Paternal programming of breast cancer risk in daughters in a rat model: opposing effects of animal- and plant-based high-fat diets

Transcription

1 Universidde de São Pulo Biliotec Digitl d Produção Intelectul - BDPI Deprtmento de Alimentos e Nutrição Experimentl - FCF/FBA Artigos e Mteriis de Revists Científics - FCF/FBA 216 Pternl progrmming of rest cncer risk in dughters in rt model: opposing effects of niml- nd plnt-sed high-ft diets Brest Cncer Reserch. 216 Jul 26;18(1):71 Downloded from: Biliotec Digitl d Produção Intelectul - BDPI, Universidde de São Pulo

2 Fontelles et l. Brest Cncer Reserch (216) 18:71 DOI /s x RESEARCH ARTICLE Open Access Pternl progrmming of rest cncer risk in dughters in rt model: opposing effects of niml- nd plnt-sed high-ft diets Cmile Cstilho Fontelles 1, Luiz Nicolosi Guido 1, Mrin Ppléo Rosim 1, Fái de Oliveir Andrde 1, Lu Jin 2, Jessic Inchuspe 2, Vness Crdoso Pires 1, Inr Alves de Cstro 1, Leen Hilkivi-Clrke 2, Soni de Assis 2 nd Thoms Prtes Ong 1,3* Astrct Bckground: Although mles contriute hlf of the emryo s genome, only recently hs interest egun to e directed towrd the potentil impct of pternl experiences on the helth of offspring. While there is evidence tht pternl mlnutrition my increse offspring susceptiility to metolic diseses, the influence of pternl fctors on dughter s rest cncer risk hs een exmined in few studies. Methods: Mle Sprgue-Dwley rts were fed, efore nd during puerty, either lrd-sed (high in sturted fts) or corn oil-sed (high in n-6 polyunsturted fts) high-ft diet (6 % of ft-derived energy). Control nimls were fed n AIN-93G control diet (16 % of ft-derived energy). Their 5-dy-old femle offspring fed only commercil diet were sujected to the clssicl model of mmmry crcinogenesis sed on 7,12-dimethylenz[]nthrcene initition, nd mmmry tumor development ws evluted. Sperm cells nd mmmry glnd tissue were sujected to cellulr nd moleculr nlysis. Results: Compred with femle offspring of control diet-fed mle rts, offspring of lrd-fed mle rts did not differ in tumor ltency, growth, or multiplicity. However, femle offspring of lrd-fed mle rts hd incresed elongtion of the mmmry epithelil tree, numer of terminl end uds, nd tumor incidence compred with oth femle offspring of control diet-fed nd corn oil-fed mle rts. Compred with femle offspring of control diet-fed mle rts, femle offspring of corn oil-fed mle rts showed decresed tumor growth ut no difference regrding tumor incidence, ltency, or multiplicity. Additionlly, femle offspring of corn oil-fed mle rts hd longer tumor ltency s well s decresed tumor growth nd multiplicity compred with femle offspring of lrd-fed mle rts. Pternl consumption of niml- or plnt-sed high-ft diets elicited opposing effects, with lrd rich in sturted ftty cids incresing rest cncer risk in offspring nd corn oil rich in n-6 polyunsturted ftty cids decresing it. These effects could e linked to ltertions in microrna expression in fthers sperm nd their dughters mmmry glnds, nd to modifictions in rest cncer-relted protein expression in this tissue. Conclusions: Our findings highlight the importnce of pternl nutrition in ffecting future genertions risk of developing rest cncer. Keywords: Pternl diet, Brest cncer, High-ft diet, Femle offspring * Correspondence: tong@usp.r 1 Deprtment of Food nd Experimentl Nutrition, Fculty of Phrmceuticl Sciences, University of São Pulo, Avenid Professor Lineu Prestes 58, Bloco 14, São Pulo, SP 558-, Brzil 3 Food Reserch Center (FoRC), São Pulo 558-, Brzil Full list of uthor informtion is ville t the end of the rticle 216 The Author(s). Open Access This rticle is distriuted under the terms of the Cretive Commons Attriution 4. Interntionl License ( which permits unrestricted use, distriution, nd reproduction in ny medium, provided you give pproprite credit to the originl uthor(s) nd the source, provide link to the Cretive Commons license, nd indicte if chnges were mde. The Cretive Commons Pulic Domin Dediction wiver ( pplies to the dt mde ville in this rticle, unless otherwise stted.

3 Fontelles et l. Brest Cncer Reserch (216) 18:71 Pge 2 of 13 Bckground Brest cncer is glol pulic helth prolem, with nerly 1.7 million new cses dignosed in 212, representing 25 % of ll cncers in women worldwide [1]. Its incidence is projected to rise significntly over the next 2 yers despite current efforts to prevent the disese [2]. Although the precise reson for this growth is still not cler, it hs een suggested tht modern women s lifestyles, including postponing first pregnncy nd hving fewer children, cn explin the increse [3]. Nutritionl hits, such s doption of Western dietry ptterns, re lso linked to incresed rest cncer risk [4]. These ptterns re chrcterized y low consumption of fruits nd vegetles, incresed energy intke, nd decresed energy expenditure, leding to oesity, s well s incresed intke of sturted ftty cids (SFA), n-6 polyunsturted ftty cids (PUFA), nd trns-ftty cids nd decresed intke of n-3 polyunsturted fts [5, 6]. While the mjority of epidemiologicl studies on nutrition nd rest cncer risk hve een focused on women s diet in dulthood, ccumulting epidemiologicl nd experimentl evidence highlights erly life experiences, including nutrition, s relevnt fctors for lter rest cncer risk determintion [7]. The developmentl origins of this cncer hve een considered predominntly from mternl perspective, with emphsis plced on the impct of high ft or energy intke during gesttion nd lcttion on femle offspring mmmry glnd development nd lter rest cncer risk [8, 9]. Although mles contriute hlf of the emryo s genome, only recently hs interest egun to e directed towrd the potentil impct of pternl experiences on the helth of offspring [1]. While experimentl studies hve shown tht pternl mlnutrition my increse the susceptiility of offspring to metolic dysregultion, oesity, nd crdiovsculr diseses [11, 12], the influence of pternl fctors on dughter s rest cncer risk hs een exmined in few studies. Among them, epidemiologicl studies show n ssocition etween higher pternl eduction level, older ge, nd smoking with incresed rte of rest cncer in the dughters [13, 14]. Unlike the femle production of germ cells tht tkes plce predominntly in erly life [15], mle production of germ cells strts in utero, with mture sperm cells eing produced throughout the entire reproductive life of the mle [16]. Becuse spermtogenesis cn e drmticlly influenced y environmentl fctors, including mlnutrition, oesity, nd n exposure to toxic compounds, the fther s helth during preconception is now cknowledged s criticl fctor in the context of the developmentl origins of helth nd disese [17]. In ddition to emryogenesis, gmetogenesis comprises intense epigenetic (DNA methyltion, histone modifiction, nd microrna [mirna or mir] expression) remodeling [18, 19]. Thus, epigeneticlly inherited incresed disese risk could e trnsmitted through the femle s well s the mle germline [2]. Specific windows within which mle gmetes would e especilly prone to environmentlly elicited epigenetic deregultion include prepuerty nd the reproductive phse [21]. Given the mrked increse in dietry ft intke over the pst three decdes [22], s well s the notion tht different kinds of dietry fts cn led to different helth outcomes [23], we designed this study to investigte whether, in rts, consumption of high levels of nimlor vegetle-sed fts y fthers would ffect their dughters risk of rest cncer. We lso investigted the underlying cellulr nd moleculr mechnisms. We fed mle Sprgue-Dwley rts, efore nd during puerty, either lrd-sed (high in SFA) or corn oil-sed (high in n-6 PUFA) high-ft diet (6 % of ft-derived energy). Control nimls were fed control AIN-93G diet contining soyen oil s ft source (16 % of ftderived energy). Mle rts were mted with femle rts tht were consuming commercil diet. We show tht pternl consumption of these high-ft diets elicited opposing effects, with niml ft incresing nd vegetle oil decresing rest cncer risk in the offspring. These effects could e linked to ltertions in mirna expression in fthers sperm nd their dughters mmmry glnds, s well s to modifictions in rest cncerrelted protein expression in this tissue. These novel dt show tht pternl high-ft diets influence their femle offspring s susceptiility to mmmry cncer, with consumption of lrd incresing nd corn oil reducing dughters mmmry cncer risk. Thus, pternl diet efore conception sets stge for dughter s risk of developing rest cncer. Methods Experimentl design This study ws pproved y the ethics committee on niml experiments of the Fculty of Phrmceuticl Sciences, University of São Pulo (protocol numer CEUA/ FCF/381). Twenty-one-dy-old mle rts were divided into three groups (n = 2 rts per group): control rts (those fed the control AIN-93G diet, with 16 % of totl clories provided y lipids), lrd-fed mles (exposed to high-sfa diet, with 6 % of totl clories provided minly from lrd), nd corn oil-fed mles (exposed to n-6 PUFA diet, with 6 % of totl clories provided minly from corn oil). At 12 weeks of ge, ll mle rts were switched to chow diet nd mted y housing one mle with one femle per cge. Pregnnt femle rts nd their offspring consumed only commercil lortory chow (Nuvitl Nutrientes, Colomo, Brzil). Body weight nd food intke were recorded two or three times per week.

4 Fontelles et l. Brest Cncer Reserch (216) 18:71 Pge 3 of 13 Determintion of the diets lipid profiles The lipid profiles of the control, lrd, nd corn oil diets were determined ccording to the methods pulished y the Assocition of Officil Anlyticl Chemists [24]. Ftty cids were esterified to ftty-cid methyl esters ccording to the method reported y Hrtmn nd Lgo [25], nd their composition ws nlyzed with gs chromtogrph (GC 17A/Clss GC 1; Shimdzu, Kyoto, Jpn) equipped with flme ioniztion detector nd SUPELCOWAX 1 fused silic cpillry column (3 mm.25 mm inner dimeter; Sigm-Aldrich, St. Louis, MO, USA). The temperture ws set t 17 C, rised to 225 C t rte of1 C/minute,ndheldfor25minutes.Thetempertures of the vporizer nd detector were 25 C nd 27 C, respectively. Helium ws used s the crrier gs (1 ml/minute). Identifiction of the ftty cids ws performed y comprison of the retention times with the stndrd mixture of ftty-cid methyl esters. Ech determintion ws performed in duplicte using two different smples for ech diet. Insulin tolernce test The tests were performed t 8 h fter the rts were fsted for 12 h, ccording to the method descried y Tkd et l. [26]. The insulin lod (75 mu/1 g ody weight) ws injected s olus, nd the lood glucose levels were determined t, 3, 6, 9, 12, nd 3 minutes fter injection in mle rts nd their 5-dy-old femle offspring. The AUC ws clculted ccording to the trpezoid rule [27]. Mture spermtozo collection nd purifiction Control diet nd lrd- nd corn oil-fed mle rts were killed once femles were pregnnt, nd the cudl epididymis ws dissected for sperm collection. The cud nd vs deferens from mle rts were collected, punctured, nd trnsferred to tissue culture dishes contining M2 medium (M2 medium with HEPES, without penicillin nd streptomycin, sterile-filtered, suitle for rt emryo; Sigm-Aldrich), where it ws incuted for 1 h t 37 C. Spermtozo smples were wshed with PBS nd then incuted with somtic cell lysis uffer (SCLB;.1 % SDS,.5 % Triton X-1 in diethylpyrocronte wter) for 1 h, ccording to protocol descried y Goodrich et l. [28]. SCLB ws rinsed off with two wshes of PBS, nd the purified spermtozo smple (t lest 95 % purity s ssessed y microscopy) ws pelleted nd used for mirna extrction. Determintion of dily sperm production The right testis ws mintined t 2 C until processing to determine the dily sperm production. The technique proposed y Ro et l. [29] is sed on the resistnce of elongted spermtids present in phses of spermtogenesis to intense mechnicl stress due to the high compction of chromtin. Sperm morphologicl nlyses According to the method of Seed et l. [3], the epididymis ws previously frozen t 2 C, underwent incision, nd ws susequently immersed in PBS to promote the dissemintion of gmetes to the queous medium. Then the otined solution ws plced on slides for exmintion y light microscopy. Two hundred sperm per niml were nlyzed microscopiclly t 4 mgnifiction. Mmmry glnd hrvesting Adominl mmmry glnds of femle offspring of control diet- nd lrd- nd corn oil-fed mle rts (n = 6 per group) were collected on postntl dy 5 nd used for prepring mmmry whole mounts nd mirna nd protein extrction. Anlysis of mmmry glnd morphology nd development Whole-mount preprtions of the fourth dominl mmmry glnd from 5-dy-old femle offspring (n =5/ group) were otined, nd the epithelil elongtion nd numer of terminl end uds (TEBs) were determined s descried y de Assis et l. [31]. Mmmry tumor induction Mmmry tumors were induced in 5-dy-old femle rt offspring (n = 24 rts/group) y dministrtion of 7,12-dimethylenz[]nthrcene (DMBA, 5 mg/kg ody weight; Sigm-Aldrich). The crcinogen ws dissolved in corn oil nd dministered y orl gvge. Animls were exmined for mmmry tumors y plption twice per week. Ltency of tumor ppernce, the numer of nimls with tumors, nd the numer of tumors per niml (multiplicity) were evluted. The tumor volume ws clculted with tumor mesures of length (), height (), nd width (c) tken with cliper rule once per week since tumor ppernce nd throughout the experiment. The formul (1/6 3.14) ( c) ws used to clculte the tumor volume, s descried y Spng-Thomsen et l. [32]. The tumor growth rte ws clculted using the mesured volumes of ech tumor t given week (d) nd the susequent week (e) of ppernce using the formul [(e d)/d] 1. Those nimls in which tumor urden pproximted 1 % of totl ody weight were killed. All others nimls were killed 19 weeks fter crcinogen dministrtion. Anlysis of mmmry glnd nd tumor cell prolifertion nd poptosis in femle offspring Cell prolifertion ws evluted in mmmry glnds (ducts nd loules) nd tumors from 5-dy-old femle offspring (n = 4/group) y Ki-67 immunohistochemistry.

5 Fontelles et l. Brest Cncer Reserch (216) 18:71 Pge 4 of 13 After eing hrvested, mmmry tissue ws directly fixed in 1 % uffered formlin, emedded in prffin, nd sectioned. Sections were then deprffinized in xylene nd hydrted through grded ethnol. Antigen retrievl ws performed with 1 mm citrte uffer, ph 6, for 2 minutes in pressure cooker. Peroxidse locking ws performed with 1 % H 2 O 2 for 1 minutes, nd nonspecific inding ws locked for 1 h with 1 % skimmed milk in PBS. Sections were incuted overnight with nti-rt Ki-67 primry ntiody (Acm, Cmridge, UK) t 1:5 dilution. After wshes, sections were incuted with the LSAB 2 System-HRP kit (Dko, Crpinteri, CA, USA) ccording to the mnufcturer s instructions, stined with 3,3 -diminoenzidine in chromogenic solution (Dko) for 1 minutes, wshed, nd counterstined for 1.5 minutes with hemtoxylin. Cell prolifertion ws quntified y ssessing the numer of Ki-67-positive cells mong 1 cells. The slides were evluted using ImgeJ softwre (Ntionl Institutes of Helth, Bethesd, MD, USA). Apoptosis nlysis ws conducted in mmmry glnds (ducts nd loules) nd tumors from 5-dy-old femle offspring (n = 4/group), ccording to the method descried y Elmore et l. [33], using ImgeJ softwre. Results re presented s men numer of poptotic cells per 1 cells. microrna expression profile nlysis Totl RNA from pternl sperm nd their femle offspring s totl mmmry glnd ws extrcted using the mirnesy Mini Kit (QIAGEN, Vlenci, CA, USA) ccording to the mnufcturer s instructions. RNA smples were quntified nd stored t 8 C until use. mirna rrys were performed t the Genomics nd Epigenomics Shred Resources t Georgetown University using Applied Biosystems TqMn Arry Rodent MicroRNA rrys (Life Technologies, Crlsd, CA, USA) to generte the mirna expression profiles for ech experimentl group. The TqMn Arry Rodent MicroRNA A + B Crds Set v3. is two-crd set contining totl of 384 TqMn Micro- RNA ssys per crd (Life Technologies). The set enles ccurte quntittion of 641 nd 373 unique mirnas for rt. There re three TqMn MicroRNA Assy endogenous controls for ech species on ech rry to id in dt normliztion [34]. The genorm lgorithm ws pplied to those endogenous controls to determine the optiml numer of stle controls. The geometric men of these selected controls ws used for rry normliztion. To conduct further sttisticl nlysis, the normlized vlue ws log-trnsformed to meet the t test requirement. Sttisticl nlysis ws conducted using the limm pckge in R [35]. mirnas tht hd flse discovery rte <.1 were considered s significntly ltered nd selected for further nlysis. Trget prediction for mirnas of interest ws conducted using TrgetScn (relese 6.2). The predicted trgeted messenger RNA (mrna) list ws then uploded to Ingenuity Pthwy Anlysis (IPA; QIAGEN Silicon Vlley, Redwood City, CA, USA) for gene set enrichment nlysis. We selected the top cnonicl pthwys for further nlysis. Anlysis of protein levels in mmmry glnds of femle offspring Protein levels of 5 ng/μl were ssessed y Western lot nlysis in totl mmmry glnds otined from 5-dyold femle rts (n = 5 per group). Totl protein ws extrcted from mmmry tissues using rdioimmunoprecipittion ssy uffer with protese inhiitor (Roche, Bsel, Switzerlnd), glycerophosphte (1 mm), sodium orthovndte (1 mm), pyrophosphte (5 mm), nd phenylmethylsulfonyl fluoride (1 mm). The smples were mixed with the uffer for 5 minutes, then incuted on ice for 3 minutes nd centrifuged for 1 minutes t high speed. Protein in the superntnt ws quntified using Pierce icinchoninic cid protein ssy regent (Thermo Scientific, Rockford, IL, USA). Protein extrcts were resolved on 4 12 % grdient denturing polycrylmide gel (SDS-PAGE). Proteins were trnsferred using the Invitrogen iblot 7-Minute Dry Blotting System (Life Technologies) nd locked with 5 % nonft dry milk for 1 h t room temperture. Memrnes were incuted with the specific primry ntiodies t 4 C overnight. After severl wshes, the memrnes were incuted with HRP-conjugted secondry ntiody (1:5; Snt Cruz Biotechnology, Dlls, TX, USA) t room temperture for 1 h. Memrnes were developed using HyGLO chemiluminescent HRP ntiody detection regent (Denville Scientific Inc., Metuchen, NJ, USA), nd exposed to Kodk utordiogrphy films (Crestrem Helth, Rochester, NY, USA). The opticl density of the nds ws quntified using Quntity One softwre (Bio- Rd Lortories, Hercules, CA, USA). To control for equl protein loding, expression of the proteins of interest ws normlized to the β-ctin signl. Sttisticl nlysis Results re expressed s the men ± SEM, nd ll nlyses were conducted with the limm pckge in R. Multiple-group comprisons were performed using onewy nlysis of vrince (ANOVA) followed y lest significnt difference (LSD) test, nd two-group comprisons were performed using Student s t test. Repetedmesures ANOVA ws pplied for cloric intke dt evlution, nd Kpln-Meier curves nd log-rnk tests were pplied for determining differences in tumor incidence. For ll dt nlyses, p.5 ws pplied s the threshold for sttisticl significnce.

6 Fontelles et l. Brest Cncer Reserch (216) 18:71 Pge 5 of 13 Results Pternl dietry nd helth prmeters Compred with control diet-fed mle rts, the ones tht were on the lrd- or corn oil-sed high-ft diets consumed more (p.5) SFA (predominntly plmitic [C16:] nd steric [C18:] cids), monounsturted ftty cids (MUFA) (predominntly oleic cid [C18:1n9c]) nd PUFA (predominntly linoleic cid [C18:2n6c]) (Fig. 1). Corn oil-fed mle rts consumed less (p.5) SFA (predominntly plmitic [C16:] nd steric [C18:] cids) nd MUFA (predominntly oleic cid [C18:1n9c]) nd more (p.5) PUFA (predominntly linoleic cid [C18:2n6c]) thn the lrd-fed mle rts (Fig. 1). Dily cloric intke ws pproximtely 7 % higher (p.5) in oth lrd- nd corn oil-fed mle rts thn in control diet-fed mle rts (dt not shown). Although lrd- nd corn oil-fed mle rts consumed nerly the sme mount of clories per dy, lrd-fed mle rts gined more (p.5) weight thn control diet- nd corn oil-fed mle rts (p.5) (Tle 1). There ws no difference (p >.5) etween control dietnd corn oil-fed mle rts regrding weight gin. Both lrd- nd corn oil-fed mle rts hd greter (p.5) dominl, retroperitonel, nd epididyml ft pd weights thn control diet-fed mle rts (Tle 1). Compred with lrd-fed mle rts, corn oil-fed mle rts hd lesser (p.5) epididyml ft pd weights, ut there ws no difference (p >.5) in dominl or retroperitonel ft pd weights (Tle 1). Further, lrd-fed mle rts hd lesser testicle (p.5), epididymis (p.5), nd seminl vesicle (p.8) weights thn the control diet- nd corn oil-fed mle rts (Tle 1). There ws no difference (p >.5) etween control diet- nd corn oil-fed mle rts regrding these prmeters (Tle 1). Lrd-fed mle rts lso hd fewer (p.5) norml sperm cells nd lower (p.5) dily sperm production thn control diet- nd corn oil-fed mle rts (Tle 1). There ws no difference (p >.5) etween control diet- nd corn oil-fed mle rts regrding these prmeters (Tle 1). Both lrd- nd corn oil-fed mle rts hd higher (p.5) fsting glucose levels thn control diet-fed mle rts (Tle 1). There ws no difference (p >.5) etween lrd- nd corn oil-fed mle rts regrding this prmeter (Tle 1). Further, in the insulin tolernce test, lrd- nd corn oil-fed mle rts hd higher (p.5) AUCs thn control diet-fed mle rts (Fig. 1), indicting tht they were insulin-intolernt. There ws no difference (p >.5) etween lrd- nd corn oil-fed mle rts regrding this prmeter (Fig. 1). Femle offspring helth prmeters Femle offspring of oth lrd- nd corn oil-fed mle rts hd greter irth weight (p.5) nd greter weight gin (p.5; p.8 for offspring of corn oil-fed mle rts) thn offspring of control diet-fed mle rts (Tle 1). There ws no difference (p >.5) etween femle offspring of lrd- nd corn oil-fed mle rts regrding irth weight (Tle 1). Femle offspring of corn oil-fed mle rts hd less (p.5) weight gin thn offspring of lrd-fed mle rts (Tle 1). Similrly to the mle rts, femle offspring of oth the lrd-fed nd the corn oil-fed mle rts hd greter (p.5) retroperitonel ft weights thn offspring of control diet-fed mle rts (Tle 1). There ws no difference (p >.5) etween femle offspring of lrdor corn oil-fed mle rts regrding this prmeter (Fig. 1c). Femle offspring of lrd-fed mle rts hd higher (p.5) fsting glucose levels (Tle 1) nd higher (p.5) AUCs thn femle offspring of control diet- or corn oil-fed Consumption of Dietry Ftty Acids (g/dy) CO LB CB, 16 CO 16 c LB, 14 CB 14, SFA MUFA PUFA Glucose (mg/dl) Glycemi (mg/dl/min) AUC Time (min) Glucose (mg/dl) Glycemi (mg/dl/min) AUC Time (min) CO BA LB OM CB Fig. 1 Pternl ftty-cid consumption nd insulin tolernce test. Mle rts fed control diet (CO) or lrd-sed (LB) or corn oil-sed (CB) high-ft diet consumption of dietry fts (sturted ftty cids [SFA], monounsturted ftty cids [MUFA], nd polyunsturted ftty cids [PUFA]) (n = 2 per group). Insulin tolernce test (ITT) of the CO-, LB-, nd CB-fed mle rts (n =6pergroup).Inset: ITT is shown s the AUC. c Fifty-dy-old femle offspring (n =6pergroup).Inset: ITT is shown s the AUC. Sttisticlly significnt difference (p.5) compred with CO nd LB, ccording to nlysis of vrince followed y lest significnt difference test. The dt re expressed s men ± SEM CO LB CB

7 Fontelles et l. Brest Cncer Reserch (216) 18:71 Pge 6 of 13 Tle 1 Helth prmeters of mle rts nd their 5-dy-old femle offspring in control diet, lrd-sed diet, nd corn oil-sed diet groups Mle rts Weight gin ± 6.4 g ± 12. g ± 7.8 g Ft weight Adominl ft 1.4 ±.1 g/1 g ody weight 2.7 ±.2 g/1 g ody weight 2.6 ±.2 g/1 g ody weight Retroperitonel ft.8 ±.1 g/1 g ody weight 1.9 ±.1 g/1 g ody weight 1.7 ±.2 g/1 g ody weight Epididyml ft 1.2 ±.1 g/1 g ody weight 2.7 ±.1 g/1 g ody weight 2. ±.2 g/1 g ody weight, Reproductive orgns Testicle.45 ±.1 g/1 g ody weight.4 ±.1 g/1 g ody weight.45 ±.1 g/1 g ody weight Epididymis.14 ±. g/1 g ody weight.13 ±. g/1 g ody weight.14 ±. g/1 g ody weight Seminl vesicle.33 ±.1 g/1 g ody weight.29 ±.2 g/1 g ody weight c.33 ±.2 g/1 g ody weight d Sperm morphology (% norml) 66.4 ± 2.8 % 5.1 ± 3. % 7.9 ± 2.9 % Dily sperm production, n/testicle/dy 31 ± ± ± Fsting glycemi 1.8 ± 3 mg/dl ± 4 mg/dl ± 3 mg/dl 5-dy-old femle offspring Birth weight 8. ± 1.5 g 8.8 ± 1. g 9.1 ± 1.5 g Weight gin ± 1.5 g 147. ± 1.2 g ± 1.1 g,c Retroperitonel ft.9 ±.1 g/1 g ody weight 1.1 ±. g/1 g ody weight 1.2 ±.1 g/1 g ody weight Fsting glycemi 16.4 ± 2. mg/dl ± 1.9 mg/dl 16.3 ± 1.2 mg/dl Arevitions: CB rts fed corn oil-sed high-ft diet nd their offspring, CO rts fed control diet nd their offspring, LB rts fed lrd-sed high-ft diet nd their offspring Sttisticlly significnt difference (p.5) compred with CO nd LB, ccording to nlysis of vrince followed y lest significnt difference test. Mrginl difference (p.8) compred with c CO nd d LB, ccording to t test. The dt re expressed s men ± SEM (n = 2 per group) mle rts (Fig. 1c). There ws no difference (p >.5) etween femle offspring of control diet- nd corn oil-fed mle rts regrding these prmeters (Tle 1 nd Fig. 1c). Femle offspring mmmry glnd morphology Mmmry glnd morphology ws ssessed on the sis of mmmry whole mounts otined from 5-dy-old femle offspring. Both elongtion of the mmmry epithelil tree (Fig. 2c) nd the numer of TEBs (Fig. 2d) were higher (p.5) in femle offspring of lrd-fed mle rts thn in femle offspring of control diet- nd corn oil-fed mle rts. There ws no difference (p >.5) etween femle offspring of control diet- nd corn oilfed mle rts regrding these prmeters (Fig. 2c nd d). Femle offspring mmmry glnd tumors dt Mmmry tumors in the femle offspring were induced y dministering the crcinogen DMBA. Femle offspring of lrd-fed mle rts hd incresed mmmry tumor incidence (p.5) compred with offspring of oth control diet- nd corn oil-fed mle rts (Fig. 3). There ws no sttisticl difference (p >.5) etween femle offspring of control diet- nd corn oil-fed mle rts regrding tumor incidence. Femle offspring of corn oil-fed mle rts exhiited longer (p.5) tumor ltency nd lower tumor multiplicity (p.5) thn femle offspring of lrd-fed mle rts (Fig. 3 nd d). Compred with femle offspring of control diet-fed mle rts, femle offspring of lrd- nd corn oil-fed mle rts did not show differences (p >.5) regrding tumor ltency nd multiplicity. Further, femle offspring of corn oil-fed mle rts showed less (p.5) tumor growth in the first week of tumor ppernce thn offspring of oth control diet- nd lrd-fed mle rts (Fig. 3c). There ws no sttisticl difference (p >.5) etween offspring of control diet- nd lrd-fed mle rts regrding tumor growth. Additionlly, there ws no sttisticl difference mong groups in the tumor growth rte for the remining experimentl weeks. Femle offspring mmmry glnd nd tumor cell prolifertion nd poptosis Femle offspring of lrd-fed mle rts exhiited n incresed (p.6) numer of prolifertive cells (Fig. 4) nd decresed (p.5) numer of poptotic cells (Fig. 4e) in mmmry glnd loules compred with femle offspring of control diet- nd corn oil-fed mle rts. There ws no difference (p >.5) etween femle offspring of control diet- nd corn oil-fed mle rts regrding these prmeters (Fig. 4 nd e). Further, there ws no difference (p >.5) in cell prolifertion nd poptosis in mmmry glnd ducts mong femle offspring of ll groups. Femle offspring of oth lrd- nd

8 Fontelles et l. Brest Cncer Reserch (216) 18:71 Pge 7 of 13 LN TEB A B Ductl Elongtion (cm) Numer of TEBs corn oil-fed mle rts exhiited decresed (p.5) numer of poptotic cells (Fig. 4f) in mmmry tumors compred with femle offspring of control diet-fed mle rts. There ws no difference (p >.5) etween femle offspring of lrd- nd corn oil-fed mle rts regrding this prmeter (Fig. 4f). In ddition, there ws no difference (p >.5) mong groups regrding cell prolifertion in the mmmry tumors (Fig. 4c). mirna expression profile in fthers sperm cells nd in their dughters mmmry glnds To compre the outcomes of the distinct pternl highft diets on the sis of mirna expression, Applied Biosystems TqMn Rodent MicroRNA rrys were used to generte the mirna profile for lrd- nd corn oil-fed fthers sperm cells, s well s for their respective dughters mmmry glnds. The microrry dt re deposited in the Gene Expression Omnius (GEO) pulic repository under ccession numer [GEO:GSE7712]. Corn oil-fed mle rts hd 89 downregulted (p.5) mirnas in the sperm compred with lrd-fed mle rts (Fig. 5). Furthermore, femle offspring of corn oil-fed mle rts hd 21 downregulted (p.5) nd 2 upregulted (p.5) mirnas in their mmmry glnds compred with femle offspring of lrd-fed mle rts (Fig. 5). There were three mirnas tht were downregulted in oth the sperm nd Fig. 2 Mmmry glnd development of 5-dy-old femle offspring of control diet (CO)-, lrd (LB)-, nd corn oil (CB)-fed mle rts. Histologicl depiction of the fourth dominl mmmry glnd showing ductl elongtion, indicted y rrow. Terminl end uds (TEBs), indicted y rrows. c Ductl elongtion. d Numer of TEBs. All vlues re expressed s the men ± SEM. Sttisticlly significnt difference (p.5) compred with CO nd LB, ccording to nlysis of vrince followed y lest significnt difference test. The dt re expressed s men ± SEM (n = 5 per group) C D Numer of tumor-free nimls First tumor ltency (dys) Numer of tumors per rt B Dys fter DMBA dministrtion A Tumor growth rte(%) D the mmmry glnds of the corn oil-fed fthers nd their dughters, respectively: mir p, mir p, nd mir-376#. IPA (Additionl file 1: Tle S1) indicted tht these mirnas could regulte signling pthwys ssocited with key physiologicl processes such s growth hormone, phosphtse nd tensin homolog (PTEN), nd prolctin signling, s well s disese processes such s Huntington s disese, crdic hypertrophy, type 2 dietes mellitus, nd rest cncer. Protein expression in femle offspring mmmry glnd Since mir p, mir p, nd mir-376# cn directly or indirectly modulte severl trgets (shown in Additionl file 1: Tle S1), we decided to perform C CO LB CB,d Fig. 3 Mmmry tumorigenesis in femle offspring of control diet (CO)-, lrd (LB)-, nd corn oil (CB)-fed mles. Numer of tumor-free rts. Numer of dys efore the ppernce of the first tumor. c Tumor growth rte in the first week of ppernce. d Totl numer of tumors per rt (multiplicity). Sttisticlly significnt difference (p.5) compred with CO nd LB, ccording to nlysis of vrince followed y lest significnt difference test. Mrginl difference (p.8) compred with d LB, ccording to t test. The dt re expressed s men ± SEM (n = 24 per group). DMBA 7,12-dimethylenz[]nthrcene

9 Fontelles et l. Brest Cncer Reserch (216) 18:71 Pge 8 of 13 A D Numer of KI67+ cells/1 cells Numer of poptotic cells/1 cells Ducts Ducts B E c Loules Loules d CO LB CB Numer of Ki67+ cells/1 cells Numer of poptotic cells/ 1 cells C F Fig. 4 Quntifiction of cell prolifertion nd poptosis in mmmry glnd nd tumors. Photomicrogrph ( 2 originl mgnifiction) of Ki-67 immunostining (cells indicted y rrows). Quntifiction of cell prolifertion in the mmmry glnd ducts nd loules of 5-dy-old femle offspring of control diet (CO)-, lrd (LB)-, nd corn oil (CB)-fed mle rts. c Quntifiction of cell prolifertion in mmmry tumors of femle offspring from CO-, LB-, nd CB-fed mle rts. d Photomicrogrph ( 4 originl mgnifiction) showing poptotic cells (indicted y rrows). e Quntifiction of poptosis in the mmmry glnd ducts nd loules of 5-dy-old femle offspring of CO-, LB-, nd CB-fed mle rts. f Quntifiction of poptosis in mmmry tumors of femle offspring of CO-, LB-, nd CB-fed mles. Sttisticlly significnt difference (p.5) compred with CO nd LB, ccording to nlysis of vrince followed y lest significnt difference test. Mrginl difference (p.6) compred with c CO nd d LB, ccording to t test. The dt re expressed s men ± SEM (n =4pergroup) CO LB CB Western lot nlysis of the following proteins linked to rest cncer: CCAAT/enhncer-inding protein et (Cepβ), cspse 3 (Csp3), insulin-like growth fctor 1 receptor (Igf1r), protein kinse D1 (Pkd1), nd trnsforming growth fctor, et receptor I (Tgfβr1). On the one hnd, there ws no difference (p >.5) mong femle offspring of the control diet-, lrd-, nd corn oilfed mle rts regrding Cepβ, Csp3, nd Igf1r levels (dt not shown). On the other hnd, femle offspring of the corn oil-fed mle rts hd higher (p.5) Pkd1 levels in the mmmry glnds thn femle offspring of lrd-fed, ut not control diet-fed, mle rts (Fig. 6). There ws no difference (p >.5) etween femle offspring of control diet- nd lrd-fed mle rts regrding this protein (Fig. 6). In ddition, Tgfβr1 levels were significntly incresed in the offspring of lrd-fed mle rts (Fig. 6) compred with offspring of oth control diet-fed (p.5) nd corn oil-fed (p.6) mle rts. There ws no difference (p >.5) etween femle offspring of corn oil-fed nd control diet-fed mle rts regrding this protein (Fig. 6). Interestingly, oth proteins re involved in regulting epithelil-to-mesenchyml trnsition (EMT): Pkd1 inhiits this process [36], nd Tgfβr1 promotes it [37]. We further explored if Tgfβ nd key regultors of its ctivity were ltered y mesuring protein levels of v-kt murine thymom virl oncogene (Akt), cofilin (Cfl), v-rf leukemi virl oncogene (c-rf), extrcellulr signlregulted kinse 1/2 (Erk1/2), phosphorylted mitogenctivted protein kinse 8 (p-jnk), mitogen-ctivted protein kinse 4 (Mkk4), mechnistic trget of rpmycin (Mtor), mitogen-ctivted protein kinse 14 (p38), phosphorylted Smd fmily memer 3/Smd fmily memer 3 (p-smd3/smd3) rtio, nd Hrvey rt srcom virus oncogene (Rs). Tgfβ protein expression ws higher (p.5) in the mmmry glnds of the femle offspring of lrd-fed mle rts thn in the offspring of control dietnd corn oil-fed mle rts. There ws no difference (p >.5) etween femle offspring of corn oil- nd control diet-fed mle rts regrding this protein (Fig. 6). Further, the levels of Akt nd p-jnk were higher (p.5) in the femle offspring of lrd-fed mle rts thn in femle offspring of control diet-fed mle rts (Fig. 6). There ws no difference (p >.5) in femle offspring of corn oil-fed mle rts nd the offspring of control diet- nd lrd-fed mle rts regrding these proteins (Fig. 6). Femle offspring of corn oil-fed mle rts hd lower levels of Mtor, Mkk4 (p.6), nd p-smd3/smd3 (p.5) thn femle offspring of lrd-fed, ut not control diet-fed, mle rts (Fig. 6). There ws no difference (p >.5)etweenfemle offspring of control diet- nd lrd-fed mle rts

10 Fontelles et l. Brest Cncer Reserch (216) 18:71 Pge 9 of 13 A B Fig. 5 Het mp of microrna (mirna or mir) expression profiles. Het mp of mirna expression profile in sperm of the mle rts from control diet-, lrd-, nd corn oil-fed mle rts. Het mp of mirna expression profile in norml mmmry tissue of 5-dy-old femle offspring (n = 3 per group)

11 Fontelles et l. Brest Cncer Reserch (216) 18:71 Pge 1 of 13 Pkd1 Akt Mtor Mkk4 p-jnk p-smd3 Smd3 Protein Level (rtio to ctin) CO LB CB d d d Pkd1 Tgf r1 Tgf Akt Mtor Mkk4 p-jnk p-smd3/ Smd3 Fig. 6 Protein ltertions ssocited with microrna (mirna) expression. Western lot nlysis of protein kinse D1 (Pkd1), trnsforming growth fctor, et receptor I (Tgfβr1), trnsforming growth fctor et (Tgfβ), v-kt murine thymom virl oncogene (Akt), mechnistic trget of rpmycin (Mtor), mitogen-ctivted protein kinse kinse 4 (Mkk4), phosphorylted mitogen-ctivted protein kinse 8 (p-jnk), nd phosphorylted Smd fmily memer 3/Smd fmily memer 3 (p-smd3/smd3) rtio protein expression in mmmry glnds of 5-dy-old femle offspring of control diet (CO)-, lrd (LB)-, nd corn oil (CB)-fed mle rts. Sttisticlly significnt difference (p.5) compred with CO nd LB, ccording to nlysis of vrince followed y lest significnt difference test. Mrginl difference (p.6) compred with d LB, ccording to t test. The dt re expressed s men ± SEM (n = 5 per group) regrding these proteins (Fig. 6). In ddition to promoting EMT, ll these ltered proteins re involved in incresing cell survivl, growth, migrtion, nd invsion. Discussion Brest cncer is complex disese with multifctoril etiology [38]. It is incresingly evident tht in utero environment cn progrm lter susceptiility to rest cncer [39]. The findings of our present study suggest tht rest cncer risk cn e determined even erlier through diet-induced chnges in pternl germ cells efore conception. Our study shows tht, compred with femle offspring of control diet-fed fthers, offspring of lrd-fed fthers did not differ in tumor ltency, growth, or multiplicity. However, femle offspring of lrd-fed fthers hd incresed elongtion of the mmmry epithelil tree, numer of TEBs, nd tumor incidence compred with oth control diet- nd corn oil-fed fthers, showing tht pternl exposure to lrd-sed high-ft diet contining SFA incresed their dughters mmmry cncer risk. TEBs re considered sites of tumor initition [4], nd incresed epithelil elongtion reflects rpid epithelil growth [41]. Additionlly, femle offspring of lrd-fed fthers showed incresed cell prolifertion nd decresed poptosis in the mmmry glnd loules compred with femle offspring of oth control diet- nd corn oil-fed fthers. Altogether, these findings support the view tht ltered mmmry glnd development represents potentil underlying mechnism of incresed rest cncer risk [42]. Compred with femle offspring of control diet-fed fthers, femle offspring of corn oil-fed fthers hd decresed tumor growth. There ws no difference in tumor incidence, ltency, or multiplicity etween femle offspring of control diet- nd corn oil-fed fthers. In ddition, femle offspring of corn oil-fed fthers hd longer tumor ltency, decresed tumor growth, nd decresed multiplicity compred with femle offspring of lrd-fed fthers. These dt show tht pternl exposure to corn oilsed high-ft diet contining n-6 PUFA hd n effect opposite tht of lrd-sed high-ft diet nd reduced their dughters mmmry cncer risk. Although mle rts tht were fed the lrd-sed nd corn oil-sed high-ft diets consumed the sme mount of clories, lrd incresed the ody weight nd size of epididyml ft pds more thn corn oil did. Thus, different ftty cids cn hve distinct effects on dipose ccumultion, s lredy shown y others [43]. Our results further show tht lrd, ut not corn oil, elicited detrimentl effects on mle reproductive prmeters (fewer norml sperm cells nd lower dily sperm production).

12 Fontelles et l. Brest Cncer Reserch (216) 18:71 Pge 11 of 13 This is in line with erlier humn nd niml dt showing tht SFA disrupt testiculr metolism nd sperm qulity, while PUFA re essentil for sperm cell memrne fluidity nd flexiility s well s fertiliztion [44]. Excessive epididyml ft in lrd-fed mles my hve een detrimentl to spermtogenesis, s epididyml tissue is n essentil depot for spermtogenesis [45]. The dverse effects of lrd my not e medited through incresed insulin resistnce. Although correltion etween insulin resistnce nd impired sperm production hs een reported in rts fed diet high in SFA [46], s lso found in the present study, corn oil-sed high-ft diet lso impired insulin tolernce ut did not ffect mle reproductive prmeters. We propose tht impired sperm qulity nd function in lrd-fed fthers could e ssocited with disruption in metolic progrmming nd incresed rest cncer risk mong their dughters. The impct of oesity in fthers leding to metolic dysfunction in their femle offspring ws previously oserved in rodent studies [1], nd it ws lso seen in our present study. Femle offspring of oth lrd- nd corn oil-fed fthers exhiited incresed ody weight nd diposity. However, only femle offspring of lrd-fed fthers displyed n impired insulin response, indicting tht the type of dietry ftty cids consumed represents key fctor in metolic progrmming through the mle germline. Epigenetic modifictions tht re necessry for chieving reproductive cpcity of mle gmetes include DNA methyltion, histone retention, nd expression of noncoding RNAs such s mirnas [47]. Becuse mirnas cn modulte the expression of hundreds of mrnas tht ffect emryonic development s well s the estlishment of the offspring s epigenome [48], they hve een proposed to medite pternl progrmming effects on the offspring [49]. The epididymis hs een implicted s the site of the ltertions of mirna signtures occurring during the mturtion of sperm cells, nd therefore n increse in epididyml ft pd size could potentilly impct inheritnce of mirna signtures nd/ or the developmentl trjectory of the offspring [5]. The impct of high-ft-diet-induced mle oesity on the mirna profile in mture spermtozo hs een exmined in rodent studies [51]. In study y Fullston et l. [52], mles fed high-sturted-ft diet exhiited chnges in mirnas in the testes nd mture spermtozo tht trget mrna ssocited with spermtogenesis, emryonic development, nd metolic diseses in the offspring. We provide further evidence tht pternl nutrition cn impct the sperm mirna profile nd possily the susequent mmmry glnd mirna profile, which in turn trgets genes implicted in rest cncer nd other diseses. Some of the mirnas tht were differentilly expressed in the lrd- nd corn oil-fed fthers germ cells lso were differentilly expressed in their dughters mmmry glnds, lthough the dughters were never directly exposed to the high-ft diets. When we compred lrd-fed fthers nd their dughters, we oserved three mirna tht were significntly ltered in oth the sperm nd mmmry glnds of corn oil-fed fthers nd their dughters: mir p, mir p, nd mir-376#. Since mirnas cn modulte gene expression y inhiiting the trnsltion of mrna or y directing their degrdtion [53], we focused on determining if the expression of the top potentilly trgeted proteins (Additionl file 1: Tle S1) ws ltered in the dughters of lrd- or corn oil-fed fthers. Among them, we highlight Pkd1 nd Tgfβr1. PKD1 is serine/threonine kinse tht is expressed in ductl epithelil cells of the mmmry glnd, mintins the epithelil phenotype, nd prevents EMT [54]. Inhiition of PKD1 cn led to pthologicl conditions such s cncer [55]. Thus, our finding of incresed Pkd1 levels in the mmmry glnds of corn oil-fed fthers offspring, compred with femle offspring of lrd-fed fthers, is in line with their lowest susceptiility to rest cncer. In ddition, compred with femle offspring of control dietnd corn oil-fed fthers, nother mirna trget, Tgfβr1, ws incresed in the dughters of lrd-fed fthers tht displyed the highest susceptiility to mmmry cncer. Tgfβr1 expression is relted to promotion of rest crcinogenesis through multiple mechnisms, including enhncing EMT [56]. We ssessed the protein levels of up- nd downstrem signling prtners of Tgfβr1. Femle offspring of lrd-fed fthers showed higher protein levels of Tgfβ thn femle offspring of oth control diet- nd corn oil-fed fthers, s well s higher protein levels of Akt nd p-jnk thn control diet-fed fthers. In ddition, femle offspring of corn oil-fed rts hd lower levels of Mtor, Mkk4, nd p-smd3/smd3 thn femle offspring of lrdfed fthers. These proteins collectively ply importnt roles in cell survivl, growth, migrtion, nd invsion [57, 58]. These findings indicte tht mechnisms other thn mirnas contriute to chnges in gene expression in the dughters mmmry tissue following pternl exposure to lrd-sed high-ft diet. Becuse fthers, mothers, nd their dughters tend to shre the sme nutritionl hits [59], it is importnt to further investigte if pternlly progrmmed rest cncer risk is ffected y mternl nd femle offspring s ft intke. Mternl intke of high corn oil diet during pregnncy increses femle offspring s mmmry cncer risk [8], while intke of lrd hs opposite effects [9]. In ddition, ecuse oesity-induced ltered sperm mirna expression in the fthers cn e normlized through exercise or dietry intervention (consumption of lnced diet), which then improves the metolic helth of femle offspring [6], the efficcy of similr intervention in reducing dughters rest cncer risk should e investigted.

13 Fontelles et l. Brest Cncer Reserch (216) 18:71 Pge 12 of 13 Conclusions In the present study, we show tht pternl intke of lrd-sed high-ft diet rich in SFA increses femle offspring s mmmry cncer risk, s indicted y the incresed elongtion of the mmmry epithelil tree, numer of TEBs, nd tumor incidence in femle offspring of lrd-fed fthers compred with femle offspring of oth control diet- nd corn oil-fed rts. However, if the pternl ft source is corn oil tht is high in n-6 PUFA, these mle rts offspring s mmmry cncer risk is reduced, s indicted y decresed tumor growth in femle offspring of corn oil-fed fthers compred with femle offspring of oth control diet- nd lrd-fed fthers, s well s y longer tumor ltency nd decresed tumor multiplicity compred with femle offspring of lrd-fed fthers. Altered mirna expression in fthers sperm nd dughters mmmry glnds my t lest underlie these effects, ut other epigenetic chnges re likely to e involved. Our findings highlight the importnce of pternl nutrition in ffecting future genertions risk of developing rest cncer. Additionl file Additionl file 1: Tle S1. Cnonicl IPA nlyses of the trget pthwys nd molecules modulted y ltered mirna from fther s sperm nd 5-dy-old femle offspring mmmry glnds from lrd-fed (LB) nd corn oil-fed (CB) mles. (DOC 31 k) Arevitions Akt, v-kt murine thymom virl oncogene; ANOVA, nlysis of vrince; Csp3, cspse 3; CB, rts tht were fed corn oil-sed high-ft diet nd their offspring; Cepβ, CCAAT/enhncer-inding protein et; Cfl, cofilin; CO, rts tht were fed control diet nd their offspring; c-rf, v-rf leukemi virl oncogene; DMBA, 7,12-dimethylenz[]nthrcene; EMT, epithelil-tomesenchyml trnsition; Erk1/2, extrcellulr signl-regulted kinse 1/2; Igf1r, insulin-like growth fctor 1 receptor; ITT, insulin tolernce test; LB, rts tht were fed lrd-sed high-ft diet nd their offspring; LSD, lest significnt difference; mirna or mir, microrna; Mkk4, mitogen-ctivted protein kinse kinse 4; mrna, messenger RNA; Mtor, mechnistic trget of rpmycin; MUFA, monounsturted ftty cid; p38, mitogen-ctivted protein kinse 14; p-jnk, phosphorylted mitogen-ctivted protein kinse 8; Pkd1, protein kinse D1; p-smd3/smd3, phosphorylted Smd fmily memer 3/Smd fmily memer 3 rtio; PTEN, phosphtse nd tensin homolog; PUFA, polyunsturted ftty cid; Rs, Hrvey rt srcom virus oncogene; SCLB, somtic cell lysis uffer; SFA, sturted ftty cid; TEB, terminl end ud; Tgfβr1, trnsforming growth fctor, et receptor I Acknowledgements We thnk the following Georgetown Lomrdi Cncer Center Shred Resources (SR) for their ssistnce: Animl Model SR, Histopthology & Tissue SR, nd Genomics & Epigenomics SR. We lso thnk Lucine F. L. Mcedo (Fculty of Phrmceuticl Sciences, University of São Pulo) for technicl ssistnce with the dietry ftty cid quntifiction, Prof. Odir Aguir Jr. (Federl University of São Pulo) for technicl support with the mle reproductive prmeters nlysis, Prof. Luis Fernndo Brisn (Institute of Biosciences, Stte University of São Pulo) for technicl support with cell poptosis nlysis, nd Prof. Fernndo Slvdor Moreno (Fculty of Phrmceuticl Sciences, University of São Pulo) for technicl support with the rt experimentl protocol. Funding This study ws supported y the Brzilin Ntionl Council for Scientific nd Technologicl Development (CNPq; process /212-8), the São Pulo Stte Reserch Funding Agency (FAPESP; process 211/ ), the Ntionl Cncer Institute (grnt R1 CA to LHC), nd the Prevent Cncer Foundtion (reserch grnt to SdA). Authors contriutions CCF conceived of the study; supervised niml work, tissue collection, nd moleculr nlysis; nd drfted the mnuscript. LNG ssisted with niml studies, tissue collection, nd moleculr ssys nd helped to revise the mnuscript. MPR ssisted with niml studies, tissue collection, nd moleculr ssys nd helped to revise the mnuscript. FdOA ssisted with niml study design nd dt nlyses nd helped to revise the mnuscript. LJ performed mirna dt nlysis nd helped to revise the mnuscript. JI ssisted with tumor nd sttisticl nlyses nd helped to revise the mnuscript. VCP conducted mle reproductive prmeter nlysis nd helped to revise the mnuscript. IAdC conducted nlysis of the diets lipid profiles nd helped to revise the mnuscript. LHC provided intellectul input nd helped to drft the mnuscript. SdA provided intellectul input nd helped to drft the mnuscript. TPO conceived of the study, oversw the reserch, nd drfted the mnuscript. All uthors red nd pproved the finl mnuscript. Competing interests The uthors declre tht they hve no competing finncil or non-finncil competing interests. Author detils 1 Deprtment of Food nd Experimentl Nutrition, Fculty of Phrmceuticl Sciences, University of São Pulo, Avenid Professor Lineu Prestes 58, Bloco 14, São Pulo, SP 558-, Brzil. 2 Georgetown Lomrdi Comprehensive Cncer Center, Wshington, DC 27, USA. 3 Food Reserch Center (FoRC), São Pulo 558-, Brzil. Received: 2 Jnury 216 Accepted: 17 June 216 References 1. Ferly J, Soerjomtrm I, Ervik M, Dikshit R, Eser S, Mthers C, Reelo M, Prkin DM, Formn D, Bry F. GLOBOCAN 212 v1.: estimted cncer incidence, mortlity nd prevlence worldwide in 212. Lyon, Frnce: Interntionl Agency for Reserch on Cncer; Accessed 17 Nov Rhi L, Smith BD, Aizenerg R, Rosenzweig AB, Fleshmn JM, Mtrisin LM. Projecting cncer incidence nd deths to 23: the unexpected urden of thyroid, liver, nd pncres cncers in the United Sttes. Cncer Res. 214;74: Howell A, Anderson AS, Clrke RB, Duffy SW, Evns DG, Grci-Closs M, Gescher AJ, Key TJ, Sxton JM, Hrvie MN. Risk determintion nd prevention of rest cncer. Brest Cncer Res. 214;16: Aluquerque RC, Bltr VT, Mrchioni DM. Brest cncer nd dietry ptterns: systemtic review. Nutr Rev. 214;72: Cordin L, Eton SB, Sestin A, Mnn N, Lindeerg S, Wtkins BA, O'Keefe JH, Brnd-Miller J. Origins nd evolution of the Western diet: helth implictions for the 21st century. Am J Clin Nutr. 25;81: Lwrence GD. Dietry fts nd helth: dietry recommendtions in the context of scientific evidence. Adv Nutr. 213;4: Colditz GA, Bohlke K, Berkey CS. Brest cncer risk ccumultion strts erly: prevention must lso. Brest Cncer Res Tret. 214;145: Hilkivi-Clrke L, Clrke R, Onojfe I, Rygd M, Cho E, Lippmn M. A mternl diet high in n-6 polyunsturted fts lters mmmry glnd development, puerty onset, nd rest cncer risk mong femle rt offspring. Proc Ntl Acd Sci U S A. 1997;94: De Oliveir AF, Fontelles CC, Rosim MP, de Oliveir TF, de Melo Loureiro AP, Mncini-Filho J, Rogero MM, Moreno FS, de Assis S, Brisn LF, Hilkivi- Clrke L, Ong TP. Exposure to lrd-sed high-ft diet during fetl nd lcttion periods modify rest cncer susceptiility in dulthood in rts. J Nutr Biochem. 214;25: Ng SF, Lin RC, Lyutt DR, Brres R, Owens JA, Morris MJ. Chronic high-ft diet in fthers progrms β-cell dysfunction in femle rt offspring. Nture. 21;467:963 6.