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., <tex-math>$D_{0}\text{'}{\rm s}$</tex-math> 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 <tex-math>$109.3\times 10^{-6}/{\rm Gy}$</tex-math>. In comparison with 60 Co γ rays (mutation frequency of <tex-math>$8.7\times 10^{-6}/{\rm Gy}$</tex-math>), 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 <tex-math>$68.6\times 10^{-6}/{\rm Gy}$</tex-math> for WR1065 present during, present immediately after, or added 3 h after irradiation, respectively; and 61.7, 47.8, and <tex-math>$68.5\times 10^{-6}/{\rm Gy}$</tex-math> for WR151326 present at the same times.

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°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.

Two thiophosphoroate radiation protectors (WR-2721 and WR-151327) were assessed for their ability to modify the effects of neutron or γ irradiation on the gastrointestinal tract. Three neutron sources (DOSAR, JANUS, and FERMILAB) were compared to the response obtained after 60 Co irradiation. The end points studied were intestinal stem cell survival and <tex-math>${\rm LD}_{50(6)}$</tex-math>. DOSAR and JANUS, both fission-spectrum neutrons, showed somewhat different gut sensitivities <tex-math>$[{\rm LD}_{50(6)}]$</tex-math> of about 240 and 400 cGy respectively. The intestinal LD 50 obtained with FERMILAB neutrons (25 meV) was closer (875 cGy) to that obtained after 60 Co (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 60 Co (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 (<tex-math>${}^{60}{\rm Co}\ \text{and}\ {\rm FERMILAB}\ \text{neutrons}$</tex-math>) protection. Possible explanations for the differing results are discussed.

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% 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 120-140 rad of fission neutrons. This resulted in a DMF between 1.4 and 1.5.

The effectiveness of S-2-(3-aminopropylamino)ethylphosphorothiotic acid (WR-2721) 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 ${\rm LD}_{50(6)}$ 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.

Intestinal crypt survival was studied in mice irradiated in air, 100% normobaric oxygen, 100% hyperbaric oxygen (30 psi), and 6% normobaric oxygen. Exposures from 900-2000 R at the rate of 47 R/min. Intestinal crypt survival was determined by the method of Hagemann, Sigdestad, and Lesher (4). The results demonstrated no difference in crypt survival for mice irradiated in air, normobaric oxygen, or hyperbaric oxygen. The crypt survival of mice was shown to have a D 0 from 377 to 380 R and a n from 7.9 to 9.7 for mice irradiated in air, 100% oxygen, or 100% oxygen at 30 psi, while mice irradiated in 6% oxygen had a <tex-math>$D_{0}=529$</tex-math> R with a n=8.1. The DMF (ratio of D 0 in 6% oxygen to D 0 in oxygen) was found to equal 1.4. The lack of difference in air and oxygen suggests the highly vascularized (oxygenated) gut is maximally sensitive. Animal lethality (<tex-math>${\rm LD}_{50(5)}$</tex-math>) is compared with crypt survival.

Intestinal crypt survival was studied using the method of Hagemann, Sigdestad and Lesher (8) in mice irradiated with 250 kVcp x-rays, cobalt-60 and fission neutrons. The D 0 's for the three treatment modalities were 377, 375 and 103 rads with extrapolation numbers of 6.2, 9.2 and 3.1, respectively. The RBE (ratio of D 0 's relative to x-rays) was 3.6 and 1.0 for neutrons and cobalt-60, respectively. The <tex-math>${\rm LD}_{50(5)}$</tex-math> values were found to be 1081 (945-1247), 1365 (1279-1456) and 252 (234-273) rads. The RBE [ratio of <tex-math>${\rm LD}_{50(5)}$</tex-math> relative to x-rays] values were found to be 4.3 and 0.8 for neutrons and cobalt-60 irradiation. Intestinal crypt survival varied from 25 to 34% of control at all of the <tex-math>${\rm LD}_{50(5)}$</tex-math> values. Total and per crypt cellularity was determined at 3 days postirradiation for each modality. The maximum proliferative response, measured at this time, occurred at or near the <tex-math>${\rm LD}_{50(5)}$</tex-math> dose for each type of radiation.

Mice were given whole-body exposures of 500 R, or abdomen-only exposures of 900 R. One or 4 days later, when crypt cellularity was either relatively low or high, respectively, a graded series of exposures was given, and crypt survival and total and per crypt levels of proliferative cellularity measured 3 days thereafter. Crypt survival was found to be markedly affected by crypt cellularity existing at the time of irradiation. Advantage may be taken of the compensatory proliferative intestinal response following a given radiation exposure to ameliorate somewhat, the efficacy of a subsequent exposure for crypt killing. A precise analysis of the relationship between proliferative and crypt progenitor compartment sizes was thwarted however, by an apparent increase in D 0 of cells surviving the conditioning exposure. The difference in D 0 's was marginally significant when determined 3 days after 500 R, and not significant 1 or 4 days after 900 R. At high radiation exposures, the proliferation rate of cells in surviving crypts was markedly reduced, in spite of a continued stimulus for accelerated proliferation. This suggests that a condition relating to growth, possibly analogous to small colony formation seen in vitro, exists in the heavily irradiated mammalian intestine.

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 damage, progression through the cell cycle, and alterations in multiplicity commencing after the G 2 block. The maximum survival ratio for that portion of the curves which probably represents repair of sublethal damage is 3-4, which is considerably less than anticipated from previous studies on single-dose crypt survival. Total and per crypt levels of proliferative cellularity have been measured 3 days following the second exposure. These curves represent the net result of perturbing a complex steady-state in vivo system and are but partially conducive to temperate analysis at this time.

A radiation survival curve for intestinal crypts has been obtained. It is characterized by a D 0 of 330 R and n of 10. Crypt survival is quantitatively similar for assays performed at 3 and 4 days following irradiation, although crypt size varies considerably during this interval. An as yet unexplained discrepancy exists between the crypt survival curve obtained experimentally and that predicted from crypt cell survival parameters and compartment size. Proliferative crypt cells appear to constitute the intestinal stem cell compartment. Recovery in terms of cellularity occurs even following exposures as high as 1400 R. It seems critical for 5-day survival of the animal that proliferative cellularity reaches some requisite level by day 3 following exposure; later increases in cellularity are not reflected by increased 5-day survival. It appears from this that crypt survival is an important feature with respect to 5-day survival of the irradiated animal.