evaluation of blueberry juice in mouse azoxymethane ... · researcharticle evaluation of blueberry...

Research ArticleEvaluation of Blueberry Juice in Mouse Azoxymethane-InducedAberrant Crypts and Oxidative Damage

Isela Aacutelvarez-Gonzaacutelez1 Fernando Garcia-Melo1 Veroacutenica R Vaacutesquez-Garzoacuten2

Sauacutel Villa-Trevintildeo2 E Osiris Madrigal-Santillaacuten3

Joseacute A Morales-Gonzaacutelez3 Jorge A Mendoza-Peacuterez4 and Eduardo Madrigal-Bujaidar1

1 Laboratorio de Genetica Escuela Nacional de Ciencias Biologicas IPN Unidad Profesional A Lopez MateosAvenida Wilfredo Massieu sn Zacatenco Colonia Lindavista CP 07738 Mexico DF Mexico

2Departamento de Biologıa Celular Centro de Investigacion y Estudios Avanzados IPN Avenida Instituto Politecnico 2508Colonia San Pedro Zacatenco Del Gustavo A Madero CP 06360 Mexico DF Mexico

3 Laboratorio de Medicina de la Conservacion Escuela Superior de Medicina IPN Plan de San Luis y Dıaz Miron snCasco de Santo Tomas Del Miguel Hidalgo CP 11340 Mexico DF Mexico

4 Laboratorio de Quımica Ambiental Escuela Nacional de Ciencias Biologicas IPN Unidad Profesional A Lopez MateosAvenida Wilfredo Massieu sn Zacatenco Colonia Lindavista CP 07738 Mexico DF Mexico

Correspondence should be addressed to Eduardo Madrigal-Bujaidar eduardomadrigallycoscom

Received 9 June 2014 Revised 7 August 2014 Accepted 9 August 2014 Published 3 September 2014

Academic Editor Jairo Kenupp Bastos

Copyright copy 2014 Isela Alvarez-Gonzalez et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

Blueberry is a plant with a number of nutritional and biomedical capabilities In the present study we initially evaluated the capacityof its juice (BJ) to inhibit the number of aberrant crypts (AC) induced with azoxymethane (AOM) in mouse BJ was administereddaily by the oral route to three groups of animals during four weeks (16 41 and 150120583Lg) respectively while AOM (10mgkg) wasintraperitoneally injected to the mentioned groups twice a week in weeks two and three of the assay We also included two controlgroups of mice one administered distilled water and the other the high dose of BJ A significant increase of AC was observed in theAOM treated animals and a mean protection of 756 was determined with the two low doses of BJ tested however the high doseof the juice administered together with AOM increased the number of crypts more than four times the value observed in animalsadministered only AOM Furthermore we determined the antioxidant potential of BJ with an ex vivo DPPH assay and found adose-dependent decrease with a mean of 195 We also determined the DNA oxidationantioxidation by identifying 8-hydroxy-21015840-deoxyguanosine adducts and found a mean decrease of 443 with the BJ administration with respect to the level induced byAOMOur results show a complex differential effect of BJ related to the tested doses opening the need to further evaluate a numberof factors so as to determine the possibility of a cocarcinogenic potential

1 Introduction

Colon cancer refers to malignant tumors originated in theinternal epithelial lining of the organ [1]This disease has beenassociated with the accumulation of mutations includingthose in the K-ras APC and p53 genes as well as withdeletions and gains in various chromosomes in additionto epigenetic changes such as those related with hypo- andhypergene methylation [2] In regard to histopathologicalfeatures of the disease one of the initial manifestations is

the presence of aberrant crypts (AC) which may be foundas a single abnormal crypt or as multiple crypts (ACF)These lesions can be observed along the colon of rodentsand humans they are characterized by a bigger size than thenormal crypts a thick layer of epithelial cells which oftenstain intensely an oval lumen and an enlarged pericryptalspace [3] One of the most used compounds for studyingAC in experimental models is azoxymethane (AOM) anintermediary metabolite of dimethylhydrazine that givesrise to methyl diazonium and methyl carbonium chemicals

Hindawi Publishing CorporationEvidence-Based Complementary and Alternative MedicineVolume 2014 Article ID 379890 8 pageshttpdxdoiorg1011552014379890

2 Evidence-Based Complementary and Alternative Medicine

which are known to damage a number of biomolecules andmay induce colon cancer [4]

During the carcinogenic process oxidative stress is anevent that may play a significant role as shown by theinduction of nitric oxide which in combination with thesuperoxide radical may generate peroxynitrite and hydroxylmolecules which in turn may cause nitration nitrosylationand oxidation in proteins DNA and other biomolecules [5]Also in concordance with these observations in a previousreport we determined an increase in the level of lipid andprotein oxidation which corresponded to the increase in thenumber of AC induced by AOM in mice [6]

Besides diet age lifestyle and other factors have beenreported as risk factors for the development of the dis-ease suggesting then the potential relevance of applyingchemopreventive measures related with the action of veg-etables and fruits because such strategy involves a varietyof actions that can be exerted in different steps of thecarcinogenic process [7] In the case of colon carcinogenesisthe comprehension of pathogenic steps such as DNA dam-age oxidative stress chronic inflammation and histologicalchanges is relevant for the selection of early biomarkersthat may show the efficacy of the studied chemopreventiveagent

In the present report we evaluated the effect of blueberryjuice (BJ) (from Vaccinium corymbosum L) on the damageinduced by AOM in the colon of mouse Blueberry is aperennial shrub 2 to 5m high which possesses almost spher-ical dark blue fruits 07 to 15 cm diameter which contain anumber of vitamins and minerals as well as phytochemicalssuch as anthocyanins flavonols flavanols stilbenes phenolicacids and proanthocyanidins [8]

The consumption of the studied plant has been increasingin natural or industrialized forms including its use inbakery products different types of syrups and alcoholicbeverages preserves and coloring additives [9]With respectto biomedical properties several effects have been reportedfor different parts of Vaccinium including antioxidantantimicrobial antiviral anti-inflammatory antihypertensiveand antiacetylcholinesterase activities [10 11] In regard toantigenotoxic and chemopreventive activities of Vacciniumreports have shown an antiproliferative potential in prostateand colon cancer cellular lines a protective effect of theirenzymatic hydrolyzates against hydrogen peroxide-inducedoxidative damage in Chinese hamster lung cell line as well asin vivo reduction of benzo(a)pyrene induced genotoxic dam-age by ethanol extracts of the plant [11ndash13] Moreover studieswith berry plants or with a number of their constituentshave suggested a potential to inhibit carcinogenic damage[14 15] In this aspect different molecular and cellular eventsinduced by berry fruits have been proposed as mediators inthe prevention of the carcinogenic process including theirantioxidant potential

The above-mentioned information suggests the perti-nence of extending studies in regard to the chemopreventivecapacity of blueberry particularly in vivo therefore the initialaim of the present report was to evaluate the capacity ofBJ to inhibit the number of AOM-induced AC in the colonof mice and secondly to explore whether such an effect

could have a relation with the BJ effect against oxidativedamage For this last purpose we determined its capacityto inhibit the percentage of the DNA adduct 8-hydroxy-21015840-deoxyguanosine (8-oxo-dG) induced by AOM as well as itscapacity to capture the 22-diphenil-1-picrilhydracil radical(DPPH) in mice

2 Material and Methods

21 Chemicals and Animals The following chemicals werepurchased from Sigma-Aldrich Chemicals (St Louis MOUSA) AOM PBS proteinase K RNAse trichloroaceticacid (TCA) trizma base albumin standard thiobarbituricacid (TBA) triethylamine hexane potassium chloride ethylacetate sodium acetate vitamin E quercetin cyanidin-3-glucoside Folin-Ciocalteu reagent andmethylene blue Anti-8-oxo-dG (4354-MC-050) was purchased from TrevigenInc (Gaithersburg MD USA) Formaldehyde HCl NaClsodium citrate xylene and ethanol were acquired fromFermont (Mexico City)

Blueberry was obtained from a pesticide-free cultivationin Zacatlan Puebla a town located 200 km north of MexicoCity The fruits were kept at minus70∘C and immediately beforethe assay the juice was mechanically obtained by pressingthe fruit with a mortar followed by filtering it in gauze toeliminate gross material

22 Phytochemical Identification Several specific groups ofphytochemicals have been identified as beingmainly involvedin the biomedical responses of blueberry therefore in thepresent report we made the identification of total phenoliccompounds flavonoids and anthocyanins

Initially BJ was treated in a batch reactor with pectinase(Pectinex 3XL 200 ppm) for 2 h at 55∘C The resultingproduct was passed through a 50 120583m filter and then it waslyophilized and powdered Lyophilized BJ extracts were frac-tionated in a silica gel chromatographic column (embeddedwith hexane) and neutralized with triethylamine Separa-tions were made with gradients ranging from a mixture ofhexaneethyl acetate (1 1) to the use of ethyl acetate withincreasing proportions of a mixture of methanolwater 1 1until only the mixture methanolwater was used Presentlyfractions of BJ are in process to be analyzed with themethodsof HPLC and 1H-NMR

221 Total Phenolic Content The concentration of phe-nolics was determined by the method of Folin-Ciocalteu[16] with some modifications The reaction mixture wasprepared with 05mL of BJ extract (02 gmL) 25mL of10 Folin-Ciocalteursquos reagent and 25mL of 75 sodiumbicarbonate The samples (in triplicate) were incubated at45∘C for 45min and left at room temperature for 2 h inthe dark before determining the absorbance at 750 nm Thesame procedure was made with vitamin E to construct acalibration curve Finally the concentration of phenolicsfor each extract was extrapolated in the calibration curveand expressed as mg of vitamin E equivalents100 g ofextract

Evidence-Based Complementary and Alternative Medicine 3

222 Total Flavonoid Content For this determination weused the aluminum chloride colorimetric method previouslyreported [17] with some modifications The tested sampleswere formed by 1mL of a methanol BJ extract solution(1mgmL) plus 1mL of 2 aluminum chloride Samples(in triplicate) were left for an hour at room temperaturebefore registering the absorbance at 510 nm With a similarprocedurewe constructed a calibration curve for the standardsolution (quercetin) where the readings of the extracts wereextrapolated to finally express the flavonoid content as mg ofquercetin equivalents100 g of extract

223 Monomeric Anthocyanin Content The anthocyaninpigment content was determined by the pH differentialmethod [18] which is based on the reversible change ofcolor with respect to the change in pH The tested fractionswere diluted with potassium chloride 0025M at pH 1 andwith sodium acetate 04M at pH 45 then absorbance wasdetermined at 520 and 700 nm after 30min Monomericanthocyanins were calculated as mg of cyanidin-3-glucosideequivalents100 g of BJ using the extinction coefficient of26900 Lmolminus1cmminus1 and a molecular weight of 4492 gmolCuvettes of 1 cm path length were used

The obtained results were the following total phenoliccontent = 350 plusmn 009mgVE equivalents100 g of BJ flavonoid= 250 plusmn 10mg of quercetin equivalents100 g of BJ andmonomeric anthocyanins = 961 plusmn 001mg of cyanidin-3-glucoside equivalents100 g of BJ These results show a sig-nificant amount of the evaluated phytochemicals and suggestthat they may be involved in the determined antioxidant andchemopreventive results

23 Experimental Protocol inMouse For the studyweused 88male mice (Swiss Webster) with a mean weight of 30 g Theywere placed in metallic cages at 22 plusmn 2∘C 50ndash60 relativehumidity and in a 12 h dark-light cycle Mice were permittedto freely consume food (Rodent Lab Chow 5001 Purina) andwater

The experiment was approved by the Committee of Ethicsof the National School of Biological Sciences Six groupswith 8 mice each were used to evaluate the number of ACand the level of 8-oxo-dG The tested doses of BJ (16 41and 15 120583Lg) correspond respectively to the consumptionof 04 12 and 3 glasses of 250mL for a human of 65 kgAccording to the selected doses of BJ and to the animalweight we administered the corresponding volume of juiceto each mouse by the intragastric route One group wasdaily administered 03mL of distilled water during the fourweeks of the assay another group was daily administered thehigh tested dose of BJ (15120583Lg) during the whole assay onemore group was intraperitoneally (ip) injected with AOM(10mgkg) twice a week the second and third week of theassay finally three other groups were daily administeredBJ with 16 41 and 15120583Lg each during the four weeks ofthe study as well as being ip injected 10mgkg of AOMtwice a week in the second and in the third week Wehad previously determined that the described experimentalmodel is appropriate for both to show the increase of AC

by AOM and their inhibition by the tested chemopreventiveagents [6]

The other 40mice were used to determine the antioxidantcapacity of BJ by applying the ex vivoDPPH assay They wereorganized in five groups with 8 individuals each and orallyadministered as follows one group 03mL of distilled wateranother group 31 120583Lg of vitamin E and the last three groupsBJ (16 41 and 15 120583Lg) Blood was collected after 2 h oftreatment by a retro-orbital puncture

231 Determination of AC Mice were cervically dislocatedat the end of the treatment then their colon was dissectedand washed in PBS The AC determination was made in thewhole colon of each of the 8 animals per group For thispurpose we extended the organ in a Petri dish with solidifiedparaffin at the bottom and fixed it in 10 formaldehydemade in PBS for 24 h After that the organs were stainedwith 4 methylene blue (diluted in PBS) for 15min and thenumber and distribution of crypts were registered at 100xmagnification

232 Immunohistochemical Determination of 8-Oxo-dG Forthis purpose we followed the method described by Moreiraet al [19] with some modifications Colon tissue sections3 120583m thick were deparaffinized and hydrated gradually Eachsample was incubated with proteinase K (50 120583gmL 1 h at37∘C)The sections were then placed in a solution made withRNAse A 100 120583gmL containing 150mM NaCl and 15mMsodium citrate at 37∘C for 1 h The tissue was washed twicewith PBS and after DNA denaturation with HCl 2N for5min the process was neutralized with trizma base 1M for5min and the tissue was washed twice in PBS Nonspecificbinding was blocked by incubating the slides for 60min in10 goat serum in PBS Primary antibodies anti-8-oxo-dGdiluted 1 250 were incubated overnight at 4∘C followed bythe secondary antibody Ig and the fluorescein-linked wholeantibody at 37∘C for 2 h Tissue images were captured by anepifluorescent microscope (Axioskop 50 C Zeiss) equippedwith a filter (120574 = 365ndash395 nm) for detecting DAPI bluestaining nuclei and another (120574 = 450ndash490 nm) for detectingthe 8-oxo-dG adducts by the FICT green color Fluorescentsamples were analyzed in a Zeiss-510 laser scanning confocalmicroscope (Carl Zeiss Inc Jena Germany) to confirm thecoexistence of 8-oxo-dG in the nucleus After confirming thecolocalization patterns we captured tissue images through anopticalmicroscope (Olympus 1X70 Olympus EuropeGmbHHamburgGermany) Subsequently tissue imageswere quan-tified in ten randomly selected fields (magnification 1000x)per individual sample Adduct positive nuclei were quantifiedwith respect to the total number of nuclei by using imageanalysis software (Analysis Soft Imaging System GmbH)

233 DPPH Assay Ex Vivo For this assay we followed themethod of Chrzczarnowicz et al [20] with some modifica-tions The blood sample from each mouse was centrifugedat 1500timesg at 4∘C for 10min to obtain the serum Then200120583L of this was mixed with 200120583L of acetonitrile 95Mand centrifuged at 9500timesg at 4∘C for 10min After this step


Table 1 Weight (g) determined in mice treated with blueberry juice (BJ) and azoxymethane (AOM)

Week Water15120583Lg

AOM10mgkg

BJ15120583Lg

BJ + AOM16120583Lg + 10mgkg

BJ + AOM41 120583Lg + 10mgkg

BJ + AOM15 120583Lg + 10mgkg

0 3025 plusmn 079 3133 plusmn 067 2972 plusmn 058 3260 plusmn 075 3235 plusmn 071 3103 plusmn 0691 3045 plusmn 090 3273 plusmn 097 2923 plusmn 068 3186 plusmn 073 3183 plusmn 074 3035 plusmn 0682 3079 plusmn 065 2772 plusmn 115a 3029 plusmn 089 3022 plusmn 077 2955 plusmn 191 2585 plusmn 107a

3 3215 plusmn 062 2584 plusmn 123a 3095 plusmn 101 2878 plusmn 062ab 2746 plusmn 068a 2416 plusmn 095a

4 3381 plusmn 062 3018 plusmn 111a 3134 plusmn 122 3124 plusmn 103 2981 plusmn 079a 2816 plusmn 095ab

Values represent the mean plusmn SD obtained in 8 mice per group The letters show significant statistical differences as follows awith respect to the control value(water) in the same week bwith respect to value in the group treated with AOM in the same week ANOVA and Student-Newman-Keuls tests 119875 le 005

Table 2 Aberrant crypts in mice treated with blueberry juice (BJ) and azoxymethane (AOM)

Group Number of aberrant crypts Mean plusmn SD1 2 3 4 5 6 7

Water15 120583Lg 0 0 0 0 0 0 0 0

AOM10mgkg 5714 plusmn 685 10 plusmn 144 328 plusmn 077 185 plusmn 041 014 plusmn 014 042 plusmn 042 042 plusmn 020 7325 plusmn 144a

BJ15 120583Lg 171 plusmn 089 028 plusmn 018 0 0 0 0 0 199 plusmn 054

BJ + AOM16 120583Lg + 10mgkg 2317 plusmn 337 183 plusmn 071 066 plusmn 049 016 plusmn 017 0 0 0 2582 plusmn 119ab

BJ + AOM41 120583Lg + 10mgkg 85 plusmn 165 075 plusmn 037 037 plusmn 026 012 plusmn 013 0 012 plusmn 013 0 986 plusmn 051ab

BJ + AOM15 120583Lg + 10mgkg 2995 plusmn 4314 12 plusmn 272 416 plusmn 109 1 plusmn 026 033 plusmn 021 0 0 31699 plusmn 948ab

Values represent the mean plusmn SD obtained in the colon of 8 mice per group aStatistically significant with respect to the mean control value and bwith respectto the value of the AOM treated group ANOVA and Student-Newman-Keuls tests 119875 le 005

25 120583L of the supernatant (deproteinized serum) was addedto 5 120583L of the radical DPPH 001M plus 970 120583L of methanoland left in the dark at room temperature for 30min Finallyabsorbance was registered at 517 nm In this assay the DPPHradical which has an unpaired electron was of a blue-violetcolor and tended to lose color turning to pale yellow

234 Statistical Analysis Statistical significance of theobtained data was determined by applying an ANOVA fol-lowed by the Student-Newman-Keuls tests For this purposewe used the software SigmaStat version 35

3 Results

Themean weight of mice along the study is shown in Table 1Control mice and those treated solely with BJ had a slightincrease at the end of the study while a 549 g decrease wasnoted in the AOM treated group at the third week of theassay with a weight recovery in the last week In the groupstreated with BJ plus the carcinogen we observed a certainweight reduction which may be related to the toxicity ofAOM although cooperation between both chemicals cannotbe discarded in light of the fact that the high reduction wasobserved with the high dose of BJ

Table 2 shows the results obtained concerning the num-ber of AC Besides Figure 1 shows various types of the

observed crypts We found no crypts in the control groupand we found a mean of 19 crypts in animals administeredthe high dose of BJ without statistical difference with respectto the previous group these results are contrary to the 732ACobserved in mice administered AOM On the other handanimals administered the low dose of the juice plus AOMhad a certain increase in the number of AC however in thisgroup we also found a reduction of 647 in the number ofAC with respect to the value obtained by administering thecarcinogen In the group treated with the intermediate doseof BJ plus AOM we observed a mean induction of 98 ACas well as an inhibition of 865 in comparison with thenumber observed in the treatment with AOM Finally micetreated with the high dose of BJ in addition to the carcinogenoriginated an unexpected increase of AC with no protectiveactivity On the contrary the mean number in this group wasmore than 300ACwhich representsmore than four times thenumber detected in the AOM treated group The same tablealso shows the distribution of AC found in our study Sevenwas observed to be the highest number of registered cryptsThis number was found only in the AOM treated group Theadministration of BJ alone gave rise to single or double cryptsonly while the combination of BJ andAOM induced up to sixcrypts

TheDNA immunohistochemical studymade in the colonof the control and treated animals is shown in Figure 2 Mice


(a) (b) (c)

Figure 1 Aberrant crypts observed in the colon of mice treated with blueberry juice and azoxymethane (a) Colon with normal crypts (b)Colon with one aberrant crypt (c) Colon with crypt foci formed by three aberrant crypts

02468

1012141618202224

1 2 3 4 5 6

8-O

xo-d

G (

)

a a

b

a ba b

a b

(1) Water 15120583Lg(2) BJ 15120583Lg(3) AOM 10mgkg(4) BJ (16120583Lg) + AOM (10mgkg)(5) BJ (41 120583Lg) + AOM (10mgkg)(6) BJ (15120583Lg) + AOM (10mgkg)

Figure 2 Quantification of 8-oxo-dG adducts in mice treated withblueberry juice (BJ) and azoxymethane (AOM) Each bar representsthe mean plusmn SD obtained in the colon of each group Eight mice pergroup aStatistically significant difference with respect to the AOMvalue bwith respect to the control value ANOVA and Student-Newman-Keuls tests 119875 le 005

of the control group and those administered BJ (150120583Lg)were observed to have 2 and 3 nuclei positively markedfor 8-oxo-dG respectively while animals treated with AOMgave rise to 212 8-oxo-dG adducts Besides in the groupsadministered AOM plus BJ (16 41 and 150 120583Lg) wedetermined a significant antioxidant effect with a decreaseof 40 51 and 42 in the level of adducts with respect to thevalue obtained in the AOM treated group

Figure 3 shows the results obtained in the DPPH assayWe found a 33 counteraction produced by vitamin E inthe presence of the DPPH radical while the three testeddoses of BJ (16 41 and 150 120583Lg) gave rise to a dose-dependent decrease which corresponded to 111 206 and269 respectively in regard to the value obtained in thenegative control

4 Discussion

In the present study we evaluated various parameters todetermine the toxic effect of AOM in mice as well as the level

05

1015202530354045

1 2 3 4 5

Radi

cal s

cave

ngin

g ac

tivity

()

a

aa

a

(1) Water 15120583Lg(2) Vitamin E 316 120583Lkg(3) BJ (16120583Lg) + AOM (10mgkg)(4) BJ (41 120583Lg) + AOM (10mgkg))(5) BJ (15120583Lg) + AOM (10mgkg))

Figure 3 Results of ex vivo DPPH assay in mice treated withblueberry juice (BJ) and azoxymethane (AOM) Data were obtainedin the serum of 8 animals per group aStatistically significantdifference with respect to the control value ANOVA and Student-Newman-Keuls tests 119875 le 005

of protection exerted by BJ In regard to animal weight itsdecrease is generally accepted to suggest a systemic deteriora-tion This effect was observed in animals administered AOMonly a result that has also been reported by other authorsalthough such decrease has not always been found [21 22]BJ alone produced a slight increase in weight along theassay however when combined with AOM the tested dosesof BJ showed no protection on the strong toxicity inducedby AOM In the evaluation of other agents (including theblueberry constituent epicatechin gallate) a similar type ofresult has been related to a deleterious effect of the carcinogento the colon epithelial cells which is insufficiently preventedby the anticarcinogen and which provokes a decrease in theappetite of the experimental animals this effect however hasnot been necessarily connected with the capacity of agents toprevent the induction of crypts [21ndash25]

Concerning the amount of AC observed in our study wefound that the administration of 15 120583Lg of BJ induced a lownumber of crypts which were in the range determined forthe control group This result clearly stated the innocuous


action of the tested juice for inducing colon preneoplasticchanges and was concordant with a number of reports thathave evaluated different types of berries administered toanimals as lyophilized products or in other forms as wellas with the negative results observed with constituents ofblueberry such as epicatechin [22 26] The described resultscontrast with the elevated number of AC as a consequenceof administering AOM a finding which is in line with thepotent colon carcinogen capacity already described for thiscompound Concerning the effect of the two low doses ofBJ (16 and 41 120583Lg) administered to animals treated withAOM we detected a significant prevention of the damageinduced by the carcinogen Such beneficial effect of blueberryhas been determined before in rats as well as with theuse of constituents of the plant including anthocyanins andpterostilbene [15 27 28] Besides the blueberry anticancerpotential has also been determined in a model of ratsgiven cyclic administrations of dextran sulphate sodiuma nongenotoxic compound that induces colitis and colitisassociated neoplasia [29]

In our study we also determined a moderate but signifi-cant antioxidant capacity by the tested juice with the DPPHassay as well as significant prevention of DNA oxidationsuggesting that this property of BJ may be involved in thereduction of genetic damage induced by AOM which insubsequent steps may give rise to preneoplastic damageThe antioxidant capacity of blueberry has been attributedto the action of chemicals such as anthocyanins catechinsquercetin derivatives and benzoic and cinnamic acids [30]However this property is only one of the various parametersto be considered in the preventive process as no change inthe level of DNA oxidative adducts has been determinedwhile other types of carcinogenic markers showed clearmodifications [15] This last information is in line with thefact that a number of molecular changes should be producedto state the neoplastic transformation including alterations ingenes involved in inflammation proliferation and apoptosis[31]

The inhibitory effect produced with the low doses of BJis highly in contrast with the damage induced when the highdose of the juice was combined with AOM Such preneoplas-tic potentiating effect deserves the mention of antecedentson the matter to put it into context In genotoxicity thistype of enhancement has been described before as well as itsrelationship with the used dose such is the case of verapamila calcium antagonist agent that significantly increased theclastogenic effect of cyclophosphamide acrylamide anddioxidine in mouse ascorbic acid also increased the numberof micronuclei and sister chromatid exchanges induced bymitomycin C in human lymphocytes [32 33] MoreoverDutra et al [34] reported an enhancement of the genotoxicpotential of doxorubicin in Drosophila by a cotreatment withtomato extract (Lycopersicon esculentum) Besides polyphe-nols and tannins contained in areca nut have been suggestedto participate in the ROS induced genotoxic preneoplasticand neoplastic effects that occur during autoxidation ofareca nut polyphenols in the betel quid chewerrsquos saliva acrucial event for the initiation and promotion of oral cancer[35]

At the level of colon preneoplastic or tumor damage afew unexpected results have also been reported Blueberrypowder (obtained from V angustifolium) failed to protectagainst AOM-induced AC in rats treated with a damagingdiet moreover authors found that blueberry increased thenumber of crypts in the distal colon of female animals[36] Likewise the administration of wheat bran was shownto enhance the rat colon carcinogenesis induced with 12-dimethylhydrazine probably due to its hyperproliferativeeffect [37] In fact experimental evidence has suggestedthat a fibre-depleted diet may have cocarcinogenic potentialin colorectal cancer while a high fibre diet may have aprotective anticarcinogenic effect [38]

In spite of the numerous reports showing antioxidation aschemopreventive activity for berries and several of their con-stituents we must recall that these chemicals may also haveopposite effects for example flavonoids such as apigenin andquercetin as well as tannins are known to have DNA strandbreaking abilities [39] Resveratrol and ascorbic acid havealso been reported to possibly cause DNA breakage throughthe generation of ROS [40 41] Heterogeneous effects ofquercetin have also been reported low doses were found toincrease the production of micronuclei and chromosomalaberrations contrary to the protective effect shown againstmitomycin C genotoxicity with a high dose [42] Moreoverthe increase in the bioavailability of carcinogens may alsomodify the effect of otherwise protective agents [43]

In this complex panorama the particular species of theplant and cultivar may play a role in the variability ofthe effect Authors have found striking differences in thechemopreventive capacity among 13 types of berries as well asno correlation between their antiproliferative and antioxidantpotential differences that were even observed in plants of cul-tivars under the same environmental growing conditions [3044] This variability was likewise observed among extractswith high proanthocyanidin content which showed positivebut also negative antiproliferative antiradical and protectiveeffects against oxidative stress in three different cell lines[45 46]

Our present results together with those mentioned byother authors point to a complex situation with respectto the BJ chemopreventive effect in which the activity ofspecific polyphenols may have a role Our report suggeststhe need to be cautious in regard to the use of BJ as wellas to perform research about the possible comutagenic andcocarcinogenic effects between the juice and AOM incor-porating a number of factors some of which have alreadybeen suggested in the above-mentioned reports such as theinfluence of different doses differences in strain sensibilityand the participation of the juice and its components in themolecular events involved in the precarcinogenesis processas various geneticepigenetic changes have a role in thetransformation of a single epithelial cell within the crypt tothe AC formation Besides other factors may participate inthe damage for example the interaction of the evaluatedagent with enzymes that catalyze the carcinogen-metabolicdetoxification the impairment of nucleotide excision repairmediated by failure of genes such as p53 or the influenceof oxidative stress that may induce genomic instability and


concur independently to generate the molecular damagerequired to induce neoplastic clones [47 48]

In fact BJ is a complex mixture in which synergisticor antagonistic interactions may occur and give differentweight to the final effect This has been suggested in regardto the content of anthocyanins proanthocyanidins andflavonol glycosides of cranberry as well as with respectto phytochemicals from the quinine tree [49 50] Finallyour results also suggest a reflection on the boundaries of achemopreventive event a mutageniccarcinogenic processand a chemotherapeutic apoptosis-mediated event

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

References

[1] S Rajamanickam and R Agarwal ldquoNatural products and coloncancer current status and future prospectsrdquo Drug DevelopmentResearch vol 69 no 7 pp 460ndash471 2008

[2] A E K Ibrahim and M J Arends ldquoMolecular typing ofcolorectal cancer applications in diagnosis and treatmentrdquoDiagnostic Histopathology vol 18 no 2 pp 70ndash80 2012

[3] P Figueiredo M Donato M Urbano et al ldquoAberrant cryptfoci endoscopic assessment and cell kinetics characterizationrdquoInternational Journal of Colorectal Disease vol 24 no 4 pp441ndash450 2009

[4] R P Bird ldquoRole of aberrant crypt foci in understanding thepathogenesis of colon cancerrdquo Cancer Letters vol 93 no 1 pp55ndash71 1995

[5] W Aoi Y Naito T Takagi et al ldquoRegular exercise reducescolon tumorigenesis associated with suppression of iNOSrdquoBiochemical and Biophysical Research Communications vol 399no 1 pp 14ndash19 2010

[6] E Madrigal-Bujaidar L Martino-Roaro K Garcıa-Aguirre SGarcıa-Medina and I Alvarez-Gonzalez ldquoGrapefruit juice sup-presses azoxymethane-induced colon aberrant crypt formationand induces antioxidant capacity in micerdquo Asian Pacific Journalof Cancer Prevention vol 14 no 11 pp 6851ndash6856 2013

[7] S de Flora and L R Ferguson ldquoOverview of mechanisms ofcancer chemopreventive agentsrdquo Mutation Research vol 591no 1-2 pp 8ndash15 2005

[8] K Skupien ldquoChemical composition of selected cultivars ofhighbush blueberry fruit (Vaccinium corymbosum L)rdquo FoliaHorticulturae vol 18 no 2 pp 47ndash56 2006

[9] R M Potter M P Dougherty W A Halteman and ME Camire ldquoCharacteristics of wild blueberry-soy beveragesrdquoLWTmdashFood Science and Technology vol 40 no 5 pp 807ndash8142007

[10] G A Garzon C E Narvaez K M Riedl and S J SchwartzldquoChemical composition anthocyanins non-anthocyanin phe-nolics and antioxidant activity of wild bilberry (Vacciniummeridionale Swartz) from Colombiardquo Food Chemistry vol 122no 4 pp 980ndash986 2010

[11] F Sanchez-Patan B Bartolome P J Martın-Alvarez M Ander-son A Howell and M Monagas ldquoComprehensive assessmentof the quality of commercial cranberry products Phenoliccharacterization and in vitro bioactivityrdquo Journal of Agriculturaland Food Chemistry vol 60 no 13 pp 3396ndash3408 2012

[12] M Senevirathne K Soo-Hyun and Y J Jeon ldquoProtective effectof enzymatic hydrolyzates fromhighbush blueberry (Vacciniumcorymbosum L) against hydrogen peroxide-induced oxidativedamage in Chines hamster lung fibroblast cell linerdquo NutritionResearch and Practice vol 4 no 3 pp 183ndash190 2010

[13] E Madrigal-Santillan S Fragoso-Antonio C Valadez-Vega etal ldquoInvestigation on the protective effects of cranberry againstthe DNA damage induced by benzo[a]pyrenerdquo Molecules vol17 no 4 pp 4435ndash4451 2012

[14] L S Adams S Phung N Yee N P Seeram L Li and S ChenldquoBlueberry phytochemicals inhibit growth and metastaticpotential of MDA-MB-231 breast cancer cells through mod-ulation of the phosphatidylinositol 3-kinase pathwayrdquo CancerResearch vol 70 no 9 pp 3594ndash3605 2010

[15] G Lala M Malik C Zhao et al ldquoAnthocyanin-rich extractsinhibit multiple biomarkers of colon cancer in ratsrdquo Nutritionand Cancer vol 54 no 1 pp 84ndash93 2006

[16] V L Singleton R Orthofer and R M Lamuela-RaventosldquoAnalysis of total phenols and other oxidation substrates andantioxidants by means of folin-ciocalteu reagentrdquo Methods inEnzymology vol 299 pp 152ndash178 1999

[17] V Dewanto W Xianzhong K K Adom and R H LiuldquoThermal processing enhances the nutritional value of tomatoesby increasing total antioxidant activityrdquo Journal of Agriculturaland Food Chemistry vol 50 no 10 pp 3010ndash3014 2002

[18] J Lee R W Durst and R E Wrolstad ldquoDetermination oftotal monomeric anthocyanin pigment content of fruit juicesbeverages natural colorants and wines by the pH differentialmethod collaborative studyrdquo Journal of AOAC Internationalvol 88 no 5 pp 1269ndash1278 2005

[19] P I Moreira L M Sayre X Zhu A Nunomura M ASmith and G Perry ldquoDetection and localization of markers ofoxidative stress by in situ methods Application in the study ofAlzheimer diseaserdquoMethods in Molecular Biology vol 610 pp419ndash434 2010

[20] J Chrzczanowicz A Gawron A Zwolinska et al ldquoSimplemethod for determining human serum 22-diphenyl-1-picryl-hydrazyl (DPPH) radical scavenging activitymdashpossible appli-cation in clinical studies on dietary antioxidantsrdquo ClinicalChemistry and Laboratory Medicine vol 46 no 3 pp 342ndash3492008

[21] M Kubota M Shimizu H Sakai et al ldquoRenin-angiotensinsystem inhibitors suppress azoxymethane-induced colonic pre-neoplastic lesions inC57BLKsJ-dbdb obesemicerdquoBiochemicaland Biophysical Research Communications vol 410 no 1 pp108ndash113 2011

[22] M Shimizu Y Shirakami H Sakai et al ldquoEGCG suppressesazoxymethane-induced colonic premalignant lesions in maleC57BL Ksj-Db Db micerdquo Cancer Prevention Research vol 1no 4 pp 298ndash304 2008

[23] I L Cameron W E Hardman and D W Heitman ldquoThenonfermentable dietary fiber lignin alters putative colon cancerrisk factors but does not protect against DMH-induced coloncancer in ratsrdquoNutrition and Cancer vol 28 no 2 pp 170ndash1761997

[24] C-K Shih W Chiang and M-L Kuo ldquoEffects of adlay onazoxymethane-induced colon carcinogenesis in ratsrdquo Food andChemical Toxicology vol 42 no 8 pp 1339ndash1347 2004

[25] Y Yasuda M Shimizu Y Shirakami et al ldquoPitavastatininhibits azoxymethane-induced colonic preneoplastic lesions inC57BLKsJ-dbdb obesemicerdquoCancer Science vol 101 no 7 pp1701ndash1707 2010


[26] J M Kim S Araki D J Kim et al ldquoChemopreventive effectsof carotenoids and curcumins on mouse colon carcinogenesisafter 12-dimethylhydrazine initiationrdquo Carcinogenesis vol 19no 1 pp 81ndash85 1998

[27] N Suh S Paul X Hao et al ldquoPterostilbene an active con-stituent of blueberries suppresses aberrant crypt foci formationin the azoxymethane-induced colon carcinogenesis model inratsrdquo Clinical Cancer Research vol 13 no 1 pp 350ndash355 2007

[28] J Boateng M Verghese L Shackelford et al ldquoSelected fruitsreduce azoxymethane (AOM)-induced aberrant crypt foci(ACF) in Fisher 344 male ratsrdquo Food and Chemical Toxicologyvol 45 no 5 pp 725ndash732 2007

[29] A Hakansson C Branning G Molin et al ldquoBlueberry husksand probiotics attenuate colorectal inflammation and oncogen-esis and liver injuries in rats exposed to cyclingDSS-treatmentrdquoPLoS ONE vol 7 no 3 Article ID e33510 13 pages 2012

[30] A Brambilla R Lo Scalzo G Bertolo and D TorreggianildquoSteam-blanched highbush blueberry (Vaccinium corymbosumL) juicephenolic profile and antioxidant capacity in relation tocultivar selectionrdquo Journal of Agricultural and Food Chemistryvol 56 no 8 pp 2643ndash2648 2008

[31] GD Stoner L-SWangN Zikri et al ldquoCancer preventionwithfreeze-dried berries and berry componentsrdquo Seminars in CancerBiology vol 17 no 5 pp 403ndash410 2007

[32] E V Nesterova A D Durnev and S B Seredenin ldquoVer-apamil contributes to the clastogenic effects of acrylamidecyclophosphamide and dioxidine on somatic cells of BALBCandC57BL6micerdquoMutationResearchmdashGenetic Toxicology andEnvironmental Mutagenesis vol 440 no 2 pp 171ndash179 1999

[33] A P Krishnaja and N K Sharma ldquoAscorbic acid potenti-ates mitomycin C-induced micronuclei and sister chromatidexchanges in human peripheral blood lymphocytes in vitrordquoTeratogenesis Carcinogenesis andMutagenesis vol 1 pp 99ndash1122003

[34] E S Dutra C D Dias B C de Araujo A J S Castroand J C Nepomucen ldquoEffect of organic tomato (Lycopersiconesculentum) extract on the genotoxicity of doxorubicin in theDrosophila wing spot testrdquo Genetics and Molecular Biology vol32 no 1 pp 133ndash137 2009

[35] J H Jeng M C Chang and L J Hahn ldquoRole of arecanut in betel quid-associated chemical carcinogenesis currentawareness and future perspectivesrdquo Oral Oncology vol 37 no6 pp 477ndash492 2001

[36] F A Simmen J A Frank XWu R Xiao L J Hennings and RL Prior ldquoLack of efficacy of blueberry in nutritional preventionof azoxymethane-initiated cancers of rat small intestine andcolonrdquo BMC Gastroenterology vol 9 article 1471 pp 67ndash742009

[37] L R Jacobs ldquoEnhancement of rat colon carcinogenesis bywheatbran consumption during the stage of 12-dimethylhydrazineadministrationrdquo Cancer Research vol 43 no 9 pp 4057ndash40611983

[38] M R Lewin ldquoIs there a fibre-depleted aetiology for colorectalcancer Experimental evidencerdquo Reviews on EnvironmentalHealth vol 9 no 1 pp 17ndash30 1991

[39] M ZHossain S F Gilbert K Patel S Ghosh A K Bhunia andS E Kern ldquoBiological clues to potent DNA-damaging activitiesin food and flavoringrdquo Food and Chemical Toxicology vol 55pp 557ndash567 2013

[40] S M Hadi M F Ullah A S Azmi et al ldquoResveratrol mobilizesendogenous copper in human peripheral lymphocytes leading

to oxidative DNA breakage a putative mechanism for chemo-prevention of cancerrdquo Pharmaceutical Research vol 27 no 6pp 979ndash988 2010

[41] M F Ullah H Y Khan H Zubair U Shamim and S M HadildquoThe antioxidant ascorbic acidmobilizes nuclear copper leadingto a prooxidant breakage of cellular DNA implications forchemotherapeutic action against cancerrdquoCancer Chemotherapyand Pharmacology vol 67 no 1 pp 103ndash110 2011

[42] M Mazumdar S Giri and A Giri ldquoRole of quercetin onmitomycin C induced genotoxicity analysis of micronucleusand chromosome aberrations in vivordquo Mutation Research vol721 no 2 pp 147ndash152 2011

[43] M E Schutte G M Alink A P Freidig et al ldquoQuercetinincreases the bioavailability of 2-amino-1-methyl-6-phenylimidazo[45-b]pyridine (PhIP) in ratsrdquo Food andChemical Toxicology vol 46 no 11 pp 3422ndash3428 2008

[44] D Boivin M Blanchette S Barrette A Moghrabi and RBeliveau ldquoInhibition of cancer cell proliferation and suppres-sion of TNF-induced activation of NF120581B by edible berry juicerdquoAnticancer Research vol 27 no 2 pp 937ndash948 2007

[45] L Meneghini L Leporini N Scanu et al ldquoEffect of phyto-chemical concentrations on biological activities of cranberryextractsrdquo Journal of Biological Regulators and HomeostaticAgents vol 25 no 1 pp 27ndash35 2011

[46] Y Yao and A Vieira ldquoProtective activities of Vaccinium antiox-idants with potential relevance to mitochondrial dysfunctionand neurotoxicityrdquo Neurotoxicology vol 28 no 1 pp 93ndash1002007

[47] G Shen J Lee MWeinfeld and X C Le ldquoAttenuation of DNAdamage-induced p53 expression by arsenic a possible mecha-nism for arsenic co-carcinogenesisrdquo Molecular Carcinogenesisvol 47 no 7 pp 508ndash518 2008

[48] F De Marco ldquoOxidative stress and HPV carcinogenesisrdquoViruses vol 5 no 2 pp 708ndash731 2013

[49] N P Seeram L S Adams M L Hardy and D HeberldquoTotal cranberry extract versus its phytochemical constituentsantiproliferative and synergistic effects against human tumorcell linesrdquo Journal of Agricultural and Food Chemistry vol 52no 9 pp 2512ndash2517 2004

[50] T K Milugo L K Omosa J O Ochanda et al ldquoAntagonisticeffect of alkaloids and saponins on bioactivity in the quinine tree(Rauvolfia caffra sond) further evidence to support biotechnol-ogy in traditional medicinal plantsrdquo BMC Complementary andAlternative Medicine vol 13 article 285 2013

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014


MEDIATORSINFLAMMATION

of


Behavioural Neurology

EndocrinologyInternational Journal of



Disease Markers


BioMed Research International

OncologyJournal of



Oxidative Medicine and Cellular Longevity


PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of



Computational and Mathematical Methods in Medicine

OphthalmologyJournal of


Diabetes ResearchJournal of



Research and TreatmentAIDS


Gastroenterology Research and Practice


Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


which are known to damage a number of biomolecules andmay induce colon cancer [4]

During the carcinogenic process oxidative stress is anevent that may play a significant role as shown by theinduction of nitric oxide which in combination with thesuperoxide radical may generate peroxynitrite and hydroxylmolecules which in turn may cause nitration nitrosylationand oxidation in proteins DNA and other biomolecules [5]Also in concordance with these observations in a previousreport we determined an increase in the level of lipid andprotein oxidation which corresponded to the increase in thenumber of AC induced by AOM in mice [6]

Besides diet age lifestyle and other factors have beenreported as risk factors for the development of the dis-ease suggesting then the potential relevance of applyingchemopreventive measures related with the action of veg-etables and fruits because such strategy involves a varietyof actions that can be exerted in different steps of thecarcinogenic process [7] In the case of colon carcinogenesisthe comprehension of pathogenic steps such as DNA dam-age oxidative stress chronic inflammation and histologicalchanges is relevant for the selection of early biomarkersthat may show the efficacy of the studied chemopreventiveagent

In the present report we evaluated the effect of blueberryjuice (BJ) (from Vaccinium corymbosum L) on the damageinduced by AOM in the colon of mouse Blueberry is aperennial shrub 2 to 5m high which possesses almost spher-ical dark blue fruits 07 to 15 cm diameter which contain anumber of vitamins and minerals as well as phytochemicalssuch as anthocyanins flavonols flavanols stilbenes phenolicacids and proanthocyanidins [8]

The consumption of the studied plant has been increasingin natural or industrialized forms including its use inbakery products different types of syrups and alcoholicbeverages preserves and coloring additives [9]With respectto biomedical properties several effects have been reportedfor different parts of Vaccinium including antioxidantantimicrobial antiviral anti-inflammatory antihypertensiveand antiacetylcholinesterase activities [10 11] In regard toantigenotoxic and chemopreventive activities of Vacciniumreports have shown an antiproliferative potential in prostateand colon cancer cellular lines a protective effect of theirenzymatic hydrolyzates against hydrogen peroxide-inducedoxidative damage in Chinese hamster lung cell line as well asin vivo reduction of benzo(a)pyrene induced genotoxic dam-age by ethanol extracts of the plant [11ndash13] Moreover studieswith berry plants or with a number of their constituentshave suggested a potential to inhibit carcinogenic damage[14 15] In this aspect different molecular and cellular eventsinduced by berry fruits have been proposed as mediators inthe prevention of the carcinogenic process including theirantioxidant potential

The above-mentioned information suggests the perti-nence of extending studies in regard to the chemopreventivecapacity of blueberry particularly in vivo therefore the initialaim of the present report was to evaluate the capacity ofBJ to inhibit the number of AOM-induced AC in the colonof mice and secondly to explore whether such an effect

could have a relation with the BJ effect against oxidativedamage For this last purpose we determined its capacityto inhibit the percentage of the DNA adduct 8-hydroxy-21015840-deoxyguanosine (8-oxo-dG) induced by AOM as well as itscapacity to capture the 22-diphenil-1-picrilhydracil radical(DPPH) in mice

2 Material and Methods

21 Chemicals and Animals The following chemicals werepurchased from Sigma-Aldrich Chemicals (St Louis MOUSA) AOM PBS proteinase K RNAse trichloroaceticacid (TCA) trizma base albumin standard thiobarbituricacid (TBA) triethylamine hexane potassium chloride ethylacetate sodium acetate vitamin E quercetin cyanidin-3-glucoside Folin-Ciocalteu reagent andmethylene blue Anti-8-oxo-dG (4354-MC-050) was purchased from TrevigenInc (Gaithersburg MD USA) Formaldehyde HCl NaClsodium citrate xylene and ethanol were acquired fromFermont (Mexico City)

Blueberry was obtained from a pesticide-free cultivationin Zacatlan Puebla a town located 200 km north of MexicoCity The fruits were kept at minus70∘C and immediately beforethe assay the juice was mechanically obtained by pressingthe fruit with a mortar followed by filtering it in gauze toeliminate gross material

22 Phytochemical Identification Several specific groups ofphytochemicals have been identified as beingmainly involvedin the biomedical responses of blueberry therefore in thepresent report we made the identification of total phenoliccompounds flavonoids and anthocyanins

Initially BJ was treated in a batch reactor with pectinase(Pectinex 3XL 200 ppm) for 2 h at 55∘C The resultingproduct was passed through a 50 120583m filter and then it waslyophilized and powdered Lyophilized BJ extracts were frac-tionated in a silica gel chromatographic column (embeddedwith hexane) and neutralized with triethylamine Separa-tions were made with gradients ranging from a mixture ofhexaneethyl acetate (1 1) to the use of ethyl acetate withincreasing proportions of a mixture of methanolwater 1 1until only the mixture methanolwater was used Presentlyfractions of BJ are in process to be analyzed with themethodsof HPLC and 1H-NMR

221 Total Phenolic Content The concentration of phe-nolics was determined by the method of Folin-Ciocalteu[16] with some modifications The reaction mixture wasprepared with 05mL of BJ extract (02 gmL) 25mL of10 Folin-Ciocalteursquos reagent and 25mL of 75 sodiumbicarbonate The samples (in triplicate) were incubated at45∘C for 45min and left at room temperature for 2 h inthe dark before determining the absorbance at 750 nm Thesame procedure was made with vitamin E to construct acalibration curve Finally the concentration of phenolicsfor each extract was extrapolated in the calibration curveand expressed as mg of vitamin E equivalents100 g ofextract















AOM10mgkg

BJ15120583Lg


BJ + AOM41 120583Lg + 10mgkg

BJ + AOM15 120583Lg + 10mgkg







Water15 120583Lg 0 0 0 0 0 0 0 0









3 Results






(a) (b) (c)


02468

1012141618202224

1 2 3 4 5 6

8-O

xo-d

G (

)

a a

b

a ba b

a b





4 Discussion


05

1015202530354045

1 2 3 4 5

Radi

cal s

cave

ngin

g ac

tivity

()

a

aa

a


















References


























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of






























AOM10mgkg

BJ15120583Lg


BJ + AOM41 120583Lg + 10mgkg

BJ + AOM15 120583Lg + 10mgkg







Water15 120583Lg 0 0 0 0 0 0 0 0









3 Results






(a) (b) (c)


02468

1012141618202224

1 2 3 4 5 6

8-O

xo-d

G (

)

a a

b

a ba b

a b





4 Discussion


05

1015202530354045

1 2 3 4 5

Radi

cal s

cave

ngin

g ac

tivity

()

a

aa

a


















References


























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















(a) (b) (c)


02468

1012141618202224

1 2 3 4 5 6

8-O

xo-d

G (

)

a a

b

a ba b

a b





4 Discussion


05

1015202530354045

1 2 3 4 5

Radi

cal s

cave

ngin

g ac

tivity

()

a

aa

a


















References


























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of





























References


























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















evaluation of blueberry juice in mouse azoxymethane ... · researcharticle evaluation of blueberry...

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