STUDIES ON THE MECHANISM OF ACTION OF IONIZING RADIATIONS. Vll. C~.LLr~LA~ RESPmATION, CELL DIVISION, AND IONIZING RADIATIONS

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1 Published Online: 20 July, 1952 Supp Ino: Downloaded rom jgp.rupress.org on January 2, 2019 STUDIES ON THE MECHANISM OF ACTION OF IONIZING RADIATIONS Vll. C~.LLr~LA~ RESPmATION, CELL DIVISION, AND IONIZING RADIATIONS BY E. S. GUZMAN BARRON ~ S. LOUISE SEKI (From the Division o Biological and Medical Research, Argonne National Laboratory, The Chemical Division, Department o Medicine, University o Chicago, Chicago, and the Marine Biological Laboratory, Woods Hole) (Received or publication, January 11, 1952) Important biological eects o small doses o ionizing radiations are inhibition o cell division (1), and oxidation o --SH groups, as shown by the reversible inhibition o --SH enzymes (2, 3), and by the high ionic yield on oxidation o glutathione and dithiols produced by ionizing radiations (4). Living ceils possess two kinds o thiol groups: non-protein thiol groups, such as glutathione and cysteine; and ixed thiol groups, such as those attached to the side chains o proteins. The irst act as regulators o cellular metabolism and are necessary or cell division and growth; the second are essential or the activity o a large number o enzymes (the--sh enzymes), and or other unctions as well (5). Thiol reagents may produce, thereore, opposite eects depending on whether the irst or the second group is destroyed. It seems that the non-protein thiol groups are more easily attacked. In act, small amounts o thiol reagents increase cellular respiration and inhibit cell division, whereas large concentrations inhibit cell respiration. The irst eect seems to be due to destruction o the non-protein thiol groups, whereas the second eect is due to destruction o the --SH enzymes (6). I the sulhydryl theory o ionizing radiations postulated by us several years ago (reports o the Metallurgical Laboratory partially published in 1946 (7)) is correct, it would be possible to ind on irradiation o cells the same eects as those ound with the thiol reagents: inhibition o respiration on irradiation with large doses o x-rays, and increase o respiration on irradiation with small doses. The experiments presented in this paper avor this contention. Small doses o x-ray irradiation o the eggs and sperm o sea urchin (Arbacia punctulata) increased their respiration, whereas large doses inhibited it. Inhibition o cell division was ound on irradiation with doses o x-rays small enough to give an increase o respiration. Thus x-rays produced eects similar to those observed with thiol reagents. EXPE]~ I~.NTAL The experiments were perormed at the Marine Biological Laboratory, Woods Hole, in the summer o 1950 and were repeated in the summer o The eggs 865 The Journal o General Physiology

2 866 ACTION OF IONIZING RADIATIONS. VII and sperm o Arbacia punctulata were obtained by stimulated shedding through the injection o 0.1 ml. o 0.5 ~s KC1. Both cells were suspended in iltered sea water at dilutions optimal or respiration measurements. They were then divided into two portions, one o which was x-ray-irradiated in a plastic dish. The eggs were also ertilized with sperm, and the time required to reach 50 per cent ceil division (two cell stage) was noted. The x-ray generator consisted o two Coolidge tubes TABLE I Eect o X-Ray Irradiation on the Respiration o Sea Urahln Sperm Temperature 23. Time 2 hours. O~ uptake Inhibition (-) or incresse (+) 25, O 0 c.mm t:.mi~ TABLE II EA~ect o X-Ray Irradiation on the Respiration o Unertilized Sea UrclKn Eggs Temperature 23. Time 2 hours. X-my dose OI uptake Inhibition (--) or increase (+) 25, c.mm r ~ operating simultaneously with a current through each o 25 ms. and an alternating voltage o 182 kv. peak. A ilter o 0.2 ram. Cu was used. The measurement o respiration started 40 minutes ater irradiation. 1. Respiration.--The respiration o dilute suspensions o sea urchin sperm is inhibited not only on direct x-ray irradiation (8) but also on suspension in sea water previously irradiated with x-rays (9). On x-ray irradiation with doses between 20,000 and r airly reproducible results may be obtained. However, at x-ray doses below r the results became erratic. On decreasing the

3 E. S. OUZMAN BARRON AND S. LOUISE SEKI 867 irradiation intensity there came a dose--between 0 and 200 r,mat which the respiration o the ceils was consistently increased (Table I). This increased TABLE HI Eject o X-Ray Irradiation on the R~ra:li~ o Fertilized Sea Urchin Eggs Temperature 23. Time 2 hours. Oi uptake Inhibition or increase I;I Commo c.u per ee~t# I TABLE IV E~e, ct o Small Doses o X-Ray Irradiation on the Cleavage o Sea Urchin Eggs (Two CdJ Stage) Eggs Irradla~ed Tim, se.c, ~o' ~ O Two cell stage per cent 5O 8O p#r r respiration, which was observed in the summer o 1950, was conirmed in the summer o Sea urchin eggs seem to be somewhat more resistant to the action o x-rays. Irradiation o unertilized sea urchin eggs with 25,000 r inhibited respiration by 11 per cent, whereas this dose o x-rays applied to sperm produced 70 per

4 868 ACTION OF IONIZING RADIATIONS. VII cent inhibition. Irradiation with 00 r had no eect at all, whereas it inhibited sperm respiration by 18 per cent. Irradiation with and 260 r increased respiration (Table II). Fertilized sea urchin eggs were more susceptible to the TABLE V Eea o Small Doses o X.Ray Irradiation on the Cleavage o the Sea Urctdn Eggs (Two Cell Stage) Sperm Irradlated Time Two cell stage $e per cent ~c u O I action o x-rays. Thus 00 r, which had no eect on unertilized eggs, inhibited 18 per cent the respiration o ertilized sea urchin eggs. Inhibition was observed even on irradiation with r. Increase o respiration came on irradiation with 200 r (Table III). 2. Cell Division.--It has been known, since the experiments o Henshaw

5 E. S. GUZMAN BARRON AND S. LOUISE SEKI 869 (, 11) that x-irradiation o sea urchin eggs produces delay in cell division. The experiments o Henshaw, however, were perormed with rather large doses o x-rays, the minimum dose being 4,000 r. On irradiation o sea urchin eggs with x-ray doses which either had no eect or increased respiration (00 and r) there was a delay in the rate o cleavage. These x-ray doses are eight 20 :r d IO u,i 8",-n m o ~O T/ME IN MINUTES Fzc. 1. Eect o HgCI2 on the respiration o unertilized sea urchin eggs. Abscissa, time in minutes. Ordinate, 02 uptake in cubic millimeters (1) ; (2) HgC12, 5 X -~ ~; (3) HgC12, 1 X -4 M. times lower than those used by Henshaw (Table IV). Similar results were obtained when eggs were ertilized with irradiated sperm (Table V). In both cases, with small doses o x-rays ( to 0 r) the only eect observed was a delay in the cleavage to the two cell stage. Eventually, the irradiated eggs and the eggs ertilized with irradiated sperm reached the same per cent o cell division as the controls. 3. Mercaptlde-Forming Agents.--Results similar to those obtained with x-irradiation were ound with thiol reagents. HgCI~ (1 X -~), a mercaptide-

6 870 ACTION OF IONIZING RADIATIONS. VII orming agent, inhibited respiration o sea urchin eggs, whereas 5 X -h~ increased it (Fig. 1). When the eggs treated with 5 X -~ HgCl~, which showed increased respiration, were ertilized with untreated sperm there was complete inhibition o cell division, an eect much more powerul than that obtained with x-irradiation with doses low enough to produce increased respiration. Inhibition o cell division by small amounts o HgCI~ was previously observed by Rapkine (12). It is known that mercaptide-orming agents (HgCI~) are more powerul thiol reagents than oxidizing agents (x-rays) (2). That soluble thiols are essential or the process o cell division was discovered by Rapkine (12), and has been conirmed repeatedly (13). Their destruction either by mercaptide ormation or by oxidation inhibits the process. Rapkine ound that inhibition o cell division by HgC12 was abolished on addition o cysteine. The resumption o cell division o x-irradiated eggs is probably due to reduction o the radiationoxidized glutathione by the enzyme, glutathione reductase. In the presence o this enzyme oxidized glutathione is reduced by reduced triphosphopyridine nucleotide (TPN+H) (14, ). It is known that the process o cellular division is controlled by the nucleus and that it involves considerable synthesis o desoxyribonucleic acid (16). Irradiation o the nucleus o the sea urchin eggs with x-ray doses above 3,000 r produces a delay in the rate o cleavage, whereas irradiation o the enucleated cells has no eect (17, 18). On irradiation with these large x-ray doses there must be, besides the oxidation o the non-protein thiol groups, also inhibition o the enzyme responsible or the synthesis o the nucleic acids. Perhaps these enzymes belong to the group o --SH enzymes whose thiol residues are very sensitive to oxidation by ionizing radiations. The sulhydryl theory o x-ray action is not in contradiction to the possibility that the locus o action o the radiation is in the nucleus or something closely associated with the nucleus, as Blum et al. (18) have postulated. It oers a possible mechanism or this action. These experiments provide urther evidence or the view maintained by us (7) that small doses o ionizing radiations act mainly by oxidizing --SH groups which are essential or cellular growth and multiplication. S~ARY On x-irradiation o the eggs and sperm o Arbacia punctulata there was inhibition o respiration with relatively large doses, whereas there was an increase with small doses. The dose required to produce an increase o respiration depended on the degree o sensitivity o the cell to the eect o ionizing radiation. Sperm cells were more sensitive; then came ertilized eggs; unertilized eggs were the least sensitive. The inhibiting eect o x-rays on cell division was observed even on irradiation with x-ray doses which produced an increase o

7 E. S. GUZMAN BARRON AND S. LOUISE SEKI 871 respiration. These results are compared to similar eects produced by thiol reagents and are attributed to oxidation o the thiol compounds in the cell. REFERENCES 1. Lea, D. E., Actions o Radiations on Living Cells, Cambridge University Press, 1947, Barron, E. S. G., Dickrnan, S., Muntz, J. A., and Singer, T. P., J. Gen. Physiol., 1949, 32, Barron, E. S. G., and Dickman, S.,.L G~. Physiol., 1949, 32, Barron, E. S. G., ~ud Flood, V., J. Gen. Physiol., 1950, 88, Barron, E. S. G., Advances Enzymol., 1951, 11, Barron, E. S. G., Nelson, L., and Ardao, M. I., J. Gen. Physiol., 1948, 32, Barron, E. S. G., November 26, 1946, U. S. Declassiied Document MDDC Barron, E. S. G., Gasvoda, B., and Flood, V., Biol. Bull., 1949, 97, Barron, E. S. G., Flood, V., and Gasvoda, B., Biol. Bull., 1949, 97, 51.. ttenshaw, P. S., Am. J. Roentgenol. and Radium Therap., 1932, 9.7, Henshaw, P. S., Am. J. Roentgenol. and Radium Therap., 1940, 43, Rapkine, L., Ann. Physiol., 1931, 7, Brachet, J., Embryologie Chimique, Paris, Ma~son et Cie., 1944, Meldrum, N. U., and Tarr, H. L. A., Biochem. J., 19, 9.9, 8.. Conn, E. E., and Vennesland, B., J. Biol. Chem., 1951, 192, Villee, C. A., Lowens, M., Gordon, M., Leonard, E., and Rich, A., J. Cell. and Comp. Physiol., 1949, 33, Henshaw, P. S., Am. J. Cancer, 1938, 33, Blum, H. F., Robinson, J. C., and Loos, G. M., J. Gen. Physiol., 1951,, 323.