Ethanol damage to rat gastric mucosa is unlikely to be mediated by ethanol

Exp. Pathol. 1988; 33: 173- 177 VEB Gustav Fischer Verlag J ena Department of Medicine, Austin Hospital, Melbourne, Victoria, Australia

Ethanol damage to rat gastric mucosa is unlikely to be mediated by ethanol By R. B. SEWELL, T. S. LING and N. D. YEOMA~S With one figure Address for correspondence: Dr. RICHARD B. Melbourne, Victoria 3084, Australia

SEWELL,

Departmcnt of Medicine, Austin Hospital,

Key W 0 r d s: ethanol; gastric mucosa; antral mucosa; lysosomes; enzymes, lysosomal; Triton WR 1339; stomach; rat

Summary During lllJury to the gastric mucosa, lysosomes become more fragile and lysosomal enzymes, which are activated at acid pH, leak into the surrounding environment. It is not clear whether these changes contribute to the mechanism of damage or are merely a secondary result of it. To test whether lysosomes modulate gastric mucosal damage, we pretreated rats with a lysosomallabilizing agent, Triton WR 1339 (1.5 g/ kg) and histologically assessed mucosal damage in vivo after challenge with 30 % ethanol. No significant differences were found in the length or depth of eroded mucosa: mean erosion length, Triton 23.9 ± 6.6 % vs. control 19.7 ± 5.2 %; mean depth (p;m), Triton 19 ± 4 vs. control 20 ± 7. After a similar pret eatment regimen, rat antral mucosa was cultured, challenged with ethanol and damage assessed by release into media of previously incorporated mucosal 51 chromium. With 15 % ethanol challenge, no change in 51 chromium release was seen: after Triton, 9.8 ± 1.4 % vs. control 10.3 ± 1.0 {Yo. Triton pretreatment perturbed gastric lysosomes as shown in organ culture by significantly raised tissue lysosomal enzyme activities and increased lysosomal enzyme release into culture media after ethanol challenge. The lack of effect of this pretreatment regimen suggests that Iysosomes do not have a major pathogenetic role in cthanolinduced gastric damage.

I ntroduct'ion Lysosomes contain an array of digestive enzymes with the capability to degrade all components of living cells (DE DUVE et al. 1966). Several authors have suggested that damage to gastric mucosallysosomes, with resulting leakage of their enzymes, might be an important pathogenetic mechanism in the gastric mucosal injury produced by a variety of agents (FERGUSON et al. 1973; PFEIFFER et al. 1980; WALDRON-EDWARD et al. 1980). Indeed, the ability of ulcerogenic agents - such as ethanol, aspirin or bile acids - to allow back diffusion of hydrogen ions from the gastric lumen (DAVENPORT 1964; 1967; HIMA1 et al. 1975). would potentially provide optimal conditions for lysosomal hydrolases, which are activated at an acidic pH (DE DuVE et al. 1966). Ethanol, for example, can readily traverse lipid-bilayer membranes and alter membrane structure (TARAS CHI et al. 1985) and also labilize the lysosomal membrane and increase release of lysosomal enzymes (WALDRON-EDWARDS et al. 1980). Several studies have demonstrated that leakage of lysosomal enzymes and/or increased lysosomal fragility occur in parallel with mucosal injury caused by such diverse stimuli as ethanol (CHIU et al. 1983; WHITTLE et al. 1985), bile acids (WALDRON-EDWARDS et al

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1977), aspirin (HIMAL et al. 1975), serotonin (FERGUSON et al. 1973), reserpine (PFEIFFER et al. 1980), restraint stress (FERGUSON et al. 1972) and sepsis (POLSON et al. 1982). Conversely, agents such as carbenoxolone (SYMONS et al. 1980) and prostanoids (FERGUSON et al. 1973), which have as one of their properties the ability to stabilize lysosomal membranes, have "cytoprotective" actions on the gastric mucosa. However, it is still not clear whether these lysosomal changes contribute to mucosal damage and protection or are merely a result of it (WHITTLE et al. 1985). To evaluate the role of Iysosomes in ethanol-induced gastric damage, we examined the effects of pretreatment with the lysosomotropic agent Triton WR 1339, which is known to enhance lysosomal fragility (NAGORNEY et al. 1983; LARusso et al. 1982; W ATTIAUX et al. 1963). The degree of mucosal ulceration was assessed firstly by quantitative light microscopy and secondly by release of 51 chromium from pre-loaded mucosal cells using a gastric organ culture preparation (SEWELL et al. 1986). If the hypothesis that lysosomes playa causative role in gastric mucosal ulceration were true, it would be predicted that mucosal damage would be greater in Triton WR 1339 pre-treated animals.

111aterial and Jl ethods :\fale Sprague-Dawley rats weighing 160-200 g were used for both in vivo and organ culture expl'riments. They were fasted overnight prior to administration of ethanol or removal of tissue for culture. The ethanol challenge for both 1:n vivo and organ culture studies was performed with relatively low ethanol concentrations which cause only mild tissue damage, in order that any accentuation of damage after Triton WR 1339 pretreatment would then be more readily apparent. In viro experiments Groups of rats were pretreated with intraperitoneal (i.p.) Triton WR 1339 prior to challenge with a single dose of ethanol. Triton WR 1339 (1.5 g/kg) was given as a single dose, then ethanol administered 4 days later, when lysosomal labilization is known to be at a maximum (NAGORNEY et al. 1983; LARl:sSO et al. 1982; WATTIAUX et al. 1963). Control animals were injected with 0.15 M NaC!. The rats were anaesthetized with sodium pentobarbitone 50 mg/kg i. p. then dosed with 30 % ethanol (1 ml) by gavage. The Triton-treated rats (n = 5) and their controls (n = 5) had the pylorus ligated immediately before the ethanol dose to prevent any variation in damage due to variable gastrie emptying. One hour after ethanol, the animals were sacrificed, the stomach removed, opened along the greater curvature and pinned out on wax. After brief rinsing with ice cold 0.15 M NaCI, stomachs were fixed by immersion in 10 % phosphate-buffered formaldehyde for 4 h. The oxyntic mucosa was ("ut into strips traversing the full width of the opened stomach, spaced 2 mm apart, parallel to the junction of forestomach and corpus. These were stored in fresh fixative overnight, then embedded in paraffin. Sections (4 ,urn) were stained with haematoxylin and eosin. Damage was quantitated by light mieroscopy, using an eyepiece micrometer, by an observer who was unaware of the experimental conditions. The total length of damaged mucosa was measured and expressed as a percentage of the length of sectioned mucosa examined (the latter averaged about 10 em per rat). The maximum depth of each sectioned erosion was also measured, and the mean erosion depth calculated for each animal.

Organ eulture studies . The effects of Triton pr?treatment on ethanol damage were also examined in organ culture, usmg gr?ups of.4 or 6 expenmental and.control stomachs per study. Antral mucosa was placed in culture m MedIUm 199 supplemented With 10 % foetal calf serum (Commonwealth Serum Laboratories, Melbourne, Australia) using our previously reported methods (YEOMANS et al. 1980). To quantitate ethanol-induced damage, tissue was loaded with 51chromium by culturing for 45 mill in 5 flCi/ml sodium 51Cr-chroma~e, followed by rinsing and culture for 2 h in two changes of plain mccllUm to remove extracellular Isotope. The method has been validated and described in more detail elsewhere (SEWE~L et al. 1986). The tissue was then injured by exposure to medium containing 15 % ethanol for 10 mm, and the damage quantitated by measuring the release of chromium into the medium over the next 60 min (expressed as percent of 51Cr in tissue). Release into the medium and tissue activ:ities of the lysosomal enzyme, N-aeetyl-fJ-glucuronidase (NAG) (EC 3.2.1.30), and of the rytosohc marker enzyme, lactate dehydrogenase (LDH) were also measured, and the results related to the protein content of the explants in each dish. To determine whether Triton WR 1339 accentuated damage, Triton (1.5 g/kg) or 0.15 M NaCl (control) was given as in a.p. injection 4 days prior to removal of tissue for culture.

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Enzyme a nd protcin assa ys N-acetyl-p-glucosaminidase, NAG (EC 3.2.1.30) , was assayed fluorometrically using 4-methyl umbelliferyl (Koch-Light Laboratories) as substrate (~{EAD ct al. 1955); LDH was determined from the rate of oxidation of NADH measured at 340 nm (REEVES et al. 1966). Onc unit of activity corresponds to the hydrolysis of 11Lmole of substrate per min. The protein content , assessed fluorom etri('ally (BOHLE N et al. 1973), and enzyme activities of the cultured explants were measured after homogenization using a motor-d riven Potter-Elvejhem homogenizer. Stat is ti ca l methods Data arc expressed as means and st andard errors of means. Experimental groups were compared with their controls using 2-tailed Student's t-tcsts.

Results 1. I n vivo hi s tolog y The mucosae of control r ats, given 15% ethanol alone, exhibited moderatel y diffuse but superficial damage. Mostly, just the top one or two cell positions in the gastric pits and the surface cells were desquamated, although occasional quite deep erosions were also produced. In many of the damaged areas, repair was already underway. The mean (± SE) length of sectioned mucosa showing damage was 19.7 ± 5.7 % of the total length examined; and since the strips cut from stomachs represented random samples of the mucosa and the lesions appeared to be randomly distributed along the sample strips, it can be concluded that the same proportion (i.e. --20'%) of the mucosal area was damaged (WEIBEL 1979). The mean depth of erosions was 20 ± 7 flm. Pre-treatment with Triton WR 1339 did not significantly alter these measures of ethanol-induced damage. The length of damaged mucosa in sections from Triton-treated rats was 23.9 ± 6.6% (p > 0.6), whil e the mean erosion depth was 19 ± 4flm (p > 0.9). 2. Organ c ulture Chromium release Release of chromium into culture medium from explants from the animals pretreated with Triton WR 1339 or 0.15 M salin e is shown in fig. 1. In control cultures not exposed to ethanol, the chromium leakage during the final hour averaged abo ut 4 'Yo, whether the rats had been pre-treated with Triton or with saline (p > 0.8). Expos ure to 15 % ethanol for 10 min resulted in substantially increased chromium leakage during th e final hour in ethanolfr ee media when compared to controls (p < 0.01). This leakage, however, was not increased by pre-treatment with Triton WR 1339 (p > 0.8). E nzyme release in organ culture The mean total NAG activity (the sum of the tissue and culture medium activities) was 12.4 ± 1.2 m"Cjg tissue protein (n = 12) in control cultures, whereas after Triton WR 1339 O/.J'tChromlum released lOr-------------------~

CJ T'"M WR 1333

10

Fig. 1. Release of 51Cr into culture medium during the 60 min after a 10 min challenge with ethanol (or normal medium ('ontrol). Triton (T) was given as a single Lp. injection 4 days before culture.

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Exp. Pathol. 33 (1988) 3

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pretreatment (n = 11) it was significantly higher - 16.5 ± 1.2 mUjg (p < 0.05). The percentage release of NAG into the culture medium in the hour after control challenge with saline for Triton-pretreated cultures was 2,0 ± 0.3 % (n = 5), not significantly different from the release from untreated cultures (1.5 ± 0.3 %; n = 6). After challenge with 15 % ethanol in Triton-pretreated cultures, the NAG release was 6.2 ± 1.3 %, significantly greater than for untreated controls (2.5 ± 0.7 %; p < 0.05). LDH was not measurable in the medium in the hour after challenge, and there were no differences in the tissue activities of LDH between the two groups (mean activity in control cultures: 0.7 Ujgm protein).

Discussion This study has failed to demonstrate increased dam age to the gastric mucosa after ethanol challenge following pretreatment with a lysoso mallahilizing agent, as assessed either in vivo histologically or in vitro using 51 chromium release as a marker of tissue damage. A number of previous studies have demonstrated cha.nges in the activities, integrity and release of lysosomal enzymes during gastric mucosal damage (FERGUSON et at. 1973; PFEIFFER et at. 1980; WHITTLE et at. 1985; FERGUSON et at. 1972; POLSON et at. 1982), but these studies could not distinguish whether the changes were causal or only secondary to the cell injury itself. Our finding that prior modulation of Iysosomes does not influ ence damage, is in accord with one other study in which Triton-pretreatment did not increase histamineinduced gastric damage in guinea pigs (NAGORNEY et al. 1983); however, since histam ineinduced damage is most probably mediated by acid hypersecretion there is less reason to expect Iysosomes to mediate damage, whereas we used ethanol as the damaging agent which is known to readily enter cells (TARASCHI et at. 1985) and release lysosomal enzymes (W ALDRON-EDWARDS et al. 1980). The current findings therefore strengthen the evidence that lysosomes do not have a major role in initiating or potentiating ethanol-induced gastric mucosal injury. It has previously been shown that Triton WR 1339 accumulates in gastric (NAGORNEY et at. 1983) as well as liver Iysoso mes (LARusso et at. 1982) and that it in creases lysosomal fragility (DE DuVE et at. 1966; NAGORNEY et at. 1983; LARusso et at. 1982; WATTIAUX et al. 1963). The increase in lysosomal enzyme activity that we noted in the cultured tissue and the increased release of lysosomal enzymes after ethanol challenge strongly suggests that the Triton WR 1339 pretreatment had specifically perturbed gastric lysosomes, and impaired their integrity. In spite of this , we found no other evidence of increased tissue damage. Other studies which have correlated histology with biochemical markers of tissu e damage, including lysosomal enzymes (CHIU et at. 1983; WHITTLE et at. 1985; NAGORNEY et at. 1983) have shown correlations after ethanol damage, but a lack of correlation when the system was perturbed, for example, with prostaglandins (WHITTLE et al. 1983) or Triton (NAGORNEY et a.l. 1983). These data are therefore in accord with our r esults which show that although the lysosomal system is perturbed, this is not reflected by a more generalized damage to the gastric mucosa. The use of 51 chromium as a marker of ethanol-induced damage in our organ culture system is sensitive and precise (SEWELL et al. 1986) and has recently been used to assess aspirin-induced damage in cell cultures from rat gastric mucosa (TERANO et al. 1984). In our hands, chromium is more sensitive than release of the cytosolic marker enzyme lactate dehydrogenase, recently, proposed as an index of gastric mucosal damage (WHITTLE et al. 1985). We did not find a close correlation between lysosomal enzyme release and chromium release. This is not surprising, however, since 51 chromium is not lysosomally bound hut probably localized in the cytosol (SANDERSON 1976). In summary, the bulk of evidence from this and previous studies suggests that the lysosomal enzyme changes demonstrated during gastric injury merely reflect the general tissue destru ction, and that Iysosomes do not play a major pathogenetic role in gastric mucosal damage.

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Acknowledgements This work was supported by grants from the National Health and Medical Research Council of Australia and by the Australian Associated Brewers' Foundation, and the manuscript was prepared by Ms. JANE BELL.

References BOHLEX, P., STEIN, S., DAIRMAN, W., UDENFRIEND, S.: Fluorometric assay of proteins in the nanogram range. Arch. Biochem. Biophys. 1973; 155: 213-220. CHIU, P. J. S., VEMULAPALLI, S., BARNETT, A.: Lysosomal release and ethanol-induced gastric lesions in rats. J. Pharm. Pharmacol. 1983; 35: 121-123. DAVENPORT, H. W.: Gastric mucosa injury by fatty and acetylsalicyclic acids. Gastroenterology 1964; 46: 245-253. - Ethanol damage to canine oxyntic glandular mucosa. Proe. Soc. Expt. BioI. Med. 1967; 126: 657-662. DEDuVE, C., WATTlAeX, R: Function of lysosomes. Ann. Rev. Physiol. 1936; 28: 435-492. FERGeSOX, W. W., EDMOND, A. W., STARLING, J. R., WANGENSTEEN, S. L.: Protective effect of prostaglandin El (PG E)l on lysosomal enzyme release in serotonin-induced gastric ulceration. Ann. Surg. 1973; 177: 648-654. - STARLIXG, J. R, WAXGENSTEEN, S. L.: Role of lysosomal enzyme release in the pathogenesis of stress-induced gastric ulceration. Surg. Forum 1972; 23: 380-382. HauL, H. S., GREENBERG, L., BOUTROS, M. I. R, WALDRON-EDWARD, D.: Effect of aspirin on ionic movement and acid hydrolase activity of explants of canine antral and duodenal mucosae. Gastroenterology 1975; 69: 439-447. LAResso, N. F., KosT, L., CARTER, J., BARIUM, S.: Triton WR 1339, a lysosomotropic compound is excreted into bile and alters excretion of lysosomal enzymes and lipids. Hepatology 1982; 2: 209-215. MEAD, J. A. R, S~lITH, J. N., WILLIAMS, R T.: Studies in detoxication. 67. The biosynthesis of the glucuronidcs of umbelliferone and 4-methylumbelliferone and their use in fluorometric determination of beta-glucuronidase. Biochem. J. 1965; 61: 569-574. NAGORNEY, D. M., LARusso, N. F., DOZOIS, R R.: Development and application of methodology for asscs~ing the role of lysosomes in experimental uleerogenesis in the guinea pig. Gastroenterology 1983; 85: 548-556. PFEIFFER, C. J., CHO, C. I., CHEEMA, A., SALTMAN, D.: Reserpine-induced gastric ulcers: protection bv lysosomal stabilization due to zinc. Eur. J. Pharmacol. 1980; 60: 347-363. POLS"Ql"·, H., MOWAT, C., HUrAL, H. S.: Increased mucosal lysosomal enzyme activity in sepsisassociated gastric lesions. Am. J. Gastroenterol. 1982; 77: 437-460. REEYES, W., FEMOGNAIU, G.: L-Iactic dehydrogenase: heart (H4). Methods Enzymology 1966; 9: 288-289. SANDEHSON, C. J.: Thc uptake and retention of chromium by cells. Transplantation. 198G; 21: 526-529. SE"'ELL, R. B., LING, T. S., YEOMANS, N. D.: Ethanol-induced damage to rat gastric mucosa mcasured by release of 5ICr in organ culture. Dig. Dis. Sci. 1986; 31: 853-868. SYMONS, A., PARKE, D.: The effects of sodium carbcnoxolone on the stability of cellular membranes. Scand. J. GastrocnteroJ. 1980; Suppl. 65: 3-10. TARASCHI, T., RUBIN, E.: Biology of disease. Effects of ethanol on the chemical and struetllral properties of biological membranes. Lab. Invest. 1986; 02: 120-131. TERANO, A., MACH, T., STACHURA, J., TARNAWSKI, A., IYEY, K.: Effect of 16,16-dim,thyl-prostaglandin E2 on aspirin induced damage to rat gastric epithelial cells in tissue culture. Gut 1934; 2ii: 19-24. WALDRON-EDWARD, D., BouTRos, M. I. R, HUUL, H. S.: Effect of bile on lysosomal stability in the mucosa of the canine gastric antrum. Gastroenterology 1977; 73: 980-984. - GREEXBERG, L.: Lysosomal stability and mucosal resistance to injury. In: HOLTERMeLLEH, K., MALAGELADA, J. (cds.). Advances in ulcer disease. Excerpta Medica. Amsterdam 1980, pp. 87-100. WATTIATX, R, Wnw, l\L, BAUDHUIN, P.: Influence of the injeetion of Triton WR 1339 on the properties of rat liver lysosomes. In: DE REUCK, A., CAMERON, M. (cds.). Lysosomcs, a CIBA Foundation Symposium. Little, Brown and Company, Boston 1963, pp. 176-196. WEIBEL, E. R: Stereological methods. In: Practical methods for biological morphometry. Academic Press, London 1979, pp. 9-203. WHITTLE, B. J. R, STEEL, G.: Evaluation of the protection of rat gastric mucosa by a prostaO'landin analogue using cellular enzyme marker and histologic techniques. Gastroenterology 1985; 88: 315-327. • YEOMANS, N. D., MILLAR, S . .T.: Synthesis and secretion of glycoproteins by mucosa of rat gastric antrum in organ culture. Dig. Dis. Sci. 1980; 2ii: 295-30l. Receivrd July 2,1987) Exp. Pathol. 33 (1988) 3

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