Combined Exposure to Alcohol and Tobacco Smoke Changes

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Combined use of alcohol and tobacco smoke change oxidative, inflammatory, and neurotrophic parameters in different brain areas of rats Dayane A. Quinteiros, Alana Witt Hansen, Bruna Bellaver, Larissa Bobermin, Rianne Remus Pulcinelli, Solange Bandiera, Greice Caletti, Paula Bitencourt, André Quincozes-Santos, and Rosane Gomez ACS Chem. Neurosci., Just Accepted Manuscript • DOI: 10.1021/acschemneuro.8b00412 • Publication Date (Web): 17 Jan 2019 Downloaded from http://pubs.acs.org on January 18, 2019

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ACS Chemical Neuroscience

Combined use of alcohol and tobacco smoke change oxidative, inflammatory, and neurotrophic parameters in different brain areas of rats

Dayane A. Quinterosa, Alana W. Hansena,*, Bruna Bellaverb, Larissa D. Boberminb, Rianne R. Pulcinellia; Solange Bandieraa, Greice Calettia, Paula E. R. Bitencourta, André QuincozesSantosb, Rosane Gomeza,*

aPrograma

de Pós-Graduação em Ciência Biológicas: Farmacologia e Terapêutica (PPGFT),

Universidade Federal do Rio Grande do Sul (UFRGS), Porto Alegre, Brazil bPrograma

de Pós-Graduação em Ciência Biológicas: Bioquímica, UFRGS, Porto Alegre,

Brazil * Both

authors contributed equally to this study

Declarations of interest: none

Corresponding author: Alana Witt Hansen Departamento de Farmacologia - ICBS -UFRGS Rua Sarmento Leite, 500/305 90050-170 – Porto Alegre – RS - Brasil Phone/fax: 55-51-992967136 E-mail: [email protected]

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Abstract Devastating effects of alcohol use and tobacco smoke habits on health are extensively reported in the literature. However, few studies have been conducted to elucidate the consequences of their combined use on the central nervous system. Here we studied the effect of this combined use on some oxidative, inflammatory, and neurotrophic parameters in the hippocampus, striatum, and frontal cortex of rats. Adult Wistar rats were allocated into control (CT), alcohol (AL), tobacco smoke (TB), or combined (ALTB) groups. Rats were exposed to environmental air (CT and AL groups) or to the smoke from 6 cigarettes (TB and ALTB groups) immediately after tap water (CT and TB), or 2 g/kg alcohol (AL and ALTB) oral gavage administration, twice a day, for 4 weeks. On day 28, rats were euthanized and brain areas were dissected for evaluating some cellular redox parameters, pro-inflammatory cytokines, and brain-derived neurotrophic factor (BDNF) levels. A One-way ANOVA analysis showed that ALTB combined treatment significantly increased oxidative stress levels in the hippocampus. ALTB also increased interleukin-1 (IL-1) levels in the striatum and frontal cortex and the tumoral necrosis factor- α (TNF-α) levels in the frontal cortex compared with AL, TB, and CT rats. Combined treatment also decreased the BDNF in the frontal cortex of rats. Oxidative damage was found, more importantly, in the hippocampus and inflammatory parameters were extended to all brain areas studied. Our results showed an interaction between alcohol and tobacco smoke according to the brain area, suggesting an additional risk of neural damage in alcoholics who smoke.

Keywords: Cytokines; cigarette; ethanol; neurotrophine; neurotoxicity; oxidative stress.


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Introduction According to the World Health Organization, harmful use of alcohol causes a large disease, social, and economic burden in society. Alcohol use disorder causes more than 60 diseases and is associated with violence and road traffic accidents related to more than 3 million death per year.1 Similarly, tobacco consumption is an epidemic and one of the biggest public health problem.1,2 Active or passive tobacco smoking is associated with more than 6 million deaths per year, related to pulmonary, cardiac, and vascular diseases, as well as different forms of cancer.2 Although these alarming numbers related to alcohol use and tobacco smoke, few studies have been elucidated the biological and functional consequences of their combined use on the central nervous system (CNS). This is a relevant problem since studies have shown that almost 80% of alcoholics smoke regularly and smokers consume more alcohol per occasion than non-smokers.3,4 Additionally, smokers consume alcohol in a higher quantity per occasion and more frequently than non-smokers.4 There is no evident reason for the prevalence of the combined use and, as above-mentioned, few studies explore the consequences of this association. In vitro and in vivo studies showed that alcohol and its metabolite, acetaldehyde, changed the cellular redox status in the brain, generating oxidative and nitrosative stress, decreasing antioxidant enzymes, with a consequence of biomolecules damage.5,6 Moreover, chronic alcohol use has been recognized as a systemic inflammatory disease.7–9 Indeed, alcohol use increases plasma levels of pro-inflammatory cytokines in humans and rodents.7,8,10 In the brain, cytokines as tumor necrosis factor alpha (TNF-) and interleukin-1β (IL-1β) enhance in microglia and astrocytes after alcohol administration, activating intracellular cascades relate to apoptotic pathways.11 Chronic alcohol use also changes brainderived neurotrophic factor (BDNF) levels, a neurotrophin that plays a key role in the synaptic plasticity, neurogenesis, and neuronal survival in humans12 and rodents.13,14 3 ACS Paragon Plus Environment


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Tobacco smoke, on the other hand, is a highly complex aerosol, composed of more than 4,700 chemical substances distributed between the gas and particulate phases.15 Tobacco smoke contains simple oxygen and carbon radicals and other reactive substances, such as polyunsaturated aldehydes and ketones, known to increase the production of reactive oxygen and nitrogen species, promoting oxidative stress in different brain areas.16,17 Some of these substances are known to directly activate intracellular signaling cascades, increasing proinflammatory mediators such as TNFα and IL-1β in peripheral tissues.17,18 Additionally, studies showed that the combined use of alcohol and tobacco smoke or nicotine decrease hippocampal neurogenesis,19–21 which may influence specific functions, changing behaviors, mood, and memory among users.22,23 Thus, our objective here was to study the effect of the combined use of alcohol and tobacco smoke on some cellular glial, redox, inflammatory, and neurotrophic parameters in the hippocampus, striatum, and frontal cortex of rats, all areas known to be related with drug addiction. To test our hypothesizes, we treated rats with alcohol (AL), tobacco smoke (TB), or combined drugs (ALTB) and compared with control (CT) rats.

Results and discussion Oxidative stress parameters Intracellular reactive oxygen species (ROS) production was measured using the 2′-7′dichlorofluorescein diacetate (DCFH-DA) assay. Results from this analysis showed that the combined use of alcohol and tobacco smoke significantly increased ROS production in the hippocampus of rats when compared with CT (P = 0.011) and TB (P = 0.001) groups (Fig. 1A). The combined ALTB use did not increase ROS in the striatum (Fig. 1B) or in the frontal cortex (Fig. 1C). Alcohol group showed lower ROS levels than CT and ALTB groups in the striatum (Fig. 1B and supplementary tables S1-3). Studies show that alcohol and its 4 ACS Paragon Plus Environment

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metabolite, acetaldehyde, increase ROS production and decrease antioxidant enzymes levels, according to doses, the regime of administration or brain area studied in rats.6,24,25 Alcohol in moderate doses exerts its own direct effect scavenging cytotoxic hydroxyl radicals produced by Fenton’s reaction.26 Different responses according to the brain area may be related to the lower antioxidant enzymes expression. However, this neuroprotective capacity might be reduced when combined use with tobacco. Antioxidant enzyme analysis showed that AL and ALTB treatment increased superoxide dismutase (SOD) levels in the hippocampus (Fig. 1D). Additionally, AL, TB and ALTB groups increased SOD levels in the striatum (Fig. 1E) and in the frontal cortex (Fig. 1F). As seen in Fig. 1G, H, and I, different treatments did not change catalase (CAT) levels in any brain area studied. We also showed that AL, TB, and ALTB treatments increased glutathione peroxidase (GPx) activity in the hippocampus (Fig. 2A). Diversely, we found that while AL and TB decreased GPx in the striatum (Fig. 2B), TB significantly increased GPx in the frontal cortex (Fig. 2C). Curiously, in these brain areas, the combined drugs did not show a difference from CT rats. Regarding the glutathione (GSH) levels, our results showed that AL, TB, and ALTB treatment significantly decreased it in the hippocampus (Fig. 2D). There were no changes in the striatum and frontal cortex after AL, TB, or ALTB treatments, except that GSH was higher in TB than CT and AL groups in the frontal cortex (Fig. 2F). Under our experimental conditions, we did not find increasing on ROS production in any brain areas after isolated AL or TB treatment. In the hippocampus, these treatments increased SOD and GPx activities and decreased GSH levels, suggesting that antioxidant defenses were efficient to decrease the redox damage. Indeed, GSH belongs to the second line of antioxidant defenses, donating an electron to GPx during its redox cycle that reduces hydroperoxides in water and oxygen, both harmless molecules.27. In addition, we did not find



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changes on glutamate cysteine ligase (GCL), except for AL group that decreased GCL activity in the striatum, or glutathione reductase (GR) activities (Supplementary Fig. S1). Concerning glial functionality, all treatments decreased glutamine synthetase (GS) activity in the hippocampus (Fig. 3A) with no effect in the striatum (Fig. 3B), but AL and TB treatments decreased GS levels in the frontal cortex and ALTB treatment restored them (Fig. 3C). Moreover, it is important to note that GS activity was closely affected by oxidative stress. We need to consider that this enzyme, exclusively expressed in astrocytes, is also associated with glutamate metabolism, protecting neurons against excitotoxicity by taking up excess glutamate and converting it into glutamine.28 Inhibition of GS in the hippocampus increases recurrent seizures of rats as a result of glutamate accumulation in the synaptic cleft.28 Studies show that alcohol administration decreases GS activity in the hippocampus and striatum of rats.6,29 In addition, the enzyme GS is very sensitive to oxidative stress30 and we suggest that it played an important role in the combined exposure to alcohol and tobacco smoke effects. Taken together (Fig. 4), our results showed that the combined treatment of alcohol and tobacco significantly increased ROS in the hippocampus of rats and antioxidant enzymes were not able to counteract the oxidative imbalance promoted by it. Pro- and antioxidant defenses are constantly recruited to restore oxidative balance more importantly when a tissue shows a potential risk of damage. Here we showed that ALTB rats increased ROS production only in the hippocampus. In this brain area, enhancing in the oxidative stress was accompanied by increasing on SOD and GPx activities (Fig. 1-2), and decreasing on GSH levels (Fig. 2). While SOD and GPx antioxidant enzymes dismutate superoxide radical and hydroperoxides produced by ALTB exposure, changes in GSH and GPx confirmed that glutathione metabolism might the main antioxidant defense recruited against ALTB damage instead of CAT - to restore the redox balance in this brain area (Fig. 4).


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Additionally, we showed that tobacco smoke, increased antioxidant enzymes in the hippocampus (GPx), striatum (SOD) and frontal cortex (GPx and SOD). Others also show that pre-exposure to cigarette smoke provides neuroprotection against the kainic acid, reducing seizures, mortality, and loss of hippocampal cells.31

Inflammatory parameters Combined use significantly increased IL-1 levels in the hippocampus (Fig. 5A), striatum (Fig. 5B), and frontal cortex (Fig. 5C). In the hippocampus, AL and TB isolated treatment also increased IL-1 levels (Fig. 5A). On the other hand, ALTB treatment increased TNF- levels in the striatum (Fig. 5E) and frontal cortex (Fig. 5F), without changes in the hippocampus (Fig. 5D). Isolated AL or TB treatment increased TNF- levels only in the striatum. Inflammatory parameters explored in this study also showed that the combined use is more deleterious than the isolated alcohol or cigarette use. In the hippocampus, a brain area where ALTB also increased ROS levels, IL-1 levels were 126% higher than CT rats, while isolated AL and TB increased them to 75% and TB 61%, respectively (Supplementary Box). Although alcohol has been considered a pro-inflammatory systemic condition8,9 we only detected increasing on IL-1 in the hippocampus and TNF- levels in the striatum and frontal cortex and for AL treated rats. Results from others show that chronic alcohol administration increases TNF-α and IL-1β levels in the hippocampus, striatum, and total cortex of rodents.9,32,33 Indeed, studies have been shown that alcohol deactivates cAMP responsive element binding protein (CREB) and activates the nuclear factor κB (NFκB), increasing the TNF-α expression, a key pro-inflammatory cytokine.24,34 Additionally, alcohol releases highmobility group box 1 (HMGB1), a toll-like receptor type 4 (TLR-4) agonist, increasing



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cytokines release as IL-1β directly from the microglia.35,36 A previous study also showed that tobacco smoke exposure increases IL-1 and TNF-α expression in the brain of rats.17

Neurotrophic parameter Combined treatment decreased BDNF levels in the hippocampus (Fig. 6A), striatum (Fig. 6B), and frontal cortex (Fig. 6C) of rats. In the hippocampus (Fig. 6A) and in the frontal cortex (Fig. 6C), AL and TB treatment also reduced BDNF levels. However, ALTB treatment decreased BDNF even more than AL and TB use in the frontal cortex (Fig. 6C) and was the only treatment reducing BDNF in the striatum, evidencing a treatment-dependent effect according to brain area. In humans, a nucleotide polymorphism in the BDNF gene has been associated with alcoholism.37,38 In rodents, a reduction in BDNF levels or inhibition of the BDNF receptors increases alcohol intake and preference,14,39 suggesting that BDNF plays an important role in drug addiction. In rodents, chronic alcohol exposure decreases the BDNF levels in the hippocampus, frontal, and prefrontal cortex.13,40 Here we reproduced these results but we could not see lower BDNF levels in the striatum, the brain area that we found lower ROS and higher antioxidant enzymes levels, suggesting that the striatum would be more resilient against brain injury from alcohol, tobacco, and their combined treatment. Regards smoke tobacco exposure, results from humans show that plasma BDNF levels are significantly lower in smokers than non-smokers.41 Tobacco smoke exposure also decreases BDNF levels in the prefrontal cortex and hippocampus of mice when they are exposed to it during prenatal, early postnatal, or infant period.42,43 In a previous study, we found that ALTB exposure decreased cell proliferation in the rat hippocampus by more than 60%19 and here we found that the association of this drugs decreased BDNF levels up to 59% (Supplementary Box), suggesting a correlation between cell proliferation and BDNF levels. In humans, magnetic resonance imaging (MRI) study showed that combined use of alcohol and cigarette 8 ACS Paragon Plus Environment

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significantly decreases thickness in different brain areas when compared with alcoholics or control individuals.44 These few studies are not conclusive and there is no study evaluating the effect of the combined use of alcohol and cigarette. Our results showed, for the first time, that tobacco smoke increases pro-inflammatory responses in different brain areas, parallel to reduced BDNF levels. Taken together, results from BDNF and cytokines release suggest that the combined use of these two drugs are more deleterious than their isolated use and may add risk for neuronal damage. Isolated tobacco use decreased BDNF levels only in the hippocampus and frontal cortex. Previous studies also showed that BDNF is lower in the plasma of human smokers or in different brain areas of rodents exposed to tobacco smoke.45 Because of the elevated morbidity and mortality associated with alcohol and tobacco abuse, it is important to address studies exploring the effect of their combined use. Here we focused on the effect of the ALTB combined use in the CNS of rats. We found that oxidative damage was more important in the hippocampus and the inflammatory response was elevated and the BDNF levels were decreased in all brain areas studied. Additionally, we showed here that the tobacco smoke alone favored the antioxidant response, but increased inflammatory response and decreased BDNF levels in all brain areas. Additional studies need to explore the mechanisms related to the interaction between alcohol and tobacco smoke in different brain areas and clarify regards the potential risk of neural damage in alcoholics who smoke.

Material and Methods Animals Adult (~ 90 days old), male Wistar rats (~ 280 g) born and reared at the Center for Reproduction and Experimentation of Laboratory Animals (CREAL- UFRGS) were housed in the animal facility of the Department of Pharmacology, in polypropylene cages (3 rats/cage, 33  40  17.8 cm). Environmental conditions were controlled (22 ± 2 °C; 12 h light/dark 9 ACS Paragon Plus Environment


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cycle; 55 ± 5% air humidity) and rats had free access to water and food (Nuvilab, Colombo, Brazil). All procedures were performed according to international and local policies for experimental animal handling and the Ethics Committee for Animal Experimentation approved this study (CEUA-UFRGS # 30088).

Alcohol solution and tobacco smoke Ethanol (98%) (Nuclear, São Paulo, Brazil) was diluted to 20% (w/v) in tap water, daily prepared, and administered at the volume of 10 mL/kg by oral gavage (2 g of alcohol per animal kg). This 2 g/kg dose increases blood alcohol concentration (BAC) up to 120 mg/dL at 60 min from the administration.46,47 Control rats received the same volume of tap water. Cigarettes from a commercial brand (0.6 mg of nicotine/cigarette, and Tar of 8 mg/cigarette, according to the manufacturer) were burned in an appropriated apparatus and smoke was dragged to a hermetically sealed glass chambers (50  30  30 cm), by a negative airflow of 10 L/min, maintained constant by a vacuum pump (Fig. 7).19

Experimental procedure Animals were allocate into control (CT) or tobacco smoke (TB) groups to receive tap water, and in alcohol (AL) or alcohol + tobacco smoke (ALTB) groups to receive 2 g/kg alcohol, via oral gavage, twice a day (9 AM and 2 PM), for 28 days (n = 12/group). Immediately after the oral administration, animals were placed in hermetic chambers (n = 6 rats/chamber), with ambient air (CT and AL groups) or tobacco smoke (6 cigarettes; TB and ALTB groups) circulation, for 2 h (Fig. 7). Cigarettes were burned with a 10 min interval between one and other to avoid intoxication of rats. The total per day treatment was 4 g/kg alcohol and 12 burning cigarettes. On day 28, rats were euthanized by decapitation 60 min after the morning treatment. Brain was carefully removed and the frontal cortex, 10 ACS Paragon Plus Environment

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hippocampus, and striatum were dissected and stored at -80 ºC for further analysis of some cellular redox parameters such as ROS, SOD, CAT, GPx, GSH, GS, GCL, and GR enzymes, as well as, TNF-, IL-1β, and BDNF levels. Trunk blood samples were centrifuged (2,5 rpm/10 min) and serum stored at -30 oC for analysis of DCFH-DA and GSH levels.

Cellular redox parameters Intracellular ROS production was measured using the non-fluorescent cell-permeating compound, 2′-7′-dichlorofluorescein diacetate (DCFH-DA) assay. Intracellular esterases hydrolyze DCFH-DA to dichlorofluorescin (DCFH) that is oxidized to fluorescent dichlorofluorescin (DCF) by reactive oxygen-containing molecules, directly measuring the redox state of a cell.48 For this assay, samples from frontal cortex, hippocampus, and striatum were treated with 10 μM DCFH-DA for 30 min at 37 oC. After, the slices were placed into a phosphate-buffered saline (PBS) solution with 0.2% Triton X-100. Fluorescence was measured in a plate reader (Spectra Max M5, Molecular Devices, USA) with excitation at 485 nm and emission at 520 nm. The ROS production was calculated as fluorescence units per milligram protein (UF/mg) and results were expressed in percentage of control. SOD activity was measured using the RANSOD® commercial kit (Randox Co, Antrim, UK), as previously described49 and the SOD activity was determined by the absorption at 505 nm in a spectrophotometer and results were expressed as UI/mg protein. The CAT activity was measured as described by Aebi (1984).50 The decrease absorbance at 240 nm was measured in brain homogenates suspended in a reaction medium containing 20 mM H2O2, 0.1% Triton X-100, 10 mM potassium phosphate buffer (pH 7.0) and 50 μg protein. Results were expressed as UI/mg protein, with one unit (UI) of enzyme activity defined as 1 μmoL of H2O2 consumed per minute.



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GCL activity was measured according to Arús et al. (2017).51 Homogenates were suspended in a sodium phosphate buffer containing 140 mM KCl, and diluted with 100 mM sodium phosphate buffer (pH 8.0) containing 5 mM EDTA. The NADH oxidation was monitored at 340 nm in sodium phosphate/KCl (pH 8.0) solution containing 5 mM Na2-ATP, 2 mM phosphoenolpyruvate, 10 mM L-glutamate, 10 mM L-α-aminobutyrate, 20 mM MgCl2, 2 mM Na2-EDTA, 0.2 mM NADH, and 17 μg pyruvate kinase/lactate dehydrogenase. Results were expressed in nmol/mg protein/min. GSH levels were assessed as previously described by Browne and Armstrong (2017).52 Briefly, frontal cortex, hippocampus, and striatum were homogenated in 20 mM sodium phosphate buffer (pH 7.4) with 140 mM KCl. Homogenates were diluted in 10 volumes of 100 mM sodium phosphate buffer (pH 8.0) containing 5 mM EDTA. The protein was precipitated with 1.7% meta-phosphoric acid and the supernatant was assayed with ophthaldialdeyde (1 mg/mL methanol) at room temperature for 15 min. Fluorescence was measured using excitation and emission wavelengths of 350 and 420 nm, respectively. A calibration curve was built with standard GSH solutions (0 – 500 μM). GSH levels were expressed as nmol/mg protein. GPx activity was measured using the RANSEL® commercial kit (Randox Co, Antrim, UK), as previously described.49 The GPx activity was determined by the absorption of NADPH at 340 nm in a spectrophotometer and results were expressed as UI/mg protein. GR activity was determined by a commercial kit (Randox Co, Antrim, UK), monitoring the oxidation of NADPH to NADP+ in the homogenate at 340 nm in solution containing 200 mM sodium phosphate buffer (pH 7.5), 6.3 mM EDTA, 1 mM GSSG, 0.1 mM NADPH. Results were expressed as U/mg protein.

Glial parameter 12 ACS Paragon Plus Environment

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GS is an enzyme exclusively expressed in the glial cells and its activity is closely related to glutamate metabolism and denotes changes on glutamatergic activity. For GS activity assay, the tissues were homogenized in a 150 mM KCl solution and assayed as previously described.49 Homogenate was added to a reaction mixture (MgCl2: 10 mM; Lglutamate: 50 mM; imidazole-HCl buffer (pH 7.4): 100 mM; 2-mercaptoethanol: 10 mM; hydroxylamine HCl: 50 mM; ATP: 10 mM) 1:1, and incubated at 37 oC for 15 min. Reaction was stopped by adding 0.4 mL of a solution with ferric chloride: 370 mM, HCl: 670 mM, and trichloroacetic acid 200 mM. Samples were centrifuged (1,000 g/10 min) and the absorbance of the supernatant was measured at 530 nm. Glutamylhydroxamate added to ferric chloride reagent was used as a standard. The results were expressed as μmol/h/mg of protein.

Neurotrophic and inflammatory parameters The BDNF levels were measured in samples from the hippocampus, striatum, and frontal cortex using a commercial ELISA kit following the manufacturer's recommendations (Promega Co., Madison, WI, USA), with a minimum detection of 15.6 pg/mL. The levels of interleukins TNF-α and IL-1β were measured in an extracellular medium using a commercial ELISA kit from Peprotech (Rocky Hill, NJ, USA) and eBioscience (Waltham, MA, USA), respectively, with the minimum detection of 0.4 ng/mL. Results were expressed as picogram per milliliter for BDNF and nanogram per milliliter for interleukines.

Statistical analysis The results were tested for normal distribution by the Shapiro-Wilk test. Normal values were analyzed using a one-way analysis of variance (ANOVA), with alcohol and tobacco smoke as independent factors, followed by the Student-Newman-Keuls post-hoc test. Non-normal values were analyzed using the Kruskal-Wallis followed by the Dunn´s test. Statistical 13 ACS Paragon Plus Environment


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significance was considered when P < 0.05. All analyses were performed using the Statistical Package for Social Sciences (SPSS) software version 21.0, and results are presented as mean  S.E.M.

Compliance with Ethical Standards All animal experiments were performed in accordance with the National Institute of Health (NIH) Guide for the Care and Use of Laboratory Animals (USA) and the National Council for the Control of Animal Experimentation (CONCEA- Brazil). The experimental protocols were approved by the Animal Care and Use Committee at Federal University of Rio Grande do Sul (CEUA-UFRGS # 30088).

Author Contributions Dayane A. Quinteros – Experiment design and execution, interpretation of the results and manuscript writing. Alana W. Hansen – Experiment execution, interpretation of the results, graphics design and manuscript writing and revision. Bruna Bellaver – Experiment execution and interpretation of the results. Larissa D. Bobermin – Experiment execution and interpretation of the results. Rianne R. Pulcinelli – Experiment execution and interpretation of the results. Solange Bandiera – Experiment execution and interpretation of the results. Greice Caletti – Experiment execution and interpretation of the results. Paula E. R. Bitencourt – Interpretation of the results, manuscript writing and revision. 14 ACS Paragon Plus Environment

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André Quincozes-Santos – Experimental design, interpretation of the results, manuscript writing and revision. Rosane Gomez – Experimental design and execution, interpretation of the results, manuscript writing and revision.

Acknowledgements This work was supported by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) and Universidade Federal do Rio Grande do Sul (UFRGS), Brazil.

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Legends of figures

Fig. 1 Reactive oxygen species (ROS) production (A, B, and C), the activities of SOD (D, E, and F) and CAT (G, H, and I) in different brain areas of rats after alcohol administration (4 g/kg/day, orally), tobacco smoke exposure (12 cigarettes/day, inhalation), or their combined use, along 30 days. The data are presented as mean ± S.E.M., (n = 8). One-way ANOVA followed by Student-Newman-Keuls post-hoc test (parametric values) or Kruskal-Wallis followed by Dunn’s post-hoc test (non-parametric values).

Fig. 2 GPx activity (A, B, and C), GSH levels (D, E, and F) in different brain areas after alcohol administration (4 g/kg/day, orally), tobacco smoke exposure (12 cigarettes/day, inhalation), or their combined use, along 30 days. The data are presented as mean ± S.E.M., (n = 8). One-way ANOVA followed by Student-Newman-Keuls post-hoc test (parametric values) or Kruskal-Wallis followed by Dunn’s post-hoc test (non-parametric values).

Fig. 3 GS activity in different brain areas (A, B, and C) after alcohol administration (4 g/kg/day, orally), tobacco smoke exposure (12 cigarettes/day, inhalation), or their combined use, along 30 days. The data are presented as mean ± S.E.M., (n = 8). One-way ANOVA followed by Student-Newman-Keuls post-hoc test (parametric values) or Kruskal-Wallis followed by Dunn’s post-hoc test (non-parametric values).

Fig. 4 Summary of the results of oxidative stress and glial functionality in groups: alcohol (AL), tobacco smoke (TB) and association of alcohol and tobacco smoke (ALTB) in different brain areas (frontal cortex, hippocampus and striatum).



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Fig. 5 Release of IL-1β (A, B, and C) and TNF-α (D, E, and F) cytokines in different brain areas after alcohol administration (4 g/kg/day, orally), tobacco smoke exposure (12 cigarettes/day, inhalation), or their combined use, along 30 days. The data are presented as mean ± S.E.M., (n = 8). One-way ANOVA followed by Student-Newman-Keuls post-hoc test (parametric values) or Kruskal-Wallis followed by Dunn’s post-hoc test (non-parametric values).

Fig. 6 BDNF levels in hippocampus (A), striatum (B) and frontal cortex (C) after alcohol administration (4 g/kg/day, orally), tobacco smoke exposure (12 cigarettes/day, inhalation), or their combined use, along 30 days. The data are presented as mean ± S.E.M., (n = 8). Oneway ANOVA followed by Student-Newman-Keuls post-hoc test (parametric values) or Kruskal-Wallis followed by Dunn’s post-hoc test (non-parametric values).

Fig. 7 Schematic representation of the experimental procedures. At 9 a.m. and 2 p.m. rats were treated with tap water or alcohol 20% (2 g/kg) and immediately after they were exposure to chambers with environmental air or tobacco smoke (6 cigarettes each exposition). Thus, they received 4 g/kg/day and exposure to the smoke from 12 cigarettes.


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Supplementary information

Supplementary Fig S1 – GCL and GR activity in hippocampus, striatum and frontal cortex. Table S1 – Results of one way ANOVA (Student-Newman-Keuls post-hoc test for parametric values) or Kruskal-Wallis (Dunn’s post-hoc test for non-parametric values) from oxidative stress parameters, TNF-α and IL-1β and BDNF in the hippocampus of rats. Table S2 – Results of one way ANOVA (Student-Newman-Keuls post-hoc test for parametric values) or Kruskal-Wallis (Dunn’s post-hoc test for non-parametric values) from oxidative stress parameters, TNF-α and IL-1β and BDNF in the striatum of rats. Table S3 – Results of one way ANOVA (Student-Newman-Keuls post-hoc test for parametric values) or Kruskal-Wallis (Dunn’s post-hoc test for non-parametric values) from oxidative stress parameters, TNF-α and IL-1β and BDNF in the frontal cortex of rats. Box – Difference of AL, TB and ALTB from control group in percentage.



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For Table of Contents Use Only

Combined use of alcohol and tobacco smoke change oxidative, inflammatory, and neurotrophic parameters in different brain areas of rats Dayane A. Quinteros, Alana W. Hansen, Bruna Bellaver, Larissa D. Bobermin, Rianne R. Pulcinelli; Solange Bandiera, Greice Caletti, Paula E. R. Bitencourt, André Quincozes-Santos and Rosane Gomez


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Fig. 1 Reactive oxygen species (ROS) production (A, B, and C), the activities of SOD (D, E, and F) and CAT (G, H, and I) in different brain areas of rats after alcohol administration (4 g/kg/day, orally), tobacco smoke exposure (12 cigarettes/day, inhalation), or their combined use, along 30 days. The data are presented as mean ± S.E.M., (n = 8). One-way ANOVA followed by Student-Newman-Keuls post-hoc test (parametric values) or Kruskal-Wallis followed by Dunn’s post-hoc test (non-parametric values). 193x197mm (300 x 300 DPI)

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Fig. 2 GPx activity (A, B, and C), GSH levels (D, E, and F) in different brain areas after alcohol administration (4 g/kg/day, orally), tobacco smoke exposure (12 cigarettes/day, inhalation), or their combined use, along 30 days. The data are presented as mean ± S.E.M., (n = 8). One-way ANOVA followed by Student-Newman-Keuls post-hoc test (parametric values) or Kruskal-Wallis followed by Dunn’s post-hoc test (non-parametric values). 190x249mm (300 x 300 DPI)


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Fig. 3 GS activity in different brain areas (A, B, and C) after alcohol administration (4 g/kg/day, orally), tobacco smoke exposure (12 cigarettes/day, inhalation), or their combined use, along 30 days. The data are presented as mean ± S.E.M., (n = 8). One-way ANOVA followed by Student-Newman-Keuls post-hoc test (parametric values) or Kruskal-Wallis followed by Dunn’s post-hoc test (non-parametric values). 118x279mm (300 x 300 DPI)


ACS Chemical Neuroscience 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47


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Fig. 5 Release of IL-1β (A, B, and C) and TNF-α (D, E, and F) cytokines in different brain areas after alcohol administration (4 g/kg/day, orally), tobacco smoke exposure (12 cigarettes/day, inhalation), or their combined use, along 30 days. The data are presented as mean ± S.E.M., (n = 8). One-way ANOVA followed by Student-Newman-Keuls post-hoc test (parametric values) or Kruskal-Wallis followed by Dunn’s post-hoc test (non-parametric values). 185x246mm (300 x 300 DPI)



Fig. 6 BDNF levels in hippocampus (A), striatum (B) and frontal cortex (C) after alcohol administration (4 g/kg/day, orally), tobacco smoke exposure (12 cigarettes/day, inhalation), or their combined use, along 30 days. The data are presented as mean ± S.E.M., (n = 8). One-way ANOVA followed by Student-NewmanKeuls post-hoc test (parametric values) or Kruskal-Wallis followed by Dunn’s post-hoc test (non-parametric values). 111x266mm (300 x 300 DPI)


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Combined Exposure to Alcohol and Tobacco Smoke Changes

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