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Drug and Alcohol Dependence 133 (2013) 52– 60

Contents lists available at ScienceDirect

Drug and Alcohol Dependence

j ourna l ho me p age: www.elsev ier .com/ locate /drugalcdep

ombined exposure to tobacco smoke and ethanol during adolescenceeads to short- and long-term modulation of anxiety-like behavior��

ael Abreu-Villac aa,∗, Cristiane C. Cavinaa, Anderson Ribeiro-Carvalhob, Moniqueorrea-Santosa, Victor F. Naiff a, Claudio C. Filgueirasa, Alex C. Manhãesa

Laboratório de Neurofisiologia, Departamento de Ciências Fisiológicas, Instituto de Biologia Roberto Alcantara Gomes, Centro Biomédico, Universidade dostado do Rio de Janeiro (UERJ), Av. Prof. Manuel de Abreu 444, 5 andar, Vila Isabel, Rio de Janeiro, RJ 20550-170, BrazilDepartamento de Ciências, Faculdade de Formac ão de Professores da Universidade do Estado do Rio de Janeiro, Rua Dr. Francisco Portela, 1470-Patronato,ão Gonc alo, RJ 24435-005, Brazil

r t i c l e i n f o

rticle history:eceived 17 January 2013eceived in revised form 10 May 2013ccepted 30 May 2013vailable online 27 June 2013

eywords:oodevelopmentobaccolcoholicotinedolescent

a b s t r a c t

Background: Tobacco smoking is associated with alcohol drinking and consumption of both drugs typicallybegins during adolescence. Since anxiety is considered a relevant factor for both smoking and drinkingdue to its motivating force for a continued consumption, anxiety alterations shared by these two drugscould explain their co-use and co-abuse.Methods: Here, we investigated the short- and long-term effects of adolescent tobacco smoke and/orethanol exposure on anxiety levels. From postnatal day 30–45, Swiss mice were exposed to tobaccosmoke (SMK – whole body exposure, 8 h/day) and/or ethanol (ETOH – 25% solution, 2 g/kg i.p. injectedevery other day) as follows: (1) SMK + ETOH exposure; (2) SMK exposure; (3) ETOH exposure; (4) Control.Anxiety levels were assessed with the elevated plus maze and open field tests.Results: By the end of exposure, SMK female mice presented an anxiolytic response in the elevated plusmaze and this response was intensified by co-exposure to ethanol. A short-term deprivation from SMKelicited an anxiogenic state in females in this maze. Although neither smoke nor ethanol effects persistedone month post-exposure, SMK + ETOH male and female mice exhibited an anxiogenic response in the

open field.Conclusion: Adolescent female mice are more susceptible to the anxiolytic effects of SMK. The strongereffect in SMK + ETOH group suggests that, in females, the combined exposure leads to lower anxietylevels. Anxiety levels do not seem to be relevant during a short-term SMK + ETOH deprivation, however,increased anxiety during long-term smoking and drinking deprivation demonstrate late-emergent effectsboth in males and females.

he A

© 2013 T

. Introduction

Both tobacco smoking and alcohol drinking are likely to initiateuring adolescence (CDC, 2010; Doubeni et al., 2010) and epi-emiological findings demonstrate a strong relationship between

moking and drinking (Dawson, 2000; Grant, 1998; Larsson andngel, 2004; Meyerhoff et al., 2006). For instance, early drinkingncreases the likelihood of poly substance use (Ellickson et al., 2003)

�� Supplementary material can be found by accessing the online version of thisaper. See Appendix A for more details.∗ Corresponding author. Tel.: +55 21 2868 8195; fax: +55 21 2860 8029.

E-mail address: yael a v@yahoo.com.br (Y. Abreu-Villac a).

376-8716 © 2013 The Authors. Published by Elsevier Ireland Ltd. ttp://dx.doi.org/10.1016/j.drugalcdep.2013.05.033

Open access under CC BY-NC

uthors. Published by Elsevier Ireland Ltd.

and alcoholism frequency increases when adolescents start smok-ing at a younger age (DiFranza and Guerrera, 1990; Grant, 1998).However, the biological bases of the co-use and co-abuse of tobaccoand alcohol are poorly understood.

There is a close relation between smoking or alcohol drinkingand mood. Tobacco smoking reduces anxiety while increased anx-iety is a symptom of tobacco withdrawal (Hughes et al., 2000).These lead to the suggestion that smokers resort to smoking inorder to modulate their anxiety levels (Gilbert et al., 1989; Picciottoet al., 2002). Regarding ethanol, its increased ingestion by anxiouspatients, a behavior possibly associated with its acute anxiolyticeffects, and the elevated anxiety during early withdrawal that occurin most alcohol-dependent patients have led to the hypothesis

Open access under CC BY-NC-ND license.

that anxiety is involved in the etiology of alcohol consumption (forreview: Heilig et al., 2010). The association between smoking andconsumption of alcoholic beverages could be explained by cumu-lative mood-altering effects of tobacco and ethanol, particularly

-ND license.

Y. Abreu-Villac a et al. / Drug and Alcohol Dependence 133 (2013) 52– 60 53

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was evaluated (Filgueiras et al., 2009). The activity in the center, inversely related tolevels of anxiety (Prut and Belzung, 2003), and assessed as the number of rectanglescrossed in the center corrected by total ambulation Cen/(Cen + Pe), was used as ameasure of anxiety-like behavior. Ethological measures – rearing, grooming and

1 Supplementary material can be found by accessing the online version of thispaper. See Appendix A for more details.

2 Supplementary material can be found by accessing the online version of this

Fig. 1. Time-lin

hose related to anxiety. Moreover, epidemiological studies andxperimental data indicate sex-dependent effects of several drugsf abuse (Dazzi et al., 2007; Lynch, 2006; Lynch and Sofuoglu, 2010;anapat et al., 1999). Despite that, there is scant information aso the consequences of tobacco and alcohol co-consumption onnxiety and on potentially sex-selective effects.

Nicotine has been described as the active component ofigarette smoke responsible for a wide variety of nervous systemffects resulting from tobacco consumption (for review: Fowlert al., 2008; Slotkin, 2002). The majority of studies in experi-ental models of adolescent exposure have specifically assessed

he consequences of nicotine exposure and, more recently, theonsequences of the combined nicotine + ethanol exposure (Abreu-illac a et al., 2007, 2008; Ribeiro-Carvalho et al., 2008, 2009, 2011;rezza et al., 2009). This combined exposure was shown to result iniochemical and behavioral effects, including anxiety-related onesAbreu-Villac a et al., 2008), that were distinct from those observedhen the drugs were used separately, indicating that nicotine and

thanol interact, affecting the functioning of the central nervousystem during this period of development.

Considering that more than 4500 substances have been identi-ed in tobacco smoke and that there is evidence that nonnicotineomponents play an important role in tobacco effects in the centralervous system (Abreu-Villac a et al., 2010; Bruijnzeel, 2012; Rose,006; Villégier et al., 2010), an alternative approach to morelosely investigate the effects of smoking would be to use animalodels of tobacco smoke exposure. Despite that, there are scant

xperimental studies on the effects of tobacco smoke and ethanolo-exposure. This lack of information is particularly disconcertinghen one considers that adolescents that both smoke cigarettes

nd drink alcoholic beverages expose themselves to a considerableumber of substances that are present in the tobacco smoke andhat may interact with nicotine and/or ethanol in affecting theentral nervous system. Accordingly, the aim of the present studyas to investigate the effects of tobacco smoke and ethanol when

dministered separately or in combination on anxiety levels. Basedn evidence that distinct behavioral tests in animal models coulde indexing different emotional aspects of anxiety-like behavior

n rodent (for review: Ramos, 2008), we opted to use two tests inhe present study: the elevated plus maze and the open field. Sincedolescence is described as a key period for initiation of tobaccond ethanol consumption, exposure occurred during this period ofevelopment. The effects of exposure and deprivation on anxiety

evels were investigated.

. Methods

.1. Animals and treatment (Fig. 1)

Experiments were carried out with the approval of the Animal Care and Useommittee of the Universidade do Estado do Rio de Janeiro (CEA/014/2011). Swiss

e experiment.

mice were bred and maintained in our vivarium (21 ± 1 ◦C, lights: on 1:00 – off13:00). Access to food and water was ad libitum. We used litters of 8–12 pups. Atweaning (postnatal day 21 = PN21) animals were separated by sex in groups of 2–5.

During adolescence (PN30 to PN45) (Spear, 2000), mice were exposed totobacco smoke and/or ethanol. Tobacco smoke was generated from referenceresearch cigarettes (University of Kentucky, Lexington, KY, USA) type 2R1F (nico-tine = 1.74 mg/cigt). Whole body exposure was for 8 h/day, from 9:00 to 17:00,7days/week in a chamber that received the smoke generated in an automaticcigarette smoking machine (Teague Enterprises, Davis, CA, USA), as a surrogatefor active smoking (Abreu-Villac a et al., 2010; Slotkin et al., 2001). Control micewere exposed to ambient air (detailed description in Supplementary Material S11).As for ethanol exposure, 25% ethanol (2 g/kg) solution (v/v) in saline was injected(i.p.) every other day in order to mimic cyclical patterns of consumption (Pascualet al., 2007; White et al., 2000). Therefore, during the period of adolescent exposure,mice presented a period of ethanol intoxication followed by deprivation every 48 h.Control mice were exposed to saline.

Pups (44 litters: 106 females and 106 males) were distributed into four treat-ment groups (detailed description in Supplementary Material S1): VEH (air + saline),SMK (smoke + saline), ETOH (air + ethanol) and those receiving the combined treat-ment: SMK + ETOH (smoke + ethanol). Behavioral tests were conducted by the endof the drug administration period (PN44-45), during a short-term (PN49-50) or along-term deprivation (PN74-75) and separate groups of mice were tested at eachtime-point. One male and one female from each litter were randomly assigned toeach treatment group/age. All mice were submitted to both behavioral tests. Bodyweights were measured every day during the exposure period (PN30-PN45) and attwo time points during deprivation (PN50 and PN75).

2.2. Behavioral testing

Elevated plus maze (detailed description in Supplementary Material S12). Thepercentage of time spent in the open arms (%Time OA: the time spent in open armsdivided by time spent in open + closed arms) and the percentage of open arms entries(%Entries OA: the number of entries in open arms divided by number of entries inopen + closed arms) were used as anxiety measures. An entry was counted when-ever the animal crossed with all four paws into an arm. Increased %Time OA and/or%Entries OA correspond to decreased anxiety-like behavior and vice versa (Rodgersand Dalvi, 1997). Ethological measures – protected head-dips and time spent in thecenter of the maze – were also quantified (detailed description in SupplementaryMaterial S23).

Open field test (detailed description in Supplementary Material S14). The testwas performed one day after the elevated plus maze. The ambulation was quantifiedon the basis of the number of rectangles crossed by the animals. Animals had to placeall four legs on a given rectangle for a crossing to be counted. Total ambulation, whichconsists of the sum of rectangles crossed in the center and in the periphery (Cen + Pe),

paper. See Appendix A for more details.3 Supplementary material can be found by accessing the online version of this

paper. See Appendix A for more details.4 Supplementary material can be found by accessing the online version of this

paper. See Appendix A for more details.

5 Alcohol Dependence 133 (2013) 52– 60

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Fig. 2. Effects of adolescent tobacco smoke and/or ethanol exposure on body massduring the exposure period (PN30-PN45) and at two time points during depriva-tion (PN50 and PN75). Values are means ± SEM. VEH, vehicle group; SMK, tobaccosmoke exposure group; ETOH, ethanol exposure group; SKM + ETOH, tobacco smoke

4 Y. Abreu-Villac a et al. / Drug and

efecation – were also quantified (detailed description in Supplementary Material25).

For mice tested by the end of drug exposure period, animals were removed fromhe exposure chamber after 3–4 h, allowed to habituate for 30 min in the testingoom prior to the elevated plus maze and the open field. After the test, the animalsere returned to the exposure chamber to complete the 8-h period of daily exposure.s for ethanol exposure, in order to avoid the acute effects of the drug, such as motor

mpairment, the last injection was administered on PN44, after the elevated plusaze.

.3. Cotinine and ethanol serum levels

Cotinine and ethanol levels were assessed in two groups specifically treated forhese analyses. To evaluate tobacco exposure, on PN45, at 4 h of exposure, animalsere individually removed from the exposure chamber, decapitated and trunk bloodas collected from VEH (females: n = 2; males: n = 2), SMK (females: n = 4; males:

= 5), ETOH (females: n = 2; males: n = 2) and SMK + ETOH (females: n = 4; males: = 5) mice for cotinine (nicotine metabolite) quantification (kit from Orasure Tech-ologies, Pennsylvania, USA). Considering cotinine relatively short half-life in mice30–50 min), blood collection was performed immediately after the animals wereemoved from the exposure chamber. Previous studies have indicated that serumicotine:cotinine ratio consistently ranges from 1:5 to 1:10, which allows for anstimation of nicotine levels in the serum of SMK mice (Trauth et al., 2000). To eval-ate ethanol exposure, on PN44, 30 min after the last injection of ethanol/saline,n interval that produces ethanol serum levels close to the highest levels iden-ified after an i.p. injection (Livy et al., 2003), animals were decapitated and thelood collected from VEH (females: n = 1; males: n = 1), SMK (females: n = 2; males:

= 2), ETOH (females: n = 5; males: n = 7) and SMK + ETOH (females: n = 5; males: = 5) mice (enzymatic kit from Alcohol Reagent Set, Pointe Scientific Inc., Michigan,SA).

.4. Data analyses

.4.1. Body mass. During exposure, a repeated-measures analysis of variancerANOVAs) was carried out - between-subjects factors: Treatment and Sex; within-ubjects factor: Day. During deprivation, ANOVAs were used.

.4.2. Elevated plus maze and open field. Two global rANOVAs were initially used -etween-subjects factors: Treatment, Age and Sex; within-subjects factor: Elevatedlus maze measures (%Time OA, %Entries OA) or open field measures [Cen + Pe,en/(Cen + Pe)]. For the sake of simplicity, results from the global rANOVAs arerovided as Supplementary Material S36.

Lower order ANOVAs and/or Fisher’s Protected Least Significant DifferenceFPLSD) were used post hoc. Figures were segmented by sex when significant Treat-ent × Sex interactions were observed. Data are compiled as means and standard

rrors. Effects were considered significant when P < 0.05 (two-tailed). For interac-ions at P < 0.10 (two-tailed), we also examined whether lower order main effectsere detectable after subdivision of the interactive variables (Snedecor and Cochran,

967). The criterion for interaction terms was not used to assign significance to theffects but rather to identify interactive factors requiring subdivision for lower-orderests of main effects of Treatment, the factor of chief interest (Snedecor and Cochran,967).

.4.3. Smoke and ethanol interactions. In addition to the one-dimensional sta-istical design described above, in which Treatment was considered a factor,

two-dimensional design was used (Abreu-Villac a et al., 2007, 2008; Ribeiro-arvalho et al., 2008, 2009). In this design, Smoke (treated: SMK and SMK + ETOH;on-treated: VEH and ETOH) was considered one of the between-subjects fac-ors. Ethanol (treated: ETOH and SMK + ETOH; non-treated: VEH and SMK) was

onsidered the other between-subjects factor. More-than-additive (synergistic)nd less-than-additive effects appear as significant interactions between the tworeatment dimensions, whereas simple, additive effects do not show significantnteractions.

.4.4. Correlation coefficients. Correlations between Cen/(Cen + Pe) in the openeld and the two anxiety-related variables in the elevated plus maze (%TimeA and %Entries OA) were analyzed by calculating Pearson’s correlation coeffi-ients.

5 Supplementary material can be found by accessing the online version of thisaper. See Appendix A for more details.6 Supplementary material can be found by accessing the online version of this

aper. See Appendix A for more details.

and ethanol exposure group. *P < 0.05, **P < 0.01, ***P < 0.001, significant differencesbetween SMK and VEH groups. #P < 0.05, ##P < 0.01, significant differences betweenSMK + ETOH and VEH groups. Differences revealed by FPLSD.

3. Results

3.1. Effects on body mass (detailed description in SupplementaryMaterial S37)

SMK + ETOH and SMK mice were lighter (P < 0.05 in both cases,FPLSD) than the VEH one from, respectively, the second and forthday of exposure onwards as a result of a reduced body mass gain(Fig. 2). SMK + ETOH and SMK mice were still lighter (P < 0.05 inboth cases, FPLSD) than the VEH ones during the short-term depri-vation (PN50; Fig. 2). One month post-treatment (PN75) differencesamong treatment groups were no longer observed.

3.2. Cotinine and ethanol serum levels

Cotinine levels did not differ between SMK (females:69.4 ± 6.8 ng/ml; males: 64.5 ± 1.8 ng/ml) and SMK + ETOH(females: 74.9 ± 13.3 ng/ml; males: 65.4 ± 16.1 ng/ml) mice.VEH and ETOH mice presented cotinine levels below the detec-tion limit of the technique (<8 ng/ml). Ethanol serum levelsdid not differ between ETOH (females: 171.9 ± 21.7 mg/dL;males: 238.0 ± 25.5 mg/dL) and SMK + ETOH mice (females:164.6 ± 36.1 mg/dL; males: 222.2 ± 21.6 mg/dL). For both cotinineand ethanol serum levels, there were no sex-dependent Treatmenteffects (no Treatment × Sex interactions). However, for ethanol inserum, females presented lower levels when compared to males(Sex effect: F = 5.1, df = 1, P = 0.04).

3.3. Elevated plus maze

By the end of exposure (PN44), the one-dimensional ANOVAsindicated sex-dependent effects (%Time OA – Treatment × Sex:F = 2.5, df = 3, P = 0.067; %Entries OA – Treatment × Sex: F = 2.5,df = 3, P = 0.067): SMK (%Time OA: P = 0.035; %Entries OA: P = 0.027;FPLSD) and SMK + ETOH females (%Time OA: P = 0.008; %Entries OA:P = 0.012; FPLSD) presented increased values (%Time OA – SMK:+130.2% and SMK + ETOH: +164.9%; %Entries OA – SMK: +59.6% andSMK + ETOH: +66.5%) when compared to VEH females, thus indi-

cating a consistent female-only anxiolytic response (Fig. 3A–B).With Smoke and Ethanol treatments considered as two dimen-sions, there were no Smoke × Ethanol interactions, which indicates

7 Supplementary material can be found by accessing the online version of thispaper. See Appendix A for more details.

Y. Abreu-Villac a et al. / Drug and Alcohol Dependence 133 (2013) 52– 60 55

Fig. 3. Effects of adolescent tobacco smoke and/or ethanol exposure on anxiety levels measured in the elevated plus maze by the end of exposure (PN44). (A) Percentageo Entrie( co sma ups as

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f time spent in the open arms (%time OA). (B) Percentage of open arms entries (%left) and females (right). Values are means ± SEM. VEH, vehicle group; SMK, tobacnd ethanol exposure group. *P < 0.05, **P < 0.01, significant difference between gro

hat the effect of combined tobacco smoke and ethanol treatmenteflected the summation of the two individual sets of effects.

During short-term deprivation (PN49), the one-dimensionalNOVAs indicated sex-dependent effects (Treatment × Sex inter-ctions) on %Entries OA (F = 3.8, df = 3, P = 0.014). After separationy sex, we found significant effects only for females. The SMKroup presented decreased values (%Entries OA: −75.4%, P = 0.006;PLSD), indicative of an anxiogenic state in females (Fig. 4A–B).n addition, ethanol treatment reversed the smoke-induced anx-ogenic effects as indicated by the significant difference betweenalues for the smoke and combined exposure groups (%Entries OA:74.6%, SMK < SMK+ETOH, P = 0.022; FPLSD) (Fig. 4A–B) and by the

ess-than-additive effect of Smoke and Ethanol detected in the two-imensional design (%Entries OA – Smoke × Ethanol: F = 7.4, df = 1,

= 0.010). There were no significant effects for males (Fig. 4A–B).During long-term deprivation (PN74), the one-dimensional

NOVAs failed to indicate alterations for both anxiety-like meas-res (Fig. 5A–B).

.4. Open field

By the end of exposure (PN45), the one-dimensional ANOVAailed to indicate treatment effects or interactions on anxiety-likeehavior (Cen/(Cen + Pe) (Fig. 6A). However, there were sex-ependent effects on locomotor activity elicited by both smokend ethanol exposures (Cen + Pe – Treatment × Sex: F = 2.6, df = 3,

= 0.058); after separation by sex, we found that only SMKP = 0.004; FPLSD) and SMK + ETOH males (P < 0.001; FPLSD) pre-ented decreased values (SMK: 22.7% and SMK + ETOH: 30.5%)hen compared to VEH males (Fig. 7A). There were no

moke × Ethanol interactions (two-dimensional analysis), whichndicates that the effect of combined tobacco smoke and ethanol

reatment reflected the summation of the two individual sets offfects.

No alterations in the open field were observed during short-termeprivation (PN50) (Figs. 6B and 7B).

s OA). For A and B, Treatment × Sex interactions allowed separate figures for malesoke exposure group; ETOH, ethanol exposure group; SKM + ETOH, tobacco smoke

revealed by FPLSD.

On month post-exposure (PN75), the one-dimensionalANOVA indicated significant effects on anxiety-like behavior(Cen/(Cen + Pe) – Treatment: F = 6.6, df = 3, P < 0.001). Althoughneither tobacco smoke nor ethanol deprivation alone elicitedsignificant changes on the anxiety measure, a long-term depriva-tion from smoke + ethanol (P = 0.005; FPLSD) elicited a reduction(−39.8%) in the activity in the center of the open field when com-pared to the VEH group (Fig. 6C), thus indicating a late-emergentanxiogenic response. We identified a significant Smoke × Ethanolinteraction (two-dimensional analysis: F = 6.9, df = 1, P = 0.011),which indicated that the reduction in the activity in the center ofthe open field due to the combined exposure reflected a synergisticeffect of Smoke and Ethanol. Regarding locomotor activity, therewere no significant differences between treatment groups (Fig. 7C).

3.5. Correlation coefficients

The Pearson correlation coefficient between Cen/(Cen + Pe) and%Time OA indicated a significant association (P < 0.001), however,the coefficient of determination was very low (r2 = 0.05). Similarresults were obtained for the correlation coefficient and coeffi-cient of determination between Cen/(Cen + Pe) and %Entries OA(r2 = 0.06; P < 0.001).

4. Discussion

Despite evidence for neuroactive properties of nonnico-tine components of tobacco smoke (Abreu-Villac a et al., 2010;Bruijnzeel, 2012; Rose, 2006; Villégier et al., 2010), most exper-imental studies examine the effects of nicotine alone. Moreover,despite the frequent association between smoking and consump-tion of alcoholic beverages (Dawson, 2000; Grant, 1998; Meyerhoff

et al., 2006), only limited information exists regarding possibleinteractions between these drugs, particularly during adolescence.The present report shows that exposure to tobacco smoke dur-ing adolescence affects brain function and provides experimental

56 Y. Abreu-Villac a et al. / Drug and Alcohol Dependence 133 (2013) 52– 60

Fig. 4. Effects of adolescent tobacco smoke and/or ethanol exposure on anxiety levels measured in the elevated plus maze during a short-term deprivation (PN49). (A)P entrif K, tobs een gr

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ercentage of time spent in the open arms (%Time OA). (B) Percentage of open armsor males (left) and females (right). Values are means ± SEM. VEH, vehicle group; SMmoke and ethanol exposure group. *P < 0.05, **P < 0.01, significant difference betw

vidence for functional interactions between tobacco smoke andthanol in the regulation of behavioral responses, particularlynxiety-related ones, during and long-after the end of exposure.

The present experimental design intended to simulate tobaccomoke and ethanol exposure levels observed for human adoles-ents. We obtained cotinine (nicotine metabolite) and ethanolerum levels comparable to those found in adolescent smokersnd drinkers (Caraballo et al., 2004; Eckardt et al., 1998; Woodt al., 2004), as well as similar levels of cotinine between SMKnd SMK + ETOH mice and similar levels of ethanol between ETOHnd SMK + ETOH mice, which further suggest that pharmacokineticnteractions are not capable of explaining our results. Interest-ngly, despite the lower ethanol serum levels in females exposed tothanol (ETOH and SMK + ETOH mice) when compared to males, byhe end of exposure, only females presented anxiety-related alter-tions. This result suggests that even lower ethanol levels in femalesre able to cause anxiety-like alterations.

.1. Effects by the end of adolescent exposure

Despite the anxiolytic properties of ethanol, here, it failed tolicit anxiety alterations. This lack of an effect could be due to theact that mice were abstinent from ethanol during the elevated plus

aze test (the penultimate injection of ethanol was administeredt PN42, approximately 46 h before the elevated plus maze test,hile the last injection was administered only after the test), which

uggests that the anxiolytic effect of ethanol had likely faded by theime of the test.

The anxiolytic effects of tobacco smoke and of smoke combinedith ethanol in females in the elevated plus maze are consis-

ent with evidence that tobacco smoking reduces stress levels (for

eview: Bruijnzeel, 2012) and that females are more susceptible toobacco and nicotine effects (for review: Lynch, 2006; Carroll et al.,009). In a recent study in which adolescent mice were offered aicotine solution to drink, there was a trend toward an anxiolytic

es (%Entries OA). For A and B, Treatment × Sex interactions allowed separate figuresacco smoke exposure group; ETOH, ethanol exposure group; SMK + ETOH, tobacco

oups as revealed by FPLSD.

effect (Abreu-Villac a et al., 2008). In contrast, Adriani et al. (2004)found that oral nicotine can increase anxiety levels. However, inthis study, mice were submitted to a 5-fold lower concentrationof nicotine. These opposing results suggest that the increase inthe nicotine dose produces a shift from an anxiogenic toward ananxiolytic effect. Interestingly, the anxiolytic effect identified inthe present study occurred in mice in which cotinine serum levelswere lower than those we obtained previously (Abreu-Villac a et al.,2008): 64.3 ± 1.9 ng/ml vs. 122.2 ± 15.5 ng/ml, respectively. Theseresults suggest that nicotine is not the only contributor to the anx-iolytic effect of tobacco smoke. In this regard, Villégier et al. (2010)showed that tranylcypromine (monoamine oxidase inhibitor) pre-treatment combined with nicotine elicits an anxiolytic effect atadulthood.

Our results in females further indicate that the anxiolytic effectsof the co-exposure were stronger than the isolated exposures. Thisresult raises the question of whether human adolescents co-useand co-abuse tobacco and ethanol in order to attain a higher level ofanxyolisis when compared to either tobacco smoke or ethanol sep-arate exposures. Considering that our previous study on nicotineand ethanol exposure during adolescence found a reduced effectelicited by the co-exposure (Abreu-Villac a et al., 2008), these con-trasting results corroborate the hypothesis that tobacco smoke andnicotine alone elicit distinct effects when combined with ethanol.

4.2. Effects during short-term deprivation

The lack of an anxiety effect during the short-term depriva-tion from ethanol could be due to the fact that adolescent rodentsare less sensitive than adults to the anxiogenic manifestations ofethanol deprivation (Doremus et al., 2003; Doremus-Fitzwater and

Spear, 2007). This result suggests that anxiogenesis is not a majorcomponent of ethanol short-term deprivation.

In contrast, anxiety levels in females are clearly increased duringshort-term tobacco smoke deprivation, a result that corroborates

Y. Abreu-Villac a et al. / Drug and Alcohol Dependence 133 (2013) 52– 60 57

Fig. 5. Effects of adolescent tobacco smoke and/or ethanol exposure on anxiety lev-els measured in the elevated plus maze during a long-term deprivation (PN74). (A)Percentage of time spent in the open arms (%Time OA). (B) Percentage of open armsentries (%Entries OA). Values are means ± SEM. VEH, vehicle group; SMK, tobaccosa

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Fig. 6. Effects of adolescent tobacco smoke and/or ethanol exposure on anxiety lev-els measured in the open field test by the end of the period of exposure (PN45) and attwo time points during deprivation (PN50 and PN75, respectively). Number of rect-angles crossed in the center corrected by total ambulation (% Ambulation in Center)

moke exposure group; ETOH, ethanol exposure group; SMK + ETOH, tobacco smokend ethanol exposure group.

revious findings (Hughes et al., 2000; Parrott, 2003). Our findingsre also similar to previous ones in rodents exposed to nicotineuring adolescence (Abreu-Villac a et al., 2008; Trauth et al., 2000),hich indicate that nicotine is the main contributor to the anxi-

ty effects of tobacco smoke during a short-term deprivation. Inact, heavy cigarette smoking during adolescence has been foundo be associated with higher risk of agoraphobia, generalized anx-ety disorder, and panic disorder during early adulthood (Johnsont al., 2000). The anxiogenic effect of tobacco smoke is consistentith the female-only increase in risk-assessment behavior during

hort-term tobacco smoke deprivation.The anxiogenic effects of tobacco smoke short-term deprivation

ere reduced in female mice previously co-exposed to ethanol dur-ng adolescence. This result suggests that ethanol has a protectiveffect on tobacco smoke anxiogenic effects that can be observedt short-term deprivation. Future studies are needed to determinehich neurochemical changes are associated with these female-

nly behavioral effects. A previous study on nicotine-ethanolnteractions in adolescent mice also demonstrated less-than-dditive effects during a similar short-term deprivation, however,espite the countermand effects of nicotine and ethanol, the anxio-enic effect in the co-exposed female mice still reached significance

Abreu-Villac a et al., 2008). These results indicate that the com-ined tobacco smoke and ethanol elicited distinct effects whenompared to nicotine and ethanol combined deprivation. If simi-ar effects occur in human adolescents who co-abuse tobacco and

by the end of the period of exposure (A) at short- (B) and long-term (C) deprivation.Values are means ± SEM. VEH, vehicle group; SMK, tobacco smoke exposure group;ETOH, ethanol exposure group; SKM + ETOH, tobacco smoke and ethanol exposuregroup. **P < 0.01, significant difference between groups as revealed by FPLSD.

58 Y. Abreu-Villac a et al. / Drug and Alcoho

Fig. 7. Effects of adolescent tobacco smoke and/or ethanol exposure on locomotoractivity (Cen + Pe) measured in the open field test by the end of the period of expo-sure (A) and at two time points during deprivation (B and C, respectively). Valuesae*

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ai

re means ± SEM. VEH, vehicle group; SMK, tobacco smoke exposure group; ETOH,thanol exposure group; SMK + ETOH, tobacco smoke and ethanol exposure group.*P < 0.01, ***P < 0.001, significant difference between groups as revealed by FPLSD.

thanol, then cumulative anxiogenic effects may not play a major

ole in relapse to drug use during a short-term withdrawal.

Adolescent female mice were more susceptible to drug-inducedlterations in anxiety levels both by the end of exposure and dur-ng the short-term deprivation. Descriptions of sex differences

l Dependence 133 (2013) 52– 60

regarding both behavioral and biochemical parameters are com-mon when it comes to the effects of drugs of abuse. In this regard,there is evidence that females are more vulnerable than males in allphases of the addiction process (for review: Lynch, 2006; Roth et al.,2004). In addition, progesterone and its metabolites were shown toaffect several neurotransmitter systems and, as a result, brain func-tion, including mood regulation (for review: Lynch and Sofuoglu,2010). Estrogen was shown to elicit anxiolytic-like behavior whileprogesterone was shown to increase anxiety levels in mice testedin the elevated plus maze, a result that, in adult mice, seems to bemodulated by �5 nAChR subunit levels (Gangitano et al., 2009).

Interestingly, in the opposite direction to the findings of anxi-ety, here we found that the decreased locomotor effect of SMK andSMK + ETOH exposure was restricted to males, which suggests atestosterone-related effect (Roth et al., 2004). Considering that thestimulant effects of a drug of abuse on locomotor activity have beenassociated to its reward effect (Kalivas et al., 1993), the decreasedlocomotor effect identified here may indicate a reduced responseto the effects of the combined tobacco smoke and ethanol in males.

4.3. Effects during long-term deprivation

There are scant studies that investigate long-term effects oftobacco smoke and ethanol co-exposure. In this regard, alcohol-dependent smokers reported higher lifetime rates of psychiatricdisorders, including anxiety disorders, than alcohol-dependentnon-smokers (Le Strat et al., 2010). Here, in contrast to the lackof effect elicited by either drug, the concomitant exposure totobacco smoke and ethanol resulted, one month post-exposure, inan increase in anxiety-like behaviors in the open field, disclosinga synergistic effect. To the best of our knowledge, the current setof results comprises the first evidence in animal models indicatingthat tobacco smoke + ethanol have a more-than-additive effect onanxiety after a long-term deprivation and is consistent with pre-vious results on nicotine and ethanol (Abreu-Villac a et al., 2008).It is conceivable that if a similar effect occurs after adolescents co-abuse of tobacco and ethanol, the synergism between drugs mayfacilitate relapse to drug use at long-term withdrawal. Future stud-ies assessing drugs self-administration in adult mice co-exposedto tobacco smoke and ethanol during adolescence are necessary tocorroborate this hypothesis.

The analysis and interpretation of emotional reactivity in exper-imental models is subject to multiple interpretations and it often isa complex task (Cryan et al., 2002; Ramos, 2008). We used the ele-vated plus maze and the open field, two established animal modelsto assess anxiety-related behavior that are based on the behav-ioral trait of rodents of avoiding open spaces. However, it shouldbe mentioned that despite the apparent coherence between meas-ures obtained in the open field and elevated plus maze, whichsuggests that measures from both tests can be used as alterna-tive indices of the same emotional construct, there is evidencefor the absence of significant inter-test correlations as well as fordifferences in the loading factors in factor analyses (Belzung andLe Pape, 1994). Indeed, our results of low coefficients of deter-mination between time spent in the center of the open field andthe two anxiety-related variables in the elevated plus maze are inaccordance with what has been demonstrated in the literature, andsupport our finding of distinct results regarding anxiety measuresin these behavioral tests. As a result, these tests have been sug-gested to measure different aspects of anxiety (for review: Ramos,2008): The open field was both suggested to model normal anxi-ety everyone is faced when confronted with a stressful/threatening

situation and as a model of generalized anxiety disorder, while theelevated plus maze was suggested to model elements of panic andgeneralized anxiety disorder in adult rodents (Prut and Belzung,2003; Cheeta et al., 2000). Accordingly, differences between tests

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ould explain why significant results in classic measures of the ele-ated plus maze were identified by the end of exposure and during

short-term deprivation while, at long-term, the anxiety alter-tions were identified only in the open field. Despite that, sinceoth behavioral tests are established models used to investigatenxiety-like behavior, we understand that the identification of aignificant treatment effect in either test is indicative of alterednxiety level.

. Conclusions

Anxiety is considered a relevant factor for both smoking andrinking due to its motivating force for a continued consump-ion (Gilbert et al., 1989; Picciotto et al., 2002; Heilig et al., 2010).ccordingly, anxiety alterations shared by these two drugs couldxplain, at least in part, their co-use and co-abuse. Here, we showhat the combined exposure to tobacco smoke and ethanol duringdolescence of mice elicits stronger anxiolytic effects than tobaccomoke or ethanol separate exposures in females. It should be notedhat in our experimental design, exposure to tobacco smoke and/orthanol was enforced, while, in contrast, human adolescents’ con-umption clearly involves the individual’s choice to engage in suchctivities. It would be interesting to study in young women whether

similar stronger anxiolytic effect occurs and whether it wouldrive higher consumption. Finally, only the combined exposureesulted in increased anxiety levels in the prolonged absence ofhese drugs. This indicates that tobacco smoke and ethanol inter-ct during adolescence altering anxiety levels throughout life. Ifhese observations can be extrapolated to human adolescents whoo-use and co-abuse tobacco and ethanol, they would indicate thathese individuals, at adulthood, would present increased anxietyevels.

ole of funding source

Funding for this study was provided by CNPq-BRAZIL Grant76991/2010-2 and Fundac ão de Amparo à Pesquisa do Estadoo Rio de Janeiro (FAPERJ-BRAZIL) Grant E-26/103.191/2011 andy fellowships from FAPERJ-BRAZIL and Sub-reitoria de Pós-raduac ão e Pesquisa da Universidade do Estado do Rio de JaneiroSR2-UERJ). CNPq-BRAZIL and FAPERJ-BRAZIL had no further role intudy design; in the collection, analysis and interpretation of data;n the writing of the report; or in the decision to submit the paperor publication.

ontributors

Authors YA-V, ARC, ACM and CCF designed the study and wrotehe protocol. Authors CCC, AR-C, MC-S and VFN were responsi-le for breeding and performed the behavioral tests. YA-V andCC managed the literature searches and summaries of previouselated work. Author YAV undertook the statistical analysis, androte the first draft of the manuscript. ARC, ACM and CCF substan-

ially helped YAV to reach the final version. All authors discussedhe results. All authors contributed to and have approved the final

anuscript.

onflict of interest

All authors declare that they have no conflict of interest.

cknowledgment

The authors are thankful to Ulisses Risso for animal care.

l Dependence 133 (2013) 52– 60 59

Appendix A. Supplementary data

Supplementary data associated with this article can befound, in the online version, at http://dx.doi.org/10.1016/j.drugalcdep.2013.05.033.

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