Archivesof
TOXICOLOGY
Arch. Toxicol. 40, 143--150 (1978)
9 Springcr-Vcrlag 1978
Selective Cytotoxic Actions of Aspirin on Parietal Cells: A Principal Factor in the Early Stages of Aspirin-Induced Gastric Damage K. D. Rainsford I and K. Brune 2 i Department of Biochemistry, University of Tasmania, Medical School, G.P.O. Box 252C Hobart, Tasmania, 7001, Australia 2 Pharmakologisches Institut, Biozentrum der Universit~it Basel, Klingelbergstral3e 70, CH-4056 Basel, Switzerland
Abstract. Electronmicroscopic observations are reported on the effects of orally administered aspirin on the rat gastric mucosa to establish the cytotoxic events that occur during the early stages of the development of gastric damage induced by this drug. The results show that aspirin, apart from damaging superficial mucous cells, causes selective damage to the acid-secreting parietal cells located deep in the fundic mucosa. It is suggested that the selective accumulation of salicylates in the parietal cells may account for the specific cytotoxic actions of aspirin on these cells. Key words: Aspirin -- Analgesics - Anti-inflammatory - Gastric mucosa -Salicylates.
Introduction Gastric mucosal damage (ulceration and haemorrhage) are amongst the most serious side-effects associated with the use of aspirin and acidic nonsteroid anti-inflammatory (NSAI) drugs (Rainsford, 1975a; Cooke, 1976). The precise mechanism(s) by which these drugs cause gastric mucosal damage has not been established. It is clear, however, that absorption of these drugs, is, a priori, a principal factor in the development of gastric irritation (Rainsford, 1975a). Recently, several studies have shown that the parietal cells deep in the gastric mucosa are selectively damaged following oral administration of salicylates. This is a major factor, along with sloughing of surface mucosal ceils, in the development of gastric damage (Rainsford, 1975b; Pfeiffer and Weibel, 1973; Schweitzer and Brune, 1978). To explain why the parietal cells are uniquely sensitive to the actions of the acidic NSAI drugs it has been suggested (Rainsford and Brune, 1976) that aspirin and other acid NSAI drugs may be selectively absorbed and subsequently entrapped in the parietal celts. Following selective uptake into parietal ceils these drugs could then exert cytotoxic Send offprint requests to K. D. Rainsford at the above address
0340-5761/78/0040/0143/$ 01.60
144
K.D. Rainsford and K. Brune
actions (e.g., disruption o f lysosomes, mitochondrial dysfunction). These specific cytotoxic effects have been observed under the electronmicroscope at time intervals o f 1 h or more following oral administration o f aspirin or salicylic acid (Rainsford, 1975b; Schweitzer and Brune, 1978). While the evidence is clear that selective absorption of salicylates does occur in the parietal cells (~< 5 min) following oral administration of these drugs (Brune et al., 1977; Schweitzer and Brune, 1978) the exact relationship o f this to development of parietal cell d a m a g e has not been elucidated. This aspect is now considered in the present study. The results show that parietal cells are specifically d a m a g e d within a few minutes following oral administration o f aspirin.
Methods Male rats (180-200 g body weight), of the Hooded strain (University of Tasmania Medical School) were starved for 24 h and allowed water ad libitum before the experiments. Some experiments were also performed in male Wistar rats (Biozentrum der Universit/it, Basel) of 120--160 g body weight. Groups of five animals were dosed orally with 100 or 150 mg/kg of aspirin (Monsanto Aust. Ltd.) and killed at time intervals of 10, 20 and 60 min later. The drug was prepared as a fine suspension in distilled water immediately before use to minimize hydrolysis. Control animals received 1 ml water alone. Sections of the fundic mucosa were fixed for 5 h at 2-4 ~ C in paraformaldehyde/glutaraldehyde fixative prepared in 0.1 M sodium cacodylate buffer pH 7.4 with 5 mM CaC1 added or 0.1 M sodium phosphate buffer pH 7.4 as described (Rainsford, 1975b). The tissues were post-fLxedin 1% OsO 4 in 0.1 M phosphate buffer for 1.5 h (Rainsford, 1975b). Thin sections of araldite embedded material (see Rainsford, 1975b) were examined under an Hitachi HU-12 electronmicroscope.
Results and Discussion Electronmicroscopic examination o f the tissues from rats which had been dosed orally with 100 and 150 m g / k g aspirin for 10 min showed that the mucous cells of the superficial and gastric pit region o f the fundic m u c o s a had been sloughed a w a y or damaged. These d a m a g e d mucous cells were evident in all aspirin-dosed rats and a p p e a r e d the same as that noted previously in the rat 1 h after 200 m g / k g aspirin treatment (Rainsford, 1975b). Others have observed these effects following similar treatment to mice (Hingston and Ito, 1971) and ferrets (Pfeiffer and Weibel, 1973). Discharge o f mucus, generalized intracellular disruption, p r o b a b l y to lysosomal-like bodies, and sloughing a w a y o f whole cells were the principal effects. Some intracellular disruption o f acid-secreting canals (canaliculi), lysosomes and mitochondria was observed in m a n y o f the parietal cells adjacent to eroded areas or regions where gastric pit m u c o u s cells had been sloughed a w a y in all rats dosed with aspirin for 10 min. F e w e r parietal cells were affected c o m p a r e d to that previously noted at times o f 1 h or later following aspirin administration (Rainsford, 1975b). Thus d a m a g e to both parietal and mucous cells in the upper gastric pit occurs as part o f the development o f focal d a m a g e near the outermost luminal areas of the gastric mucosa. Attention was particularly directed to the parietal cells located in deeper regions o f the gastric pit o f aspirin-treated animals. While in thick sections these cells had a n o r m a l a p p e a r a n c e under the light microscope, m a r k e d intracellular changes were
Parietal Cell Cytotoxicity of Aspirin
145
Fig. 1. Disruption of the acid-secreting canals, or canaliculi (C), in a parietal cell located in the midgastric pit of a Hooded rat following oral administration of 150 mg/kg aspirin p.o. for 10 min. Mitochondria (M) appear well preserved. Magnification 30,000 •
observed in parallel thin sections examined under the electronmicroscope. These changes were observed in an appreciable number of parietal cells located in all regions ranging from the basal region (Fig. 1 and 2 compare with control in Fig. 3) through to the middle and upper gastric pit regions (Fig. 4). However, the damage involved more parietal cells and was more extensive in those cells located in the midupper than in the lower gastric pit regions.
146
K.D. Rainsford and K. Brune
Fig. 2. Appearance of large number of vacuoles (V) (which may be disrupted lysosomes) in parietal cells located deep in the gastric pit of a Hooded rat dosed with 150 mg/kg aspirin for 10 min. The rough endoplasmic reticulum and other intracellular components of the neighbouring zymogen (Chief) cell are unaffected. Magnification 75,000 x
Fig. 3. Parietal cell from a control rat (Hooded strain) given an oral dose of lml water and killed 10 min later. Magnification 22,000 x
7~
rl.
O
O
Fig. 4. Extensive disruption to mitochondria (M) in a parietal cell (20 min) following the oral administration to a rat of 150 mg/kg aspirin to a male Wistar rat. Magnification 11,600 x
r~
Parietal Cell Cytotoxicity of Aspirin
149
The first signs of damage to the parietal cells in aspirin-dosed rats are disruption of canaliculi (Fig. 1) and dense bodies (Fig. 2), which are presumably secondary lysosomes. Disruption of the secretory canaliculi seems to precede the appearance of large vacuoles and damaged mitochondria (Fig. 1, c.f. Fig. 2 and 4). The large vacuoles (Fig. 2) may be derived from lysosomes and so could represent sites of autolytic activity. It should be emphasized that it was only the parietal cells located in the deeper regions of the gastric mucosa that were damaged at the early times ( 1 0 - 2 0 rain) following aspirin administration. The neighbouring zymogen (Chief) cells at the base of the gastric pit (Fig. 1 and 2) and fibroblast-like cells in the midupper pit region (Fig. 4) were unaffected. The actions of aspirin appear quite selective on parietal ceils in the gastric pit region. Additional cellular changes observed were the ingress of some eosinophil cells and occasional opening of the junctions between endothelial cells. These effects were more evident at later time intervals (1 h) but did not appear to be related to the development of parietal cell damage. It is possible from these observations to reconstruct a series of events which may be taking place in parietal cells following the accumulation of high concentrations of aspirin (i.e. according as suggested to the pH-partition hypothesis). Firstly, aspirin could cause disruption of the membranes of the parietal cell canaliculi following the actions of salicylates on membrane systems (Inglot and Wolna, 1968; Levitan and Barker, 1972; McLaughlin, 19 73). Similarly, accumulation of the drug in the lysosomal membrane could lead to "labilization" of this membrane and subsequent release of hydrolytic enzymes in the presence of high concentrations of aspirin and salicylate (Tanaka and Iizuka, 1968, Harford and Smith, 1970) such as would be expected in the stomach. The release of hydrolytic enzymes (of acid pH optima) from lysosomes, and acid from within the secretory canaliculi could be responsible for the disruptive changes and autolytic events that were observed in these cells (Figs. 1, 2, and 4). Following cell death these regions of the gastric mucosa might serve as foci for further damage. Thus the parietal cells could be regarded as susceptible cells behind the normal defensive barrier (comprising the mucus layer and underlying mucous cells). A strategic weakening of the defence line against irritant drugs such as aspirin could be a consequence of specific drug actions on parietal cells.
Acknowledgements. The authors thank Miss Kathy Wagner of the Pharmacology Department (Biozentrum der Universit~itBasel) and Miss Barbara Greenstreet of the Department of Biochemistry (University of Tasmania) for expert technical assistance. This study was supported by the National Health and Medical Research Council (Australia), the Swiss National Foundation of Scientific Research and the University of Tasmania.
References Brune, K., Graf, P., Rainsford, K. D.: A pharmacokinetic approach to the understanding of therapeutic effects and side effects of salicylates. In: Proceedings of a Symposium on "Aspirin and Related Drugs: Their actions and uses". Hobart 1976 (K. D. Ralnsford, K. Brune, M. W. Whitehouse, eds.). Agents and Actions, Suppl. 1, 9--26 (1977) Brune, K., Schweitzer, A,, Eckert, H.: Parietal cells of the stomach trap salicylatesduring absorption. Biochem. Pharmacol. 26, 1735--1740 (1977) Cooke, A. R.: Drugs and gastric damage. Drugs 11, 36-44 (1976)
150
K.D. Ralnsford and K. Brune
Harford, D. J., Smith, M. J. H.: The effect of sodium salicylate on the release of acid phosphatase activity from rat liver lysosomes. J. Pharm. Pharmacol. 22, 578-583 (1970) Hingson, D. J., Ito, S.: Effect of aspirin and related compounds on the fine structure of mouse gastric mucosa. Gastroenterology (Basel) 61, 156-177 (1971) Inglot, A. D., Wolna, E.: Reactions of non-steroid anti-inflammatory drugs with the erythrocyte membrane. Biochem. Pharmacol. 17, 269-279 (1968) Levitan, H., Barker, J. L." Salicylate: A structure activity study of its effects on membrane permeability. Science, 176, 1423-1425 (1972) McLaughlin, S.: Salicylates and phospholipid bilayer membranes. Nature (Lond.) 243, 234--237 (1973) Pfeiffer, C. J., Weibel, J.: The gastric mucosal responses to acetysalicylic acid in the ferret. An ultrastructural study. Amer. J. dig. Dis., 15~ 834--846 (1973) Rainsford, K. D.: The biochemical pathology of aspirin induced gastric damage. Agents and actions 5, 326-344 (1975a) Rainsford, K. D.: Electronmicroscopic observations on the effects of orally administered aspirin and aspirin-bicarbonate mixtures on the development of gastric mucosal damage in the rat. Gut 16, 514-527 (1975b) Rainsford, K. D.: The comparative gastric ulcerogenic activities of non-steroid anti-inflammatory drugs. Agents and Actions 7, 573-577 (1977) Rainsford, K. D., Brune, K.: Role of the parietal cell in gastric damage induced by aspirin and related drugs: Implications for safer therapy. Med. J. Aust. 1, 881--883 (1976) Schweitzer, A., Brune, K." Salicylic acid and proquazone: The differences in absorption and biodistribution explain their different profile of side-effects. Persp. in Inflammation (in press) (1978) Tanaka, K., Iizuka, Y.: Suppression of enzyme release from isolated rat liver lysosomes by nonsteroidal anti-inflammatory drugs. Biochem. Pharmacol. 17, 2023--2032 (1968) Received October 18, 1977
TOXICOLOGY
Arch. Toxicol. 40, 143--150 (1978)
9 Springcr-Vcrlag 1978
Selective Cytotoxic Actions of Aspirin on Parietal Cells: A Principal Factor in the Early Stages of Aspirin-Induced Gastric Damage K. D. Rainsford I and K. Brune 2 i Department of Biochemistry, University of Tasmania, Medical School, G.P.O. Box 252C Hobart, Tasmania, 7001, Australia 2 Pharmakologisches Institut, Biozentrum der Universit~it Basel, Klingelbergstral3e 70, CH-4056 Basel, Switzerland
Abstract. Electronmicroscopic observations are reported on the effects of orally administered aspirin on the rat gastric mucosa to establish the cytotoxic events that occur during the early stages of the development of gastric damage induced by this drug. The results show that aspirin, apart from damaging superficial mucous cells, causes selective damage to the acid-secreting parietal cells located deep in the fundic mucosa. It is suggested that the selective accumulation of salicylates in the parietal cells may account for the specific cytotoxic actions of aspirin on these cells. Key words: Aspirin -- Analgesics - Anti-inflammatory - Gastric mucosa -Salicylates.
Introduction Gastric mucosal damage (ulceration and haemorrhage) are amongst the most serious side-effects associated with the use of aspirin and acidic nonsteroid anti-inflammatory (NSAI) drugs (Rainsford, 1975a; Cooke, 1976). The precise mechanism(s) by which these drugs cause gastric mucosal damage has not been established. It is clear, however, that absorption of these drugs, is, a priori, a principal factor in the development of gastric irritation (Rainsford, 1975a). Recently, several studies have shown that the parietal cells deep in the gastric mucosa are selectively damaged following oral administration of salicylates. This is a major factor, along with sloughing of surface mucosal ceils, in the development of gastric damage (Rainsford, 1975b; Pfeiffer and Weibel, 1973; Schweitzer and Brune, 1978). To explain why the parietal cells are uniquely sensitive to the actions of the acidic NSAI drugs it has been suggested (Rainsford and Brune, 1976) that aspirin and other acid NSAI drugs may be selectively absorbed and subsequently entrapped in the parietal celts. Following selective uptake into parietal ceils these drugs could then exert cytotoxic Send offprint requests to K. D. Rainsford at the above address
0340-5761/78/0040/0143/$ 01.60
144
K.D. Rainsford and K. Brune
actions (e.g., disruption o f lysosomes, mitochondrial dysfunction). These specific cytotoxic effects have been observed under the electronmicroscope at time intervals o f 1 h or more following oral administration o f aspirin or salicylic acid (Rainsford, 1975b; Schweitzer and Brune, 1978). While the evidence is clear that selective absorption of salicylates does occur in the parietal cells (~< 5 min) following oral administration of these drugs (Brune et al., 1977; Schweitzer and Brune, 1978) the exact relationship o f this to development of parietal cell d a m a g e has not been elucidated. This aspect is now considered in the present study. The results show that parietal cells are specifically d a m a g e d within a few minutes following oral administration o f aspirin.
Methods Male rats (180-200 g body weight), of the Hooded strain (University of Tasmania Medical School) were starved for 24 h and allowed water ad libitum before the experiments. Some experiments were also performed in male Wistar rats (Biozentrum der Universit/it, Basel) of 120--160 g body weight. Groups of five animals were dosed orally with 100 or 150 mg/kg of aspirin (Monsanto Aust. Ltd.) and killed at time intervals of 10, 20 and 60 min later. The drug was prepared as a fine suspension in distilled water immediately before use to minimize hydrolysis. Control animals received 1 ml water alone. Sections of the fundic mucosa were fixed for 5 h at 2-4 ~ C in paraformaldehyde/glutaraldehyde fixative prepared in 0.1 M sodium cacodylate buffer pH 7.4 with 5 mM CaC1 added or 0.1 M sodium phosphate buffer pH 7.4 as described (Rainsford, 1975b). The tissues were post-fLxedin 1% OsO 4 in 0.1 M phosphate buffer for 1.5 h (Rainsford, 1975b). Thin sections of araldite embedded material (see Rainsford, 1975b) were examined under an Hitachi HU-12 electronmicroscope.
Results and Discussion Electronmicroscopic examination o f the tissues from rats which had been dosed orally with 100 and 150 m g / k g aspirin for 10 min showed that the mucous cells of the superficial and gastric pit region o f the fundic m u c o s a had been sloughed a w a y or damaged. These d a m a g e d mucous cells were evident in all aspirin-dosed rats and a p p e a r e d the same as that noted previously in the rat 1 h after 200 m g / k g aspirin treatment (Rainsford, 1975b). Others have observed these effects following similar treatment to mice (Hingston and Ito, 1971) and ferrets (Pfeiffer and Weibel, 1973). Discharge o f mucus, generalized intracellular disruption, p r o b a b l y to lysosomal-like bodies, and sloughing a w a y o f whole cells were the principal effects. Some intracellular disruption o f acid-secreting canals (canaliculi), lysosomes and mitochondria was observed in m a n y o f the parietal cells adjacent to eroded areas or regions where gastric pit m u c o u s cells had been sloughed a w a y in all rats dosed with aspirin for 10 min. F e w e r parietal cells were affected c o m p a r e d to that previously noted at times o f 1 h or later following aspirin administration (Rainsford, 1975b). Thus d a m a g e to both parietal and mucous cells in the upper gastric pit occurs as part o f the development o f focal d a m a g e near the outermost luminal areas of the gastric mucosa. Attention was particularly directed to the parietal cells located in deeper regions o f the gastric pit o f aspirin-treated animals. While in thick sections these cells had a n o r m a l a p p e a r a n c e under the light microscope, m a r k e d intracellular changes were
Parietal Cell Cytotoxicity of Aspirin
145
Fig. 1. Disruption of the acid-secreting canals, or canaliculi (C), in a parietal cell located in the midgastric pit of a Hooded rat following oral administration of 150 mg/kg aspirin p.o. for 10 min. Mitochondria (M) appear well preserved. Magnification 30,000 •
observed in parallel thin sections examined under the electronmicroscope. These changes were observed in an appreciable number of parietal cells located in all regions ranging from the basal region (Fig. 1 and 2 compare with control in Fig. 3) through to the middle and upper gastric pit regions (Fig. 4). However, the damage involved more parietal cells and was more extensive in those cells located in the midupper than in the lower gastric pit regions.
146
K.D. Rainsford and K. Brune
Fig. 2. Appearance of large number of vacuoles (V) (which may be disrupted lysosomes) in parietal cells located deep in the gastric pit of a Hooded rat dosed with 150 mg/kg aspirin for 10 min. The rough endoplasmic reticulum and other intracellular components of the neighbouring zymogen (Chief) cell are unaffected. Magnification 75,000 x
Fig. 3. Parietal cell from a control rat (Hooded strain) given an oral dose of lml water and killed 10 min later. Magnification 22,000 x
7~
rl.
O
O
Fig. 4. Extensive disruption to mitochondria (M) in a parietal cell (20 min) following the oral administration to a rat of 150 mg/kg aspirin to a male Wistar rat. Magnification 11,600 x
r~
Parietal Cell Cytotoxicity of Aspirin
149
The first signs of damage to the parietal cells in aspirin-dosed rats are disruption of canaliculi (Fig. 1) and dense bodies (Fig. 2), which are presumably secondary lysosomes. Disruption of the secretory canaliculi seems to precede the appearance of large vacuoles and damaged mitochondria (Fig. 1, c.f. Fig. 2 and 4). The large vacuoles (Fig. 2) may be derived from lysosomes and so could represent sites of autolytic activity. It should be emphasized that it was only the parietal cells located in the deeper regions of the gastric mucosa that were damaged at the early times ( 1 0 - 2 0 rain) following aspirin administration. The neighbouring zymogen (Chief) cells at the base of the gastric pit (Fig. 1 and 2) and fibroblast-like cells in the midupper pit region (Fig. 4) were unaffected. The actions of aspirin appear quite selective on parietal ceils in the gastric pit region. Additional cellular changes observed were the ingress of some eosinophil cells and occasional opening of the junctions between endothelial cells. These effects were more evident at later time intervals (1 h) but did not appear to be related to the development of parietal cell damage. It is possible from these observations to reconstruct a series of events which may be taking place in parietal cells following the accumulation of high concentrations of aspirin (i.e. according as suggested to the pH-partition hypothesis). Firstly, aspirin could cause disruption of the membranes of the parietal cell canaliculi following the actions of salicylates on membrane systems (Inglot and Wolna, 1968; Levitan and Barker, 1972; McLaughlin, 19 73). Similarly, accumulation of the drug in the lysosomal membrane could lead to "labilization" of this membrane and subsequent release of hydrolytic enzymes in the presence of high concentrations of aspirin and salicylate (Tanaka and Iizuka, 1968, Harford and Smith, 1970) such as would be expected in the stomach. The release of hydrolytic enzymes (of acid pH optima) from lysosomes, and acid from within the secretory canaliculi could be responsible for the disruptive changes and autolytic events that were observed in these cells (Figs. 1, 2, and 4). Following cell death these regions of the gastric mucosa might serve as foci for further damage. Thus the parietal cells could be regarded as susceptible cells behind the normal defensive barrier (comprising the mucus layer and underlying mucous cells). A strategic weakening of the defence line against irritant drugs such as aspirin could be a consequence of specific drug actions on parietal cells.
Acknowledgements. The authors thank Miss Kathy Wagner of the Pharmacology Department (Biozentrum der Universit~itBasel) and Miss Barbara Greenstreet of the Department of Biochemistry (University of Tasmania) for expert technical assistance. This study was supported by the National Health and Medical Research Council (Australia), the Swiss National Foundation of Scientific Research and the University of Tasmania.
References Brune, K., Graf, P., Rainsford, K. D.: A pharmacokinetic approach to the understanding of therapeutic effects and side effects of salicylates. In: Proceedings of a Symposium on "Aspirin and Related Drugs: Their actions and uses". Hobart 1976 (K. D. Ralnsford, K. Brune, M. W. Whitehouse, eds.). Agents and Actions, Suppl. 1, 9--26 (1977) Brune, K., Schweitzer, A,, Eckert, H.: Parietal cells of the stomach trap salicylatesduring absorption. Biochem. Pharmacol. 26, 1735--1740 (1977) Cooke, A. R.: Drugs and gastric damage. Drugs 11, 36-44 (1976)
150
K.D. Ralnsford and K. Brune
Harford, D. J., Smith, M. J. H.: The effect of sodium salicylate on the release of acid phosphatase activity from rat liver lysosomes. J. Pharm. Pharmacol. 22, 578-583 (1970) Hingson, D. J., Ito, S.: Effect of aspirin and related compounds on the fine structure of mouse gastric mucosa. Gastroenterology (Basel) 61, 156-177 (1971) Inglot, A. D., Wolna, E.: Reactions of non-steroid anti-inflammatory drugs with the erythrocyte membrane. Biochem. Pharmacol. 17, 269-279 (1968) Levitan, H., Barker, J. L." Salicylate: A structure activity study of its effects on membrane permeability. Science, 176, 1423-1425 (1972) McLaughlin, S.: Salicylates and phospholipid bilayer membranes. Nature (Lond.) 243, 234--237 (1973) Pfeiffer, C. J., Weibel, J.: The gastric mucosal responses to acetysalicylic acid in the ferret. An ultrastructural study. Amer. J. dig. Dis., 15~ 834--846 (1973) Rainsford, K. D.: The biochemical pathology of aspirin induced gastric damage. Agents and actions 5, 326-344 (1975a) Rainsford, K. D.: Electronmicroscopic observations on the effects of orally administered aspirin and aspirin-bicarbonate mixtures on the development of gastric mucosal damage in the rat. Gut 16, 514-527 (1975b) Rainsford, K. D.: The comparative gastric ulcerogenic activities of non-steroid anti-inflammatory drugs. Agents and Actions 7, 573-577 (1977) Rainsford, K. D., Brune, K.: Role of the parietal cell in gastric damage induced by aspirin and related drugs: Implications for safer therapy. Med. J. Aust. 1, 881--883 (1976) Schweitzer, A., Brune, K." Salicylic acid and proquazone: The differences in absorption and biodistribution explain their different profile of side-effects. Persp. in Inflammation (in press) (1978) Tanaka, K., Iizuka, Y.: Suppression of enzyme release from isolated rat liver lysosomes by nonsteroidal anti-inflammatory drugs. Biochem. Pharmacol. 17, 2023--2032 (1968) Received October 18, 1977
