Original Paper Digestion 1992:53:108-113
N.S. Mann S.K. Mann P.N. Brawn B. Weaver
Effect of Zinc Sulfate and Acetylcysteine on Experimental Gastric Ulcer: In vitro Study
Keywords
Abstract
Organ culture In vitro chronic ulcer Sulfhydryl compounds N-Ethylmalcimidc N-(4-aminophenyl)maleimide stain
A small ulcer produced in vitro by monopolar electrocoagula tion on endoscopically obtained human antral biopsies and incubated in Trowel T-8 medium at 37 °C for 8 h has manyhistologic features of chronic gastric ulcer in man. Zinc sulfate and acetylcysteine in low concentrations had a significant healing effect in this ulcer model. Since the beneficial effect of zinc sulfate and acetylcysteine was counteracted by N-ethylmaleimide, a known blocker of sulfhydryl compounds, the beneficial effect of these two compounds probably was me diated through sulfhydryl compounds. Using special stain, N(4-aminophenyl)maleimide, the sulfhydryl groups were local ized in the epithelial cells of the surface layer and gastric glands.
Chronic gastric ulcer (GU) is a common clinical problem [1], In contrast to duodenal ulcer, the medical management of GU is un satisfactory, and recurrences are common [2, 3]. To evaluate the efficacy of various an tiulcer drugs, many in vivo experimental models have been used [4-8]. Although acute injury to the gastric epithelial cells has been studied extensively by in vitro cell culture technique [9-11], no suitable models are
available for in vitro study of chronic GU. This was probably due to the fact that no suit able culture medium was available to main tain the histologic and functional integrity of excised tissues in organ culture experiments for a reasonable duration of time. With the availability of newer culture media, it is possi ble to maintain the anatomic and functional integrity of various tissues up to 24 h or even longer [12]. Browning and Trier [13] have
Presented in part at the Annual National Meetings AAP/ASCI/AFCR, Washington, D.C., USA. May 3, 1986. and April 30, 1989, and published as abstracts.
N.S. Mann. MD Gastrocntcrology-Hepatology-Nutrition Section 1901 South First Street Temple. TX 76504 (USA)
Received: February 18, 1992 Received in revised form: August 13, 1992
©1992 S. Karger AG. Basel 0012-2823/92/ 0532-0108S2.75/0
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Departments of Medicine and Pathology, Texas A & M University College of Medicine, and Veterans Administration Medical Center, Temple, Tex., USA
Table 1. Effect of ZnS04 and NAC on healing score (means ±
— ----------Healing score
Groups I II III IV V VI VII VIII IX X XI
TM + 0.2 ml saline (control) TM + NEM 0.01 mg (control) TM ± NAC 0.1 mg TM + NAC 0.2 mg TM + NAC 0.5 mg TM + NAC 0.2 mg + NEM 0.01 mg TM + ZnSOj 0.01 mg TM + ZnSO.i 0.02 mg TM + ZnSC>4 0.05 mg TM + ZnS04 0.02 mg + NEM 0.01 mg TM + NAC 0.2 mg + ZnSQ) 0.02 mg
l.2±0.4 0.7 + 0.3 3.0 ±0.3* 3.4 ±0.3* 1.2 ± 0.2 0.8 ±0.1 3.2 ±0.4* 3.3 ±0.3* 1.5 ± 0.2 1.0±0.1 3.8 ±0.3*
established the viability of human small bowel mucosa in an organ culture experiment: Eastwood and Trier [14] have done the same with human rectal mucosa. The functional capacity of hamster intestine has been dem onstrated in similar experiments [ 15]. We have shown [ 16] that endoscopically obtained human antral biopsy specimens can maintain histologic integrity for at least 8 h in organ culture medium. We have also established [ 17] that an ulcer produced by electrocoagula tion on these specimens, after incubation for 8 h. has many histologic features of chronic human GU. In the present study, we evalu ated the effect of zinc sulfate (ZnSOj) and Nacetylcysteine (NAC) on this ulcer model and the possible role of sulfhydryl (SH) com pounds in the beneficial effect of ZnSC>4 and NAC.
Materials and Methods The study was approved by the Human Studies Subcommittee of the Olin E. Teague Veterans Admin istration Medical Center. The subjects underwent gas troscopy for gastrointestinal complaints, but on endos copy the gastric mucosa appeared to be normal. One
hundred and ten antral biopsy specimens from endo scopically normal stomachs were obtained. The endo scopic biopsies were deep enough to include the muscularis mucosae. Three to five biopsy specimens were obtained from each subject. Each biopsy measured about 4 mm in size. They were divided into 11 groups of 10 each. On each of the biopsy specimens, a 2-mm ulcer was produced by monopolar electrocoagulation by using a specially designed electrode applied for 2 s at a setting of 3 (Cameron-Miller) as already published [17-19]. Immediately after electrocoagulation, the specimens were put in 5 ml of Trowcll T-8 medium (TM) containing 0.9 part fetal calf scrum and 0.1 part crystalline penicillin G (10,000 U/ml) and streptomy cin sulfate (10 mg/ml). TM. fetal calf serum and antibi otics are commercially available (Grand Island Bio logic, N.Y., USA). In addition to TM, the specimen tubes either contained normal saline (control) or Nethylmaleimide (NEM) or various amounts of NAC, ZnS04, NAC + NEM, ZnS04 + NEM or NAC + ZnSCb (table 1). The specimens were gassed for 5 min with 95% O: + 5% CO2 , sealed and incubated at 37 °C for 8 h. At the end of 8 h. TM was discarded, and the speci mens were fixed in 10% formalin. The specimens were stained with HE and were evaluated blindly. Mean healing score for each group was calculated by using the healing score criteria already described [ 18]. Brief ly. the healing score points were as follows: (a) preser vation of the epithelial layer in the rest of the specimen = 3: (b) presence of binudeated cells in the gastric glands = 1, and (c) presence of mitotic figures in gastric glands = 1. Mean healing scores ± SEM of various
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* p < 0.025 vs. control.
healing effect compared to the control (p > 0.05). The beneficial effect of NAC and ZnS04 was countered by NEM (group VI and X, p < 0.025). NAC and ZnS04 when used together in a lower concentration (group XI) had a synergistic effect on the healing score, compared to NAC alone (group IV) or ZnS04 alone (group VIII). but it was not statistically significant (p > 0.05). The ulcer produced in this experiment had histological features resembling chronic GU (fig. 1). In groups III. IV. VII and VIII. there was preservation of the surface epithelium and presence of binucleated cells and mitotic figures in the gastric glands (fig. 2). The APM stain (fig. 3) showed that the SH compounds were located in the epithelial cells of the sur face layer and the gastric glands rather than in the cells of the lamina propria or the blood vessels. By this method SH compounds were stained bluish-green.
Discussion We have previously shown [7] that GU produced by electrocoagulation in the rat stomach and studied histologically after 1 week has the characteristic histologic features of chronic human GU. The ulcer had four his tologic zones, i.e. (a) acute inflammatory exu groups were compared using Student's t test. Slides date, (b) fibrinoid necrosis, (c) granulation tis were also stained with N-(4-aminophenyl)malcimide sue with lymphocytes and fibroblasts, and (APM) to localize the SH compounds [20-23]. (d)scar tissue. In the present study we have adapted that in vivo model to our in vitro organ culture technique. The histologic fea Results tures (fig. 1) which are similar to human GU The results are summarized in table 1. In are acute inflammatory' exudate, fibrinoid ne this ulcer model, lower concentrations of crosis and margination of the ulcer base with NAC and ZnS04 (groups III. IV. VII and lymphocytes. Obviously, in this model, there VIII) had significant (p < 0.025) healing could be no scar tissue. In this model, change effect compared to the controls (groups I and in ulcer size could not be used as a sign of II). At higher concentrations (groups V and healing, but other parameters of repair, e.g. IX) , NAC and ZnS04 showed no beneficial preservation of the surface epithelium over
no
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Fig. 1. The histologic features of chronic GU arc seen. i.e. inflammatory exudate, fibrinoid necrosis and margination of the ulcer base with lymphocytes. HE. X 75.
Fig. 2. A group III ulcer show ing preserv ation of the surface epi thelium: presence of binucleated cell (arrow) and mitotic figure (arrowhead) in gastric glands. HE. X 250.
Fig. 3.Special stain with APM showed SH-containing cells stained bluish-green. X 100.
I ll
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the nonulcerated portion of the specimen and the presence of binucleated cells and mitotic figures in the gastric glands, were used. NAC has been shown to be cvtoprotective against ethanol-induced acute gastric injury in rats as reported by Szabo and Trier [24], Elson and Soli [25] have shown that NAC can reverse oxygen radical injury in glutathionedepleted cultured canine chief cells. Zn is an important component of many metalloenzymes including DNA and RNA polymerase. It is involved in cell replication, cell growth and immune defenses. Impaired wound heal ing occurs when there is Zn deficiency [26]. Zn and other heavy metals have been shown to have a healing and protective effect on the gastric mucosa [18. 27]. NAC and ZnSOa are known to activate and generate SH com pounds. SH compounds have been shown to play an important role in gastric cytoprotection in numerous in vitro and in vivo studies [9. 10. 24.25,27-39], NEM is a known blocker and scavenger of SH compounds: it has countered the benefi cial effect of endogenous and exogenous SH on the gastric mucosa and other tissues [27, 28.31.32. 34. 37.40-42]. In the present study
low concentrations of Z11SO4 and NAC had a beneficial healing effect on the GU which was countered by NEM suggesting that the benefi cial effect was mediated by SH radicals which were found to be present in the epithelial cells as shown by the APM stain. SH compounds may represent a class of cytoprotective agents with wide spectrum of activity as they also are required in the synthesis of prostanoids and for prostaglandin receptor activation: their protective effect has also been seen in other organs, e.g. liver and kidney [43-47].
In the present study high concentrations of NAC or Z 11 SO4 had adverse effects on the healing of this experimental ulcer. The reason for this is not clear, but it is probably not due to excess SH generation because when lower concentrations of NAC and ZnSC>4 were given together, there was some synergistic effect. At higher concentrations, ZnSOa and NAC may cause local toxic damage. The mechanism of protection by SH compounds is not known but oxygen free radical scaveng ing is a possibility [24],
1 Kukral JC: Gastric ulcer: An ap praisal. Surgery 1968:63:1024— 1036. 2 Scheurer U. Witzek l., Halter F. Keller HM. Huber R. Galeazzi R: Gastric and duodenal ulcer healing under placebo treatment. Gastroen terology 1977;72:838-841. 3 Hanscom DH. Buchman E: VA Cooperative Study of Gastric Ulcer: The follow-up period. Gastroenter ology 1971;61:585-591. 4 Okabe S. Pfeiffer CJ: Chronicity of acetic acid ulcer in the rat stomach. AmJ DigDis 1972:17:619-629. 5 Takagi K. Okabe S. Saz.iki R: A new method for the production of chronic gastric ulcer in rats and the effect of several drugs on its healing. Jpn J Pharmacol 1969:19:418-426. 6 Brodie DA: Experimental peptic ul cer. Gastroenterology 1968:55:125— 134. 7 Mann NS. Reddy AB, Khan JM. Lathon PV: Experimental gastric ulcer produced by electrocoagulation. Am .1 Proctol Gastroenterol Colored Surg 1982:33:14-20. 8 Mann NS. Mann SK, Reddy AB, Malhotra KK, Lathon PV: The cffed of prostaglandin, sucralfate, cimetidine and antacid on gastric ul cer. Milit Med 1984:149:677-679.
9 Romano M. Razandi M. Raza A. Szabo S: Sulfhvdryls may mediate acetaminophen protection of hu man gastric mucosal cells in vitro. Gastroenterology !988;94:A385. 10 Romano M, Razandi M. Krause WJ. Ivey KJ: The calcium channel blocker diltiazem docs not protect rat gastric cpithelia cells against drug-induced damage in vitro: Ef fect on prostaglandin production and sulfhydryl level. Gastroenterol ogy 1988:94:A385. 11 Romano M. Razandi M. Ivey KJ: Cimetidine and ranitidine: Do they protect cells in vitro? Gastroenterol ogy 1988;94:A383. 12 Trowell OA: The culture of mature organs in a synthetic medium. Exp Cell Res 1959;16:118-147. 13 Browning TH. Trier JS: Organ cul ture of mucosal biopsies of human small intestine. J Clin Invest 1969: 48:1423-1432. 14 Eastwood GL. Trier JS: Organ cul ture of human rectal mucosa. Gas troenterology 1973:64:375-382. 15 Strauss EW: The absorption of fat by intestine of golden hamster in vitro. J Cell Biol 1963:17:597-603. 16 Mann NS. Mann SK. Tsai MF: His tologic integrity of human gastric mucosa obtained by gastroscopy in organ culture experiment. Gastrointest Endosc 1986:32:178.
17 Mann NS. Tsai MF. Mann SK: His tologic feature of experimental hu man gastric ulcer produced by in vitro electrocoagulation maintained in organ culture. Gastrointest En dosc 1986:32:178. 18 Mann NS. Mann SK. Tsai MF: Ex perimental human gastric ulcer pro duced by in vitro electrocoagula tion: Effect of sodium oxyferriscorbone. Clin Res !986:34:442A. 19 Mann NS. Mann SK. Brawn PN: Effect of acetylcysteine on experi mental human gastric ulcer pro duced by in vitro electrocoagula tion. Clin Res I989;37:370A. 20 Sippel TO: The histochemistry of thiols and disulphides. I. The use of N-(4-aminophenyl)maleimide for demonstrating thiol groups. HistochemJ 1973:5:413-423. 21 Sippel TO: The histochemistry' of thiols and disulphides. II. Method ology of differential staining. HistochcmJ 1978:10:585-595. 22 Sippel TO: The histochemistry of thios and disulphides. III. Staining patterns in rat tissues. Histochem J 1978:10:597-609. 23 Sippel TO: The histochemistry of thiols and disulphides. IV. Protec tive fixation by organomercurialformalin mixtures. Histochem J 1980;12:107-117.
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References
32 Romano M. Razandi M. Ivey KJ: Role of endogenous and exogenous sulfhydryls in the protection of rat gastric epithelial cells gainst oxygen radical-induced damage. Gastroen terology I989;96:A423. 33 Romano M, Razandi M, Raza A. Szabo S. Ivey KJ: Protection of hu man gastric cells in vitro by the sulfhydryl agent cystcaminc is not re lated to stimulation of prostaglandin synthesis. Gastroenterology 1987; 92:1600. 34 Majzoubi D. Brown A. Szabo S: Su cralfate protects the gastric mucosa even after parenteral administra tion: Possible role of sulfhydryls. Dig Dis Sci 1985:30:387. 35 Miller TA, Smith GS. Kuo YJ. Schmidt KL, Livingston L: The in fluence of prostaglandin and ethanol on tissue levels of non-protein sulfhvdryl compounds in canine gastric mucosa. Dig Dis Sci 1985:30:389. 36 Szabo S. Trier JS. Pihan G. Raza A. Dupuy D: The role of endogenous non-protein and protein sulfhydryls in gastric mucosal injury and protec tion. Dig Dis Sci 1985:30:402. 37 Raza A, Szabo S: Biochemical changes in endogenous sulfnvdryls in gastric mucosal injury and protec tion: Studies with diethylmaleate and N-ethylmaleimide. Dig Dis Sci 1985:30:397. 38 Szabo S. Trier JS. Frankel PW: Sulfhydrvl compounds may mediate gastric cytoprotection. Science 1981:214:200-202.
39 Silen W. Walsh JH. Garner A. Ro bert A, Pfeiffer CJ. Szabo S: Lessons from experimental ulcers: 'Take home messages' from the 5th Inter national Conference on Experimen tal Ulcer. Dig Dis Sci 1986:31: 1265-1268. 40 Kohama K. Kohama T. KendrickJones J: Effect of N-ethylmalcimide on Ca-inhibition of phvsarum myo sin. J Biochem (Tokyo) 1987; 102: 17-23. 41 Elferink JG. Deierkauf M: Involve ment of membrane sulfhvdryl groups in apomorphine-induced in hibition of neutrophil migration. Res Commun Chem Pathol Phar macol 1987:57:187-196. 42 Gysin R. Flanagan SD: Alkylation of free sulfhydryls fortifies electroplax subsynaptic structures. J Neurochcm 1987:49:452-459. 43 Wolfe LS: Eicosanoids: Prostaglan dins. thromboxanes, leukotrienes, and other derivatives of carbon-20 unsaturated fatty acids. J Neurochem 1982;38:1-14. 44 Johnson M. Jessup R. Ramwell PW: The significance of protein disulfide and sulfhvdryl groups in prostaglan din action. Prostaglandins 1974:5: 125-136. 45 Szabo S. Ganon MJ: The protective effect of sulfur-containing steroids against nephrocalcinosis by mer curic chloride in rats. Gen Pharma col 1983:14:269-272. 46 Prescott LF: Paracetamol overdos age. Pharmacological consider ations and clinical management. Drugs 1983;25:290-314. 47 Szabo S. Jaeger RJ. Moslen MT. Reynolds ES: Modification of 1. /dichlorethylene hepatotoxicity by hypothyroidism. Toxicol Appl Pharmacol 1977:42:367-376.
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24 Szabo S. Trier JS: Pathogenesis of acute gastric mucosal injurs" Sulfhydryls as a protector, adrenal cortex as a modulator and vascular endo thelium as a target: in Allen A. et al (cds): Mucosal Protection in the Up per Gastrointestinal Tract. New York. Raven. 1984. pp 287-293. 25 Elson CE, Soil AH: Oxygen radical injuiy in glutathione-depleted cul tured canine chief cells is reversible by N-acetyl-cysteine. Gastroenterol ogy 1987:92:1561. 26 Solomons NW: Zinc: in Shils and Young (eds): Modern Nutrition in Health and Disease. Philadelphia. Lea & Febiger. 1988. pp 238-248. 27 Dupuy D. Szabo S: Protection by metals against ethanol-induced gas tric mucosal injury in the rat: Com parative biochemical and pharma cologic studies implicate protein sulfhydryls. Gatroenterology 1986: 91:966-974. 28 Szelenyi 1. Brune K: Possible role of sulfhydryls in mucosal protection induced by aluminum hydroxide. DigDis Sci 1986:31:1207-1210. 29 Szabo S. Trier JS. Brown A. LaRoeque M: Protection against aspirininduced hemorrhagic gastric ero sions and mucosal vascular injury by coadministration of sulihydryl drugs. Gastroenterology 1985:88: 1604. 30 Szabo S. Trier JS. Brown A. Schnoor J: Early vascular injury and in creased vascular permeability in gastric mucosal injury caused by ethanol in the rat. Gastroenterology 1985;88:228-236. 31 Tackcuchi K. Okada M. Niida H. Okabe S: Endogenous sulfhydryls (SH) in healing process of gastric mucosal lesions induced by HCI in rats. Gastroenterology 1989:96: A501.
N.S. Mann S.K. Mann P.N. Brawn B. Weaver
Effect of Zinc Sulfate and Acetylcysteine on Experimental Gastric Ulcer: In vitro Study
Keywords
Abstract
Organ culture In vitro chronic ulcer Sulfhydryl compounds N-Ethylmalcimidc N-(4-aminophenyl)maleimide stain
A small ulcer produced in vitro by monopolar electrocoagula tion on endoscopically obtained human antral biopsies and incubated in Trowel T-8 medium at 37 °C for 8 h has manyhistologic features of chronic gastric ulcer in man. Zinc sulfate and acetylcysteine in low concentrations had a significant healing effect in this ulcer model. Since the beneficial effect of zinc sulfate and acetylcysteine was counteracted by N-ethylmaleimide, a known blocker of sulfhydryl compounds, the beneficial effect of these two compounds probably was me diated through sulfhydryl compounds. Using special stain, N(4-aminophenyl)maleimide, the sulfhydryl groups were local ized in the epithelial cells of the surface layer and gastric glands.
Chronic gastric ulcer (GU) is a common clinical problem [1], In contrast to duodenal ulcer, the medical management of GU is un satisfactory, and recurrences are common [2, 3]. To evaluate the efficacy of various an tiulcer drugs, many in vivo experimental models have been used [4-8]. Although acute injury to the gastric epithelial cells has been studied extensively by in vitro cell culture technique [9-11], no suitable models are
available for in vitro study of chronic GU. This was probably due to the fact that no suit able culture medium was available to main tain the histologic and functional integrity of excised tissues in organ culture experiments for a reasonable duration of time. With the availability of newer culture media, it is possi ble to maintain the anatomic and functional integrity of various tissues up to 24 h or even longer [12]. Browning and Trier [13] have
Presented in part at the Annual National Meetings AAP/ASCI/AFCR, Washington, D.C., USA. May 3, 1986. and April 30, 1989, and published as abstracts.
N.S. Mann. MD Gastrocntcrology-Hepatology-Nutrition Section 1901 South First Street Temple. TX 76504 (USA)
Received: February 18, 1992 Received in revised form: August 13, 1992
©1992 S. Karger AG. Basel 0012-2823/92/ 0532-0108S2.75/0
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Departments of Medicine and Pathology, Texas A & M University College of Medicine, and Veterans Administration Medical Center, Temple, Tex., USA
Table 1. Effect of ZnS04 and NAC on healing score (means ±
— ----------Healing score
Groups I II III IV V VI VII VIII IX X XI
TM + 0.2 ml saline (control) TM + NEM 0.01 mg (control) TM ± NAC 0.1 mg TM + NAC 0.2 mg TM + NAC 0.5 mg TM + NAC 0.2 mg + NEM 0.01 mg TM + ZnSOj 0.01 mg TM + ZnSO.i 0.02 mg TM + ZnSC>4 0.05 mg TM + ZnS04 0.02 mg + NEM 0.01 mg TM + NAC 0.2 mg + ZnSQ) 0.02 mg
l.2±0.4 0.7 + 0.3 3.0 ±0.3* 3.4 ±0.3* 1.2 ± 0.2 0.8 ±0.1 3.2 ±0.4* 3.3 ±0.3* 1.5 ± 0.2 1.0±0.1 3.8 ±0.3*
established the viability of human small bowel mucosa in an organ culture experiment: Eastwood and Trier [14] have done the same with human rectal mucosa. The functional capacity of hamster intestine has been dem onstrated in similar experiments [ 15]. We have shown [ 16] that endoscopically obtained human antral biopsy specimens can maintain histologic integrity for at least 8 h in organ culture medium. We have also established [ 17] that an ulcer produced by electrocoagula tion on these specimens, after incubation for 8 h. has many histologic features of chronic human GU. In the present study, we evalu ated the effect of zinc sulfate (ZnSOj) and Nacetylcysteine (NAC) on this ulcer model and the possible role of sulfhydryl (SH) com pounds in the beneficial effect of ZnSC>4 and NAC.
Materials and Methods The study was approved by the Human Studies Subcommittee of the Olin E. Teague Veterans Admin istration Medical Center. The subjects underwent gas troscopy for gastrointestinal complaints, but on endos copy the gastric mucosa appeared to be normal. One
hundred and ten antral biopsy specimens from endo scopically normal stomachs were obtained. The endo scopic biopsies were deep enough to include the muscularis mucosae. Three to five biopsy specimens were obtained from each subject. Each biopsy measured about 4 mm in size. They were divided into 11 groups of 10 each. On each of the biopsy specimens, a 2-mm ulcer was produced by monopolar electrocoagulation by using a specially designed electrode applied for 2 s at a setting of 3 (Cameron-Miller) as already published [17-19]. Immediately after electrocoagulation, the specimens were put in 5 ml of Trowcll T-8 medium (TM) containing 0.9 part fetal calf scrum and 0.1 part crystalline penicillin G (10,000 U/ml) and streptomy cin sulfate (10 mg/ml). TM. fetal calf serum and antibi otics are commercially available (Grand Island Bio logic, N.Y., USA). In addition to TM, the specimen tubes either contained normal saline (control) or Nethylmaleimide (NEM) or various amounts of NAC, ZnS04, NAC + NEM, ZnS04 + NEM or NAC + ZnSCb (table 1). The specimens were gassed for 5 min with 95% O: + 5% CO2 , sealed and incubated at 37 °C for 8 h. At the end of 8 h. TM was discarded, and the speci mens were fixed in 10% formalin. The specimens were stained with HE and were evaluated blindly. Mean healing score for each group was calculated by using the healing score criteria already described [ 18]. Brief ly. the healing score points were as follows: (a) preser vation of the epithelial layer in the rest of the specimen = 3: (b) presence of binudeated cells in the gastric glands = 1, and (c) presence of mitotic figures in gastric glands = 1. Mean healing scores ± SEM of various
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* p < 0.025 vs. control.
healing effect compared to the control (p > 0.05). The beneficial effect of NAC and ZnS04 was countered by NEM (group VI and X, p < 0.025). NAC and ZnS04 when used together in a lower concentration (group XI) had a synergistic effect on the healing score, compared to NAC alone (group IV) or ZnS04 alone (group VIII). but it was not statistically significant (p > 0.05). The ulcer produced in this experiment had histological features resembling chronic GU (fig. 1). In groups III. IV. VII and VIII. there was preservation of the surface epithelium and presence of binucleated cells and mitotic figures in the gastric glands (fig. 2). The APM stain (fig. 3) showed that the SH compounds were located in the epithelial cells of the sur face layer and the gastric glands rather than in the cells of the lamina propria or the blood vessels. By this method SH compounds were stained bluish-green.
Discussion We have previously shown [7] that GU produced by electrocoagulation in the rat stomach and studied histologically after 1 week has the characteristic histologic features of chronic human GU. The ulcer had four his tologic zones, i.e. (a) acute inflammatory exu groups were compared using Student's t test. Slides date, (b) fibrinoid necrosis, (c) granulation tis were also stained with N-(4-aminophenyl)malcimide sue with lymphocytes and fibroblasts, and (APM) to localize the SH compounds [20-23]. (d)scar tissue. In the present study we have adapted that in vivo model to our in vitro organ culture technique. The histologic fea Results tures (fig. 1) which are similar to human GU The results are summarized in table 1. In are acute inflammatory' exudate, fibrinoid ne this ulcer model, lower concentrations of crosis and margination of the ulcer base with NAC and ZnS04 (groups III. IV. VII and lymphocytes. Obviously, in this model, there VIII) had significant (p < 0.025) healing could be no scar tissue. In this model, change effect compared to the controls (groups I and in ulcer size could not be used as a sign of II). At higher concentrations (groups V and healing, but other parameters of repair, e.g. IX) , NAC and ZnS04 showed no beneficial preservation of the surface epithelium over
no
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Experimental Human GU
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Fig. 1. The histologic features of chronic GU arc seen. i.e. inflammatory exudate, fibrinoid necrosis and margination of the ulcer base with lymphocytes. HE. X 75.
Fig. 2. A group III ulcer show ing preserv ation of the surface epi thelium: presence of binucleated cell (arrow) and mitotic figure (arrowhead) in gastric glands. HE. X 250.
Fig. 3.Special stain with APM showed SH-containing cells stained bluish-green. X 100.
I ll
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the nonulcerated portion of the specimen and the presence of binucleated cells and mitotic figures in the gastric glands, were used. NAC has been shown to be cvtoprotective against ethanol-induced acute gastric injury in rats as reported by Szabo and Trier [24], Elson and Soli [25] have shown that NAC can reverse oxygen radical injury in glutathionedepleted cultured canine chief cells. Zn is an important component of many metalloenzymes including DNA and RNA polymerase. It is involved in cell replication, cell growth and immune defenses. Impaired wound heal ing occurs when there is Zn deficiency [26]. Zn and other heavy metals have been shown to have a healing and protective effect on the gastric mucosa [18. 27]. NAC and ZnSOa are known to activate and generate SH com pounds. SH compounds have been shown to play an important role in gastric cytoprotection in numerous in vitro and in vivo studies [9. 10. 24.25,27-39], NEM is a known blocker and scavenger of SH compounds: it has countered the benefi cial effect of endogenous and exogenous SH on the gastric mucosa and other tissues [27, 28.31.32. 34. 37.40-42]. In the present study
low concentrations of Z11SO4 and NAC had a beneficial healing effect on the GU which was countered by NEM suggesting that the benefi cial effect was mediated by SH radicals which were found to be present in the epithelial cells as shown by the APM stain. SH compounds may represent a class of cytoprotective agents with wide spectrum of activity as they also are required in the synthesis of prostanoids and for prostaglandin receptor activation: their protective effect has also been seen in other organs, e.g. liver and kidney [43-47].
In the present study high concentrations of NAC or Z 11 SO4 had adverse effects on the healing of this experimental ulcer. The reason for this is not clear, but it is probably not due to excess SH generation because when lower concentrations of NAC and ZnSC>4 were given together, there was some synergistic effect. At higher concentrations, ZnSOa and NAC may cause local toxic damage. The mechanism of protection by SH compounds is not known but oxygen free radical scaveng ing is a possibility [24],
1 Kukral JC: Gastric ulcer: An ap praisal. Surgery 1968:63:1024— 1036. 2 Scheurer U. Witzek l., Halter F. Keller HM. Huber R. Galeazzi R: Gastric and duodenal ulcer healing under placebo treatment. Gastroen terology 1977;72:838-841. 3 Hanscom DH. Buchman E: VA Cooperative Study of Gastric Ulcer: The follow-up period. Gastroenter ology 1971;61:585-591. 4 Okabe S. Pfeiffer CJ: Chronicity of acetic acid ulcer in the rat stomach. AmJ DigDis 1972:17:619-629. 5 Takagi K. Okabe S. Saz.iki R: A new method for the production of chronic gastric ulcer in rats and the effect of several drugs on its healing. Jpn J Pharmacol 1969:19:418-426. 6 Brodie DA: Experimental peptic ul cer. Gastroenterology 1968:55:125— 134. 7 Mann NS. Reddy AB, Khan JM. Lathon PV: Experimental gastric ulcer produced by electrocoagulation. Am .1 Proctol Gastroenterol Colored Surg 1982:33:14-20. 8 Mann NS. Mann SK, Reddy AB, Malhotra KK, Lathon PV: The cffed of prostaglandin, sucralfate, cimetidine and antacid on gastric ul cer. Milit Med 1984:149:677-679.
9 Romano M. Razandi M. Raza A. Szabo S: Sulfhvdryls may mediate acetaminophen protection of hu man gastric mucosal cells in vitro. Gastroenterology !988;94:A385. 10 Romano M, Razandi M. Krause WJ. Ivey KJ: The calcium channel blocker diltiazem docs not protect rat gastric cpithelia cells against drug-induced damage in vitro: Ef fect on prostaglandin production and sulfhydryl level. Gastroenterol ogy 1988:94:A385. 11 Romano M. Razandi M. Ivey KJ: Cimetidine and ranitidine: Do they protect cells in vitro? Gastroenterol ogy 1988;94:A383. 12 Trowell OA: The culture of mature organs in a synthetic medium. Exp Cell Res 1959;16:118-147. 13 Browning TH. Trier JS: Organ cul ture of mucosal biopsies of human small intestine. J Clin Invest 1969: 48:1423-1432. 14 Eastwood GL. Trier JS: Organ cul ture of human rectal mucosa. Gas troenterology 1973:64:375-382. 15 Strauss EW: The absorption of fat by intestine of golden hamster in vitro. J Cell Biol 1963:17:597-603. 16 Mann NS. Mann SK. Tsai MF: His tologic integrity of human gastric mucosa obtained by gastroscopy in organ culture experiment. Gastrointest Endosc 1986:32:178.
17 Mann NS. Tsai MF. Mann SK: His tologic feature of experimental hu man gastric ulcer produced by in vitro electrocoagulation maintained in organ culture. Gastrointest En dosc 1986:32:178. 18 Mann NS. Mann SK. Tsai MF: Ex perimental human gastric ulcer pro duced by in vitro electrocoagula tion: Effect of sodium oxyferriscorbone. Clin Res !986:34:442A. 19 Mann NS. Mann SK. Brawn PN: Effect of acetylcysteine on experi mental human gastric ulcer pro duced by in vitro electrocoagula tion. Clin Res I989;37:370A. 20 Sippel TO: The histochemistry of thiols and disulphides. I. The use of N-(4-aminophenyl)maleimide for demonstrating thiol groups. HistochemJ 1973:5:413-423. 21 Sippel TO: The histochemistry' of thiols and disulphides. II. Method ology of differential staining. HistochcmJ 1978:10:585-595. 22 Sippel TO: The histochemistry of thios and disulphides. III. Staining patterns in rat tissues. Histochem J 1978:10:597-609. 23 Sippel TO: The histochemistry of thiols and disulphides. IV. Protec tive fixation by organomercurialformalin mixtures. Histochem J 1980;12:107-117.
I 12
Mann/Mann/Brawn/Weaver
Experimental Human GU
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References
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