OXYGEN is known to increase the effectiveness of X-rays in killing bacteria[1,2] and cells from many other organisms[3] by a factor of 2 or 3. There have been numerous reports of substances which protect against the effects of irradiation under aerobic conditions[4], but there does not appear to be any report of a substance which, in complete anoxia, raises the radiosensitivity of bacteria to the level normally associated with full aeration. Before giving the evidence which suggests that nitric oxide may be such a substance, we may consider the factors which first directed attention to this gas.
The observed effect of oxygen on radiosensitivity implies that ionizing radiation produces lesions in the bacteria, some of which are converted to lethal injuries only if oxygen is present. It has been postulated that such a lesion is a radical, probably on a carbon atom in one of the molecules which are essential to cell survival[5]. It may be supposed, there-fore, that oxygen affects radiosensitivity by virtue of its affinity for carbon radicals. Molecular oxygen owes its reactivity and its affinity for free radicals to the unusual configuration of electron spins in the outer orbitals. Of the sixteen electrons in oxygen, fourteen fill the dg (2p) and lower energy orbitals which form the almost inert structure of molecular nitrogen. The remaining two electrons in molecular oxygen are in two pg (2p) orbitals. There is spectroscopic evidence that these electrons have unpaired spins, a finding which explains the paramagnetic properties of molecular oxygen[6]. The two unpaired electrons make oxygen a biradical and give it an affinity for other radicals[7].
Fig. 1. Survival curves for Shigella flexneri irradiated with 250 kV. X-rays while the suspension was bubbled with either nitrogen or nitrogen containing 0.8 per cent nitric oxide. The upper line shows the effect of the nitric oxide mixture on the cells in the absence of any radiation
Another paramagnetic molecule which has an unpaired electron in the pg (2p) orbital is nitric oxide, the most stable molecule with an odd number of electrons. Nitric oxide is a stable gas with little tendency to dimerize. It is more soluble than oxygen in water and it does not hydrolyse or dissociate to any great extent. Nitric oxide shows an affinity comparable to that of oxygen, for carbon radicals7. It combines readily with oxygen to form nitrogen peroxide, which hydrolyses in water to form nitric acid, so that nitric oxide can be used only in the absence of oxygen. Nitric oxide combines reversibly with hĉmatin[8] and cytochrome oxidase[9], forming compounds similar to those of carbon monoxide. That nitric oxide is not toxic in small amounts is shown by the observation that certain bacteria form it in the course of the reduction of nitrites to nitrogen[10]. It would seem from this list of properties that nitric oxide might affect radiosensitivity in much the same way that oxygen docs, while at the same time having a contrasting action on cell metabolism.
A preliminary investigation has been made into the effects of nitric oxide on the radiosensitivity of bacteria which were kept free from oxygen while in contact with the gas. The methods used follow those of a previous investigation into the effect of oxygen on radiosensitivity[2]. An overnight broth culture of Shigella flexneri was washed, resuspended and diluted for use in buffered saline. Samples were placed in a glass irradiation vessel with a glass filter base, through which oxygen-free nitrogen was passed. The suspension was vigorously bubbled with nitrogen for an initial period of equilibriation, as well as throughout the course of the irradiation carried out at 25° C. with 250 kV. X-rays filtered with 0.5 mm. aluminum, at a dose-rate of 1,500 rads per min. The top of the irradiation vessel was closed except for a narrow capillary outlet, through which the gas escaped. The suspension was sampled through the same capillary by tilting the vessel. The samples taken before and during the course of the irradiation were plated out on nutrient agar and incubated. The number of bacteria surviving was found from tile visible colony counts. When required, nitric oxide was mixed with oxygen-free nitrogen in amount determined by a flow-meter consisting of a glass tube along which a mercury pellet was pushed by the gas. When starting an experiment, the cell suspension was bubbled with nitrogen for a period of at least 5 min. at a flow-rate of 5 ml. per sec. before admitting nitric oxide. Samples were withdrawn when required and were passed from the irradiation vessel into a vial and bubbled with nitrogen to remove the nitric oxide before there was appreciable exposure to air. In addition, control samples were bubbled with nitric oxide for a period comparable with the time of the irradiation to test the effect of the gas alone upon the survival of the bacteria.
The results are shown in Fig. 1. It was found that. 0.8 per cent of nitric oxide in the gas mixture, which gave a concentration of 15 µM, was sufficient to double the sensitivity of the bacteria to radiation, while in the absence of any irradiation, exposure to nitric oxide at a concentration of 20 µM reduced the number of survivors by 10 per cent in a comparable time. The increase in sensitivity observed with nitric oxide is about the same as that observed with oxygen at a comparable concentration2. Further experiments will be needed to test whether oxygen and nitric oxide are comparable, molecule for molecule, in their effect upon the sensitivity of bacteria and other micro-organisms.
It is of interest that the bacteria given nitric oxide in place of oxygen should show the radiosensitivity normally associated with aerobic conditions, while all aerobic metabolism has been halted.
P. HOWARD-FLANDERS*
Donner Laboratory, University of California, Berkeley, Calif.
* On leave of absence from the Experimental Radiopathology Research Unit, Hammersmith Hospital, London, W.12.
1 Hollaender, A., and Stapleton, G. E., Physiol. Rev., 33, 77 (1953).
2 Howard-Flanders, P., and Alper, T., Rad. Res. (in the press).
3 Gray, L. H., Conger, A. D., Ebert, M., Hornesey, S., and Scott, O.C.A., Brit. J. Radiol., 26, 638 (1953).
4 Hollaender, A., and Stapleton, G. E., "Ionizing Radiation and Cell Metabolism", edit. G.E.W. Wolstenholme, 120-135 (J. a. A. Churchill, London, 1956).
5 Alper, T., Rad. Res., 5, 573 (1956). Howard-Flanders, P., and Moore . D. (unpublished work).
6 Herzberg, G., "Spectra of Diatomic Molecules", 343 (1950).
7 Dewar, M. J. S., "Electronic Theory of Organic Chemistry", 246 (Clarendon Press, Oxford, 1949).
8 Keilin, J., Biochem. J., 59, 571 (1955).
9 Waino, W. W., "Reactions of Cytochrome Oxidase", J. Biol. Chem. 212, 723 (1955).
10 Chung, C. W., and Najjar, V. A., J. Biol. Chem., 218, 617 (1956)
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