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Effects of cyclosporine a on the functions of submandibular and parotid glands of rats
Gen. Pharmac. Vol. 27, No. 5, pp. 887490, 1996 Copyright © 1996 Elsevier Science Inc. Printed in the USA.
ISSN 0306-3623/96 $15.00 + .00 SSDI 0306-3623(95)02097-7 All rights reserved ELSEVIER
Effects of Cyclosporine A on the Functions of Submandibular and Parotid Glands of Rats A. R. Dehpour, 1. N. Shirzad, 1 P. Ghafourifar 1 and M . AbdoUahi 2 IDEPARTMENT OF PHARMACOLOGY,SCHOOL OF MEDICINE, AND 2 DEPARTMENT OF TOXICOLOGY AND PHARMACOLOGY,FACULTY OF PHARMACY, TEHRAN
UNIVERSI~ OF MEDmAL SCIENCES, TEHRAN, IV.AN.[TEL: 0"98-21-6112316; FAX: 0-98-21-6461178] ABSTRACT. 1. The present study was designed to investigate the possible effects of long-term (45 days) administration of therapeutic doses of cyclosporine A (25 mg/kg/day), on the functions of submandibular and parotid glands of rats. Pure submandibular and parotid saliva were collected intraorally by microcannulation of the ducts. 2. The weight gains of the treated animals during the study and the weights of the salivary glands at the end of 45 days were reduced significantly as compared with those of controls. 3. Sialochemistry studies revealed a marked decrease in total protein concentration in saliva obtained from submandibular glands (P<0.05). 4. Determination of electrolyte concentrations in saliva of submandibular gland and serum showed considerable differences between treated and control groups. 5. Significant elevation of amylase activity in serum and parotid saliva was observed in the treated rats in comparison with controls (P<0.001). 6. Data presented here indicates that long-term administration of therapeutic doses of cyclosporine A causes significant alterations in salivary output and composition. GEN PHARMAC27;5:887--890, 1996. KEY WORDS. Cyclosporine A, submandibular, parotid, saliva, rats INTRODUCTION Cyclosporine A (CSA) is a cyclic endecapeptide of the family of fungal metabolites that can affect the activated helper-inducer T-lymphocytes by inhibiting the release of interleukin-II, and has proved to be a potent and effective immunosuppressant. It is being used with increasing frequency for organ transplantation (Tejani et al., 1988; Lustig et al., 1987; Wong and Dirks, 1988; Barton et al., 1989). One of the major side effects of C S A is its nephrotoxicity (Wong and Dirks, 1988; Sabbatini et al., 1991; Mason, 1989; Devarajan et al., 1989; B~ickman et al., 1990; Andreucci and Fuiano, 1989). Several serum electrolyte disturbances induced by C S A were reported in human and animal experiments (Kinson, 1990; Ihara et al., 1990; Nozue et al., 1992; Rahman and Ing, 1989; Barton et al., 1989; Wong and Dirks, 1988). The adverse effects of C S A on exocrine pancreatic functions have been shown previously (Doi et al., 1990). Autoradiographic studies revealed the appearance of 3H-CSA in mice salivary glands after intravenous injection of the drug (B~ickman et al., 1987). Because of its high lipophilicity, CSA is transferred from blood to saliva in measurable amounts (McGaw et al., 1987; Coates et al., 1988). Whether or not this transfer across the salivary glands may cause any alteration in their functions, and also in salivary composition, was investigated in the present study. MATERIALS A N D METHODS Chemicals
CSA was provided from Sandoz Ltd., Baseli Switzerland and dissolved in olive oil, to which ethanol was add¢d (90% olive oil, 10% *To whom all correspondence should be addressed. Received 18 May 1995; accepted 31 October 1995.
ethanol). The final solution contained 25 mg of CSA per ml. All other chemicals were purchased from Sigma Chemical Co. (St. Louis, MO). Animals and treatment
Male albino rats (4 months old) weighing 180-190 g were divided into 2 groups with 8 animals in control and 16 animals in CSAtreated groups. Some animals died during treatment. The rats were housed in a controlled-temperature and humidity room in separate cages for control and treated groups, and allowed free access to standard rat chow and tap water. The CSA-treated group received a daily dose of CSA (25 mg/kg) (Thomson et al., 1981) by gastric intubation. The control group received the vehicle alone and this was continued for 45 consecutive days. At the end of 45 days, animals were starved for 24 hours prior to anesthesia, when they were allowed free access to water. Under anesthesia, induced by intraperitoneal injection of sodium pentobarbital (50 mg/kg), the trachea was cannulated with a short length polyethylene tube. Both ducts of the submandibular gland (SMG) were cannulated with micropolyethylene cannulas as described by Yoshida eta/. (1967). Parotid saliva was collected with a micropipette, directly from Stensen's duct according to the method of Yu (1990). Intraperitoneal injection of pilocarpine nitrate (8 mg/kg) was used as a secretagogue. After the first 2 drops were discarded, pure saliva was collected for 30 min into preweighed tubes, then weighed and frozen at -20°C until analytical procedures. Blood samples were collected by cardiac puncture and serum was immediately separated. Assays
At the end of the experiment, salivary glands were removed, dissected free from extraneous tissues and weighed. The amylase activ-
888
A . R . Dehpour et al. 300
either alanine or aspartate aminotransferase activities in serum of the CSA-treated group were 6-+0.31 and 11.65+0.41 U/L, respectively, which were not significantly different from those of control rats, with 6.28-+0.28 for alanine and 12.08+0.36 for aspartate aminotransferase.
250
DISCUSSION
.,.s 200
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10
r 20
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p 40
i 50
Days of t r e a t m e n t F I G U R E 1. Cyclosporine was given orally (25 mg/kg/day) for 45 days. Data points are means-+ SEM of 8 control ( B ) and 14 treated (A) animals. The body weights of the treated rats are significantly ( P < 0 . 0 5 ) lower than those of controls.
ity in parotid saliva and serum was determined by the method of Bemfeld (1951), using starch suspension as substrate. The major electrolytes, those regulated or largely influenced by CSA treatment, were estimated in saliva and serum. Sodium, potassium, calcium and magnesium concentrations were measured by atomic absorption spectrophotometry (Shimadzu, model 160A in background correction mode). Total protein in saliva was determined by the method of Lowry et al. (1951) using bovine serum albumin as the standard. G O T and GPT were measured in serum using the method of Reitman and Frankel (1957). Results are expressed as means-+ SEM, compared by student's t-test for unpaired data and considered significantly different when P<0.05. All computations were performed by a computer using SPSS software. RESULTS The gain in body weight of the rats during the study was appreciably affected by treatment with CSA. The body weights of the treated rats were significantly (P<0.05) lower than those of controls (Fig. I ). The weight of SMG normalized for body weights was 1.84-+0.07 g/kg in controls and 1.46+0.04 in treated animals. The relative weights of parotid glands (PG) were 1.38-+0.04 and 1.12-+0.08 g in control and treated rats, respectively. These results indicate that significant differences had occurred during the course of treatment with CSA on the weights of submandibular (P<0.001) and parotid (P<0.02) glands of the treated rats. W h e n controls were compared to CSA-treated animals, there was a significant reduction in the total protein concentration in SMG saliva after CSA administration (P<0.05), (Table 1). A comparison between the control and treated groups revealed that the amylase activity was significantly increased in saliva obtained from PG and serum by CSA administration (Table 2). The plasma and salivary electrolyte status after CSA treatment, is shown in Table 3, where Na + and Ca 2+ concentrations in saliva derived from SMG were suppressed significantly after C S A therapy. Considerable elevation in serum K +, Mg 2+ and Ca 2+ concentrations occurred after the period of treatment. Mean values for
Consistent with previous studies (Tejani et al., 1988; Farthing et al., 1981; Sigalet et al., 1992; Wong and Dirks, 1988), the weight gains of animals treated with CSA were less than those of the controls (Fig. 1). This is probably due to adverse effects of CSA on emulsification of dietary fat and the intraluminal digestion of proteins and carbohydrates (Sigalet et al., 1992). Our results also indicate that CSA caused a considerable decrease in weights of SMG and PG. In previous studies, it has been shown that there is a high accumulation of CSA in the pancreas and salivary glands and that excretory cells become atrophic after CSA administration (Ihara et al., 1989; B~ickman et al., 1987). However, the question remains as to whether it is the manifestation of an acute toxic effect of CSA on the pancreas and/or the salivary glands, and that means a susceptibility to chronic CSA toxicity. CSA with its high lipophilicity can be transferred from blood to saliva in a measurable amount (McGaw et al., 1987; Coates et al., 1988). In the current study, we have shown that CSA-treated rats exhibited marked alterations in electrolyte concentrations as well as protein content and amylase levels in saliva, when compared with animals that received the vehicle alone (Tables 1, 2, 3). Also, there were several changes in electrolyte concentrations and amylase levels in the serum of CSA-treated animals. The concentrations of electrolytes in pure SMG saliva have shown a decrease in Na + and Ca 2+ levels accompanied by unchanged K + and Mg > concentrations of treated rats, in comparison with that of the controls. This could be attributed to the changes in metabolism of these cations during the course of treatment or be part of the adverse effects of CSA on the excretory function of pancreas and salivary glands that disturbed the composition of electrolytes in saliva. A significant decrease in the content of the total protein in SMG saliva, even when normalized for the weights of the glands, was observed (Table 1). Levels of proteins in biological fluids are a consequence of several interrelated processes, including protein synthesis and breakdown, cell damage and membrane permeability. CSA may affect one or a combination of some of these processes (Thomson et al., 1981 ). Consistent with previous studies, which have shown that protein synthesis by the liver (Mason, 1989) and excretion by exocrine pancreatic tissues (Hirano et al., 1992) was decreased with CSA therapy, our data revealed a significant decrease in the total protein output of SMG saliva. Which of these processes of protein TABLE 1. Comparison of salivary total protein concentration in CSA-treated and control animals Total protein (mg/100ml) in SMG saliva Control n= 8 Treated n= 9
Total protein (mg/100 ml/g of gland weight) in SMG saliva
291.7 -+ 38.42
697.85 -+ 92.6
173.3 + 23.452
481.34 -+ 43.67 a
Walues significantly differing from controls at P < 0.05; duration of treatment: 45 days; dose of CSA: 25 mg/kg/day;secretagogue: pilocarpine nitrate (8 mg/kg); SMG, submandibular gland.
Cyclosporin A in Rat Glands
889
TABLE 2. Comparison of salivary and serum amylase activity in CSA-treated and control animals Amylase (U/ml/g
Control n = 8 Treated
Amylase (U/ml) in P G saliva
of gland weight) in P G saliva
Amylase (U/ml) in serum
744.91 ± 42.17
2172.73 ± 161.4
10.92 ± 0.47
1031.99 m 30.67 a
4318.69 ± 340.7 ~
16.31 ± 0.64 a
n
11
"Values significantly differing from controls at P < 0.001; duration of treatment: 45 days; dose of CSA: 25 mg/kg/day; secretagogue: pilocarpine nitrate (8 mg/kg); PG, parotid gland.
synthesis or excretion were affected by CSA in SMG needs further investigation. The amounts of electrolytes in serum are unlikely to occur as a simple overflow from the salivary glands, because the pattern of release into saliva and serum differs. Determination of cations in serum showed marked elevation of K ÷, Ca 2+ and Mg > concentrations in CSA-treated rats in comparison with those of controls. Occurrence of hyperkalemia after C S A therapy has been reported by many investigators in humans and rats (Foley et al., 1985; Adu et al., 1983). As etiological factors of hyperkalemia, Adu et al. (1983) implicated a tubular defect of potassium and hydrogen ion secretion caused by renal tubular damages. CSA-induced distal renal tubular acidosis of the hyperkalemic type, has been reported in the presence or absence of hyporeninemic hypoaldosteronism (Rahman and Ing, 1989). The appearance of hypocalcemia and reversible hypomagnesemia after administration of CSA was reported previously (Barton et al., 1989; Wong and Dirks, 1988; Nozue et al., 1992; Kinson, 1990; Thompson et al., 1984). However, our data have demonstrated marked elevations in serum Ca 2+ and Mg 2+ levels. The reason for this discrepancy remains unclear. These conflicting results may reflect differences in animal species, as well as differences in mode of C S A administration and duration of treatment. It is well known that CSA accumulates in pancreatic tissues (Hirano et al., 1992). This accumulation of the drug may lead to pancreatic injuries in normal (Hirano et al., 1992) and transplant rats (Ihara et al., 1989; Elalamy et al., 1988). In this regard, a pancreatitis-induced hyperamylasemia was previously reported by several investigators (Hirano et al., 1992; Ihara et al., 1989; Elalamy et al., 1988). These results are in a good agreement with our findings, which show a significant increase in serum amylase activity. Amylase is also synthesized in acinar cells of parotid glands (Beeley and
Chisholm, 1976). Our results also revealed a marked elevation of amylase activity in parotid saliva of CSA-treated rats (Table 2). Ikeno et al. (1982) suggest that organs synthesizing amylase may be divided into 2 groups depending on the amylase activity in the unstimulated gland and on the effect of pilocarpine on the amylase activity in these organs. In this regard, they placed the parotid gland and the pancreas in the same group. So, we suggest that any mechanism that leads to an increase in pancreas amylase secretions influences the secretion of the enzyme in the parotid gland. In addition, it was shown that most of the amylase activity in serum appears to be due to enzyme release from the parotid gland instead of the pancreas (Ikeno et al., 1982). Therefore, we suggest that CSA-associated hyperamylasemia may not only be a consequence of its increased pancreatic secretion, but also of its elevated parotid secretion. Correspondingly, serum transaminase, aspartate aminotransferase and alanine aminotransferase activities have not shown statistically significant differences between CSA-treated and control groups. This finding is in accord with that of other investigators, who demonstrated that these enzymes are generally unaltered during CSA therapy in humans and rats (Farthing et al., 1981; Mason, 1989). In the present study we have indicated that long-term administration (45 consecutive days) of therapeutic doses of CSA (25 mg/kg/ day) causes considerable changes of the secretory function of PG and SMG and causes several disturbances in electrolyte concentrations, as well as amylase activity, in the serum of animals treated with this drug. References Adu D., Tumey J., Michael J. and McMaster P. (1983) Hyperkalemia in cyclosporine-treated renal allograft recipient. Lancet 2, 370-372. Andreucci V. E. and Fuiano G. (1989) Cyclosporine-A nephrotoxicity, in:
TABLE 3. Comparison of electrolytes concentrations of SMG saliva and serum in CSA-treated and control animals Concentration (mEq/L) in SMG saliva Control n=8 Na ÷ K+ Ca ++ Mg ++
55.94 96.15 7.1 1.87
± ± -+ ±
0.95 13.b5 1.29 0.18
Treated n=6 40.87 118.8 3.7 1.73
-+ 1.78"* _+ 5.26 ± 0.54* ± 0.13
Concentration (mEq/L) in serum Control n=8 240.22 7.95 6 1.28
_+ 7.39 + 0.29 + 0.57 ± 0.18
Treated n=5 252.17 11.72 10.3 2.26
± + + ±
5.85 1.13" 0.22*** 0.25**
Values significantly differing from controls at *P < 0.05; **P < 0.01; ***P < 0.001; duration of treatment: 45 days; dose of CSA: 25 mg/kg/day; secretagogue: pilocarpine nitrate (8 mg/kg); SMG, submandibular gland.
890 International year book of nephrology (ed. Andreucci V. E.) pp. 171-203. Kluwer-Academic Press, Boston. B~ickman L., Bran& I., Ringd~n O. and Dallner G. (1987) Distribution of ~H-Cyclosporine-A in mice by autoradiography. Transplant Proc. 19, 1236-1239. B/ickman L., Sundelin B. and Bohman S. O., (1990) Chronic renal lesions in rats after long-term treatment with cyclosporine-A. Transplant Proc. 22, 133-134. Barton C. H., Vaziri N. D., Mina-Araghi S., Crosby S. and Seo M. I. (1989) Effects of cyclosporine-A on magnesium metabolism in rats. J. Lab. Clin. Med. 114, 232-236. Beeley J. A. and Chisholm D.M. (1976) Sarcoidosis with salivary gland involvement, biochemical studies on parotid saliva. J. Lab. Clin. Med. 88, 276-281. Bemfeld P. (1951) Enzymes of starch degradation and synthesis. Adv. Enzymol. 12, 379-428. Coates J., Lain S. and McGaw T. (1988) Radioimmunoassay of salivary cyclosporine with use of 1251-labelled cyclosporine. Clin. Chem. 34, 1545-1550. Devarajan P., Kaskel F.J., Arbeit L.A. and Moore L.C. (1989) Cyclosporine nephrotoxicity: Blood volume, sodium conservation and renal hemodynamics. Am. J. Physiol. 256, F71-F78. Doi R., Tangoku A., Chowdhury P., Blevins G. T., Pasley J. N. and Rayford L. (1990) Effects of FK506 and cyclosporine-A on exocrine pancreas in rats. Pancreas 5, 704. Elalamy I., Myam I., Casson C., Fabre M. and Moatti N. (1988) Amylase isoamylase and lipase activities in cyclosporine- treated rots. Clin. Chem. 34, 2582. Farthing M. J. G., Clark M.L., Pendry A., Sloane J. and Alexander P. (1981) Nature of the toxicity of cyclosporine-A in the rat. Biochem. Pharmac. 30, 3311-3316. Foley R. J., Hamner R. W. and Weinman E. J. (1985) Serum potassium concentrations in cyclosporine-A and azathioprine treated renal transplant patients. Nephron 40, 280-285. Hirano T., Manabe T., Ando K. and Tobe T. (1992) Acute cytotoxic effect of cyclosporine-A on pancreatic acinar cells in rats. Scand. J. Gastroenterol. 27, 103-107. Ihara H., Hosokawa S., Ogino T., Arima M. and Ikoma F. (1990) Activation of K+ channel and inhibition of Na+-K + ATPase of human erythrocytes by cyclosporine: Possible role in hyperpotassemia in kidney transplant recipients. Transplant Proc. 22, 1736-1739. lhara H., Ogino T., Hosokawa S., Yabumoto H., Arima M. and Ikoma F.(1989) Perioperative hyperamylasemia in cyclosporine-treated renal transplant recipients: Does it have clinical significance? Transplant Proc. 21, 2147-2150. Ikeno T., Nasu J., Hashimoto S. and Kuzuya H. (1982) Mechanisms of in-
A . R . Dehpour et al. crease in amylase activity in rat submandibular and sublingual glands after administration of pilocarpine. Archs. Oral Biol. 27, 597-601. Kinson G. A. (1990) Effects of steroids and cyclosporine-A on compensatory renal growth and plasma electrolytes in rats. Pharmac. Res. 22, 455-462. Lowry O., Rosebrough N., Farr A. and Randall R. (1951) Protein measurement with the Folin-phenol reagent. J. Biol. Chem. 193, 265-275. Lustig S., Stem N., Eggena P., Tuck M. L. and Lee D. B. N. (1987) Effects of cyclosporine on blood pressure and renin-aldosterone axis in rats. Am. J. Physiol. 253, 1596-1600. Mason J. (1989) Pharmacology of cyclosporine (Sandimmune)R, Vll. Pathophysiology and toxicology of cyclosporine in humans and animals. Pharmac. Rev. 42, 423-434. McGaw T., Lam S. and Coates J. (1987) Cyclosporine induced gingival overgrowth: Correlation with dental plaque scores, gingivitis scores and cyclosporine levels in serum and saliva. Oral Surg. Oral Med. Oral Pathol. 64, 293-299. Nozue T., Kobayashi A., Kodama T., Uemasu F., Endoh H., Sako A. and Takagi Y. (1992) Pathogenesis of cyclosporine induced hypomagnesemia. J. Pediatr. 120, 638-640. Rahman M. A. and lng T. S. (1989) Cyclosporine-A and magnesium metabolism. J. Lab. Clin. Med. 114, 213-214. Reitman S. and Frankel S. (1957) A colorimetric method for the determination of serum oxaloacetic and glutamic pyruvic transaminases. Am. J. Clin. Pathol. 28, 56-61. Sabbatini M., Denicola L., Ucceleo F., Ramano G., Papaccio G., Esposito V., Sepe V., Come G. and Fuiano G. (1991) Medium-term cyclosporine renal dysfunction and its reversibility in rats. Am. Physiol. Soc. F, 898905. Sigalet D. L., Knetman N. M. and Thomson A. B. R. (1992) Reduction of nutrient absorption in normal rats by cyclosporine. Transplantation 53, 1103-1107. Tejani A., Lancman I., Pompantz A., Khawar M. and Chen C. (1988) Nephrotoxicity of cyclosporine-A and cyclosporine-G in rat model. Transplantation 45, 184-188. Thompson C. B., Sullivan K. M., June C. H. and Thomas E. D. (1984) Association between cyclosporine neurotoxicity and hypomagnesemia. Lancet 17, 1116-1120. Thomson A. W., Whiting P. H., Cameron I. D., Lessels S. E. and Simpson J. G. (1981) A toxicological study in rats receiving immunotherapeutic doses of cyclosporine-A. Transplantation 31, 121-124. Wong N. L. M. and Dirks J. H. (1988) Cyclosporine-induced hypomagnesemia and renal magnesium wasting in rats. Clin. Sci. 75, 509-514. Yoshida Y., Sprecher R., Schenyer C. and Schenyer L. (1967) Role of [3-receptors in sympathetic regulation of electrolytes in rat submaxillary saliva. Proc. Soc. Exp. Biol. Med. 126, 912-916. Yu J. H. (1990) The influence of varying the electrical frequency of sympathetic nerve stimulation on fluid and calcium secretion of the rat parotid salivary glands. Arch. Oral Biol. 35,639-643.
ISSN 0306-3623/96 $15.00 + .00 SSDI 0306-3623(95)02097-7 All rights reserved ELSEVIER
Effects of Cyclosporine A on the Functions of Submandibular and Parotid Glands of Rats A. R. Dehpour, 1. N. Shirzad, 1 P. Ghafourifar 1 and M . AbdoUahi 2 IDEPARTMENT OF PHARMACOLOGY,SCHOOL OF MEDICINE, AND 2 DEPARTMENT OF TOXICOLOGY AND PHARMACOLOGY,FACULTY OF PHARMACY, TEHRAN
UNIVERSI~ OF MEDmAL SCIENCES, TEHRAN, IV.AN.[TEL: 0"98-21-6112316; FAX: 0-98-21-6461178] ABSTRACT. 1. The present study was designed to investigate the possible effects of long-term (45 days) administration of therapeutic doses of cyclosporine A (25 mg/kg/day), on the functions of submandibular and parotid glands of rats. Pure submandibular and parotid saliva were collected intraorally by microcannulation of the ducts. 2. The weight gains of the treated animals during the study and the weights of the salivary glands at the end of 45 days were reduced significantly as compared with those of controls. 3. Sialochemistry studies revealed a marked decrease in total protein concentration in saliva obtained from submandibular glands (P<0.05). 4. Determination of electrolyte concentrations in saliva of submandibular gland and serum showed considerable differences between treated and control groups. 5. Significant elevation of amylase activity in serum and parotid saliva was observed in the treated rats in comparison with controls (P<0.001). 6. Data presented here indicates that long-term administration of therapeutic doses of cyclosporine A causes significant alterations in salivary output and composition. GEN PHARMAC27;5:887--890, 1996. KEY WORDS. Cyclosporine A, submandibular, parotid, saliva, rats INTRODUCTION Cyclosporine A (CSA) is a cyclic endecapeptide of the family of fungal metabolites that can affect the activated helper-inducer T-lymphocytes by inhibiting the release of interleukin-II, and has proved to be a potent and effective immunosuppressant. It is being used with increasing frequency for organ transplantation (Tejani et al., 1988; Lustig et al., 1987; Wong and Dirks, 1988; Barton et al., 1989). One of the major side effects of C S A is its nephrotoxicity (Wong and Dirks, 1988; Sabbatini et al., 1991; Mason, 1989; Devarajan et al., 1989; B~ickman et al., 1990; Andreucci and Fuiano, 1989). Several serum electrolyte disturbances induced by C S A were reported in human and animal experiments (Kinson, 1990; Ihara et al., 1990; Nozue et al., 1992; Rahman and Ing, 1989; Barton et al., 1989; Wong and Dirks, 1988). The adverse effects of C S A on exocrine pancreatic functions have been shown previously (Doi et al., 1990). Autoradiographic studies revealed the appearance of 3H-CSA in mice salivary glands after intravenous injection of the drug (B~ickman et al., 1987). Because of its high lipophilicity, CSA is transferred from blood to saliva in measurable amounts (McGaw et al., 1987; Coates et al., 1988). Whether or not this transfer across the salivary glands may cause any alteration in their functions, and also in salivary composition, was investigated in the present study. MATERIALS A N D METHODS Chemicals
CSA was provided from Sandoz Ltd., Baseli Switzerland and dissolved in olive oil, to which ethanol was add¢d (90% olive oil, 10% *To whom all correspondence should be addressed. Received 18 May 1995; accepted 31 October 1995.
ethanol). The final solution contained 25 mg of CSA per ml. All other chemicals were purchased from Sigma Chemical Co. (St. Louis, MO). Animals and treatment
Male albino rats (4 months old) weighing 180-190 g were divided into 2 groups with 8 animals in control and 16 animals in CSAtreated groups. Some animals died during treatment. The rats were housed in a controlled-temperature and humidity room in separate cages for control and treated groups, and allowed free access to standard rat chow and tap water. The CSA-treated group received a daily dose of CSA (25 mg/kg) (Thomson et al., 1981) by gastric intubation. The control group received the vehicle alone and this was continued for 45 consecutive days. At the end of 45 days, animals were starved for 24 hours prior to anesthesia, when they were allowed free access to water. Under anesthesia, induced by intraperitoneal injection of sodium pentobarbital (50 mg/kg), the trachea was cannulated with a short length polyethylene tube. Both ducts of the submandibular gland (SMG) were cannulated with micropolyethylene cannulas as described by Yoshida eta/. (1967). Parotid saliva was collected with a micropipette, directly from Stensen's duct according to the method of Yu (1990). Intraperitoneal injection of pilocarpine nitrate (8 mg/kg) was used as a secretagogue. After the first 2 drops were discarded, pure saliva was collected for 30 min into preweighed tubes, then weighed and frozen at -20°C until analytical procedures. Blood samples were collected by cardiac puncture and serum was immediately separated. Assays
At the end of the experiment, salivary glands were removed, dissected free from extraneous tissues and weighed. The amylase activ-
888
A . R . Dehpour et al. 300
either alanine or aspartate aminotransferase activities in serum of the CSA-treated group were 6-+0.31 and 11.65+0.41 U/L, respectively, which were not significantly different from those of control rats, with 6.28-+0.28 for alanine and 12.08+0.36 for aspartate aminotransferase.
250
DISCUSSION
.,.s 200
/ A--#
0
150 - 1@
i
r
@
10
r 20
, 50
p 40
i 50
Days of t r e a t m e n t F I G U R E 1. Cyclosporine was given orally (25 mg/kg/day) for 45 days. Data points are means-+ SEM of 8 control ( B ) and 14 treated (A) animals. The body weights of the treated rats are significantly ( P < 0 . 0 5 ) lower than those of controls.
ity in parotid saliva and serum was determined by the method of Bemfeld (1951), using starch suspension as substrate. The major electrolytes, those regulated or largely influenced by CSA treatment, were estimated in saliva and serum. Sodium, potassium, calcium and magnesium concentrations were measured by atomic absorption spectrophotometry (Shimadzu, model 160A in background correction mode). Total protein in saliva was determined by the method of Lowry et al. (1951) using bovine serum albumin as the standard. G O T and GPT were measured in serum using the method of Reitman and Frankel (1957). Results are expressed as means-+ SEM, compared by student's t-test for unpaired data and considered significantly different when P<0.05. All computations were performed by a computer using SPSS software. RESULTS The gain in body weight of the rats during the study was appreciably affected by treatment with CSA. The body weights of the treated rats were significantly (P<0.05) lower than those of controls (Fig. I ). The weight of SMG normalized for body weights was 1.84-+0.07 g/kg in controls and 1.46+0.04 in treated animals. The relative weights of parotid glands (PG) were 1.38-+0.04 and 1.12-+0.08 g in control and treated rats, respectively. These results indicate that significant differences had occurred during the course of treatment with CSA on the weights of submandibular (P<0.001) and parotid (P<0.02) glands of the treated rats. W h e n controls were compared to CSA-treated animals, there was a significant reduction in the total protein concentration in SMG saliva after CSA administration (P<0.05), (Table 1). A comparison between the control and treated groups revealed that the amylase activity was significantly increased in saliva obtained from PG and serum by CSA administration (Table 2). The plasma and salivary electrolyte status after CSA treatment, is shown in Table 3, where Na + and Ca 2+ concentrations in saliva derived from SMG were suppressed significantly after C S A therapy. Considerable elevation in serum K +, Mg 2+ and Ca 2+ concentrations occurred after the period of treatment. Mean values for
Consistent with previous studies (Tejani et al., 1988; Farthing et al., 1981; Sigalet et al., 1992; Wong and Dirks, 1988), the weight gains of animals treated with CSA were less than those of the controls (Fig. 1). This is probably due to adverse effects of CSA on emulsification of dietary fat and the intraluminal digestion of proteins and carbohydrates (Sigalet et al., 1992). Our results also indicate that CSA caused a considerable decrease in weights of SMG and PG. In previous studies, it has been shown that there is a high accumulation of CSA in the pancreas and salivary glands and that excretory cells become atrophic after CSA administration (Ihara et al., 1989; B~ickman et al., 1987). However, the question remains as to whether it is the manifestation of an acute toxic effect of CSA on the pancreas and/or the salivary glands, and that means a susceptibility to chronic CSA toxicity. CSA with its high lipophilicity can be transferred from blood to saliva in a measurable amount (McGaw et al., 1987; Coates et al., 1988). In the current study, we have shown that CSA-treated rats exhibited marked alterations in electrolyte concentrations as well as protein content and amylase levels in saliva, when compared with animals that received the vehicle alone (Tables 1, 2, 3). Also, there were several changes in electrolyte concentrations and amylase levels in the serum of CSA-treated animals. The concentrations of electrolytes in pure SMG saliva have shown a decrease in Na + and Ca 2+ levels accompanied by unchanged K + and Mg > concentrations of treated rats, in comparison with that of the controls. This could be attributed to the changes in metabolism of these cations during the course of treatment or be part of the adverse effects of CSA on the excretory function of pancreas and salivary glands that disturbed the composition of electrolytes in saliva. A significant decrease in the content of the total protein in SMG saliva, even when normalized for the weights of the glands, was observed (Table 1). Levels of proteins in biological fluids are a consequence of several interrelated processes, including protein synthesis and breakdown, cell damage and membrane permeability. CSA may affect one or a combination of some of these processes (Thomson et al., 1981 ). Consistent with previous studies, which have shown that protein synthesis by the liver (Mason, 1989) and excretion by exocrine pancreatic tissues (Hirano et al., 1992) was decreased with CSA therapy, our data revealed a significant decrease in the total protein output of SMG saliva. Which of these processes of protein TABLE 1. Comparison of salivary total protein concentration in CSA-treated and control animals Total protein (mg/100ml) in SMG saliva Control n= 8 Treated n= 9
Total protein (mg/100 ml/g of gland weight) in SMG saliva
291.7 -+ 38.42
697.85 -+ 92.6
173.3 + 23.452
481.34 -+ 43.67 a
Walues significantly differing from controls at P < 0.05; duration of treatment: 45 days; dose of CSA: 25 mg/kg/day;secretagogue: pilocarpine nitrate (8 mg/kg); SMG, submandibular gland.
Cyclosporin A in Rat Glands
889
TABLE 2. Comparison of salivary and serum amylase activity in CSA-treated and control animals Amylase (U/ml/g
Control n = 8 Treated
Amylase (U/ml) in P G saliva
of gland weight) in P G saliva
Amylase (U/ml) in serum
744.91 ± 42.17
2172.73 ± 161.4
10.92 ± 0.47
1031.99 m 30.67 a
4318.69 ± 340.7 ~
16.31 ± 0.64 a
n
11
"Values significantly differing from controls at P < 0.001; duration of treatment: 45 days; dose of CSA: 25 mg/kg/day; secretagogue: pilocarpine nitrate (8 mg/kg); PG, parotid gland.
synthesis or excretion were affected by CSA in SMG needs further investigation. The amounts of electrolytes in serum are unlikely to occur as a simple overflow from the salivary glands, because the pattern of release into saliva and serum differs. Determination of cations in serum showed marked elevation of K ÷, Ca 2+ and Mg > concentrations in CSA-treated rats in comparison with those of controls. Occurrence of hyperkalemia after C S A therapy has been reported by many investigators in humans and rats (Foley et al., 1985; Adu et al., 1983). As etiological factors of hyperkalemia, Adu et al. (1983) implicated a tubular defect of potassium and hydrogen ion secretion caused by renal tubular damages. CSA-induced distal renal tubular acidosis of the hyperkalemic type, has been reported in the presence or absence of hyporeninemic hypoaldosteronism (Rahman and Ing, 1989). The appearance of hypocalcemia and reversible hypomagnesemia after administration of CSA was reported previously (Barton et al., 1989; Wong and Dirks, 1988; Nozue et al., 1992; Kinson, 1990; Thompson et al., 1984). However, our data have demonstrated marked elevations in serum Ca 2+ and Mg 2+ levels. The reason for this discrepancy remains unclear. These conflicting results may reflect differences in animal species, as well as differences in mode of C S A administration and duration of treatment. It is well known that CSA accumulates in pancreatic tissues (Hirano et al., 1992). This accumulation of the drug may lead to pancreatic injuries in normal (Hirano et al., 1992) and transplant rats (Ihara et al., 1989; Elalamy et al., 1988). In this regard, a pancreatitis-induced hyperamylasemia was previously reported by several investigators (Hirano et al., 1992; Ihara et al., 1989; Elalamy et al., 1988). These results are in a good agreement with our findings, which show a significant increase in serum amylase activity. Amylase is also synthesized in acinar cells of parotid glands (Beeley and
Chisholm, 1976). Our results also revealed a marked elevation of amylase activity in parotid saliva of CSA-treated rats (Table 2). Ikeno et al. (1982) suggest that organs synthesizing amylase may be divided into 2 groups depending on the amylase activity in the unstimulated gland and on the effect of pilocarpine on the amylase activity in these organs. In this regard, they placed the parotid gland and the pancreas in the same group. So, we suggest that any mechanism that leads to an increase in pancreas amylase secretions influences the secretion of the enzyme in the parotid gland. In addition, it was shown that most of the amylase activity in serum appears to be due to enzyme release from the parotid gland instead of the pancreas (Ikeno et al., 1982). Therefore, we suggest that CSA-associated hyperamylasemia may not only be a consequence of its increased pancreatic secretion, but also of its elevated parotid secretion. Correspondingly, serum transaminase, aspartate aminotransferase and alanine aminotransferase activities have not shown statistically significant differences between CSA-treated and control groups. This finding is in accord with that of other investigators, who demonstrated that these enzymes are generally unaltered during CSA therapy in humans and rats (Farthing et al., 1981; Mason, 1989). In the present study we have indicated that long-term administration (45 consecutive days) of therapeutic doses of CSA (25 mg/kg/ day) causes considerable changes of the secretory function of PG and SMG and causes several disturbances in electrolyte concentrations, as well as amylase activity, in the serum of animals treated with this drug. References Adu D., Tumey J., Michael J. and McMaster P. (1983) Hyperkalemia in cyclosporine-treated renal allograft recipient. Lancet 2, 370-372. Andreucci V. E. and Fuiano G. (1989) Cyclosporine-A nephrotoxicity, in:
TABLE 3. Comparison of electrolytes concentrations of SMG saliva and serum in CSA-treated and control animals Concentration (mEq/L) in SMG saliva Control n=8 Na ÷ K+ Ca ++ Mg ++
55.94 96.15 7.1 1.87
± ± -+ ±
0.95 13.b5 1.29 0.18
Treated n=6 40.87 118.8 3.7 1.73
-+ 1.78"* _+ 5.26 ± 0.54* ± 0.13
Concentration (mEq/L) in serum Control n=8 240.22 7.95 6 1.28
_+ 7.39 + 0.29 + 0.57 ± 0.18
Treated n=5 252.17 11.72 10.3 2.26
± + + ±
5.85 1.13" 0.22*** 0.25**
Values significantly differing from controls at *P < 0.05; **P < 0.01; ***P < 0.001; duration of treatment: 45 days; dose of CSA: 25 mg/kg/day; secretagogue: pilocarpine nitrate (8 mg/kg); SMG, submandibular gland.
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A . R . Dehpour et al. crease in amylase activity in rat submandibular and sublingual glands after administration of pilocarpine. Archs. Oral Biol. 27, 597-601. Kinson G. A. (1990) Effects of steroids and cyclosporine-A on compensatory renal growth and plasma electrolytes in rats. Pharmac. Res. 22, 455-462. Lowry O., Rosebrough N., Farr A. and Randall R. (1951) Protein measurement with the Folin-phenol reagent. J. Biol. Chem. 193, 265-275. Lustig S., Stem N., Eggena P., Tuck M. L. and Lee D. B. N. (1987) Effects of cyclosporine on blood pressure and renin-aldosterone axis in rats. Am. J. Physiol. 253, 1596-1600. Mason J. (1989) Pharmacology of cyclosporine (Sandimmune)R, Vll. Pathophysiology and toxicology of cyclosporine in humans and animals. Pharmac. Rev. 42, 423-434. McGaw T., Lam S. and Coates J. (1987) Cyclosporine induced gingival overgrowth: Correlation with dental plaque scores, gingivitis scores and cyclosporine levels in serum and saliva. Oral Surg. Oral Med. Oral Pathol. 64, 293-299. Nozue T., Kobayashi A., Kodama T., Uemasu F., Endoh H., Sako A. and Takagi Y. (1992) Pathogenesis of cyclosporine induced hypomagnesemia. J. Pediatr. 120, 638-640. Rahman M. A. and lng T. S. (1989) Cyclosporine-A and magnesium metabolism. J. Lab. Clin. Med. 114, 213-214. Reitman S. and Frankel S. (1957) A colorimetric method for the determination of serum oxaloacetic and glutamic pyruvic transaminases. Am. J. Clin. Pathol. 28, 56-61. Sabbatini M., Denicola L., Ucceleo F., Ramano G., Papaccio G., Esposito V., Sepe V., Come G. and Fuiano G. (1991) Medium-term cyclosporine renal dysfunction and its reversibility in rats. Am. Physiol. Soc. F, 898905. Sigalet D. L., Knetman N. M. and Thomson A. B. R. (1992) Reduction of nutrient absorption in normal rats by cyclosporine. Transplantation 53, 1103-1107. Tejani A., Lancman I., Pompantz A., Khawar M. and Chen C. (1988) Nephrotoxicity of cyclosporine-A and cyclosporine-G in rat model. Transplantation 45, 184-188. Thompson C. B., Sullivan K. M., June C. H. and Thomas E. D. (1984) Association between cyclosporine neurotoxicity and hypomagnesemia. Lancet 17, 1116-1120. Thomson A. W., Whiting P. H., Cameron I. D., Lessels S. E. and Simpson J. G. (1981) A toxicological study in rats receiving immunotherapeutic doses of cyclosporine-A. Transplantation 31, 121-124. Wong N. L. M. and Dirks J. H. (1988) Cyclosporine-induced hypomagnesemia and renal magnesium wasting in rats. Clin. Sci. 75, 509-514. Yoshida Y., Sprecher R., Schenyer C. and Schenyer L. (1967) Role of [3-receptors in sympathetic regulation of electrolytes in rat submaxillary saliva. Proc. Soc. Exp. Biol. Med. 126, 912-916. Yu J. H. (1990) The influence of varying the electrical frequency of sympathetic nerve stimulation on fluid and calcium secretion of the rat parotid salivary glands. Arch. Oral Biol. 35,639-643.