Radiation-induced Reproductive Death Research Articles

Radiosensitivity of normal and polyunsaturated fatty acid supplemented fibroblasts after depletion of glutathione.

Mouse fibroblast LM cells have been modified with respect to their phospholipid composition in all subcellular fractions, including the nuclear membrane. The content of polyunsaturated fatty acids (PUFA) was significantly increased but no difference in cell survival after X-irradiation could be observed between the normal and PUFA enriched cells. It is concluded that the radiosensitive PUFAs in the membranes are well protected against radiation damage. This protection of the PUFA cells could not be caused by vitamin E, because this membrane protector was not present in these fibroblasts. The content of glutathione (GSH) was about the same in the normal and the modified cells. Reduction of the cellular GSH content to less than 5 per cent of that for non-treated cells did not alter cellular survival after radiation of either normal or PUFA enriched cells under oxic or anoxic conditions. The radiosensitive lipids present in the membranes of the PUFA enriched cells proved to be vulnerable to radiation-induced lipid peroxidation when extracted from the cells and reconstituted into liposomes, indicating that the fatty acids per se are peroxidizable. It is concluded that the lipids in the membranes of mammalian cells are not the principal target in radiation-induced reproductive death, and that no generalization is permitted with respect to glutathione, as being the major hydrogen donating species in mammalian cells responsible for the repair of those target molecules responsible for cell survival after radiation.

Studies on the Nuclear Envelope as a Radiation-Sensitive Site in Mammalian Cells: The Absence of an Effect on the Binding of Nuclear Membrane-Associated DNA

Experiments were performed to distinguish between two possible critical sites for radiation-induced reproductive death: (1) DNA-nuclear membrane attachment points and (2) peripheral DNA. Two different procedures varying in the degree of mechanical disruption during and following nuclear lysis were employed. In no case was any quantitative change in the nuclear membrane-associated DNA observed for cells irradiated with 500 to 20,000 rad. Furthermore, when samples of purified nuclear membrane from irradiated cells were analyzed for evidence of radiation-induced covalent cross linking of DNA to membrane protein or lipid, no qualitative effect of irradiation on the interaction was detected. The results suggest that radiation does not disrupt the DNA-membrane association points; therefore, the nuclear peripheral DNA is implicated as a critical target for reproductive death.

Spin-quenching by oxygen of a radiosensitizing nitroxide free radical in aqueous solution.

Stable organic nitroxide free radicals have recently become of considerable interest in radiobiology, since they have been found to increase the sensitivity of anoxic cells to radiation-induced reproductive death without affecting the aerobic sensitivity (Emmerson and Howard-Flanders 1964, 1965, Emmerson 1967 and Brustad 1968). Such characteristics make these compounds of potential interest in radiotherapy. Among the nitroxide free radicals studied to date, 2,2,6,6-tetramethyl-4-piperidone-N-oxyl (often referred to as triacetoneamine-N-oxyl or TAN) appears to combine the most desirable qualities of relatively low toxicity with efficient radiosensitization. Emmerson and Howard-Flanders (1965) and Emmerson (1967) have suggested that TAN exerts its sensitizing effect by reacting with radiationinduced radicals in vital cellular constituents in the same way as does oxygen. Pulse radiolysis experiments have revealed that TAN interacts rapidly with OH-induced DNA transients (Willson and Emmerson 1970). We have recently investigated the nature of this radiation-induced interaction in dilute neutral aqueous solution. Our results show that TAN becomes covalently bonded to OH-induced DNA transients under anoxic conditions, whereas no binding occurs when irradiation is performed in the presence of oxygen. This lack of binding, as well as the absence of a radiosensitizing effect upon oxygenated cells, might most obviously be explained by competition between TAN and 02 for reaction with radiation-induced DNA transients. An alternative explanation, which so far has not been considered, could involve an interaction of oxygen with TAN to render the latter ineffective. However, it has been pointed out that the nitroxide free radicals do not react with oxygen, at least to form peroxides (Emmerson and Howard-Flanders 1964, Hoffmann and Henderson 1961). Nevertheless, since TAN is a free radical, its ability to react with radiation-induced DNA transients may conceivably be related to the unpaired electron localized on the NO group. We therefore decided to conduct an electron spin resonance (ESR) study to determine whether oxygen alters the free radical properties of TAN. TAN was synthesized from 2,2,6,6-tetramethyl-4-piperidone-HCl (Aldrich Chemical Company) according to published procedures (Briere, Lemarie and Rassat 1965), with some minor modifications. Our X-band ESR spectrometer with transmission H102 mode cavity has been described previously (Henriksen 1962). All E.S.R. measurements described here were carried out at 77°K. All

A comparison of radiation-induced reproductive death and chromosome exchanges in Chinese hamster cells at room temperature and in liquid nitrogen.

SummaryReproductive cell death and chromosome exchanges produced in primary Chinese hamster cells after exposure to 250 kV x-rays were studied with irradiation performed at room temperature and in liquid nitrogen. In the cell population irradiated are room temperature, the mean lethal dose was 179 rads, and there were 0·38 chromosome exchanges per cell. With irradiation in liquid nitrogen, the mean lethal dose was 977 rads, and there were 0·19 exchanges per cell. Thus, irradiation at the low temperature resulted in one-half as many chromosome exchanges per cell at the mean lethal dose. This suggests that chromosome exchanges are not a direct cause of radiation-induced cell reproductive death. At low temperatures, survival curves were exponential and the dose power for the production of chromosome exchanges per cell was less than one. The significance of these findings is discussed.

Study of Mechanisms of Radiation-Induced Reproductive Death of Mammalian Cells in Culture: Estimation of Stage at Cell Death and Biological Description of Processes Leading to Cell Death

Lethally irradiated cells undergo one of two types of death, the so-called death or interphase death (1, 2). Since L5178Y cells exposed to moderately large doses of ionizing radiation have been shown to undergo reproductive death (3) (as do most cultured mammalian cells), this cell line appears to be suitable for the studies of the mechanisms leading to this type of death. As the first step in such a study, we believed that there was a great need for a better and more complete description of the process of reproductive death than those currently available (4-9). As early as 1937, Tansley, Spear, and Gliicksmann observed that, in irradiated rat retina, the peak of cell degeneration was always preceded by the peak of mitosis. This finding was extended to other cell systems, mainly by Spear and his colleagues (4-7). Lea interpreted this as suggesting that the degenerative process leading to cell death begins at mitotic division (1). In spite of the importance of such a statement, no serious effort has been made to elucidate this phenomenon. With the new concept of life cycle of cells introduced by Howard and Pelc (10) and Lajtha et al. (11), it has become obvious that the process of reproductive death of irradiated cells can be studied in terms of biological events occurring throughout the life cycle. In the present paper, we have placed special emphasis on the question: At what stage of its life cycle does an irradiated cell die? The answer to this basic question would seem essential for the understanding of the exact biological mechanisms leading to reproductive cell death.

A Comparison of Mammalian Cell-killing by Incorporated3H-thymidine and3H-uridine

SummaryThe fact that the β-radiation from 3H atoms incorporated into cellular DNA is absorbed largely in the nucleus, while more of that from 3H incorporated into RNA is absorbed in the cytoplasm than in the nucleus, provides a means of determining the relative importance of nuclear and cytoplasmic damage in radiation-induced mammalian cell-death. Synchronously-dividing populations of Chinese hamster fibroblasts were pulse-labelled with variosu doses of either 3H-thymidine or 3H-uridine. The labelled cells were plated, and survival-curves obtained. Autoradiographic silver grain-counts and liquid scintillation counting were used to measure the nuclear β-radiation doses. It was found that the slope of the 3H-uridine survival-curve was somewhat greater than that of the 3H-thymidine curve. Since the β-radiation dose to the cytoplasm was larger than that to the nucleus, however, it is concluded that the contribution of cytoplasmic damage to radiation-induced reproductive death of mammalian cells is very small.