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Effect of endotoxin on the rat colon glutathione level
Vol. 151, No. 2, 1988
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 844-850
March 15, 1988
EFFECT OF ENDOTOXIN ON THE RAT COLON GLUTATHIONE LEVEL M.F. Chen, L.T. Chen', and HoW. Boyce, Jr.
Departments of Internal Medicine and Anatomy* University of South Florida College of Medicine Tampa, Florida 33612-4799 Received February 3, 1988
The effect of endotoxin on the colon glutathione level was studied in male rats. Endotoxin (Escherichia cold from 25 ug to 1000 ug/100g body weight was administered intravenously. The Glutathione level was measured 16 hours after endotoxin was given. Results showed that endotoxin signficantly enhanced the colon glutathione concentration as measured by 5,5'-dithiobis (2-nitrobenzoic acid). The increase which ranged from 11% to 50% was dose dependent. At an endotoxin dose of 1000 ug/100g body weight, colon glutathione level was found to be enhanced from 2 hours up to 48 hours. In contrast, the duodenum and jejunum glutathione levels were found to be significantly reduced. The increase in the colon glutathione level may have a protective effect against oxidative damage to the colon. ®1988AcademicPress, Znc
High levels of reduced glutathione (GSH) (L-7-glutamyl-L-cysteinyl-glycine) has been reported to be potentially useful as an anticarcinogen(1,2).
Perchellet
and associates(l) showed that in mice, intraperitoneal administration of GSH before treatment with 12-0-tetradecanoyl-phorbol-13-acetate (TPA) inhibited the formation of skin papillomas by more than 50 percent.
Novi(2) showed that
administration of GSH by stomach intubation to rats bearing aflatoxin B 1 induced liver tumors, regressed tumor growth. The mechanism (s) by which GSH treatments inhibit the development of skin papilloma or cause the regression of hepatoma is not clear.
The compound, however, has been shown to inhibit
ornithine decarboxylase (L-ornithine carboxy-lyase (E.C.4.1.1.17) activity induced by TPA(1). Endotoxin has been shown, clinically and experimentally, to elicit damage in certain types of tumors(3-6).
Among the malignant tumors which have been
reported to respond to toxin therapy include carcinoma of the cervix and intestines, carcinoma of the ovary with metastases to the intestines, peritoneum, liver and colon, and carcinoma of the nasopharynx(3). In rats, peritoneal carcinoma0006-291X/88 $1.50 Copyright © 1988 by Academic Press, Inc. All rights of reproduction in any form reserved.
844
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BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
tosis(6), and in mice, several subcutaneous transplanted tumors e.g. SA-1 sarcoma and sarcoma 37 have been shown to regress with endotoxin treatment(4,5). The mechanisms by which endotoxin causes necrosis and/or regression of these tumors are not clear, but the complex immune system has been suggested to play a role(7-10). Since increased GSH and endotoxin therapy have both been shown to cause the regression of certain types of tumor, it is probable t h a t endotoxin t r e a t m e n t may result in an increase in the GSH level in certain tissues or organs with or without tumors in association. The purpose of this study therefore, is to investigate the e f f e c t of endotoxin on the colon GSH level in rats.
The concentration of GSH in the duodenum and
jejunum were also measured for comparison.
If endotoxin is found to raise the
colon GSH level, the next phase of the study will be to investigate the e f f e c t of endotoxin on colon carcinoma. Material and Methods
Animals: Male Sprague Dawley rats (200-250 g) were obtained from Holtzman laboratory. Prior to the experimental t r e a t m e n t , rats were maintained on Purina Chow and w a t e r ad libitum for one week following r e c e i p t from the supplier. Endotoxin: A 0.6¢ solution of endotoxin (Escherichia coli 055:B5, Difco Laboratories, Detroit, MI) was prepared in sterile pyrogen-free saline. Rats under light ether anesthesia were injected through the penis vein with endotoxin solution (1 mg or less/100 g body weight). Rats injected with pyrogen f r e e saline served as controls. Glutathione Measurement: At specified intervals, rats were lightly anesthesized and then killed by decapitation. The duodenum, jejunum and colon were removed, opened longitudinally, cleansed with cold saline, b l o t t e d dry, weighed and homogenized in 20 times the volume (w/v) of ice cold 5~ t r i c h l o r o a c e t i c acid. The homogenates were centrifuged the supernatants f i l t e r e d through Whatman #42 f i l t e r paper, and the f i l t r a t e s analyzed for GSH according to the method of Ball (11). In this method, tissue cysteine content was first removed by reaction with glyoxylic acid. For the measurement of the colon or duodenum mucosal GSH level, the mucosal was gently scraped off with a scapel and processed as above. Results were expressed as means +_ 1S.D. and s t a t i s t i c a l significance was determined by Student's t - t e s t . P-values a t or less than 0.05 were considered significant. Results The e f f e c t of d i f f e r e n t doses of endotoxin on the colon, duodenum, and j e junum GSH level is shown in Table 1.
Rats were injected with varying doses of 845
Vol. 151, No. 2, 1988
Table 1.
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Effect of various doses of endotoxin on the colon, jejunum and duodenum glutathione levels Tissue glutathione (mg/g wet weight)
Endotox|n-"
0ug
25ug
50ug
100ug
500ug
1000ug
Colon
0.445±0.034 (16)
0.492±0.05* 0.550±0.018" (6) (6)
0.586±0.066* (6)
0.659±0.078* (6)
0.668±0.045* (8)
Jejunum
0.584,0.048 (11)
0.510±0.053" 0.534±0.036* (6) (6)
0.540±0.044 (6)
0.459±0.046* (5)
0.463±0.040* (4)
Duodenum
0.693,0.048 (ii)
0.621±0.07" 0.652±0.055 (5) (6)
0.621±0.046" 0.610±0.049" (6) (6)
0.610±0.029" (4)
Numbers in parentheses are numbers of animals used. Endotoxin dose: ug/100g body weight administered intravenously. Rats were fasted following endotoxin or saline injection. Colon, jejunum, and duodenum glutathione levels were determined 16 hrs after endotoxin administration. Values are means ±1 S.D. Asterisks indicate that values are significantly different (p < 0.05} from control group.
endotoxin (25 ug t o 1000 ug/100g body weight), f a s t e d f o l l o w i n g endotoxin or saline i n j e c t i o n and w e r e killed 16 hours a f t e r endotoxin a d m i n i s t r a t i o n .
Endotoxin
is found t o i n c r e a s e the colon GSH level, and this e f f e c t is dose r e l a t e d .
In con-
t r a s t , both t h e duodenum and jejunum GSH l e v e l s w e r e s i g n i f i c a n t l y lowered. Table 2 shows t h a t the colon GSH l e v e l is s i g n i f i c a n t l y i n c r e a s e d a t 2 hours a f t e r endotoxin a d m i n i s t r a t i o n and r e m a i n e d i n c r e a s e d up to 48 hours.
Both t h e
duodenum and jejunum w e r e s i g n if i can t l y reduced. To d e t e r m i n e w h e t h e r f a s t i n g has any e f f e c t on the duodenum, j e j u m u m , and colon GSH levels, we c o m p a r e d the GSH c o n c e n t r a t i o n in t h e s e s e c t i o n s of t h e gut
Table 2.
Glutathione levels of rat colon, jejunum and duodenum at various time intervals after endotoxin administration Tissue glutathione (mg/g wet weight) Control
2 hrs
6 hrs
16 hrs
Colon
0.445,0.034 (16)
0.465±0.105"* (9)
0.525±0.13"* 0.614±0.096" 0.608,0.113" (4) (8) (4)
0.698±0.095* (6)
Jejunum
0.583±0.048 (II)
0.471,0.051" (5)
0.463±0.04* (4)
0.466±0.093** (7)
Duodenum
0.693,0.048 (II)
0.586,0.012" (5)
0.535±0.025* (4)
0.566±0.061" (7)
Numbers in parentheses are numbers of animals used. Endotoxin dose: 1000ug/100g body weight administered intravenously. Values are m e a n s , 1 S.D. Asterisks indicate that values are significantly different *(p < 0.001). ** (p < 0.05) from control group. 846
24 his
48 hrs
Vol. 151, No. 2, 1988
Table 3.
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Effect of fasting on the rat liver, duodenum, jejunum and colon glutathione level Tissue glutathione (m~/g wet weight) Liver
Fastinga
Duodenum
Jejunum
Colon
1.57+0.15" 0.683_+0.093 0.545+0.078 0.432+0.027
n=6
Non-fasting n=6
2.24+0.11
0.668_+0.051
0.572+0.041
0.454+0.031
Asterisk indicates that the value is significantly different (p < 0.001) from nonfasting value. Values are mean + 1 S.D. a Rats were fasted for 24 hours.
in r a t s which have b e e n f a s t e d for 24 hours to t h a t of n o n - f a s t e d rats. The results are shown in Table 3.
Unlike the liver, gut GSH levels do n o t appear to be af-
f e c t e d by fasting. Table 4 shows t h a t the increase in colon GSH level following endotoxin a d m i n i s t r a t i o n occur mainly in the mucosal layer and to a lesser degree in the muscle layer. Discussion This study showed t h a t the r a t colon GSH level can be s i g n i f i c a n t l y e n h a n c e d by endotoxin t r e a t m e n t . This e n h a n c e m e n t is dose d e p e n d e n t and persists up to 48 hours a t the t e s t e d dose of l m g / 1 0 0 g body weight. In c o n t r a s t , the duodenum and j e j u n u m GSH c o n c e n t r a t i o n is significantly reduced.
Table 4.
In control rats t r e a t e d with
Colon and duodenum mucosal and muscle glutathione concentration after endotoxin administration Tissue glutathione (mg/g wet weight) Colon
Colon
mucosal
muscle
Duodenum mucosal
Duodenum muscle
Endotoxin treated n=4
0.951+0.099"
0.490±0.028
0.676+0.026**
0.474_+0.034
Control
0.730+0.074
0.418+0.081
0.795+0.029
0.564+0.069
n=3 Endotoxin dose: 1000ug/100g body weight administered intravenously. Colon and duodenum glutathione levels were determined 16 hours after endotoxiu administration. Values are means + 1 S.D. Asterisks indicate that values are significantly different * (p < 0.025), • *(p < 0.005) from control group. 847
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BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
pyrogen-free saline, the colon is found to have a lower GSH level than the jejunum which in turn has a lower GSH level than the duodenum. The biological implication of the differences in GSH concentration in the large and small intestine in response to endotoxin is not known at the present time.
However, an enhanced GSH level in the colon may be a protective
mechanism to help maintain the integrity of the colon mucosa and thus prevent the release of potentially pathogenic bacteria and their degradative products which are normally found in the large intestine of mammals. In contrast to the colon, the GSH level in the small intestine decreases after endotoxin injection. Reduced GSH level in the small intestine may contribute to hyperemia and hemorrhage observed in the jejunum and ileum when rats were infused with endotoxin(12). A decrease in the GSH level may result in an increase in the level of lipid peroxidation in tissues.
This is suggested by the fact that the GSH level in the
fasting liver is reduced by 30 percent while the level of lipid peroxidation is increased(13,14). Lipid peroxidation, mediated by H20 2 and free radicals of 02 which are secreted by macrophages in response to endotoxin, is believed to be one of the important
causes of cell
membrane destruction and cell damage(15); cell
membrane contains much lipid, especially unsaturated f a t t y acids(15).
GSH, one
of the major antioxidants in the soluble fraction of cells, plays an important role in the prevention of lipid peroxidation through the action of GSH peroxidase(16). Therefore, an increase in the GSH level in the colon may be a natural response to maintain the integrity of the gut mucosal barrier.
Yoshikawa(17) and associates
reported no significant increase in lipid peroxidation level in the colon, whereas in the ileum mucosa the level was significantly increased in rats infused with 100 mg/kg of endotoxin for 4 hours as compared to control rats infused with physiological saline. The mechanism(s) by which endotoxin causes an increase in the colon GSH level is not known, but interorgan translocation may be a possibility(18), together with increased synthesis.
The loss of GSH in the duodenum and jejunum could be 848
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BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
due to its oxidation to the disulfide form (GSSG) as a result of an increased level of lipid peroxidation and consequent inability to catalyze its reduction to GSH. Some of the loss of GSH in endotoxin shock may also be due to inhibition of synthesis of this compound(19). We tested the effect of fasting on the gut GSH level..We found the GSH levels in the colon, jejunum and duodenum appear to be unaffected by 24 hour fasting while the GSH level in the liver is reduced by 30¢.
Therefore, the
decreased GSH level observed in the jejunum and duodenum after endotoxin treatment is not due to reduced food intake but is likely due to endotoxin. Enhanced GSH level has been shown to regress aflatoxin Bl-induced liver tumors(2).
In the present study, we found that the colon GSH level can be
enhanced by endotoxin treatment. Whether or not GSH plays a role, together with the complex immune system, in endotoxin-induced damage to certain types of tumors remains to be studied. It is probable that endotoxin could be used to treat colon tumors. Acknowledgements This project was supported in part by BRSG S07 RR05749 awarded by the Biomedical Research Support Grant Program, Division of Research Resources, National Institute of Health, and the American Cancer Society, Florida Division Grant No. F87USF-1. References 1. Perchellet, J.P., Owen, M.D., Posey, T.D., Orten, D.K., Schneider, B.A. (1985) Carcinogenesis. 6:567-573. 2. Novi, A.M. Science. (1981) 212:541-542. 3. Nauts, H.C., Fowler, G.A., Bogatho, F.H. (1953) Acta. Med. Scand. Suppl. 276,5-103. 4. Carswell, E.A., Old, L.F., Kassel, R.L., et al. {1975) Proc. Nat. Acad. Sci. 72:3666-3670. 5. O'MaUey, W.E., Achinstein, B., Shear, M.J. (1962) J. Natl. Cancer. Inst. 29:1161-1168. 6. Lagadec, P., Jeannin, J.F., Reisser, D., et al. (1987) Invasion Metastasis. 7:83-95. 7. Berendt, M.J., North, R.J., Kirstein, D.P. (1978) J. Expt. Med. 148, 15501559. 849
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8. Berendt, M.J., North, R.J., Kirstein, D.P. (1978) J. Expt. Med. 148, 15601569. 9. Mannel, D.N., Moore, Immunity. 30, 523-530.
R.N., Mergenhagen, S.E. (1980) Infection and
10. Flick, D.A., Gifford, G.E. (1986) Immunobiol. 171,320-328. 11. Ball, C.R. (1966) Biochem. Pharm. 15, 809-816. 12. Wallace, J.L., Whittle, B.J.R. (1986) European J. Pharmacology 124, 209210. 13. Tateishi, N., Higashi, T., Shinya, S., et al. (1984) J. Biochem. 75, 93-103. 14. Hogberg, J., Moldeus, P., Arborgh, B., et al. (1975) Europ. J. Biochem. 59, 457-462. 15. Tappel, A.L. (1973) Fed. Proc. 32, 1870-1874. 16. Arias, I.M., Jakoby, W.B. (1976) Raven Press, New York.
Glutathione:
Metabolism and function.
17. Yoshikawa, T., Murakami, M., Furukawa, Y., et al. Haemostas. 49,214-216.
(1983) Thromb.
18. Curthoys, N.P. (1986) Fed. Proc. 45, 2166-2169. 19. Beck, L.V., Linkenheimer W. (1952) Proc. Soc. Exp. Biol. Med. 81,291-294.
850
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 844-850
March 15, 1988
EFFECT OF ENDOTOXIN ON THE RAT COLON GLUTATHIONE LEVEL M.F. Chen, L.T. Chen', and HoW. Boyce, Jr.
Departments of Internal Medicine and Anatomy* University of South Florida College of Medicine Tampa, Florida 33612-4799 Received February 3, 1988
The effect of endotoxin on the colon glutathione level was studied in male rats. Endotoxin (Escherichia cold from 25 ug to 1000 ug/100g body weight was administered intravenously. The Glutathione level was measured 16 hours after endotoxin was given. Results showed that endotoxin signficantly enhanced the colon glutathione concentration as measured by 5,5'-dithiobis (2-nitrobenzoic acid). The increase which ranged from 11% to 50% was dose dependent. At an endotoxin dose of 1000 ug/100g body weight, colon glutathione level was found to be enhanced from 2 hours up to 48 hours. In contrast, the duodenum and jejunum glutathione levels were found to be significantly reduced. The increase in the colon glutathione level may have a protective effect against oxidative damage to the colon. ®1988AcademicPress, Znc
High levels of reduced glutathione (GSH) (L-7-glutamyl-L-cysteinyl-glycine) has been reported to be potentially useful as an anticarcinogen(1,2).
Perchellet
and associates(l) showed that in mice, intraperitoneal administration of GSH before treatment with 12-0-tetradecanoyl-phorbol-13-acetate (TPA) inhibited the formation of skin papillomas by more than 50 percent.
Novi(2) showed that
administration of GSH by stomach intubation to rats bearing aflatoxin B 1 induced liver tumors, regressed tumor growth. The mechanism (s) by which GSH treatments inhibit the development of skin papilloma or cause the regression of hepatoma is not clear.
The compound, however, has been shown to inhibit
ornithine decarboxylase (L-ornithine carboxy-lyase (E.C.4.1.1.17) activity induced by TPA(1). Endotoxin has been shown, clinically and experimentally, to elicit damage in certain types of tumors(3-6).
Among the malignant tumors which have been
reported to respond to toxin therapy include carcinoma of the cervix and intestines, carcinoma of the ovary with metastases to the intestines, peritoneum, liver and colon, and carcinoma of the nasopharynx(3). In rats, peritoneal carcinoma0006-291X/88 $1.50 Copyright © 1988 by Academic Press, Inc. All rights of reproduction in any form reserved.
844
Vol. 151, No. 2, 1988
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
tosis(6), and in mice, several subcutaneous transplanted tumors e.g. SA-1 sarcoma and sarcoma 37 have been shown to regress with endotoxin treatment(4,5). The mechanisms by which endotoxin causes necrosis and/or regression of these tumors are not clear, but the complex immune system has been suggested to play a role(7-10). Since increased GSH and endotoxin therapy have both been shown to cause the regression of certain types of tumor, it is probable t h a t endotoxin t r e a t m e n t may result in an increase in the GSH level in certain tissues or organs with or without tumors in association. The purpose of this study therefore, is to investigate the e f f e c t of endotoxin on the colon GSH level in rats.
The concentration of GSH in the duodenum and
jejunum were also measured for comparison.
If endotoxin is found to raise the
colon GSH level, the next phase of the study will be to investigate the e f f e c t of endotoxin on colon carcinoma. Material and Methods
Animals: Male Sprague Dawley rats (200-250 g) were obtained from Holtzman laboratory. Prior to the experimental t r e a t m e n t , rats were maintained on Purina Chow and w a t e r ad libitum for one week following r e c e i p t from the supplier. Endotoxin: A 0.6¢ solution of endotoxin (Escherichia coli 055:B5, Difco Laboratories, Detroit, MI) was prepared in sterile pyrogen-free saline. Rats under light ether anesthesia were injected through the penis vein with endotoxin solution (1 mg or less/100 g body weight). Rats injected with pyrogen f r e e saline served as controls. Glutathione Measurement: At specified intervals, rats were lightly anesthesized and then killed by decapitation. The duodenum, jejunum and colon were removed, opened longitudinally, cleansed with cold saline, b l o t t e d dry, weighed and homogenized in 20 times the volume (w/v) of ice cold 5~ t r i c h l o r o a c e t i c acid. The homogenates were centrifuged the supernatants f i l t e r e d through Whatman #42 f i l t e r paper, and the f i l t r a t e s analyzed for GSH according to the method of Ball (11). In this method, tissue cysteine content was first removed by reaction with glyoxylic acid. For the measurement of the colon or duodenum mucosal GSH level, the mucosal was gently scraped off with a scapel and processed as above. Results were expressed as means +_ 1S.D. and s t a t i s t i c a l significance was determined by Student's t - t e s t . P-values a t or less than 0.05 were considered significant. Results The e f f e c t of d i f f e r e n t doses of endotoxin on the colon, duodenum, and j e junum GSH level is shown in Table 1.
Rats were injected with varying doses of 845
Vol. 151, No. 2, 1988
Table 1.
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Effect of various doses of endotoxin on the colon, jejunum and duodenum glutathione levels Tissue glutathione (mg/g wet weight)
Endotox|n-"
0ug
25ug
50ug
100ug
500ug
1000ug
Colon
0.445±0.034 (16)
0.492±0.05* 0.550±0.018" (6) (6)
0.586±0.066* (6)
0.659±0.078* (6)
0.668±0.045* (8)
Jejunum
0.584,0.048 (11)
0.510±0.053" 0.534±0.036* (6) (6)
0.540±0.044 (6)
0.459±0.046* (5)
0.463±0.040* (4)
Duodenum
0.693,0.048 (ii)
0.621±0.07" 0.652±0.055 (5) (6)
0.621±0.046" 0.610±0.049" (6) (6)
0.610±0.029" (4)
Numbers in parentheses are numbers of animals used. Endotoxin dose: ug/100g body weight administered intravenously. Rats were fasted following endotoxin or saline injection. Colon, jejunum, and duodenum glutathione levels were determined 16 hrs after endotoxin administration. Values are means ±1 S.D. Asterisks indicate that values are significantly different (p < 0.05} from control group.
endotoxin (25 ug t o 1000 ug/100g body weight), f a s t e d f o l l o w i n g endotoxin or saline i n j e c t i o n and w e r e killed 16 hours a f t e r endotoxin a d m i n i s t r a t i o n .
Endotoxin
is found t o i n c r e a s e the colon GSH level, and this e f f e c t is dose r e l a t e d .
In con-
t r a s t , both t h e duodenum and jejunum GSH l e v e l s w e r e s i g n i f i c a n t l y lowered. Table 2 shows t h a t the colon GSH l e v e l is s i g n i f i c a n t l y i n c r e a s e d a t 2 hours a f t e r endotoxin a d m i n i s t r a t i o n and r e m a i n e d i n c r e a s e d up to 48 hours.
Both t h e
duodenum and jejunum w e r e s i g n if i can t l y reduced. To d e t e r m i n e w h e t h e r f a s t i n g has any e f f e c t on the duodenum, j e j u m u m , and colon GSH levels, we c o m p a r e d the GSH c o n c e n t r a t i o n in t h e s e s e c t i o n s of t h e gut
Table 2.
Glutathione levels of rat colon, jejunum and duodenum at various time intervals after endotoxin administration Tissue glutathione (mg/g wet weight) Control
2 hrs
6 hrs
16 hrs
Colon
0.445,0.034 (16)
0.465±0.105"* (9)
0.525±0.13"* 0.614±0.096" 0.608,0.113" (4) (8) (4)
0.698±0.095* (6)
Jejunum
0.583±0.048 (II)
0.471,0.051" (5)
0.463±0.04* (4)
0.466±0.093** (7)
Duodenum
0.693,0.048 (II)
0.586,0.012" (5)
0.535±0.025* (4)
0.566±0.061" (7)
Numbers in parentheses are numbers of animals used. Endotoxin dose: 1000ug/100g body weight administered intravenously. Values are m e a n s , 1 S.D. Asterisks indicate that values are significantly different *(p < 0.001). ** (p < 0.05) from control group. 846
24 his
48 hrs
Vol. 151, No. 2, 1988
Table 3.
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Effect of fasting on the rat liver, duodenum, jejunum and colon glutathione level Tissue glutathione (m~/g wet weight) Liver
Fastinga
Duodenum
Jejunum
Colon
1.57+0.15" 0.683_+0.093 0.545+0.078 0.432+0.027
n=6
Non-fasting n=6
2.24+0.11
0.668_+0.051
0.572+0.041
0.454+0.031
Asterisk indicates that the value is significantly different (p < 0.001) from nonfasting value. Values are mean + 1 S.D. a Rats were fasted for 24 hours.
in r a t s which have b e e n f a s t e d for 24 hours to t h a t of n o n - f a s t e d rats. The results are shown in Table 3.
Unlike the liver, gut GSH levels do n o t appear to be af-
f e c t e d by fasting. Table 4 shows t h a t the increase in colon GSH level following endotoxin a d m i n i s t r a t i o n occur mainly in the mucosal layer and to a lesser degree in the muscle layer. Discussion This study showed t h a t the r a t colon GSH level can be s i g n i f i c a n t l y e n h a n c e d by endotoxin t r e a t m e n t . This e n h a n c e m e n t is dose d e p e n d e n t and persists up to 48 hours a t the t e s t e d dose of l m g / 1 0 0 g body weight. In c o n t r a s t , the duodenum and j e j u n u m GSH c o n c e n t r a t i o n is significantly reduced.
Table 4.
In control rats t r e a t e d with
Colon and duodenum mucosal and muscle glutathione concentration after endotoxin administration Tissue glutathione (mg/g wet weight) Colon
Colon
mucosal
muscle
Duodenum mucosal
Duodenum muscle
Endotoxin treated n=4
0.951+0.099"
0.490±0.028
0.676+0.026**
0.474_+0.034
Control
0.730+0.074
0.418+0.081
0.795+0.029
0.564+0.069
n=3 Endotoxin dose: 1000ug/100g body weight administered intravenously. Colon and duodenum glutathione levels were determined 16 hours after endotoxiu administration. Values are means + 1 S.D. Asterisks indicate that values are significantly different * (p < 0.025), • *(p < 0.005) from control group. 847
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BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
pyrogen-free saline, the colon is found to have a lower GSH level than the jejunum which in turn has a lower GSH level than the duodenum. The biological implication of the differences in GSH concentration in the large and small intestine in response to endotoxin is not known at the present time.
However, an enhanced GSH level in the colon may be a protective
mechanism to help maintain the integrity of the colon mucosa and thus prevent the release of potentially pathogenic bacteria and their degradative products which are normally found in the large intestine of mammals. In contrast to the colon, the GSH level in the small intestine decreases after endotoxin injection. Reduced GSH level in the small intestine may contribute to hyperemia and hemorrhage observed in the jejunum and ileum when rats were infused with endotoxin(12). A decrease in the GSH level may result in an increase in the level of lipid peroxidation in tissues.
This is suggested by the fact that the GSH level in the
fasting liver is reduced by 30 percent while the level of lipid peroxidation is increased(13,14). Lipid peroxidation, mediated by H20 2 and free radicals of 02 which are secreted by macrophages in response to endotoxin, is believed to be one of the important
causes of cell
membrane destruction and cell damage(15); cell
membrane contains much lipid, especially unsaturated f a t t y acids(15).
GSH, one
of the major antioxidants in the soluble fraction of cells, plays an important role in the prevention of lipid peroxidation through the action of GSH peroxidase(16). Therefore, an increase in the GSH level in the colon may be a natural response to maintain the integrity of the gut mucosal barrier.
Yoshikawa(17) and associates
reported no significant increase in lipid peroxidation level in the colon, whereas in the ileum mucosa the level was significantly increased in rats infused with 100 mg/kg of endotoxin for 4 hours as compared to control rats infused with physiological saline. The mechanism(s) by which endotoxin causes an increase in the colon GSH level is not known, but interorgan translocation may be a possibility(18), together with increased synthesis.
The loss of GSH in the duodenum and jejunum could be 848
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BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
due to its oxidation to the disulfide form (GSSG) as a result of an increased level of lipid peroxidation and consequent inability to catalyze its reduction to GSH. Some of the loss of GSH in endotoxin shock may also be due to inhibition of synthesis of this compound(19). We tested the effect of fasting on the gut GSH level..We found the GSH levels in the colon, jejunum and duodenum appear to be unaffected by 24 hour fasting while the GSH level in the liver is reduced by 30¢.
Therefore, the
decreased GSH level observed in the jejunum and duodenum after endotoxin treatment is not due to reduced food intake but is likely due to endotoxin. Enhanced GSH level has been shown to regress aflatoxin Bl-induced liver tumors(2).
In the present study, we found that the colon GSH level can be
enhanced by endotoxin treatment. Whether or not GSH plays a role, together with the complex immune system, in endotoxin-induced damage to certain types of tumors remains to be studied. It is probable that endotoxin could be used to treat colon tumors. Acknowledgements This project was supported in part by BRSG S07 RR05749 awarded by the Biomedical Research Support Grant Program, Division of Research Resources, National Institute of Health, and the American Cancer Society, Florida Division Grant No. F87USF-1. References 1. Perchellet, J.P., Owen, M.D., Posey, T.D., Orten, D.K., Schneider, B.A. (1985) Carcinogenesis. 6:567-573. 2. Novi, A.M. Science. (1981) 212:541-542. 3. Nauts, H.C., Fowler, G.A., Bogatho, F.H. (1953) Acta. Med. Scand. Suppl. 276,5-103. 4. Carswell, E.A., Old, L.F., Kassel, R.L., et al. {1975) Proc. Nat. Acad. Sci. 72:3666-3670. 5. O'MaUey, W.E., Achinstein, B., Shear, M.J. (1962) J. Natl. Cancer. Inst. 29:1161-1168. 6. Lagadec, P., Jeannin, J.F., Reisser, D., et al. (1987) Invasion Metastasis. 7:83-95. 7. Berendt, M.J., North, R.J., Kirstein, D.P. (1978) J. Expt. Med. 148, 15501559. 849
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8. Berendt, M.J., North, R.J., Kirstein, D.P. (1978) J. Expt. Med. 148, 15601569. 9. Mannel, D.N., Moore, Immunity. 30, 523-530.
R.N., Mergenhagen, S.E. (1980) Infection and
10. Flick, D.A., Gifford, G.E. (1986) Immunobiol. 171,320-328. 11. Ball, C.R. (1966) Biochem. Pharm. 15, 809-816. 12. Wallace, J.L., Whittle, B.J.R. (1986) European J. Pharmacology 124, 209210. 13. Tateishi, N., Higashi, T., Shinya, S., et al. (1984) J. Biochem. 75, 93-103. 14. Hogberg, J., Moldeus, P., Arborgh, B., et al. (1975) Europ. J. Biochem. 59, 457-462. 15. Tappel, A.L. (1973) Fed. Proc. 32, 1870-1874. 16. Arias, I.M., Jakoby, W.B. (1976) Raven Press, New York.
Glutathione:
Metabolism and function.
17. Yoshikawa, T., Murakami, M., Furukawa, Y., et al. Haemostas. 49,214-216.
(1983) Thromb.
18. Curthoys, N.P. (1986) Fed. Proc. 45, 2166-2169. 19. Beck, L.V., Linkenheimer W. (1952) Proc. Soc. Exp. Biol. Med. 81,291-294.
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