breast cancer is the most common cancer 
 among american women and 
 the second leading cause of cancer deaths . it is estimated that 232,340 
 women will be diagnosed with breast cancer and 39,620 will die in 
 2013 . accumulating data from epidemiological 
 and experimental studies have shown that reproductive hormones , particularly 
 e2 , play a significant role in breast cancer etiology . 
 breast carcinogenesis is highly interlinked with the high levels of 
 e2 and its metabolism to catechols mediated by cytochrome 
 p450 enzymes . other phase i and phase 
 ii enzymes involved in the metabolic activation and deactivation include 
 estrone sulfatase , sulfotransferases , catechol - o - methyltransferase , 
 and uridine-5-diphosphate glucuronosyltransferase . the biological effects of e2 and its 
 metabolites are mediated by two distinct e2 receptors ( er- 
 and er- ) by binding with varying affinities . micrornas ( mirnas ) are small noncoding rnas , and 
 their expressions 
 have been correlated with specific breast cancer pathological features 
 such as e2 and progesterone receptor expression , proliferation 
 index , and tumor stage . in an independent 
 study , the expression of 667 mirnas in 29 breast tumor and 21 adjacent 
 normal tissues demonstrate 130 mirnas to have significant differential 
 expressions in breast tumors when compared to the normal adjacent 
 tissues . mirna expression is suppressed 
 or stimulated by e2 and other e2 receptor ligands 
 in human breast cancer cells , rat mammary gland , and other e2 responsive organs such as the endometrium and uterus . there is growing evidence from in vitro 
 and in vivo studies that 
 consumption of fruits and vegetables is associated with reduced risk 
 of developing cancer . berries are rich 
 in anthocyanins , which are considered to be a good candidate for preventing 
 the development of cancer by protecting cells from the damage caused 
 by reactive oxygen species . there are 
 over 500 anthocyanins characterized , but a great majority of them 
 contain a core structure of six anthocyanidins , namely , cyanidin , 
 delphinidin , malvidin , pelargonidin , peonidin , and petunidin . anthocyanins have mono- , di- , and tricyclic 
 sugars attached to the anthocyanidin core structure and possess high 
 antioxidant properties . blueberry ( bb ) is among the few fruits 
 that contain five of the major anthocyanidins ( cyanidin , delphinidin , 
 malvidin , peonidin , and petunidin ) . anthocyanins 
 activate phase ii enzymes and induce apoptosis as well as demonstrate 
 antiproliferative , anti - inflammatory , and antiangiogenesis properties . in our recent study , an equimolar mixture of 
 five major anthocyanidins was found to synergistically inhibit proliferation 
 of two non - small - cell lung cancer cell lines both in cell culture 
 and in vivo . in another study , we have 
 demonstrated that dietary blueberry ( berkley ) when provided in chemoprevention 
 mode ( 5% , w / w ) inhibited e2-mediated mammary tumorigenesis 
 in aci rats . in this study , we 
 report on both chemopreventive and therapeutic 
 potential of highbush blueberry powder ( 50:50 blend of tifblue and 
 rubel ) . freeze - dried highbush whole blueberry powder ( 50:50 
 blend of tifblue and rubel ) was received from the u.s . this blend contains 38 mg / g total phenolics 
 and 21 mg / g total anthocyanins . ain-93 m diet and ain-93 m diet supplemented 
 with 2.5 and 5% bb were prepared in pellet form by harlan - teklad ( madison , 
 wi , usa ) and stored at 4 c in vacuum - sealed bags until use . bb diet was customized to have the same calorific value as ain-93 
 m. animal experiments were executed in agreement 
 with an approved protocol from the institutional animal care and use 
 committee at the university of louisville . female aci rats ( 56 
 weeks old ) were purchased from harlan sprague  dawley , inc . groups 
 1 , 4 , and 7 were provided with ain-93 m diet , and groups 2 and 5 were 
 fed a diet supplemented with bb ( 2.5% w / w ) . after 2 weeks of experimental diet intervention , 
 groups 47 were implanted subcutaneously with a 1.2 cm silastic 
 implant containing 9 mg of 17-estradiol ( e2 ) as 
 described . group 7 received control ain-93 
 m diet for 14 weeks and then was changed to bb diet ( 5% w / w ) after 
 the appearance of first palpable tumor in control group . as the bb 
 diet in group 7 started after the appearance of tumors , this was considered 
 as therapeutic intervention . starting from 12 weeks of e2 treatment , animals were palpated weekly for tumor incidence 
 and multiplicity . when the palpable tumors reached > 90% in group 
 4 , 
 animals in all groups were euthanized by co2 asphyxiation . lung , liver , brain , mammary tissue , and pituitary gland were collected , 
 weighed , and snap frozen for further analysis . small pieces of mammary tissue 
 and pituitary gland were fixed in 10% formalin for histopathological 
 analysis . to assess the presence of microscopic 
 tumors and pathological changes in the mammary glands of rats treated 
 with e2 for 12 weeks , 5 m mammary tissue sections 
 ( n = 21 ) from multiple previous studies were stained 
 with hematoxylin and eosin and analyzed by a board - certified veterinary 
 pathologist . tissue sections 
 from five randomly 
 selected animals from each group were stained for proliferating cell 
 nuclear antigen ( pcna ) using a pcna staining kit ( life technologies , 
 grand island , ny , usa ) following the manufacturer s protocol . five to six different sites in each slide were scored for deeply stained 
 epithelial nuclei by two cytopathologists and represented as percent 
 deeply stained cells . plasma prolactin 
 levels were quantified 
 using a rat prolactin elisa immuno - assay kit ( alpco , salem , nh , usa ) 
 following the manufacturer s instructions . briefly , plasma 
 samples ( n = 5 ) from e2-treated groups 
 were diluted 100- or 200-fold , and plasma from groups that did not 
 receive e2 was diluted 2-fold prior to the analysis . the 
 samples were added to a 96-well plate incubated with rat prolactin 
 sample buffer followed by enzyme - labeled anti - rat prolactin antibody . 
 then samples were incubated with 3,3,5,5-tetramethylbenzidine 
 ( tmb ) and substrate solution ( provided in the kit ) followed by measurement 
 of the optical density at 450 nm immediately after the addition of 
 stop solution . about 25 l of diluted plasma samples was measured 
 against various concentrations of calibrator samples ranging from 
 5 to 80 ng / ml of prolactin levels . briefly , whole - cell 
 lysates were prepared from mammary tissues using radioimmunoprecipitation 
 assay ( ripa ) lysis buffer ( santa cruz biotechnology inc . , dallas , 
 tx , usa ) , and protein concentrations were measured by using the bicinchoninic 
 acid ( bca ) method . proteins were separated on mes - sds - bis - tris 412% 
 gradient gel ( invitrogen , carlsbad , ca , usa ) . the proteins were transferred 
 to pvdf membranes and probed for cyclin d1 , pcna , estrogen receptor - alpha 
 ( er- ) , c - myc , and progesterone receptor ( pr ) . total rna 
 from frozen mammary tissues was isolated using trizol reagent ( invitrogen ) . qrt - pcr was performed with 100 ng of rna using 
 a qscript one - step sybr green qrt - pcr kit ( quanta biosciences , gaithersburg , 
 md , usa ) with 500 pmol of forward and reverse primers for each gene . the genes and primer sequences are as follows : cyp1a1 ( forward , 5 
 gggatatagaagccattcagacttg 
 3 ; reverse , 5 tggagaccttccgacattcat 
 3 ) ; er- ( forward , 5 ggcacatgagtaacaaaggca 
 3 ; reverse , 5 ggcatgaagacgatgagcat 
 3 ) ; 18s ( forward , 5 gggaggtagtgacgaaaaataacaat 
 3 ; reverse , 5 ttgccctccaatggatcct 
 3 ) . quantitative pcr was carried out on a 7500 fast - real time 
 pcr instrument ( applied biosystems , green island , ny , usa ) , following 
 the manufacturer s guideline . the gene expressions were calculated 
 by relative quantification method ( 2 ) comparing 
 with control and normalizing to 18s rna . small rnas ( < 200 
 bp ) were isolated from mammary tissues using a mirvana microrna isolation 
 kit ( invitrogen ) following the manufacturer s instruction . the concentration of the small rna was quantified by bioanalyzer ( agilent 
 technologies , santa clara , ca , usa ) . qpcr analysis of mir-18a , mir20a , 
 mir25 , and mir-34c was performed using a taqman mirna reverse transcription 
 kit and taqman gene - specific mirna assays ( applied biosystems , foster 
 city , ca , usa ) according to the manufacturer s instructions . relative changes in mirna expressions were quantified by comparison 
 with the control and normalization to 5s rna . whole blood was collected at the 
 time of euthanasia to analyze serum chemistry and hematological parameters , 
 as described previously . liver functions 
 were analyzed by measuring the levels of aspartate transaminase , alanine 
 aminotransferase , alkaline phosphatase , -glutamyl transpeptidase , 
 and amylase in serum . kidney functions were analyzed by measuring 
 blood urea nitrogen , creatinine , and various electrolytes in serum . various hematological parameters such as white blood cells ( wbcs ) , 
 red blood cells , hemoglobin , hematocrit , mean corpuscular volume , 
 mean corpuscular hemoglobin , mean corpuscular hemoglobin concentration , 
 and platelet count were analyzed from whole blood . all statistical comparisons between 
 controls and e2-treated groups were made using an unpaired 
 one - tailed student s t test . diet consumption 
 was analyzed using an unpaired two - tailed student s t test , and body weight was analyzed by two - way anova . all 
 statistical analyses were done in graphpad prism software , version 
 4.3 ( san diego , ca , usa ) . there was no significant 
 difference in diet consumption among groups receiving either control 
 diet or ain-93 m diet supplemented with 2.5 and 5% bb , in the absence 
 of e2 treatment . the diet consumption , however , increased 
 in e2-treated animals fed control diet or bb - supplemented 
 diets ( figure 1a ) . the increase was significant 
 in animals receiving the 5% bb diet in both preventive and therapeutic 
 mode ; the increase was insignificant in the 2.5% bb diet group . at 
 the end of the study ( 25 weeks ) , one - way anova analysis showed no 
 significant difference in body weights among the control and e2-treated animals ( figure 1b ) . ( a ) average diet consumption 
 by aci rat per day fed with ain-93 m diet and diet supplemented with 
 2.5 and 5% bb in the presence and absence of e2 . values 
 denote mean  sd of diet consumption over a period of 25 weeks . asterisk ( a ) indicates significant difference of e2 control 
 from untreated control ( p = 0.0001 ) . asterisk ( b ) 
 indicates significant difference of bb diets from e2 control 
 ( p = 0.001 and 0.044 ) . ( b ) average body weight of 
 rats receiving either ain-93 m or bb diet . one - way anova analysis 
 showed that there was no significant difference in body weight after 
 25 weeks of e2 treatment . bb diet was 
 highly effective in favorably modulating the various tumor indices 
 ( latency , incidence , multiplicity , and burden ) . the groups receiving 5% bb diet in both 
 the chemopreventive and therapeutic modes had a significant delay 
 with the first tumor appearance in the control group by 28 and 37 
 days , respectively , as compared to the age - matched control animals 
 ( figure 2a ) . the group that received 2.5% bb 
 diet also had delayed tumor latency but only slightly , about 7 days . 
 at the end of the study , the control group developed palpable mammary 
 tumors in 96% animals , whereas animals provided with the 5% bb diet 
 in chemopreventive and therapeutic groups had palpable tumors only 
 in 60% ( p = 0.0109 ) and 55% ( p = 
 0.0021 ) of animals , respectively ; the 2.5% bb diet also showed some 
 ( 11% ) but insignificant reduction in tumor incidence compared to the 
 control group . animals receiving control diet had an 
 average tumor volume of 979  218 mm , whereas animals 
 on the 5% bb diet had significant reductions , 512  130 mm ( p = 0.0398 ) and 452  144 mm ( p = 0.0106 ) , for chemopreventive and therapeutic 
 mode , respectively ( figure 2b ) . animals on 
 the 2.5% bb diet showed a modest but insignificant ( 0.1435 ) reduction 
 with an average tumor volume of 698  145 mm . tumor 
 multiplicity in control animals was 4.17  0.48 per animal , and 
 it was reduced to 2.17  0.52 ( p = 0.0039 ) and 
 1.75  0.45 ( p = 0.0004 ) by the 5% bb diet given 
 in chemopreventive and therapeutic mode , respectively ( figure 2c ) ; the 2.5% bb diet also showed a significant reduction 
 in tumor multiplicity , 2.75  0.45 ( p = 0.0199 ) . effect 
 of blueberry diet on ( a ) tumor incidence , ( b ) tumor volume , 
 and ( c ) tumor multiplicity . ( a ) tumor incidence was calculated from 
 the weekly palpation report and analyzed using a nonparametric log - rank 
 test . asterisk indicates significant difference of 5% bb diet [ preventive 
 ( p = 0.0109 ) and therapeutic mode ( p = 0.0021 ) ] from e2-treated control . ( b ) tumor volume 
 and multiplicity were calculated at the time of euthanasia and analyzed 
 using unpaired one - tailed student s t test . asterisk indicates that the tumor volume of both 5% bb diets ( p = 0.0398 and p = 0.0106 ) was significantly 
 different from e2-treated control . at the end of 
 the study , the liver , mammary , and pituitary tissue weights were not 
 significantly different among the groups without e2 treatment . mammary tissue weight with 5% bb diet in chemopreventive mode was 
 modestly offset ( p = 0.0486 ) from e2-treated 
 control . there was a 45-fold increase in the pituitary weight 
 in e2-treated animals ; however , this was significantly 
 offset with the 5% bb diet in preventive ( 33.9  5.6 mg vs 42.2 
  10.7 mg ; p = 0.0031 ) and therapeutic ( 34.4 
  5.3 mg ; p = 0.0031 ) modes ( table 2 ) . pituitary weights in animals receiving the 2.5% 
 bb diet also showed significant decrease ( 34.4  8.0 mg ; p = 0.0070 ) . average body weight of animals 
 is measured at the time of euthanasia . in all animals that were treated with 
 e2 for 12 weeks ( n = 21 ) , there was expansion 
 of mammary tissue with proliferation of terminal buds that differentiated 
 into acini . the acinar epithelial cells were frequently vacuolated , 
 and the acini contained amphophilic inspissated secretory material 
 filling the lumina ( figure 3a ) . concomitantly , 
 there was proliferation of ductules and ducts with variable grades 
 of epithelial dysplasia ( n = 13 ) . the ductular epithelial 
 cells were cuboidal with distinct cell borders and moderate amounts 
 of eosinophilic cytoplasm . often ( n = 4 ) , the nuclei 
 showed karyomegaly and were hyperchromatic , containing one to three 
 distinct nucleoli ( figure 3b ) . mitotic figures 
 were not present in the sections examined . in > 50% of cases ( n = 11 ) , the ducts were ectatic and lined by hyperplastic 
 epithelium that formed two or more layers ( figure 3a ) . ductular adenomas ( n = 3 ) were characterized 
 by the development of a 100250 m long papillary projection 
 from the ductular walls that narrowed the lumina ( figure 3a , inset ) . the neoplastic cells contained scant 
 cytoplasm and round nuclei with one to three distinct nucleoli . in 
 all cases , desquamated epithelial cells were present within the ductular 
 lumina . multifocal perivascular mast cell infiltration correlated with the 
 severity of the lesion in all cases . in addition , the ductular epithelial 
 dysplasia correlated with the size of expansion of the terminal bud . presence 
 of epithelial dysplasia and ductular papillomas from mammary 
 tissue of rats treated with e2 implant for 12 weeks . ( a ) 
 image of mammary section at 40 magnification showing long papillary 
 projections from ductular walls ( thin arrows ) and ducts lined with 
 hyperplastic epithelium with two or more layers ( bold arrows ) . ( b ) mammary 
 section at 600 magnification with nuclei showing karyomegaly , 
 which are hyperchromatic containing one to three distinct nucleoli 
 ( thin arrows ) . cell proliferation was analyzed 
 by staining tumor and adjacent mammary tissue for pcna protein markers 
 using antibody - based assay . after 25 weeks , there was no significant 
 difference in cell proliferation among animals receiving either control 
 or 2.5 and 5% bb diets . however , the e2 treatment increased 
 the cell proliferation to nearly 6-fold ( p = 0.0095 ) 
 ( figure 4 ) . this increase in the cell proliferation 
 was significantly offset by 34 and 35% with 5% bb diet administered 
 in chemopreventive and therapeutic mode , respectively ( figure 4 ) ; the effect was insignificant with the 2.5% bb 
 diet . proliferative index and effect of blueberry diet with or without 
 estrogen ( e2 ) treatment . ( b ) graph denotes the percentage of deeply stained cells for pcna 
 in mammary tissues ( n = 5 ) . statistically significant from e2-treated 
 control ( p = 0.0373 and p = 0.0251 ) . no significant difference was observed 
 in the levels of plasma prolactin between untreated groups and bb 
 diet groups . on the other hand , there was a substantial increase ( p = 0.0035 ) in prolactin levels with e2 treatment 
 ( 6973  2443 ng / ml ) compared to untreated control ( 11.8  
 1.8 ng / ml ) . after 25 weeks , animals provided with 5% bb diet in chemopreventive 
 ( 2205  438 ng / ml ; p = 0.05 ) and therapeutic 
 mode ( 1950  285 ng / ml ; p = 0.01 ) demonstrated 
 significant reduction in the elevated levels of plasma prolactin ( figure 5 ) . a modest but insignificant reduction in prolactin 
 levels ( 4193  1509 ng / ml ) was also observed with the 2.5% bb 
 diet . circulating plasma prolactin levels among rats receiving either 
 control diet or diet supplemented with 2.5 and 5% bb . values represent 
 the average of six samples  se done in duplicates .  statistically significant from untreated control ( p = 0.0035 ) .  statistically significant 
 from e2-treated control (  , p = 
 0.05 ;  , p = 0.01 ) . there was a pronounced 
 effect of e2 on the expressions of cytochrome cyp 1a1 and 
 er- at the mrna levels . all groups were compared against the 
 untreated control , and the fold change was expressed as mean  
 standard error . the 5% bb intervention provided in chemopreventive 
 and chemotherapeutic modes both significantly offset the e2-associated increase in cyp1a1 to 28.3  6-fold ( p = 0.0011 ) and 27.6  10.3-fold ( p = 0.0102 ) , 
 respectively . similarly , the mrna expression level of er- in 
 e2-treated animals was up - regulated by 19.7  2-fold 
 compared to untreated control , and this increase was also significantly 
 offset by the 5% bb diet , chemopreventive mode ( 14.2  2-fold ; p = 0.0201 ) and therapeutic mode ( 11.1  4-fold ; p = 0.05 ) ( figure 6a ) . western blot 
 analysis showed that e2 treatment up - regulated both cyclin 
 d1 and pcna . intervention of 5% bb diet in both preventive and therapeutic 
 modes down - regulated cyclin d1 and pcna at protein levels . however , 
 er- protein level was not modulated with either e2 treatment or administration of bb diet . asterisk represents 
 significant difference from e2-treated control ( p = 0.0011 and 0.01 ) . ( b ) protein expression of cyclin d1 , 
 pcna , and er- in mammary tissue . the total cellular lysate 
 isolated from the mammary tissues was analyzed by western blot . we analyzed 
 four e2-specific mirnas ( mir-18a , -20a , -25 , and -34c ) 
 on the basis of our previous study in this animal model to determine the effect of bb treatment on their 
 modulation . all four mirnas were significantly up - regulated under 
 the influence of e2 intervention in agreement with our 
 previous findings . mir-18a and mir-34c were up - regulated about 13- 
 and 11-fold , respectively , compared to untreated control . the effect 
 of e2 on mir-18a and mir-34c was reduced to 6.4-fold ( p = 0.0013 ) and 4.9-fold ( p = 0.0033 ) , 
 respectively , with the 5% bb diet in therapeutic mode ( figure 7 ) . berry diet given in chemopreventive mode also 
 offset these two elevated mirnas , but the effect was not as pronounced . there was no significant effect of bb treatment on the up - regulated 
 mir-20a and mir-25 expression levels . the small 
 rna was isolated by mirvana microrna kit and quantified by bioanalyzer . qpcr analysis was performed using a taqman microrna reverse triscription 
 kit and taqman gene - specific microrna assays . asterisk indicates 
 significant difference from e2-treated control ( p = 0.0013 and 0.0033 ) . analysis of serum for liver enzymes such as aspartate 
 transaminase , alanine aminotransferase , alkaline phosphatase , -glutamyl 
 transpeptidase , amylase , and lipase indicated that they were within 
 the normal range , and no difference was found with e2 or 
 bb diet or in combination compared to control , suggesting that berry 
 diets had no toxic effects ( table 3 ) . serum 
 was analyzed for plasma proteins ( albumin and globulin ) , glucose , 
 cholesterol , and triglycerides ( table 3 ) . analysis 
 of blood urea nitrogen , creatinine , and various electrolytes showed 
 no significant changes , indicating normal kidney functions with bb 
 treatment ( table 3 ) . whole blood from all of 
 the groups was analyzed for hematological parameters such as white 
 blood cells , red blood cells , hemoglobin , hematocrit , mean corpuscular 
 volume , mean corpuscular hemoglobin , mean corpuscular hemoglobin concentration , 
 and platelets . no difference in any of the hematological parameters 
 was found in any group compared to aged - matched control animals ( table 3 ) . the levels of neutrophils and lymphocytes measured 
 in whole blood showed no significant difference among all groups ( table 3 ) . cancer 
 chemopreventive agents are either natural products or their 
 synthetic analogues , which inhibit the transformation of normal cells 
 to premalignant cells or the progression of premalignant cells to 
 malignant cells . it is believed that 
 chemopreventive agents can inhibit oncogenic pathways , inhibit growth , 
 and induce apoptosis in cancer cells . conventional chemopreventive agents are administered long - term and 
 believed to block or delay the progression of transformed cells by 
 modulating cell proliferation or differentiation . they also prevent activation of carcinogen , trap carcinogen 
 before reaching the active site , and enhance detoxification systems . on the other hand , standard therapeutic drugs 
 are toxic to both cancer and normal cells , which results in therapeutic 
 activity and side effects to normal cells . this study was conducted to test both the chemopreventive and therapeutic 
 potential of tifblue and rubel blueberry blend against e2-mediated mammary tumorigenesis . we had previously reported on the 
 chemopreventive effect of berkley blueberry on mammary tumorigenesis . this is the first study of its kind in which the therapeutic potential 
 of blueberry has been shown . we also confirmed the microscopic tumors 
 and precancerous conditions by histological evaluation of mammary 
 gland sections from rats treated and necropsied at 12 weeks of e2 treatment . variable ductular epithelial changes including 
 dysplasia , hyperplasia , and adenoma formation were found , suggesting 
 various stages of carcinogenesis . in addition , periductular fibroplasia 
 as well as mast cell infiltration positively correlated with degree 
 of dysplasia . rubel 
 blueberry blend at 2.5 and 5% given in the chemopreventive mode was 
 highly effective in increasing the tumor latency by 7 and 28 days , 
 respectively . the administration of 2.5% bb diet showed significant 
 reduction in tumor multiplicity and pituitary weight . in our earlier 
 studies , 2.5% bb diet made from the mixture of three blueberries was 
 not effective against e2 treatment ; this could be because 
 the e2 implant size was 3 cm long containing 27 mg of e2 . in another study with a similar 
 9 mg e2 implant , a 2.5% diet showed protective effects 
 that were in the same vicinity as reported by us previously . in both studies , the 2.5% bb diet had only modest 
 effect and did not significantly reduce the tumor volume , tumor latency , 
 and molecular markers investigated . the higher dose of bb ( 5% ) administered 
 in chemopreventive mode was highly protective in reducing tumor volume 
 and tumor multiplicity and increased tumor latency to 28 days , in 
 agreement with our previous findings . however , the protective effect of blueberry was slackened after 
 20 weeks of e2 treatment , likely due to the exponential 
 growth rate of the cells to reach the malignant stage . for the first 
 time bb diet was administered after 12 weeks of e2 treatment 
 to determine the therapeutic effect against precancerous and microscopic 
 adenomas . when compared with animals that received 5% bb diet in chemopreventive 
 mode , the therapeutic group showed a significantly enhanced effect 
 by reducing tumor volume and multiplicity and increasing the tumor 
 latency to 37 days , that is , 9 days more than the preventive groups . 
 even when the bb diet was initiated after 14 weeks of e2 treatment , the therapeutic group elicited protective effects . the 
 therapeutic potential of bb anthocyanidins has been shown by others 
 against human prostate cancer and by 
 us against lung cancer xenograft . however , 
 there is no study for direct comparison in e2-induced mammary 
 tumorigenesis . interestingly , the present data also suggest that bb 
 diet can be started in the postinitiation stage of tumorigenesis and 
 still elicit a response similar to that of chemopreventive mode . yet > 50% of the ingested anthocyanins are found in the cecum , implying poor absorption . anthocyanin structure 
 exists as a flavilium cation in acidic conditions , which is stable , 
 but converts to unstable quinoidal or carbinol structure at neutral 
 or alkaline conditions . however , it has 
 also been shown that anthocyanins are stabilized by flavanols and 
 ascorbic acid present in the food matrix . this may explain the higher bioavailability of anthocyanins when 
 given as a whole fruit powder relative to individual anthocyanins , indicating strong synergism occurs in absorption 
 between coexisting molecules in fruit . furthermore , in a study with 
 healthy subjects with ileostomy , nearly 40% of anthocyanins were recovered 
 in the ileal fluid , indicating that despite the difference in ph , 
 the anthocyanins were stable and large amounts will pass from the 
 small intestine to the large intestine . yet , all of the bioavailability studies were done with bolus doses 
 of pure anthocyanins or fruit powder / extracts . anthocyanins were not detected in the plasma of animals 
 fed a diet mixed with sources rich in anthocyanins or the pure compound . however , lack of detection of anthocyanins in 
 the plasma does not indicate lack of tissue bioavailability . when 
 anthocyanins were delivered intravenously , a biphasic decay of plasma 
 levels was observed , indicating tissue distribution . in a parallel publication in this issue , we have discussed 
 in detail the bioavailability of anthocyanins and its levels in lung 
 tissue . we have demonstrated the presence 
 of all five anthocyanidins , delphinidin , cyanidin , peonidin , petunidin , 
 and malvidin , in lung tissues . thus , anthocyanins are bioavailable 
 and bioaccumulate in non - gastrointestinal tissues . in agreement 
 with our previous studies , the effect of e2 was reflected 
 in the body , liver , mammary , and pituitary weights 
 of the animals . the transitory increase 
 in body weight and organ weight is majorly due to the e2 and may not be due to the diet consumption . moreover , control ain-93 
 m diet and diets supplemented with 2.5 and 5% bb were isocaloric . although there was no effect of bb diet on body weight gain and organ 
 weights , it was found to significantly offset e2-associated 
 elevation in the weight of pituitary tumors . the pituitary weight 
 was affected due to the increase in plasma prolactin levels induced 
 by e2 , resulting in pituitary prolactinomas . however , the e2-mediated mammary 
 tumor model was well studied and standardized to reduce the side effect 
 to the pituitary gland . earlier studies 
 have shown that prolonged supply of e2 increases the production 
 of prolactin in e2-sensitive rat strains . high circulatory levels of prolactin in serum are one of the major 
 reasons for animals to develop e2-induced mammary tumors . berry diet at 2.5 and 5% significantly offset 
 the prolactin levels elevated by e2 . it catalyzes the conversion of e2 into its metabolites 
 ( 2e2 and 4e2 ) , which further undergo redox cycle 
 leading to the formation of reactive oxygen species . in this study , we showed that bb diet significantly reduced 
 the expression of cyp1a1 , suggesting its protective role against mammary 
 carcinogenesis . studies have shown that cyclin d1 and e2 receptors have important roles in regulating proliferation of breast 
 epithelial cells . it is well established that e2 via er- elicits rapid signals driving cancer cells 
 to proliferation . this biological effect of e2-mediated 
 by er- was reduced significantly in 5% bb diet provided in 
 therapeutic mode . regulation of cyclin d1 gene expression has been 
 associated with changes in the proliferation rate of breast cancer 
 cells . induction of cyclin d1 expression 
 occurs rapidly and is a critical feature of the mitogenic action of 
 e2 . the expression of cyclin d1 was down - regulated by 22.6-fold 
 with 5% bb diet in chemopreventive and therapeutic modes , respectively . investigation of pcna in nuclear proteins revealed that dietary bb 
 reduced the expression levels significantly . pcna was shown to interact 
 with er- both in the absence and in the presence of dna , to 
 enhance the interaction of er- with ere - containing dna , and 
 associate with endogenous e2-responsive genes . these findings clearly indicate that bb diet 
 could modulate major e2-associated proliferation markers 
 that lead to protective effects against mammary tumorigenesis in animals 
 treated with e2 . another interesting finding in this 
 study is from the analysis 
 of mirnas related to the er- family . aberrant mirna expression 
 is implicated in e2-related breast , uterine , and ovarian 
 cancer development and progression . hormones 
 play a crucial role in regulating mirnas by both genomic ( transcriptional ) 
 and nongenomic mechanisms . identified mirnas having expression correlated with specific breast 
 cancer biopathologic features , such as e2 and progesterone 
 receptor expression , tumor stage , vascular invasion , or proliferation 
 index . targeting specific mirnas for inhibiting 
 specific stages of a cancer is being 
 explored . several mirnas that regulate er are involved in synchronized 
 feedback mechanisms as a component of er activation . many of these 
 er - regulating mirnas therefore are either e2-inducible 
 ( mir-18a and let-7 ) or subject to e2-mediated repression 
 ( mir-206 , mir-221/222 , and mir-145 ) . this 
 laboratory has recently identified mirnas that modulated under the 
 influence of e2 in this animal model and report aberrant 
 changes in mirna expression profile as early as 3 weeks of e2 treatment . we chose four mirnas , mir-18a , 
 -20a , -25 , and -34c , on the basis of our previous study where these 
 mirnas were up - regulated with e2 to understand the influence 
 of bb diet on their modulation . increases in mir-18a and mir-20a mature 
 forms were observed after e2 stimulation . in this study , bb diet administered in therapeutic mode significantly 
 reduced mir-18a and mir-34c , whereas chemopreventive mode showed modest 
 but significant reduction . these findings provide initial insights 
 of bb diet in decreasing the e2-induced mirna levels . however , 
 the bb diet failed to modulate mir-20a and -25 expression levels , 
 which remain the subject of further investigation . in the chemopreventive 
 mode , the relative enhanced effect observed with 
 the therapeutic approach could be due to the adaptive response of 
 the rats to the bb phytochemicals in the chemopreventive mode . for 
 example , the cyp1a1 mrna level in mammary tissue was highest at 3 
 weeks after e2 implant and gradually decreased with time . the effect of the bb phytochemicals was more pronounced in the inhibition 
 of conversion of normal cells to precancerous or precancerous frank 
 tumors , but were not as effective once the tumors reached the exponential 
 growth rate . thus , by the time the mammary cells started exhibiting 
 precancerous conditions , the efficacy was better when bb phytochemicals 
 were introduced into the system than when they were already in the 
 circulation . nevertheless , both approaches were effective in delaying 
 the onset and growth of palpable tumors . in summary , this is 
 the first demonstration indicating that bb 
 diet is highly protective even in preinitiated e2-mediated 
 mammary tumors in aci rats through its effects by modulating cell 
 proliferation and molecular targets . thus , the consumption of bb can 
 be used as an effective strategy for the treatment of e2-associated breast cancer and possible prevention of its relapse 
 and metastasis . our study demonstrates the chemopreventive and therapeutic 
 potential of blueberry and advances our understanding of the working 
 mechanisms in search of potential future drugs .
berries are gaining increasing importance 
 lately for their chemopreventive 
 and therapeutic potential against several cancers . in earlier studies , 
 a blueberry - supplemented diet has shown protection against 17-estradiol 
 ( e2)-mediated mammary tumorigenesis . 
 this study tested 
 both preventive and therapeutic activities of diet supplemented with 
 whole blueberry powder ( 50:50 blend of tifblue and rubel ) . 
 animals 
 received 5% blueberry diet , either 2 weeks prior to or 12 weeks after 
 e2 treatment in preventive and therapeutic groups , respectively . 
 both interventions delayed the tumor latency for palpable mammary 
 tumors by 28 and 37 days , respectively . 
 tumor volume and multiplicity 
 were also reduced significantly in both modes . 
 the effect on mammary 
 tumorigenesis was largely due to down - regulation of cyp 1a1 and er- 
 gene expression and also favorable modulation of microrna ( mir-18a 
 and mir-34c ) levels . 
 these data suggest that the blueberry blend tested 
 is effective in inhibiting e2-mediated mammary tumorigenesis 
 in both preventive and therapeutic modes .