Split-dose Irradiation Research Articles

Use of High Dose Rate Electron Beams to Study Cellular Repair Mechanisms

Repair of sublethal damage produced by ionizing radiation is an important factor influencing cellular survival. This is particularly so when low dose rates, as encountered in environmental exposures, are involved. Since repair processes may have characteristic times ranging from nanoseconds to hours, conventional radiation sources are not capable of giving the range of dose rates needed for a systematic investigation. A 2 MV electrostatic accelerator has been modified to generate an electron beam to satisfy this requirement. This accelerator and associated electronic controls have been used to demonstrate that the repair process responsible for the altered survival observed after split-dose irradiations is the same as that responsible for the shoulder of the survival curve for Chlamydomonas reinhardi. Furthermore, it has made possible the identification and measurement of the rates of two repair processes which function concurrently in these cells.

A Study of the Repair of Potentially Lethal and Sublethal Radiation Damage in Chinese Hamster Cells Exposed to Extremely Hypo- or Hypertonic NaCl Solutions

The effect of 0.05 and 1.5 M NaCl solutions on the repair of potentially lethal damage (PLD) and sublethal damage (SLD) in irradiated Chinese hamster ovary (CHO) cells has been examined. A 20-min exposure to the 0.05 or 1.5 M salt solutions which was terminated immediately before irradiation had no effect on the repair of PLD. However, salt treatment during or after irradiation caused an inhibition of repair of PLD. The possibility that 0.05 or 1.5 M treatment irreversibly affected repair enzymes could be ruled out. Exposure of cells to 0.05 or 1.5 M NaCl solutions before or during the first x-ray dose of a split-dose regimen decreased the ability of the cell to repair SLD. The recovery observed in the experiments was characterized by a change in D/sub 0/ of the survival curves and was related to the loss of interaction of radiation-induced lesions and salt effects as the incubation time between the second x-ray dose and salt treatment increased. However, a partial return of the shoulder of the survival curve when salt treatment preceded split-dose irradiation indicated that some repair of SLD does occur. In summary, salt treatment prior to irradiation does not inhibit repair of either SLDmore » or PLD, whereas, salt treatment during or after irradiation inhibits repair of both SLD and PLD.« less

Repair of Sublethal Damage in Mammalian Cells Irradiated at Ultrahigh Dose Rates

The lethal response of asynchronous Chinese hamster ovary (CHO) cells exposed to single and split doses of radiation at conventional or ultrahigh dose rates has been examined to determine whether repair of sublethal damage occurs in cells irradiated at ultrahigh dose rates. The high-intensity irradiations were performed with electrons delivered in single 3-nsec pulses from a 600-kV field emission source under medium-removed, thin-layer conditions. Conventional dose-rate experiments were done under identical thin-layer conditions with 50-kVp x rays, or under full-medium conditions with 280-kVp x rays. Oxygenated cells were irradiated and maintained at 22 to 24/sup 0/C between exposures. Survival did not increase as the time between two doses of pulsed electrons increased from 0 to 4 min, indicating no evidence of fast repair. However, increased survival was observed when 30 to 90 min was allowed to elapse between the split doses. The half-time for maximum repair was approx. = 30 min irrespective of the exposure conditions and radiation modality used. Observed repair ratios increased from approx. = 2 to 4 as the single-dose surviving fraction decreased from 10/sup -2/ to 5 x 10/sup -4/. Over this survival range the repair ratios, measured at the same value of surviving fraction, weremore » independent of dose rate. The observed repair ratios imply that the shoulder regions of the nonfractionated x-ray and pulsed-electron survival curves were not completely restored between the split doses. However, the fraction of the shoulder restored between split doses of radiation was dose-rate-independent. It is concluded that sublethal damage can be repaired in oxygenated CHO cells irradiated at dose rates of the order of 10/sup 11/ rad/sec.« less

Lens Opacification in Mice Exposed to 14-MeV Neutrons

The induction of lens opacification in mice exposed to 14-MeV neutrons was evaluated by counting the number of opacities observed in the posterior region of the lens using a slit-lamp microscope. The incidence of cataract formation in the anterior region of the lens was also observed. Serial observations were carried out from 3 up to 25 months after irradiation. Male hybrid mice (C54B1 X C3H), 75 days old at the time of the exposure, have been used. A linear progression of opacities was observed with age at a rate depending on the dose. By comparison with 250-kVp X rays the RBE was found to increase, with decreasing dose, from 9 to 21. The incidence of opacification in the anterior region of the lens gives an RBE value of 8. Split-dose irradiations were carried out with an interval of 24 hr between doses. There was very little difference in the number of opacities observed after single or fractionated neutron doses of approximately the same total amount. In contrast, fractionation of the X-ray dose reduced the effectiveness of the irradiation.

Survival of Spermatogonial Stem Cells in the Mouse after Split-Dose Irradiation with Fission Neutrons of 1-MeV Mean Energy or 300-kV X Rays

Radioresistant, colony forming stem cells in the mouse testis move into a much more radiosensitive phase of their cell cycle shortly after irradiation. This is demonstrated in experiments in which total doses of 300 rad of neutrons and 1200 rad of x rays were split into two equal fractions. With neutron fractionation a rapid decrease of stem cell survival as a function of the interval between dose fractions was observed, and survival was reduced to less than 3 percent of the single-dose value for intervals ranging from 16 to 40 hr. At that time the radiosensitivity of the survivors of the first dose is greatly increased. The D/sub 0/ value of the neutron dose--response curve 24 hr after a first dose of 150 rad is 25 +- 1 rad, much less than the D/sub 0/ value for single exposure which is equal to 77 +- 3 rad. Between 44 and 72 hr after the first dose, increased survival levels were found, on the average amounting to 15 percent of the single dose level. The increased survival is caused by an entrance of first-dose survivors into a cell cycle phase with a D/sub 0/ value higher than 25 rad of neutrons.more » With x-ray fractionation a similar decrease of stem cell survival in the 16 to 40-hr period was observed, although the decrease is not as large as with neutron fractionation. For the 4-hr interval, however, an increase in cell survival was found, amounting to 160 percent of the single-dose value. This increase can be quantitatively explained by the repair of induced sublethal damage. Evidence was obtained that the radiosensitivity of stem cells is not influenced by nembutal anaesthesia. Furthermore, no apparent diurnal rhythm in radiosensitivity was observed. The results indicate that the irradiation triggers a shorter cell cycle for the surviving stem cells.« less

Change in Radiosensitivity of Sea-urchin Eggs during Early Cleavage Stages

When sea-urchin eggs were irradiated with 137Cs gamma-rays, their radiosensitivity, expressed by the percentage which formed pluteus larvae, fluctuated during the early cleavage cycle. Split-dose irradiations were made both in the sensitive and resistant phases. For eggs in the sensitive phase, the effect of the first exposure of 500 rad was not diminished during the interval before the second exposure. Eggs irradiated in the resistant phase were only slightly damaged. Results imply that fluctuations in radiosensitivity of sea-urchin eggs are caused mainly by different degrees of non-repairable damage in each phase of cleavage rather than by different recovery abilities.

Protection of normal tissues by WR2721 during fractionated irradiation

The radioprotective effect of S-2-(3-aminopropylamino)ethyl phosphorothioate (WR2721) was studied in two normal mouse tissues-skin and small intestine—that were given single and fractionated doses of 137Cs radiation. A total of two-thirds of the toxic LD 50 of WR2721 could be given before single-dose irradiation. However, toxicity increased when the same drug dose was given in two or more fractions before split-dose irradiation: less than one-half of the single drug dose could he administered safely. In the skin, WR2721 was slightly less protective with fractionated irradiation (DMF = 1.3–1.5) than with single-dose irradiation (DMF = 1.5–1.7). The DMF decreased with increased radiation dose in both cases. In the small intestine, WR2721 protection was essentially the same with single-dose irradiation (DMF = 1.6) and fractionated doses (DMF = 1.7). This degree of radioprotection in skin and intestine, in spite of a greatly reduced drug concentration, supports the possibility of clinical use.

Pluripotential stem cell differentiation in hemopoietic colonies

To determine if mononuclear cells proliferating in murine hemopoietic spleen colonies were pluripotential in addition to possessing kinetic features of stem cells, we performed sequential studies of mice during their recovery from a split-dose irradiation regimen of 850 roentgens leg shielded-3-hr interval-850 roentgens leg irradiated (850R L.S. 3- L.I.). Injecting tritiated thymidine during stem cell compartment repletion 3 and 4 days after 850R L.S. 3- L.I. resulted in heavily labeled mononuclear cells resembling medium to large leptochromatic lymphocytes in the portion of spleen removed an hour after injection. The splenic remnant obtained from the same mouse 24–48 hr later contained lightly labeled erythroblasts, myeloid cells, and lymphoid cells. Grain counts suggested that erythroblasts and their precursors had undergone about four divisions, myeloid cells and their precursors two to three divisions, and lymphoid cells and their precursors two to three divisions during the 48-hr period. Similar studies in plethoric mice demonstrated the labeling of mononuclear cells on day 4 and their differentiation to myeloid and lymphoid cells by day 6. This finding confirmed that the labeled mononuclear cells were not exclusively erythroblast progenitors. On the basis of these and previous studies of post-irradiation survival and erythropoietic recovery, we conclude that these endogenous monomuclear cells, which resemble medium to large leptochromatic lymphocytes and replicate during stem cell compartment repletion, are pluripotential hemopoietic stem cells.

Pluripotential Stem Cell Differentiation in Hemopoietic Colonies

AbstractTo determine if mononuclear cells proliferating in murine hemopoietic spleen colonies were pluripotential in addition to possessing kinetic features of stem cells, we performed sequential studies of mice during their recovery from a split-dose irradiation regimen of 850 roentgens leg shielded-3-hr interval-850 roentgens leg irradiated (850R LS. 3 LI.)- Injecting tritiated thymidine during stem cell compartment repletion 3 and 4 days after 850R LS. 3 L.I. resulted in heavily labeled mononuclear cells resembling medium to large leptochromatic lymphocytes in the portion of spleen removed an hour after injection. The splenic remnant obtained from the same mouse 24-48 hr later contained lightly labeled erythroblasts, myeloid cells, and lymphoid cells. Grain counts suggested that erythroblasts and their precursors had undergone about four divisions, myeloid cells and their precursors two to three divisions, and lymphoid cells and their precursors two to three divisions during the 48-hr period. Similar studies in plethoric mice demonstrated the labeling of mononuclear cells on day 4 and their differentiation to myeloid and lymphoid cells by day 6. This finding confirmed that the labeled mononuclear cells were not exclusively erythroblast progenitors. On the basis of these and previous studies of post-irradiation survival and erythropoietic recovery, we conclude that these endogenous mononuclear cells, which resemble medium to large leptochromatic lymphocytes and replicate during stem cell compartment repletion, are pluripotential hemopoietic stem cells.

Recovery in Burkitt lymphoma cells after X-ray irradiation.

The radiation response of cultured Burkitt lymphoma cell, known as a particular cell line of high radiosensitivity, was studied by the colony formation method. The values of n, Dq, and Do derived from the survival curve after a single dose X-irradiation resulted in small values as 1.1, 10 rad, and 125 rad. The maximum recovery of about 5% was observed 3 hr after the irradiation by a split-dose experiment. In the survival curve derived from a split-dose irradiation, n and Dq increased while Do remained unchanged. The reason for these increases in n and Dq may be attributed to the nonhomogeneous or mixed population response.

Effects of x-radiation on growth and function of the repair blastema (granulation tissue). II. Measurements of angiogenesis in the Selye pouch in the rat.

SummaryThe Selye pouch technique has been used to stimulate growth of the repair blastema and angiogenesis and measure radiosensitivity of capillary blood vessels in the subcutis. Measurements of radiation damage to the tissue were based on (i) weight per unit area and corresponding DNA content of the repair blastema, (ii) the volume of blood produced in the pouch by angiogenesis and (iii) vascular macrocolony counts. Colony counts gave a D0 value of ∼240 rads for single-dose and ∼180 rads recovery for split-dose irradiation. No significant difference was observed for single doses of irradiation given shortly before or after raising the pouches. Increasing the interval between irradiation of intact skin and raising of the pouch, was associated with rapid and marked repair of radiation damage; a delay of 2–3 weeks resulted in a dose-reduction factor of 500–625 rads for single doses of 1500–1800 rads given to intact skin. Local irradiation of an established pouch with a single large dose (2–4 krads) rapidly...

Absence of a fractionation effect in irradiated HeLa cells.

SummaryAerated and hypoxic HeLa cells have been exposed to single and split doses of radiation using pulses of fast electrons and conventional X-radiation. With aerated cells the single dose survival curves exhibited a shoulder with both types of radiation, before becoming exponential, but no fractionation effect was observed in the experiments with fast electrons, although one was found with X-rays. For cells under conditions of extreme hypoxia the single-dose survival curves were exponential both with electron pulses and with X-rays, and split-dose experiments showed no fractionation effect.

Differential split-dose radiation response of resting and regenerating haemopoietic stem cells.

SummaryHaemopoietic stem cells are less sensitive to killing by a dose of radiation, if it is delivered in two fractions separated in time. The magnitude of this decrease in sensitivity is greater when the cells are irradiated in the normal steady state, compared with a regenerating state. However, the effect was reversed when donor mice received two radiation doses, separated in time by 24 hours, and this was due to effects (on differentiation and on proliferation) occuring between the first and second doses. These observations are discussed in relation to the therapeutic practice of daily dose treatments.

The Effect of Postirradiation Bleeding or Endotoxin on Proliferation and Differentiation of Hematopoietic Stem Cells

Abstract Bleeding after irradiation failed to affect the time of onset or rate of regeneration of the hematopoietic stem cell compartment as measured by a split-dose irradiation method. Bleeding after a single 600 rad exposure hastened the time of onset of erythropoiesis as measured by spleen colonies and 59Fe uptake, but the early rise was abortive and the second increase began at the same time as the first increase seen in irradiated controls. The largest abortive increase was seen when bleeding occurred within 4 hr of irradiation. Lesser effects were seen when bleeding occurred 12 or 24 hr after and no effect 48 hr after irradiation. A generation time of 6.4 hr for a colony-forming cell that forms a colony in 4 days was calculated from the estimated number of cells in one such colony. Injection of cytosine arabinoside at the time of bleeding reduced colony numbers and 59Fe uptake equally in bled and control mice. Injection of 25 µg Salmonella typhosa endotoxin just after irradiation produced a similar early abortive rise in microscopic granulocytic colonies. These results are compatible with a model for pluripotential stem cell compartment in which: (1) differentiation and self-replication occur concomitantly unless the compartment is reduced below approximately 1O% of normal size; then self-replication occurs without appreciable differentiation; (2) for a short time (∼ 24 hr) after depletion below the threshold, differentiation can occur in response to strong stimuli.

Recovery and reoxygenation of murine squamous-cell cancer by split-dose irradiation.

A study of radiation cure of murine squamous-cell cancer with presumed normal oxygenation shows that less dose is required when the dose is delivered in two fractions rather than one, probably because the sparing effect of fractionation is masked by a shift of cancer cells to a more oxygenated state between treatment fractions.

Recruitment and altered recovery in noncycling bone marrow stem cells.

The recruitment of noncycling bone marrow stem cells into mitosis over a seventy-two hour period was observed in mice populations injected with vinblastine. One per cent of the total bone marrow stem cell population does not enter mitosis over this time period. Single and split radiation dose studies on noncycling bone marrow stem cells demonstrate single-hit kinetics with reduced or absent capacity for sublethal injury repair. These observations are of value in planning treatment regimens involving cycle-active chemotherapeutic agents.

PECULIAR IMMUNOBIOLOGY OF BONE MARROW ALLOGRAFTS

F(1) hybrid mice are capable of rejecting inbred parental strain bone marrow grafts after a single lethal exposure to X-rays. The incompatibility is genetically controlled by the Hybrid-histocompatibility-1 (Hh-1) locus in or near the D end of the Histocompatibility-2 (H-2) region. The onset of parental graft rejection begins 9-12 hr after transplantation and is completed by 24 hr. Maturation of hybrid resistance does not occur until the 22nd day of life. In adults, the resistance to parental marrow grafts can be temporarily abrogated or weakened by administration of cyclophosphamide or dead cultures of Corynebacterium parvum, acute supralethal exposures to radiation, or by split-dose irradiation with 6-37-day intervals. Parental marrow grafts elicit a transplantation reaction in irradiated F(1) mice which is indistinguishable from that elicited in irradiated allogeneic (H-2-incompatible) hosts. Because of this immunogenetic similarity, the following question is raised: are the same or different alloantigens responsible for rejection of parental and allogeneic marrow grafts? In the first case, Hh-1 alleles would be recessive determinants of tissue-specific transplantation antigens, whereas in the second case they would be the determinants of parental- and tissue-specific antigens subject to genetic suppression in Hh-1 heterozygotes. Although the available evidence is not conclusive in excluding one of the two possibilities, it favors the concept that allograft reactivity to hemopoietic cells is elicited by recessive tissue-specific antigens.

Patterns of Proliferation and Differentiation of Hematopoietic Stem Cells After Compartment Depletion

Abstract The time of onset and rate of self-replication in the irradiation depleted pluripotential hematopoietic stem cell compartment of mice was measured by a split-dose irradiation method. Mice were irradiated to reduce the compartment size and at daily intervals thereafter were again irradiated; 10 days after the second irradiation the number of endogenous spleen colonies was determined. As irradiation interval was lengthened, colonies increased in an exponential fashion with no apparent lag interval after the first irradiation. The doubling time of the colony-forming cell compartment was calculated as 16 hr if severely reduced in size by 300 R or more but was slower if less irradiation was given. The time of resumption of erythropoiesis following irradiation was measured after 100-900 R by determining uptake of radioactive iron into marrow, spleen, and circulating red cells each day after irradiation. With 200 R or less, erythropoiesis was not abolished. With higher doses there was an interval of no apparent erythropoiesis which increased by 1.6 days for each increment of 100 R. These results are compatible with a pluripotential stem cell compartment, which (1) begins self replication almost immediately following compartment size reduction, and (2) will not differentiate if reduced in size below approximately 10% of normal.

Split-dose irradiation of Escherichia coli in the absence and presence of mercaptoethylamine.

SUMMARY A single dose of γ-radiation to Escherichia coli b gave a semi-logarithmic dose-response curve. A second irradiation 3, 6, or 18 hr after a priming dose of 8 krad showed that the cells had become more radio-sensitive during the incubation between irradiations. When mercaptoethylamine was present during irradiation, protection against the lethal effects of the γ-radiation occurred. After a priming dose to the bacteria in the presence of the radio-protective chemical, and incubations for 3,6, or 18 hr in its absence, a second irradiation in the absence of the chemical showed them to be much more radio-resistant than the normal unprotected Escherichia coli b. This indicates that the presence of the protector during irradiation alters the type of damage induced or the development and repair of that damage.

Recovery and Repopulation in Mouse Skin as a Function of Time after X-Irradiation

Gross skin reactions of mouse feet have been studied after one or two doses of x-rays. For split-dose irradiations a first dose of 1000 rads was used. The total dose for various fractionation intervals to produce a chosen level of skin reaction (integrated over 8 to 30 days) was determined. The dose increment ( $D_{2}-D_{1}$ ) necessary to produce the same effect with a split dose (D2) as with a single dose (D1) was determined for the various intervals. The value of $D_{2}-D_{1}$ is interpreted as being due to (1) prompt intracellular recovery from sublethal injury; (2) progression through phases of different sensitivity in the cell cycle of cells which had been partially synchronized by the first dose; and (3) an increase in basal cell number by repopulation. The dose increment in the first 24 hours was found to be 500 to 550 rads, and a further increment of 580 rads was required from 1 to 21 days. These values are in close agreement with the results of clone-count...