- Email: [email protected]
Effect of total body x-irradiation on some enzymes of rat tissues
Effect of Total Body X-Irradiation on Some Enzymes of Rat Tissues1 Jay S. Roth, Herbert J. Eichel, Arthur Wase, Carl Alper and M. John Boyd Prom the William Goldman Isotope Laboratory, Division of Biological Hahn.emann Medical College, Philadelphia, Pennsylvania
Chemistry,
Received November 19, 1952
Studies on x-irradiated animals have shown that alterations may occur in the activity of enzyme systems. Many of these studies employed widely varying techniques and different experimental conditions; thus it is not unusual that in some casescontradictory results were obtained. In order to learn more about’ the extent of the changes which occur at the enzymatic level in x-irradiated animals, it appeared desirable to carry out further experiments of this nature. In this paper, therefore, we have investigated the effect of 600 roentgens (1.) of total body xirradiation on the activity of several enzymes in rat tissues. EXPERIMENTAL Male Wistar rats weighing from 225 to 275 g. were divided into two groups. One group consisting of 47 animals was irradiated with 600 r. of total body irradiat,ion using 200 kv., 20 ma., 50 cm. target distance, and a 0.5-mm. Cu filter and a l-mm. Al filter; half-value layer 0.9 mm. Cu. Radiation was delivered at Ehor./min. Six animals were placed in the bottom of a metabolism cage and held in position by a light cardboard cover while being irradiated. The rats were then placed four in a cage and paired with suitable controls, the latter being pair-fed against t,he experimental animals. All animals were given a stock diet of Foxchow meal and water ad libitum. One day after irradiation, one control and five experimental animals were sacrificed by exsanguination. Blood was obtained by cardiac puncture for serum studies. The livers and kidneys were removed, homogenized immediately with 5 vol. of ice-cold distilled water in an all-glass homogenizer at 5”C., and then diluted in various ways depending on the enzyme being assayed. -. 1 This project was supported by a grant from the U. S. Atomic Energy Commission, Contract AT (30-l) 1069. 95
96
ROTH,
EICHEL,
WASE,
SLPER
BND
BOYD
On the 2, 3, 5, 8, 10 and 12th days, three control and three irradiated animals were sacrificed, and the tissues were prepared as before. Ribonucleaae was determined by the method of Roth and Milstein (1) on 1:lO dilutions of the original liver homogenate. Assays were run at pH 6.0. All tests were made within 4 hr. of autopsy since it has been shown that liver ribonuclease activity may increase on standing.* Lipase was determined by the method of Alper and Polakoff (2) ; catalase, by the method of Perlmann and Lipmann (3) ; choline oxidase, by the method of Williams, Litwack, and Elvehjem (4) ; xanthine oxidase, by the method of Axelrod and Elvehjem (5); and cathepsin, by the method of Tallan, Jones, and Fruton (6). All of these assays were performed on the original homogenates. In the case of lipase, catalase, and xanthine oxidase, the homogenates were stored in the frozen,state until the determinations could be made. These enzymes have been found to be quite stable under such conditions.2 Oxygen uptake was determined on liver and kidney homogenates within l-2 hr. after the tissues were removed from the rats. The homogenates were assayed for nitrogen by the micro-Kjeldahl method, and.all enzyme activities, with the exception of serum lipase, are expressed as activity/mg. N. RESULTS
Of 47 irradiated rats, 13 of those that were not autopsied died by the end of the experimental period. All of the irradiated animals showed the usual symptoms associated with excessive radiation. Changes in body weights were not determined, but at the end of the experimental period of 12 days, the control and irradiated animals were of the same average weight, as was the case at the beginning of the experiment. Enzyme Changes In describing the changes in the x-irradiated animals, comparisons have been made to the daily control values and not the average value of the 19 controls (see figures). Liver ribonuclease (Fig. 1) increased somewhat on the 2nd and 3rd day, decreased to 25 y0 of normal by the 8th day, and returned to a value significantly above the control level on the 12th day. Inasmuch as the function of ribonuclease is not known, it is diacult to interpret these results in terms of the biological effects of radiation. Changes in ribonuclease activity of bone marrow and spleen in irradiated rabbits have been observed (8) with a large decrease in activity being reported in bone marrow and a smaller one in spleen. Xanthine oxidase activity [Qo2 (N)] for the 12 day period is plotted in Fig. 2. There was no significant change in activity during the first 5 days after irradiation. By the 8th day, however, the xanthine oxidase 2 J. S. Roth, H. J. Eichel, and C. Alper, unpublished results.
97
FIQ. 1. The effect, of 600 r. total body x-irradiation rat liver.
on ribonuclease activit!.
24221
of
Y x AVERAGE X-RAY
t 9 0 18i
x
x
\
5 , ,I;* ‘\ ‘. /
FIG. 2. The effect of 600 r. total body x-irradiation tivit,y of rat liver.
3
1’
//’
,; x O
on xanthine oxidase ac-
activity of the irradiated animals rose significantly above the control level. This elevation was still present on the 12th day and represented
98
ROTH,
EIcHEL,
WASE,
c
ALPER
AND
AVERAGE CONTROLS
BOYD
O
200 1 I 80’ I 60I AO1.20LOO-
FIG. 3. The effect of 600 I. total body x-irradiation tivity.
140
on rat serum lipase ac-
AVERAGE CONTROLS
L
x I
8 D&S FIG. 4. The effect of 600 r. total body x-irradiation liver.
IO’
I
I2
on catalase activity
of rat
an increase in Q,,(N) of 40 T0 over the control value. Considerable variability was noted in both experimental and control activities. Serum lipase activity of irradiated and control animals is plotted in Fig. 3. The activity is reported in terms of units of lipolytic activity
X-IRRADIATION
AND
ENZYMES
99
per milliliter of serum. There was a marked decrease in serum lipase activity of the irradiated animals from 3 to 5 days, followed by a return nearly to the control level on the 8th day. Goldstein and Neworaschkin (9) reported an increase, followed by a decrease,in blood lipase activity in humans treated locally with x-radiation. Liver catalase activities (units/mg. N) for the 12 day period are plotted in Fig. 4. After 3 days the activities of the irradiated animals averaged approximately 30% less than the controls. This pattern was maintained throughout the remainder of the experimental period. Divergent resu1t.shave been reported on the effects of x-radiation on catalase activity in wivo. In mice, Kazuo et al. (10) noted fairly extensive fluctuations while Feinstein and co-workers (11) reported a steady decline in activity in, the same animal. In the rat, Ludewig and others (12) observed no change in liver catalase activity in contrast, to om results. In addition to these results, no significant changes were observed in the activities of liver choline oxidase, cathepsin, and lipase or in the oxygen consumption of liver and kidney homogenates during the la-day experimental period. DISCUSSION
The effects of total body x-irradiation on enzymes in zrivo,as reported in this paper, present no clear-cut pattern. It is somewhat doubtful if one would be expected or will be obtained on further study. Aside from the direct effects of ionizing radiation or the products produced by it in water, many secondary disturbances in the irradiated animal’s metabolism are to be expected in view of the severe damage that results from high levels of total body x-irradiation. It is possible to make a few generalizations, but it should be emphasized that there are many exceptions to these and that the animal, the level of irradiation, and many other factors may influence the results materially. I. X-radiation appears to have little effect on oxidative enzymes within the cell. Those enzymes that have been studied, including succinic dehydrogenase and oxidase (7, 14-16), choline oxidase (17), and cytochrome oxidase (14, 16), have in most instances shown no changes. One exception is in x-irradiated bone marrow (13). Also xanthine oxidase activity as reported here was found to increase after 5 days. These observations are reflected in the over-all oxidative metabolism of the ani-
100
ROTH, EICIIEL,
WASE, ALPER AND BOYD
mals which is not appreciably altered even by exposure to lethal doses of x-radiation (‘25). 2. With some exceptions, the hydrolytic enzymes which have been studied seem to be more generally affected in wivo by x-radiation; thus pseudocholinesterase (18), lipase, ribonuclease, adenosinetriphosphatase (16), 5-nucleotidase (IS), alkaline phosphatase (16, B-21), and some cathepsins (22, 23) showed changes in activity either of a positive or negative nature. Some of these changes may be due to destruction of naturally occurring inhibitors or activators by the x-radiation. The activity of the nonoxidative enzyme, transaminase (24), has also been found to change under certain conditions. Several of the reports are still conflicting and further studies will be required to substantiate this general conclusion. The correlation of the biological effects of x-radiation on animals with the changes in enzyme activity observed will probably be difficult until more knowledge is available on the mechanisms and sites of enzyme synthesis within the cell. SUMMARY
Rats were subjected to 600 roentgens of total body x-irradiation, and changes in enzyme activities and oxygen uptake were studied for a 12day period. Changes were observed in liver cat&se, xanthme oxidase, ribonuclease, and serum lipase. No significant alterations were observed in oxygen uptake of liver and kidney, or in choline oxidase, lipase, and cathepsin activity of liver. REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10.
ROTH, J. S., AND MILSTEIN, S. W., J. Biol. Chem. 196,489 (1952). ALPEB, C., AND POLAKOFF, P. P., Js., Federation Proc. 10,155 (1951). PERLMANN, G. E., AND LIPMANN, F., Arch. Biochem. 7, 169 (1945). WILLIAMS, J. N., JR., LITWACK, G., AND ELVEHJEM, C. A., J. Biol. Chem. 133, 73 (1951). AXELROD, A. E., AND ELVEHJEM, C. A., J. Biol. Chem. 140,725 (1941). TALLAN, H. H., JONES, M. E., AND FRUTON, J. S., J. Biol. Chem. 194, 793 (1952). ROTH, J. S., WASE, A., EICHEL, H. J., AND ALPER, C., Abstracts 121st Meeting Am. Chem. Sot. Buffalo, N. Y., p. 3C, 1952. ZITTLE, C. A., in E. MCDONALD, ed., Neutron Effects on Animals. p. 79. Williams and Wilkins Co., Baltimore, Md., 1947. GOLDSTEIN, M. J., AND NEWORASCHHIN, J. P., Strahlentherupie 36,736 (1930). Razuo, M., MOMOKI, S., AND ITO, H., Zgaku to Seibutsugaku 18,303 (1951).
11. FEINSTEIN, R. N., BVTLER, C. I,., .~ND HENDLEY, 11. C., Rcience 111, 14!) (1950). 12. LUDEWIU, S., AND CHANUTIN, A., Arch. Biochem. 29, 441 (1956). 13. DINNING, J. S., MESCHAN, I., KEITH, C. K., AND Day, I’. I,., Proc. Sot. Espi/. Biol. Med. 74, 776 (1950). l-1-. LEMAY, M., Proc. Sot. Exptl. Biol. Med. 77, 337 (1951). 15. BARRON, E. S. G., Declsssified Document AECD-2316 (CH-3654). I’. S. ,4tomic Energy Commission, 1946. 16. Aslfwm,L, G., AND HTCKUAN, J., Proc. Sot. Exptl. Biol. Med. 80, 407 (1952). IT. KUNKEL, H. O., .~ND FHILLIPS, P. H., Arch. Biochem. and Biophys. 37, 366 (1952). 1X. BURN, J. H., KORDIK, I’., AND ~IOLE, R. H., Brit. d. Pharmacol. 7, 58 (1952). 19. FETRAKIS, N. L., ASHLER, F. hr., AND FERKEL, R. T,., S;~v:ll Rsdiol. Defense Report, AD 126B, 1919. 20. LUDEWIG, S., AND CHANUTIN, A., Am. J. Physiol. 163, 6# (1950). 21. LUDEWJG, S., Federation Proc. 9, 198 (1950). 22. BALLJN, J. C., AND FEINSTEIN, R. N., Federation Proc. 11. 184 (1952). 23. T.~NzI, B., Boll. sot. ital. biol. sper. 14, 732 (1939). 24. BRIN, M., Federation Proc. 11, 190 (1952). 25. SMITH, D. E., TYREE, E. B., PATT, H. M., .4x1) J.WKSOK, I,:., Proc. Sot. Es@. Biol. Med. 78, 774 (1951).
Chemistry,
Received November 19, 1952
Studies on x-irradiated animals have shown that alterations may occur in the activity of enzyme systems. Many of these studies employed widely varying techniques and different experimental conditions; thus it is not unusual that in some casescontradictory results were obtained. In order to learn more about’ the extent of the changes which occur at the enzymatic level in x-irradiated animals, it appeared desirable to carry out further experiments of this nature. In this paper, therefore, we have investigated the effect of 600 roentgens (1.) of total body xirradiation on the activity of several enzymes in rat tissues. EXPERIMENTAL Male Wistar rats weighing from 225 to 275 g. were divided into two groups. One group consisting of 47 animals was irradiated with 600 r. of total body irradiat,ion using 200 kv., 20 ma., 50 cm. target distance, and a 0.5-mm. Cu filter and a l-mm. Al filter; half-value layer 0.9 mm. Cu. Radiation was delivered at Ehor./min. Six animals were placed in the bottom of a metabolism cage and held in position by a light cardboard cover while being irradiated. The rats were then placed four in a cage and paired with suitable controls, the latter being pair-fed against t,he experimental animals. All animals were given a stock diet of Foxchow meal and water ad libitum. One day after irradiation, one control and five experimental animals were sacrificed by exsanguination. Blood was obtained by cardiac puncture for serum studies. The livers and kidneys were removed, homogenized immediately with 5 vol. of ice-cold distilled water in an all-glass homogenizer at 5”C., and then diluted in various ways depending on the enzyme being assayed. -. 1 This project was supported by a grant from the U. S. Atomic Energy Commission, Contract AT (30-l) 1069. 95
96
ROTH,
EICHEL,
WASE,
SLPER
BND
BOYD
On the 2, 3, 5, 8, 10 and 12th days, three control and three irradiated animals were sacrificed, and the tissues were prepared as before. Ribonucleaae was determined by the method of Roth and Milstein (1) on 1:lO dilutions of the original liver homogenate. Assays were run at pH 6.0. All tests were made within 4 hr. of autopsy since it has been shown that liver ribonuclease activity may increase on standing.* Lipase was determined by the method of Alper and Polakoff (2) ; catalase, by the method of Perlmann and Lipmann (3) ; choline oxidase, by the method of Williams, Litwack, and Elvehjem (4) ; xanthine oxidase, by the method of Axelrod and Elvehjem (5); and cathepsin, by the method of Tallan, Jones, and Fruton (6). All of these assays were performed on the original homogenates. In the case of lipase, catalase, and xanthine oxidase, the homogenates were stored in the frozen,state until the determinations could be made. These enzymes have been found to be quite stable under such conditions.2 Oxygen uptake was determined on liver and kidney homogenates within l-2 hr. after the tissues were removed from the rats. The homogenates were assayed for nitrogen by the micro-Kjeldahl method, and.all enzyme activities, with the exception of serum lipase, are expressed as activity/mg. N. RESULTS
Of 47 irradiated rats, 13 of those that were not autopsied died by the end of the experimental period. All of the irradiated animals showed the usual symptoms associated with excessive radiation. Changes in body weights were not determined, but at the end of the experimental period of 12 days, the control and irradiated animals were of the same average weight, as was the case at the beginning of the experiment. Enzyme Changes In describing the changes in the x-irradiated animals, comparisons have been made to the daily control values and not the average value of the 19 controls (see figures). Liver ribonuclease (Fig. 1) increased somewhat on the 2nd and 3rd day, decreased to 25 y0 of normal by the 8th day, and returned to a value significantly above the control level on the 12th day. Inasmuch as the function of ribonuclease is not known, it is diacult to interpret these results in terms of the biological effects of radiation. Changes in ribonuclease activity of bone marrow and spleen in irradiated rabbits have been observed (8) with a large decrease in activity being reported in bone marrow and a smaller one in spleen. Xanthine oxidase activity [Qo2 (N)] for the 12 day period is plotted in Fig. 2. There was no significant change in activity during the first 5 days after irradiation. By the 8th day, however, the xanthine oxidase 2 J. S. Roth, H. J. Eichel, and C. Alper, unpublished results.
97
FIQ. 1. The effect, of 600 r. total body x-irradiation rat liver.
on ribonuclease activit!.
24221
of
Y x AVERAGE X-RAY
t 9 0 18i
x
x
\
5 , ,I;* ‘\ ‘. /
FIG. 2. The effect of 600 r. total body x-irradiation tivit,y of rat liver.
3
1’
//’
,; x O
on xanthine oxidase ac-
activity of the irradiated animals rose significantly above the control level. This elevation was still present on the 12th day and represented
98
ROTH,
EIcHEL,
WASE,
c
ALPER
AND
AVERAGE CONTROLS
BOYD
O
200 1 I 80’ I 60I AO1.20LOO-
FIG. 3. The effect of 600 I. total body x-irradiation tivity.
140
on rat serum lipase ac-
AVERAGE CONTROLS
L
x I
8 D&S FIG. 4. The effect of 600 r. total body x-irradiation liver.
IO’
I
I2
on catalase activity
of rat
an increase in Q,,(N) of 40 T0 over the control value. Considerable variability was noted in both experimental and control activities. Serum lipase activity of irradiated and control animals is plotted in Fig. 3. The activity is reported in terms of units of lipolytic activity
X-IRRADIATION
AND
ENZYMES
99
per milliliter of serum. There was a marked decrease in serum lipase activity of the irradiated animals from 3 to 5 days, followed by a return nearly to the control level on the 8th day. Goldstein and Neworaschkin (9) reported an increase, followed by a decrease,in blood lipase activity in humans treated locally with x-radiation. Liver catalase activities (units/mg. N) for the 12 day period are plotted in Fig. 4. After 3 days the activities of the irradiated animals averaged approximately 30% less than the controls. This pattern was maintained throughout the remainder of the experimental period. Divergent resu1t.shave been reported on the effects of x-radiation on catalase activity in wivo. In mice, Kazuo et al. (10) noted fairly extensive fluctuations while Feinstein and co-workers (11) reported a steady decline in activity in, the same animal. In the rat, Ludewig and others (12) observed no change in liver catalase activity in contrast, to om results. In addition to these results, no significant changes were observed in the activities of liver choline oxidase, cathepsin, and lipase or in the oxygen consumption of liver and kidney homogenates during the la-day experimental period. DISCUSSION
The effects of total body x-irradiation on enzymes in zrivo,as reported in this paper, present no clear-cut pattern. It is somewhat doubtful if one would be expected or will be obtained on further study. Aside from the direct effects of ionizing radiation or the products produced by it in water, many secondary disturbances in the irradiated animal’s metabolism are to be expected in view of the severe damage that results from high levels of total body x-irradiation. It is possible to make a few generalizations, but it should be emphasized that there are many exceptions to these and that the animal, the level of irradiation, and many other factors may influence the results materially. I. X-radiation appears to have little effect on oxidative enzymes within the cell. Those enzymes that have been studied, including succinic dehydrogenase and oxidase (7, 14-16), choline oxidase (17), and cytochrome oxidase (14, 16), have in most instances shown no changes. One exception is in x-irradiated bone marrow (13). Also xanthine oxidase activity as reported here was found to increase after 5 days. These observations are reflected in the over-all oxidative metabolism of the ani-
100
ROTH, EICIIEL,
WASE, ALPER AND BOYD
mals which is not appreciably altered even by exposure to lethal doses of x-radiation (‘25). 2. With some exceptions, the hydrolytic enzymes which have been studied seem to be more generally affected in wivo by x-radiation; thus pseudocholinesterase (18), lipase, ribonuclease, adenosinetriphosphatase (16), 5-nucleotidase (IS), alkaline phosphatase (16, B-21), and some cathepsins (22, 23) showed changes in activity either of a positive or negative nature. Some of these changes may be due to destruction of naturally occurring inhibitors or activators by the x-radiation. The activity of the nonoxidative enzyme, transaminase (24), has also been found to change under certain conditions. Several of the reports are still conflicting and further studies will be required to substantiate this general conclusion. The correlation of the biological effects of x-radiation on animals with the changes in enzyme activity observed will probably be difficult until more knowledge is available on the mechanisms and sites of enzyme synthesis within the cell. SUMMARY
Rats were subjected to 600 roentgens of total body x-irradiation, and changes in enzyme activities and oxygen uptake were studied for a 12day period. Changes were observed in liver cat&se, xanthme oxidase, ribonuclease, and serum lipase. No significant alterations were observed in oxygen uptake of liver and kidney, or in choline oxidase, lipase, and cathepsin activity of liver. REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10.
ROTH, J. S., AND MILSTEIN, S. W., J. Biol. Chem. 196,489 (1952). ALPEB, C., AND POLAKOFF, P. P., Js., Federation Proc. 10,155 (1951). PERLMANN, G. E., AND LIPMANN, F., Arch. Biochem. 7, 169 (1945). WILLIAMS, J. N., JR., LITWACK, G., AND ELVEHJEM, C. A., J. Biol. Chem. 133, 73 (1951). AXELROD, A. E., AND ELVEHJEM, C. A., J. Biol. Chem. 140,725 (1941). TALLAN, H. H., JONES, M. E., AND FRUTON, J. S., J. Biol. Chem. 194, 793 (1952). ROTH, J. S., WASE, A., EICHEL, H. J., AND ALPER, C., Abstracts 121st Meeting Am. Chem. Sot. Buffalo, N. Y., p. 3C, 1952. ZITTLE, C. A., in E. MCDONALD, ed., Neutron Effects on Animals. p. 79. Williams and Wilkins Co., Baltimore, Md., 1947. GOLDSTEIN, M. J., AND NEWORASCHHIN, J. P., Strahlentherupie 36,736 (1930). Razuo, M., MOMOKI, S., AND ITO, H., Zgaku to Seibutsugaku 18,303 (1951).
11. FEINSTEIN, R. N., BVTLER, C. I,., .~ND HENDLEY, 11. C., Rcience 111, 14!) (1950). 12. LUDEWIU, S., AND CHANUTIN, A., Arch. Biochem. 29, 441 (1956). 13. DINNING, J. S., MESCHAN, I., KEITH, C. K., AND Day, I’. I,., Proc. Sot. Espi/. Biol. Med. 74, 776 (1950). l-1-. LEMAY, M., Proc. Sot. Exptl. Biol. Med. 77, 337 (1951). 15. BARRON, E. S. G., Declsssified Document AECD-2316 (CH-3654). I’. S. ,4tomic Energy Commission, 1946. 16. Aslfwm,L, G., AND HTCKUAN, J., Proc. Sot. Exptl. Biol. Med. 80, 407 (1952). IT. KUNKEL, H. O., .~ND FHILLIPS, P. H., Arch. Biochem. and Biophys. 37, 366 (1952). 1X. BURN, J. H., KORDIK, I’., AND ~IOLE, R. H., Brit. d. Pharmacol. 7, 58 (1952). 19. FETRAKIS, N. L., ASHLER, F. hr., AND FERKEL, R. T,., S;~v:ll Rsdiol. Defense Report, AD 126B, 1919. 20. LUDEWIG, S., AND CHANUTIN, A., Am. J. Physiol. 163, 6# (1950). 21. LUDEWJG, S., Federation Proc. 9, 198 (1950). 22. BALLJN, J. C., AND FEINSTEIN, R. N., Federation Proc. 11. 184 (1952). 23. T.~NzI, B., Boll. sot. ital. biol. sper. 14, 732 (1939). 24. BRIN, M., Federation Proc. 11, 190 (1952). 25. SMITH, D. E., TYREE, E. B., PATT, H. M., .4x1) J.WKSOK, I,:., Proc. Sot. Es@. Biol. Med. 78, 774 (1951).