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Increase in glutathione and glutathione reductase in livers of rats fed ethionine
ARCHIVES
OF
Increase
BIOCHEMISTRY
AND
BIOPHYSICS
in Glutathione
and
118,
85-89
(1967)
Glutathione
Reductase
in Livers
of
Rats Fed Ethionine JENG IV. HSU
AND
SIGMUND
GELLER
Biochemistry Research Laboratory, Veterans Administration
Hospital, and the Department of Biochemistry, School of Hygiene and Public Health, The Johns Hopkins University, Baltimore, Maryland Received May 18, 1966
The effect of feeding a stock diet to which had been added 0.25yo of DL-ethionine on liver-reduced glutathione (GSH), glutathione reductase (GR), glucose 6-phosphate dehydrogenase (G-6-PD), and 6-phosphogluconate dehydrogenase (6-PGD) was studied in male and female rats. Liver GSH concentration was consistently in-
creased in rats of both sexes fed ethionine. This effect could not be prevented by methionine, which, in fact, also increased liver GSH, but was reversible by feeding an ethionine-free
diet. Cysteine, added to the stock diet in place of ethionine, did not
increase hepatic GSH. Activities of the enzymes GR and G-6-PD, but not of 6-PGD, were significantly higher in the livers of the ethionine fed rats. Additional studies indicated that ascorbic acid concentrations in blood, liver, and adrenals were reduced after ethionine feeding. ditioned room in individual screen-bottom cages, were employed. Controls received a stock diet of Purina Laboratory Chow, and the various experimental animals, the same diet supplemented with 0.250jo each of DL-ethionine or methionine, or both, or cysteine. Experimental and control animals were offered equal weighed amounts of their respective diets (except those in the reversibility experiment, which were fed ad libitum). Water was supplied ad libitum. At appropriate intervals the rats were anesthetized with Nembutal and exsanguinated by heart puncture with a heparinized syringe. In certain experiments, a decapitator was used to sacrifice the animals without anesthesia. Tissues were excised as soon as possible after sacrifice. GSH assay. GSH in blood and liver was measured by the method of Patterson and Lazarow (5). The reaction of GSH with alloxan was used to produce a substance with a maximum absorbance at 305 rnp. Blood was prepared for assay as indicated by the authors with the exception that the precipitated proteins were removed from the hemolyzate by filtration through Whatman No. 2 filter paper rather than by centrifugation. Liver was saturated with NaCl and homogenized in 3040 volumes of ice-cold 5yo metaphosphoric acid in a Potter-Elvehjem homogenizer. The
The numerous morphological and biochemical effects of the ethyl analogue of methionine, ethionine, have been reviewed comprehensively by Farber (1) and also by Stekol (2). Among these are production of fatty liver, inhibition of protein synthesis in vitro and in vivo, and reductions in hepatic phosphorylase activity and ATP concentration, each of which can be counteracted not only by methionine but, also by ATP or adenine. This depression of ATP level led us to study the effect of ethionine on the metabolism of the tripeptide glutathione, which requires 1 mole of ATP in each of its two stages of synthesis (3). The present report, concerns the effect of ethionine feeding on GSH and ascorbic acid concentrations in tissues and on the activities in the liver of GR, G-6-PD, and 6-PGD. A preliminary communication has been published (4). EXPERIMENTAL
PROCEDURE
McCollum strain rats, matched with respect t.o sex, age, and weight, and housed in an air-con85
86
HSU
AND
GELLER
TABLE EXPECT OF ETHIONINE
Diet”
Body
No. rats
FEEDING wt.
Liver
Males Stock +E Females Stock +E
AND
T
(pm)
Initial
I
ON GROWTH
Finn1
wt.
(gm)
ORGAN
WEIGHTS
wt. (gm)/lOl gm body wt.
Liver
Kidney
374 f 305 f
18b 20
330 f 292 f
226 f 228 f
7 6
210 Zt 7 199 f 5
TABLE
-
Diet
10 11
8.2 f 10.9 f
-
i
0.02 0.52c
5.9 zt 0.27 8.2 f 0.25d
,n diet
Males Stock SE Stock +E Stock +E Stock +E
1 1 2 2 3 3 7 7
GSHO
Females Stock +E Stock +E Stock i-E
3 3 4 4 8 8
.-
5 5 5 6 6 6 5 5
179 211 221 268 182 263 198 272
7 8 5 6 5 6
173 249 202 287 198 237
f f f f f f f f
f f f f f f
337 492 367 530 381 454 393 556
359 675 465 654 433 770
9 21” 10 20” 16 7
- - u pmoles per 100 ml erythrocytes liver. * Mean f SE. c p < 0.05. dp < 0.01.
b~$,($~/
0.18 0.09
0.6 f 0.8 f
0.03 0.03”
2.8 f 4.2 f
1.3 f 1.4 *
0.04 0.05
0.6 f 0.7 *
0.01 0.04
0.08 0.12d
f f f f f f f f
Diet’
f f f f f f
17 23d 24 21d 11 29d 21 14d
14 lgd 12 16d 28 gd
or IW
gm
homogenate was kept in the cold for an hour or more, shaken occasionally, and filtered as above. Ascorbic acid assay. Total ascorbic acid in blood, liver, and adrenal glands was assayed according to the Schaffert and Kingsley (6) modification of the dinitrophenylhydrazine method of
Each male rat received
III
REVERSIBILITY OF EFFECT OF ETHIONINE ON GSH IN RAT ERYTHROCYTES AND LIVER BY OMITTING ETHIONINE FROM DIET
Liver
20b 19 7 23 5 23” 26 20
Eodig
2.1 f 2.4 f
TABLE
Erythrocytes
(pm)
2.4 f O.OG 3.5 f: 0.16”
II
FEEDING ON GSH LEVELS AND LIVER IN THE RAT NO. rats
Weeks
wt
RAT
_-
a Stock: Purina Laboratory Chow. + E: Stock diet + 0.25% nn-ethionine. 14 gm, and each female? 12 gm of diet daily for 4 weeks. b Mean f SE. c p < 0.05. d p < 0.01.
EFFECT OF ETHIONINE IN ERYTHROCYTES
IN THE
Experiment Stock
No. rats
GSHb
Erythrocytes
Liver
A
+E Experiment B Stock 1 1 then 1 stock SE I
6 6
226 f 319 f
llc 15d
5
196 f
6
5
189 i
2
a Female rats employed. For mals received 12 gm daily of their For an additional 4 weeks, rats fed stock diet ad libitum. b ~moles/lOO ml erythrocytes c Mean f SE. d p < 0.01.
441 rt 22 649 & 22d 500 f
11
524 f
22
3 weeks, all anirespective diets. in Expt. B were or 100 gm liver.
Roe and Kuether (7). Blood was prepared for assay as indicated by the authors; liver and adrenals; by homogenizing with 6% trichloroacetic acid. Enzyme assays. Liver was homogenized in 19 volumes of ice-cold 0.25 M sucrose-O.001 M EDTA; a motor-driven Teflon pestle tissue grinder was used. The supernatant fluid obtained by centrifuging the homogenate at 105,000 g for 20 minutes at 0” in a Spinco model L ultracentrifuge was assayed for GR by a modification of the method of Manso and Wroblewski (8). The activities of G-6-PD and 6PGD in the
ETHIONINE
FEEDING
AND
cantly. However, there was a marked increase in liver weight and in the ratio of liver t’o body weight. Weight of kidneys showed no appreciable change. The effect of ethionine on GSH concentrations in erythrocytes and liver of male and female rats is shown in Table II. In both sexes, liver GSH levels were sign%cantly elevated, and erythrocyte GSH was somewhat increased. The data summarized in Table III demonstrate that the feeding of stock diet to rats previously fed ethionine completely restored to normal both erythrocyte and liver GSH levels. To determine whether methionine would prevent the ethionine-induced increment in GSH, this amino acid was fed simultaneously. Table IV shows that hepatic GSH remained elevated. Furthermore, feeding a supplement to the stock diet of methionine alone resulted in a significant rise in GSH. We have also confirmed the finding (12) that a low-methionine soy prot,ein based diet results in lower hepatic GSH levels than does a methionine-supplemented regimen. Addition of cysteine to the stock diet did not affect the concentration of GSH in liver or erythrocyt’es (Table IV). The act,ivities of the enzymes GR, G-6I’D, and 6-PGD, as affected by ethionine, are presented in Table V. The activity of GR increased markedly, as did the concentration of GSH. The activit’y of G-6-PD (but not of 6-PGD) also rose substantially. Ingestion of ethionine, while increasing
supernatant fluid were assayed by the method of Glock and McLean (9) as modified by Fitch et al. (10). Absorbances for all enzyme assays were measured at 340 rnp at 25” in a Beckman Model DU spectrophotometer equipped with thermospacers. Protein was estimated by the met,hod of Lowry et al. (ll), and crystalline bovine plasma albumin was used as the standard. RESULTS
Table I indicates that the ingestion of ethionine by male and female rats for 4 weeks did not change body weight’ signifiTSBLE
IV
EFFECT OF FEEDING ETHIONINE OR METHIONINE, OR BOTH OR CYSTEINE ON GSH LEVELS IN ERYTHROCYTES AND LIVER IN THE RAT DieP
\Teeks No. rats
Stock i-E + EM Stock + M + EM Stock fC
3 3 3 3 3 3 2 2
6 G G 4 6 G 6 6
GSHb Erythrocytes
204 220 235 192 190 260 177 194
Liver
zk llc z!z 15 f 17 f 27 f l-1 f 14 f 10 f 5
321 533 514 355 451 588 422 415
& 22 f 23d h 7d f 9 f 9d & 14d i 5 ZIX 14
a Male rats employed. f E: 0.25% DL-ethionine, + 11: 0.25%‘, nL-methionine, + EM: 0.250/;, nL-ethionine and 0.25yC nL-methionine, + C: 0.25y0 nL-cysteine-HCl. b ~moles/lOO ml erythrocytes or 100 gm liver. c Mean i SE. dp < 0.01. TABLE ACTIVITIES Dieta
Experiment Stock SE Experiment Stock +E
OF G.R.,
G-6-PD,
GSHb
87
GLUTATHIONE
V
SND 6-PGD
IN LIVERS
GRC
OF Ram
FED
ETHIONINE
G-6.l’Dd
6-PGDd
I 410 f 640 f
20d 35’
37 * 67 *
3.1 3.9f
57 * 92 f
482 f 638 f
18 23’
33 f 81 f
1.5 5.6f
65 f 127 f
7.8 6.3”
30 f 23 f
3.2 1.3
5.1 19.3e
36 f 32 f
1.5 2.4
II
a Female rats on diet for 3 weeks; 4 per group in Expt. b pmoles/lOO gm liver. c pmoles of substrate utilized/minute/gm protein. d Mean f SE. op < 0.05.
‘p < 0.01.
I and 5 per group
in Expt.
II.
HSU AND GELLER
88
TABLE EFFECT OF ETHIONINE DieP
Stock ; Ehange
FEEDING
ON
VI
ASCORBIC ACID
AND
GSH* Liver
421 f 21” 630 f+4921’
GSH
LEVELS
IN RAT
TISSUES
Ascorbic acida Erythrocytes
226 f 11 319 f+4115d
Liver
136 f 11 79 f-424”
Blood
3.00 f 0.16 1.70 f-430.29”
Adrenals
1830 f 120 1110 f -39see
a 6 female rats per group on diet for 3 weeks. * ~moles/lOO gm tissue, 100 ml erythrocytes, or 100 ml blood. c Mean f SE. d p < 0.05. “p < 0.01.
hepatic GSH, caused ascorbic acid to decrease by about the same percentage. Ascorbic acid concentrations in the adrenals and in blood were similarly depressed (Table VI). DISCUSSION
As part of a study of the effect of amethopterin on mice implanted with lymphocytic leukemias, Schachter and Law (13) observed that injection of ethionine caused liver GSH (which had been depressed by the tumor) to increase 50% above the level in controls (tumor-bearing mice not injected with ethionine). The effect on normal mice was not tested. Our finding that ethionine feeding not only did not depress, but actually increased, hepatic GSH probably indicates that an ATP-trapping effect of S-adenosylethionine (14) is not inhibiting GSH synthesis. Furthermore, the facts that simultaneous supplementation of the stock diet with methionine and ethionine did not prevent the increase in GSH and that the methionine supplement alone caused an increase in GSH imply that ethionine and methionine are undergoing the same reactions and producing similar, rather than antagonistic, effects. One possible explanation for the increased level of GSH is that ethionine is being converted to cysteine, the increased availability of which results in increased synthesis of the tripeptide. Simpson et al. (15) had shown that ethionine interferes with the conversion of methionine S to cysteine; Stekol and Weiss (16), that only a small
amount of 36S-labeled ethionine was converted to cysteine; and Wu and Bollman (17), that ethionine injection increased the level of free cysteine in the liver of the rat. Recently, it was reported (18, 19) that feeding or injecting rats with either ethionine or methionine increased the cystathionase activity and the free homoserine level in the liver. No mention was made of the amount of the co-product of the cystathionase reaction, cysteine. As can be seen from Table IV, a dietary supplement of cysteine had not effect on GSH concentrations. Ghosh (20), using the guinea pig, found that a dietary supplement of 2 % cysteineHCl did not affect the content of GSH in most tissues and lowered it slightly in the liver. In general, cysteine, unlike methionine, has been found to have little or no ability to counteract the effects of ethionine (1). The fact that GSH and ascorbic acid are inversely affected by ethionine is of interest because of their common biological role as reducing agents. The increase in activity of GR serves this reduction function by maintaining glutathione in the reduced state, as does the augmented activity of G-6-PD by its production of TPNH. This increase in activity of G-6-PD, but not of 6-PGD, confirms t’he findings of Sie and Hablanian with rats (21) and mice (22) fed 0.5% ethionine. Ascorbic acid levels have previously been reported (23, 24) to be unaffected by ethionine treatment, although the induction of ascorbic acid synthesis by drugs was suppressed by it.
ETHIONINE
FEEDING
Recently, Kremzner and Starr (25) reported an ethionine-induced increase in liver spermidine in male and female rats and in liver histamine in females. It is of interest that Tabor et al. (26) demonstrated in E. coli the synthesis of spermine from methionine, and that Dubin (27) and Tabor et al. (28) isolated from this organism a spermidine-“glutathione” compound (in which cysteine is replaced by cysteic acid) and purified an enzyme catalyzing its synthesis requiring glutathione, ATP and Mg++ . It is certain that mode, dosage, and time relationships of administration affect the actions of ethionine reported here. In a preliminary communication (29) we have reported that injection of rats with ethionine (1 mg/gm of body weight) resulted in a sharp drop in hepatic GSH at 5 hours. By 24 hours, this effect was reversed and GSH was increased (30). Undoubtedly, also involved are the complex problems of ATP priorities (14) and of differential cell growth (1, 31, 32).
AND
10. 11.
12. 13. 14.
15. 16. 17. 18. 19. 20. 21. 22.
L.
REFERENCES 1. FARBER, E., Advan. Cancer Res. 7, 383 (1963). 2. STEHOL, J. A., Advan. Enzymol. 26,369 (1963). 3. SNOKE, J. E., AND BLOCH, K., J. Biol. Chem. 199, 407 (1952). 4. Hsu, J. M., GELLER, S., -44~~ WANG, C. L., Federation hoc. 25, 363 (1966). 5. PATTERSON, J. W., AND LAZARO~, A., in “Methods of Biochemical Analysis” (D. Glick, ed.), Vol. 2, p. 274. Wiley (Interscience), New York (1955). 6. SCHAFFERT, R. R., AND KINGSLEY, G. R., J. Biol. Chem. 212, 59 (1955). 7. ROE, J. H., AND KUETHER, C. A., J. Biol. Chem. 147, 399 (1943). 8. MANSO, C., AND WROBLEWSKI, F., J. Clin. Invest. 37, 214 (1958).
23. 24. 25. 26. 27. 28. 29.
30. 31. 32.
89
G. E., AND MCLEAN, P., Biochem. J. 66, 400 (1953). FITCH, W. M., HILL, R., AND CHAIKOFF, I. L., J. Biol. Chem. 234, 1048 (1959). LOWRY, 0. H., ROSEBROIJGH, N. J., FARR,. A. L., AND RANDALL, R. J., J. Biol. Chem. 193, 265 (1951). MORTENSEN, R. A., J. Biol. Chem. 204, 239’ (1953). SCHACHTER, B ., AND LAW, L. W., J. Natl. Cancer Inst. 17, 391 (1956). FARBER, E., SHULL, K. H., VILLA-TREVINO, S., LOMBARDI, B., AND THOMAS, M., Nature 203, 34 (1964). SIMPSON, M. V., FARBER, E., AND TARVER, If., J. Biol. Chem. 182, 81 (1950). STEKOL, J. A., AND WEISS, K., J. Biol. Chem.. 186, 577 (1950). Wu, C., AND BOLLMAN, J. L., J. Biol. Chem210, 673 (1954). CHATAGNER, F., AND TRAUTMANN, O., Nature 200, 75 (1963). CHATAGNER, F., Nature 203, 1177 (1964). GHOSH, B. K., Ann. Biochem. Exptl. Med. 23, 595 (1963). SIE, H. G., AND HABLANIAN, A., Nature 206, 1317 (1965). SIE, H. G., AND HABLANIAN, A., Biochem. J. 97, 32 (1965). BERNHEIM, M. L. C., Biochem. Pharmacol. 7, 59 (1961). TOUSTER, O., .~ND HOLLMANN, S., Ann. N.Y. Acad. Sci. 92, 318 (1961). KREMZNER, L. T., AND STARR, R. M., Federation hoc. 26, 559 (1966). TABOR, H., ROSENTHAL, S. M., AXD TABOR, C. W., J. Biol. Chem. 233, 907 (1958). DUBIN, D. T., Biochem. Biophys. Res. Commun. 1, 262 (1959). TABOR, C. W., TABOR, H., BND DEMEIS, L., Federation Proc. 26, 709 (1966). Hsu, J. M., GELLER, S., AND WANG, C. L., Abstr. Am. Chem. Xoc. 150th Meeting, C243 (September 1965). HSU, J. M., GELLER, S., AND WANG, C. L., unpublished observations (1965). RUBIN, E., HUTTERER, F., GALL, E. C., AND POPPER, H., Nature 192, 886 (1961). RUBIN, E., Federation Proc. 26, 479 (1966).
9. GLOCK,
ACKNOWLEDGMENTS The authors are grateful to Mrs. Chun Wang for her excellent technical assistance.
GLUTATHIONE
OF
Increase
BIOCHEMISTRY
AND
BIOPHYSICS
in Glutathione
and
118,
85-89
(1967)
Glutathione
Reductase
in Livers
of
Rats Fed Ethionine JENG IV. HSU
AND
SIGMUND
GELLER
Biochemistry Research Laboratory, Veterans Administration
Hospital, and the Department of Biochemistry, School of Hygiene and Public Health, The Johns Hopkins University, Baltimore, Maryland Received May 18, 1966
The effect of feeding a stock diet to which had been added 0.25yo of DL-ethionine on liver-reduced glutathione (GSH), glutathione reductase (GR), glucose 6-phosphate dehydrogenase (G-6-PD), and 6-phosphogluconate dehydrogenase (6-PGD) was studied in male and female rats. Liver GSH concentration was consistently in-
creased in rats of both sexes fed ethionine. This effect could not be prevented by methionine, which, in fact, also increased liver GSH, but was reversible by feeding an ethionine-free
diet. Cysteine, added to the stock diet in place of ethionine, did not
increase hepatic GSH. Activities of the enzymes GR and G-6-PD, but not of 6-PGD, were significantly higher in the livers of the ethionine fed rats. Additional studies indicated that ascorbic acid concentrations in blood, liver, and adrenals were reduced after ethionine feeding. ditioned room in individual screen-bottom cages, were employed. Controls received a stock diet of Purina Laboratory Chow, and the various experimental animals, the same diet supplemented with 0.250jo each of DL-ethionine or methionine, or both, or cysteine. Experimental and control animals were offered equal weighed amounts of their respective diets (except those in the reversibility experiment, which were fed ad libitum). Water was supplied ad libitum. At appropriate intervals the rats were anesthetized with Nembutal and exsanguinated by heart puncture with a heparinized syringe. In certain experiments, a decapitator was used to sacrifice the animals without anesthesia. Tissues were excised as soon as possible after sacrifice. GSH assay. GSH in blood and liver was measured by the method of Patterson and Lazarow (5). The reaction of GSH with alloxan was used to produce a substance with a maximum absorbance at 305 rnp. Blood was prepared for assay as indicated by the authors with the exception that the precipitated proteins were removed from the hemolyzate by filtration through Whatman No. 2 filter paper rather than by centrifugation. Liver was saturated with NaCl and homogenized in 3040 volumes of ice-cold 5yo metaphosphoric acid in a Potter-Elvehjem homogenizer. The
The numerous morphological and biochemical effects of the ethyl analogue of methionine, ethionine, have been reviewed comprehensively by Farber (1) and also by Stekol (2). Among these are production of fatty liver, inhibition of protein synthesis in vitro and in vivo, and reductions in hepatic phosphorylase activity and ATP concentration, each of which can be counteracted not only by methionine but, also by ATP or adenine. This depression of ATP level led us to study the effect of ethionine on the metabolism of the tripeptide glutathione, which requires 1 mole of ATP in each of its two stages of synthesis (3). The present report, concerns the effect of ethionine feeding on GSH and ascorbic acid concentrations in tissues and on the activities in the liver of GR, G-6-PD, and 6-PGD. A preliminary communication has been published (4). EXPERIMENTAL
PROCEDURE
McCollum strain rats, matched with respect t.o sex, age, and weight, and housed in an air-con85
86
HSU
AND
GELLER
TABLE EXPECT OF ETHIONINE
Diet”
Body
No. rats
FEEDING wt.
Liver
Males Stock +E Females Stock +E
AND
T
(pm)
Initial
I
ON GROWTH
Finn1
wt.
(gm)
ORGAN
WEIGHTS
wt. (gm)/lOl gm body wt.
Liver
Kidney
374 f 305 f
18b 20
330 f 292 f
226 f 228 f
7 6
210 Zt 7 199 f 5
TABLE
-
Diet
10 11
8.2 f 10.9 f
-
i
0.02 0.52c
5.9 zt 0.27 8.2 f 0.25d
,n diet
Males Stock SE Stock +E Stock +E Stock +E
1 1 2 2 3 3 7 7
GSHO
Females Stock +E Stock +E Stock i-E
3 3 4 4 8 8
.-
5 5 5 6 6 6 5 5
179 211 221 268 182 263 198 272
7 8 5 6 5 6
173 249 202 287 198 237
f f f f f f f f
f f f f f f
337 492 367 530 381 454 393 556
359 675 465 654 433 770
9 21” 10 20” 16 7
- - u pmoles per 100 ml erythrocytes liver. * Mean f SE. c p < 0.05. dp < 0.01.
b~$,($~/
0.18 0.09
0.6 f 0.8 f
0.03 0.03”
2.8 f 4.2 f
1.3 f 1.4 *
0.04 0.05
0.6 f 0.7 *
0.01 0.04
0.08 0.12d
f f f f f f f f
Diet’
f f f f f f
17 23d 24 21d 11 29d 21 14d
14 lgd 12 16d 28 gd
or IW
gm
homogenate was kept in the cold for an hour or more, shaken occasionally, and filtered as above. Ascorbic acid assay. Total ascorbic acid in blood, liver, and adrenal glands was assayed according to the Schaffert and Kingsley (6) modification of the dinitrophenylhydrazine method of
Each male rat received
III
REVERSIBILITY OF EFFECT OF ETHIONINE ON GSH IN RAT ERYTHROCYTES AND LIVER BY OMITTING ETHIONINE FROM DIET
Liver
20b 19 7 23 5 23” 26 20
Eodig
2.1 f 2.4 f
TABLE
Erythrocytes
(pm)
2.4 f O.OG 3.5 f: 0.16”
II
FEEDING ON GSH LEVELS AND LIVER IN THE RAT NO. rats
Weeks
wt
RAT
_-
a Stock: Purina Laboratory Chow. + E: Stock diet + 0.25% nn-ethionine. 14 gm, and each female? 12 gm of diet daily for 4 weeks. b Mean f SE. c p < 0.05. d p < 0.01.
EFFECT OF ETHIONINE IN ERYTHROCYTES
IN THE
Experiment Stock
No. rats
GSHb
Erythrocytes
Liver
A
+E Experiment B Stock 1 1 then 1 stock SE I
6 6
226 f 319 f
llc 15d
5
196 f
6
5
189 i
2
a Female rats employed. For mals received 12 gm daily of their For an additional 4 weeks, rats fed stock diet ad libitum. b ~moles/lOO ml erythrocytes c Mean f SE. d p < 0.01.
441 rt 22 649 & 22d 500 f
11
524 f
22
3 weeks, all anirespective diets. in Expt. B were or 100 gm liver.
Roe and Kuether (7). Blood was prepared for assay as indicated by the authors; liver and adrenals; by homogenizing with 6% trichloroacetic acid. Enzyme assays. Liver was homogenized in 19 volumes of ice-cold 0.25 M sucrose-O.001 M EDTA; a motor-driven Teflon pestle tissue grinder was used. The supernatant fluid obtained by centrifuging the homogenate at 105,000 g for 20 minutes at 0” in a Spinco model L ultracentrifuge was assayed for GR by a modification of the method of Manso and Wroblewski (8). The activities of G-6-PD and 6PGD in the
ETHIONINE
FEEDING
AND
cantly. However, there was a marked increase in liver weight and in the ratio of liver t’o body weight. Weight of kidneys showed no appreciable change. The effect of ethionine on GSH concentrations in erythrocytes and liver of male and female rats is shown in Table II. In both sexes, liver GSH levels were sign%cantly elevated, and erythrocyte GSH was somewhat increased. The data summarized in Table III demonstrate that the feeding of stock diet to rats previously fed ethionine completely restored to normal both erythrocyte and liver GSH levels. To determine whether methionine would prevent the ethionine-induced increment in GSH, this amino acid was fed simultaneously. Table IV shows that hepatic GSH remained elevated. Furthermore, feeding a supplement to the stock diet of methionine alone resulted in a significant rise in GSH. We have also confirmed the finding (12) that a low-methionine soy prot,ein based diet results in lower hepatic GSH levels than does a methionine-supplemented regimen. Addition of cysteine to the stock diet did not affect the concentration of GSH in liver or erythrocyt’es (Table IV). The act,ivities of the enzymes GR, G-6I’D, and 6-PGD, as affected by ethionine, are presented in Table V. The activity of GR increased markedly, as did the concentration of GSH. The activit’y of G-6-PD (but not of 6-PGD) also rose substantially. Ingestion of ethionine, while increasing
supernatant fluid were assayed by the method of Glock and McLean (9) as modified by Fitch et al. (10). Absorbances for all enzyme assays were measured at 340 rnp at 25” in a Beckman Model DU spectrophotometer equipped with thermospacers. Protein was estimated by the met,hod of Lowry et al. (ll), and crystalline bovine plasma albumin was used as the standard. RESULTS
Table I indicates that the ingestion of ethionine by male and female rats for 4 weeks did not change body weight’ signifiTSBLE
IV
EFFECT OF FEEDING ETHIONINE OR METHIONINE, OR BOTH OR CYSTEINE ON GSH LEVELS IN ERYTHROCYTES AND LIVER IN THE RAT DieP
\Teeks No. rats
Stock i-E + EM Stock + M + EM Stock fC
3 3 3 3 3 3 2 2
6 G G 4 6 G 6 6
GSHb Erythrocytes
204 220 235 192 190 260 177 194
Liver
zk llc z!z 15 f 17 f 27 f l-1 f 14 f 10 f 5
321 533 514 355 451 588 422 415
& 22 f 23d h 7d f 9 f 9d & 14d i 5 ZIX 14
a Male rats employed. f E: 0.25% DL-ethionine, + 11: 0.25%‘, nL-methionine, + EM: 0.250/;, nL-ethionine and 0.25yC nL-methionine, + C: 0.25y0 nL-cysteine-HCl. b ~moles/lOO ml erythrocytes or 100 gm liver. c Mean i SE. dp < 0.01. TABLE ACTIVITIES Dieta
Experiment Stock SE Experiment Stock +E
OF G.R.,
G-6-PD,
GSHb
87
GLUTATHIONE
V
SND 6-PGD
IN LIVERS
GRC
OF Ram
FED
ETHIONINE
G-6.l’Dd
6-PGDd
I 410 f 640 f
20d 35’
37 * 67 *
3.1 3.9f
57 * 92 f
482 f 638 f
18 23’
33 f 81 f
1.5 5.6f
65 f 127 f
7.8 6.3”
30 f 23 f
3.2 1.3
5.1 19.3e
36 f 32 f
1.5 2.4
II
a Female rats on diet for 3 weeks; 4 per group in Expt. b pmoles/lOO gm liver. c pmoles of substrate utilized/minute/gm protein. d Mean f SE. op < 0.05.
‘p < 0.01.
I and 5 per group
in Expt.
II.
HSU AND GELLER
88
TABLE EFFECT OF ETHIONINE DieP
Stock ; Ehange
FEEDING
ON
VI
ASCORBIC ACID
AND
GSH* Liver
421 f 21” 630 f+4921’
GSH
LEVELS
IN RAT
TISSUES
Ascorbic acida Erythrocytes
226 f 11 319 f+4115d
Liver
136 f 11 79 f-424”
Blood
3.00 f 0.16 1.70 f-430.29”
Adrenals
1830 f 120 1110 f -39see
a 6 female rats per group on diet for 3 weeks. * ~moles/lOO gm tissue, 100 ml erythrocytes, or 100 ml blood. c Mean f SE. d p < 0.05. “p < 0.01.
hepatic GSH, caused ascorbic acid to decrease by about the same percentage. Ascorbic acid concentrations in the adrenals and in blood were similarly depressed (Table VI). DISCUSSION
As part of a study of the effect of amethopterin on mice implanted with lymphocytic leukemias, Schachter and Law (13) observed that injection of ethionine caused liver GSH (which had been depressed by the tumor) to increase 50% above the level in controls (tumor-bearing mice not injected with ethionine). The effect on normal mice was not tested. Our finding that ethionine feeding not only did not depress, but actually increased, hepatic GSH probably indicates that an ATP-trapping effect of S-adenosylethionine (14) is not inhibiting GSH synthesis. Furthermore, the facts that simultaneous supplementation of the stock diet with methionine and ethionine did not prevent the increase in GSH and that the methionine supplement alone caused an increase in GSH imply that ethionine and methionine are undergoing the same reactions and producing similar, rather than antagonistic, effects. One possible explanation for the increased level of GSH is that ethionine is being converted to cysteine, the increased availability of which results in increased synthesis of the tripeptide. Simpson et al. (15) had shown that ethionine interferes with the conversion of methionine S to cysteine; Stekol and Weiss (16), that only a small
amount of 36S-labeled ethionine was converted to cysteine; and Wu and Bollman (17), that ethionine injection increased the level of free cysteine in the liver of the rat. Recently, it was reported (18, 19) that feeding or injecting rats with either ethionine or methionine increased the cystathionase activity and the free homoserine level in the liver. No mention was made of the amount of the co-product of the cystathionase reaction, cysteine. As can be seen from Table IV, a dietary supplement of cysteine had not effect on GSH concentrations. Ghosh (20), using the guinea pig, found that a dietary supplement of 2 % cysteineHCl did not affect the content of GSH in most tissues and lowered it slightly in the liver. In general, cysteine, unlike methionine, has been found to have little or no ability to counteract the effects of ethionine (1). The fact that GSH and ascorbic acid are inversely affected by ethionine is of interest because of their common biological role as reducing agents. The increase in activity of GR serves this reduction function by maintaining glutathione in the reduced state, as does the augmented activity of G-6-PD by its production of TPNH. This increase in activity of G-6-PD, but not of 6-PGD, confirms t’he findings of Sie and Hablanian with rats (21) and mice (22) fed 0.5% ethionine. Ascorbic acid levels have previously been reported (23, 24) to be unaffected by ethionine treatment, although the induction of ascorbic acid synthesis by drugs was suppressed by it.
ETHIONINE
FEEDING
Recently, Kremzner and Starr (25) reported an ethionine-induced increase in liver spermidine in male and female rats and in liver histamine in females. It is of interest that Tabor et al. (26) demonstrated in E. coli the synthesis of spermine from methionine, and that Dubin (27) and Tabor et al. (28) isolated from this organism a spermidine-“glutathione” compound (in which cysteine is replaced by cysteic acid) and purified an enzyme catalyzing its synthesis requiring glutathione, ATP and Mg++ . It is certain that mode, dosage, and time relationships of administration affect the actions of ethionine reported here. In a preliminary communication (29) we have reported that injection of rats with ethionine (1 mg/gm of body weight) resulted in a sharp drop in hepatic GSH at 5 hours. By 24 hours, this effect was reversed and GSH was increased (30). Undoubtedly, also involved are the complex problems of ATP priorities (14) and of differential cell growth (1, 31, 32).
AND
10. 11.
12. 13. 14.
15. 16. 17. 18. 19. 20. 21. 22.
L.
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89
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9. GLOCK,
ACKNOWLEDGMENTS The authors are grateful to Mrs. Chun Wang for her excellent technical assistance.
GLUTATHIONE