Curtis P. Sigdestad

Intestinal Crypt Survival and Total and Per Crypt Levels of Proliferative Cellularity Following Irradiation: Fractionated X-Ray Exposures

The split-dose response of jejunal crypt survival has been measured for first exposures of 1000 or 600 R (1400 R total exposure). The curves obtained are analyzed in terms of repair of sublethal da...

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.

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.

Effects of radioprotectors on DNA damage and repair, proteins, and cell-cycle progression

The impetus for investigating the application of phosphorylthioate drugs for use in cancer therapy arose from the early observation by Yuhas and Storer (1969) that these drugs might differentially protect normal as compared to tumor tissue. Recent reports now suggest that these agents may have a more general role with respect to the cancer problem, especially in the area of cancer prevention. Following the initial observation by Milas et al. (1984) that S-2(3-aminopropylamino)ethylphosphorothioic acid (WR-2721, ethiofos) significantly protected rodents against the induction of tumors by irradiation, a series of reports have appeared in the literature that describe the antimutagenic (Grdina et al., 1985a; Nagy et al., 1986; Nagy and Grdina, 1986), antitransformation (Hill et al., 1986), and the anticarcinogenic properties (Grdina et al., 1985b) of WR-2721 and its corresponding free thiol 2-(3-aminopropylamino)ethanethiol (WR-1065). These studies have helped to stimulate a strong interest in characterizing selected molecular and cellular end points affected by radiation such as DNA damage and repair, effects on proteins, and cell-cycle progression.

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.

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., D0s and ns 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.

The effect of s-2-(3-aminopropylamino)ethylphosphorothioic acid (wr-2721) on intestinal crypt survival. I. 4 Mev x-rays.

SIGDESTAD, C. P., CONNOR, A. M., AND SCOTT, R. M. The Effect of S-2- (3-aminopropylamino)ethylphosphorothioic Acid (WR-2721) on Intestinal Crypt survival I. 4 MeV X-Rays. Radiat. Res. 62, 267-275 (1975). The effectiveness of S-2-(3-aminopropylamino)ethylphosphorothiotic acid (WR2721) to protect against 4 MeV X-irradiation was tested on the intestinal epithelium of the mouse. The agent was found to be most effective when injected 15 min prior to irradiation. The protective agent increased the LDso(6) by 816 rads. This resulted in a dose-modification factor of 1.64. WR-2721 did not modify the inherent radiosensitivity of the intestinal crypts, but displaced the curve to the right by 758 rads. The total and per crypt cellularity in protected and unportected mice exposed to X-irradiation is described.

Radioprotectors in treatment therapy to reduce risk in secondary tumor induction.

Radiation and chemotherapy are two effective modalities in the arsenal of cancer therapy. Because of increased awareness by the general public and earlier detection and improved diagnosis, more and more patients are being effectively treated and cured of cancer. Unfortunately, most of these therapeutic agents are, for the most part, extremely mutagenic and/or carcinogenic. When multiple agents such as combined radiation and chemotherapy are used, the carcinogenic potential may rise sharply. For example, patients treated for Hodgkins disease appear to exhibit a relatively high risk for therapy-induced acute myelogenous leukemia (Bartolucci et al . , 1983) and non-Hodgkins lymphoma (Jacquillat et al . , 1984). Thus, the risk of therapy-induced secondary tumors in patients having potentially curable disease is becoming a significant health problem (Rowley et al . , 1981; Penn, 1982; Chak et al . , 1984; Jacobs and Gale, 1984; Dorr and Coltman, 1985). Considerable effort has been devoted to developing treatment protocols that maximize therapeutic gain. Emphasis has been directed toward improving the ability of patients to withstand the acute deleterious effects of these therapeutic agents. The intent has been to attempt to enhance a sparing or differential protective effect to dose-limiting normal tissues. It is this rationale that has prompted considerable study of a class of chemical agents collectively referred to as radioprotectors. Specifically, the compound S-2(3-aminopropylamino)ethylphosphorothioic acid (WR-2721, ethiofos) has been investigated because of its reported ability to differentially protect normal tissues compared with neoplastic tissues (Yuhas et al. , 1980; Phillips, 1980). Evaluation of this compound for use in radiation therapy is currently under way at a number of institutions. Recently, however, investigators have reported that WR-2721 can also significantly protect against the induction of tumors in rodents by ionizing radiation (Milas et al . , 1984). This observation is especially intriguing since it suggests that this class of agents might also be effective fi~r use in reducing the risk of late-effects damage such as the formation of therapy-induced secondary tumors.

The Effect of S-2-(3-Aminopropylamino)ethylphosphorothioic Acid (WR-2721) on Intestinal Crypt Survival: II. Fission Neutrons

The effect of S-2-(3-aminopropylamino)ethylphosphorothioic acid (WR-2721) was tested in the intestine for its ability to protect against fission neutron irradiation. The parameters tested were lethality, intestinal crypt survival, and DNA synthesizing cellularity. The results showed a dose modification factor (DMF) of 1.6 for lethality in mice treated with WR-2721 prior to fission neutron irradiation. This resulted in the shifting of the dose--mortality probit curve to the right by 149 rad. The crypt survival curve in protected mice was found to have a slope which was significantly different from unprotected controls. The DMF calculated at 50 percent crypt survival was found to be 1.3. The dose-dependent DNA synthesizing cellularity in the intestine 3 days after irradiation was tested in protected and unprotected animals. WR-2721 was shown to protect the intestine from about 120 to 140 rad of fission neutrons. This resulted in a DMF between 1.4 and 1.5. (auth)