E.L. Powers

Nitrous Oxide as a Sensitizer of Bacterial Spores to X-rays

SummaryIn N2O-saturated water, spores of Bacillus megaterium exhibit sensitivity to x-rays that is higher than that in N2-saturated water, but lower than that in O2-saturated water. O2-N2O mixtures show O2 sensitivity. Ethyl alcohol present simultaneously at 0·8 Molar prevents the N2O sensitization completely. These results can be interpreted as due to an increase in ·OH radical yield by N2O; and removal of ·OH by ethanol. In dilute solutions of ethanol only partial reversal (or competition) of the N2O effect is observed. This effect is concentration-independent over a wide range of dilute concentrations. An apparent exhaustion of the alcohol is observed at higher doses, producing a two-component survival curve, each component being exponential. The break-points are dose-dependent, with only one being expected on the basis of measured yields in chemical systems; and the slope beyond the break-point is always that of O2-saturated suspensions. The last results can be interpreted as indicating two actions of...

Some Effects of Accelerated Charged Particles on Bacterial Spores

SummaryDry spores of Bacillus megaterium were irradiated with stripped atoms accelerated in the heavy ion linear accelerator. Nine different ions were used, each at 8·3 MeV/nucleon, with specific energy losses (LET) varying from 5 keV/µ (total) for the ionized deuteron up to 500 keV/µ for the Ne10+ ion. Survival curves were constructed for each ion in three experimental conditions allowing distinction among three classes of damage; class I, the completely oxygen-independent damage; class II, that seen only when oxygen is present during irradiation; and class III, that produced by interactions between oxygen and free radicals.Class I increased with increasing LET up to 190 keV/µ while classes II and III are constant in this range. Beyond this, II and III decrease to zero approximately at 500 keV/µ, with I falling slightly. Cross-sectional values correspond with actual geometric dimensions of the spore: a high LET particle is lethal in any part of the protoplast; lower LET particles yield cross-sectional va...

H2O2 and ·OH Radicals in Radiation Inactivation of Bacterial Spores

SummarySuspensions of Bacillus megaterium spores, when irradiated in the presence of N2O and limiting concentrations of ethanol, produce bi-phasic response curves with the high-dose line showing increased sensitivity. Experiments correlating sensitivity to concentrations of H2O2 implicate this chemical species as one sensitizing agent. H2O2 is not sensitizing, however, in the absence of ·OH, suggesting that two actions involving both species must take place for inactivation to occur. A chemical mechanism competent to explain these facts is presented. Sensitization seen in O2-saturated solutions may not be explained by this mechanism.

Responses of Cells to Radiation Sensitizers: Methods of Analysis

Several well-known transforms of the widely-used Michaelis-Menten function linearize the kinetics of many competition reactions such as enzymatic processes. These transforms allow easy and accurate evaluation of the mathematical constants of the system, as well as giving clues to the various mechanisms involved in these competitions. In this paper these linearization techniques are applied to several sets of data from several authors that describe the radiation sensitivity determined by varied concentrations of two sensitizers--O2 and misonidazole. It is shown that, when the increment in sensitivity determined at the various concentrations of sensitizer is used as the dependent variable, straight lines are obtained from various sets of data when either the so-called Lineweaver-Burk or the Eadie-Hofstee transform is used. The E-H transform results in a better distribution of data points and, accordingly, is preferred. The transform allows recognition of two oxygen-dependent processes (one at low [O2] and one at high [O2]) in data apparently demonstrating but one; and, as well, two processes determined at two levels of misonidazole from data that appeared to describe one. These results support the evidence given earlier for two oxygen effects in other cells. Also, the transform reveals that in different cell systems two inhibitors of the oxygen effect appear to act in the same manner on one oxygen effect and in a different way on the other. In discussion the value of the transform in analysing mechanisms of sensitization is examined, and its further potential use in understanding the action of chemical protective agents is pointed out.

2,3-Butanedione, an Electron-stabilizing Compound, as a Modifier of Sensitivity of Bacillus Megaterium Spores to X-rays

SummaryThe organic compound 2,3-butanedione (diacetyl) has a high affinity for free electrons, and can stabilize electrons produced by high-energy radiations in aqueous solutions. In the non-metabolizing spore of Bacillus megaterium (buffer suspension at pH 7·0), this compound increases radiation sensitivity in the absence of oxygen as concentration increases up to approximately 4 × 10−3 M. The maximal increase represents 40 per cent of the total observed in the full oxygen effect. Since the compound at maximally effective concentration in the presence of oxygen does not increase radiation sensitivity above that level seen in oxygen alone, it is allowed that the potentiating action of the compound represents one of the actions of oxygen, i.e. oxygen may act in part by stabilizing electrons. Above about 6 × 10−3 M the effectiveness of the compound as a sensitizer decreases, until the anoxic level of sensitivity is reached at 6 × 10−2 M. No suggestions concerning this reversal of effect at high concentratio...

Radiation Sensitivity of Transforming DNA

Biologically active DNA isolated from Bacillus subtilis was exposed in vitro to X-rays at a concentration of 10 microgram/ml in 29 mM phosphate buffer. Radiation-induced damage to the DNA was quantitatively determined by measuring the decrease in its transforming activity (try2 locus) using B. subtilis 168M (try-) as recipient. In O2, which removes .H and eaq-, the radiation sensitivity of the DNA is less than that in N2-saturated water. In N2O, which has been shown to increase yields of .OH in irardiated aqueous solutions, the radiation sensitivity of Transforming DNA is twice that observed in O2 and 1.5 times that in N2. Addition of 5 X 10(-2) M ethanol or 1.7 X 10(-1) M t-butanol, both .OH scavengers, causes large (about tenfold) reduction in the radiation sensitivity in all three saturating gases. These results suggest the importance of the .OH radical in the loss of biological activity of DNA.

Water-derived Radicals and Radiation Sensitivity of Bacteriophage T7

SummaryThe coliphage T7 was x-irradiated in the presence and absence of ethanol, an effective ·OH and ·H scavenger, in buffered suspensions saturated with N2, N2O and O2. In 10−3 × dilution of the original suspending organic medium, ethanol was protective in all three experimental conditions. In 10−5 × dilution of the original medium (equivalent to contaminant free), ethanol is also protective in all three experiments and to a greater extent; but the relative order of radiation sensitivities changes. It is concluded that, when irradiated in pure suspension, T7 phage is inactivated partly by ·OH, and that ·H and eaq− play small, if any, roles as inactivating species. Additional observations were that the ‘O2-protection’ phenomenon absent in purified suspension is observed in 10−3 M ethanol solutions; and that there is evidence that exhaustion of additives and, perhaps, build-up of products are responsible for the biphasic natures of some response lines. Also, there is evidence that radiation-induced sub-le...

Effects of varying O2 concentration on the X-ray sensitivity of transforming DNA.

The X-ray-induced inactivation of the biological activity of Bacillus subtilis transforming DNA in dilute aqueous solution has been studied over a wide range of O2 concentrations in an attempt to elucidate the mechanisms involved in O2 action. When the DNA is irradiated in the presence of 100 per cent O2 there is a protection of the transforming DNA compared to the sensitivity in N2-saturated or in N2O-saturated solutions. When the equilibrating gas contains intermediate concentrations of O2 (1 per cent--90 per cent) in N2 or N2O, the DNA sensitivity is equivalent to that in pure N2 or N2O respectively. At low O2 concentrations (approximately 0.14 per cent O2 in N2 or in N2O) there is a sensitization of the DNA and this sensitization can be prevented by .OH scavengers. Possible mechanisms for these actions of O2 on the radiation sensitivity of transforming DNA are discussed.

Radiation Sensitivity of DNA-metal Complexes: A Pulse Radiolysis Study

The effect of silver(I) and mercury(II) complexing upon the rate of reaction of both the hydrated electron and the OH radical with DNA has been investigated using the technique of pulse radiolysis. The results presented do not support the hypothesis that the mechanism of radiation sensitivity by these metals is mediated through increased rate of free radical attack upon DNA. Also, there is evidence presented that neither metal, when complexed with DNA, reacts at a significant rate with the free radical species e-aq and OH. The technique of pulse radiolysis was also used to study the complexation of both silver(I) and mercury(II) with DNA (calf-thymus, 40 per cent GC). Scatchard plots, derived from the experimental data, gave association constants of 1.85 x 10(5) dm3 mol-1 and 9.9 x 10(4) dm3 mol-1 and n values (number of complexing sites per nucleotide) of 0.71 and 0.31 for silver(I) and mercury(II) respectively.

Modification of the Radiation Sensitivity of Bacteriophage T7 by O2 and N2O

SummaryThe radiation sensitivity of T7 suspensions is strongly dependent on chemical additives. We have compared the response to x-irradiation in the presence of 100 per cent N2, 100 per cent O2 (eaq− and H· scavenger) and 100 per cent N2O an eaq−(scavenger that increases ·OH concentration), in the presence of a 10−2 dilution of medium and a 10−5 dilution (equivalent to medium free).Survival curves are frequently multicomponent, with sharp break points, and radiation sensitivity must be measured at varying dosage levels. In the 10−3 dilution, the order of radiation sensitivity at low doses is, kL = N2 > N2O > O2, and at high doses, kH = O2 ≅ N2O > N2. In the 10−5 dilution, or medium free, the order at low doses is kL = N2O > N2 ≅ O2 with kH difficult to define.It is concluded: (1) the OH radical is an important damaging species for T7, and (2) ‘O2 protection’ is an experimental artifact associated with impurities in the suspension.