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Gastric cytoprotection by sodium salicylate
PROSTAGLANDINS
GASTRIC CYTOPROTECTION BY SODIUM SALICYLATE Andre Robert Department of Experimental Biology The Upjohn Company Kalamazoo, Michigan 49001 ABSTRACT Sodium salicylate (SA), contrary to acetylsalicylic acid (ASA, aspirin), was not ulcerogenic in rats. SA was also found to be cytoprotective: it prevented formation of gastric mucosal necrosis produced by either absolute ethanol or 0.6 M HCl, and formation of gastric ulcers produced by acidified ASA. The degree of protection was dose dependent. The mechanism of this cytoprotection is unknown, but unlike cytoprotection elicited by mild irritants, e.g., 20% ethanol or 0.35 M HCl, whose effects appear to be due to endogenous formation of PG by the stomach, SA acts through a different mechanism, since its protective effect was not blocked by indomethacin. INTRODUCTION Acetylsalicylic acid (ASA, aspirin) and salicylic acid or its sodium salt (SA) are nonsteroidal antiinflammatory compounds. They are believed to exert their effect by inhibiting cyclooxygenase activity in inflamed tissues, and thus prostaglandin formation (1). However, they differ from each other in other aspects. In animals, ASA is ulcerogenic whereas SA is not (2). ASA inhibits prostaglandin cyclooxygenase activity in rat gastric mucosa, thereby eliciting a gastric depletion of prostaglandins (PG), whereas SA does not affect gastric PG cyclooxygenase (3). This different effect on the gastric content of PG may explain why only ASA is ulcerogenic. On the other hand, both ASA and SA, when introduced with acid in a dog gastric pouch (Heidenhain type), break the gastric mucosal barrier (4). This fact suggests that, at least in the dog, SA can irritate the stomach. We observed earlier that a variety of "mild" irritants, when given orally to rats minutes earlier, prevent development of gastric mucosal necrosis otherwise produced by "strong" irritants. Thus, 20% ethanol, a mild acid or a mild base (0.35 M HCl, or 0.075 M NaOH), a slightly hypertonic solution (4% NaCl) or a dilute bile salt solution (5 mM sodium taurocholate in 0.2 M HCl) protect the gastric mucosa against massive necrosis produced by absolute ethanol, 0.6 M HCl, 0.2 M NaOH, 25% NaCl, 80 mM Na taurocholate, and even boiling water (5,6). We wondered whether SA might also act as a mild irritant and thus afford gastric cytoprotection.
SUPPLEMENT TO VOL. 21
139
PROSTAGLANDINS
METHODS Animals. Male Sprague-Dawley rats of 200-240 g were used. Food, but notzwater, was removed in the morning. At 3:00 PM, water was also withheld, and the animals were placed in individual cylindrical stainless steel cages with flat bottom and perforations to allow ventilation. These cages, by limiting their movements, prevented the ingestion of hair and feces. This procedure, described earlier (7), does not appear to be stressful; the animals are often observed to be asleep in these cages. Production of gastric necrosis. On the following morning, after pretreatment with SA or saline, one ml of either absolute ethanol or 0.6 M HCl was administered orally, and the animals were killed one hour later. Their stomachs were dissected out, opened along the greater curvature and randomized. The mucosa was then examined with a 2X binocular magnifier by an observer who was unaware of the treatment given. The number of necrotic lesions for each stomach was counted. In other animals, gastric ulcers were produced by oral administration of 150 mg/kg of ASA suspended in one ml of 0.1 M HCl. One drop of Tween 80 was added for each 20 ml and the mixture was shaken with glass beads for 3 hours to obtain a fine suspension. The animals were killed with CO2 one hour after ASA administration, the stomachs were randomized and the number of ulcers for each stomach was counted. Treatment with SA. Sodium salicylate (SA) was administered either orally in one ml of water, 20 minutes before the necrotizing agents, or subcutaneously in one ml of saline, one hour before the necrotizing agents. In some experiments, the pH of the solution was adjusted to either 7.0 or 2.8; at pH 2.8, part of the SA precipitated to produce a milky suspension. For comparison, in some experiments ASA was also given under the same conditions as SA to other groups of animals. Ulcerogenicity of SA and ASA. SA and ASA (150 mg/kg) were administered orally in one ml of various concentrations 0":HCl, from 0.025 to 0.125 M. The animals were killed one hour later. The number of gastric ulcerations were counted in each stomach a, veraged in each group. The Student t test was used for statistical evaluation. The number of animals per group is indicated in the Figures. The EDs,, i.e., dose reducing average number of lesions per group by 50%, was calculated after the doses were plotted on a log scale. RESULTS Appearance of gastric lesions The gastric lesions produced by absolute ethanol, 0.6 M NC1 and acidified ASA were located in the corpus, i.e., the oxyntic portion secreting acid and pepsin. The antrum and forestomach looked intact;
SUPPLEMENT TO VOL. 21
PROSTAGLANDINS mucosal damage of the antrum after ethanol and HCl was detected only histologically. Lesions produced by ethanol and HCl consisted of elongated necrotic bands, black or red, l-10 mm long by l-3 l~lil wide, parallel to the long axis of the stomach. ASA-induced lesions were black spots in the corpus, penetrating not so deeply into the mucosa as the lesions produced by ethanol and HCl. SA protects against absolute ethanol and 0.6 M HCl SA prevented gastric necrosis produced by absolute ethanol and the degree of protection was dose dependent (Fig. 1). The effect was more pronounced after oral (EDso: 15 mg/kg) than subcutaneous (EDso: 150 mg/kg) administration. ASA, given under similar conditions, was not cytoptotective (Fig. 2). SA also protected against necrosis produced by 0.6 M HCl, and the degree of protection was near maximal at the doses used (Fig. 3). ~S~RICLESIONS/STOMACH 100 c
Figure 1. Sodium salicylate (SA) prevents lesions. SA given either 20 min (oral) or ministration of 1 ml of absolute ethanol. ethanol. Ten rats per group (oral), 8 rats
ethanol-induced gastric 1 hr (SC) before oral adRats killed 1 hr after per group (SC). *:P
SA protects against ASA SA reduced the number of ulcers produced by acidified ASA, and the effect was dose dependent (Fig. 4). The ED,, was 40 mg/kg orally and 100 mg/kg subcutaneously.
SUPPLEMENT TO VOL. 21
141
PROSTAGLANDINS
SASVRIC LESIOKS:
0
10
16
26 M6/K606ALLV
50
166
Figure 2. Aspirin (ASA) does not prevent ethanol-induced gastric lesions. ASA and SA given orally 20 min before oral administration of 1 mllof absolute ethanol. Rats killed 1 hr after ethanol. Eight rats per group. *: P
Figure 3. Sodium salicylate (SA) prevents HCl-induced gastric lesions. SA given either 20 min (oral) or 30 min (SC) before oral administration of 1 ml of 0.6 M HCl. Rats killed 1 hr after HCl. Eight rats per group. *: P
142
SUPPLEMENT TO VOL. 21
PROSTAGLANDINS
GASTRICLESIONS:
\
25
50 15 MCIYC
100
150
\
\
\
200
\
\ .* 300
Figure 4. Sodium salicylate (SA) prevents aspirin-induced gastric ulcers. SA given either 20 min (oral) or 60 min (SC) before oral administration of 150 mg/kg of aspirin in 0.1 M HCl. Rats killed 1 hr after aspirin. Eight to ten rats per group. *: PcO.01. Prior treatment with indomethacin Indomethacin, 5 mg/kg, was given orally one hour before administration of SA (100 mg/kg, orally). Twenty minutes after SA, absolute ethanol (one ml) was given orally, and the animals were killed one hour later. As shown in Fig. 5, the cytoprotection afforded by SA was not influenced by prior treatment with indomethacin. In the same study, other rats received 20% ethanol 20 minutes before absolute ethanol. This treatment reduced the number of necrotic lesions by 73% (from 13.0 lesions per stomach to 3.6) (Fig. 5). Indomethacin abolished the protection due to 20% ethanol (Fig. 5). Relative ulcerogenicity of SA and ASA Fio. 6 shows that SA was not ulcerooenic, even when diluted in UD to 0.125 M HCl. ASA, on the other hand,-was ulcerogenic, even when ’ given in water suspension. DISCUSSION These studies show that SA, in addition to not being ulcerogenic, is actually cytoprotective. The effect was shown after either oral or subcutaneous administration; it was more pronounced by the oral route. In separate studies, not reported here, salicylic acid itself was administered: it was not ulcerogenic and was as cytoprotective as the
SUPPLEMENT TO VOL. 21
143
PROSTAGLANDINS
Figure 5. Indomethacin does not affect cytoprotection by sodium salicylate ( A). Indomethacin: 5 mg/kg oral'ly, 1 hr before oral administration of either 100 mg/kg of SA or 1 ml of 20% ethanol. Absolute ethanol (1 ml) given orally 20 min after SA. Rats killed 1 hr after absolute ethanol. Indomethacin blocked cytoprotective effect of 20% ethanol but not that of SA. Ten rats per group. *: P
I
I ‘.t__--+_-~~, 0
25
50
100125
MI, mM
Figure 6. Relative ulcerogenicity of aspirin (ASA) and sodium salicylate (SA). ASA and SA: 150 mg/kg given orally in 1 ml of various concentrations of HCl. Rats killed 1 hr later. Six rats per group.
144
SUPPLEMENT TO VOL. 21
PROSTAGLANDINS
sodium salt. The mechanism of cytoprotection by SA is unknown. Unlike cytoprotection elicited by mild irritants, which is presumed td be due to endogenous formation of PG ("adaptive cytoprotection"), SA exerts this effect by another mechanism, since blocking the biosynthesis of PG by indomethacin did not prevent the protective action of SA. Unlike SA, ASA is not cytoprotective. Even when given orally at pH 7, ASA failed to inhibit mucosal necrosis produced by absolute ethanol (data not included). Interestingly, SA blocked the ulcerogenic activity of ASA. The fact that by itself SA was not ulcerogenic, even when administered with acid, is further indication that it does not irritate rat gastric mucosa. In the case of ASA, even an aqueous solution was ulcerogenic, probably because there was sufficient acid in the stomach to keep ASA in a unionized form and thus allow enough penetration into the mucosa to produce damage. Both SA and ASA were found earlier to be cytoprotective for the small intestine in rats. Oral and subcutaneous administration of these compounds prevented formation of perforating ulcerations of the small intestine produced by a large dose of indomethacin (8). FOOTNOTE This work was performed while the author was spending a year as a visiting scientist at the V.A. Center, CURE, Los Angeles, California. REFERENCES 1.
Vane, J. R. Inhibition of prostaglandin synthesis as a mechanism of action for aspirin-like drugs. Nature (New Biol.) 231:232, 1971.
2.
Glenn, E. M., B. J. Bowman, and N. A. Rohloff. Anomalous biological effects of salicylates and prostaglandins. Agents Actions 9:257, 1979.
3.
Whittle, B.J.R., G. A. Higgs, K. E. Eakins, S. Moncada, and J. R. Vane. Selective inhibition of prostaglandin production in inflammatory exudates and gastric mucosa. Nature -284:271, 1980.
4.
Davenport, H. W. Damage to the gastric mucosa: effect of salicylates and stimulation; Gastroenterology 49:189, 1965.
5.
Robert, A., C. Lancaster, A. J. Hanchar, and J. E. Nezamis. Mild irritants prevent gastric necrosis through prostaglandin forma74:1086, 1978. tion: histological study. Gastroenterology -
6.
Chaudhury, T. K., and A. Robert. Prevention by mild irritants of gastric necrosis produced in rats by sodium taurocholate.
SUPPLEMENT TO VOL. 21
145
PROSTAGLANDINS
Dig. Dis. SC. -25:830, 1980. 7.
Robert, A., J. E. Nezamis, and J. P. Phillips. Effect of prostaglandin El on gastric secretion and ulcer formation in the rat. Gastroenterology -55:481, 1968.
8.
Robert, A., A. J. Hanchar, C. Lancaster, and J. E. Nezamis. Cytoprotective effect of aspirin and aminopyrine. Gastroenterology -72:1120, 1977.
146
SUPPLEMENTTOVOL.21
GASTRIC CYTOPROTECTION BY SODIUM SALICYLATE Andre Robert Department of Experimental Biology The Upjohn Company Kalamazoo, Michigan 49001 ABSTRACT Sodium salicylate (SA), contrary to acetylsalicylic acid (ASA, aspirin), was not ulcerogenic in rats. SA was also found to be cytoprotective: it prevented formation of gastric mucosal necrosis produced by either absolute ethanol or 0.6 M HCl, and formation of gastric ulcers produced by acidified ASA. The degree of protection was dose dependent. The mechanism of this cytoprotection is unknown, but unlike cytoprotection elicited by mild irritants, e.g., 20% ethanol or 0.35 M HCl, whose effects appear to be due to endogenous formation of PG by the stomach, SA acts through a different mechanism, since its protective effect was not blocked by indomethacin. INTRODUCTION Acetylsalicylic acid (ASA, aspirin) and salicylic acid or its sodium salt (SA) are nonsteroidal antiinflammatory compounds. They are believed to exert their effect by inhibiting cyclooxygenase activity in inflamed tissues, and thus prostaglandin formation (1). However, they differ from each other in other aspects. In animals, ASA is ulcerogenic whereas SA is not (2). ASA inhibits prostaglandin cyclooxygenase activity in rat gastric mucosa, thereby eliciting a gastric depletion of prostaglandins (PG), whereas SA does not affect gastric PG cyclooxygenase (3). This different effect on the gastric content of PG may explain why only ASA is ulcerogenic. On the other hand, both ASA and SA, when introduced with acid in a dog gastric pouch (Heidenhain type), break the gastric mucosal barrier (4). This fact suggests that, at least in the dog, SA can irritate the stomach. We observed earlier that a variety of "mild" irritants, when given orally to rats minutes earlier, prevent development of gastric mucosal necrosis otherwise produced by "strong" irritants. Thus, 20% ethanol, a mild acid or a mild base (0.35 M HCl, or 0.075 M NaOH), a slightly hypertonic solution (4% NaCl) or a dilute bile salt solution (5 mM sodium taurocholate in 0.2 M HCl) protect the gastric mucosa against massive necrosis produced by absolute ethanol, 0.6 M HCl, 0.2 M NaOH, 25% NaCl, 80 mM Na taurocholate, and even boiling water (5,6). We wondered whether SA might also act as a mild irritant and thus afford gastric cytoprotection.
SUPPLEMENT TO VOL. 21
139
PROSTAGLANDINS
METHODS Animals. Male Sprague-Dawley rats of 200-240 g were used. Food, but notzwater, was removed in the morning. At 3:00 PM, water was also withheld, and the animals were placed in individual cylindrical stainless steel cages with flat bottom and perforations to allow ventilation. These cages, by limiting their movements, prevented the ingestion of hair and feces. This procedure, described earlier (7), does not appear to be stressful; the animals are often observed to be asleep in these cages. Production of gastric necrosis. On the following morning, after pretreatment with SA or saline, one ml of either absolute ethanol or 0.6 M HCl was administered orally, and the animals were killed one hour later. Their stomachs were dissected out, opened along the greater curvature and randomized. The mucosa was then examined with a 2X binocular magnifier by an observer who was unaware of the treatment given. The number of necrotic lesions for each stomach was counted. In other animals, gastric ulcers were produced by oral administration of 150 mg/kg of ASA suspended in one ml of 0.1 M HCl. One drop of Tween 80 was added for each 20 ml and the mixture was shaken with glass beads for 3 hours to obtain a fine suspension. The animals were killed with CO2 one hour after ASA administration, the stomachs were randomized and the number of ulcers for each stomach was counted. Treatment with SA. Sodium salicylate (SA) was administered either orally in one ml of water, 20 minutes before the necrotizing agents, or subcutaneously in one ml of saline, one hour before the necrotizing agents. In some experiments, the pH of the solution was adjusted to either 7.0 or 2.8; at pH 2.8, part of the SA precipitated to produce a milky suspension. For comparison, in some experiments ASA was also given under the same conditions as SA to other groups of animals. Ulcerogenicity of SA and ASA. SA and ASA (150 mg/kg) were administered orally in one ml of various concentrations 0":HCl, from 0.025 to 0.125 M. The animals were killed one hour later. The number of gastric ulcerations were counted in each stomach a, veraged in each group. The Student t test was used for statistical evaluation. The number of animals per group is indicated in the Figures. The EDs,, i.e., dose reducing average number of lesions per group by 50%, was calculated after the doses were plotted on a log scale. RESULTS Appearance of gastric lesions The gastric lesions produced by absolute ethanol, 0.6 M NC1 and acidified ASA were located in the corpus, i.e., the oxyntic portion secreting acid and pepsin. The antrum and forestomach looked intact;
SUPPLEMENT TO VOL. 21
PROSTAGLANDINS mucosal damage of the antrum after ethanol and HCl was detected only histologically. Lesions produced by ethanol and HCl consisted of elongated necrotic bands, black or red, l-10 mm long by l-3 l~lil wide, parallel to the long axis of the stomach. ASA-induced lesions were black spots in the corpus, penetrating not so deeply into the mucosa as the lesions produced by ethanol and HCl. SA protects against absolute ethanol and 0.6 M HCl SA prevented gastric necrosis produced by absolute ethanol and the degree of protection was dose dependent (Fig. 1). The effect was more pronounced after oral (EDso: 15 mg/kg) than subcutaneous (EDso: 150 mg/kg) administration. ASA, given under similar conditions, was not cytoptotective (Fig. 2). SA also protected against necrosis produced by 0.6 M HCl, and the degree of protection was near maximal at the doses used (Fig. 3). ~S~RICLESIONS/STOMACH 100 c
Figure 1. Sodium salicylate (SA) prevents lesions. SA given either 20 min (oral) or ministration of 1 ml of absolute ethanol. ethanol. Ten rats per group (oral), 8 rats
ethanol-induced gastric 1 hr (SC) before oral adRats killed 1 hr after per group (SC). *:P
SA protects against ASA SA reduced the number of ulcers produced by acidified ASA, and the effect was dose dependent (Fig. 4). The ED,, was 40 mg/kg orally and 100 mg/kg subcutaneously.
SUPPLEMENT TO VOL. 21
141
PROSTAGLANDINS
SASVRIC LESIOKS:
0
10
16
26 M6/K606ALLV
50
166
Figure 2. Aspirin (ASA) does not prevent ethanol-induced gastric lesions. ASA and SA given orally 20 min before oral administration of 1 mllof absolute ethanol. Rats killed 1 hr after ethanol. Eight rats per group. *: P
Figure 3. Sodium salicylate (SA) prevents HCl-induced gastric lesions. SA given either 20 min (oral) or 30 min (SC) before oral administration of 1 ml of 0.6 M HCl. Rats killed 1 hr after HCl. Eight rats per group. *: P
142
SUPPLEMENT TO VOL. 21
PROSTAGLANDINS
GASTRICLESIONS:
\
25
50 15 MCIYC
100
150
\
\
\
200
\
\ .* 300
Figure 4. Sodium salicylate (SA) prevents aspirin-induced gastric ulcers. SA given either 20 min (oral) or 60 min (SC) before oral administration of 150 mg/kg of aspirin in 0.1 M HCl. Rats killed 1 hr after aspirin. Eight to ten rats per group. *: PcO.01. Prior treatment with indomethacin Indomethacin, 5 mg/kg, was given orally one hour before administration of SA (100 mg/kg, orally). Twenty minutes after SA, absolute ethanol (one ml) was given orally, and the animals were killed one hour later. As shown in Fig. 5, the cytoprotection afforded by SA was not influenced by prior treatment with indomethacin. In the same study, other rats received 20% ethanol 20 minutes before absolute ethanol. This treatment reduced the number of necrotic lesions by 73% (from 13.0 lesions per stomach to 3.6) (Fig. 5). Indomethacin abolished the protection due to 20% ethanol (Fig. 5). Relative ulcerogenicity of SA and ASA Fio. 6 shows that SA was not ulcerooenic, even when diluted in UD to 0.125 M HCl. ASA, on the other hand,-was ulcerogenic, even when ’ given in water suspension. DISCUSSION These studies show that SA, in addition to not being ulcerogenic, is actually cytoprotective. The effect was shown after either oral or subcutaneous administration; it was more pronounced by the oral route. In separate studies, not reported here, salicylic acid itself was administered: it was not ulcerogenic and was as cytoprotective as the
SUPPLEMENT TO VOL. 21
143
PROSTAGLANDINS
Figure 5. Indomethacin does not affect cytoprotection by sodium salicylate ( A). Indomethacin: 5 mg/kg oral'ly, 1 hr before oral administration of either 100 mg/kg of SA or 1 ml of 20% ethanol. Absolute ethanol (1 ml) given orally 20 min after SA. Rats killed 1 hr after absolute ethanol. Indomethacin blocked cytoprotective effect of 20% ethanol but not that of SA. Ten rats per group. *: P
I
I ‘.t__--+_-~~, 0
25
50
100125
MI, mM
Figure 6. Relative ulcerogenicity of aspirin (ASA) and sodium salicylate (SA). ASA and SA: 150 mg/kg given orally in 1 ml of various concentrations of HCl. Rats killed 1 hr later. Six rats per group.
144
SUPPLEMENT TO VOL. 21
PROSTAGLANDINS
sodium salt. The mechanism of cytoprotection by SA is unknown. Unlike cytoprotection elicited by mild irritants, which is presumed td be due to endogenous formation of PG ("adaptive cytoprotection"), SA exerts this effect by another mechanism, since blocking the biosynthesis of PG by indomethacin did not prevent the protective action of SA. Unlike SA, ASA is not cytoprotective. Even when given orally at pH 7, ASA failed to inhibit mucosal necrosis produced by absolute ethanol (data not included). Interestingly, SA blocked the ulcerogenic activity of ASA. The fact that by itself SA was not ulcerogenic, even when administered with acid, is further indication that it does not irritate rat gastric mucosa. In the case of ASA, even an aqueous solution was ulcerogenic, probably because there was sufficient acid in the stomach to keep ASA in a unionized form and thus allow enough penetration into the mucosa to produce damage. Both SA and ASA were found earlier to be cytoprotective for the small intestine in rats. Oral and subcutaneous administration of these compounds prevented formation of perforating ulcerations of the small intestine produced by a large dose of indomethacin (8). FOOTNOTE This work was performed while the author was spending a year as a visiting scientist at the V.A. Center, CURE, Los Angeles, California. REFERENCES 1.
Vane, J. R. Inhibition of prostaglandin synthesis as a mechanism of action for aspirin-like drugs. Nature (New Biol.) 231:232, 1971.
2.
Glenn, E. M., B. J. Bowman, and N. A. Rohloff. Anomalous biological effects of salicylates and prostaglandins. Agents Actions 9:257, 1979.
3.
Whittle, B.J.R., G. A. Higgs, K. E. Eakins, S. Moncada, and J. R. Vane. Selective inhibition of prostaglandin production in inflammatory exudates and gastric mucosa. Nature -284:271, 1980.
4.
Davenport, H. W. Damage to the gastric mucosa: effect of salicylates and stimulation; Gastroenterology 49:189, 1965.
5.
Robert, A., C. Lancaster, A. J. Hanchar, and J. E. Nezamis. Mild irritants prevent gastric necrosis through prostaglandin forma74:1086, 1978. tion: histological study. Gastroenterology -
6.
Chaudhury, T. K., and A. Robert. Prevention by mild irritants of gastric necrosis produced in rats by sodium taurocholate.
SUPPLEMENT TO VOL. 21
145
PROSTAGLANDINS
Dig. Dis. SC. -25:830, 1980. 7.
Robert, A., J. E. Nezamis, and J. P. Phillips. Effect of prostaglandin El on gastric secretion and ulcer formation in the rat. Gastroenterology -55:481, 1968.
8.
Robert, A., A. J. Hanchar, C. Lancaster, and J. E. Nezamis. Cytoprotective effect of aspirin and aminopyrine. Gastroenterology -72:1120, 1977.
146
SUPPLEMENTTOVOL.21