David Ewing

Anoxic radiation protection of bacterial spores in suspension.

Several compounds (nine alcohols, sodium formate, and CO2 gas) have been tested for an ability to alter the anoxic radiation sensitivity of Bacillus megaterium spores, irradiated in suspension. Som...

Synergistic damage from H2O2 and OH radicals in irradiated cells.

The anoxic sensitization of bacterial spores by added H2O2 has been studied. Two mechanistic pathways for damage from H2O2 were found; one of these requires the presence of OH radicals. For this kind of damage, the relationship between H2O2 and OH appears to be that they are reactants. O-2 (and/or HO2), the product of such a reaction, is likely the agent which actually causes damage. These results with reagent H2O2 are compared with results of experiments in which H2O2 and OH are present as radiolytic products.

Radiation Sensitization of E. coli B/r by Nitrous Oxide

E. coli B/r have been used to study radiation sensitization by nitrous oxide (N2O). Cells suspended in Sörensens phosphate buffer show a large amount of sensitization by N2O (relative to the response in 100% N2). Cells in McIlvaines phosphate-citric acid buffer, however, show no sensitization by N2O. Sensitization in Sörensens buffer can be prevented by hydroxyl radical (.OH) removal or by catalase. Chemical assays for the amounts of H2O2 formed under various conditions provide the basis for the conclusion that the high concentration of the citrate ion in McIlvaines buffer does not allow the build-up of H2O2. Sensitization by N2O requires that both H2O2 and OH radicals be present.

Do.OH scavenger secondary radicals protect by competing with oxygen for cellular target sites

Recently, using Chinese hamster V79 cells, we found no relationship between the level of protection and the overall rate for .OH removal [Ewing and Walton, Radiat. Res. 126, 187-197 (1991)]. We offered several possible interpretations for this observation, including that the scavengers may actually have multiple ways to protect, ways that would occur in addition to, or instead of, simple .OH removal. With bacterial spores, we had noted that protection occurs only with those .OH scavengers that are able to react and form secondary, reducing radicals (alpha-hydroxy radicals, RCOH), and we suggested that protection might occur if these radicals reduced cellular radical sites in competition with (damaging) reactions of O2. We have now tested that hypothesis with four .OH scavengers (DMSO, ethanol, glycerol, and methanol), and Chinese hamster V79 cells, irradiated while equilibrated with 0.9% O2 and 100% O2; our recent experiments with these scavengers in air provide data for a third O2 concentration. If these scavengers protect in vitro mammalian cells by forming secondary reducing radicals which compete with O2 for damaged cellular sites, we expect that when we reduce the O2 concentration, we will concomitantly reduce the scavenger concentrations needed for protection. If the proposed competition occurs, we expect the scavenger concentrations for 50% maximum effect to occur in the ratio of the three O2 concentrations used approximately 1:20:100. We found no evidence for such a competition as the mechanism of protection for these four .OH scavengers.

Radiation Protection of Escherichia coli B/r by Hydroxyl Radical Scavengers

We have used Escherichia coli B/r to test the proposal that hydroxyl radicals (.OH) are major contributors to lethal damage when bacteria in equilibrium with air or 100% nitrogen are exposed to ionizing radiation. In addition, we have tested the hypothesis that oxygen sensitizes bacterial cells to radiation by reacting at radical sites previously formed by reactions of .OH. Our results with B/r indicate that the involvement of OH radicals in damage may have been overestimated. We believe that simple .OH removal provides B/r with only a relatively small amount of protection in N2 and air. Although some .OH scavengers can have large protective effects in air, evidence supports the tentative conclusion that these effects are not based on simple .OH removal. If this conclusion is correct, then radiation sensitization by oxygen--at least of this bacterial strain--would be unrelated to reactions of .OH.

Radiation sensitization by oxygen of in vitro mammalian cells: is .O-2 involved?

Oxygen is a potent sensitizer of cells exposed to ionizing radiation, and, although the exact chemical mechanisms are not fully understood, some evidence suggests that this sensitization may involve the formation of superoxide anion radicals (.O-2) [F. Lavelle, A. M. Michelson, and L. Dimitrijevic, Biochem. Biophys. Res. Commun. 55, 350-357 (1973); A. Petkau and W. S. Chelack, Int. J. Radiat. Biol. 26, 421-426 (1974); L. W. Oberley, A. L. Lindgren, S. A. Baker, and R. H. Stevens, Radiat. Res. 68, 320-328 (1976)] To test this hypothesis, we compared the sensitivity of Chinese hamster V79 cells irradiated in O2/N2 and O2/N2O gas mixtures with and without the addition of other radical scavenging agents. In these tests, although oxygen was present, be blocked the radiation-induced reactions of O2 which produce .O-2. We found that the total amount of biological damage depends simply on the concentration of O2 that is present; the overall sensitivity is not reduced when .O-2 cannot be formed. Thus radiation sensitization by O2--at least of this cell line--does not require the formation of superoxide anion radicals.

Additivity in the sensitizing effects of nitrous oxide and oxygen

In earlier work, we proposed that nitrous oxide (N2O) and low concentrations of oxygen (10(-6) less than [O2] less than 10(-4) mol dm-3) share a common sensitizing mechanism. We also proposed that the basis for sensitization by N2O is different from that by high concentrations of oxygen ([O2] greater than 10(-4) mol dm-3). We have now tested these proposals with several Escherichia coli strains using mixtures of O2 and N2O. In the strains that are sensitized by N2O, we found that damage from low concentrations of O2 does not add to that from N2O. In contrast, we did find additivity in the sensitizing effects of N2O and high concentrations of O2. In those E. coli strains that are not sensitized by N2O, the effects of any concentration of O2 are the same in either N2 or N2O. These results are qualitatively the same as those from our previous study with E. coli B/r, and they support our proposals concerning similarities and differences in sensitizing mechanisms of N2O and O2.

The Effects of Dimethylsulfoxide (DMSO) on the Radiation Sensitivity of Bacterial Spores

Dimethylsufoxide (DMSO) is a potent sensitizer of irradiated bacterial spores (Bacillus megaterium). It is effective under either anoxic or well-oxygenated conditions; in both cases, DMSO increases the response by a factor of 3 to 4. In water, the oxygen enhancement ratio (OER) with 260-kVp X rays is 1.9, while in pure DMSO, which is not toxic to spores, the OER is reduced to 1.1. Spores exposed to DMSO and then washed and resuspended in water still show most of the DMSO-characteristic sensitization, even when the spores are soaked in water for 24 hr before irradiation. This effect is not attributable to DMSO retention by the spore; instead, we suggest that DMSO causes a long-lasting change in a critical spore component that changes the way the entire cell responds to radiation.

H/sub 2/O/sub 2/: A radiolytic product that can function as a spore radiation sensitizer

Aqueous suspensions of bacterial spores, irradiated in air, show an increased sensitivity when they are subsequently irradiated under anoxic conditions. This sensitization is experimentally attributed to the action of H/sub 2/O/sub 2/, formed during the X-ray exposure in air. However, H/sub 2/O/sub 2/ formed in this way cannot function as an anoxic spore sensitizer if 10/sup -1/ M t-butanol is present. The results indicate that OH radicals must be present for sensitization by H/sub 2/O/sub 2/ to occur.

Sensitization of bacterial spores by p-nitroacetophenone (PNAP) and 0.8% O2. Choice of suspending fluid.

In the bacterial spore system, both oxygen and p-nitroacetophenone (PNAP) function as radiation sensitizers. Previous work had shown that PNAP has a protective effect in 0.8%O/sub 2/. These results, obtained in phosphate buffer (pH 7.0), have now been compared with results from water-suspension experiments. In water, mixtures of PNAP and oxygen show the same response as 0.8%O/sub 2/ alone; i.e., PNAP does not protect. Although the mechanisms are not understood, the previously observed protection by PNAP in 0.8%O/sub 2/ must be designated as buffer dependent. Therefore, spore studies to examine chemical sensitizing processes of PNAP and O/sub 2/ should be in water, not phosphate buffer.