- Email: [email protected]
Opposite effects of antidepressants on unstimulated and stimulated salivary flow
Archives of Oral Biology (2005) 50, 17—21
www.intl.elsevierhealth.com/journals/arob
Opposite effects of antidepressants on unstimulated and stimulated salivary flow Luciane Kopittke, Rosane Gomez, Helena M.T. Barros* ˜o Faculdade Federal de Cie ˆncias Me ´dicas de Porto Alegre (FFFCMPA), Division of Pharmacology, Fundac¸a Rua Sarmento Leite 245, 3rd Floor, 90050-170 Porto Alegre, RS, Brazil Accepted 24 August 2004
KEYWORDS Saliva; Antidepressants drugs; Hyposalivation
Summary In this study, effects on both stimulated and non-stimulated salivary flow as well as salivary components of different antidepressant drugs were compared. Rats received imipramine (IMI; 10 mg/ml), fluoxetine (FLU; 20 mg/ml) or moclobemide (MOC; 30 mg/ml) by gavage. The drugs were administered 24, 5 and 1 h before saliva collection (sub-acute treatment) or as a once a day treatment for 14 days (chronic treatment). Animals were sedated with thiopental and saliva was collected using preweighed cotton balls inserted in the mouth for 1 min before and after pilocarpine stimulus. Pilocarpine-stimulated saliva was also collected for biochemical assays of total proteins, amylase, phosphate and calcium, performed through automated colorimetric methods. Non-stimulated salivary flow was decreased by sub-acute IMI 10 mg/kg treatment. Pilocarpine-stimulated salivary flow was significantly increased by acute treatments with IMI, FLU and MOC in comparison to the control group. The same opposite pattern of effects on non-stimulated and pilocarpinestimulated salivation was seen after chronic treatment with the antidepressants. Increased levels of calcium following sub-acute treatment with IMI and after prolonged treatment with FLU and MOC were detected. In the assayed samples, phosphate was found to be increased following chronic treatment with FLU or MOC. These results may explain the discrepant effects of the antidepressants on salivation described in pre-clinical and clinical studies. # 2004 Elsevier Ltd. All rights reserved.
Saliva and its components are indispensable for maintenance of oral health.1 Many drugs affect the output or composition of saliva.2 Psychoactive agents are among the most probable to induce * Corresponding author. Tel.: +55 51 32248822x129; fax: +55 51 32248822x129. E-mail address: [email protected] (Helena M.T. Barros).
salivary output changes. Reduction in measured salivary output is referred to as hyposalivation and may be perceived by the patients as xerostomia, while increased salivary output, hypersalivation, may be described as sialorrhea.3 Antidepressants are the most frequently prescribed psychoactive substances, nowadays, and as much as 30—60% of the patients taking these medications refer xerostomia as an important side effect. Reduction of
0003–9969/$ — see front matter # 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.archoralbio.2004.08.006
18
salivary flow may vary between patients who receive different antidepressant classes. Those with xerostomia present 30—60% reduction of salivary flow.4—8 The xerostomic effect of antidepressants is related to reduction of stimulated salivary flow7 or to basal salivation.8 In experimental studies with laboratory animals, there is a large variability in saliva flow rates after antidepressant treatments, according to the type of antidepressant agent used, the method of evaluation (non-stimulated or stimulated saliva collection) or by considering wholesaliva or parotid flow.9 In pre-clinical assays, non-stimulated or stimulated whole-saliva or individual gland flow are usually performed in rats through dissection of the parotid, sub-mandibular, and sublingual glands,10—12 after ligation of salivary gland duct13 or by its cannulation.14—19 For all of these methods, invasive surgical procedures are required. There are also other non-surgical techniques for saliva collection, which often require tracheobronchial intubation of the animals.20 Therefore, besides being invasive many procedures cause destruction or irreversible damage to the tissues. As in the clinical studies,21 a non-invasive sialometric method in experimental laboratory uses cotton balls placed on the floor of the mouth. The salivary flow in rats may be determined through the difference in weight of the cotton before and after collection from either non-stimulated or stimulated salivary flow. However, it can only be used for salivary flow determination as the amount collected is not enough for analysis. Another non-invasive method for saliva collection in rats was proposed, in which anaesthetized animals, receiving a sialogogue agent are placed heads-down onto a downward-sloped bench and saliva flows into flasks. This method allows a high amount of saliva collection for analysis of its constituents.22 One concern about this method is related to a possible interaction between the sialogogue agent and the antidepressant drug. Because of the great variability of effects of antidepressant drugs in clinical research, our aim was to analyze and compare the effects on nonstimulated and stimulated salivary flow of different antidepressant drugs and its effects on salivary components, using a pre-clinical model. Male Wistar rats (n = 40) were maintained in groups of five animals per cage (polyethylene 50 cm 36 cm 18 cm). The animals were kept at room temperature of 22 2 8C and light cycle from 7 a.m. to 7 p.m., receiving rat chow (Nutrilab1, Brazil) and water ad libitum. The experimental procedures comply with national and international guidelines for the use of animals.
L. Kopittke et al.
The Ethics Committee for Animal Research of FFFCMPA approved the research. The animals were divided into four treatment groups, 10 animals each. Saline, imipramine (IMI; Tofranil1, Biogale ˆnica) 10 mg/ml, fluoxetine (FLU; Prozac1, Elli Lilly) 20 mg/ml or moclobemide (MOC; Aurorix1, Roche) 30 mg/ml, suspended in saline. All solutions were administered by oral gavage, 1 ml/ kg. These doses were chosen because they show anti-immobility action in rats submitted to the forced-swimming test commonly used in antidepressant agents’ trials. To follow the same administration schedule used in antidepressant screening trials with the forced-swimming test27, drugs were administered 24, 5 and 1 h before saliva collection. Following the sub-acute salivation test, the animals received treatment with the same drugs once a day, for 14 days, until the next salivation testing. The same intensive schedule of drug administration was given on the 15th day, before chronic salivation testing. On the day of the experiments, 0.5 cm cotton balls were prepared and individually placed into previously labelled closed glass flasks, which were weighed in an analytic electronic scale (Sartorius 2662). For each animal, two such sets were prepared, one for non-stimulated salivation and another for pilocarpine-stimulated salivation measurements. An hour after the last antidepressant dose in subacute administration, animals were sedated with thiopental 30 mg/kg, intraperitoneally, and kept in lateral decubitus. The first cotton ball was inserted under the rat’s tongue for 1 min. The cotton was then returned to its flask and weighed. Seventy-five minutes after the last treatment dosing, pilocarpine 0.1 mg/kg (Merck1, Brazil) was administered subcutaneously, and after 10 min the procedure of saliva collection with the cotton was repeated. The dose of pilocarpine was selected in a pilot study and corresponds to the maximum sialogogue dose with minimum systemic toxicity for the rats. Twenty minutes after pilocarpine administration, the animals were placed heads-down on a 20 cm deep acrylic bench divided into four 12 cm (wide) sections, separated by 4 cm (high) divisions. The height of the bench in the anterior sides is 8 cm and the posterior side is inclined upward in 88. With the animals’ heads lower than their bodies the saliva drops fell directly into the collecting flasks, placed in front of the bench (modified from Bernarde et al.22). The saliva collected in the flasks was stored at 30 8C until biochemical assays. Quantitative determination of total proteins, amylase, phos-
Opposite effects of antidepressants
Figure 1 Unstimulated salivary flow (A) and pilocarpinestimulated salivary flow (B) of rats treated with antidepressants: imipramine (IMI; 10 mg/kg), fluoxetine (FLU; 20 mg/kg), moclobemide (MOC; 30 mg/kg) or saline (SAL). Sub-acute treatment followed the classical administration schedule used in tests to screen for antidepressant effects (i.e. 24, 5 and 1 h before the test) and chronic treatment was given for 15 days, one dose daily. Results are mean S.E.M. (n = 5—10 in each group). The asterisk denotes difference from the respective group saline (p < 0.05) and the horizontal curly bracket denotes difference from subacute treatment (p < 0.05).
phate and calcium were performed using automated colorimetric methods (Express Plus1, Bayer). After this first day test, the animals were removed from the bench and placed into their home cages after recovery from anaesthesia. For 14 subsequent days, they were treated with their respective treatments. On the 15th day of treatment, they were submitted to the same above-mentioned procedures for treatment and salivary collection. Salivary flow after sub-acute or prolonged treatment was compared through a one-way ANOVA. Several animals treated with fluoxetine or moclobemide died during the pilocarpine test after prolonged treatment, leaving only 5 out of the 10 animals in each group. Fifty percent of the animals in these two groups died during this last procedure. The amount of saliva and the biochemical values of the survivors were also assessed with two-way
19
ANOVA for repeated measures, considering duration and drug treatment as factors. When appropriate, multiple comparisons were performed with the Student—Newman—Keuls test. The amount of non-stimulated salivary flow and pilocarpine-stimulated salivary flow and the levels of proteins, amylase, calcium, and phosphate are represented as x S.E.M. Differences were considered statistically significant if p < 0.05. When the data were analyzed with two-way repeated measures ANOVA for non-stimulated salivary flow, they showed a decrease in non-stimulated salivary flow by IMI 10 mg/kg treatment (F(3,51) = 3.635, p < 0.05) and a significant decrease in salivation flow due to chronic treatments (F(1,51) = 78.92, p < 0.05), as seen in Fig. 1A. Pilocarpine-stimulated salivary flow increased up to 50 times in comparison to non-stimulated salivation values. Pilocarpine-stimulated salivary flow was significantly increased by sub-acute treatments with IMI 10 mg/kg, FLU 20 mg/kg and MOC 30 mg/kg in comparison to the control group (F(3,51) = 9.553, p < 0.05) as seen in Fig. 1B. When duration of treatments were compared there was a significant decrease in the amount of salivation after prolonged treatment (F(1,51) = 88.778, p < 0.05). No significant interaction between treatments and their duration was seen. The amount of stimulated salivation varied from 20 to 50 ml and was sufficient for biochemical assays. In some occasions, we found ‘‘crystallization’’ of the collected material, precluding biochemical assays. There were no statistical differences between treatments regarding the number of samples that could not be assayed. The sub-acute or chronic treatments with antidepressants did not affect total protein or amylase levels (Table 1). Increased levels of calcium following sub-acute treatment with IMI (p < 0.05) and after prolonged treatment with FLU and MOC were detected (p < 0.05). In the assayed samples, phosphate was found to be increased following chronic treatment with FLU or MOC (p < 0.05). These results agree with other pre-clinical and clinical studies which demonstrate that antidepressant drugs affect salivation flow and change saliva composition24,7 that may explain why patients using these agents refer to xerostomia.23 The tricyclic antidepressants may reduce salivary flow rate by modulating a- and b-adrenergic, and muscarinic— cholinergic neural transmission (as reviewed by Koller et al.24 and Szabadi and Tavernor3). This study also shows that the effects of antidepressants upon salivation vary if the individuals are under the influence of a sialogogue. Increased pilocarpine-induced salivation by antidepressants
20
L. Kopittke et al.
Table 1 Total proteins, amylase, phosphate, and calcium in saliva of rats after sub-acute and chronic use of imipramine (IMI; 10 mg/kg), fluoxetine (FLU; 20 mg/kg), moclobemide (MOC; 30 mg/kg) or saline (SAL). Total proteins (mg/ml) SAL sub-acute (8) SAL chronic (2) IMI sub-acute (3) IMI chronic (1) FLU sub-acute (4) FLU chronic (2) MOC sub-acute (5) MOC chronic (2) Ftreatment (3,51) Ftime (1,51) Fintegration (3,19)
33.75 25.00 33.67 30.00 57.50 50.00 40.00 60.00
1.4 0.005 0.5
65.3 50 176.4 0 154.8 300 83.7 0
Amylase (U/l) 0.120 0.038 0.204 0.125 0.130 0.037 0.232 0.122 0.9 2.6 0.02
38.3 9.6 116.3 0 32.2 2.3 160.4 0.7
Phosphate (mg/mM) 1.3 0.8 2.1 1.5 2.2 3.0 1.8 2.6
4.34 0.1 1.1
0.3 0.6 0.5 0 0.4 0.8** 0.2 0.4**
Calcium (mg/mM) 1.1 1.5 3.0 2.3 1.7 5.0 1.9 5.9
0.2 0.5 0.7** 0 0.4 1.9**,# 0.2 0.3**,#
9.48 17.8 7.2
The values given in parentheses represent the number of samples. Values are mean standard error (Student—Newman—Keuls test, p < 0.05). The ‘‘**’’ denotes difference from the respective group saline and ‘‘#’’ denotes difference from the imipramine. Bold represents significant variation (p < 0.05).
is unexpected, since in comparison to clinical information, patients treated with antidepressants usually complain of dry mouth. The present preclinical data do not support such hypothesis, because all antidepressant agents used increased pilocarpine-induced salivation. The main difference between the two studies is that the clinical results were obtained with citric acid stimulated salivation, whereas we used pilocarpine to increase salivation. In another laboratory study, the increased pilocarpine-stimulated salivation by desipramine, another tricyclic antidepressant, was tentatively explained by cholinergic and adrenergic feedback mechanisms24 that may also be involved in the effects of MAO inhibitors and selective serotonin reuptake inhibitors. This observation may be clinically useful, since patients with hyposalivation symptoms due to drug treatments would need small doses of pilocarpine to return to a normal salivation level. One could also consider that patients with a higher cholinergic tonus could present sialorhoea instead of hyposalivation when treated with antidepressants. Controlled clinical studies are unquestionably needed to elucidate these matters. The methods herein adapted for saliva collection are simple, fast, and easily performed, without initial invasive procedures. It may also be recommended for repeated assays. Even though the volume of salivation is reduced after a previous test, this reduction does not seem to compromise the results of subsequent tests. The other asset of the method is that it allows saliva collection for biochemical assays. In this case, there might also be different effects on salivary composition whether it has been stimulated or not. Other studies of toxicological nature should be performed to elucidate
the high mortality rate after combined treatment with antidepressants and pilocarpine. It has been seen that antidepressant agents increase basal non-stimulated salivary total protein concentrations and decreases amilase levels25 while we could not replicate such finding. It is possible that because pilocarpine acts upon the cholinergic system it increases the water content of saliva8 and subtle drug effects on salivary content may not be evident. The only changes in saliva content induced by antidepressant agents are in calcium and phosphate, inducing higher levels of these ions. This change might be protective towards teeth demineralization.26 In short, from this experimental study, it is possible to conclude that the antidepressant agent IMI decreases salivation, while fluoxetine and moclobemide only induce tendency to decrease salivation. The overall picture mirrors the clinical data, since much more patients receiving IMI complain of xerostomia in comparison to patients who use the SSRI or IMAO agents. On the other hand, there is a potentiation type interaction between pilocarpine and the antidepressants that increases salivation. In the clinical setting, this interaction could be represented by a much higher increase in pilocarpineinduced salivation in antidepressant-induced xerostomic patients. An important question in connection to this would be to verify if there are increased cardiovascular, gastrointestinal or respiratory adverse effects due to the combination of treatments for depression and for hyposalivation induced by the antidepressant agent used. Pre-clinical observations of effects by antidepressants in stimulated and non-stimulated salivation should be further evaluated to determine their relevance from a clinical standpoint.
Opposite effects of antidepressants
References 1. Mandel ID. Sialochemistry in diseases and clinical situations affecting salivary glands. Crit Rev Clin Lab Sci 1980;12: 321—66. 2. Lindhe J. Tratado de periodontologia clı´nica. Rio de Janeiro: Guanabara Koogan; 1988. 3. Szabadi E, Tavernor S. Hypo- and hypersalivation induced by psychoactive drugs: Incidence, mechanisms and therapeutic implications. CNS Drugs 1999;11:449—66. 4. Yu JH, Chen YY, Suarez K. Acute amitriptyline effects on parasympathetic-evoked rat saliva. Neuropsychobiology 1989;20:132—5. 5. Mo ¨rnstad H, Von Knorring L, Forsgren L, Holmgren S. Longterm effects of two principally different antidepressant drugs on saliva secretion and composition. Scand J Dent Res 1986; 94:461—70. 6. Mo ¨rnstad H, Von Knorring L, Forsgren L, Holmgren S. Acute effects of some different antidepressant drugs on saliva composition. Neuropsychobiology 1986;15:73—9. 7. Hunter KD, Wilson WS. The effects of antidepressant drugs on salivary flow and content of sodium and potassium ions in human parotid saliva. Arch Oral Biol 1995;40:983—9. 8. Peeters FPML, deVries MW, Vissink DDS. Risks for oral health with the use of antidepressants. Gen Hosp Psychiatry 1998; 20:150—4. 9. Wang SL, Zhao ZT, Li J, Zhu XZ, Dong H, Zhang YG. Investigation of the clinical value of total saliva flow rates. Arch Oral Biol 1998;43:39—43. 10. Schneyer CA, Schneyer LH. Electrolyte levels of rat salivary secretions. Proc Soc Exp Biol Med 1959;101:568—9. 11. Hooloway PJ, Williams RAD. A study of the oral secretions of rats stimulated by pilocarpine. Arch Oral Biol 1965;10: 237—44. 12. Mangos JA, Braum G. Excretion of total solute, sodium and ¨gers Arch potassium in the saliva of the rat parotid gland. Pflu Gen Physiol 1966;290:184—92. 13. Carlsson GE, Hugoson A, Persoon G. Dental abrasion and alveolar bone loss in the white rat: V. Calcium ion concentration in saliva after selective desalivation. Odontol Rev 1968;19:311—6.
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14. Drum DE. Simple technique for direct cannulation of rat salivary ducts. J Dent Res 1963;42:892. 15. Yoshida Y, Sprecher RL, Schneyer CA, Schneyer LH. Role of beta-receptors in sympathetic regulation of electrolytes in rat submaxillary saliva. Proc Soc Exp Biol Med 1967;126: 912—6. 16. Ulmansky M, Sela J, Dishon T, Rosenmann E, Boss JH. A technique for the intubation of the parotid duct in rats. Arch Oral Biol 1972;17:609—12. 17. Omnell K, Qwarntro ¨m EE. A technique for intraoral cannulation and infusion of the rat submandibular gland. Dentomaxillofac Radiol 1983;12:13—5. 18. Dreisbach RH. Secretion of calcium by rat submandibular gland. Am J Physiol 1959;196:645—8. 19. Wolf RO, Kakehashi S. Rat parotid saliva collection technique. J Dent Res 1966;45:979. 20. Vissink A, Gravenmade EJ, Konings AWT, Ligeon EE. An adaptation of the lashley cup for use in rat saliva collection. Arch Oral Biol 1989;34:577—8. 21. Bolwig TG, Rafaelsen OJ. Salivation in affective disorders. Physiol Med 1972;2:232—8. 22. Bernarde MA, Fabian FW, Rosen S, Hoppert CA, Hunt HR. A method for the collection of large quantities of rat saliva. Dent Res 1956;35:326—7. 23. Leipzig RM. Gastrointestinal and hepatic effects of psychotropic drugs. In: Kane JM, Lieberman JA, editors. Adverse effects of psychotropic drugs. New York: The Guilford Press; 1992. 24. Koller MM, Purushotham KR, Maeda N, Scarpace PJ, Humphreys-Beher MG. Desipramine induced changes in salivary proteins, cultivate oral microbiota and gingival health in aging female NIA Fisher 344 rats. Life Sci 2000;68:445—55. 25. Abe K, Yokota Y, Dawes C. Effects of parasympathomimetic and sympathomimetic drugs on the secretion and composition of rat sublingual saliva. J Dent Res 1982;61:52—6. 26. Bardow A, Nyvad B, Nauntofte B. Relationships between medication intake, complaints of dry mouth, salivary flow rate and composition, and the rate of tooth demineralization in situ. Arch Oral Biol 2001;46:413—23. 27. Porsolt RD, Le Pichon M, Jalfre M. Depression: a new animal model sensitive to antidepressant treatments. Nature 1977; 266(5604):730—2.
www.intl.elsevierhealth.com/journals/arob
Opposite effects of antidepressants on unstimulated and stimulated salivary flow Luciane Kopittke, Rosane Gomez, Helena M.T. Barros* ˜o Faculdade Federal de Cie ˆncias Me ´dicas de Porto Alegre (FFFCMPA), Division of Pharmacology, Fundac¸a Rua Sarmento Leite 245, 3rd Floor, 90050-170 Porto Alegre, RS, Brazil Accepted 24 August 2004
KEYWORDS Saliva; Antidepressants drugs; Hyposalivation
Summary In this study, effects on both stimulated and non-stimulated salivary flow as well as salivary components of different antidepressant drugs were compared. Rats received imipramine (IMI; 10 mg/ml), fluoxetine (FLU; 20 mg/ml) or moclobemide (MOC; 30 mg/ml) by gavage. The drugs were administered 24, 5 and 1 h before saliva collection (sub-acute treatment) or as a once a day treatment for 14 days (chronic treatment). Animals were sedated with thiopental and saliva was collected using preweighed cotton balls inserted in the mouth for 1 min before and after pilocarpine stimulus. Pilocarpine-stimulated saliva was also collected for biochemical assays of total proteins, amylase, phosphate and calcium, performed through automated colorimetric methods. Non-stimulated salivary flow was decreased by sub-acute IMI 10 mg/kg treatment. Pilocarpine-stimulated salivary flow was significantly increased by acute treatments with IMI, FLU and MOC in comparison to the control group. The same opposite pattern of effects on non-stimulated and pilocarpinestimulated salivation was seen after chronic treatment with the antidepressants. Increased levels of calcium following sub-acute treatment with IMI and after prolonged treatment with FLU and MOC were detected. In the assayed samples, phosphate was found to be increased following chronic treatment with FLU or MOC. These results may explain the discrepant effects of the antidepressants on salivation described in pre-clinical and clinical studies. # 2004 Elsevier Ltd. All rights reserved.
Saliva and its components are indispensable for maintenance of oral health.1 Many drugs affect the output or composition of saliva.2 Psychoactive agents are among the most probable to induce * Corresponding author. Tel.: +55 51 32248822x129; fax: +55 51 32248822x129. E-mail address: [email protected] (Helena M.T. Barros).
salivary output changes. Reduction in measured salivary output is referred to as hyposalivation and may be perceived by the patients as xerostomia, while increased salivary output, hypersalivation, may be described as sialorrhea.3 Antidepressants are the most frequently prescribed psychoactive substances, nowadays, and as much as 30—60% of the patients taking these medications refer xerostomia as an important side effect. Reduction of
0003–9969/$ — see front matter # 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.archoralbio.2004.08.006
18
salivary flow may vary between patients who receive different antidepressant classes. Those with xerostomia present 30—60% reduction of salivary flow.4—8 The xerostomic effect of antidepressants is related to reduction of stimulated salivary flow7 or to basal salivation.8 In experimental studies with laboratory animals, there is a large variability in saliva flow rates after antidepressant treatments, according to the type of antidepressant agent used, the method of evaluation (non-stimulated or stimulated saliva collection) or by considering wholesaliva or parotid flow.9 In pre-clinical assays, non-stimulated or stimulated whole-saliva or individual gland flow are usually performed in rats through dissection of the parotid, sub-mandibular, and sublingual glands,10—12 after ligation of salivary gland duct13 or by its cannulation.14—19 For all of these methods, invasive surgical procedures are required. There are also other non-surgical techniques for saliva collection, which often require tracheobronchial intubation of the animals.20 Therefore, besides being invasive many procedures cause destruction or irreversible damage to the tissues. As in the clinical studies,21 a non-invasive sialometric method in experimental laboratory uses cotton balls placed on the floor of the mouth. The salivary flow in rats may be determined through the difference in weight of the cotton before and after collection from either non-stimulated or stimulated salivary flow. However, it can only be used for salivary flow determination as the amount collected is not enough for analysis. Another non-invasive method for saliva collection in rats was proposed, in which anaesthetized animals, receiving a sialogogue agent are placed heads-down onto a downward-sloped bench and saliva flows into flasks. This method allows a high amount of saliva collection for analysis of its constituents.22 One concern about this method is related to a possible interaction between the sialogogue agent and the antidepressant drug. Because of the great variability of effects of antidepressant drugs in clinical research, our aim was to analyze and compare the effects on nonstimulated and stimulated salivary flow of different antidepressant drugs and its effects on salivary components, using a pre-clinical model. Male Wistar rats (n = 40) were maintained in groups of five animals per cage (polyethylene 50 cm 36 cm 18 cm). The animals were kept at room temperature of 22 2 8C and light cycle from 7 a.m. to 7 p.m., receiving rat chow (Nutrilab1, Brazil) and water ad libitum. The experimental procedures comply with national and international guidelines for the use of animals.
L. Kopittke et al.
The Ethics Committee for Animal Research of FFFCMPA approved the research. The animals were divided into four treatment groups, 10 animals each. Saline, imipramine (IMI; Tofranil1, Biogale ˆnica) 10 mg/ml, fluoxetine (FLU; Prozac1, Elli Lilly) 20 mg/ml or moclobemide (MOC; Aurorix1, Roche) 30 mg/ml, suspended in saline. All solutions were administered by oral gavage, 1 ml/ kg. These doses were chosen because they show anti-immobility action in rats submitted to the forced-swimming test commonly used in antidepressant agents’ trials. To follow the same administration schedule used in antidepressant screening trials with the forced-swimming test27, drugs were administered 24, 5 and 1 h before saliva collection. Following the sub-acute salivation test, the animals received treatment with the same drugs once a day, for 14 days, until the next salivation testing. The same intensive schedule of drug administration was given on the 15th day, before chronic salivation testing. On the day of the experiments, 0.5 cm cotton balls were prepared and individually placed into previously labelled closed glass flasks, which were weighed in an analytic electronic scale (Sartorius 2662). For each animal, two such sets were prepared, one for non-stimulated salivation and another for pilocarpine-stimulated salivation measurements. An hour after the last antidepressant dose in subacute administration, animals were sedated with thiopental 30 mg/kg, intraperitoneally, and kept in lateral decubitus. The first cotton ball was inserted under the rat’s tongue for 1 min. The cotton was then returned to its flask and weighed. Seventy-five minutes after the last treatment dosing, pilocarpine 0.1 mg/kg (Merck1, Brazil) was administered subcutaneously, and after 10 min the procedure of saliva collection with the cotton was repeated. The dose of pilocarpine was selected in a pilot study and corresponds to the maximum sialogogue dose with minimum systemic toxicity for the rats. Twenty minutes after pilocarpine administration, the animals were placed heads-down on a 20 cm deep acrylic bench divided into four 12 cm (wide) sections, separated by 4 cm (high) divisions. The height of the bench in the anterior sides is 8 cm and the posterior side is inclined upward in 88. With the animals’ heads lower than their bodies the saliva drops fell directly into the collecting flasks, placed in front of the bench (modified from Bernarde et al.22). The saliva collected in the flasks was stored at 30 8C until biochemical assays. Quantitative determination of total proteins, amylase, phos-
Opposite effects of antidepressants
Figure 1 Unstimulated salivary flow (A) and pilocarpinestimulated salivary flow (B) of rats treated with antidepressants: imipramine (IMI; 10 mg/kg), fluoxetine (FLU; 20 mg/kg), moclobemide (MOC; 30 mg/kg) or saline (SAL). Sub-acute treatment followed the classical administration schedule used in tests to screen for antidepressant effects (i.e. 24, 5 and 1 h before the test) and chronic treatment was given for 15 days, one dose daily. Results are mean S.E.M. (n = 5—10 in each group). The asterisk denotes difference from the respective group saline (p < 0.05) and the horizontal curly bracket denotes difference from subacute treatment (p < 0.05).
phate and calcium were performed using automated colorimetric methods (Express Plus1, Bayer). After this first day test, the animals were removed from the bench and placed into their home cages after recovery from anaesthesia. For 14 subsequent days, they were treated with their respective treatments. On the 15th day of treatment, they were submitted to the same above-mentioned procedures for treatment and salivary collection. Salivary flow after sub-acute or prolonged treatment was compared through a one-way ANOVA. Several animals treated with fluoxetine or moclobemide died during the pilocarpine test after prolonged treatment, leaving only 5 out of the 10 animals in each group. Fifty percent of the animals in these two groups died during this last procedure. The amount of saliva and the biochemical values of the survivors were also assessed with two-way
19
ANOVA for repeated measures, considering duration and drug treatment as factors. When appropriate, multiple comparisons were performed with the Student—Newman—Keuls test. The amount of non-stimulated salivary flow and pilocarpine-stimulated salivary flow and the levels of proteins, amylase, calcium, and phosphate are represented as x S.E.M. Differences were considered statistically significant if p < 0.05. When the data were analyzed with two-way repeated measures ANOVA for non-stimulated salivary flow, they showed a decrease in non-stimulated salivary flow by IMI 10 mg/kg treatment (F(3,51) = 3.635, p < 0.05) and a significant decrease in salivation flow due to chronic treatments (F(1,51) = 78.92, p < 0.05), as seen in Fig. 1A. Pilocarpine-stimulated salivary flow increased up to 50 times in comparison to non-stimulated salivation values. Pilocarpine-stimulated salivary flow was significantly increased by sub-acute treatments with IMI 10 mg/kg, FLU 20 mg/kg and MOC 30 mg/kg in comparison to the control group (F(3,51) = 9.553, p < 0.05) as seen in Fig. 1B. When duration of treatments were compared there was a significant decrease in the amount of salivation after prolonged treatment (F(1,51) = 88.778, p < 0.05). No significant interaction between treatments and their duration was seen. The amount of stimulated salivation varied from 20 to 50 ml and was sufficient for biochemical assays. In some occasions, we found ‘‘crystallization’’ of the collected material, precluding biochemical assays. There were no statistical differences between treatments regarding the number of samples that could not be assayed. The sub-acute or chronic treatments with antidepressants did not affect total protein or amylase levels (Table 1). Increased levels of calcium following sub-acute treatment with IMI (p < 0.05) and after prolonged treatment with FLU and MOC were detected (p < 0.05). In the assayed samples, phosphate was found to be increased following chronic treatment with FLU or MOC (p < 0.05). These results agree with other pre-clinical and clinical studies which demonstrate that antidepressant drugs affect salivation flow and change saliva composition24,7 that may explain why patients using these agents refer to xerostomia.23 The tricyclic antidepressants may reduce salivary flow rate by modulating a- and b-adrenergic, and muscarinic— cholinergic neural transmission (as reviewed by Koller et al.24 and Szabadi and Tavernor3). This study also shows that the effects of antidepressants upon salivation vary if the individuals are under the influence of a sialogogue. Increased pilocarpine-induced salivation by antidepressants
20
L. Kopittke et al.
Table 1 Total proteins, amylase, phosphate, and calcium in saliva of rats after sub-acute and chronic use of imipramine (IMI; 10 mg/kg), fluoxetine (FLU; 20 mg/kg), moclobemide (MOC; 30 mg/kg) or saline (SAL). Total proteins (mg/ml) SAL sub-acute (8) SAL chronic (2) IMI sub-acute (3) IMI chronic (1) FLU sub-acute (4) FLU chronic (2) MOC sub-acute (5) MOC chronic (2) Ftreatment (3,51) Ftime (1,51) Fintegration (3,19)
33.75 25.00 33.67 30.00 57.50 50.00 40.00 60.00
1.4 0.005 0.5
65.3 50 176.4 0 154.8 300 83.7 0
Amylase (U/l) 0.120 0.038 0.204 0.125 0.130 0.037 0.232 0.122 0.9 2.6 0.02
38.3 9.6 116.3 0 32.2 2.3 160.4 0.7
Phosphate (mg/mM) 1.3 0.8 2.1 1.5 2.2 3.0 1.8 2.6
4.34 0.1 1.1
0.3 0.6 0.5 0 0.4 0.8** 0.2 0.4**
Calcium (mg/mM) 1.1 1.5 3.0 2.3 1.7 5.0 1.9 5.9
0.2 0.5 0.7** 0 0.4 1.9**,# 0.2 0.3**,#
9.48 17.8 7.2
The values given in parentheses represent the number of samples. Values are mean standard error (Student—Newman—Keuls test, p < 0.05). The ‘‘**’’ denotes difference from the respective group saline and ‘‘#’’ denotes difference from the imipramine. Bold represents significant variation (p < 0.05).
is unexpected, since in comparison to clinical information, patients treated with antidepressants usually complain of dry mouth. The present preclinical data do not support such hypothesis, because all antidepressant agents used increased pilocarpine-induced salivation. The main difference between the two studies is that the clinical results were obtained with citric acid stimulated salivation, whereas we used pilocarpine to increase salivation. In another laboratory study, the increased pilocarpine-stimulated salivation by desipramine, another tricyclic antidepressant, was tentatively explained by cholinergic and adrenergic feedback mechanisms24 that may also be involved in the effects of MAO inhibitors and selective serotonin reuptake inhibitors. This observation may be clinically useful, since patients with hyposalivation symptoms due to drug treatments would need small doses of pilocarpine to return to a normal salivation level. One could also consider that patients with a higher cholinergic tonus could present sialorhoea instead of hyposalivation when treated with antidepressants. Controlled clinical studies are unquestionably needed to elucidate these matters. The methods herein adapted for saliva collection are simple, fast, and easily performed, without initial invasive procedures. It may also be recommended for repeated assays. Even though the volume of salivation is reduced after a previous test, this reduction does not seem to compromise the results of subsequent tests. The other asset of the method is that it allows saliva collection for biochemical assays. In this case, there might also be different effects on salivary composition whether it has been stimulated or not. Other studies of toxicological nature should be performed to elucidate
the high mortality rate after combined treatment with antidepressants and pilocarpine. It has been seen that antidepressant agents increase basal non-stimulated salivary total protein concentrations and decreases amilase levels25 while we could not replicate such finding. It is possible that because pilocarpine acts upon the cholinergic system it increases the water content of saliva8 and subtle drug effects on salivary content may not be evident. The only changes in saliva content induced by antidepressant agents are in calcium and phosphate, inducing higher levels of these ions. This change might be protective towards teeth demineralization.26 In short, from this experimental study, it is possible to conclude that the antidepressant agent IMI decreases salivation, while fluoxetine and moclobemide only induce tendency to decrease salivation. The overall picture mirrors the clinical data, since much more patients receiving IMI complain of xerostomia in comparison to patients who use the SSRI or IMAO agents. On the other hand, there is a potentiation type interaction between pilocarpine and the antidepressants that increases salivation. In the clinical setting, this interaction could be represented by a much higher increase in pilocarpineinduced salivation in antidepressant-induced xerostomic patients. An important question in connection to this would be to verify if there are increased cardiovascular, gastrointestinal or respiratory adverse effects due to the combination of treatments for depression and for hyposalivation induced by the antidepressant agent used. Pre-clinical observations of effects by antidepressants in stimulated and non-stimulated salivation should be further evaluated to determine their relevance from a clinical standpoint.
Opposite effects of antidepressants
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