OXYGEN AND NITRIC OXIDE AS MODIFIERS OF RADIATION INJURY IN SPORES OF BACILLUS MEGATERIUM.

Among recent observations on the oxygen effect in radiation biology, the one that the gas nitric oxide (NO) appears to be able to substitute for oxygen in enhancing the effects of ionizing radiations in some biological systems is of particular importance. This observation was made originally by Howard-Flanders in bacteria,1 and was later confirmed by Kihlman2 for chromosome breaks in Vicia and by Gray et al.3 for lethal effects in ascites tumor cells. In a more recent paper, HowardFlanders and Jockey4 claim molecule for molecule substitution of NO for 02, and suggest that the mode of action of both substances is the same, probably the reaction of the gases with radiation-induced radicals to form a reaction product lethal to the cell. Observations that appear to be contrary have been recorded. NO reduces radiation sensitivity of dry seeds of Agrostis (Sparrman et al.5), and of dry bacterial spores (Powers et al.6). Since the latter report,6 we have extended our studies and in this communication shall give evidence that the action of NO in our dry bacterial system is a double one-enhancing and protecting. In addition, extension of the studies previously reported7 on the action of oxygen are included herein. All of our results with NO and 02 lead to an explanation of their actions in this single dry system that constitutes for us the basis of a general explanation of the oxygen effect. Methods.-The spores were those of a nonlysogenic strain of Bacillus megaterium (ATTC #8245). This strain sporulates very well, yielding 100 per cent spore suspensions when grown on potato extract agar. The spores were mounted on cellulose membrane filters (Millipore filters) in our usual manner,8 dried in vacuo, and exposed to X-rays in gastight containers of either of two types. In some instances, cylindrically shaped plastic containers with a 0.08-mm thick plastic window were used. In others, the containers were gastight stainless steel cylinders attached to the X-ray tube with a manifold connected to pumps and gas sources. The temperature under different exposure conditions was measured with thermocouples as explained before.7 After the experimental treatment the filters were placed on paper pads saturated with a buffered peptone-glucose medium and incubated 16 hr at 350C. The ability of the spores to form visible colonies was the criterion of survival. The X-ray source was a Machlett OEG 60 tube with a tungsten target and beryllium window, operated at 50 kv and 45 ma (HVL 0.070 mm Al) with no added filtration except for the thin plastic window of the gas chamber. The filters were irradiated in stacks of five at a dose rate of about 15,000 r/min in the steel gas chambers. The dose rate was 43,310 r/min in the plastic chambers. The chambers containing the filters were pumped to less than 1 mm Hg, and then 02-free N2 (or at times He) was introduced to one atmosphere. This process was repeated twice. Then N2 (or at times He) was introduced to one-half atmosphere, NO or 02 was allowed to flow to the pressure proper for the concentration