24 h later were irradiated, whole body, with doses ranging from 7 to 11 Gy. The LD50 in 30 days was calculated using Probit analysis. The results indicated that the water soluble extract of whole ginseng gave the best protection against gamma radiation. The isolated protein and carbohydrate fractions gave less protection, while the saponin fraction did not protect.

The effect of 2-[(aminopropyl)amino] ethanethiol (WR-1065) on radiation induced DNA double strand damage and repair in V79 cells.

Radiation induced DNA double strand breaks are believed to be important lesions involved in processes related to cell killing, induction of chromosome aberrations and carcinogenesis. This paper reports the effects of the radioprotector 2-[(aminopropyl)amino]ethanethiol (WR-1065) on radiation-induced DNA damage and repair in V79 cells using the neutral elution method performed at pH 7.2 or pH 9.6. WR-1065 (4 mM) was added to the culture medium either 30 minutes prior to and during irradiation with Cobalt-60 gamma rays (for dose response experiments) or during the repair times tested (for DNA rejoining experiments). The results indicate that WR-1065 is an effective protector against the formation of radiation-induced double-strand breaks in DNA as measured using a neutral elution technique at either pH. The protector reduced the strand scission factors by 1.44 and 1.77 in experiments run at pH 9.6 and pH 7.2, respectively. The kinetics of DNA double-strand rejoining were dependent upon the pH at which the neutral elution procedure was performed. Unlike the results obtained with alkaline elution, rejoining of DNA breaks was unaffected by the presence of WR-1065 at either pH.

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.

Chemical protection against gastrointestinal radiation injury in mice by WR 2822, WR 2823, or WR 109342 after 4 MeV X ray or fission neutron irradiation.

Three compounds were tested for their radioprotective properties against the effects of 4 MeV X rays or fission neutron irradiation. The endpoints tested were lethality, intestinal crypt survival, and DNA synthesizing cellularity. Two of the compounds tested; S-2(4-aminobutylamino) ethylphosphorothioic acid (WR 2822) and the aminopentylamino derivative (WR 2823) are closely related to WR 2721. The third agent was the iminothiol derivative of 1-methyl-aminoadamantine (WR 109342). All drugs were administered via intraperitoneal injections at their approximately maximum tolerated dose. WR 2822 was shown to have a slight protective effect against X rays and neutrons. The dose modification factor (DMF) for gastrointestinal death (LD50(6)) was 1.23 for X rays and 1.51 for neutrons. The assay for intestinal crypt survival produced DMF's of 1.44 (X rays) and 1.4 (neutrons). Wr 2823 also showed a protective action in these assays. The DMF for LD50(6) was 1.32 (X rays) and 1.42 (neutrons). WR 109342 was found to be extremely toxic and had no significant protective effects. All three drugs were more toxic and demonstrated less protection in most of these assays than the benchmark radioprotective agent WR 2721, although WR 2822 protected against lethal effects of fission neutrons almost as well as WR 2721. Both WR 2822 and WR 2823 produced greater protection in the crypt survival assays for fission neutron irradiation than WR 2721.

Density centrifugation of murine fibrosarcoma cells following in situ labelling with tritiated thymidine.

Murine fibrosarcoma (FSa) cells form at least five unique subpopulations after centrifugation in linear Renografin density gradients. Each of these subpopulations has been characterized with respect to selected kinetic parameters using pulse-labelling techniques and flow microfluorometry (FMF) analysis. Tumour-bearing mice were first injected intraperitoneally with a pulse label of tritiated thymidine ([3H]TdR, 50 microCi). Following 15, 30, 60 min or 24 hr these animals were injected with cold thymidine. Animals were killed, their tumours removed and made into suspension, and separated by density gradient centrifugation. Each gradient was fractionated and the density, cell number, tritium activity, and labelling index (LI) per fraction were determined. These data were then compared to FMF data for selected cell density, cell number, tritium activity, and labelling index (LI) per fraction were determined. These data were then compared to FMF data for selected cell density bands. The results indicated a relatively higher uptake of [3H]TdR in the cells recovered at the lighter (1.06-1.12 g/cm3) as compared to the heavier (greater than 1.12 g/cm3) densities. Following a 30-min pulse, the LI's of light cells (less than 1.12 g/cm3) ranged from 25 to 30%, while the heavier cells (greater than 1.12 g/cm3) had LI's between 10 and 15%. The unseparated control cells had an LI of 19%. comparable results were found at the other times tested. In contrast, the FMF profiles describing the DNA contents of the cells banding in the gradient showed no difference in proportion of S-phase cells among the separated subpopulations. This lack of correlation between the FMF determination of S-phase cells and labelling index for the denser cell populations implies that DNA content alone is not an effective measurement of the functional activity of cells in solid tumours. Finally, the relatively reduced uptake of [3H]TdR by these denser cells suggests that they may have resided at relatively large distances from the functional vasculature in the tumour.