Reduction of the concentration of oxygen in bacterial suspensions has been

Transcription

1 THE ROLE OF ENZYMATIC OXYGEN REMOVAL IN CHEMICAL PROTECTION AGAINST X-RAY INACTIVATION OF BACTERIA' G. E. STAPLETON, DANIEL BILLEN, AND ALEXANDER HOLLAENDER Biology Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee Received for publication November 29, 1951 Reduction of the concentration of oxygen in bacterial suspensions has been shown to result in a several-fold reduction in the X-ray sensitivity of Escderidia coli (Hollaender, Stapleton, and Martin, 1951). Addition of a wide variety of chemical compounds to supensions of E. coli prior to X irradiation afforded similar protection (Burnett, Stapleton, Morse, and Hollaender, 1951; Hollaender, Burnett, and Stapleton, 1951). Pyruvate, added to suspensions of this organism before irradiation, has been found to reduce the lethal and mutagenic effect of X rays (Thompson el al., 1951). Several explanations have been offered for the protection of bacterial cells by either the removal of oxygen from the suspension or by the action of chemical agents, namely: (1) reduction of the ionic yield of oxidizing radiodecomposition products of water in the aqueous suspension; (2) protection of sulfhydryl groups of cellular proteinr; and (3) modification of the reductive state of the organism. Some basis for the first explanation is available from the investigations of Dale (1947), Weiss (1947), and Barron et al. (1949) which suggest that X-ray production of NO, and H20,, in aqueous solutions, is dependent on the presence of dissolved oxygen. If these highly reactive producta are toxic agents, then the removal of dissolved oxygen, by any means, should reduce the lethal effect per unit dose. Too little experimental evidence is available to allow an assessment of the relative importance of the various suggested mechanisms. We have investigated the conditions which affect the protection given by some of the chemical compounds in an effort to elucidate the mechanism of chemical protection. It was found that removal of oxygen either by cellular enzyme action or autooxidation in the presence of these compounds can explain the protection afforded againt X-ray inactivation of E. coli. METHODS Cultures of E. coli, stran B/r, were grown for 18 hours under constant aeration at 37 C in Difco 0.8 per cent nutrient broth. Suspensions, harvested by centrifugation and washed in M/15 phosphate buffer (ph 6.8), were brought to one-tenth the broth volume in phosphate buffer. Final suspensions contained approximately 2 X 1010 cells per ml. In the irradiation studies 0.1 ml of the suspension was added to 0.9 ml of buffered solution of each chemical to be studied. Preirradiation treatment consisted of holding cells with or without the chemical for 30 minutes prior to irradiation at either ice bath temperature or at 37 C, as indicated in table 1. 1 Work performed under Contract No. W-7405-Eng-26 for the Atomic Energy Commisoion. 805

2 806 G. E. STAPLETON, D. BILLEN, AND A. HOLLAENDER [VOL. 63 Suspensions were then irradiated in the same thin walled volumetric tubes in which they had been incubated. Eight of these vessels could be exposed simultaneously in a ring-type holder in a uniform radiation field, one tube being used for each dose level. The holder was so designed that the tubes could be immersed either in an ice water mixture or in water at room temperature during the exposure. The source of X rays was a "maxitron 250" unit operated at 250 kvp and 30 ma with a 3-mm alumin filter added. The dose measured at 20 cm from the target, in the position.occupied by the tubes, was 100,000 r per hour. Immediately after irradiation, the suspensions were appropriately diluted in sterile water. One-tenth ml of the final dilutions was pipetted onto the surface of three nutrient agar plates and spread uniformly over the entire agar surface. The plates were incubated overnight at 37 C, and the macroscopically visible TABLE 1 Effect of preincubation temperature on protective capacity of various chemical agents PROTECTIVE AGENT MOLA CONCEN- TRATI _ suvivwonc?raction X 10-5 wrm 30 MNUTEs INCUBATION AT 1ONN 2 C 37 C 30,000 r 60,000 r 30,000 r 60,000 r Buffer... _ 1, , Formate , ,000 3,100 Succinate , ,000 2,600 Pyruvate... 0O.01 3, ,000 1,700 Berine , ,000 5,000 BAL Cysteine ,000 6,900 26,000 7, ,000 11,000 35,000 12,000 Ethanol , ,000 1, , ,000 4,600 Sodium hydrosulfite ,000 8,000 29,000 10,500 *Surviving fraction = N/No = number of cells surviving any X-ray dose original number of cells colonies counted to determine the surviving fraction. Further incubation did not increase the number of colonies per plate. In respiration studies to be reported, oxygen uptake was measured by the usual Warburg manometric techniques (Umbreit et al., 1949). Each manometer cup contained the cell suspension in M/15 buffer; the same cell concentration was employed as in the radiation studies. In experiments in which inhibitors were used in conjunction with a protective chemical, the per cent inhibition of oxidation of the chemical by the inhibitor was obtained by a comparison of the rate of oxygen uptake at the end of 30 minutes with that of a control sample. This period represented the time used in the incubation treatment prior to irradiation. RESULTS AND DIISCUSSION Investigation of the conditions affecting chemical protection revealed that protection given by certain compounds (formate, succinate, pyruvate, serine,

3 1952] CHEMICAL PROTECTION AGAINST X-RAY INACTIVATION 80fl7 a-alanine, and ethanol) was greatly increased when they were incubated with the cells at 37 C for 30 minutes before irradiation (table 1). This finding suggested that the mechanism of protection by the amino and carboxylic acids was enzymatic in nature. It was also observed, through respiration studies, that those compounds which are utilized as oxidative substrates by the cells showed protective properties.,b- and a-alanine are good examples of this observation. A comparison of respiration and protective capacity of these two compounds revealed that fl-alanine, which was oxidized at an insignificant rate as compared with a-alanine, offered no protection, while a-alanine gave marked protection if the cells were incubated with it prior to X irradiation. The utilization of oxygen by the cells on these substrates suggested that enzymatic reduction of the oxygen TABLE 2 Relationship of respiratory inhibition to loss of protection PROTECTIVE AGENT PROTECTION PER CENT LOSS PER CENT PROTEC- FACTOR WITH OF PROTECTION INHIBITION OF TION AGENT WITH RESPIRATION WITH FACTOR WITH AGENT Cyanide lodo- Cyanide.Iodo Cya AGENT ~~acetate acetate Cyanide tade Formate Succinate Pyruvate Serine Cysteine BAL Ethanol Sodium hydrosulfite The loss of protection and respiratory inhibition by malonate (0.03 M) on succinate was similar to that obtained with cyanide. Cyanide and iodoacetate concentration = M. * Protection factor = ratio of X-ray dose required to inactivate a given fraction of cells in the presence of a protective agent to that required in its absence. concentration in or around the bacterial cells might give a partial explanation of the mechanism of protection of the amino and carboxylic acids. In order to test this hypothesis further, respiratory inhibitors were used to determine if the protection could be eliminated by blocking oxygen consumption on those chemical compounds utilized as respiratory substrates by the cells. Sodium cyanide, iodoacetate, and malonate were used because of their known inhibitory action on respiration. Since the toxicity of these inhibitors permitted the use of only limited concentrations, complete inhibition of respiration was not obtained. Results of the inhibition studies on those compounds requiring incubation for protection indicated that, when the rate of oxygen uptake on the compound is sufficiently lowered, the protective capacity is reduced and in some cases eliminated completely (tables 1 and 2). A representative plot of survival fraction at 60,000 r with and without inhibitor, in the presence and absence of succinate, is given in figure 1. It may be noted (figure 1) that the presence of the inhibitors alone increased slightly the radiosensitivity of the cells. This effect Iodo-

4 808 G. E. STAPLETON, D. BDILN, AND A. HOLLANDER [VOL. 63 may be due to blocking of endogenous respiration, thereby preventing the removal of oxygen from critical sites within or on the cell. An experiment designed to show the relationship between the respiration rate on a particular compound and its protective capacity was conducted ng succinate as the substrate and sodium cyanide, in several concentrations, as the inhibitor. It can be seen (figure 2) that, with increasing concentrations of cyanide, I09 EFFECT OF TEMPERATURE AND INHIBITORS ON SURVIVAL OF E.COLI B/r WITH AND WITHOUT PROTECTIVE COMPOUND INHIBITOR CONCENTRATION o CYANIDE M 2 IO MALONATE 0.03 ol M IODOACETATE M PREINCUBATIO BUFFER SPENION SUCCINATE (0.01 M) TEMPERATURE SUSPENSION cc) 2 3t ' CN MAL I'AC CN MAL I'AC Figure 1. Effect of preincubation with and without respiratory inhibitors on succinate protection against X-ray inactivation of Escherichia coli, strain B/r. Survival fraction at an X-ray dose of 60,000 r is shown for the various condition. 0 buffer suspensions, U buffered succinate mspensions (0.01 x). Concentration: cyanide and iodoacetate mx; malonate = 0.03 x. the loss of succinate protection follows closely the reduction of respiration rate. The finding that there is an apparent threshold dose of cyanide required to eliminate succinate protection indicates that a critical amount of oxygen must be removed from the cell or the vicinity of the cell in order that the protective effect be demonstrable. Indications of a "critical oxygen concentration" in bacterial X-ray inactivation have been demonstrated by the use of a different technique. The nature of the protection with cysteine and ethanol appeared to be at variance with the general finding that rate of respiration and protection could

5 1952] 15]HEMICAL PROTECTION AGAINST X-RAY INACTIVATION 809 be correlated since, with both compounds (table 2), cyanide reduced the protective capacity without measurable inhibition of respiration at the end of the 30-minute period. However, cyanide inhibition of respiration on cysteine was observed for the first 15 minutes; this might account for the decrease in protection with this compound in the presence of cyanide. The nature of the loss of ethanol protection in the presence of cyanide was not determined in these experiments. Downloaded from O CYANIDE MOLAR CONCENTRATION Figure B. Comparison of the loss of protective capacity of succinate with the inhibition of oxygen uptake on this substrate by cyanide. Left ordinate = surviving fraction at 60,000 r; right ordinate = per cent inhibition of respiration as a function of molar cyanide concentration. In contrast to those chemicals which require preincubation with the cells, dimercaptopropanol (BAL) and sodium hydrosulfite (Na2S204) (Burnett, Morse, Burke, and Hollaender, 1952) gave simnlar protection with incubation at 2 and 37 C (table 1). Since both these compounds were found to remove oxygen from supensions, independently of enzyme action, it seems that reduction of oxygen concentration in the suspension can explain the nature of the protection afforded by both these compounds. It is possible that hydrogen donation by the chemical, under the conditions on September 22, 2018 by guest

6 810 G. E. STAPLETON, D. BILLEN, AND A. HOLLAENDER [VOL. 63 required for protection, may be an important mechanism of protection. The presence of hydrogen or reduced substances within the bacterial cell at the time of irradiation might result in a sparing effect on important biological systems by competition for oxidizing radiodecomposition products. This possibility was not eliminated by use of inhibitors, since the respiratory inhibitors used might also have affected hydrogen transfer systems of the bacterium. In order to investigate the relative importance of hydrogen transfer in altering the state of reduction within the cells, the hydrogenase system of these cells was utilized. Molecular hydrogen was used as the substrate, thereby eliminating an organic compound as a hydrogen donator. E. coli cells grown in tryptone broth, without aeration, posse an active hydrogenase system (Ordal, 1951). This enzyme enables the bacterium to utilize molecular hydrogen in the reduction of a number of compounds including oxygen (Stephenson, 1947). Cells containing an active hydrogenase, irradiated after having been flushed with 100 per cent hydrogen for 15 minutes at 37 C, were equally as sensitive as similar cells which had been flushed under the same conditions with 100 per cent nitrogen. Moreover, addition of methylene blue or fumarate, which are readily used as hydrogen acceptors, did not increase the survival over that obtained without these acceptors. This apparently indicates that neither hydrogen donation nor production of a highly reduced state is a major mechanism in chemical protection. Since the hydrogenase system in this organism is capable of removing oxygen from the suspension by utilizing it as a hydrogen acceptor, it was decided to compare the X-ray sensitivity of cells possessing this enzyme, after flushing with a hydrogen-oxygen mixture, with similr cells after treatment with a nitrogenoxygen mixture. Mixtures of 10 per cent oxygen in hydrogen and nitrogen were prepared by water displacement (Umbreit et al., 1949) and were bubbled through suspensions of cells having the hydrogenase system. The survival of cells was much greater at 40 and 80 kr in the hydrogen-oxygen mixture than in the nitrogen-oxygen mixture. The reduction of X-ray sensitivity by oxygen reduction through the hydrogenase sytem was similar to that obtained by oxygen removal through bubbling with another gas or by addition of a chemical protective agent. Cells grown under constant aeration or in the presence of nitrate (Billen, 1951), and not possessing the hydrogenase system, showed no significant difference in either mixture. Thus, it would seem that removal of oxygen from within the suspended bacteria can explain the protection afforded by this system. An arbitrary distinction is made here between the protection afforded by reduction of oxygen concentration by enzymatic utilization of oxygen in the presence of an oxidizable substrate, and that brought about passively, either by oxygen replacement with another gas (Hollaender, Stapleton, and Martin, 1951) or by addition of autooxidizable compounds to the suspension. It may very well be that the end result obtained through any of the mentioned systems is a reduction of intracellular oxygen concentration, and thereby a reduction of the yield of toxic radiodecomposition products per unit dose. It is of interest that we have been able to demonstrate that the bacterial cells, under the proper conditions, have the ability to alter their own radiosensitivity, by virtue of their respiratory enzyme systems.

7 19521 CHEMICAL PROTECTION AGAINST X-RAY INACTIVATION 811 SUMIARY Compounds found to be protective against the lethal action of X rays on bacterial cells can be divided into two classes on the basis of whether or not preincubation with the cells was required for protection. A decrease in protective capacity was correlated with a decrease in respiration rate on those compounds which require preincubation for protection. Cells possessng an active hydrogenase system, exposed to X rays in a hydrogen-oxygen mixture, were found to be more radioresistant than similar cells exposed in a nitrogen-oxygen mixture. Production of a highly reductive state in the cells and suspensions did not alter their radioresistance. The data presented are consistent with the hypothesis that oxygen removal rather than hydrogen donation is the major mode of protection afforded by chemical protective agents. REFERENCES BAunON, E. S. G., DICKMAN, S., MUNTZ, J. A., AND SINGER, T. P Mechanism of action of ionizing radiations. J. Gen. Physiol., 32, BILLEN, DANIEL 1951 The inhibition by nitrate of enzyme formation during growth of Escherichia coli. J. Bact., 62, BURNETT, W. T., JR., MORSE, M. L., BuRK, A. W., JR., AND HOLLAENDER, A Reduction of X-ray sensitivity of Escherichia coli by sodium hydrosulfite and certain other inorganic sulfur compounds. J. Bact., 63, BURNETT, W. T., JR., STAPLETON, G. E., MORSE, M. L., AND HOLLAENDER, A Reduction of X-ray sensitivity of Escherichia coli B/r by sulfhydryl compounds, alcohol, glycols, and sodium hydrosulfite. Proc. Soc. Exptl. Biol. Med., 77, DALE, W. M Action of radiations on aqueous solutions. Experimental work with enzymes in solution. Brit. J. Radiology, Suppl. 1, HOLLAENDER, A., BURNETT, W. T., JR., AND STAPLETON, G. E The modification of X-ray sensitivity by chemicals. Isotopes in Chemistry Symposium (Ciba Foundation), Churchill, Ltd., London. HOLLAENDER, A., STAPLETON, G. E., AND MARTIN, F. L X-ray sensitivity of E. coli as modified by oxygen tension. Nature, 167, ORDAL, E. J On hydrogenase. Bact. Proc., 1951, STEPHENSON, M Some aspects of hydrogen tranfer. Antonie van Leeuwenhoek. J. Microbiol. Serol., 12, THOMPSON, T. L., MEFFERD, R. B., JR., AND WYSS, The protection of bacteria by pyruvate against radiation effects. J. Bact., 62, UMBREIT, W. W., BuRRIS, R. H., AND STAuFFER, J. F Manometric techniques and related methods for the study of tissue metabolism. Second edition, Burgess Publishing Co., Minneapolis, Minn. WEISS, JOSEPH 1947 Some aspects of the action of radiations on aqueous solutions. Brit. J. Radiology, Suppl. 1, 56,58.