Dose Protraction Research Articles

Effect of X-ray dose protraction and a tumor promoter on transformation induction in vitro.

We have investigated the effects of X-rays given in a brief exposure (1 min or less) or protracted over 5 h, on cell survival and the induction of neoplastic transformation in C3H/10T1/2 cells with an emphasis on latent transformation damage remaining after protracted irradiation. This latent damage and its expression were investigated at accumulated doses of 0.25 to 4 Gy by chronic treatment with TPA (12-O-tetradecanoyl-phorbol-13-acetate or phorbol myristate acetate) at 0.1 microgram/ml beginning after irradiation. Transformation incidence from protracted as well as brief X-irradiations was linearly related to X-ray dose in the presence of 0.1 microgram/ml TPA/ml. In the absence of TPA, the best fits were obtained with cubic rather than quadratic functions. The effect-modifying factors due to dose protraction were similar with or without TPA and averaged 4.6 at low doses (up to 2 Gy). Also within this dose range average transformation enhancement due to TPA was approximately 4. Our results indicate that dose protraction does not change the shape the dose-response curve for transformation, and that the shape change induced by TPA is also independent of dose protraction.

Organs and Cells at Risk after Inhalation of Insoluble Beta-Emitting Radionuclides: Lessons From Experimental Studies

Lifespan studies of Beagle dogs that inhaled beta-emitting radionuclides in a relatively insoluble form have been conducted at the Lovelace Inhalation Toxicology Research Institute. These studies were designed to determine the types and locations of radiation-induced tumors as well as the influence of dose protraction on the incidence of such tumors. Although not all these studies are complete, data on organs and cells at risk are available. Histologic tumor types of the lung indicate that numerous cell types are probably at risk after inhalation of beta-emitting radionuclides. Tumors of blood vessels, connective tissue, and pulmonary epithelium have been seen. The numerous cell types involved in tumors of the pulmonary epithelium make it difficult to designate one cell type at risk. The tumors do originate, however, in the terminal airway and alveolar portions of the lung, not the larger airways. Organs other than lung developed tumors through direct irradiation of the tissues. These are tracheobronchial lymph nodes and heart. Neither of these organs has been considered targets for radionuclide-induced tumors. Results from these studies indicate that attention to dose effect relationships in these non-pulmonary tissues may be warranted.

Radiocarcinogenesis: Results fromin vitro, experiments

Transformation frequencies induced in cell systems by low- and high-LET radiations are compared and analysed as a function of LET and dose protraction. At acute dose rates, the dose-response curve with low-LET radiations shows a complex shape: the transformation frequency increases with the dose by a power of less than 1 in the interval (0.25÷1.5) Gy; a power of about 2 from 1.5 Gy to 4.5 Gy, where it reaches its maximum value and then remains constant. High-LET radiation transformation frequencies increase with dose by a power of 2 from 0.1 to 2 Gy after which they remain constant. RBE values increase with LET up to 140 ke V/μm and then decrease. The transformation frequency is reduced following dose fractionation or dose rate reduction with low-LET radiation, whereas it is enhanced with high-LET radiation, such as 0.85 MeV neutrons. Values of RBE for transformation up to 50 can be evaluated for neutrons. Various models that have been proposed to explain these results are analysed and compared.

Effects of Protraction of the a Dose to the Lungs of Mice by Repeated Inhalation Exposure to Aerosols of 239 PuO 2

To determine the long-term biological effects of protracted alpha irradiation of the lung, 84-day-old C57BL/6J mice were repeatedly exposed by inhalation to aerosols of 239PuO2 every other month for up to six exposures in 10 months to reestablish lung burdens of 20, 90, or 460 Bq. Other mice were exposed only once when either 84 or 460 days of age to achieve desired initial lung burdens of 20, 90, 460, or 2300 Bq. Suitable control groups were maintained. Groups of mice with similar cumulative alpha doses to the lung had 3.4 to 4.4 times greater incidence of pulmonary tumors (adenomas and adenocarcinomas) when the dose to the lung was protracted by the repeated inhalation exposures compared to mice that received a single inhalation exposure. Excess pulmonary tumors per unit dose to the lung were also greater in groups of repeatedly exposed mice compared to those exposed only once. Repeatedly exposed mice also died earlier with pulmonary tumors than did those exposed once. It appears that protraction of an alpha dose to lungs increases the carcinogenic risk of inhaled 239PuO2 in mice.

Repair time for oncogenic transformation in C3H/10T1/2 cells subjected to protracted X-irradiation.

With exponential cultures of C3H/10T1/2 cells, we have investigated the effect of X-ray dose protraction on oncogenic cell transformation in the dose range 0.25-2 Gy. Within a particular experiment a constant exposure time was used. In different experiments exposure time varied between 1 and 5h. Cell transformation was analysed using the linear-quadratic relation, gamma (D) = alpha 1D + alpha 2D2, between transformation frequency per surviving cell and X-ray dose. Based on values of the linear coefficients, we developed an empirical formula for relating slopes of dose induction curves obtained at high or reduced dose rate condition. Our estimate of repair half-time for cell transformation with 95 per cent confidence limits is 2.4 (1.8, 3.0) h.

Cataract analysis and the assessment of radiation risk in space

Radiation cataract, a non-stochastic effect on the lens, is readily amenable to non-invasive analysis. Thus, it provides the means to assess radiation risk in space and for long-term monitoring of those who frequent that environment. The importance of such evaluations are underscored by the uncertainties associated with the assignment of quality factors for the effects of heavy charged particles constituting cosmic and solar radiation. Experimental studies were conducted using albino rats to evaluate the cataractogenic potential of 570 MeV/amu Argon ions administered as both single and protracted doses. The cataract studies and investigations of quantitative cytopathological changes associated with them indicate that as the dose of heavy particles decreases, the relative biological effectiveness, compared to X rays, increases. Fractionating the exposures not only failed to reduce the cataractogenic effect but caused a dose- dependent enhancement in the time of onset of opacification. Cytopathologically, the damage caused by heavy particles, when compared to low-LET radiation was found to be quantitatively dissimilar but qualitatively identical. In addition, damage which might be consistent with microlesions was not evident. The data indicates that as regards the cataractogenic potential of heavy particles at low doses an assignment of a Quality Factor (QF) of at least 40 may be in order.

Dose protraction studies with low- and high-LET radiations on neoplastic cell transformation in vitro

A major objective of our heavy-ion research is to understand the potential carcinogenic effects of cosmic rays and the mechanisms of radiation-induced cell tranformation. During the past several years, we have studied the relative biological effectiveness of heavy ions with various atomic numbers and linear energy transfer on neoplastic cell transformation and the repair of transformation lesions induced by heavy ions in mammalian cells. All of these studies, however, were done with a high dose rate. For risk assessment, it is extremely important to have data on the low-dose-rate effect of heavy ions. Recently, with confluent cultures of the C3 H10 T 1 2 cell line, we have initiated some studies on the low-dose-rate effect of low- and high-LET radiation on cell transformation. For low-LET photons, there was a decrease in cell killing and cell transformation frequency when cells were irradiated with fractionated doses and at low dose rate. Cultured mammalian cells can repair both subtransformation and potential transformation lesions induced by X rays. The kinetics of potential transformation damage repair is a slow one. No sparing effect, however, was found for high-LET radiation. There was an enhancement of cell transformation for low-dose-rate argon (400 MeV/u; 120 keV/μm) and iron particles (600 MeV/u; 200 keV/μm). The molecular mechanisms for the enhancement effect is unknown at present.

Survival and Oncogenic Transformation of C3H/10T 1/2 Cells after Extended X Irradiation

We performed two independent series of experiments aiming at establishing dose-response curves for lethality or oncogenic transformation in vitro following acute and protracted X-ray doses between 0.25 and 2 Gy. In the first series of experiments, we measured the survival of C3H/10T1/2 CL8 fibroblasts and their transformed counterparts (MCA TCL15) as a function of X-ray dose delivered at 0.49 Gy/min. In addition, 1- and 2-Gy doses were split into four fractions separated by 3-h intervals. The accuracy of survival fraction measurements was about 2%. We found that the dose-response curve at 0.49 Gy/min was a linear function of the dose for both cell lines with a negative slope of 0.171 +/- 0.007 Gy-1 or 0.160 +/- 0.003 Gy-1 for 10T1/2 or MCA cells, respectively, indicating a similar initial radiosensitivity of normal and transformed 10T1/2 cells. Dose fractionation resulted in a significant increase of the survival, relative to that measured when X-ray dose was delivered in a single fraction. The enhancement of survival was not, however, significantly different for 10T1/2 and MCA cells, indicating similar cellular repair abilities. In the second series of experiments, we measured oncogenic transformation in vitro (along with the survival) of C3H/10T1/2 cells, using a constant exposure time technique in which the dose rate was proportional to the total dose so that the repair time was equal at all dose levels. The dose-response curves for oncogenic transformation in the low-dose range between 0.25 and 2 Gy were consistent with a linear response with a positive slope 2.50 +/- 0.11 X 10(-4), 1.50 +/- 0.03 X 10(-4), or 0.87 +/- 0.05 X 10(-4) Gy-1, for acute, 1-h, or 3-h protracted exposures, respectively. Hence, relative to the acute irradiation, the 1- or 3-h protraction of the X-ray dose reduced the slopes by 0.60 +/- 0.03 or 0.35 +/- 0.03, respectively. These results indicate that in the dose range between 0.25 to 2 Gy, the dose-response curves for survival or oncogenic transformation were linear and can be modified by the temporal distribution of the X-ray dose.

Repair Processes in Radiation Biology

Three dualities underscore the role of repair in radiobiology: the discrete and random nature of energy deposition; the coupled processes of damage and repair; and the harmful and beneficial properties of radiation in connection with issues of public health and the radiation therapy of cancer, respectively. Starting with the survival curve of HeLa S3 cells, published by T. T. Puck and P. I. Marcus [J. Exp. Med. 103, 653-666 (1956)], the survival response of repair-competent mammalian cells is discussed and is related to clinical experience. This is followed by an examination of DNA repair in surviving cells from which it is inferred that even in repair-deficient cells, very likely large numbers of single- and double-strand DNA breaks are repaired because the progeny of surviving cells are essentially free of such damage. These observations support the idea that even when single-hit dose-effect relationships are involved (e.g., survival of ataxia telangiectasia cells), repair processes may play a role. Lastly, single-hit kinetics connected with the induction of neoplastic transformation are discussed. The data demonstrate that linear dose-response dependencies due to ionizing radiation (i.e., gamma rays and fission-spectrum neutrons) can be modulated by dose protraction, but that neither the direction nor the extent of such modulations can be predicted on theoretical grounds. To be applied more effectively for the benefit of mankind, therefore, it remains necessary to understand fully the biological mechanisms of the action of ionizing radiation.

The dose response relationship obtained at constant irradiation times for the induction of chromosome aberrations in human lymphocytes by cobalt-60 gamma rays.

The induction of unstable chromosome aberrations in human peripheral blood lymphocytes exposed in vitro to protracted doses of cobalt-60 radiation is presented. Four dose response curves have been produced using constant exposure times of 1,3,6, and 12 h. The data fit well to the linear quadratic model and the yield coefficients have been compared with those obtained for acute (less than 10 min) exposure. The quadratic coefficient is dependent on irradiation time and decreases approximately as predicted by Lea and Catcheside's G-function hypothesis. The possibility of a small proportion of much longer lived breaks is discussed. For purposes of biological dosimetry it is sufficient to assume a mean time of 2 h and a single exponential function for the repair of lesions when relating the effects of brief and protracted exposure.

Repair Processes and Radiation Quality in Neoplastic Transformation of Mammalian Cells

Appropriate in vitro mammalian cell systems facilitate the study of cellular mechanisms of radiation oncogenesis. C3H 10T1/2 cells derived from mouse embryo have been used to study the effects of protracted irradiation on cell killing and incidence of neoplastic transformation. Neoplastic transformation in these cells results in the appearance of colonies of densely piled up, disorganized cells that grow on top of the contact inhibited monolayer of nontransformed cells. Protracted exposures of 60Co gamma rays or fission-spectrum neutrons (from the JANUS reactor at the Argonne National Laboratory) were given either at low dose rates or as multifractionated regimens using high dose rates. Dose protraction of gamma rays by multifractionation at a high dose rate (50 rad/min) results in appreciable reduction in cell killing and also significantly reduces the incidence of neoplastic transformation. Irradiation at a low dose rate has the same qualitative effect. In contrast, protracted exposures of fission-spectrum neutrons, given at a low dose rate (0.086 or 0.43 rad/min), result in significant enhancement of the frequency of transformation in the dose region of up to 80 rad. However, the survival of cells is essentially the same as that for the same single dose of neutrons given at a high dose rate. These observations are consistent with net "error-free" repair of transformational damage following protracted exposure of a low LET radiation and possibly a net "error-prone" repair of transformational damage after protracted irradiations with fission-spectrum neutrons.

Dosimetry of genotoxic agents and dose-response relationships of their effects

Dose-response relationships and determination of dose of mutagens and carcinogens are summarized and discussed on the basis of conceptual and kinetic aspects. Different dose definitions may be referred to steps in the chain of events from exposure (or emission) to observed effects. A system is applied to show the influence of various processes on the kinetics of the transfers between consecutive steps. The same system illustrates processes influenced by protraction and fractionation of dose, synergists, comutagens/cocarcinogens, heritable factors, etc. The response at a given dose is expected to depend on the product of consecutive transfer functions. An application of general rules of chemical kinetics shows that when a chemical is introduced at a sufficiently low level, all processes affecting the transfers and therefore the transfer functions themselves become first-order, provided the induction status of enzymes and the cell-division rate remain constant. Under the same conditions, dose-response relationships are expected to be linear, i.e. without “safe” thresholds. However, present knowledge of the kinetics of repair at low levels of DNA damage and of the kinetics of induction of repair functions is not enough complete to be decisive. These considerations and the fact that observed dose-response data in some cases indicate the existence of thresholds but in others appear able to reject the threshold hypothesis lead to the conclusion that, generally, dose-response curves are most probably linear down to dose zero. However, certain mutagens/carcinogens give rise to lesions repaired so effectively that quasi-thresholds appear in certain subpopulations or organs.

Investigations on the mutagenic activity of fractions from diesel exhaust particulate matter in the Salmonella typhimurium test system (Ames test)

The inducibility of heritable mutations in female mammals has been measured in the mouse specific-locus test (SLT). For radiation-induced mutations, a large body of data has been accumulated that includes information about biological and physical factors that influence mutation yields. However, relatively few SLT studies in females have been conducted with chemicals to date.A single estimate of the spontanous mutation rate in oocytes, 6536,207, has been derived as the most appropriate one to subtract from experimental rates. This rate is highly significantly below the spontaneous mutation rate in males.Mutations recovered from females mutagenized at any time after about the 12th day post-conception are induced in non-dividing cells. In adult females, most oocytes are arrested in small follicles; maturation from this stage to ovulation takes several weeks. High-dose-rate radiations are more mutagenic in mature and maturing oocytes than in spermatogonia of the male; on the other hand, no clearly induced mutations have been recovered from irradiated arrested oocytes. Efficient repair processes have been invoked to explain the latter finding as well as the upward-curving dose-effect relation for acute irradiation, and the fact that dose protraction drastically reduces mutation yield from mature and maturing oocytes. The dose-protraction effect is much greater than that found in spermatogonia. Radiation-induced mutation rates in embryonic, fetal, and newborn females are overall lower than those in the mature and maturing oocytes of adults. A dose-protraction effect has also been demonstrated at an early developmental stage when the nuclear morphology of mouse oocytes most resembles that of the human.Of only 5 chemicals so far explored for their effect in oocytes, 2 (ethylnitrosourea, ENU, and triethylenemelamine, TEM), and possibly a third (procarbazine hydrochloride, PRC), are mutagenic — with at least one of these (ENU) mutagenic in arrested as well as maturing oocytes. However, the mutation rate is, in each case, lower than for treated male germ cells. By contrast, ENU-induced mutation yield for the maternal genome of the zygote is an order of magnitude higher than that for the zygote's paternal genome or for spermatogonia. A high proportion of mutants derived from chemical treatment of oocytes (including the oocyte genome in zygotes) are mosaics, probably owing to lesions affecting only 1 strand of the DNA.A characteristic of specific-locus mutations induced in oocytes is that they include a considerably higher percentage of large (multi-locus) lesions (LLs) than do mutations induced in spermatogonia. For each germ-cell type, the frequency of LLs appears lower for the chemicals so far tested than for radiations.

Effects of the single or repeated inhalation exposure of Syrian hamsters to aerosols of 239PuO2.

Male Syrian hamsters were scheduled to be exposed by inhalation approximately every 60 days for 1 year (7 exposures) to aerosols of 239PuO2 beginning at 84 days of age. Other hamsters were exposed once when 84 or 320 days of age. Plutonium-239 deposited in the lungs by the repeated inhalation exposures was cleared from the lungs at a rate similar to that following a single inhalation exposure. The incidence of radiation pneumonitis, bronchiolar epithelial hyperplasia, and alveolar squamous metaplasia were the only lesions that were related to radiation dose. Only two primary lung tumours were found among the hamsters exposed to 239PuO2. No primary lung tumours were found in the control hamsters. It was concluded that the incidence of lung tumours was not increased by the protraction of the alpha radiation dose to the lungs from repeated inhalation exposure.

The Role of Time-factor and Rbe for the Induction of Osteosarcomas by Incorporated Short-lived Bone-seekers

In a large series of experiments, fractionate-d injections of short-lived bone-seekers have been shown in many cases to cause a remarkable increase of the osteosarcoma incidence compared with a single administration of the same total skeletal dose. This effect has been observed with both alpha- and beta-emitters. In addition the latency period was shortened by protracting the dose. The total skeletal doses investigated ranged between 0.9 and 20 Gy for alpha-emitters (224Ra and 227Th) and between 28 and 112 Gy for the beta-emitter (177Lu). In all cases the protracted dose had higher or at least equal effects when compared with a single application. Reference experiments with long-lived alpha- and a-emitting bone-seeking nuclides (226Ra and 90Sr) showed that the incidence of osteosarcomas per Gy was sometimes lower than that observed when the same skeletal dose was applied by protraction of short-lived radionuclides. The dependence of osteosarcoma incidence on dose-time distribution, duration of internal irradiation, and radiation quality is discussed. In this context the possibility that the critical initial dose rate may be related to the initiating event within the multi-stage hypothesis of carcinogenesis is considered.

Repeated Inhalation Exposure of Mice to 144 CeO 2 : II. Biologic Effects

Groups of mice (C57Bl/6J strain) were repeatedly exposed by inhalation to aerosols of ${}^{144}{\rm CeO}{}_{2}$ at 60-day intervals for seven consecutive exposures to reestablish lung burdens of ${}^{144}{\rm Ce}$ of 0.2, 1.0, or 4.5 μCi. Additional groups of mice were exposed only once when 70 days old to achieve desired initial lung burdens of 0.2, 1.0, or 4.5 μCi. Control mice consisted of those exposed once or repeatedly to stable ${\rm CeO}_{2}$ , sham-exposed once or repeatedly, and unexposed mice. Protraction of the absorbed radiation dose by repeated inhalation exposures resulted in a sparing from the life-shortening effects of ${}^{144}{\rm Ce}$ pulmonary irradiation. The protraction of dose had no effect on the total number of lung tumors seen or their time of onset. The total incidence of lung tumors, benign and malignant, was correlated with cumulative dose, not dose rate.

Intrinsic and extrinsic variables affecting sensitivity to radiation carcinogenesis

Mathematical models relating observed yield of cancers vs administered radiation dose have become popular in recent years, especially as means of predicting hazards associated with exposure conditions which are beyond the realm of practical experimentation. While the validity of these predictions remains a controversy, these models, especially the αD + βD 2 model, have more recently been used to infer the mechanism(s) underlying the carcinogenic process. Through the analysis of simple experimental systems, we demonstrate in this report that aD + bD 2 kinetics can result from injury to the cells which eventually develop into the cancer (target cells) or from injury to those cells which affect target cell survival. Further, these kinetics can fail to predict the consequences of dose protraction, largely due to the fact that transformation increases with dose, while survival decreases. The role of these models in helping to develop an understanding of mechanisms should be restricted, therefore, to the formulation of basic hypotheses which are subject to direct testing in the laboratory.

The use of urinary N-acetyl-β-glucosaminidase in human renal toxicology II. Elevation in human excretion after aspirin and sodium salicylate

The use of human urinary N-acetyl-β-glucosaminidase in the detection of drug nephrotoxicity was investigated using aspirin as a toxic probe with which to exert a subtle stress upon the tubular epithelium. Aspirin, which is nephrotoxic only at much higher or protracted doses, caused an increase in the cumulative excretion of N-acetyl-β-glucosaminidase that was dose and dose-rate dependent. Using each subject as his own control, 3.6 g of aspirin caused a 15% average increase in the cumulative excretion at 24–32 hr, and the elevated excretory rate returned to control at 32–40 hr; 4.8 g of aspirin administered over 16 hr caused a 54% increase in cumulative excretion at 16–24 hr, and the excretory rate returned to control by 56 hr. When the first dose rate was doubled to 3.6 g administered over 8 hr, the cumulative enzyme release was 170% above control at 16–24 hr, and at 72 hr the excretory rate was still slightly elevated. Since sodium salicylate caused a similar effect, the initial presence of the acetyl group is not a structural determinant of the toxicity of aspirin to the tubular epithelium. It is concluded that the excretory rate of human urinary N-acetyl-β-glucosaminidase will serve as a sensitive and predictive indicator of drug toxicity to the tubular epithelium and structure-activity modification of such.

Radiation protection of the lens for patients and users.

The problem of radiation-induced cataract development in the lenses of patients and users exposed to diagnostic-quality radiation is reviewed. A cataractogenic dose appears to be about 200 rads from a single exposure and up to 750 rads in protracted doses. Routine use of new protective glasses developed by the authors is recommended for patients. For users, any of the commercially available high lead-content glasses will reduce doses to safe levels. About 70% reduction of dose is afforded by the new photochromic sunglasses.

Late effects after incorporation of the short-lived alpha-emitters 224Ra and 227Th in mice.

Abstract 224Ra (half-life 3.64 days) or 227Th (half-life 18.7 days) were intraperitoneally injected in 3–4 week old NMRI mice. The spontaneous osteosarcoma incidence was about 1% in females and 0% in males. The distribution of 224Ra within the skeleton showed the highest concentration in the long bones, somewhat lower values in the vertebral column, increasing towards the lower part. Single injections of 224Ra with activities between 1 and 100 μCi/kg, corresponding to mean skeletal doses (m.s.d.) of 30–3000 rad resulted in an osteosarcoma incidence of 7% in females at the lowest activity applied and reached a maximum of 22% at higher activities, whereas in males the maximum was 8.5%. Single injections of 227Th with activities between 0.1 and 50 μCi/kg of 227Th (corresponding to m.s.d. of 20–10,000 rad) produced an osteosarcoma incidence of 3% at the lowest activity applied and a maximum incidence of 60% at 5 μCi/kg corresponding to 1000 rad. Dose protraction by fractionated application of 224Ra with individual injections of 0.5 or 1.5 or 4.5 μCi/kg twice weekly and treatment periods of 4, 12, 24 and 36 weeks reaching total doses of 12 μCi/kg (m.s.d. 360 rad), 36 μCi/kg (m.s.d. 1080 rad) and 72 μCi/kg (m.s.d. 2160 rad) resulted in an increased osteosarcoma incidence with protraction time and total dose. In this series the highest osteosarcoma incidence (more than 9(1%′0) was observed after a protraction of 36 μCi/kg (m.s.d. 1080 rad) over 36 weeks. After single injection of 224Ra or 227Th in the higher dose range the head bones represented the main location of the osteosarcoma. In addition an increased occurrence of ossifying fibromas of the jaws was observed in the higher dose groups. Fractionated application of 224Rad did not increase the tumor risk of the head bones. After 50 μCi/kg of 227Th (m.s.d. 10,000 rad) bone necrosis preceded the appearance of bone tumors. A somewhat earlier occurrence of leukemia was seen in certain groups after single injection of 227Th and one group of fractionated application of 224Ra.