Gastric mucosal secretions and lesions by different doses of streptozotocin in rats

Toxicology Letters, 55 (1991) 21-29 Elsevier

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TOXLET 02486

Gastric mucosal secretions and lesions by different doses of streptozotocin in rats

Pawinee Piyachaturawat, Jiranud Poprasit and Thirayudh Glinsukon Department of Physiology, Faculty of Science, Mahidol University, Bangkok (Thailand)

(Received 3 1 July 1989) (Accepted 6 August 1990) Key words: Gastric lesion; Gastric H+ secretion; Gastric emptying rate; Streptozotocin;

Insulin

SUMMARY The effects of various doses of streptozotocin (STZ) on gastric mucosal secretions and lesions were investigated in rats. STZ at a dose as low as 30 mg/kg significantly increased plasma glucose (P-C 0.05). The elevation of plasma glucose was dependent on the dose on STZ (3&65 mg/kg) administered. The levels of SGOT, SGPT, bilirubin and BUN also increased with the dose of STZ. The secretory rate of gastric H+ decreased whereas the degree of mucosal hyperemia increased with increasing levels of plasma glucose. Treatment with insulin improved all abnormal conditions except the gastric mucosal lesions. The lesions occurred with low H+-secretory rate and low gastric emptying rate. It is suggested that STZ might act directly on the gastric mucosa. Its action depended on the dose administered and was not primarily related to the insulin deficit.

INTRODUCTION

Streptozotocin (STZ), an N-nitroso-N-methylurea derivative of 2-deoxy-D-glucase, has been reported to selectively destroy pancreatic beta-cells with production of permanent diabetes [l-5]. The selectivity of the drug for beta-cells is due to the glucose moiety of STZ which facilitates its uptake into the islets [6]. STZ has also been reported to possess nephrotoxic and hepatotoxic activities. Its effect on the kidneys and liver was suggested to be due to its function in excretion and metabolism, respectively [4, 7, 81. In addition to those organs, it was recently found that lesions

Address for correspondence:

Dr. Pawinee Piyachaturawat, Department of Physiology, Faculty of Science, Mahidol University, Rama VI Road, Bangkok 10400, Thailand.

0378-4274/91/%3.50 @ 1991 Elsevier Science Publishers B.V. (Biomedical Division)

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of the gastric mucosa occurred in STZ-induced diabetes [9]. At present, the mechanism of STZ-diabetes in inducing gastric mucosal lesions is not clear. As the drug can affect several organ functions and shows a dose-dependent effect [4, 51, it is not known whether the gastric mucosal lesions are due to the direct action of STZ or are caused indirectly as a consequence of impairments in other organs by STZ. The aim of the present study was therefore to investigate the effect of different doses of STZ and insulin treatment on gastric secretory function and lesions. Possible involvement of abnormalities in other organ functions were also investigated. MATERIALS AND METHODS

Animals Adult male Wistar rats weighing 180-200 g were obtained from the National Animal Production Center, Mahidol University (Bangkok, Thailand). They were fed standard rat chow (Gold Coin, Singapore) and water ad libitum. The animals were divided into 5 subgroups. After overnight fasting, they were injected with STZ at doses of 0, 30,40, 50 or 65 mg/kg body wt., respectively, into the jugular vein under ether anesthesia. One week post-treatment the animals were sacrificed for study of the morphology of the gastric mucosa, gastric secretory and motor function, and determination of plasma glucose, serum glutamic-pyruvic transaminase, serum glutamic oxaloacetic transaminase, bilirubin and blood urea nitrogen (BUN). Insulin treatment was carried out only in rats which received 65 mg/kg body wt. of STZ. It was started on the same day as the STZ injection and performed daily until sacrifice. The insulin (NPH insulinium, Organon, Netherlands) was given subcutaneously at a dose of 10 units/kg body wt. twice a day. Gastric content analysis Animals were prepared for collection of gastric contents and analysis of the contents for H+, pepsin and soluble mucus secretion. Observation of mucosal lesions was done as previously described [9]. To investigate a possible direct effect of STZ on the gastric mucosa, 2 ml of STZ solution at a concentration of 65 mg/ml in unbuffered normal saline (pH 5) was instilled into the stomach. In control rats, unbuffered saline was administered instead of the STZ solution; 30 min later, H+-secretion was stimulated by histamine. The capacity of the STZ-exposed mucosa to secrete H+ and the occurrence of lesions were examined. Gastric emptying determination Polyethylene glycol (PEG 4000), a non-absorbable marker, was used as an indicator to assess gastric motility. The rats were fasted for 16 h and anesthetized with ether. A polyethylene tube was passed orally into the stomach and the gastric contents were washed out. Thereafter, 2 ml of 1 gs PEG in normal saline was gently

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instilled and the rat was returned to the cage. The sequential propulsion of PEG from the stomach at 15, 30, 45 or 60 min after administration was determined. Each rat was anesthetized again. The cardiac and pyloric sphincters were ligated and the stomach was excised. The contents of the stomach were emptied into graduated centrifuge tubes and centrifuged to separate solids. The volumes of supernatants were recorded and removed for determination of PEG by the method of Malawer and Powell [lo]. Blood biochemical analysis

A blood sample was collected from the abdominal aorta of each rat fasted overnight under ether anesthesia. It was allowed to clot at room temperature and the serum was separated by centrifugation and stored at - 20°C until assayed. Activities of serum glutamic oxaloacetic transaminase (SGOT) and serum glutamic pyruvic transaminase (SGPT), and level of bilirubin and blood urea nitrogen (BUN) were determined by the methods of Reitman and Frankel [l 11, Jendrassik and Grof [12] and Wybenga and Inkpen [ 131,respectively. An unpaired Student’s t-test was used to determine the effects of STZ. A P-value < 0.05 was considered to be significant. RESULTS

Figure 1 shows the effect of different doses of STZ on the plasma glucose level and the gastric secretory function at 1 week post-treatment. STZ at 30 mg/kg significantly elevated plasma glucose (P 0.05, Fig. 1b). The gastric mucosa of the STZ-treated animals was apparently hyperemic. The extent of the effect increased with the dose of STZ. Insulin treatment of the high-dose STZ-treated animals (65 mg/kg) effectively reduced the level of plasma glucose. It was reduced from 439.6 + 5.5 mg% in untreated diabetes to 193.8 + 10.4 mg% in insulin-treated diabetes as shown in Table I. The insulin treatment of control rats did not significantly affect the level of plasma glucose or the secretory rate of gastric H+, pepsin or soluble mucus in comparison with the untreated controls. However, it could significantly reverse all gastric secretions to the control level, although the plasma glucose level was still significantly higher (P < 0.05). Figure 2 illustrates the effect of STZ treatment on the gastric emptying rate. The percent PEG remaining in the stomach of the STZ-treated rats was higher at all examination times (15, 30, 45 and 60 min) in comparison with the controls. The slight

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t

* h b-

0

*

*

-

(mg/kg

STi!

STZ

)

(mg/kg)

Fig. 1. Levels of plasma glucose, secretory rate of histamine-stimulated gastric H+, pepsin and soluble mucus in rats at 1 week after a single intravenous administration of streptozotocin at doses of 30, 40, 50 and 65 mg/kg body wt., respectively. Each bar represents mean f SEM of8-10 animals. *P
100

a@

0

ST2

0

ST2

l

Control

l

Control tInsulin

*Insulin -

100

6

PI 40

-

-40

$

30

30-

0 -. 2. z

20

-

20

5 2

IO -

IO

&&; 0

S 0” J

0 0

I5

30

45

50 min

0

I5

30

45

50 min

Fig. 2. Percent polyethlene glycol (PEG) remaining in the stomach at 15, 30,45 and 60 mm after its administration into control, STZ-diabetic, insulin-treated control and insulin-treated diabetic rats. Each bar represents mean f SEM of 8-12 animals.

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TABLE

I

EFFECT RATE

OF INSULIN OF

CONTROL

AND

Substances

measured

Plasma (mg/lm

TREATMENT

ON THE

HISTAMINE-STIMULATED STZ-TREATED

H+,

RATS (65 mg/kg) Control

glucose

OF PLASMA PEPSIN

AT 1 WEEK

+ insulin

193.8k

31.9+

Insulin treatment

stomach/h)

was immediately

mean k SEM and number

7.4*

started

after giving STZ and continued

in parentheses.

1.1

(10)

(11)

of animals

3.1

(9)

9.lkl.3 sulfate/g

10.4*

936.6 f 79.4

(8)

Soluble mucus

IN

(8)

1008.7*6.15

(,ug chondroitin

MUCUS

(8)

(8)

stomach/h)

SECRETORY

ST2 + insulin

38.0+ 6.9

Pepsin

SOLUBLE

POST-TREATMENT

(10)

@Eq H+/g stomach/h)

GLUCOSE,

AND

117.Ok6.2

ml)

Acid

(units/g

LEVEL

GASTRIC

daily until sacrifice.

Values are

*PC 0.05.

delay in gastric emptying was not significantly different from that of the controls (P> 0.05). Insulin treatment slightly enhanced gastric emptying in both STZ-treated rats and controls, but there was no statistically significant difference between them. Changes in liver and kidney function after receiving different doses of STZ and insulin are shown in Table II. As in the case of plasma glucose, a significant,elevation of liver enzyme activity of both SGOT and SGPT was observed at a dose of 30 mg/kg (PC 0.05), and the effect was found to be dose-dependent. However, a significant increase in serum bilirubin was observed only at the high dose of 65 mg/kg, but this is still within the normal range. The level of serum BUN also varied according to the dose of the drug. A significant elevation was observed at doses of 40 mg/kg and higher. Insulin treatment of STZ-treated rats effectively reduced the activities of SGOT and SGPT and the levels of bilirubin and BUN to those of control rats. Although insulin treatment improved all measured parameters in the STZ-treated animals, only mild gastric lesions associated with damage by STZ were ameliorated, whereas the formation of severe lesions was aggravated, as shown in Figure 3. Desquamation of surface epithelial cells and a dilated blood vessel at the apical surface are evident in the STZ-treated mucosa. The insulin-treated gastric mucosa showed focal bleeding with dilatation of glandular lumina and necrotic lesions. The possibility of STZ action directly on the gastric mucosa was examined: 90 min of exposure of the stomach to STZ (65 mg/ml) did not affect the secretory capacity of gastric H+ or gastric mucosal morphology (data not shown).

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xd +I

a d

Fig. 3. Gastric fundic muscosa obtained from: (a) control rat; (b) 1 week post-streptozotocin treatment (65 mg/kg) showing desquamation of surface epithelial cells and a dilated blood vessel; (c) I week of insulin treatment in STZ-treated rat. Note bleeding and necrotic lesions in the mucosa. (a) and (b) x 155; (c) x 124.

DISCUSSION

Streptozotocin (STZ) is known to possess diabetogenic properties by causing selective destruction of pancreatic B-cells [3,4]. In the present study, a single intravenous injection of STZ at a dose as low as 30 mg/kg was effective in inducing diabetes mellitus, though the diabetic state was less pronounced than that induced by the high dose (Fig. 1). These results confirm a previous report that demonstrated a progressive increase in mean plasma glucose following administration of STZ greater than 20 mg/kg [l]. A relationship between the amount of STZ injected and the number of B-cells destroyed has also been reported by Junode et al. [4]. As in our earlier study, a period of 1 week post-treatment was sufficient for the observation of gastric mucosal lesions and secretory changes in STZ-induced diabetes [9]; the present study were carried out at 1 week post-treatment. During the increase in the level of hy~rgly~~a the secretory rate of gastric H+ was progressively reduced. However, pepsin secretion was not affected and mucus secretion was increased. In spite of the low gastric H+ output, gastric mucosal hyperemia developed in all of the STZ-treated animals, particularly at high doses. This apparent reduction of gastric H+ by STZ might be explained by two possible m~hanisms. First, there might have been an actual reduction in secretion as a result of diabetes [14].

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Alternately, the reduced amount of H+ detected might have been only the consequence of back-diffusion of H+ through the damaged mucosa. In the stomach both secretory capacity and motor activity are dependent on neural activity [15]. The delay in gastric emptying rate in the STZ-treated animals (Fig. 2), which indicates a low neural activity as well as secretory capacity, might therefore have excluded the possibility of loss of H+ secretion by back-diffusion. Thus, the reduction in gastric H+ in the present study, which was related to the level of plasma glucose, could have resulted from the.hyperglycemic effect of STZ. Insulin treatment reduced the changes in the secretory rate of gastric H+ and all blood biochemical parameters including SGOT, SGPT, bilirubin and BUN to near control levels (Table II). However, insulin treatment did not completely prevent STZ from causing gastric mucosa lesions. It improved only mild gastric damage, whereas severe damage in some mucosae was aggravated. These results suggest that the formation of lesions was not a primary result of insulin deficit, but might have been due to the direct action of STZ on the gastric mucosa. Insulin treatment cannot completely reverse the effect of STZ when the mucosal lesions caused are excessive and irreparable, and beyond the capacity for regeneration. In addition, deterioration of the lesions with insulin might be the result of an increase in acid secreted in the lumen through its hypoglycemic effect [ 161.In support of this, marked mucosal necrosis was evident in the area of the lesion. However, direct exposure of the gastric mucosa to STZ neither altered the gastric H+ secretory rate nor the gross appearance of the mucosal surface. It is possible that STZ has no acute toxic effect and that detectable effects take a longer time to develop. A period of 90 min of exposure is not long enough to produce visible effects. STZ is a nitroso-compound known to have powerful biological effects, including the production of acute and chronic cellular injury. The effects of this compound are generally not produced by its own properties but are induced by decomposition products [2, 5, 171. STZ has been shown to be a methylating or alkylating agent which can cause cell necrosis in the gastrointestinal tract [17]. In the present study, the gastric mucus-secreting cells on the luminal surface which are renewed rapidly [ 181, might be initially damaged by the alkylating action of STZ. An increase in mucus secretion with increasing dose of STZ was evident in this study (Fig. l), and was probably related to the degree of surface epithelial cell damage as reported over a longer period of observation in our previous study [9]. Destruction of the cells in the proliferative zone by the alkylating action of STZ together with the diabetogenic effect would result in the delay or absence of epithelial repair. The impairment of liver and kidney function by STZ might not have been its major effect in the formation and development of gastric mucosal lesions in the present study, as seen in the insulin-treated animals. The mucosal lesions were well developed, whereas liver and kidney functions were comparable to those in control rats. Therefore, the development of gastric mucosal lesions by STZ can be attributed to the direct alkylating action of STZ which did not produce other acute effects. The lesions were further aggravated by diabetes.

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ACKNOWLEDGEMENT

This research work was partly supported by a Mahidol University Grant. REFERENCES 1 Ganda,

O.P.,

Rossini,

A. and Arthur,

A.L. (1976) Studies

on streptozotocin

diabetes.

Diabetes

25,

595a3. 2 Johansson,

E.B. and Tjalve, H. (1978) Studies on the tissue disposition

with special reference 3 Junod,

A., Lamber,

to the pancreatic

A.E., Oci, L., Pictet, R., Gonet,

togenic action of streptozotocin. 4 Junod,

A., Lamber,

tin: relationship 5 Raymon,

islets. Acta Endocrinol.

and fate of 14C-streptozotocin

89, 339-351.

A.E. and Renold,

A.E. (1967) Studies of the diabe-

Proc. Sot. Exp. Biol. Med. 126,201-205.

A.E., Stauffacher,

of dose to metabolic

W. and Renold, response.

B.W. (1982) Streptozotocin.

A.E. (1969) Diabetogenic

action ofstreptozoto-

J. Clin. Invest. 48, 129-139.

A review of its pharmacology,

efficacy

and toxicity.

Cancer

Treat. Rep. 6,427438. 6 Anderson, relation

T., Schein, P.S., McMenamin, with extent of depression

M.G. and Cooney,

of pancreatic

P.A. (1974) Streptozotocin

islet nicotinamide

adenine

diabetes:

dinucleotide.

cor-

J. Clin. Invest.

54,672-671. 7 Evan, A.P., Mong, of streptozotocin 8 Rakieten,

S.A., Gattone,

N., Gordon,

B.S., Cooney,

action of streptozotocin. 9 Piyachaturawat, diabetic

V.H., Comors,

and streptozotocin-induced Cancer

P., Poprasit,

D.A.,

Chemother.

B.A., Aronoff,

diabetes Davis,

G.R. and Luft, F.C. (1984) The effect

on the kidney.

Renal Physiol.

R.D. and Schein,

tumorigenic

Rep. 52, 563-567.

J. and Glinsukon,

T. (1988) Gastric

mucosal

lesions in streptozotocin-

rats. Cell Biol. Int. Rep. 12, 5363.

10 Malawer,

S.J. and Powell, D.W. (1967) An improved

lizing an emulsifier. 1 I Reitman, oxaloacetic 12 Jendrassik, Bilirubins. 13 Wybenga, 1467. 14 Rabinowith,

Gastroenterology

S. and Frankel,

S. (1957) A calorimetric pyruvic

L. and Grof,

P. (1938) Vereinfachte

Biochem.

turbidimetric

analysis

of polyethylene

transaminase.

method

for the determination

Am. J. Clin. Pathol. photometrische

of serum glutamic

28, 5663.

Methoden

zur Bestimmung

D.R. and Inkpen, Chemistry,

J.A. (1974) Lipids.

Principles

J.M. and Fowler,

In: R.J. Henry,

and Technics,

A.I. (1931) Gastric

Gastroenterology 28,493499. 16 Stadil, F. (1972) Effect of vagotomy J. Gastroenterol.

D.C. Cannon

2nd edn. Harper acidity

in diabetes

on gastrin

release during

and J.W. Winkelman

and Row, Hagerstown, mellitus:

its clinical of gastric

insulin hypoglycaemia

Anat. Rec. 115,231. 19 Rakieten, N., Rakieten, Treat.

pp. 1443significance

acid secretion.

in ulcer patients.

7,225231.

17 Magee, P.N. and Swann, P.F. (1969) Nitroso compounds. Br. Med. Bull. 25,240-244. 18 Steven, C.E. and Leblund, C.P. (1953) Renewal of the mucous cells in the gastric mucosa

Cancer.

des Blut-

Z. 297,81-89.

based on study of 100 cases. Arch. Intern. Med. 47,38&390. 15 Pevsner, L. and Grossman, M.I. (1955) The mechanism of vagal stimulation

tozotocin.

glycol uti-

53,25&256.

and glutamic

(Eds.), Clinical

Stand.

7, 78-89.

P.S. (1968) Renal

M.L. and Nadkarni, Rep. 29,91-98.

M.V. (1963) Studies on the diabetogenic

of the rat.

action of strep-