Addition of açaí ( Euterpe oleracea) to cigarettes has a protective effect against emphysema in mice

Addition of açaí ( Euterpe oleracea) to cigarettes has a protective effect against emphysema in mice

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  Addition of açaí ( Euterpe oleracea ) to cigarettes has a protective effectagainst emphysema in mice Roberto Soares de Moura a, ⇑ , Karla Maria Pereira Pires b , Thiago Santos Ferreira b,c , Alan Aguiar Lopes b,c ,Renata Tiscoski Nesi c , Angela Castro Resende a , Pergentino Jose Cunha Sousa d ,Antonio Jorge Ribeiro da Silva e , Luis Cristóvão Porto b , Samuel Santos Valenca c a Laboratório de Farmacologia Cardiovascular e Plantas Medicinais, Departamento de Farmacologia e Psicobiologia, IBRAG – UERJ, Rio de Janeiro, Brazil b Laboratório de Reparo Tecidual, IBRAG – UERJ, Rio de Janeiro, Brazil c Laboratório de Inflamação, Estresse Oxidativo e Câncer, ICB – UFRJ, Rio de Janeiro, Brazil d Faculdade de Farmácia, ICS – UFPA, Belém, Brazil e Núcleo de Pesquisa de Produtos Naturais – UFRJ, Rio de Janeiro, Brazil a r t i c l e i n f o  Article history: Received 21 June 2010Accepted 7 December 2010Available online xxxx Keywords: Cigarette smokeAçai ( Euterpe oleracea )EmphysemaOxidative stressInflammationMouse a b s t r a c t Chronic inhalation of cigarette smoke (CS) induces emphysema by the damage contributed by oxidativestress during inhalation of CS. Ingestion of açai fruits ( Euterpe oleracea ) in animals has both antioxidantand anti-inflammatory effects. This study compared lung damage in mice induced by chronic (60-day)inhalation of regular CS and smoke from cigarettes containing 100mg of hydroalcoholic extract of açaiberry stone (CS+A). Sham smoke-exposed mice served as the control group. Mice were sacrificed onday 60, bronchoalveolar lavage was performed, and the lungs were removed for histological and bio-chemical analyses. Histopathological investigation showed enlargement of alveolar space in CS micecompared to CS+A and control mice. The increase in leukocytes in the CS group was higher than theincrease observed in the CS+A group. Oxidative stress, as evaluated by antioxidant enzyme activities,mieloperoxidase, glutathione, and 4-hydroxynonenal, was reduced in mice exposed to CS+A versusCS. Macrophage and neutrophil elastase levels were reduced in mice exposed to CS+A versus CS. Thus,the presence of açai extract in cigarettes had a protective effect against emphysema in mice, probably byreducing oxidative and inflammatory reactions. These results raise the possibility that addition of açaíextract to normal cigarettes could reduce their harmful effects.   2010 Elsevier Ltd. All rights reserved. 1. Introduction Cigarette smoke (CS) is usually associated with development of chronic obstructive pulmonarydisease (COPD), a pathological con-ditioncharacterizedbyaslow, progressive, andlargelyirreversibleairflow limitation due to chronic bronchitis and/or emphysema(O’Donnell and Parker, 2006). COPD is a major and increasing glo-bal health problem: It is currently the 4th leading cause of deathworldwide, and is projected to become the 3th leading cause of death and the 5th most common cause of disability by the year2020 (Lopez and Murray, 1998). Airway oxidative stress, inflam-mation, andproteolysisare intimatelyrelated tothe pathophysiol-ogy of CS-induced COPD. The increase in oxidative stress in thelungs is due to an imbalance in the production of oxidants andreactive oxygen species (ROS) as well as to a reduction of antioxi-dant defense mechanisms (Church and Pryor, 1985; Pryor et al.,1983; Schumacher et al., 1977). ROS are constituents of both the tar and gas phases of smoke, which include reactive aldehydes,quinones, and benzo(a)pyrene. These agents, plus reduced pul-monary antioxidant defense mechanisms, result in an oxidativeburden that along with chronic inflammation leads to cellulardamage and lung remodeling. To reduce the lung damage inducedby smoke, the cigarette industry has produced low tar and lownicotine cigarettes; however, it is not clear that reduction of tarandnicotinehavedecreasedtheincidenceofCOPDamongsmokers(Dobson, 2008; Dunsby and Bero, 2004). Experimental data in animals show that systemically administered antioxidants may 0278-6915/$ - see front matter   2010 Elsevier Ltd. All rights reserved.doi:10.1016/j.fct.2010.12.007  Abbreviations:  CS, cigarette smoke; COPD, chronic obstructive pulmonarydisease; ROS, reactive oxygen species; ASE, açaí stone extract; COHb, carboxyhe-moglobin; H&E, hematoxylin and eosin; BAL, bronchoalveolar lavage; SOD, super-oxide dismutase; CAT, catalase; GP  x , glutathione peroxidase; GSH, reducedglutathione; GSSG, glutathione disulfide; MPO, myeloperoxidase; WB, Westernblotting; 4-HNE, 4-hydroxynonenal; MMP-12, metalloelastase. ⇑ Corresponding author. Address: Laboratório de Farmacologia Cardiovascular ePlantas Medicinais, Departamento de Farmacologia e Psicobiologia, IBRAG – UERJ,Av. 28 de Setembro 87 fundos, Vila Isabel, CEP, 20.551-030 Rio de Janeiro, Brazil.Tel.: +55 21 25 87 63 99; fax: +55 21 25 87 68 08. E-mail address: (R.S. de Moura).Food and Chemical Toxicology xxx (2011) xxx–xxx Contents lists available at ScienceDirect Food and Chemical Toxicology journal homepage: Please cite this article in press as: de Moura, R.S., et al. Addition of açaí ( Euterpe oleracea ) to cigarettes has a protective effect against emphysema in mice.Food Chem. Toxicol. (2011), doi:10.1016/j.fct.2010.12.007  reduce lung damage. Specifically, oral administration of antioxi-dants such as vitamin C (Banerjee et al., 2008; Panda et al., 2000;Silva Bezerra et al., 2006), vitamin E (Graziano et al., 1985; Uneri et al., 2006), black tea (Banerjee et al., 2007), mate tea (Lanzetti et al., 2008), and curcumin (Shishodia et al., 2003; Vanisree and Sudha, 2006) reduce lung damage induced by CS in experimentalanimal models. Euterpe oleracea  Mart., popularly known as the açaí berry, iswidely cultivated in the Amazon region of Brazil. Chemical stud-ies have shown that the açaí berry has a diverse composition thatincludes hydroxybenzoic acids, antioxidant polyphenolics, flavan-3-ols, and anthocyanins, predominantly cyanidin 3- O -rutinosideand cyanidin 3- O -glucuronide (Del Pozo-Insfran et al., 2004; Kanget al., 2010; Lichtenthaler et al., 2005; Rodrigues et al., 2006;Schauss et al., 2006b). Other studies have shown that consump-tion of the açai berry has other beneficial effects due to its highsuperoxide anion scavenging properties, its anti-inflammatory ef-fect via inhibition of cyclooxygenases 1 and 2 (Schauss et al.,2006a), its vasodilator effect (Rocha et al., 2007), and its inhibition of nitric oxide production and iNOS activity andexpression (Matheus et al., 2006). The antioxidant effect of a fruit and berry juice blended containing açai as the predominantingredient was demonstrated in an  In vivo  study performed inhealth subjects ( Jensen et al., 2008). The present study was undertaken to determine whether adding a hydroalcoholic ex-tract of açai berry stone to cigarettes reduces the harmful effectsof CS in mice. 2. Methods  2.1. Reagents The following reagents were purchased from Sigma Chemical(St. Louis, MO, USA): acrylamide, adrenaline, bovine serumalbumin, bromophenol blue, calcium chloride dehydrate, 5,5 0 -dithiobis-(2-nitrobenzoic acid) (DTNB), dipotassium hydrogenphosphate, eosine, glutathione, glutathione disulfide, glycerol,hematoxylin, hexadecyltrimethylammonium bromide (HTAB), 2-mercaptoethanol, monopotassium phosphate, myeloperoxidase,nicotinamide adenine dinucleotide phosphate (NADPH), 2-nitro-5-thiobenzoate (TNB), sodium acetate buffer, sodium chloride,sodium dodecylsulfate, 3,3 0 ,5,5 0 -tetramethylbenzidine (TMB), thio-barbituric acid, triethanolamine, Tris–HCl, TritonX-100, Tween-20,2-vinylpyridine, and zinc chloride. Panótico was purchased fromLaborclin (Pinhais, Paraná, Brazil). Bradford assay reagents werepurchased from Bio-Rad (Hercules, CA, USA). Acetic acid, ethanol,formalin, and hydrogen peroxide were purchased from Vetec(Duque de Caxias, Rio de Janeiro, Brazil).  2.2. Animals C57BL/6 male mice (8 weeks old; 20–24 g) were purchasedfrom the Veterinary Institute at Fluminense Federal University(Niterói, Rio de Janeiro, Brazil). Mice were housed 5 per cage in aroom with a controlled environment, a 12-h light/12-h dark cycle(lights off at 6 pm), and an ambient temperature of 25 ± 2   C(humidity around 40–60%). The animals had free access to filteredtap water and food (™Nuvilab for rodents, Nuvital Nutrients S.A.,Colombo, Paraná, Brazil). Acclimatization was performed for twoweeks before the experimental period began. All procedures werecarried out in accordance with The Ethics Committee for Experi-mental Animals Use and Care (CEA) of Instituto de Biologia RobertoAlcântara Gomes/Universidade do Estado do Rio de Janeiro. TheCEA follow guidelines from Intramural Animal Care and Use(ACU) program of the National Institutes of Health (NIH). The cig-arette smoke protocol was adapted from OECD guidelines for inha-lation studies (Draft Updated Test Guideline 413, 2009).  2.3. Preparation and characterization of açaí stone extract (ASE)E. Oleracea  Mart. fruits were obtained from the Amazon Bay(Belém do Pará, Pará, Brazil) excicata number 29052 MuseuGoeldi–Belem do Para. Many readers may not be aware that theseed of the acai fruit constitutes as much as 80% of the fruit mass.On that matter, some characteristics of this berry are describednext. The açai fruits ( n  = 10) are black, weight 1.612 ± 0.072 gand have an oval shape (1.456 ± 0.032  1.215 ± 0.036 cm). Theaçai stones obtained by removal of the skin of açai fruit arebrown ochre, weigh 1.308 ± 0.051 g and have an oval shape(1.310 ± 0.031  1.090 ± 0.034 cm). Hydroalcoholic extracts wereobtained from a decoction of the berry stone. Approximately200 g of açaí stone were boiled in 400 mL of water for 5 min, mixedfor 2 min, and then boiled again for 5 min. The decoction wascooled to room temperature and then extracted by addition of 400 mL of ethanol with shaking for 2 h. The extract was stored indark bottles inside a refrigerator (4   C) for 10 days. After this mac-eration period, the hydroalcoholic extracts of açaí were filteredthrough #1 Whatman filter paper, and the ethanol was evaporatedusing a rotary evaporator (Fisatom Equipamentos Científicos LtdaSão Paulo, São Paulo, Brazil) under low pressure at 55   C. Theextract was then lyophilized (LIOTOP model 202, FisatomEquipamentos Científicos Ltda São Paulo, São Paulo, Brazil) withtemperature from   30 to   40   C and vacum of 200 mm Hg andfrozen at   20   C until use. The concentration of polyphenols inthe ASE, as measured by analysis of total phenolic content usingthe Folin–Ciocalteau procedure (George et al., 2005), was 255.7 ± 15.3 mg/g ( n  = 4, expressed in gallic acid equivalent) of extract. Typically 100 g of stone yielded approximately 5 g of lyophilized extract.We intended analyzing the composition of ASE by mass spec-troscopy, but the results obtained from this equipment in our insti-tution were not reliable. Thus, we decided to use HPLC instead.Therefore ASE was analyzed on a RP-18 column (250  4 mm,5 l m particles) according to a procedure reported by Peng et al.(2001). Elution was made with solvents A [0.2% (v/v) phosphoricacid] and B [82% (v/v) acetonitrile, 0.04% (v/v) phosphoric acid].Flow rate was 1 ml/min. DAD UV–Vis absorption spectra were re-corded on-line during HPLC analysis. The HPLC elution profile ob-served is strongly indicative of the presence of proanthocyanidins(Peng et al., 2001). The peak eluting at 37.2 min corresponds to cat- echin as confirmed by co injectionof a standard and by comparisonof the ultraviolet absorption spectrum. The late elution (at54.7 min) and UV spectrum of the main peak are consistent withthe presence of polymeric proanthocyanidins (Fig. 1).  2.4. CS exposure The mice were divided into three groups of 20 animals: a con-trol group, a CS group, and a CS + A group. The control group wassham-smoked, i.e. exposed to ambient air using a smoking cham-ber. The CS group was exposed to smoke from 12 commercially-obtained full-flavor (Marlboro – Philips Morris, Santa Cruz, RioGrande do Sul, Brazil) filtered Virginia cigarettes (10 mg of tar,0.9 mg of nicotine, and 10 mg of carbon monoxide) each day for60 days using a smoking chamber as described previously(Menegali et al., 2009; Valenca et al., 2008a, 2004; Valenca and Porto, 2008). Briefly, mice were placed in the inhalation chamber(40 cm long, 30 cm wide, and 25 cm high) inside a laboratory fumehood. A cigarette was coupled to a plastic 60 mL syringe so thatpuffs could be drawn in and subsequently expelled into the expo-sure chamber. One Liter of the puff was expelled into the chamber. 2  R.S. de Moura et al./Food and Chemical Toxicology xxx (2011) xxx–xxx Please cite this article in press as: de Moura, R.S., et al. Addition of açaí ( Euterpe oleracea ) to cigarettes has a protective effect against emphysema in mice.Food Chem. Toxicol. (2011), doi:10.1016/j.fct.2010.12.007  The animals were maintained in this smoke-air condition for 6 min(  3%), and the inhalation chamber was opened, by removing itscover, and the smoke evacuated for 1 min. There was thus expo-sure to four cigarettes i.e. 4  6-min exposures that were separatedby 1-min intervals in which the chamber was cleared of smoke.This procedure was repeated three times per day (morning, noon,and afternoon) for a total of 72 min/day of exposure to CS fromtwelve cigarettes. Each cigarette produced 300 mg/m 3 of total par-ticulate matter in the chamber as measured by weighing the mate-rial collected on Pallflex filters. Immediately after CS exposure,carboxyhemoglobin (COHb) levels were measured using the meth-od described by Beutler and West (1984). Briefly, 1.2 mL hemolyz-ing solution (phosphate buffer, pH 6.85, in water, 1:10) is added to10 mL blood, homogenized and left to stand for 10 min; 100 mL of this solution are pipetted into a test tube containing 2.3 mL reduc-ing solution (25 mg of sodium dithionite in 20 mL of phosphatebuffer, pH 6.85, prepared immediately before use), homogenizedand left to stand for 10 min. Absorbance at 420 and 432 nm is readusing the reducing solution as a reference. Carboxyhemoglobinconcentration was obtained by an equation that considered theAR—the ratio of the absorbance values of the solution at 420 and432 nm considering the factors (reference values) from blood bub-bled with 100% carbon monoxide or 100% oxygen. CS-exposedmice had a mean COHb of 8.9 (± 0.96 [SEM]) versus air-exposedmice with a mean COHb of 1.9 (± 0.31 [SEM]).The CS + A mice were exposed to smoke from 12 commercially-obtained full-flavor filtered Virginia cigarettes plus açaí each dayfor 60 days using the same smoking chamber described above.ASE (100 mg) was mixed with 1 mL saline and injected into eachcigarette (100 mg/cigarette). The cigarettes with açaí were driedat 37   C for 24 h. Exposure to smoke from the cigarettes withaçaí was performed as for CS without açaí. When ‘smoked,’ eachcigarette with açaí produced 370 mg/m 3 of total particulate matterin the chamber as measured by weighing material collected onPallflex filters. The COHb concentration from the CS-A cigaretteswas similar to that from regular CS. The choice of this dose(100 mg/cigarette) was made based on previous data from ourgroup in which 100 mg of açaí (orally) reduced both the influx of inflammatory cells into the mouse lungs and the TNF- a  expressionafter acute cigarette smoke exposure (short-term 5 days, data notpublished yet). The experimental protocol used in the present pa-per was very similar to that one, except by the time of exposure(long-term 60 days and short-term 5 days). We performed thisprotocol with ASE into the cigarettes since we believe that thepresent result is a novelty on this subject.  2.5. Histology and morphometry Twenty-four hours after the final exposure to CS, the mice weresacrificed by cervical displacement and the right ventricle was per-fused with saline to remove blood from the lungs. The lungs from10 mice from each group were fixed with 10% buffered formalinpumped through a tube inserted into the trachea using continu-ously maintained pressure of 25 cm H2O (AVS Projetos, Campinas,São Paulo, Brazil). After addition of formalin, the lungs wereclamped and removed en bloc, after which they were immersedin fixing solution for 48 h, processed in increasing alcohol concen-trations, cleared in xylene, and embedded in paraffin to obtain sec-tions from the apex, middle third, and base of the lungs. Sectionsfrom the right lung (5 l m thick) were stained with hematoxylinand eosin (H&E).Pulmonary emphysema was quantified based on the degree of alveolar destruction as determined by measuring the mean linearintercept in micrometers ( L m ; mean alveolar diameter) as de-scribed previously (Valenca and Porto, 2008). In brief, this involves determining the number of times the gas exchange structures inthe parenchyma intersect a series of grid lines. When emphysemais present,there arefewer intercepts of the alveolarstructureswiththe grid, indicating alveolar destruction. The  L m  is obtained usingthe equation  L m  =  L tot / L i , where  L tot  is the total length of the linesin the microscope field and  L i  is the number of intercepts of thealveolar structures with the grid lines. To obtain the  L m , 16 fields Fig. 1.  HPLC analysis of ASE. We used a RP-18 column (250  4 mm, 5 l m particles). Elution was made with solvents A [0.2% (v/v) phosphoric acid] and B [82% (v/v)acetonitrile, 0.04% (v/v) phosphoric acid]. Flow rate was 1 ml/min. The peak eluting at 37.2 min corresponds to catechin as confirmed by co injection of a standard and bycomparison of the ultraviolet absorption spectrum. The late elution (at 54.7 min) and UV spectrum of the main peak are consistent with the presence of polymericproanthocyanidins. R.S. de Moura et al./Food and Chemical Toxicology xxx (2011) xxx–xxx  3 Please cite this article in press as: de Moura, R.S., et al. Addition of açaí ( Euterpe oleracea ) to cigarettes has a protective effect against emphysema in mice.Food Chem. Toxicol. (2011), doi:10.1016/j.fct.2010.12.007  in each section were counted and observed at 200  magnificationthrough a grid attached to the monitor. We used three sections permouse and five mice per group.  2.6. Bronchoalveolar lavage The lung air spaces were washed three times with buffered sal-ine solution (500 l L) for a final bronchoalveolar lavage (BAL) fluidvolume of 1.2–1.5 mL). The collected BAL fluid was stored on ice.The total number of cells in the BAL fluid was determined usinga Neubauer chamber. Differential cell counts were performed oncytospin cell preparations (Cientec CT 2000, Piracicaba, São Paulo,Brazil) stained with Panótico. At least 200 cells per BAL samplewere counted using standard morphologic criteria. After BAL, thelungs were removed immediately, homogenized on ice with 10%(w/v) 0.1 M potassium phosphate buffer (pH 7.4) using a tissuehomogenizer (NT136; Campinas, São Paulo, Brazil), and centri-fuged at 800  g   for 5 min. The supernatants were stored at   20   Cfor biochemical analysis. The protein concentration in the lunghomogenate samples was determined by the Bradford method(Bradford, 1976).  2.7. Superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx) activity determination SOD, CAT, and GP  x  activities were determined in the lunghomogenates as follows. SOD activity was assayed by measuringinhibition of adrenaline auto-oxidation as absorbance at 480 nm(Bannister and Calabrese, 1987) using a Beckman spectrophotom-eter (model DU 640; Beckman Instruments, Fullerton, CA, USA).CAT activitywasmeasured bythe rateof decrease of hydrogenper-oxide concentration at 240 nm (Aebi, 1984). GP  x  activity was mea-sured by monitoring the oxidation of NADPH at 340 nm in thepresence of hydrogen peroxide (Flohe and Gunzler, 1984).  2.8. Reduced glutathione (GSH) and glutathione disulfide (GSSG) assay This assay was based on the reaction of GSH or GSSG withDTNB, which produces the TNB chromophore (Rahman et al.,2006). To determine GSSG, lung homogenate samples were treatedwith 2-vinylpyridine, which covalently reacts with GSH (but notGSSG). The excess 2-vinylpyridine is neutralized with triethanola-mine. The rate of formation of TNB, measured at 412 nm, is propor-tional to the concentration of GSH or GSSG in the sample. Theconcentration of an unknown sample is determined using the lin-ear equation or the regression curve generated by measuring sev-eral knownconcentrations of GSH orGSSG. The finaldeterminationis presented as the GSH/GSSG ratio.  2.9. Myeloperoxidase (MPO) assay MPO activity was measured using hydrogen peroxide, HTAB,and TMB. Initially 100 l L of a lung homogenate sample was centri-fuged with 900 l L of HTAB at 14,000g for 15 min. The supernatant(75 l L) was incubated with 5 l L of TMB for 5 min at 37   C. Themixture was then incubated with 50 l L of hydrogen peroxide for10 min at 37   C, after which 125 l L of sodium acetate buffer wasadded. The reaction was read using a microplate reader (Bio-Radmodel 550, Hercules, CA, USA) at 630 nm (Suzuki et al., 1983). The concentration of MPO in the samples was determined usinga standard curve established using purified MPO.  2.10. Western blotting (WB) Proteins from lung homogenate samples were denatured byaddition of sample buffer (50 mM Tris–HCl, pH 6.8, 1% sodiumdodecylsulfate, 5% 2-mercaptoethanol, 10% glycerol, 0.001%bromophenol blue) and heating in boiling water for 3 min. Proteins(50 l g) were resolved by 15% sodium dodecylsulfate polyacryl-amide gel electrophoresis, and the proteins were transferred tonitrocellulose membranes(Invitrogen, Carlsbad,CA, USA). Rainbowmarkers (Amersham Pharmacia Biotech, Piscataway, NJ, USA) wererun in parallel for estimation of molecular weights. The mem-branes were blocked with Tween-TBS (20 mM Tris–HCl, pH 7.5,500 mM sodium chloride, 0.5% Tween-20) containing 2% bovineserum albumin and probed with specific primary antibodies (SantaCruz Biotechnology, Santa Cruz, CA, USA): goat anti-mouse4-hydroxynonenal (1:2000), goat anti-mouse metalloelastase(1:2000), and goat anti-mouse neutrophil elastase (1:3000). Afterextensive washing in TBS-Tween, the membranes were incubatedwith biotin-conjugated donkey anti-goat immunoglobulin G(1:10000) for 1 h (SantaCruz Biotechnology, Santa Cruz, CA, USA)and then developed with the ECL Western Detection Reagent(Amersham Pharmacia Biotech, Piscataway, NJ, USA) according tothe manufacturer’s instructions. The bands were assessed by den-sitometry using Scion Image Software (Scion Co., Frederick, MD,USA).  2.11. Statistical analysis The data are expressed as mean ± SEM. For comparison of   L m ,BAL cells, antioxidant enzyme activities, MPO, and densitometry(WB) in the control, CS, and CS + A groups, the data were analyzedwith one-way analysis of variance (ANOVA) followed by Tukey’spost hoc test (  p  < 0.05). To compare the GSH/GSSG ratio in thecontrol, CS, and CS + A groups, the data were analyzed by theKruskal–Wallis test followed by Dunn’s post hoc test (  p  < 0.05).Statistical analyses were performed using GraphPad Prism soft-ware (GraphPad Prism version 5; GraphPad Software, SanDiego,CA, USA). 3. Results  3.1. Histopathological investigation Pulmonary tissue analysis showed that the control group mice,who were exposed to ambient air, had normal-sized air spaces andnormal alveolar septa (Fig. 2a). The alveolar spaces were enlargedin mice in the CS group, and leukocytes were numerousin the alve-oli (Fig. 2b). The lung parenchyma of the CS + A group had someenlarged areas and some leukocytes in the alveoli (Fig. 2c). Thelung histology in the CS + A group mice was not to the same asin the control group mice, but appeared less affected by emphy-sema than did the lungs of mice in the CS group.Alterations in the lung parenchyma were quantified based onthe  L m  (Fig. 3). The  L m  was 38% greater in the CS group comparedto the control group (  p  < 0.001). The  L m  was 25% lower in theCS + A group compared to the CS group (  p  < 0.01) but was not sig-nificantly different from the  L m  in the control group.  3.2. CS+A modulates cell influx BAL fluid from mice exposed to CS showed a 400% increase inleukocytes compared with the control group (  p  < 0.001) (Fig. 4).Mice exposed to CS + A showed 65% fewer leukocytes in the BAL fluid compared to the CS group (  p  < 0.001). There were 140% morealveolar macrophages (Fig. 5) in the BAL fluid of CS mice comparedwith the control group (  p  < 0.001), and 100x more neutrophils(Fig. 6) (  p  < 0.001). Mice exposed to CS + A showed 44% fewer mac-rophages in the BAL fluid compared with the CS group (  p  < 0.001),while there were 83% fewer neutrophils (  p  < 0.001). However, 4  R.S. de Moura et al./Food and Chemical Toxicology xxx (2011) xxx–xxx Please cite this article in press as: de Moura, R.S., et al. Addition of açaí ( Euterpe oleracea ) to cigarettes has a protective effect against emphysema in mice.Food Chem. Toxicol. (2011), doi:10.1016/j.fct.2010.12.007  there were more neutrophils in BAL fluid from CS + A mice thanfrom control mice (  p  < 0.01).  3.3. CS+A reduces oxidative stress Results of biochemical analysis of oxidative stress parametersare shown in Table 1. SOD activity decreased 34% (  p  < 0.001) inthe CS group and 50% (  p  < 0.001) in the CS + A group comparedwith the control group. CAT activity decreased 16% (  p  < 0.001) inthe CS group and 28% in the CS + A group compared with the con-trol group. GP  x  activity decreased 74% (  p  < 0.001) in the CS groupcompared with the control group. There was a 5   increase inGP  x  activity in the CS + A group (  p  < 0.001) than in the CS group.The GSH/GSSG ratio decreased 48% (  p  < 0.01) in the CS group com-pared with the control group and increased 78% (  p  < 0.01) in theCS + A group compared with the CS group. Finally, MPO increased123% (  p  < 0.001) in the CS group compared with the control group.MPO was reduced 43% in the CS + A group compared with the con-trol group (  p  < 0.001). Western blotting for 4-hydroxynonenal (4-HNE) expression (Fig. 7) was performed to confirm the biochemicalanalysis of oxidative stress markers. Expression of 4-HNE was in-creased in the CS group compared with the control group(  p  < 0.05), but not significantly different in the CS + A group com-pared to the control group.  3.4. CS+A reduces elastase expression Metalloelastase (MMP-12 or macrophage elastase) and neutro-phil elastase levels were examined using Western blotting; these Fig. 2.  Photomicrographs of lung sections stained with hematoxylin and eosin (400  magnification). (a) Control group mice were exposed to ambient air. (b) CS group micewere exposed to smoke from twelve cigarettes per day for 60 days. (c) CS + A group mice were exposed to smoke from twelve açaí cigarettes per day for 60 days. Açaí extract(100 mg) was mixed with 1 mL of saline and injected into each cigarette. A = alveoli; AD = alveolar duct; EA = enlarged areas; arrows = leukocytes. Fig. 3.  Morphometry of lung parenchyma. Control group mice were exposed toambient air. CS group mice were exposed to smoke from twelve cigarettes per dayfor 60 days. CS + A group mice were exposed to smoke from twelve açaí cigarettesper day for 60 days. Açaí extract (100 mg) was mixed with 1 mL of saline and theninjected into each cigarette.  L m , the mean linear intercept, indicates the degree of alveolar destruction. Data are expressed as means ± SEM ( n  = 10 for each group) andwere analyzed by the Kruskal–Wallis test followed by Dunn’s post hoc test(  p  < 0.05).  ⁄⁄⁄  p  < 0.001 compared with the control group.  ##  p  < 0.01 compared withthe CS group. Fig. 4.  Leukocytes in bronchoalveolar lavage fluid (BALF). Control group mice wereexposed to ambient air. CS group mice were exposed to smoke from twelvecigarettes per day for 60 days. CS + A group mice were exposed to smoke fromtwelve açaí cigarettes per day for 60 days. Açaí extract (100 mg) was mixed with1 mL of saline and injected into each cigarette. Data are expressed as means ± SEM( n  = 10 for each group) and were analyzed by one-way ANOVA followed by Tukey’spost hoc test (  p  < 0.05).  ⁄⁄⁄  p  < 0.001 compared with the control group.  ###  p  < 0.001compared with the CS group. R.S. de Moura et al./Food and Chemical Toxicology xxx (2011) xxx–xxx  5 Please cite this article in press as: de Moura, R.S., et al. Addition of açaí ( Euterpe oleracea ) to cigarettes has a protective effect against emphysema in mice.Food Chem. Toxicol. (2011), doi:10.1016/j.fct.2010.12.007
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