ISPUB.COM The Internet Journal of GastroenterologyVolume 8 Number 1

1 of 7

Effect Of Ovariectomy And Of Estrogen AdministrationUpon Duodenal Ulceration Induced By CysteamineK Ashokan, M Kurane, M Pillai

Citation

K Ashokan, M Kurane, M Pillai. Effect Of Ovariectomy And Of Estrogen Administration Upon Duodenal Ulceration InducedBy Cysteamine. The Internet Journal of Gastroenterology. 2008 Volume 8 Number 1.

Abstract

Duodenal ulcers were induced in ovariectomized, intact and old mice using cyteamine hydrochloride. Under these experimentalconditions ovariectomy and old age strikingly increased sensitivity to ulcer induction while estrogen administration showed adecrease in sensitivity to ulcer induction. Nevertheless, the administration of estrogen in old showed no effects in either intact oroveriectomized mice. This change in ulcer sensitivity reflected from histological, histochemistry and biochemical studies. Thehistological study was performed by using haematoxylin-eosin staining technique. The histochemistry of the duodenal regionwas studied by using periodic acid Schiff reaction (PAS) for glycoproteins. The biochemical study was performed to studyvarious constituents of glycoproteins like hexose, fucose, sialic acid and the protein. The result showed that ulcer severity wasmore in overiectomized cyteamine treated mice and old mice treated with cyteamine. The histological studies showed theovariectomy decreased or not shown any change in the ulcer sensitivity considering cryptus Lieburkuhn and bruner’ glands. Thesame result reflected in histochemistry study by differential intensity in the staining property of the bruners gland. Thebiochemical study showed that the glycoprotein contents were reduced many folds in overiectomized cyteamine treated miceand their reversal in estrogen administered ovariectomized cysteamine injected mice. These findings prove that estrogen protectthe duodenal ulcer form cysteamine administration.

INTRODUCTION

Duodenal ulcer is a mucosal erosion of duodenum, due tomultiple causes, including bacteria (Marshall and Warren1984; Lykoudes 1958), chewing gum tobacco smoking, noteating properly, blood group, spice (NADDIC), chronicstress (Kim et al. 2007) and gender differences (Andes et al.2008). Specific protection of estrogen against gastric –acidinduced duodenal injury was reported (Andes et al. 2008).Peptic ulcer occurs more frequently in men than in women(Grey 1929). The sex differences are less marked after 45years of age probably because the incidence of ulcerincreases in post menopausal women (Crean 1963;Watkinson 1960). The general assumption is that the ulcerdifferences between sexes are related in some way to sexhormones and that the female sex hormones protect againstulceration (Crean 1963; Kyle et al. 1963). The immunityagainst duodenal ulcer is increase in females duringpregnancy (Dey and Dey 1974). It was shown that thechance of men developing duodenal ulcer remainremarkably constant between the age of 20 years and 65years. In contrast the chances of women developingduodenal ulcer remains relatively low throughout the wholelife of her active reproductive life (Truelove 1960).

Nevertheless, female sex hormones have been reported topromote ulceration (Antonsen 1955; Guerrine et al. 1967) orat best to have no effect the disease. Increased susceptibilityto gastric ulceration during the late pregnancy was alsoreported (Kelly and Robert 1969). Duodenal ulcer sensitivityincreases with age (Christensen et al. 2006). The presentstudy was undertaken therefore, to investigate in mice theeffect of ovariectomy on sensitivity of duodenal ulcerationinduced by using cysteamine –HCl and to determine whetherestrogen administration reverse the effects of ovariectomyand aging. The study was also carried out by histological,histochemical and biochemical methods in the duodenalregion of the normal, overiectomized, cyteamine injected.ovariectomized – cysteamine injected, old female mice andcysteamine injected old female mice.

MATERIALS AND METHODS

EXPERIMENTAL ANIMALS

There were 60 mice in the present study. The mice weredivided into two groups- 2 months old females (40 numbers)and 3 years old females (20 numbers). Ten mice were usedas control in both groups. The 2 months old female micewere treated with cysteamine-HCl divided into tow groups.

Effect Of Ovariectomy And Of Estrogen Administration Upon Duodenal Ulceration Induced ByCysteamine

2 of 7

The group one contain 10 female mice, were used ascysteamine –HCl treated one, the second group contain 20female mice were used for ovariectomy.

OVARIECTOMY AND ADMINISTRATION OFESTROGEN

The ovariectomy was done under mild ether anesthesia. Theoperated mice were maintained fro 15 days in separate agewith optimum care of light, temperature, humidity, food andwater. On the 16th day half the number of ovariectomizedcysteamine treated mice were injected (i.p) consecutively for3 days with 2 mg/body weight estradiol- 17β (Sigma, Batchno. E9505) in olive oil. The remaining ovariectomized micewere injected with olive oil only. On the 4th day of the 1stinjection the ovariectomized, ovariectomized cyteamineinjected and ovariectomized cysteamine injected andestrogen administered mice were used for duodenal ulcerinduction (Szabo 1978). Ten 3 years old mice were used foradministration of cysteamine, after three days it wassubjected for ulcer induction.

HISTOLOGY

The pyloro-duodenal junctions were fixed in 10% neutralbuffered fromaline, washed and routinely processed forhistological technique. The sections were stained withhaematoxyline-eosine (Gurr1962). Histology of pyloricglands, duodenal villi, crypts of Lieberkuhn and Brunner’sglands was studied.

HISTOCHEMISTRY

To study the changes in the duodenal mucosa glycoproteinsof crypts of Lieberkuhn, goblet cells, pyloric gland cells andBrunner’s glands of cyteamine treated and control mice PAStechniques (McManus 1946) were used.

BIOCHEMISTRY

The glycoprotein from Brunner’s gland was isolated by themethod of Satakopan and Kurup (1977).To study variousconstituents of glycoproteins biochemical estimations offucose (Dische and Shettles 1977), hexose (Dubois et al.1956), sialic acid (Warren 1959) and protein (Lowry et al.1951) were used.

DATA ANALYSIS

Statistical analyses were performed using the StatisticalPackage for Social Science (version 13.0, SPSS, Inc)software. Results were expressed as means ± SE (standardError). All reported p-values were made on the basis of 2-sided tests and compared to a significance level of 5%,

differences were considered statistically significant at p<0.05.

RESULTS

The ovariectomized mice showed little ulceration (Ulcerindex 2.6) or no ulceration. The overiectomozed + estradiol-17β injected mice showed ulceration very low compared tothe overiectomized mice (ulcer index 1.64) (Table No.1) Theovariectomized + Cysteamine administered mice showedhigher ulceration (Ulcer index 5.1) (Table No.1).Theadministration of estradiol- 17β to cysteamine injectedovariectopmized mice showed little recovery of ulceration(Ulcer index 4.2) (Table No.1). The old mice administeredwith cysteamine showed ulceration as high asovariectomized + cysteamine administrated mice (Ulcerindex 4.8) (Table No.2).

Figure 1

Table No.1: Ulcer severity in normal with and withoutAdministration of estradiol

Figure 2

Table No.2. Ulcer severity in Ovariectomized with andwithout cysteamine and estrogen administration

Effect Of Ovariectomy And Of Estrogen Administration Upon Duodenal Ulceration Induced ByCysteamine

3 of 7

Figure 3

Table 3. Carbohydrates and protein contents of solubleglycoprotein isolated from Brunner’s glands of female mice

* Values are mean ± Standard error, p< 0.05 is significant

Administration of estradiol- 17β to cysteamine treated oldmice fails to recover the severity of ulceration (Table No.2).The histology of ovariectomized mice (Fig.1) showed thatthe pyloric glands were simple tubular and situated deep inthe sub mucosa. The duodenal villi were tall, leaf like anduniformly arranged with desquamation intermittently. Thecrypts of Lieburkuhn and Brunner’s glands are unaffected.The cysteamine administration to the ovariectomized mice(Fig.2) cause pyloric glands with dilated lumen and picnoticnuclei and increased eosinophilia. The pyloric villi showedfissures and ramifications, and ulcer formation. The gobletscells are less in it. The Brunner’s gland acini showedreduced height, dilated lumen and nuclei with abnormal sizeand shape. The histochemistry showed strong PAS reactionin pyloric glands, crypts of Lieberkuhn, Brunner’s glands inovariectomized mice but less in pyloric pit (Fig.3).

Figure 4

Fig 1: Micrograph of duodenum of Ovariectomized mice

Figure 5

Fig 2: Micrograph of duodenum of Ovariectomized-cyteamine injected mice

Effect Of Ovariectomy And Of Estrogen Administration Upon Duodenal Ulceration Induced ByCysteamine

4 of 7

Figure 6

Fig 3: Micrograph of duodenum of Ovariectomized micePAS stain

Figure 7

Fig 4: Micrograph of duodenum of Ovariectomized-Cyteamine Injected Mice stained with PAS

The duodenal villi showed PAS activity but other cells arePAS negative. The cyteamine treated overiectomized miceshowed reduction in PAS reactivity in all cells of pyloricglands, pyloric pits, goblet cells and Brunner’s glands (Fig4). The biochemical studies showed that the hexose contentsreduce 3 folds in cyteamine injected mice comparing to thenormal (Table 3). In ovariectomized it was less than that ofnormal but more than that of cyteamine treated mice. But inovariectomized cysteamine treated mice it was 6 times lessthan that of normal mice. The fucose contents, sialic acidcontents and protein contents (Table 3) also showed thesame trend as hexose.

DISCUSSION

Peptic duodenal and gastric ulcers raise serious healthproblems and significant economic cost worldwide. Thereare approximately 500,000 new cases and 4.5 million peoplesuffering from these diseases each year in USA (Valle et al.2003). Duodenal ulcer is three times more common thangastric ulcer. Available evidence suggests that duodenalulcer most likely results from an imbalance between“aggressive” factors, such as infection of Helicobacter pylori(H pylori), gastric acid and pepsin, and “defensive” factors,such as duodenal mucosal bicarbonate (HCO37 -) secretion8(DMBS) (Valle et al. 2003; Hogan et al. 1996) a and sexhormones . It has long been observed that the ratio betweenmen and women who develop duodenal ulcer is 1.9:1 in theUS, whereas in Europe and in Asia this ratio is 2.2: 1(Kurata et al. 1985; Rosenstock and Jorgensen 1995;Ostensen et al. 1985; Bonnevie, 1975) and 3.1: 1(Wu HC etal. 2008), respectively. These clinical observations suggest agender difference in the incidence and severity of duodenalulcer; however, the underlying mechanism(s) are currentlyunknown. As far as gender differences are concerned, sexhormones have been often evaluated as the causative factors.For example, numerous studies have suggested a protectiverole of estrogen in the development of various diseasesincluding cardiovascular diseases (Gerhard and Ganz, 1995;Grodstein et al. 1996; Orshal and Khalil 2004), cerebraldamage and mortality (Zhang et al., 2004; Hurn and Macrae,2000), and osteoporosis (Gerhard and Ganz 1995; Grodsteinet al. 1996; Orshal et al. 2004; Popp et al. 2006). In contrast,information regarding the protective effects of sex hormonein the gastrointestinal tract is very limited (Furner et al.1989). The present investigation showed that cysteamineinduced duodenal ulcer severity decreased on theadministration of estrogen. But estrogen failed to recover theulcer incidence in old females. This indicates that estrogenitself is not the reason for the low incidence of duodenalulcer in female during her reproductively active period. Thehigh incidence of duodenal ulcer in overiectomized –cysteamine injected mice may be due to the lack of estrogenin association with other factors. Anders et al. (2008)revealed the gender specific duodenal protection by estrogenin terms of HCO3 secretion and the underlying molecularmechanisms of estrogen stimulation of DMBS that is linkedto ER-Ca2+-CFTR and Cl-/HCO3exchanger pathways. Weobserved that the incidence of duodenal ulcer is more or lessidentical in overiectomized –cysteamine injected femalesand old –cysteamine injected females. The result wasreflected in histological, histochemical, and biochemical

Effect Of Ovariectomy And Of Estrogen Administration Upon Duodenal Ulceration Induced ByCysteamine

5 of 7

studies. Histological changes that took place in goblet cells,pyloric gland cells and Brunner’s gland cells inovariectomized cyteamine treated mice were not observed inovariectomized estrogen injected cysteamine treated mice.Sugars from glycoprotein of Brunner’s gland were depletedin cysteamine treated females. These findings indicate thatthe estrogen is able to protect the duodenal mucosa fromdamaging effects of cysteamine. Manekar and Namaji(1977) suggested that female sex hormones have protectiveeffects against ulcer formation. It has been established thatglycoprotein containing bicarbonate is mainly responsiblefor the protection of duodenal mucosa. The glycoprotein andbicarbonate are mainly secreted by Brunner’s glands. Thus itcan be concluded that estrogen may be assisting in thesecretion of glycoprotein from Brunner’s glands. Increase inthe secretion of glycoprotein from Brunner’s gland acini andduct cells has been shown in the present investigation inestrogen treated mice. Secretion of glycoprotein of otherexocrine cells of the duodenum is also influenced by theestrogen. This substantiates the role of estrogen in protectingthe duodenal mucosa, but the exact mechanism is to beexplored further.

References

r-0. Anders S., Cheyanne C., Kwang H. K; Jimmy C; Xia D.,Biguang T., Hong-hai Z., Dong-bao C., Hui andD.(2008)Gender Specific Protection of Estrogen againstGastric Acid-induced Duodenal Injury: Stimulation ofDuodenal Mucosal Bicarbonate Secretion.Endocrino, 22:1222-12234.r-1. Antonsen S. (1955) The influence of sex hormones onexperimentally gastric ulcers in rats. Acta Endocrinol, 19:203-212.r-2. Bonnevie O. (1975) The incidence in CopenhagenCounty of gastric and duodenal ulcers 32 in the same patient.Scand J Gastroenterol, 10:529-36.r-3. Furner S.E., Davis F.G., Nelson R.L., Haenszel W.(1989) A case-control study of large bowel cancer andhormone exposure in women. Cancer Res, 49:4936-40.r-4. Christensen S., Riis A., Norgaard M., Thomsen R.W.,Tonnesen E.M., Larsson A.(2006) Perforated peptic ulcer:use of pre-admissio oral glucocorticoids and 30-daymortality. Aliment Pharmac Ther, 23(1):45-52.r-5. Crean G.P. (1963). The endocrine system and thestomach. Vit and Horm, 21:215-219.r-6. Dey N.C. and Dey T.K. (1974) Disease of the digestivesystem. In: A text book of pathology. Allied Agency,Calcutta, India. Pp 354.r-7. Dische Z., Shetttles T.K. (1977) A specific colourreaction of methyl pentoses and a specificspectrophotomentric micro method for their determination.JBiol Chem, 175: 595-603.r-8. Dubois M., Cilles K.A., Hamilton J.K., Rebers D.A.,Smith F. (1956) Anal Chem, 28:350-356.r-9. Furner S.E., Davis F.G., Nelson R.L, Haenszel W.(1989) A case-control study of large bowel cancer andhormone exposure in women. Cancer Res, 49:4936-40.r-10. Gerhard M.., Ganz P. (1995) How do we explain the

clinical benefits of estrogen? From bedside to bench.Circulation, 92:5-8.r-11. Grey J. (1929). Tobacco smoking and gastricsymptoms. Anna Intern Med, 3: 267-277.r-12. Grodstein F., Stampfer M.J., Manson J.E., ColditzG.A., Willett W.C., Rosner B, Speizer F.E., Hennekens C.H.(1996) Postmenopausal estrogen and progestin use and therisk of cardiovascular disease. N Engl J Med, 335:453-61.r-13. Guerrine F., Merveille P., L’hermine C., DecoulxM.(1967)Action dela castration sur les lesion gastriques parin phenylbutazone chez le rat. CR Soc Biol, 161:195-206.r-14. Gurr E.(1962) In: Staining animal tissues practical andtheoretical. Leonard Hill Ltd, London.r-15. Hogan D.L., Rapier R.C., Dreilinger A., Koss M.A.,Basuk P.M., Weinstein W.M.,r-16. Nyberg L.M., Isenberg J.I. (1996) Duodenalbicarbonate secretion: eradication of Helicobacter pylori andduodenal structure and function in humans.Gastroenterology, 110:705-716.r-17. Hurn P.D., Macrae I.M. (2000) Estrogen as aneuroprotectant in stroke. JCereb Blood Flow Metab, 20:631-52.r-18. Kelly P., Robert A.(1969) Inhibition by pregnancy andlactation of steroid induced ulcer in rat. Gastroentrol, 56:24-36.r-19. Kim Y.H., Lee J.H., Lee S.S. (2007).Long term stressand helicobacter pylori infection independently inducegastric mucosal lesions in C57B/6 mice. Scand.J.Gastroentrol, 37 (11): 1259-1264.r-20. Kurata J.H., Honda G.D., Frankl H. (1985). Theincidence of duodenal and gastric ulcers in a large healthmaintenance organization. Am J Public Health, 75:625-9.r-21. Kyle J., Clark S, Waro J.T., Adesola A.O., WelbournR.B.(1963) The relationship of the major endocrine glands toexperimental peptic ulceration. In: Pathophysiology ofpeptic ulcer. (ed. S.C.Skoryna), Lippincott, Philadelphia.r-22. Lowry O.H., Rosenbrough N.J., Farr A.L., RandallR.J.(1951)Protein measurement with with folin phnolreagent.J Biol Chem, 193: 265-275.r-23. Lykoudes J.(1958)The general practitioner in Greecewho in 1958 discovered etiology of, and a treatment for,peptic ulcer disease. Helicobacter Pioneers, 3: 75-84.r-24. Manekar M.S., Namaji K.I.(1977) Effect of female sexhormones in experimentally induced acute ulceration. IndianJ Med Res, 65: 894-899.r-25. Marshal B. J and Warren J.R.(1984) Unidentifiedcurved bacilli in the stomach patients with gastritis. Lancet,1: (8336): 1273-1275.r-26. McManus J.F.A.(1946) Histological demonstration ofmucin after periodic acid. Nature, 158: 202-212.r-27. Orshal J.M., Khalil R.A. (2004) Gender, sex hormones,and vascular tone. Am J Physiol Regul Integr Comp Physiol,286: 233-49.r-28. Ostensen H., Gudmundsen T.E., Bolz K.D., BurholP.G., Bonnevie O. (1985) The incidence of gastric ulcer andduodenal ulcer in north Norway. A prospectiveepidemiological study. Scand J Gastroenterol, 20:189-92.r-29. Popp A.W., Bodmer C., Senn C., Fuchs G., KraenzlinM.E., Wyss H., Birkhaeuser MH., Lippuner K. (2006)Prevention of postmenopausal bone loss with long-cyclehormone replacement therapy. Maturitas, 53:191-200.r-30. Rosenstock S.J., Jorgensen T. (1995) Prevalence andincidence of peptic ulcer disease in a Danish County--aprospective cohort study. Gut, 36:819-24.r-31. Satakopan V.M., Kurup P.A.(1977) Changes incarbohydrate components of glycoprotein in tissues otherthan vascular tissue in Atheromomatons rats. Indian J Exp

Effect Of Ovariectomy And Of Estrogen Administration Upon Duodenal Ulceration Induced ByCysteamine

6 of 7

Biol, 15: 776-779.r-32. Speizer F.E., Hennekens C.H.(1996) Postmenopausalestrogen and progestin use and the risk of cardiovasculardisease. N Engl J Med, 335:453-61.r-33. Szabo S. (1978) Animal model for human disease:duodenal ulcer disease. Am J Pathol, 93: 273-276.r-34. Truelove S.C.(1960) Stibestrol, phenobarbitone anddiet in chronic duodenal ulcer, a fractional therapeutic trial.Brit.Med.J, 30: 559-566.r-35. Valle J.D., Chey W.D., Scheiman J.M.(2003) Acidpeptic disorders. In: Textbook of Gastroenterology. (4th ed.T. Yamada), Lippincott Williams & Wilkins, Philadelphia,PA, pp 1322-1376.

r-36. Warren L. (1959) Thiobarbituric acid assay of sialicacids. Biol Chem, 358: 73-120.r-37. Watkinson G. (1960) The incidence of chronic ulcerfound at necropsy. Gut, 1:14-19.r-38. Wu H.C., Tuo B.G., Wu W.M., Gao Y., Xu Q.Q., ZhaoK.(2008) Prevalence of Peptic Ulcer in Dyspeptic Patientsand the Influence of Age, Sex, and Helicobacter pyloriInfection. Dig Dis Sc, 265-2656.r-39. Zhang Y., Champagne N., Beitel L.K., Goodyer C.G.,Trifiro M., LeBlanc A. (2004) Estrogen and androgenprotection of human neurons against intracellular amyloidbeta1- toxicity through heat shock protein 70. J Neurosci,24:5315-21.

Effect Of Ovariectomy And Of Estrogen Administration Upon Duodenal Ulceration Induced ByCysteamine

7 of 7

Author Information

KV Ashokan, PhDDepartment of biology, P.V.P.Colleeg, Kvathe Mahankal, Sangli, Maharashtra, India

MM Kurane, PhDDepartment of zoology, Willingdon College, Sangli, Maharashtra, India

MM Pillai, PhDDepartment of Biotechnology, KIIT’s engineering college, Gokul Shirgaon, Kolhapur Mahrashtra, India