INTRODUCTION

Chemical dependency, which in- cludes both alcoholism and drug addiction, is a primary illness char- acterized by the dependence to a mood-altering chemical (Kalivas & Volkow, 2005). A chemically depen- dent person is not able to stop drink- ing or using a drug despite serious social and health consequences.

It is possible that chemically depen- dent subjects may present relevant changes in the volume and compo- sition of the salivary fluid because the secretion of the salivary glands is controlled by the parasympa- thetic and sympathetic systems, simultaneously (Carpenter, 2013; Proctor & Carpenter, 2007) which can be affected by alcohol, drugs or both (Aps & Martens, 2005; Enberg, Alho, Loimaranta, & Lenander-Lu- mikari, 2001; Newlin, 1995). Those changes can be responsible for the disturbances on the integrity of the enamel, periodontal tissues and oral mucosa (Antoniazzi et al., 2018; Cho, Hirsch, & Johnstone, 2005; da Fonseca, 2009; Rawal, Tatakis,

&Tipton, 2012; Reddy et al., 2012; Sordi, Massochin, Camargo, Lemos,

&Munhoz, 2017).

Some studies have shown that sali- vary constituents, such as proteins, calcium, phosphate, potassium, bicarbonate, IgA, lisozyme and lactoferrin may undergo changes in their concentrations due to the abuse of alcohol (Enberg et al., 2001; Waszkiewicz et al., 2017; Waszkiewicz, Zalewska-Szajda, Zalewska, Waszkiewicz, Szajda, Repka, Szulc, Kpka, et al., 2012; Waszkiewicz, Zalewska-Szajda, Za- lewska, Waszkiewicz, Szajda, Rep- ka, Szulc, Kepka, et al., 2012; Wasz- kiewicz, Zalewska, Szajda, Szulc, et al., 2012; Waszkiewicz, Zalewska, Szajda, Waszkiewicz, et al., 2012).

Nevertheless, there are no studies evaluating if the salivary composi-

tion of patients with an alcohol or illicit drug dependency is altered. The objective of this study was to compare the salivary concentra- tion of proteins, amylase, urea, calcium, phosphate and flow rate between chemically dependent and non-chemically dependent subjects.

MATERIALS AND METHODS

Ethical approval

The study´s protocol was approved by the Research Ethics Committee of the Federal University of Paraná, Brazil (Approval number: 84071). All the participants received de- tailed information concerning the nature and the procedures in- volved in the study and signed in- formed consent forms.

Subject´s selection

Twenty-seven volunteers, males, aged between 18 and 50 years old, with alcohol dependence and drug addiction, attending the Institute for Research and Treatment of Alcohol (Campo Largo, Paraná, Brazil) were recruited. All the par- ticipants were going to begin the detoxification program.

The control group consisted of 27 healthy males, 18-50 years of age, recruited from the Police Academy of Curitiba, Paraná, Brazil. These participants did not have alcohol dependence nor reported the use of drugs. Only healthy volunteers were accepted for this group, and individuals with any regular medi- cation, substance-related addic- tion or illness were excluded from the study.

Saliva Collection

Saliva samples, from the chemi- cally dependent group, were taken one day before the detoxification treatment started.

Stimulated saliva was collected be- tween 09:00 A.M. and 11:00 A.M. in a quiet room, free from external in- terferences. The participants were previously instructed to refrain from eating, drinking, or cleaning their teeth for 2 hours before the collec- tion process. The saliva sample was obtained with the use of paraffin film in order to perform a five-minute stimulation. Then, they spitted their saliva into a sterile container (Sterile Universal Collector - J.PROLAB 80 ml). The sample volumes were mea- sured gravimetrically according to the method of Banderas-Tarabay et al., (1997) using a precision balance (MARTE AM200, Santa Rita do Sa- pucaí/MG, Brazil) and the samples were immediately frozen at -20 °C until further analysis. All the samples were processed within 7 to 10 days.

Biochemical Analysis

All the samples were centrifuged at 2,600g for 10 min at 4°C to remove cellular and food debris and none of them were contaminated with blood. Calcium and phosphate concentrations were determined by colorimetric testing. (Calcio Liquiform, Labtest diagnostica, Lagoa Santa/MG, Brazil; Fósforo, Labtest diagnostica, Lagoa Santa/ MG, Brazil). Determination of pro- tein concentration was carried out using Coomassie blue with bovine serum albumin as the standard. Urea and amylase concentrations were analyzed using enzymatic colorimetric test kits (Urea UV Liquiform, Labtest diagnostica, La- goa Santa/MG, Brazil and Amilase, Labtest diagnostica, Lagoa Santa/ MG, Brazil). All biochemical analy- ses were done 3 times for each sali- va sample using a spectrophotom- eter (S-2000 UV - VIS, SP, Brazil).

Statistical analysis

The results were expressed as mean ± standard deviation. Sta- tistical analysis was undertaken

Table 1. Age, alcohol and illicit drugs use

(quantity and duration) by groups

Table 2. Comparison of the salivary variables between the groups.

using Student’s t test for indepen- dent samples. A p-value <0.05 was accepted to be statistically signifi- cant. Statistical analysis was per- formed with Statistical Package for the Social Sciences for Windows (SPSS, version 20.0, SPSS Inc., Chi- cago, IL, USA).

RESULTS

The mean age of the chemically dependent group was 37.56 ± 10.70 years, while the control group showed 38.15 ± 11.12 years. None of the participants of the control group reported the use of alcohol or illicit drugs. On the other hand, all subjects in the addiction group confirmed using alcohol and co- caine. Also, 48% of these partici- pants used to smoke cocaine or cannabis. The quantity and dura- tion of the consumption are pre- sented in Table 1.

Salivary flow and urea concen- tration did not show statistically significant differences between groups, however, total proteins, amylase, calcium and phosphate concentration were higher on the chemically dependent group. Ta- ble 2 shows the comparison of the analyzed variables.

DISCUSSION

The results of the study show alter- ations of the salivary composition among the chemically dependent subjects. Several studies have re- ported the dangerous character of alcohol and illicit drugs, as well as their physiological consequences in short and long terms (Cho et al., 2005; Gossop, Manning, & Ridge, 2006; Pateria, de Boer, & MacQuil- lan, 2013; Singh et al., 2017). On the other hand, there are no studies evaluating the salivary composi- tion on chemically dependent sub- jects, or they were not found dur- ing the literature review made for this research.

Alcohol acts as a central nervous system (CNS) depressant and it´s effects are potentially deleterious and irreversible to the CNS (Ron & Barak, 2016). There is an increase in the synthesis and release of noradrenaline through the blood vessels (Koob, 1992). The salivary fluid becomes thicker due to the sympathetic adrenergic stimulus by increasing the effects of sympa- thetic nervous activity (Carpenter, 2013). Consequently, saliva may present lower fluidity and higher secretion of proteins and calcium, as observed in the study.

Cannabis also affects the CNS in a similar way to alcohol. It has depres- sant and psychomimetic effects. Muscarinic receptors, from acetyl- choline, are coupled to G protein and produce excitatory and stimu- latory effects of salivary gland se- cretion, known as parasympathetic stimulation (Proctor & Carpenter, 2014). The abusive use of canna- bis results in anticholinergic activ- ity such as a blockage of the effects of acetylcholine on muscarinic M3 receptors (Ralevic, 2003), that may decrease salivary secretion. There- fore, this constant sympathetic stimulation leads to the production of viscous saliva, low in quantity and but rich in proteins and inorganic electrolytes (Aps & Martens, 2005). This may also explain the difference found between groups, in the total protein and calcium concentration.

On the other hand, cocaine (crack) is a sympathomimetic drug of in- direct action, since it blocks the transport of noradrenaline, sero- tonin and dopamine in the syn- aptic cleft, occurring prolongation of the user’s euphoric sensation (Dackis & O’Brien, 2001). It inhib- its the capture of catecholamines by noradrenaline and dopamine transporters to noradrenergic nerve terminals, intensifying the effects of sympathetic nervous ac- tivity (Riezzo et al., 2012). These

may induce changes in the saliva flow rate. While some authors re- ported that saliva flow does not change (Woyceichoski et al., 2013), but others had found a significant association between the use of crack and a reduced salivary flow (Antoniazzi et al., 2018). In the present study we did not see any difference on stimulated salivary flow between groups.

Stimulated saliva provides infor- mation about the secretory capac- ity of the salivary glands. The sali- vary composition is influenced by the taste and mechanical stimulus (Carpenter, 2013). The acid stimu- lus may interfere with the buffer capacity and cause precipitation of certain salivary proteins and calcium (Dawes, 1984). In order to avoid alterations in the salivary composition in this study it was used only a mechanical stimula- tion with insipid wax.

The significant increase of total proteins, amylase, calcium and phosphate found in the saliva of the chemically dependent group may be explained by the control of the central and autonomic nervous system in the salivary glands (Proc- tor & Carpenter, 2014). Secretion of the salivary glands is qualitatively and quantitatively modified in the presence of sympathetic and para- sympathetic nervous stimulus (Aps

&Martens, 2005) and both, alcohol and illicit drugs influence the re- sponse of the sympathetic system (Koob, 1992; Magura & Rosen- blum, 2000; Olière, Joliette-Riopel, Potvin, & Jutras-Aswad, 2013).

The salivary parameters that pre- sented statistical differences (to- tal proteins, amylase, calcium and phosphate) are related to the au- tonomic nervous mechanism in the salivary secretion (Proctor & Carpenter, 2014). The nerve fibers of the sympathetic system release noradrenalin that binds α and β

adrenergic receptors. β -receptors, whose second messenger is cyclic AMP (cyclic adenosine monophos- phate) stimulates the precipitation of enzymes and proteins. There is an increase in permeability of the membranes of acinar cells, which contain zymogen granules that stores proteins that are liberated in high amounts (Castle & Castle, 1998; Proctor, 2016; Turner & Sugi- ya, 2002). That is why β-adrenergic stimulus results in increased pro- tein (Turner & Sugiya, 2002) and salivary phosphate concentration (Beal, 1991).

The α-adrenergic receptors activate P substance, a peptidergic receptor that acts as mediator of the nervous stimulus, present in the acinar cell membrane. This increases the lev- els of calcium and the quantity of salivary, by potentiating the effect of acetylcholine (Aps & Martens, 2005; Proctor, 2016), which can explain why the salivary calcium concen- tration in the chemical dependent group was higher.

Urea is a normal component of saliva and it is passively diffused from blood (Macpherson & Dawes, 1991; Thorn, Prause, & Oxholm, 1989). The results of the biochemi- cal analysis of urea showed no significant difference between the two groups. The urea concentra- tion is dependent on the salivary flow (Thorn et al., 1989) and since there was no variation of the sali- vary flow between the groups, it can be expected that urea concen- tration remained the same.

In this study, all the participants of the chemically dependent group reported that they used cocaine as well as alcohol and sometimes can- nabis. This is an important limita- tion of the study, because it makes very difficult to draw specific con- clusions for each substance and it is impossible to discern if the modifications in saliva composi-

tion are due to the use of alcohol, a specific drug or the combination of all of them. On the other hand, it is extremely difficult to select chemi- cally dependent users who con- sume only one kind of drug.

CONCLUSION

Saliva composition can have an impact in oral health, and it seems to be altered on the chemically de- pendent subjects. Future studies with larger samples and analysis of other salivary constituents should be performed, as well as longitudi- nal follow-up to verify if the chang- es continue in the long term.

Acknowledgments: We thank the Coordination for the Improvement of Higher Level Education Person- nel (CAPES) for the postgraduate scholarship for LL, WB and CEM.

Autors

Luciana Lyraa, William Bellania, Carolina E. Mazura, João A. Brancherb, Antonio Adilson S. de Limaa, José Miguel Amená- bara

a)Stomatology Department, Universida- de Federal do Paraná, Curitiba, Paraná, Brazil.

b)Department of Biological Sciences, Pontifícia Universidade Católica do Para- ná, Curitiba, Paraná, Brazil

Luciana Lyra: lulyra@hotmail.com

William Bellani: lulyra@hotmail.com Carolina E. Mazur: carolmazur6@hotmail.com João A. Brancher: jabrancher@hotmail.com Antonio Adilson S. de Lima: antollima@gmail.com José Miguel Amenábar: jamenaba@gmail.com

Corresponding Author:

José Miguel Amenábar

Posgraduate Program in Dentistry, Uni- versidade Federal do Paraná, Curitiba, Paraná, Brazil Av. Pref. Lothário Meissner, 632 – Jardim Botânico 80210-170 Curitiba, PR. Tel: +55(41) 3360-4024 / Fax: +55(41) 3360-4134. jamenaba@gmail.com

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