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Lipid peroxidation: A possible role in gastric damage induced by ethanol in rats
Life Sciences, Vol. 38, pp. 2163-2167 Printed in the U.S.A.
LIPID PEROXIDATION:
Pergamon Press
A POSSIBLE ROLE IN GASTRIC DAMAGE INDUCED BY ETHANOL IN RATS
Takuji Mizui and Masami Doteuchi Shionogi Research Laboratories, Shionogi & Co., Ltd., Fukushima-ku, Osaka, 553 Japan (Received in final form March 19, 1986)
Summary Gastric mucosal lipid peroxide levels, based on the amounts of thiobarbituric acid reacting substances, increased soon after oral application of absolute ethanol. On the other hand, gastric mucosal nonprotein sulfhydryl levels slightly but significantly decreased. Administration of 20% ethanol, a mild irritant which can hardly produce gastric lesions, did not influence either level. Pretreatment with prostaglandin E 2 or F2a, in a dose that offered protection of the gastric mucosa, prevented the increase of mucosal lipid peroxides after absolute ethanol administration. These observations suggest that lipid peroxidation in the gastric mucdsa may be closely related to production of the gastric damage by ethanol. Various types of exogenous and/or endogenous prostaglandins (PGs) have been shown to protect gastric mucosa against necrotizing agents such as absolute ethanol (1,2). The mechanisms have not been elucidated although many hypotheses have been proposed (3). Recently, nonprotein sulfhydryl compounds, mainly reduced glutathione (GSH), have been suggested as having roles in the gastric protective mechanisms (4,5,6). GSH is important in the cellular protective mechanisms against a number of noxious reactants including oxygen-derived free radicals, and reduction of cellular GSH contents have been observed when lipid peroxidation occurs in tissues (7,8). Szabo and his associates found that gastric mucosal nonprotein sulfhydryls decreased rapidly after administration of ethanol in ulcerative doses (6). Pretreatment with cytoprotective agents such as PGE 2 and spermine can prevent the decrease of nonprotein sulfhydryls (9,10). Furthermore, the protection offered by PGF28 against absolute ethanol disappears in the presence of sulfhydryl blockers such as N-ethylmaleimide or iodoacetamide (6). The decrease of nonprotein sulfhydryls in the gastric mucosa after ethanol administration may result from propagation of lipid peroxidation in the tissue. The present study examined whether or not lipid peroxidation in the gastric mucosa is related to pathogenesis of the gastric damage induced by ethanol. Materials and Methods Male Jcl Sprague-Dawley rats, weighing 200-300 g, were deprived of food, but not water for 24 hr before the experiments. Animals were killed by decapitation at various times after oral administration of 20 or 100% ethanol (I ml/animal). The stomachs were quickly removed. The mucosal 0024-3205/86 $3.00 + .00 Copyright (c) 1986 Pergamon Press Ltd.
2164
Lipid Peroxidation
and Gastric Damage
Vol.
38, No.
23, iq86
layer in the glandular part of the stomach was separated from the muscle layer by blistering with injection of ice-cold saline under the serosa through a 26-gauge needle, washed with ice-cold saline and then frozen on liquid nitrogen. The samples were stored at -80°C until biochemical determination. Lipid peroxides in the gastric mucosa were determined fluorometrically as thiobarbituric acid-reactive substances according to Ohkawa et al. (II). The tissue samples were placed in 19 volumes of ice-cold saline and homogenized with a high speed homogenizer (ULTRA-TURRAX) for 15 sec. The homogenate (0.4 ml) was mixed with 0.I ml of 8.1% sodium dodecyl sulfate, 0.75 ml of 20% acetic acid of pH 3.5, and 0.75 ml of 0.8% thiobarbituric acid. The mixture was heated in a boiling water bath for 120 min and after cooling, 0.5 ml of distilled water was added. The pink pigment was extracted with 2.5 ml of n-butanol-pyridine (15:1) and fluorescence intensity was determined (excitation: 515 nm; emission: 553 nm). Malondialdehyde bis(dimethylacetal) was used as a standard. Nonprotein sulfhydryls in the gastric mucosa were determined by the Ellman procedure (12), as described previously (9). Protein was determined
according
to Lowry et al.
(13).
In the ulcer study, the animals were killed with an overdose of ether and the stomachs were removed, inflated with 6 ml of 1% buffered formalin and opened along the greater curvature after fixation by immersion in 1% buffered formalin. The gastric lesion index was evaluated from the total length of the lesion produced. PGE 2 or PGF2e (Wako Pure Chemical Industries, Ltd.) at 0.2 and 1 mg/kg in 1% ethanol, respectively, was administered orally 30 min before absolute ethanol application. The data were analyzed by means of Student's 100
t-test.
10
Fig. i
8O
o o.
60
c
o
4 "';
E r"-"
lO0% E t O H 20
•
I
I
I
0
5
3O
6O
Time
(rain)
._= g
1
Changes of the lipid peroxide level and nonprotein sulfhydryl level in the gastric mucosa after absolute ethanol administration. Each point represents the mean ± S.E. of 6 animals. Statistical significance of difference from the nontreated group: * P < 0.05, ** P < 0.02 *** P < 0.01.
Vol. 38, No. 23, 1986
Lipid Peroxidation and Gastric Damage
2165
Results As shown in Figure i, when absolute ethanol (I ml/animal) was given orally, the concentration of lipid peroxides in the gastric mucosa rapidly increased within 5 min, remained at the increased level for almost 30 min, and then returned nearly to the normal level 1 hr later. In contrast to the increase of lipid peroxides, nonprotein sulfhydryls in the gastric mucosa slightly, but significantly, decreased at 5 min after absolute ethanol administration, and the decrease was still observed 1 hr later. Under comparable treatments the lesion formation (mean ± S.E.) was 117.4 ± 14.1, 103.9 ± 22.4 and 81.2 ± 22.5 ~ at 5, 30 and 60 min, respectively, after administration of absolute ethanol; no significant difference were found among the groups. When the oral application was done with 20% ethanol (i ml/animal), a level that could hardly produce lesions, the lipid peroxide and nonprotein sulfhydryl levels showed no or little change, respectively (Figure 2). Table 1 shows the effects of PGE 2 or PGF^e on lipid peroxidation at 5 z min after administration of absolute ethanol. The increments of lipid peroxides could be completely counteracted by PGE 2 (0.2 mg/kg) or PGF2e (i mg/kg) given orally 30 min before absolute ethanol. In a separate experiment, PGE 2 or PGF2~ remarkably prevented the lesion formation at 1 hr after absolute ethanol application.
Discussion The present study shows that lipid peroxidation is induced in the gastric mucosa soon after oral administration of absolute ethanol. Lipid peroxidation may be associated with the pathological processes in ethanolinduced gastric damage as in injury to various tissues: liver necroses (14,15), lung damage (15), ischemic disease of the heart (16) or brain
100
10
Fig. 2
80
8
++!
o-
-
Changes of the lipid peroxide level and nonprotein sulfhydryl level in the gastric mucosa after 20% ethanol administration. Each point represents the mean + S.E. of 4-6 animals. Statistical significance of difference from the nontreated group: * P < 0.05.
.O6o 0,.
=.
4o
4
20% EtOH
0
L,
2
I
0 5 Time
I
I
30
60 (min)
=
:k
2166
Lipid Peroxidation and Gastric Damage
Vol. 38, No. 23, 1986
TABLE 1 Relationship between Changes in Lipid Peroxide Level and Severity of Absolute Ethanol-Induced Gastric Lesions
Control
Lipid peroxides (nmoles/100 mg ~rotein) 50.4 ± 2.1 (5)
100% Ethanol
72.4 + 3.5*
Treatment
(6)
PGE 2 0.2 mg/kg + 200% E t h a n o l
47.8 ± 2.8+%%
(5)
Gastric lesions (mm)
89.5 ± 22.5 10.5 ±
7.4%
(5) (5)
PGF2~ 1 mg/kg 48.8 ± 1.5++% (5) Ii.0 + 4.0T+ (5) + 100% Ethanol PGE2 or PGF2e was administered orally 30 min 5efore absolute ethanol. Each value represents the mean ± S.E. and the number of animals is given in parentheses. Statistical significance of difference from the control: P < 0.001. Statistical significance of difference from the group given absolute ethanol alone: + P < 0.02, ++ P < 0.01, %++ P < 0.001. (17). As gastric lesions are produced very quickly after administration of absolute ethanol (18,19), we could not establish whether lipid peroxidation precedes lesion formation. However, here we would like to discuss its possible role in lesion formation. A recent paper reported that vascular damage in the gastric mucosa occured immediately after administration of ethanol in ulcerative doses while pretreatment with PGs could prevent this (18,19). Ischemic hypoxia due to circulatory injuries in the gastric mucosa may induce oxygen-derived free radicals that could produce lipid peroxidation in the membranes of various cells or cellular organella, such as the plasma membrane, endoplasmic reticulum, mitochondria or lysosome. Furthermore, iron and copper compounds from plasma and erythrocytes which had been extravasated, could produce metal-catalyzed autoxidation of unsaturated membrane lipids (17). A more recent report suggests the involvement of oxygen-derived free radicals in ischemia-induced gastric damage in rats subjected to hemorrhagic shock (20). Free radicals may also be generated in the oxidation process of ethanol. Alcohol dehydrogenase, an enzyme which catalyzes the oxidation of ethanol to acetaldehyde, has been shown to be located in human gastric epithelial cells (21). Superoxide radicals could be produced in the oxidation of acetaldehyde by xanthine oxidase, an enzyme secondarily responsible for the metabolism of acetaldehyde (8). In addition, the metabolism of ethanol by the microsomal ethanol-oxidizing system in which NADPH-cytochrome c reductase and cytochrome P-450 are involved, leads to the generation of superoxide radicals (22,23). Recently, several PGs have been shown to offer protection against various injuries in tissues other than the gastrointestinal tract. PGI 2 protects against hypoxia in cat heart (24), rat brain (25) and even isolated perfused cat liver in which a vasodilator effect is minimized and platelet aggregation is eliminated (26). 16,16-Dimethyl PGE 2 prevents carbon tetrachloride- or bromobenzene-induced liver necrosis (27,28). Interestingly, lipid peroxidation has been suggested to be related to the
Vol.
38, No.
23, 1986
pathogenesis
Lipid Peroxidation
and Gastric Damage
2167
of the injuries prevented by PGs.
At present, the mechanisms by which PGE 2 or PGF2~ prevent lipid peroxidation in the gastric mucosa after administration of absolute ethanol are not clear, but inhibition of lipid peroxidation may be one of the many beneficial effects of PGs [others being stimulation of mucus (3), bicarbonate secretion (3), vascular protection (18,19)]. Acknowledgement The authors thank Miss N. Shimono
for her excellent
technical
assist-
ance. References I. A. ROBERT, J.E. NEZAMIS, C. LANCASTER and A.J. HANCHAR, Gastroenterology 77 433-443 (1979). 2. A. ROBERT, J.E. NEZAMIS, C. LANCASTER, J.P. DAVIS, S.O. FIELD and A.J. HANCHAR, Am. J. Physiol. 245 GII3-GI21 (1983). 3. T.A. MILLER, Am. J. Physiol. 245 G601-G623 (1983). 4. S.C. BOYD, H.A. SASAME and M.R. BOYD, Science 205 1010-1012 (1979). 5. S.C. BOYD, H.A. SASAME and M.R. BOYD, Life Sci. 28 2987-2992 (1981). 6. S. SZABO, J.S. TRIER and P.W. FRANKEL, Science 214 200-202 (1981). 7. N.S. KOSOWER and E.S. KOSOWER, Int. Rev. Cytol. 54 109-160 (1978). 8. L.A. VIDELA and A. VALENZUELA, Life Sci. 31 2395-2407 (1982). 9. T. MIZUI and M. DOTEUCHI, Jpn. J. Pharmacol. 33 939-945 (1983). i0. R. FUMAGALLI, R. CAPONI, A. CORSINI, A. BRAMBILLA, M. PALMIRA, F. BERNINI and U. VALCAVI, Prostaglandins 29 467-474 (1985). ii. H. OHKAWA, N. OHISHI and K. YAGI, Anal. Biochem. 95 351-358 (1979). 12. G. L. ELLMAN, Arch. Biochem. Biophys. 82 70-77 (1959). 13. O.H. LOWRY, N.I. ROSEBROUGH, A.L. FARR and R.J. RANDAL, J.Biol. Chem. 193 265-275 (1951). 14. N.R. DI LUZIO, Fed. Proc. 32 1875-1881 (1973). 15. G.L. PLAA and H. WITSHI, Ann. Rev. Pharmacol. Toxicol. 16 125-141 (1976). 16. M.L. HESS, N.H. MANSON and E. OKABE, Can. J. Physiol. Pharmacol. 60 1382-1389 (1982). 17. H.B. DEMOPOULOS, E.S. FLAMM, D.D. PIETRONIGRO and M.L. SELIGMAN, Acta Physiol. Scand. 492 (suppl.) 91-119 (1980). 18. P.H. GUTH, G. PAULSEN and H. NAGATA, Gastroenterology 87 1083-1090 (1984) . 19. S. SZABO, J.S. TRIER, A. BROWN and J. SCHNOOR, Gastroenterology 88 228-236 (1985). 20. M. ITOH and P.H. GUTH, Gastroenterology 88 1162-1167 (1985). 21. D.M. PESTALOZZI, R. BUHLER, J.P. von WARTBURG and M. HESS, Gastroenterology 85 1011-1016 (1983). 22. R. TESCHKE, Y. HASUMURA and C.S. LIEBER, Arch. Biochem. Biophys. 163 404-415 (1974). 23. C. LAI, T.A. GROVER a~d L.H. PIETTE, Arch. Biochem. Biophys. 193 373-378 (1979). 24. M.L. OGLETREE, A.M. LEFER, J.B. SMITH and K.C. NICOLAOU, Eur. J. Pharmacol. 56 95-103 (1979). 25. Y. MASUDA, Y. SHIRAISHI, Y. OCHI, T. KARASAWA, T. KADOKAWA and M. SHIMIZU, Jpn. J. Pharmacol. 36 (suppl.) 330P (1984). 26. H. ARAKI and A.M. LEFER, Am. J. Physiol. 238 HI76-HI81 (1980). 27. J. STACHURA, A. TARNAWSKI, K.J. IVEY, T. MACH, J. BOGDAL, J. SZCZUDRAWA and B. KLIMCZYK, Gastroenterology 8 1 211-217 (1981). 28. C. FUNCK-BRENTANO, M. TINEL, C. DEGOTT, P. LETTERON, G. BABANY and D. PESSAYRE, Biochem. Pharmacol. 33 89-96 (1984).
LIPID PEROXIDATION:
Pergamon Press
A POSSIBLE ROLE IN GASTRIC DAMAGE INDUCED BY ETHANOL IN RATS
Takuji Mizui and Masami Doteuchi Shionogi Research Laboratories, Shionogi & Co., Ltd., Fukushima-ku, Osaka, 553 Japan (Received in final form March 19, 1986)
Summary Gastric mucosal lipid peroxide levels, based on the amounts of thiobarbituric acid reacting substances, increased soon after oral application of absolute ethanol. On the other hand, gastric mucosal nonprotein sulfhydryl levels slightly but significantly decreased. Administration of 20% ethanol, a mild irritant which can hardly produce gastric lesions, did not influence either level. Pretreatment with prostaglandin E 2 or F2a, in a dose that offered protection of the gastric mucosa, prevented the increase of mucosal lipid peroxides after absolute ethanol administration. These observations suggest that lipid peroxidation in the gastric mucdsa may be closely related to production of the gastric damage by ethanol. Various types of exogenous and/or endogenous prostaglandins (PGs) have been shown to protect gastric mucosa against necrotizing agents such as absolute ethanol (1,2). The mechanisms have not been elucidated although many hypotheses have been proposed (3). Recently, nonprotein sulfhydryl compounds, mainly reduced glutathione (GSH), have been suggested as having roles in the gastric protective mechanisms (4,5,6). GSH is important in the cellular protective mechanisms against a number of noxious reactants including oxygen-derived free radicals, and reduction of cellular GSH contents have been observed when lipid peroxidation occurs in tissues (7,8). Szabo and his associates found that gastric mucosal nonprotein sulfhydryls decreased rapidly after administration of ethanol in ulcerative doses (6). Pretreatment with cytoprotective agents such as PGE 2 and spermine can prevent the decrease of nonprotein sulfhydryls (9,10). Furthermore, the protection offered by PGF28 against absolute ethanol disappears in the presence of sulfhydryl blockers such as N-ethylmaleimide or iodoacetamide (6). The decrease of nonprotein sulfhydryls in the gastric mucosa after ethanol administration may result from propagation of lipid peroxidation in the tissue. The present study examined whether or not lipid peroxidation in the gastric mucosa is related to pathogenesis of the gastric damage induced by ethanol. Materials and Methods Male Jcl Sprague-Dawley rats, weighing 200-300 g, were deprived of food, but not water for 24 hr before the experiments. Animals were killed by decapitation at various times after oral administration of 20 or 100% ethanol (I ml/animal). The stomachs were quickly removed. The mucosal 0024-3205/86 $3.00 + .00 Copyright (c) 1986 Pergamon Press Ltd.
2164
Lipid Peroxidation
and Gastric Damage
Vol.
38, No.
23, iq86
layer in the glandular part of the stomach was separated from the muscle layer by blistering with injection of ice-cold saline under the serosa through a 26-gauge needle, washed with ice-cold saline and then frozen on liquid nitrogen. The samples were stored at -80°C until biochemical determination. Lipid peroxides in the gastric mucosa were determined fluorometrically as thiobarbituric acid-reactive substances according to Ohkawa et al. (II). The tissue samples were placed in 19 volumes of ice-cold saline and homogenized with a high speed homogenizer (ULTRA-TURRAX) for 15 sec. The homogenate (0.4 ml) was mixed with 0.I ml of 8.1% sodium dodecyl sulfate, 0.75 ml of 20% acetic acid of pH 3.5, and 0.75 ml of 0.8% thiobarbituric acid. The mixture was heated in a boiling water bath for 120 min and after cooling, 0.5 ml of distilled water was added. The pink pigment was extracted with 2.5 ml of n-butanol-pyridine (15:1) and fluorescence intensity was determined (excitation: 515 nm; emission: 553 nm). Malondialdehyde bis(dimethylacetal) was used as a standard. Nonprotein sulfhydryls in the gastric mucosa were determined by the Ellman procedure (12), as described previously (9). Protein was determined
according
to Lowry et al.
(13).
In the ulcer study, the animals were killed with an overdose of ether and the stomachs were removed, inflated with 6 ml of 1% buffered formalin and opened along the greater curvature after fixation by immersion in 1% buffered formalin. The gastric lesion index was evaluated from the total length of the lesion produced. PGE 2 or PGF2e (Wako Pure Chemical Industries, Ltd.) at 0.2 and 1 mg/kg in 1% ethanol, respectively, was administered orally 30 min before absolute ethanol application. The data were analyzed by means of Student's 100
t-test.
10
Fig. i
8O
o o.
60
c
o
4 "';
E r"-"
lO0% E t O H 20
•
I
I
I
0
5
3O
6O
Time
(rain)
._= g
1
Changes of the lipid peroxide level and nonprotein sulfhydryl level in the gastric mucosa after absolute ethanol administration. Each point represents the mean ± S.E. of 6 animals. Statistical significance of difference from the nontreated group: * P < 0.05, ** P < 0.02 *** P < 0.01.
Vol. 38, No. 23, 1986
Lipid Peroxidation and Gastric Damage
2165
Results As shown in Figure i, when absolute ethanol (I ml/animal) was given orally, the concentration of lipid peroxides in the gastric mucosa rapidly increased within 5 min, remained at the increased level for almost 30 min, and then returned nearly to the normal level 1 hr later. In contrast to the increase of lipid peroxides, nonprotein sulfhydryls in the gastric mucosa slightly, but significantly, decreased at 5 min after absolute ethanol administration, and the decrease was still observed 1 hr later. Under comparable treatments the lesion formation (mean ± S.E.) was 117.4 ± 14.1, 103.9 ± 22.4 and 81.2 ± 22.5 ~ at 5, 30 and 60 min, respectively, after administration of absolute ethanol; no significant difference were found among the groups. When the oral application was done with 20% ethanol (i ml/animal), a level that could hardly produce lesions, the lipid peroxide and nonprotein sulfhydryl levels showed no or little change, respectively (Figure 2). Table 1 shows the effects of PGE 2 or PGF^e on lipid peroxidation at 5 z min after administration of absolute ethanol. The increments of lipid peroxides could be completely counteracted by PGE 2 (0.2 mg/kg) or PGF2e (i mg/kg) given orally 30 min before absolute ethanol. In a separate experiment, PGE 2 or PGF2~ remarkably prevented the lesion formation at 1 hr after absolute ethanol application.
Discussion The present study shows that lipid peroxidation is induced in the gastric mucosa soon after oral administration of absolute ethanol. Lipid peroxidation may be associated with the pathological processes in ethanolinduced gastric damage as in injury to various tissues: liver necroses (14,15), lung damage (15), ischemic disease of the heart (16) or brain
100
10
Fig. 2
80
8
++!
o-
-
Changes of the lipid peroxide level and nonprotein sulfhydryl level in the gastric mucosa after 20% ethanol administration. Each point represents the mean + S.E. of 4-6 animals. Statistical significance of difference from the nontreated group: * P < 0.05.
.O6o 0,.
=.
4o
4
20% EtOH
0
L,
2
I
0 5 Time
I
I
30
60 (min)
=
:k
2166
Lipid Peroxidation and Gastric Damage
Vol. 38, No. 23, 1986
TABLE 1 Relationship between Changes in Lipid Peroxide Level and Severity of Absolute Ethanol-Induced Gastric Lesions
Control
Lipid peroxides (nmoles/100 mg ~rotein) 50.4 ± 2.1 (5)
100% Ethanol
72.4 + 3.5*
Treatment
(6)
PGE 2 0.2 mg/kg + 200% E t h a n o l
47.8 ± 2.8+%%
(5)
Gastric lesions (mm)
89.5 ± 22.5 10.5 ±
7.4%
(5) (5)
PGF2~ 1 mg/kg 48.8 ± 1.5++% (5) Ii.0 + 4.0T+ (5) + 100% Ethanol PGE2 or PGF2e was administered orally 30 min 5efore absolute ethanol. Each value represents the mean ± S.E. and the number of animals is given in parentheses. Statistical significance of difference from the control: P < 0.001. Statistical significance of difference from the group given absolute ethanol alone: + P < 0.02, ++ P < 0.01, %++ P < 0.001. (17). As gastric lesions are produced very quickly after administration of absolute ethanol (18,19), we could not establish whether lipid peroxidation precedes lesion formation. However, here we would like to discuss its possible role in lesion formation. A recent paper reported that vascular damage in the gastric mucosa occured immediately after administration of ethanol in ulcerative doses while pretreatment with PGs could prevent this (18,19). Ischemic hypoxia due to circulatory injuries in the gastric mucosa may induce oxygen-derived free radicals that could produce lipid peroxidation in the membranes of various cells or cellular organella, such as the plasma membrane, endoplasmic reticulum, mitochondria or lysosome. Furthermore, iron and copper compounds from plasma and erythrocytes which had been extravasated, could produce metal-catalyzed autoxidation of unsaturated membrane lipids (17). A more recent report suggests the involvement of oxygen-derived free radicals in ischemia-induced gastric damage in rats subjected to hemorrhagic shock (20). Free radicals may also be generated in the oxidation process of ethanol. Alcohol dehydrogenase, an enzyme which catalyzes the oxidation of ethanol to acetaldehyde, has been shown to be located in human gastric epithelial cells (21). Superoxide radicals could be produced in the oxidation of acetaldehyde by xanthine oxidase, an enzyme secondarily responsible for the metabolism of acetaldehyde (8). In addition, the metabolism of ethanol by the microsomal ethanol-oxidizing system in which NADPH-cytochrome c reductase and cytochrome P-450 are involved, leads to the generation of superoxide radicals (22,23). Recently, several PGs have been shown to offer protection against various injuries in tissues other than the gastrointestinal tract. PGI 2 protects against hypoxia in cat heart (24), rat brain (25) and even isolated perfused cat liver in which a vasodilator effect is minimized and platelet aggregation is eliminated (26). 16,16-Dimethyl PGE 2 prevents carbon tetrachloride- or bromobenzene-induced liver necrosis (27,28). Interestingly, lipid peroxidation has been suggested to be related to the
Vol.
38, No.
23, 1986
pathogenesis
Lipid Peroxidation
and Gastric Damage
2167
of the injuries prevented by PGs.
At present, the mechanisms by which PGE 2 or PGF2~ prevent lipid peroxidation in the gastric mucosa after administration of absolute ethanol are not clear, but inhibition of lipid peroxidation may be one of the many beneficial effects of PGs [others being stimulation of mucus (3), bicarbonate secretion (3), vascular protection (18,19)]. Acknowledgement The authors thank Miss N. Shimono
for her excellent
technical
assist-
ance. References I. A. ROBERT, J.E. NEZAMIS, C. LANCASTER and A.J. HANCHAR, Gastroenterology 77 433-443 (1979). 2. A. ROBERT, J.E. NEZAMIS, C. LANCASTER, J.P. DAVIS, S.O. FIELD and A.J. HANCHAR, Am. J. Physiol. 245 GII3-GI21 (1983). 3. T.A. MILLER, Am. J. Physiol. 245 G601-G623 (1983). 4. S.C. BOYD, H.A. SASAME and M.R. BOYD, Science 205 1010-1012 (1979). 5. S.C. BOYD, H.A. SASAME and M.R. BOYD, Life Sci. 28 2987-2992 (1981). 6. S. SZABO, J.S. TRIER and P.W. FRANKEL, Science 214 200-202 (1981). 7. N.S. KOSOWER and E.S. KOSOWER, Int. Rev. Cytol. 54 109-160 (1978). 8. L.A. VIDELA and A. VALENZUELA, Life Sci. 31 2395-2407 (1982). 9. T. MIZUI and M. DOTEUCHI, Jpn. J. Pharmacol. 33 939-945 (1983). i0. R. FUMAGALLI, R. CAPONI, A. CORSINI, A. BRAMBILLA, M. PALMIRA, F. BERNINI and U. VALCAVI, Prostaglandins 29 467-474 (1985). ii. H. OHKAWA, N. OHISHI and K. YAGI, Anal. Biochem. 95 351-358 (1979). 12. G. L. ELLMAN, Arch. Biochem. Biophys. 82 70-77 (1959). 13. O.H. LOWRY, N.I. ROSEBROUGH, A.L. FARR and R.J. RANDAL, J.Biol. Chem. 193 265-275 (1951). 14. N.R. DI LUZIO, Fed. Proc. 32 1875-1881 (1973). 15. G.L. PLAA and H. WITSHI, Ann. Rev. Pharmacol. Toxicol. 16 125-141 (1976). 16. M.L. HESS, N.H. MANSON and E. OKABE, Can. J. Physiol. Pharmacol. 60 1382-1389 (1982). 17. H.B. DEMOPOULOS, E.S. FLAMM, D.D. PIETRONIGRO and M.L. SELIGMAN, Acta Physiol. Scand. 492 (suppl.) 91-119 (1980). 18. P.H. GUTH, G. PAULSEN and H. NAGATA, Gastroenterology 87 1083-1090 (1984) . 19. S. SZABO, J.S. TRIER, A. BROWN and J. SCHNOOR, Gastroenterology 88 228-236 (1985). 20. M. ITOH and P.H. GUTH, Gastroenterology 88 1162-1167 (1985). 21. D.M. PESTALOZZI, R. BUHLER, J.P. von WARTBURG and M. HESS, Gastroenterology 85 1011-1016 (1983). 22. R. TESCHKE, Y. HASUMURA and C.S. LIEBER, Arch. Biochem. Biophys. 163 404-415 (1974). 23. C. LAI, T.A. GROVER a~d L.H. PIETTE, Arch. Biochem. Biophys. 193 373-378 (1979). 24. M.L. OGLETREE, A.M. LEFER, J.B. SMITH and K.C. NICOLAOU, Eur. J. Pharmacol. 56 95-103 (1979). 25. Y. MASUDA, Y. SHIRAISHI, Y. OCHI, T. KARASAWA, T. KADOKAWA and M. SHIMIZU, Jpn. J. Pharmacol. 36 (suppl.) 330P (1984). 26. H. ARAKI and A.M. LEFER, Am. J. Physiol. 238 HI76-HI81 (1980). 27. J. STACHURA, A. TARNAWSKI, K.J. IVEY, T. MACH, J. BOGDAL, J. SZCZUDRAWA and B. KLIMCZYK, Gastroenterology 8 1 211-217 (1981). 28. C. FUNCK-BRENTANO, M. TINEL, C. DEGOTT, P. LETTERON, G. BABANY and D. PESSAYRE, Biochem. Pharmacol. 33 89-96 (1984).