J. M. Nelson

Clastogenic effects of defined numbers of 3.2 MeV alpha particles on individual CHO-K1 cells

Research to determine the effects of defined numbers of alpha particles on individual mammalian cells is helpful in understanding risks associated with exposure to radon. This paper reports the first biological data generated using the single-particle/single-cell irradiation system developed at Pacific Northwest Laboratory. Using this apparatus, CHO-K1 cells were exposed to controlled numbers of 3.2 MeV alpha particles, and biological responses of individual cells to these irradiations were quantified. Chromosomal damage, measured by the induction of micronuclei, was evaluated after no, one, two, three or five particle traversals. Exposures of up to five alpha particles had no influence on the total numbers of cells recovered for scoring. With increased numbers of alpha particles there was a decrease in the ratio of binucleated to mononucleated cells of 3.5%/hit, suggesting that alpha particles induced dose-dependent mitotic delay. A linear hit-response relationship was observed for micronucleus induction: Micronuclei/binucleated cell = 0.013 +/- 0.036 + (0.08 +/- 0.013) x D, where D is the number of particles. When the estimated dose per alpha-particle traversal was related to the frequency of induced micronuclei, the amount of chromosomal damage per unit dose was found to be similar to that resulting from exposures to alpha particles from other types of sources. Approximately 72% of the cells exposed to five alpha particles yield no micronuclei, suggesting the potential for differential sensitivity in the cell population. Additional studies are needed to control biological variables such as stage of the cell cycle and physical parameters to ensure that each cell scored received the same number of nuclear traversals.

Dose-Rate Evidence for Two Kinds of Radiation Damage in Stationary-Phase Mammalian Cells

Survival based on colony formation was measured for starved plateau-phase Chinese hamster ovary (CHO) cells exposed to 250 kVp X rays at dose rates of 0.0031, 0.025, 0.18, 0.31, and 1.00 Gy/min. A large dose-rate effect was demonstrated. Delayed plating experiments and dose response experiments following a conditioning dose, both using a dose rate of 1.00 Gy/min and plating delays of up to 48 hr, were also used to investigate the alternative repair hypotheses. There is clearly a greater change in survival in dose-rate experiments than in the other experiments. Thus we believe that a process which depends on the square of the concentration of initial damage, and which alters the effect of initial damage on cell survival is being observed. We have applied the damage accumulation model to separate the single-event damage from this concentration-dependent form and estimate the repair rate for the latter type to be 70 min for our CHO cells. Use of this analysis on other published dose-rate studies also yields results consistent with this interpretation of the repair mechanisms.

Ferritin-iron increases killing of Chinese hamster ovary cells by X-irradiation

Abstract. Stationary‐phase Chinese hamster ovary cells were cultured in medium containing ferritin (‐19% iron by weight) added at concentrations ranging from 0 to 128 μg/ml. One set of cultures was unirradiated, and another set was exposed to 4.0 Gy of X‐ray. Clonogenic cell survival was assessed in each set of cultures. In the absence of added ferritin, 4.0 Gy killed approximately 50% of the cells. In the absence of radiation, ferritin was not toxic at less than 48 μg/ml; above 48 μg/ml, toxicity increased with concentration. Apoferritin was not toxic at any concentration tested (up to 1000 μg/ml). Although 32 μg/ml ferritin, reflecting only a 3–6 fold increase in iron concentration over normal serum, was not toxic, it reduced the survival of X‐irradiated cells by an additional 75%. These results indicate that a sublethal concentration of ferritin can be a potent radiosensitizer. This suggests the possibility that high body iron stores may increase susceptibility to radiation injury in humans.

Multiple components of split-dose repair in plateau-phase mammalian cells: a new challenge for phenomenological modelers.

Split-dose experiments using starved plateau-phase Chinese hamster ovary cells have been used to investigate the kinetics of repair, expressed in terms of enhancement of reproductive survival. The results show two distinct components of repair, one having a characteristic time of just over 1 h for the removal of a lesion, the other, about 18 h. The rate at which each component removes damage and the fraction of the total damage that each removes appear to be independent of the initial amount of damage produced, i.e., dose. This lack of dose dependence is not consistent with some simple models of ionizing radiation damage and repair, such as those which assume that saturation of a repair process, depletion of enzyme pools, or the interaction of pairs of sublesions is responsible for the curvature in the dose-response relationship. However, the relationship between the amounts of each type of damage and dose appears to be consistent with models that assume that only a portion of the initial damage is directly accessible to the repair systems or that the initial damage consists of a mixture of potentially lethal and sublethal lesions.

Kinetic Differences Between Fed and Starved Chinese Hamster Ovary Cells

When Chinese hamster (CHO‐K1) cells are grown as monolayer cultures, they eventually reach a population‐density plateau after which no net increase in cell numbers occurs. the kinetics of aged cells in nutritionally deprived (starved) or density‐inhibited (fed) late plateau‐phase cultures were studied by four methods: (i) Reproductive integrity and cell viability were monitored daily by clonogenic‐cell assay and erythrosin‐b dye‐exclusion techniques. (ii) Mitotic frequencies of cells from 18 day old cultures were determined during regrowth by analysing time‐lapse video microscope records of dividing cells. (iii) Tritiated‐thymidine ([3H]TdR) auto‐radiography was used to determine the fractions of DNA‐synthesizing cells in cultures entering plateau phase and during regrowth after harvest. (iv) the rate of labelled nucleoside uptake and incorporation into DNA was measured using liquid scintillation or sodium iodide crystal counters after labelling with [3H]TdR or [125]UdR.

Rapid repair of ionizing radiation injury in Chlamydomonas reinhardi.

Repair of sublethal damage is an important factor affecting the survival of irradiated cells. An understanding of this phenomenon is fundamental to understanding the action of ionizing radiations. Although multiple components of such repair have been detected in plateau-phase mammalian cells, processes having different repair rates have never been observed previously in proliferating eucaryotic cell systems. This report describes experiments used to study repair of sublethal damage in cells exposed to fast electrons. Split-dose techniques, utilizing two equal doses given at 100 krad/min and separated by intervals as short as 15 sec, followed by clonogenic cell assay, have been used throughout. These experiments demonstrate the existence of at least two independent repair processes in synchronous, exponentially growing cultures of Chlamydomonas reinhardi. Furthermore, each process probably represents repair of a distinctly different kind of damage. The rapid process described in this report is characterized by a mean repair time on the order of 2 to 4 min in contrast to 20 to 30 min for the well-known Elkind-Sutton type repair. Both processes are nearly temperature independent at temperatures above 25/sup 0/C.

Effects of Torsional Stress on the Interaction of DNA with Radiation and Chemicals

Abstract The purpose of this work is to investigate a mechanism for synergistic interaction between exposures to radiation and chemicals. The mechanism that we propose is based on the torsional stress that DNA sequences in active genes experience as a consequence of gene expression. Our approach involves experiments on an in vitro plasmid DNA system in conjunction with computer modeling to aid in the interpretation of experimental findings. We have observed that the torsional stress from normal physiological levels of negative supercoiling increases the susceptibility of plasmid pIBI 30 to the induction of single-strand breaks by X rays. Calculations of the conformational equilibrium of pIBI 30 at various levels of torsional stress identified a region of the plasmid that was rich in adenine-thymine (A-T) base pairs and particularly sensitive to melting of the double helix. In this article we present experimental evidence that this region of pIBI 30 is hypersensitive to radiation-induced strand scission. S...

Irradiation of Single Cells with Individual High-Let Particles

The dose-limiting normal tissue of concern when irradiating head and neck lesions is often the vascular endothelium within the treatment field. Consequently, the response of capillary endothelial cells exposed to moderate doses of high LET particles is essential for establishing exposure limits for neutron-capture therapy. In an effort to characterize the high-LET radiation biology of cultured endothelial cells, we are attempting to measure cellular response to single particles. The single-particle irradiation apparatus, described below, allows us to expose individual cells to known numbers of high-LET particles and follow these cells for extended periods, in order to assess the impact of individual particles on cell growth kinetics. Preliminary cell irradiation experiments have revealed complications related to the smooth and efficient operation of the equipment; these are being resolved. Therefore, the following paragraphs deal primarily with the manner by which high LET particles deposit energy, the requirements for single-cell irradiation, construction and assembly of such apparatus, and testing of experimental procedures, rather than with the radiation biology of endothelial cells.

Interpreting Survival Observations Using Phenomenological Models

It is generally accepted that the dose-rate dependence of the mammalian-cell survival curve reflects the modification of initial damage by cellular biochemical processes. However, these processes have not been adequately described to allow predictions of the effects at low doses or of radiations having higher stopping powers. The kinetics of damage induction and removal have been studied extensively, even though specific mechanisms have yet to be demonstrated. For example, it can be shown that one component of cell killing depends on the square of the concentration of radiation damage, a dependence that can be explained by sublethal damage models as well as by models featuring misrepair of potentially lethal damage. We have demonstrated two components of radiation-damage repair in plateau-phase Chinese hamster ovary cells. Analysis of repair rates shows that one component has a mean repair time of less than 1 hour, and another has a time of 18 hours. Repair rates, and fractions of damage repaired, appear independent of the initial amounts of damage produced. We have compared our data with the predictions of several different phenomenological models. These observations do not appear compatible with simple models that assume either the saturation of a rapid repair process or the interaction of pairs of sublesions. Rather, they support more-complex models that consider combinations of both sublethal and potentially lethal damage or multiple-step processes. Our observations are also consistent with models that consider the accessibility of damage to the repair enzymes.

Body Iron Stores May Modify Sensitivity to Occupational Radiation Exposure

Abstract Iron is the most abundant transition metal in humans, and it plays a central role in metabolism. However, excess physiological iron may be toxic; it may increase the risk of cancer; and it has now been shown to increase cellular sensitivity to radiation injury. Increased available iron stores may enhance cell killing and genetic alterations through catalysis of free-radical reactions. This may lead to direct DNA damage or depletion of cellular reserves of reducing equivalents, thereby interfering with the maintenance of genomic integrity. Human epidemiological studies are being conducted to determine the effect of excess iron on cancer risk; experimental laboratory studies are probing the increased risk of radiation injury. In prospective studies of a sample of the U.S. population, high serum transferrin saturation was found to correlate directly with cancer risk over a 10-year period. In experimental studies using Chinese hamster ovary cells, ferritin (∼19% iron by weight) added to the growth me...