Prolonged caffeine intake decreases alveolar bone damage induced by binge-like ethanol consumption in adolescent female rats

Con en s lis s a ailable a ScienceDi ec Biomedicine & Pha maco he apy jou nal homepage: www.else ie .com/loca e/biopha O iginal a icle P olonged caffeine in ake dec eases al eola bone damage induced by binge- like e hanol consump ion in adolescen emale a s C is iane Maia a, *, B uno Gonçal es Pinhei o a , Ca la C is iane Soa es da Sil a a , Rod igo A. Cunha b , Deiweson Souza-Mon ei o c , Ma ia Ka olina Ma ins Fe ei a c , Tuany Ra aeli Schmid d , Gab iela de Souza Balbino e , Fab ício Mezzomo Colla es e , Manoela Domingues Ma ins d , Ra ael Rod igues Lima c a Labo a o y o Pha macology o Inflamma ion and Beha io , Facul y o Pha macy, Ins i u e o Heal h Science, Fede al Uni e si y o Pa á, Belém, Pa á, B azil b CNC-Cen e o Neu osciences and Cell Biology, Facul y o Medicine, Uni e si y o Coimb a, Po ugal c Labo a o y o Func ional and S uc u al Biology, Biological Science Ins i u e, Fede al Uni e si y o Pa á, Belém, Pa á, B azil d Depa men o O al Pa hology, School o Den is y, Fede al Uni e si y o Rio G ande do Sul, Po o Aleg e, B azil e Depa men o Den al Ma e ials, School o Den is y, Uni e sidade Fede al do Rio G ande do Sul, Po o Aleg e, B azil ARTICLE INFO Keywo ds: Binge d inking Caffeine Adenosine A 2A ecep o Adolescence Al eola bone loss Pe iodon i is ABSTRACT E hanol consump ion has been epo ed o nega i ely impac on pe iodon al disease. In pa icula , o al ca i y diso de s occu upon e hanol exposu e du ing adolescence, a li e pe iod associa ed wi h pa icula pa e ns o sho and in ense (‘binge-like’) e hanol consump ion ha is mos dele e ious o o al heal h. The haza dous cen al effec s o e hanol ha e been linked o he o e unc ion o adenosine ecep o s, which a e an agonized by caffeine, a bioac i e subs ance p esen in nume ous na u al nu ien s, which can also modi y bone me abolism. The aim o his s udy was o in es iga e he effec s o caffeine on al eola bone damage induced by an e hanol binge d inking pa adigm du ing adolescence. Female Wis a a s (35 days old; n = 30) we e alloca ed o six g oups: con ol ( ehicle), e hanol (3 g/kg/day; 3 days On-4 days Offchallenge), caffeine (10 mg/kg/day), caffeine plus e hanol, SCH58261 (0.1 mg/kg/day, an an agonis o A 2A ecep o s), and SCH58261 plus e hanol. Bone mic omo phology and e ical bone loss we e analyzed by compu ed mic o omog aphy. Ou da a showed ha e hanol binge d inking educed al eola bone quali y, wi h epe cussion on al eola bone size. This e hanol- induced al eola bone de e io a ion was ab oga ed upon ea men wi h caffeine, bu no wi h SCH58261. This shows ha caffeine p e en ed he pe iodon al diso de caused by e hanol binge d inking du ing adolescence, an effec ha was no media ed by adenosine A 2A ecep o blockade. 1. In oduc ion Alcohol consump ion among adolescen s affec s 58 % o indi iduals be ween 17–18 yea s old [1]. In he Uni ed S a es, nea 401,000 ado- lescen s (12–17 yea s old) suffe ed om Alcohol Use Diso de s (AUD) in 2018 [2]. Al hough emale consume lowe amoun s o alcohol pe occasion, young women consume inc easing amoun s o e hanol in some coun ies, wi h ends o d inking like men bu wi h mo e medical p oblems han men [2–6]. High alcohol consump ion is known o affec he neu obeha io al sys em [1], wi h epe cussions on o he body sys ems [7]. In pa icula , i has p e iously been p oposed ha alcohol consump ion is a p ominen ac o impac ing on al eola bone heal h since he exposu e o alcohol can igge o agg a a e pe iodon i is [8,9]. The ampli ude o he damaging effec o alcohol exposu e on bones is dependen on he dose, he d inking pa e n, and he age o he subjec s [10,11]. Al hough li le is known abou he mechanisms un- de lying he effec s o alcohol exposu e on al eola bone loss in he absence o o he damaging ac o s [12,13], i is wo h no ing ha an inc eased ulne abili y o he ha m ul effec s alcohol occu s du ing adolescence [14,15]; howe e , ew s udies ha e ocused on he ado- lescence pe iod. Since 2015, ou g oup has been s udying he consequences o di - e en pa e ns o alcohol exposu e on he o al ca i y du ing adoles- cence in animal models [14,16,17]. Fi s , we employed a hea y d inking exposu e pa adigm in adolescen a s (6.5 g/kg/day om 35 days o age un il 90 days) and obse ed ha alcohol adminis a ion h ps://doi.o g/10.1016/j.biopha.2020.110608 Recei ed 31 May 2020; Recei ed in e ised o m 16 July 2020; Accep ed 2 Augus 2020 ⁎ Co esponding au ho a : Labo a ó io de Fa macologia da Inflamação e do Compo amen o, Ins i u o de Ciências da Saúde, Uni e sidade Fede al do Pa á, Rua Augus o Co êa 1, Campus do Guamá, Belém, Pa á, 66075-900, B azil. E-mail add esses: [email p o ec ed],[email p o ec ed] (C. Maia). Biomedicine & Pha maco he apy 130 (2020) 110608 A ailable online 09 Augus 2020 0753-3322/ © 2020 The Au ho s. Published by Else ie Masson SAS. This is an open access a icle unde he CC BY-NC-ND license (h p://c ea i ecommons.o g/licenses/by-nc-nd/4.0/). T induced al eola bone loss e en in he absence o expe imen ally-in- duced pe iodon i is [14]. Nex , we es ed a binge d inking p o ocol (3 days on- 4 days off), which models he pa e n o alcohol exposu e among adolescen emale subjec s, and we ep oduced he p e iously desc ibed pa e n o de e io a ion o sali a y glands and al eola bone heal h [17–19]. The exac mechanism ha unde lies he nega i e influences o al- cohol in ake on al eola bone heal h is unclea . P o-inflamma o y e - ec s, oxida i e damage, as well as a nega i e ac ion on bone e- modeling ha e been p oposed in he pa hogenesis o he al eola bone damage [9,10,20,21]. Among se e al p o-inflamma o y pa hways, in- ducible ni ic oxide syn hase (iNOS) o e exp ession and i s final p o- duc ni ic oxide (NO) ha e been obse ed in inflamed pe iodon al issue [22]. One modula ion sys em able o con ol he o ma ion o NO is ope a ed by adenosine, as epo ed in se e al cells [23–25]. Ac- co dingly, adenosine can influence inflamma o y p ocesses in pe iod- on al diseases [26–28]. Gingi al issues o humans ha e been ound o possess diffe en adenosine ecep o s, namely A 1 R, A 2A R, and A 2B R [28]. A 2A R ha e been associa ed wi h appa en ly con adic o y effec s, igge ing o educing p o-inflamma o y signaling [29,30]. In an ex- pe imen al pe iodon i is s udy, A 2A R ac i a ion educed pe iodon al inflamma ion p ocesses, dec easing he elease o p o-inflamma o y media o s [31]. On he con a y, he ac i a ion o os eoblas A 2A R igge s os eoclas ogenic cy okine p oduc ion, associa ed wi h educed bone u no e and educed bone quali y [32]. These pa adoxical effec s o adenosine ecep o s on inflamma ion p obably esul om he up- egula ion o he adenosine pa hways, which igge p o-inflamma o y cascades and issue damage in ch onic inflamma ion o upon inju y [33]. Thus, he e idences sugges ha he ac i a ion o adenosine e- cep o s may play a pi o al ole on epi helial issues diso de s, including ch onic pe iodon i is [34]. Alcohol in ake can also al e adenosine signaling in nume ous is- sues [35–37]. Howe e , he in ol emen o adenosine ecep o s in e hanol-induced pe iodon i is has no been es ed in spi e o he known p esence o adenosine signaling in he o al ca i y. One way o gauge he in ol emen o he adenosine modula ion sys em is o s udy he effec o he bioac i e compoud caffeine (1,3,7- ime hylxan hine), which main mechanism o ac ion is he an agonism o adenosine ecep o s ( o e iew see [38]). The effec o caffeine on bone me abolism has been desc ibed, wi h a nega i e impac obse ed a high doses (equi alen o 6 cups o co ee pe day o mo e [39,40];). Thus, high doses o caffeine agg a a e al eola bone loss in pe iodo al disease, bu do no induce al eola bone eso p ion in he absence o expe imen al pe iodon i is in adul o aged a s [41,42]. Impo an ly, an epidemiological s udy sugges ed ha he dose o caffeine is a key a iable ha de e mines he posi i e e sus nega i e effec s o caffeine on pe iodon al heal h [43]. Howe e , he impac o lowe doses o caffeine, equi alen o he mos common pa e n o in ake in humans (i.e. 2–3 cups o coffee/day) on o al heal h is s ill o be adequa ely cha ac e ized, a ques ion o pa icula ele ance, since i is hese lowe doses o caffeine (10 mg/kg/day in oden s) ha affo d a obus p o- ec ion agains diffe en heal h ea hs ( e iewed in [44]). We now hypo hesize ha he daily in ake o low doses o caffeine in emale a s could dampen he al eola bone loss caused by alcohol exposu e du ing adolescence. 2. Ma e ial and me hods 2.1. Animals Adolescen emale Wis a a s (28 days-old) om E and o Chagas Ins i u e (IEC/PA) animal acili y, we e andomly g ouped and kep in polyca bona e cages (n = 3–4 a s pe cage) in a oom unde con olled en i onmen al condi ion ( empe a u e 25 ± 1 °C and 12 h ligh /da k cycle, wi h ligh s on 6:00 a.m. Animals ecei ed ood and wa e ad libi um. All p ocedu es ollowed he NIH Guide o he Ca e and Use o Labo a o y Animal and we e app o ed by he E hics Commi ee on Expe imen al Animals o he UFPA (license numbe 8324250716). 2.2. Expe imen al ea men s Expe imen al p ocedu es s a ed when a s we e 35 days old. The binge d inking pa adigm consis ed o 4 sessions o e hanol binge-like exposu e (3 days On-4 days Off; 20 % w/ ) o dis illed wa e (con ol g oup) by ga age [45,46]. E hanol o dis illed wa e was adminis e ed once a day in he mo ning. Twen y- ou hou s a e he fi s binge Fig. 1. Schema ic expe imen al design. C. Maia, e al. Biomedicine & Pha maco he apy 130 (2020) 110608 2 d inking session, he g oups exposed o e hanol o dis illed wa e we e u he di ided in o 3 subg oups. Two subg oups challenged wi h e hanol as well he wo subg oups exposed o dis illed wa e ecei ed daily by o al ou e ei he caffeine (Sigma-Ald ich), a a low o mode a e dose (10 mg/kg/day), o dis illed wa e o 21 days (59-days old). The final subg oups de i ed om e hanol o dis illed wa e g oups ecei ed in ape i oneally he A 2A R an agonis SCH58261 (0.1 mg/kg/day) un il hey we e 59-days old (30 min be o e eu hanasia; Fig. 1). Body weigh gain (%) was measu ed h oughou he expe imen al ea men s. This expe imen al design esul ed in 6 g oups o a s: con ol, e hanol, ca - eine, e hanol plus caffeine, SCH58261, and e hanol plus SCH58261. The dose o caffeine was based on he ela ionship es ablished by [47], defining ha a dose o 10 mg/kg in a s is equi alen o 250 mg/ kg in humans (2–3 cups o a be e age). This limi s he pha macological ac ion o caffeine o he non-selec i e an agonism o adenosine e- cep o s [47]. The dose o SCH58261 (0.1 mg/kg) is defined as sup a- maximal and selec i e o an agonize A 2A R[48], affo ding obus p o- ec i e h ough A 2A R an agonism in diffe en animal models ( e iewed in [49]). On he 59 h pos na al day, a s we e anes he ized by in ape i oneal injec ion o xylazine 2% (2 mg/mL) plus ke amine 10 % (10 mg/mL) and hei jaws we e collec ed. The le hemimandibules we e main- ained in 4% o mol solu ion o mic o-compu ed omog aphy (mic o- CT) e alua ion. 2.3. Mic o-compu ed omog aphy (mic o-CT) analysis The le hemimandibules we e submi ed o x- ay mic o-CT (Mic oCT.SMX-90 CT; Shimadzu Co p., Kyo o, Japan). Each sample was posi ioned e ically on a o a o y de ice and he images we e ob ained unde a o a ion o 360° (in ensi y 70 kV; 100 mA) and econs i u ed by inspeXio SMX-90CT so wa e (Shimadzu Co p., Kyo o, Japan), om a o al o 541 images pe sample ( oxel 10 μm; esolu ion 1024 × 1024; hickness 14 μm). Heigh e alua ion o al eola bone loss was analyzed using he RadiAn DICOM Viewe 5.0.1 (Medixan , Poznan, Poland) so wa e o 3D econs uc ion. T idimensional models we e se led on a s anda d posi ion (i.e., lingual and es ibula oo h ace isible). The e ical bone loss was measu ed by he dis ance be ween he cemen um-enamel junc ion and he al eola bone c es dis ance. Six poin s we e e alua ed on he fi s in e io mola , (i.e., mesiolingual, midlingual, dis olingual, mesio es ibula , mid es ibula , dis o es ibula ). The a e age alues o each oo h we e conside ed [50]. Al eola bone issue quali y was analyzed by ImageJ®so wa e (Na ional Ins i u es o Heal h, Be hesda, MD, USA) on a se ies o 50 images om he in e io fi s mola al eola bone egion. The in e - adicula egion o he in e io fi s mola wi h a mean leng h o 0.200 mm 2 om he ce ical hi d un il he middle hi d o he oo , was chosen as he egion o in e es (ROI). Segmen a ion was pe o med on he images wi h a g ay scale h eshold anging om 120 o 250. Mo phome ic da a was collec ed by measu ing he abecula numbe , abecula hickness, abecula sepa a ion, and bone olume ac ion (bone olume/ issue olume) using he BoneJ plug-in [51]. 2.4. Raman spec oscopy To analyze he chemical s uc u e o al eola bone a e he ea - men s, he PO 43− /AmideI a io was calcula ed by Raman spec oscopy. The hemimandibles we e sec ioned pe pendicula ly o he bone a he cen e o he in e io fi s mola . A 1 mm- hick slice was placed on a Raman spec ome e (Sen e a, B uke Op ics, E ingen, Ge many). Analysis was pe o med wi h a 785 nm lase a 100 mW on a s an- da dized a ea on he al eola bone c es . Fo y-fi e poin s we e e al- ua ed om he al eola c es o he cen e o he al eola bone wi h a s anda dized dis ance (100 μm). The Raman spec a we e ob ained and he PO 43− peak a 960 cm -1 as well as he amideI peak a 1660 cm -1 we e in eg a ed and used o calcula e he PO 43- :amideI a io. 2.5. S a is ical analyses Da a a e shown as mean ± S.E.M. (n = 5 animals pe g oup o mic o-CT e alua ion and n = 3 o Raman analysis). A Shapi o-Wilk es was pe o med o e i y no mali y o da a dis ibu ion. The es powe was calcula ed h ough he diffe ence be ween wo mean by OpenEpi (Ve sion 2.3.1), wi h a ype I e o o 5% and a powe o 80 %. S a is ical compa ison was conduc ed by one-way ANOVA ollowed by Sidak's mul iple compa isons pos hoc es . The analyses o body weigh gain (%) we e ca ied ou using a wo-way ANOVA epea ed measu e ollowed by Tukey’spos hoc es . P < 0.05 was conside ed as s a is i- cally significan diffe ences. 3. Resul s 3.1. E hanol binge d inking and/o caffeine in ake du ing adolescence do no modi y body weigh The exposu e o ei he e hanol binge-like wi hou o wi h ei he caffeine o he A 2A R an agonis SCH58261, did no modi y he a e age body weigh gain o emale a s du ing he ea men pe iod (Fig. 2). 3.2. Long- e m caffeine in ake p e en s he dec ease o al eola bone quali y and densi y caused by e hanol binge d inking du ing adolescence Ini ially, we explo e he effec s o caffeine on he al eola bone de imen al consequences o adolescence binge d inking. Repea ed exposu e o e hanol binge in ake du ing adolescence caused a educ ion Fig. 2. Effec s o caffeine o o he adenosine A 2A ecep o (A 2A R) an agonis (SCH58261) on adolescen binge-like e hanol challenge on he body weigh gain o emale Wis a a s (59-days-old, n = 5 animals/g oup). Expe imen al g oups consis ed o Con ol g oup (dis illed wa e ; 3 days on-4 days off; 35 h o 59 h pos na al days); E hanol g oup (3.0 g/kg/ day; 3 days on-4 days off; 35 h o 59 h pos na al days); A 2A R an agonis g oup (SCH58261 0.1 mg/kg/day daily; 38 h o 59 h pos na al days; An ); A 2A R an agonis plus e hanol g oup (SCH58261 0.1 mg/kg/day daily plus binge-like ea men ; 38 h o 59 h pos na al days; An + E hanol); Caffeine g oup (caffeine 10 mg/kg/day daily; 38 h o 59 h pos na al days; CAF); and Caffeine plus e hanol g oup (caffeine plus binge- like ea men ; CAF + E hanol). Values a e exp essed as he pe cen age o body weigh gain om day 0 (100 %) un il he 4 h week o ea men . No s a is ical diffe ences a p < 0.05 using a wo-way ANOVA epea ed measu e ollowed by Tukey’spos hoc es s. C. Maia, e al. Biomedicine & Pha maco he apy 130 (2020) 110608 3 o abecula hickness compa ed o con ol a s (p = 0.0037). Caffeine ea men mi iga ed his dec eased abecula hickness, which eached alues equi alen o hese o con ol animals (Fig. 3A). In addi ion, no s a is ically significan diffe ence o abecula hickness (p > 0.05) was ound o caffeine g oup when compa ed o he con ol subjec s. T abecula sepa a ion was inc eased in a s subjec o binge e hanol d inking (p < 0.0001). Ch onic adminis a ion o caffeine o e hanol- challenged a s amelio a ed abecula sepa a ion inc emen , bu his pa ame e did no each he alues measu ed in he con ol g oup (p = 0.0086, e hanol s. e hanol plus caffeine; p = 0.0279, con ol s. e hanol plus caffeine; Fig. 3C). In addi ion, no s a is ically significan diffe ence o abecula sepa a ion (p > 0.05) was ound o caffeine g oup when compa ed o he con ol subjec s. The abeculae numbe was no modified by ei he e hanol binge d inking o caffeine, since all es ed g oups p esen ed alues simila o he con ol g oup (p > 0.05; Fig. 3B). The pe cen age o bone olume ac ion (BV/TV) was educed by e hanol binge d inking (p < 0.0001). No ably, caffeine in ake by e hanol-challenged a s a enua ed his bone loss caused by e hanol binge d inking du ing adolescence, esul ing in bone densi y le els si- mila o con ol a s (p < 0.0001, con ol s. e hanol; p = 0.0023, e hanol s. e hanol plus caffeine; Fig. 3D). In addi ion, no s a is ically significan diffe ence o bone densi y (p > 0.05) was ound o caffeine g oup when compa ed o he con ol subjec s. 3.3. The selec i e an agonism o A 2A R does no ep oduce he posi i e effec s o ch onic caffeine ea men on binge e hanol d inking du ing adolescence To es i A 2A R we e media ing he benefi s o caffeine on he ne- ga i e effec s on bone mic omo phology caused by e hanol binge in ake du ing adolescence, we es ed he A 2A R an agonis SCH58261. Fig. 4 shows ha all he pa ame e s o bone quali y ha we e de e io a ed by e hanol binge-d inking (i.e., abecula hickness, abecula sepa a ion, and bone densi y) we e no eco e ed by ea men wi h he A 2A R an- agonis , SCH58261. In e es ing, SCH58261 pe se (i.e. in he absence o e hanol challenge) educed abecula hickness (p = 0.0001), bu no abecula sepa a ion and bone densi y (Fig. 4). 3.4. Caffeine eco e s al eola bone loss elici ed by e hanol binge d inking du ing adolescence Fig. 5(AEe) shows ep esen a i e 3D econs uc ions o hemi- mandibules om each expe imen al g oup, and also displays he Fig. 3. Effec s o caffeine on he impac o adolescen binge-like e hanol challenge on he al eola bone quali y o emale Wis a a s (59-days-old, n = 5 animals/ g oup). Expe imen al g oups consis ed o he Con ol g oup (dis illed wa e ; 3 days on-4 days off; 35 h o 59 h pos na al days); E hanol g oup (3.0 g/kg/day; 3 days on-4 days off; 35 h o 59 h pos na al days); Caffeine g oup (caffeine 10 mg/kg/day daily; 38 h o 59 h pos na al days; CAF); and Caffeine plus e hanol g oup (caffeine plus binge-like ea men ; CAF + E hanol). The ep esen a i e image o he in e adicula egion, close o he u ca ion a ea, was chosen as he s anda dized egion o in e es (ROI) o he analyses (le posi ion). Abo e, ep esen a i e ROI h ee-dimensional images o he analyzed g oups. Values a e mean ± SEM o mic o-CT pa ame e s: (A) abecula hickness (Tb.Th; mm); (B) mean numbe o abecula pe uni leng h (Tb.N); (C) abecula sepa a ion (Tb.Sp; mm); and (D) he pe cen age o bone olume in ela ion o he o al measu ed a ea (BV/TV; %). *p < 0.05 compa ed o con ol g oup; **p < 0.01 compa ed o con ol g oup; ****p < 0.0001 compa ed o con ol g oup; ##p < 0.01 compa ed o e hanol g oup. One-way ANOVA ollowed by Sidak’spos hoc es , p < 0.05. Scale ba : 1 mm. C. Maia, e al. Biomedicine & Pha maco he apy 130 (2020) 110608 4 a e age quan ifica ions o he dis ance be ween he cemen um-enamel junc ion and he al eola bone c es o he fi s in e io mola (Fig. 5F). The da a show ha e hanol binge d inking du ing adolescence educed hese quan ified dis ances, implying ha he e was an al eola bone loss (p = 0.0330). Impo an ly, ch onic adminis a ion o caffeine de- c eased his al eola bone loss induced by binge-like e hanol d inking (p = 0.0272), whe eas he e was no modifica ion in he p esence o he selec i e A 2A R an agonis SCH58261 (p > 0.005). 3.5. Mine al con en was influenced by he p esence o e hanol Fig. 6 shows he mine al:ma ix a io (PO4 3− :amideI) ob ained in he Raman measu emen s o he al eola bone c es o ea ed animals (Fig. 6A). Regions wi h yellow/o ange in ensi y indica e highe alues o he mine al/ma ix a io, whe eas egions wi h blue in ensi y ep esen lowe alues. In he con ol g oup, highe alues we e mo e equen ly obse ed in compa ison o he o he g oups ha p esen ed mos ly g een/blue egions. Howe e , no s a is ically significan diffe - ence was obse ed on he a e age mine al:ma ix a io be ween g oups (Fig. 6B). The Raman spec oscopical mapping was used o p obe po- en ial diffe ences in he mine al:ma ix a io a he al eola c es (Fig. 6C). 4. Discussion This s udy assessed he effec s o low o mode a e caffeine in ake on al eola bone damage induced by e hanol binge d inking in adolescen Wis a a s. Ou esul s demons a e, in an unp eceden ed way, ha caffeine mi iga es he nega i e effec s on al eola bone induced by e hanol exposu e du ing adolescence. Ou da a also indica ed ha his beneficial effec o caffeine was unlikely o be media ed by he ade- nosine A 2A ecep o pa hway. Al hough solely based on he analysis o an animal model, ou findings highligh he possible damage o binge-like e hanol in ake o he o al heal h o adolescen s, ce ainly a p oblem o socie al ele ance. Fi s ly, he Na ional Ins i u e on Alcohol Abuse and Alcoholism has s essed he ex en o e hanol exposu e du ing adolescence and i s ne- ga i e epe cussion on nume ous issues and o gans [2]. In he Uni ed S a es, nea ly 401,000 adolescen s (12–17 yea s old) suffe ed om Alcohol Use Diso de s (AUD) in 2018 [2]. The consequences o his o e sp ead in oxica ion o he s oma ogna hic sys em is unce ain. On he o he side, caffeine is p esen in se e al be e ages consumed in nume ous cul u ally diffe en socie ies [52]. I s heal h benefi s ha e been b oadly epo ed, which implies ha a nu i ional s yle o li e can coun e balance haza dous ou comes elici ed by oxican s [52], e en- ually wi h he excep ion o p egnancy whe e caffeine in ake has been epo ed o al e me abolism [53] and b ain wi ing [54] in animal models. The abili y o e hanol exposu e o damage al eola bone has p e- iously been epo ed [8,9]. In ac , s udies om ou g oup ha e con- cluded ha alcohol exposu e causes damage and al eola bone loss in he absence o addi ional damaging ac o s upon hea y o binge d inking pa adigms, espec i ely in a s [14,19]. Ou esul s co obo- a e hese findings, demons a ing ha ou sessions, each o 3 days ON- 4 days OFF, o e hanol in ake du ing adolescence un il adul hood, can igge al eola bone damage in emale a s. In addi ion, he body weigh gain was no modified by e hanol o adenosine ecep o ligands adminis e ed in he p esen s udy, which excludes malnu i ion as a con ibu ing ac o o he obse ed esul s. Al eola bone p esen s unique ea u es wi h specific cha ac e is ics ha dis inguishes i om o he bones, which a e equi ed o p o ide physiological suppo o oo h ac i i y [55]. The analysis o mic o- a chi ec u al pa ame e s cons i u es he main me hodology o e alua e al eola bone mo phology and heal h [56]. Specifically, al eola bone is a compac bone wi h la ge abecula hickness, and educed abe- cula numbe and abecula sepa a ion, which eflec s an high bone mass and high mine aliza ion [55]. In he p esen s udy, binge-like e hanol in ake du ing adolescence educed he abecula hickness and inc eased he abecula sepa a ion, which impac ed on bone mass (bone ac ion mass). Such al eola bone al e a ion may gene a e ne- ga i e eflexes on emodeling p ocessess elici ed by local and sys emic s imuli [55]. I is well known ha e hanol affec s bone emodeling, leading o changes in he abecula a angemen bu li le is known abou i s effec on he balance be ween o ganic and ino ganic con en [21]. Changes in he biochemical composi ion o bones may be explained by enhanced os eoclas ogenesis and educed os eoblas ac i i y, and hese ac i i ies a e known o be affec ed by e hanol in diffe en bone ypes ( o e iew see [57]). The egula ion o he ac i i y o bone cells could lead o a lowe mine aliza ion o collagen fib ils on he newly o med bone when emodeling akes place [58]. Raman analysis was used in Fig. 4. Effec s o he adenosine A 2A ecep o an agonis (SCH58261) on he impac o adolescen binge-like e hanol challenge on he al eola bone quali y o emale Wis a a s (59-days-old, n = 5 animals/g oup). Expe imen al g oups consis ed o he Con ol g oup (dis illed wa e ; 3 days on-4 days off; 35 h o 59 h pos na al days); E hanol g oup (3.0 g/kg/day; 3 days on-4 days off; 35 h o 59 h pos na al days); A 2A R an agonis g oup (SCH58261 0.1 mg/kg/day daily; 38 h o 59 h pos na al days; An ); and A 2A R an agonis plus e hanol g oup (SCH58261 0.1 mg/kg/day daily plus binge-like ea men ; 38 h o 59 h pos - na al days; An + E hanol). Values a e mean ± SEM o mic o-CT pa ame e s: (A) abecula hickness (Tb.Th; mm); (B) abecula sepa a ion (Tb.Sp; mm); and (C) he pe cen age o bone olume in ela ion o he o al measu ed a ea (BV/TV; %). **p < 0.01 compa ed o con ol g oup; ***p < 0.001 compa ed o con ol g oup. One-way ANOVA ollowed by Sidak’spos hoc es , p < 0.05. C. Maia, e al. Biomedicine & Pha maco he apy 130 (2020) 110608 5 he p esen s udy o quan i y he mine al con en o bone issue by he measu emen o phospha e ib a ion on he bone samples, while he amide peak was used o quan i y he o ganic con en [59]. Al hough he mic os uc u e o bone was modified by e hanol challenging, he chemical analyses showed ha he o ganic:ino ganic con en o he al eola c es was no affec ed by he es ed ea men s. This leads o he conclusion ha he p esen ly used binge d inking p o ocol may no be sufficien o induce modifica ions o he ac i i y o bone cells o Fig. 5. Effec s o caffeine and o he adenosine A 2A ecep o an agonis (SCH58261) on adoles- cen binge-like e hanol challenge on he al eola bone quali y o emale Wis a a s (59-days-old, n = 5 animals/g oup). Rep esen a i e h ee-di- mensional images o hemimandibles o he (A) Con ol g oup (dis illed wa e ; 3 days on-4 days off; 35 h o 59 h pos na al days); (B) E hanol g oup (3.0 g/kg/day; 3 days on-4 days off; 35 h o 59 h pos na al days); (C) A 2A R an agonis g oup (SCH58261 0.1 mg/kg/day daily; 38 h o 59 h pos na al days; An ); (D) A 2A R an agonis plus e hanol g oup (SCH58261 0.1 mg/kg/day daily plus binge-like ea men ; 38 h o 59 h pos na al days; An + E hanol); (E) Caffeine g oup (caffeine 10 mg/kg/day daily; 38 h o 59 h pos na al days; CAF); and (F) Caffeine plus e hanol g oup (caffeine plus binge-like ea - men ; CAF + E hanol). Panel (G) ep esen s he dis ance be ween he cemen um-enamel junc ion and al eola bone c es (CEJ-ABC; mm). Red do ed lines highligh he diffe ences be ween g oups. Values a e mean ± SEM. **p < 0.01 compa ed o con ol g oup; ##p < 0.01 com- pa ed o e hanol g oup. One-way ANOVA ol- lowed by Sidak’spos hoc es , p < 0.05. Scale ba : 1 mm. C. Maia, e al. Biomedicine & Pha maco he apy 130 (2020) 110608 6 modi y bone composi ion. We ha e al eady epo ed ha he exposu e o a 4-pe iods o binge d inking p o ocol in adul hood (s a ing a 90 days old) esul ed in educed al eola bone quali y, bu no al eola bone loss [19]. How- e e , in con as o wha occu s du ing adul hood, we ha e now de- mons a ed ha e hanol exposu e du ing adolescence elici ed al eola bone loss. In o he wo ds, e hanol binge d inking dec eases he quali y o he al eola bone mic os uc u e bo h in adolescence and adul hood, bu he e is only an al eola bone loss (i.e., educ ion o e ical di- mension) in adolescen subjec s. He e, we canno in e ha in he long- e m, addi ional binge d inking exposu e migh u he in ensi y al- eola bone loss, since bone quali y is al eady affec ed a he onse o adul hood. Besides, his s udy canno answe i he impai men o bone quali y can spon aneouly eco e upon wi hd awal o e hanol. I is impo an o s ess ha he es ed e hanol consump ion pa e n, which consis s o in ense and episodic e hanol in ake (i.e., high doses du ing a ew days and hou s ollowed by an abs inence pe iod) ep esen s he mos equen consump ion pa e n by adolescen s and younge people [3,60]. The e o e, we belie e ha al eola bone mic os uc u e damage esul ing om binge-like e hanol exposu e om mid-adolescence in o adul hood is a aluable model o be e comp ehend he impac o e hanol in ake by adolescen s. Con adic o y findings ha e been epo ed wi h espec o he e- la ionship be ween caffeine in ake and bone heal h. A hea y in ake o caffeine has been linked o haza dous effec s on bone, including al- eola bone [40–42]. In e es ingly, ou esul s demons a e ha low o mode a e doses o caffeine du ing adolescence no only did no cause any al eola bone mic os uc u e damage, bu e en a oided he e- duc ion o al eola bone quali y induced by e hanol exposu e in ado- lescen a s. Since mode a e doses o caffeine ac h ough he an ag- onism o adenosine ecep o s [43], his implies ha he adenosine modula ion sys em is no engaged in physiological condi ions in he al eola bone sys em o adolescen a s, bu become ac i e unde s ess ul condi ions, in acco dance wi h he p edominan allos a ic a- he han homeos a ic ole o he adenosine sys em [61]. Few s udies ha e ocused on he effec s o caffeine on al eola bone, and all s udies es ed caffeine a high doses [41,42]. The p esen s udy is he fi s s udy showing ha a low o mode a e doses, caffeine displays beneficial e - ec s agains an e hanol challenge (i.e., sys emic s imuli) conce ning al eola bone emodeling. In con as o o he skele al bone ha o igina es in he mesode m, al eola bone is de i ed p ima ily om he neu oec ode m [62]. This emb yologic diffe ence con e s o al eola bone a dynamic me abolism and a diffe en esponse o nu i ion o bone oxic s imuli [63]. We also di ec ed expe imen al effo s o in es iga e he mos likely mechanism ope a ed by caffeine o affo d he obse ed p o ec ion o he in eg i y o al eola bone upon exposu e o e hanol. The p incipal mechanism o ac ion o caffeine, especially a low doses, consis s o he an agonism o adenosine ecep o s [38,47]. Fu he mo e, i is well documen ed ha inflamma o y p ocesses unde lie pe iodon al disease, which can be modula ed by he pu ine gic sys em [22–24]. Among hese adenosine ecep o s, adenosine A 2A ecep o s (A 2A R) ha e been pa icula ly in ol ed in he con ol o inflamma o y p ocesses [64] and al hough he impac o e hanol on A 2A R exp ession in pe iodon al is- sues emains unknown, he exp ession o A 2A R has been shown in ch onic pe iodon i is [30]. Thus, we now hypo hesized ha caffeine in ake could a enua e al eola bone damage induced by e hanol binge d inking in adolescen a s h ough an an agonism o A 2A R con olling inflamma ion. To es i A 2A R we e esponsible o he effec s o ca - eine, we would expec ha he selec i e blockade o A 2A R using a po en and selec i e an agonis , SCH58261, should affo d he same beneficial effec s o caffeine agains e hanol-induced de e io a ion o al eola bone. We ailed o demons a e his hypo hesis, since a p e- iously alida ed effec i e dose o SCH58261 ( e iewed in [49]) did no mimic he p o ec i e effec o caffeine I is no ewo hy ha he A 2A R an agonis did no inc ease he al- eola bone loss elici ed by e hanol in ake, which also speaks agains a pu a i e beneficial ole o he A 2A R pa hway on e hanol-induced pe i- odon al disease du ing adolescence. In addi ion, SCH58261 pe se ad- minis a ion educed solely abecula hickness, wi h no epe cussion on al eola bone mass. Al hough he exac con ibu ion o A 2A R-sub ype on he pa hophy- siology o o al diseases is no ye well unde s ood, he adenosine modula ion sys em is s ill conside ed as an impo an a ge o o al heal h ( o e iew see [65]), as bes he alded by he benefi s now e- po ed o caffeine. The e o e, i can be specula ed ha o he adenosine ecep o s, such as A 2B R, may play an impo an ole in pe iodon al disease elici ed by e hanol exposu e du ing adolescence, a hypo hesis ha dese es o be u he s udied. Fig. 6. Effec s o caffeine and he adenosine A 2A ecep o an agonis (SCH58261) on adolescen binge-like e hanol challenge on he al eola bone chemical s uc u e (PO 43− /AmideI) o emale Wis a a s (59-days-old, n = 3 animals/g oup). Raman spec oscopic analyzes o al eola bone c es (panel A) in g aphs (panel B) o colo ime ic ep esen a i e (panel C) o he Con ol g oup (dis illed wa e ; 3 days ON-4 days OFF; 35 h o 59 h pos na al days); E hanol g oup (3.0 g/kg/day; 3 days on-4 days off; 35 h o 59 h pos na al days); A 2A R an agonis g oup (SCH58261 0.1 mg/kg/day daily; 38 h o 59 h pos na al days; An ); A 2A R an agonis plus e hanol g oup (SCH58261 0.1 mg/kg/day daily plus binge-like ea men ; 38 h o 59 h pos na al days; An + E hanol); Caffeine g oup (caffeine 10 mg/kg/day daily; 38 h o 59 h pos na al days; CAF); and Caffeine plus e hanol g oup (caffeine plus binge-like ea men ; CAF + E hanol). Values a e mean ± SEM. No s a is ical diffe ences a p < 0.05 using a one-way ANOVA ollowed by Sidak’spos hoc es . C. Maia, e al. Biomedicine & Pha maco he apy 130 (2020) 110608 7 5. Conclusion The p esen s udy e ealed ha e hanol binge d inking du ing adolescence causes al eola bone damage, e en in he absence o ex- pe imen al pe iodon i is in a s. In addi ion, he daily in ake o low o mode a e doses o caffeine p e en ed he ha m ul effec s on al eola bone o e hanol binge d inking du ing adolescence. We also showed ha he beneficial effec s o caffeine do no in ol e adenosine A 2A R an agonism. Decla a ion o Compe ing In e es RAC is a scien ific consul an o he Ins i u e o Scien ific In o ma ion on Coffee. All o he au ho s decla e no conflic o in e es s. Acknowledgmen s This s udy was ca ied ou wi h suppo om he P og ama Nacional de Coope ação Acadêmica na Amazônia –PROCAD/Amazônia da Coo denação de Ape eiçoamen o de Pessoal de Ní el Supe io –CAPES/B asil and by Coo denação de Ape eiçoamen o de Pessoal de Ní el Supe io - B azil (CAPES) Finance Cod 001. This wo k was also suppo ed by Resea ch P o-Rec o y o he Fede al Uni e si y o Pa á (PROPESP, UFPA, B azil). 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