INFLUENCE OF ADRENOCORTICOTROPIN, CORTISONE, ASPIRIN, AND PHENYLBUTAZONE ON THE RATE OF EXFOLIATION AND THE RATE OF RENEWAL OF GASTRIC MUCOSAL CELLS

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1 GASTROENTEROLOGY Copyright 1970 by The Williams & Wilkins Co. Vol. 58, No.3 Printed in U.S.A. INFLUENCE OF ADRENOCORTICOTROPIN, CORTISONE, ASPIRIN, AND PHENYLBUTAZONE ON THE RATE OF EXFOLIATION AND THE RATE OF RENEWAL OF GASTRIC MUCOSAL CELLS MARTIN MAX, M.D., AND RENE MEN GUY, M.D. Department of Surgery, University of Chicago, Division of Biological Sciences and Pritzker School of Medicine, Chicago, Illinois The rate of exfoliation of canine gastric surface epithelial cells was estimated by measuring the concentration of deoxyribonucleic acid in Heidenhain pouch washings before, during, and after the administration of cortisone, adrenocorticotropic hormone (ACTII), aspirin, and phenylbutazone. Also, the mitotic frequency in gastric mucosal biopsies was determined before and during the administration of ACTH and aspirin. In addition, the influence of aspirin on the rate of uptake of thymidine-h3 by the gastric mucosa of rats was evaluated by radioautography. The rate of exfoliation of surface epithelial cells was reduced by ACTH and cortisone and was increased by aspirin and phenylbutazone. Whereas ACTH decreased the mitotic frequency in gastric mucosal biopsies, aspirin had no significant effect; moreover, aspirin did not affect the rate of uptake of thymidine-h3 by gastric mucosa of rats. The data suggest that one of the mechanisms of gastric mucosal injury by ACTH and cortisone may be a reduced rate of renewal of surface epithelial cells. Aspirin and phenylbutazone, on the other hand, appear to affect the gastric mucosa in such a way that shedding of surface cells increases without a concomitant increase in the rate of cell renewal. The ability of the anti-inflammatory drugs, cortisone, adrenocorticotropic hormone (ACTH), aspirin, and phenylbutazone, to cause gastric mucosal injury through erosions and hemorrhage is an important and as yet unexplained phenomenon. While it would appear attractive to explain the action of these drugs to Received August 6, Accepted October 1, Address requests for reprints to: Dr. Rene Menguy, Department of Surgery, University of Chicago, 950 East 59th Street, Chicago, Illinois This work was supported in part by Grant AM from the National Institute of Arthritis and Metabolic Diseases and Training Grant 5T1-GM01720 from the National Institute of General Medical Sciences, National Institutes of Health, United States Public Health Service, and in part by the George A. Bernard Surgical Research Fund. hypersecretion of acid and/or pepsin, it has proven difficult to substantiate this thesis experimentally. Studies of the influence of ACTH and cortisone on gastric acid secretion have yielded contradictory results, with some workers reporting an increase,!' 2 and others no change or a decrease.3, 4 The administration of aspirin to man5 or to dogs6 has been shown to decrease gastric acid secretion even when the contact of aspirin with the acid secreting mucosa was avoided. Bonfils et al. found a decrease in gastric acidity in subjects receiving phenylbutazone. 7 Moreover, states of gastric hyperacidity in man usually cause duodenal ulceration and almost never, in our experience, gastric ulceration or diffuse erosive gastritis, which appear to characterize the mucosal injury by these drugs. Since the role of the ulcer-abetting, acid-pepsin agency is 329

2 330 MAX AND MEN GUY Vol. 58, No.8 in doubt, it seemed appropriate to examine any influence that these drugs may have on the mechanisms which normally protect the gastric mucosal membrane from the action of its own secretions. In 1954, Hollander formulated the concept of a protective two-component gastric mucosal barrier consisting of a layer of mucus adherent to the underlying layer of superficial epithelial cells, with the former being continuously renewed by secretions from the latter.8 The ability of this gastric mucosal barrier to accomplish its protective function should depend upon a normal rate of mucus secretion on the one hand and on a normal rate of epithelial cell renewal on the other. Previous studies from this laboratory have shown that cortisone,9 ACTH,lO aspirin,ll and phenylbutazone I2 all decrease the rate of mucus secretion by the canine gastric antrum, and have formed the basis for the suggestion that altered rates of mucus secretion may, in part, e x p l the a i ~ phenomenon of gastric mucosal injury by these drugs. In addition, however, the influence of these drugs on the rate of renewal of the cellular component of the gastric mucosal barrier must be determined. This avenue already has been explored partly. Rasanen found that short term administration of ACTH to rats reduced the mitotic rate of the gastric mucosa.is However, prolonged administration of ACTH has been found to increase the number of mitoses in the gastric mucosa of the rat.14 The administration of glucocorticoids to rats reduced the frequency of gastric mucosal mitoses.1s Similar studies in larger animals have not been done. Croft found that human gastric washings contained larger concentrations of deoxyribonucleic acid (DNA) after the stomach had been irrigated with solutions of aspirin.15 Although this indicates that the rate of exfoliation of gastric epithelial cells increases under these conditions, it does not reveal whether the increase results from direct contact of acetylsalicy lic acid with the gastric mucosa or from some action of circulating aspirin on cellular metabolism and it does not provide information on the actual rate of cell renewal in the gastric mucosa. The purpose of the presently reported studies is to evaluate the influence of cortisone, ACTH, aspirin, and phenylbutazone on the rate of exfoliation of surface epithelial cells of the canine gastric mucosa into the lumen of the stomach and to determine what influence these drugs have on the rate of renewal of gastric mucosal cells. Materials and Methods Evaluation of rate of exfoliation of gastric mucosal cells. Heidenhain pouches were constructed in mongrel dogs weighing from 18 to 25 kg. The animals were maintained on a high protein laboratory diet supplemented with salt. After a 30-day recovery period from the operation, experiments were started. To minimize the factor of HCI secretion, all collections from the pouches were made after the animals had been fasted for 30 hr. Phosphate buffer (ph 7.4, 0.06 M), in an amount adjusted to the volume of each pouch, was instilled by gravity into the pouches and was withdrawn after 10 min. Three such consecutive 1O-min washings were pooled to be analyzed for concentration of DNA. The samples were transferred quantitatively into a centrifuge bottle containing a small amount of 2.0 M ethylenediaminetetra acetate to minimize destruction of DNA by deoxyribonucleases. Samples were maintained on ice at all times. After measuring the volume of the sample, 1 ml of 30% albumin per 100 ml of sample and 10 ml of 50% trichloroacetic acid per 100 ml of sample were added to the latter to precipitate the DNA and the sample was frozen for 1 to 7 days. The thawed sample was centrifuged (1800 g) and the supernatant fluid was discarded. The precipitate was suspended in cold 5% trichloroacetic acid, transferred to a 50-mI centrifuge tube, and centrifuged (2500 g). The supernatant fluid was discarded. The sediment was resuspended in 5% trichloroacetic acid and was hydrolyzed twice at 90 C for 15 min. The hydrolysates were saved and combined. The volume was recorded and the sample was frozen to precipitate any remaining solids. After thawing and centrifugation at 2500 g, the final solution was kept at 4 C until analyzed for DNA. To avoid interference of sialic acid in gastric washings, the Croft modification of the method of Burton was used.. " Hydrolysates prepared from calf thymus DNA served as standards. The standard DNA solution contained 40 mg of DNA per 100 mi in 5.0 X 10-3 M NaOH and

3 March 1970 RATE OF EXFOLIATION AND RENEWAL 331 was stored at 4 C. The solution used for the analyses contained 836 mg atoms of P per m!. To determine the amount of DNA in the sample, 2 ml of the hydrolysate followed by 2 ml of diphenylamine reagent were added to 5 ml of perchloric acid. The reaction was carried out at 6 to 13 C for 48 to 54 hr. The optical density of the samples was read at 600 mft and the amount of DNA in the unknown samples was calculated by comparison with the standard. The amount of DNA in gastric washings was expressed as millimicrogram atoms of P per minute and was considered to be a measurement of the rate of exfoliation of surface epithelial cells. The influence of various drugs on this rate of exfoliation was evaluated as follows. The influence of aspirin was studied by giving each dog 1.2 g of aspirin orally 4 hr before each collection during a test period of at least 16 collections. The latter was preceded by a control period of at least 16 collections. The dogs were allowed to recover from the effects of aspirin. During this 5- to 6-week recovery period the collections were continued. The first 16 collections made during the recovery period served as a recontrol-recovery observation for the aspirin experiment. The next 16 collections served as a control for the subsequent experiment in which the animals received phenylbutazone (50 to 70 mg per kg twice a day for 14 days). The dogs were allowed to recover for 3 months, after which they received in the same sequence as above, 5 U per kg of ACTH gel daily for 14 days followed by cortisone acetate (10 mg per kg per day) for 14 days. One dog was added to the previously used 3 for the cortisone experiment. Student's t-test was used to evaluate the significance of differences between control and test mean values. Evaluation of the influence of drugs on the rate of cell renewal in the gastric mucosa. 1. Stainless steel cannulae were placed in the stomachs of mongrel dogs at the junction of the body and the antrum. The cannulae were brought out through a stab wound in the anterior abdominal wall. Through the cannulae and using a small proctoscope, biopsy specimens were taken at intervals from the posterior wall of the fundus. Tissues were fixed in a flat position in buffered formalin, and sections 4 ft thick were stained with hematoxylin and eosin. One slide was prepared from each biopsy specimen. At the completion of the entire experiment the slides were coded by someone not involved in the study and then were read by one of the authors. The code was broken only after all of the counts had been recorded. Mitoses were counted in the neck stratum of the gastric glands and only in glands that were sectioned longitudinally to the glandular lumen. In each section 2000 cells belonging to the neck stratum of the glands were counted and the results were expressed as mitoses per 2000 cells. Drugs were given for the following periods: ACTH (5 U per kg daily) for 8 days; aspirin (1.2 g per kg in 50% dimethyl sulfoxide (DMSO) subcutaneously; 50% DMSO alone during the control period) for 10 days. Mucosal biopsies were taken on the 4th and last day of drug administration. Two control biopsies, 4 days apart, were taken prior to the administration of each drug. The average of the two control and test counts were taken to express the control and test mitotic index for each dog and for each experiment. The significance of the difference between control and test mitotic indices was evaluated by the paired difference test. 2. To determine the influence of aspirin on the rate of renewal of mucosal cells in the gastric mucosa of the rat, 48 male rats weighing from 180 to 200 g were paired by weight. Test rats received 300 mg per kg per day of aspirin dissolved in 50% DMSO subcutaneously in three divided doses. The first dose given 3 hr prior to the injection of thymidine-h", the second at the time of thymidine-h" administration, and the third 3 hr later. Subsequent injections of aspirin were 8 hr apart. The paired control animals received similar volumes of DMSO alone. Both test and control rats received 200 p..c of thymidine-h" intraperitoneally. Eight pairs of rats were killed 7 hr after the administration of thymidine-h" (during which time they had received two doses of aspirin plus a loading dose). Another 8 pairs were killed 24 hr after the injection of thymidine and the remaining 8 pairs were killed at 48 hr after the administration of thymidine-h". The stomachs were opened along the greater curvature, pinned flat to cardboard, and Immersed overnight in Carnoy's fixative in the cold. Thin longitudinal sections including the forestomach and antrum were cut from the paraffin-imbedded sections (two from each stomach). The slides were coated with radiosensitive emulsion (Kodak Nuclear Track Emulsion, type NTB2 ) according to the method of Kopriwa and Leblond17 and were exposed for 14 days. The exposed emulsions were de-

4 332 MAX AND MEN GUY Vol. 58, No.3 veloped in Kodak Dektol D-72 developer and fixed in Kodak fixative 2F-5. After drying in a dust-free atmosphere, the tissues were stained with Feulgen's stain. The slides were coded as described in the previous paragraph and were studied blind. Slides were studied at approximately 600 magnification and the number of cells per crypt was counted for a total of 20 crypts per slide. Crypts cut tangentially were disregarded. The rate of cell renewal was expressed as the number of labeled cells percentage of the total crypt cell count. Analysis of variance (pair comparison) was used to evaluate the difference between cell labeling of control and test rats. Results Influence of aspirin, phenylbutazone, ACTH, and cortisone on the rate of exfoliation of gastric mucosal cells. The results of these experiments are summarized in table 1. The effects of aspirin and phenylbutazone differed sharply from those of ACTH and cortisone. During the administration of aspmn and of phenylbutazone the rate of cell exfoliation from the mucosa of the Heidenhain pouches increased sharply, as evidenced by the increased DNA content of the gastric washings. After withdrawal of as- TABLE 1. Influence of aspirin, phenylbutazone, ACTH, and cortisone on the rate of exfoliation of gastric mucosal cezzsg Drug Dog no. No. of tests Control Testb Recovery Aspirin ± ± 5.1 A = +52% P < ± ± 2.0 A = +110% P < ± ± 14.5 A = +272% P < Phenylbutazone ± ± 2.1 A = +178% P < ± ± 7.2 A = +51% P < ± ± 15.1 A = +88% P < ACTH ± ± 0.59 A= -41% P < ± ± 7.7 A= -34% P < ± ± 1.8 A = -29% P < 0.05 Cortisone ± ± 1.4 A= -34% P < ± ± 5.7 A = -19% P < ± ± 1.6 A= -32% P < ± ± 4.8 A= -66% P < ± ± ± ± ± 13.5c 5.2 ± ± ± ± ± ± ± 2.5 G All values expressed as mj.!g atoms of DNA-P/min ± SD. b A, deviation from control value; P, significance of A. c This dog began to bleed at the end of test period with phenylbutazone which probably explains the failure to return to control values during the recovery period.

5 March 1970 RATE OF EXFOLIATION AND RENEWAL 333 TABLE 2. Influence of ACTH and aspirin on the mitotic activity of the canine gastric mucosa G Dog no. ACTH Aspirin Control Test Ab Control Test A C! 7.5 ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± G Average ± SD mitotic index from two biopsies, control or test. b Mean difference for ACTH, ± 1.07 (95% confidence limits). C Mean difference for aspirin, ± 1.35 (95% confidence limits). TABLE 3. Rate of cell renewal in the gastric mucosa of rats receiving aspirin 7-Hr renewal 24-Hr renewal 48-Hr renewal Control Test pb Control Test P Control Test P > > >0.8 G All test and control rats were paired. Values represent percentage of crypt cells that were labeled. b P, significance of variation between paired control and test rats. pirin and phenylbutazone, rates of cell exfoliation returned to control levels except for one dog (no. 2) who began to have mild bleeding from the pouch on the last test day with phenylbutazone. The reverse obtained when the dogs received ACTH and cortisone. In all instances, the rate of cell exfoliation fell significantly during the test period and returned to control levels during the recov ~ rperiod. y Influence of ACTH and aspirin on the mitotic activity of the canine gastric mucosa. The data are summarized in table 2. As in the previous experiment, the effects of ACTH differed from those of aspirin. Under the influence of ACTH the measured mitotic index decreased. The 95% confidence interval around the mean difference between control and test values was below zero. One can conclude from these data that ACTH (and presumably cortisone) reduces mitotic activity in the canine gastric mucosa. On the other hand, the 95% confidence interval around the mean difference between the paired control and test aspirin values included zero. The latter suggests that aspirin has no influence on the mitotic activity of the gastric mucosa. Phenylbutazone similarly was evaluated in 3 dogs. Because of the smaller sampling, the data are not reported in detail. However, the results were similar to those obtained with aspirin in that no consistent change in mitotic activity was produced by the drug. Influence of aspirin on the rate of cell renewal in the gastric mucosa. The data are summarized in table 3. They show quite clearly that the percentage of labeled cells was not different in the aspirintreated rats than in the paired control rats. This suggests that aspirin does not affect the mitotic activity of the rat gastric mucosa, an observation which confirms the one made above in the dog. Discussion It should be noted that one possible source of error in the estimation of the rate of cell exfoliation lies in degradation of DNA by deoxyribonucleases in gastric juice. This was minimized by limiting the duration of each pouch washing to 10

6 334 MAX AND MENGUY Vol. 58, No.3 min, by cooling the collected material immediately, and by the addition of ethy lenediaminetetraacetate. Moreover, any loss of DNA during the collection should remain constant and should not affect relative changes brought about by the drugs studied. Also, the estimation of cellular renewal by determination of the mitotic index could be affected by an increase or a decrease in the duration of mitosis. However, none of the drugs that we studied is known to alter the duration of mitosis. The surface epithelium of the alimentary tract undergoes continuous renewal. In the rat, the animal in which most of such cell kinetic studies have been done, the diurnal renewal of the surface epithelium of the stomach has been estimated at 35%.13 The regeneration of surface epithelial cells results from undifferentiated cells of the neck stratum of the gastric glands. The latter dividing mitotically differentiate into mucous neck cells or surface epithelial cells, which migrate up the crypts and, upon reaching the surface epithelium, are shed into the lumen. It seems reasonable to assume that the stomach of most large mammals including man repeatedly is subjected to multiple minute traumata because of the large amount of roughage in most mammalian diets. The repair of such microtraumata must depend upon the rate of renewal of surface epithelial cells. A decrease in the latter should retard the healing of microdefects in the monocellular surface epithelial layer which could, conceivably, serve as loci minoris resistentiae for the subsequent formation of erosions or ulcers. Our data suggest that ACTH and adrenocortical steroids have this influence on the gastric mucosa. Under the influence of ACTH and cortisone, the concentration of DNA in gastric pouch washings decreased, suggesting a reduced rate of exfoliation of surface epithelial cells. In turn, the latter probably resulted from a diminished rate of renewal of surface epithelial cells in view of the reduced mitotic index in the gastric mucosa that we observed during ACTH administration. These findings confirm those of Rasanen who found that ACTH and cortisone reduced the mitotic frequency in the gastric mucosa of the rat. 13 He did not find similar changes in the intestinal mucosa which may explain why steroid lesions are limited largely to the gastric mucosa. The idea that a reduced number of mitoses in the gastric mucosa may affect adversely the ability of the mucosal membrane to protect itself against or to repair an injury is borne out by the elegant experiments of Janowitz et al. who produced excision ulcers in gastric mucosal explants to the abdominal wall of dogs.ls Healing of the lesions was retarded significantly by the administration of ACTH or cortisone, a finding which may be explained by the reduced rate of renewal of the surface epithelium, a possibility which Janowitz and co-workers did consider. Our results using aspirin and phenylbutazone are less easy to interpret. Both agents caused a significant rise in the rate of exfoliation of surface epithelial cells as evidenced by the higher concentration of DNA in gastric pouch washings during the test period than during the control or recovery periods. Although our findings agree with those of Croft/5 who reported larger amounts of DNA in the gastric washings of human subjects after the stomachs had been irrigated with solutions of aspirin, the changes that we observed had to result from some action of the drugs in the circulation since at no time did they come into direct contact with the mucosa of the Heidenhain pouches. Why does exfoliation of surface epithelial cells increase in dogs receiving aspirin or phenylbutazone? A priori, one might attribute this to an influence on the renewal rate of surface epithelial cells contrary to that produced by ACTH, i.e., an increased number of mitoses in the gastric mucosa. This hypothesis seems untenable since in neither the rat nor the dog did aspirin alter the mitotic frequency in the gastric mucosa. Although only three experiments with phenylbutazone were done in dogs, the results did not reveal any influence of phenylbutazone on the mitotic rate. How does one explain, then, the increased in-

7 March 1970 RATE OF EXFOLIATION AND RENEWAL 335 traluminal shedding of surface epithelial cells produced by these two drugs. Quite possibly these drugs alter the consistency of interstitial, intercellular mucopolysaccharides, thus reducing intercellular adhesiveness. This, coupled with a reduced coat of surface mucus, conceivably could enhance shedding of cells. The fact that we have found reduced levels of glycoproteins in gastric secretions of dogs re CeIVIng aspirinll and phenylbutazone!2 and that BolleV9 noted impaired mucopolysaccharide synthesis in connective tissue under the influence of aspirin and phenylbutazone support this thesis. In turn, an increased loss of mucosal cells without a concomitant increase in cell replacement could lead to patchy mucosal denudation, erosions, and bleeding. In previous publications from this laboratory, it was reported that cortisone9 ACTH,!o aspirin,!! and phenylbutazone12 all reduce the rate of secretion of mucus by the canine stomach and reduce the concentration of protein-bound carbohydrate in these secretions. On the basis of these observations it was postulated that these agents injure the gastric mucosa by reducing its ability to form a protective layer of mucus. Although this thesis remains valid, it would seem reasonable, on the basis of the data in this report, to postulate an additional mechanism in the case of the lesions due to ACTH and cortisone, namely an impaired rate of renewal of the layer of surface epithelial cells. With respect to aspirin, we have emphasized the influence of parenteral aspirin on the gastric mucosa. Davenport20 has shown that the topical application of aspirin to the gastric mucosa affects the permeability of the mucosal barrier. Which of these routes, topical and parenteral, is the more important in the clinical phenomenon of gastric mucosal injury by aspirin remains to be determined. REFERENCES 1. Villareal, R., W. F. Ganong, and S. J. Gray Effect of adrenocorticotropic hormone upon the gastric secretion of hydrochloric acid, pepsin and electrolytes in the dog. Amer. J. Physiol. 183: Cooke, A. R., and M. 1. Grossman Effect of hydrocortisone on secretion of acid and pepsin by Heidenhain pouches. Proc. Soc. Exp. Bioi. M ed. 123: Hirschowitz, B. 1., D. H. P. Stretten, H. M. Pollard, and J. A. Boldt, Jr Role of gastric secretions in activation of peptic ulcers by corticotropin. J. A. M. A. 168: Dragstedt, L. R., H. Ragins, L. R. Dragstedt, Jr., and S. O. Evans Stress and duodenal ulcer. Ann. Surg. 144: Winkelman, E. 1., and W. H. J. Summerskill Gastric secretion in relation to gastrointestinal bleeding from salicylate compounds. Gastroenterology 40: Lynch, A., H. Shaw, and G. W. Milton Effect of aspirin on gastric secretion. Gut 6: Bonfils, S., J. P. Hardouin, C. Richir, and F. Delbarre Considerations cliniques et experimentales sur la genese des lesions gastriques (ulceres, hemorragies) provoquees par la phenylbutazone. Bull. Soc. Med. Hop. Paris 71: Hollander, F The two-component mucous barrier. Its activity in protecting the gastroduodenal mucosa against peptic ulceration. A. M. A. Arch. Intern. Med. 93: Menguy, R., and Y. F. Masters Effect of cortisone on mucoprotein secretion by gastric antrum of dogs: pathogenesis of steroid ulcer. Surgery 54: Desbaillets, L., and R. Menguy Inhibition of gastric mucous secretion by ACTH: an experimental study. Amer. J. Dig. Dis. 12: Menguy, R., and Y. F. Masters Effects of aspirin on gastric mucous secretion. Surg. Gynec. Obstet. 120: Menguy, R., and L. Desbaillets Influence of phenylbutazone on gastric secretion of mucus. Proc. Soc. Exp. Bioi. M ed. 125: Rasanen, T Fluctuations in the mitotic frequency of the glandular stomach and intestine of rat under the influence of ACTH, glucocorticoids, stress and heparin. Acta Physiol. Scand. 58: Rasanen, T., and H. Teir Adrenocorticotrophin and mitotic activity. Growth 25: Croft, D. N Aspirin and the exfoliation of gastric epithelial cells. Cytological and

8 336 MAX AND MENGUY Vol. 58, No.3 biochemical observations. Brit. M ed. J. 2: Croft, D. N., and M. Lubran The estimation of deoxyribonucleic acid in the presence of sialic acid: application to analysis of human gastric washings. Biochem. J. 95: 612-.Q Kopriwa, B. M., and C. P. Leblond Improvements in the coating technique of radioautography. J. Histochem. Cytochem. 10: Janowitz, H. D., V. A. Weinstein, R. G. Shaer, J. F. Cereghini, and F. Hollander The effects of cortisone and corticotropin on the healing of gastric ulcer: an experimental study. Gastroenterology 34: Bollet, A. J Inhibition of glucosamine- 6-PO. synthesis by salicylate and other anti-inflammatory agents in vitro. Arthritis Rheum. 4: Davenport, H. W Gastric mucosal hemorrhage in dogs. Effects of acid, aspirin, and alcohol. Gastroenterology 56: