Research Article Tualang Honey Protects against BPA ... · PDF fileEvidence-Based Complementaryand AlternativeMedicine accordingly,dehydratedthroughagradedseriesofincreasing concentration

Research ArticleTualang Honey Protects against BPA-Induced MorphologicalAbnormalities and Disruption of ER120572 ER120573 and C3 mRNA andProtein Expressions in the Uterus of Rats

Siti Sarah Mohamad Zaid12 Normadiah M Kassim1 and Shatrah Othman3

1Department of Anatomy Faculty of Medicine University of Malaya 50603 Kuala Lumpur Malaysia2Department of Environmental Sciences Faculty of Environmental Studies Universiti Putra Malaysia43400 Serdang Selangor Malaysia3Department of Molecular Medicine Faculty of Medicine University of Malaya 50603 Kuala Lumpur Malaysia

Correspondence should be addressed to Shatrah Othman shatrahoumedumy

Received 8 September 2015 Accepted 12 November 2015

Academic Editor Omer Kucuk

Copyright copy 2015 Siti Sarah Mohamad Zaid et al This is an open access article distributed under the Creative CommonsAttribution License which permits unrestricted use distribution and reproduction in any medium provided the original work isproperly cited

Bisphenol A (BPA) is an endocrine disrupting chemical (EDC) that can disrupt the normal functions of the reproductive systemThe objective of the study is to investigate the potential protective effects of Tualang honey against BPA-induced uterine toxicityin pubertal rats The rats were administered with BPA by oral gavage over a period of six weeks Uterine toxicity in BPA-exposedrats was determined by the degree of the morphological abnormalities increased lipid peroxidation and dysregulated expressionand distribution of ER120572 ER120573 and C3 as compared to the control rats Concurrent treatment of rats with BPA and Tualang honeysignificantly improved the uterine morphological abnormalities reduced lipid peroxidation and normalized ER120572 ER120573 and C3expressions and distribution There were no abnormal changes observed in rats treated with Tualang honey alone comparablewith the control rats In conclusion Tualang honey has potential roles in protecting the uterus from BPA-induced toxicity possiblyaccounted for by its phytochemical properties

1 Introduction

Bisphenol A (BPA) (22-bis(4-hydroxyphenyl)) is one of themost ubiquitous environmental endocrine disrupting chem-icals (EDC) in the world It is widely used in industries asplasticizer for the production of polycarbonate plastics epoxyresins and as nonpolymer additive to other plastics [1] It hasreceived significant worldwide attention due to its exposureto human through leaches from the inner lining of foodand beverage containers plastic bottles dental sealants watersupply pipes and adhesives [2]

Many studies have revealed the negative effects of BPAon the development of the reproductive system in humansand laboratory animals [3 4] BPA has been detected in theserum and follicular and amniotic fluids [5] fetal serum [6]milk of nursing mothers [7] and urine [8 9] These reportssuggest that humans are routinely exposed to BPA Thus it

has fueled additional concern in humanminds about BPA ontheir health

Numerous studies have reported that BPA could inducemorphological and functional alterations of the female geni-tal system particularly on the uterus [3 10] The main func-tion of uterus is regulated by cyclic changes of sexual steroidhormones and for this reason it is widely used as a classicaltarget organ to determine estrogenic effects of BPA [11] BPAwas shown to cause uterine disruptions by influencing theexpression and distribution of estrogen receptor-120572 (ER120572)and estrogen receptor-120573 (ER120573) [12] BPA was also reportedto reduce uterine immunity via dysregulation of comple-ment C3 expression that caused ascending infections in thefemale reproductive tract [13] Study by Xiao et al (2011)has found that prenatal exposure to BPA could adverselyaffect transportation andpreimplantation of the embryo fromdisturbance in the uterine receptivity that lead to infertility

Hindawi Publishing CorporationEvidence-Based Complementary and Alternative MedicineVolume 2015 Article ID 202874 18 pageshttpdxdoiorg1011552015202874

2 Evidence-Based Complementary and Alternative Medicine

in female rats [14] Furthermore BPA has been reportedto promote oxidative stress and inflammation in the femalereproductive tract whichmight contribute to themorpholog-ical and functional abnormalities [15]

Natural compounds with antioxidant properties havebeen extensively studied as a means to counter disease-associated oxidative stress [16] With these concerns in mindwe used a natural product with high antioxidant contentnamely Tualang honey (Agromas Malaysia) as a possiblepotential therapeutic agent to counter the deleterious effectsof BPA Previous scientific studies have claimed that Tualanghoney has the capability to ameliorate oxidative stress inrenal and pancreas of streptozotocin-induced diabetic rats[17] reverse atrophic uterus and vagina [18] and to improveosteoporotic bone [19] in postmenopausal animal model Inaddition Tualang honey has the capability to protect againstcigarette-induced damage on testis [20] and to counter theproliferation of oral squamous cell carcinomas (OSCC) [21]human osteosarcoma [21] and keloid fibroblasts [22]

Tualang honey a dark brownish honey has been reportedto containmore than 200 substances including sugars aminoacids vitamins minerals enzymes organic acids and phyto-chemicals The primary content of Tualang honey is invertedsugars that consist of fructose 296 glucose 30 sucrose06 and maltose 79 as well as a small amount of complexmixture of other saccharides (disaccharides trisaccharidesand oligosaccharides) [23] It also contains high total pheno-lic compounds (gallic syringic benzoic trans-cinnamic p-coumaric and caffeic acids) and flavonoids (catechin kaemp-ferol naringenin luteolin and apigenin) [24] It has tremen-dous antioxidant properties with high radical scavengingand antioxidant activities [25]The other compounds presentin Tualang honey are stearic acids furfural acetic acid 2-furylmethylketone palmitic acid ethyl linoleate oleic acid 2-hyroxy-2-cyclopenten-1-one hyacinthine and 5-methyl fur-fural [23]

The aims of our studywere to investigate the effects of sub-chronic administration of BPA on the uterine morphologyand function by determining the lipid peroxidation proteindistribution and mRNA expressions of estrogen receptor-120572 estrogen receptor-120573 and complement C3 Consequentlyinvestigation of the possible protective effects of Tualanghoney against BPA-induced uterine toxicity is executed

2 Material and Methods

21 Tualang Honey (Agromas Malaysia) Tualang honey waspurchased from the Federal Agricultural Marketing Author-ity (FAMA) under the Ministry of Agriculture and Agro-Based Industry Malaysia Tualang honey is wild multifloralhoney collected from Apis dorsatarsquos beehive that is built ona giant tree Koompassia excelsa (locally known as Tualangtree) in the rain forest of Kedah Malaysia At Honey Proces-singCentre inKualaNerang Kedah the honeywas processedthrough several stages (quality inspection dehydration pack-aging and labeling) In brief the honeywas filtered to removesolid particles concentrated in an oven at 40∘C and subjectedto 120574 irradiation at 25 kGy at Sterilgamma (M) Sdn Bhd

(Selangor Malaysia) The water concentration of the honeywas standardized by FAMA at 18

22 AnimalModel and Experimental Design Female Spraguerats (P21) were obtained from theAnimalHusbandry Facultyof Medicine University of Malaya All the experimentaldesign and procedures were conducted according to theNational Institutes of Health guide for the care and use ofLaboratory animals (NIH Publications number 8023 revised1978) which has been approved by the Animal Care andCommittee (ACUC) of the University ofMalayaThroughoutthe experimental period the rats were maintained underthe standard laboratory conditions (temperature 25 plusmn 2∘C50 plusmn 15 relative humidity and normal photoperiod of12 h dark and 12 h light) and supplied ad libitum with waterand commercial pellet diet (Gold Coin Feedmills Pte LtdMalaysia) The rats were placed in stainless steel cages withwood bedding and water was supplied in glass bottles tominimize additional exposures endocrine disruptors Theywere acclimatized to the laboratory environment for sevendays prior to the commencement of the experiment At 28days of age the rats were randomly divided into four groups(119899 = 8 in each group)

NC group (negative control) was administered with thevehicle (02mL of corn oil) PC group (positive control) wasadministered with BPA (10mgkg body weight suspended incorn oil) THC group (Tualang honey control) was admin-istered with Tualang honey (200mgkg body weight 30minbefore administration of corn oil) TH group (Tualang honey+ BPA) was administered with Tualang honey (200mgkgbodyweight 30min before administration of BPA at 10mgkgbody weight) The procedure was performed in the morning(between 0900 and 1000 AM) once daily by oral gavage(to mimic the most likely route of human exposure) for sixconsecutive weeks Throughout the administration perioddaily body weight was recorded After the last treatment therats were sacrificed during their diestrous phase

Once the rat was sacrificed the wet weight of the wholeuterus was recorded for uterotrophic response evaluationThe left horn of the uterus was immediately fixed in 10buffered formalin for histopathological analysis One half ofthe right horn of uterus was kept in phosphate buffer formalondialdehyde (MDA) determination while the other halfwas kept in RNAlater for mRNA extraction and subsequentlystored in minus80∘C freezer until further analysisThe dose selec-tion of BPA at 10mgg body weight was based on previousstudies where BPA at this dose was reported to induce disrup-tion on themorphological and biochemical parameters in thereproductive system [3 26ndash28] As for Tualang honey it wasfreshly prepared daily to avoid oxidation of the antioxidantscontentThe dose of Tualang honey at 200mgkg bodyweightwas based on previous study that showed positive biologicaleffects on female reproductive organs and the dose was equalto one tablespoonwhich is routinely taken by an adult human[18]

23 Histopathological Evaluation Theuteri were fixed in 10buffered formalin for 24 hours prior to further processingfor histopathological examination The uteri were trimmed

Evidence-Based Complementary and Alternative Medicine 3

accordingly dehydrated through a graded series of increasingconcentration of ethanol cleared in xylene and finallyembedded in paraffin to form paraffin block Subsequentlytissue sections of 5 120583m thicknesses were obtained andmounted onto glass slides deparaffinized in xylene hydratedwith water and stained with hematoxylin and eosin (Sigma-Aldrich USA) Once again sections were dehydrated in agraded series of ethanol cleared in xylene andmounted withCanada Balsam (Sigma-Aldrich) All sections were analyzedfor any morphological changes under a light microscope(Olympus CH-B145-2) attached to an image analyzer (NIS-Elements Advanced Research Nikon Japan) Representativemicrographs were taken for future reference

24 Histomorphometry For histomorphometric analysis ofthe uterus the tissue sections were reviewed and the clearestrepresentative sections on each slide were photographed attimes20 and times40 magnifications The mean values of height ofthe luminal epithelial cells thickness of the endometriumand the myometrium layers were measured in six randomlychosen areas of the sections All measurements (in 120583m)were manually determined by tracing the on-screen imagesusing a computer-image analyzing program (NIS-ElementsAdvanced Research Nikon Japan)

25 Immunohistochemistry

251 Estrogen Receptor-120572 and Complement C3 The distribu-tion of estrogen receptor-120572 and complement C3 in rat uteriwas evaluated using ImmunoCruz Rabbit ABC staining sys-tem sc-2018 kit and ImmunoCruz Goat ABC staining systemsc-2023 kit respectively

Briefly tissue sections were deparaffinized hydrated towater boiled in 01M sodium citrate buffer (pH 60) for 15minutes and incubated with 01ndash1 of hydrogen peroxidefor 10 minutes to quench the endogenous peroxidase activityNonspecific staining was blocked by incubating it in 15blocking serum Subsequently the sections were incubatedwith primary antibody at 1 100 dilution of ER120572 (MC-20sc-542 Santa Cruz Biotechnology USA) or 1 100 dilution of C3(V-20sc-14612 Santa Cruz Biotechnology USA) overnightat 4∘C On the following day the sections were incubatedwith biotinylated secondary antibody for one hour and thenincubated with AB enzyme reagent for another 30 minutesPositive staining appeared (bluish in color) after incubationwith peroxidase abstract for 10 minutes and finally thesections were counterstained with hematoxylin The sectionswere then dehydrated through a graded series of ethanolcleared in xylene and mounted with Canada Balsam (Sigma-Aldrich) and covered with glass coverslips before examina-tion under light microscopy The negative control tissue (notincubated with primary antibody) was included to ensure nofalse positive staining and for accurate interpretation of thestaining results

252 Estrogen Receptor-120573 The distribution of estrogenreceptor-120573 in uterus rats was evaluated using Rabbit SpecificHRPDAB (ABC) Detection IHC Abcam kit

Briefly the sections were deparaffinized hydrated boiledin 01M sodium citrate buffer (pH 60) for 15 minutes andincubated with hydrogen peroxide for 20 minutes to quenchthe endogenous peroxidase activity Nonspecific staining wasblocked by incubation in protein block Subsequently thesections were incubated with primary antibody at 1 200dilution (ER120573 antibody ab3576 Abcam USA) overnight at4∘C Next day the sections were incubated with biotinylatedsecondary goat anti-polyvalent antibody for 30 minutesat room temperature Positive staining developed after theincubation with DAB chromogen for 10 minutes followedby counterstaining with haematoxylin The sections weredehydrated in 2x 95 of ethanol 2x 100 and 3x xylenesfor 10 seconds each Finally the sections were mounted withCanada Balsam (Sigma-Aldrich) and covered with glass cov-erslips for observation under light microscopy The negativecontrol tissue (not incubated with primary antibody) wasincluded to ensure no false positive staining and accurateinterpretation of the staining results

253 Determination of Malondialdehyde (MDA) LevelsMDA levels of uterus were measured by the double heatingmethod [29] using Thiobarbituric Acid Reactive Substances(TBARS) assay (OxiSelect TBARS assay kit Cell BiolabsUSA) The uterus of each animal was homogenized in phos-phate buffered saline (PBS) containing butylated hydroxytol-uene (BHT) Subsequently the uterine homogenate was cen-trifuged at 10000 g for 5 minutes to collect the supernatantfor the TBARS assay

Briefly 100120583L of samples and standards was mixedwith 100 120583L of SDS lysis solution in appropriate tubes andincubated for 5 minutes at room temperature Then 250120583Lof TBA reagent was added to each tube followed by 60minutes of incubation at 95∘CThe tubes were cooled at roomtemperature in ice bath for five minutes All the tubes werecentrifuged at 3000 rpm for 15 minutes and 200120583L of thesupernatant was transferred to a 96-well microplate for mea-surement of absorbance using a spectrophotometer at 532 nmwavelength The concentration of MDA was calculated usingthe absorbance coefficient of the MDA-TBA complex andexpressed as micromoles per microgram protein (120583M120583gprotein)

26 mRNA Expression of ER120572 ER120573 and Complement C3

261 Purification of Total RNA Purification of total RNA ofuteruswas performedusingRNeasy ProtectMiniKitQiagenUSA 30mg of tissue samples of uteri was disrupted andhomogenized in Buffer RLT The lysate was centrifuged atmaximum speed (12000 rpm) The supernatant were trans-ferred into a new 15mL tube and one volume of 70 ofethanol was added This sample was transferred into anRNeasy spin column and centrifuged and the flow-throughwas discarded Subsequently buffer RPE was added to theRNeasy spin column and centrifuged for one minute at10000 rpm and the flow-through was again discarded Thisstep was repeated but centrifugation was performed for alonger period of two minutes


Table 1 Sequences of primers and references for TaqMan-PCR

Gene Forward primer Reverse primer References(accession number)

Estrogen receptor-120572 51015840-AAGCTGGCCTGACTCTGCAG-31015840 51015840-GCAGGTCATAGAGAGGCACGA-31015840 Spreafico et al [31](X61098)

Estrogen receptor-120573 51015840-CTCTGTGTGAAGGCCATGAT-31015840 51015840-GGAGATACCACTCTTCGCAATC-31015840 Kuiper et al [32](U57439)

Complement C3 51015840-CTGTACGGCATAGGGATATCACG-31015840 51015840-ATGCTGGCCTGACCTTCAAGA-31015840 Misumi et al [33](X52477)

Finally the spin column was placed in a new 15mL tubeadded with 30 120583L of RNase-free water and centrifuged at10000 rpm for one minute to elute the RNA Finally mea-surement of the concentration and purity of RNA was doneby using a NanoDrop (BioTek USA) at OD

260280while the

quality assessment was performed via denaturing agarose gelelectrophoresis The RNA was kept at minus20∘C until furtheranalysis

262 Reverse Transcription of RNA to cDNA Reverse tran-scription (RT) of RNA to single-stranded cDNA was doneusing the Applied Biosystems Kit USA Equal amounts ofRNA (300 ng) from each sample were reverse-transcribedinto cDNA Each RT reaction mix consisted of 2x RT buffer(10 120583L) 1 120583L of reverse transcriptase nuclease-free water andRNA samplemade up to a total volume of 20 120583LThe reactionmixes were briefly centrifuged to spin down the contentsand eliminate air bubbles Finally the tubes were placed ina programmed thermal cycler (Thermo Scientific) for 60minutes at 37∘C followed by five minutes at 95∘C

263 Quantitative Real-Time PCR of Selected Genes Quan-titative real-time PCR of selected genes was carried outusing Applied Biosystems Kit USA The PCR reaction mix(reaction size of 20120583L) was prepared in triplicate Each PCRreaction mix consists of 20x TaqMan Gene Expression Assay(1 120583L) 2x TaqMan Gene Expression Master Mix (10 120583L)cDNA template (4 120583L) and RNase-free water (5 120583L) Theendogenous control gene used in this study was 120573-actinmRNA In order to ensure specific amplification three typesof controls (water only reaction without primers and tem-plates derived without reverse transcriptase) were includedin the PCR reaction The prepared PCR reaction mix wasinverted to mix the reaction components followed by briefcentrifugation Twenty 120583L of PCR reaction mix was transferto each appropriate 96-well reaction plate and briefly cen-trifuged

Finally the plate was placed in the StepOne Plus Real-Time PCR system (Applied Biosystems USA) followed byautomated amplification of the genes of interest The point atwhich exponential amplification of the PCR products begins(values for cycle threshold CT) was determined using theApplied Biosystems software Relative abundances of the tar-getmRNAwere calculated using the 2minusΔΔCTmethod [30]Theexperiment was repeated three times to ensure the validityof the results The relative mRNA expression levels for each

selected gene were calculated in terms of the 120573-actin internalcontrol The sequences of primer used for the amplificationof gene are shown in Table 1

27 Statistical Analysis All statistical evaluations were per-formed with Statistical Package for Social Sciences (SPSS IncChicago Illinois USA version 180 for Windows) FirstlyShapiro-Wilk 119882 test was performed to test whether allresults followed a normal distribution (normally distributedif 119875 value was greater than 005) Parametric variables wereanalyzed using One-way analysis of variance (ANOVA) fol-lowed by Bonferroni test formultiple comparisons to identifysignificant differences between groups Values were reportedas Mean plusmn SEM 119875 lt 005 was considered significant

3 Results

31 BodyWeight andUterineWeight In toxicological studiesanalysis of body weight and the weight of selected organs is asensitive indicator for adverse effects of chemical exposure Inparticular analysis of normalization of absolute organ weightto body weight (relative organ weight) is a more accurateanalytical endpoint for the identification of harmful effectsof a toxic agent on the organ weights The changes in bodyweight gain anduterine relativeweight of animals in all experi-mental groups are shown in Table 2 and Figures 1(a) and 1(b)respectively

To determine whether BPA exposure induces changes inbody weight as a result of toxicity effects the body weight ofall rats was monitored throughout the experimental periodAlthough changes in the body weight gain were not statisti-cally significant betweenBPA-exposed group and all the othergroups six weeks of subchronic exposure to BPA in the PCgroup had caused a slight increment (173) in the bodyweight gain compared to the control animals (NC group)Similar increment (1655) in body weight gain was alsonoted in rats concurrently treated with BPA and Tualanghoney (TH group) The changes in body weight gain forTualang honey treated alone (THC group) rats were compa-rable to the control rats (NC group)

Similarly the changes in uterine weight were recorded atthe end of the administration period since this could be usedas an indicator for any changes in the normal functions Aftersixweeks of BPA exposure (PC group) significant declinewasobserved in the uterine relative weight by 25 compared tothe control rats (NC group) However concurrent treatment


Table 2 Body weight gain uterine relative weight malondialdehyde (MDA) level and uterine histomorphometry parameters in all experi-mental groups

Group Body weightgain (g)

Uterine relativeweight (wetweightbody

weight)

Malondialdehyde (MDA)level (120583M120583 protein)

Height of luminalepithelial cells

(120583m)

Thickness ofendometrium (120583m)

Thickness ofmyometrium (120583m)

NC 7888 plusmn 1461 189 plusmn 012bbb 00037 plusmn 000026bbb 3051 plusmn 137bbbc 57187 plusmn 1914bb 29918 plusmn 1686bb

PC 9925 plusmn 990 141 plusmn 004aaacd 00074 plusmn 000053aaacddd 1888 plusmn 081aaaccddd 48674 plusmn 1015aaccddd 22839 plusmn 891aacdd

THC 895 plusmn 1064 183 plusmn 007bb 00036 plusmn 000089bbb 2823 plusmn 048abbb 57425 plusmn 1945bbb 29926 plusmn 992bb

TH 925 plusmn 462 173 plusmn 004b 00049 plusmn 000064b 2686 plusmn 068bbb 54630 plusmn 378b 28451 plusmn 1663bb

Data are expressed as Mean plusmn SEM(1) a119875 lt 005 and aa

119875 lt 001 versus NC (negative control)(2) b119875 lt 005 bb119875 lt 001 and bbb

119875 lt 0001 versus PC (BPA 10mgkg)(3) c119875 lt 005 and cc

119875 lt 001 versus TH (Tualang honey 200mgkg + BPA 10mgkg)(4) d119875 lt 005 dd119875 lt 001 and ddd

119875 lt 0001 versus THC (Tualang honey 200mgkg)

0

20

40

60

80

100

120

140

()

173088 1655

THNC PC THC(a)

bbbb

0

20

40

60

80

100

120

()

PC THC THNC

aaa c dd(minus2539)

bb(minus60) (minus16)

(b)

Figure 1 (a) Changes in body weight gain Body weight gains were not statistically different among all experimental groups (b) Uterirelative weights in all experimental groups BPA exposure which caused a significant decline in body weight was observed (PC group) whileconcurrent treatment of BPA with Tualang honey (TH group) significantly prevented the reduction in the relative body weight Data areexpressed as Mean plusmn SEM (1) aaa

119875 lt 0001 versus NC (negative control) (2) b119875 lt 005 bb

119875 lt 001 and bbb119875 lt 0001 versus PC (BPA

10mgkg) (3) c119875 lt 005 versus TH (Tualang honey 200mgkg + BPA 10mgkg) (4) dd


of rats with BPA and Tualang honey (TH group) had signifi-cantly prevented this effectThe uterine relative weight in ratstreated with Tualang honey alone (THC group) was compa-rable to the control rat (NC group) reflecting that Tualanghoney itself has no deleterious effect on the uterus weight

32Malondialdehyde (MDA)Level Lipidperoxides are unsta-ble primary products of lipid peroxidation which decomposeto the stable formmalondialdehyde (MDA) Measurement ofthe MDA levels as the end product of lipid peroxidation is awidely accepted assay and is considered to be a crucial indexof oxidative stress associated with organ pathophysiology inanimals

As shown in Table 2 and Figure 2 a significant increase intheMDA level by 497was noted in rats exposed to BPA (PCgroup) as compared to the control rats (NC group) Howeverconcurrent treatment of BPA with Tualang honey had led

to significant reduction in the MDA levels (TH group) TheMDA level was not affected byTualang honey treatment alone(THC group) and the value is comparable to the normal rats(NC group)

33 Uterine Histomorphometry Analysis As shown in Table 2and Figures 3(a) 3(b) and 3(c) BPA exposure had causeda significant reduction in all histomorphometric parame-ters (including luminal epithelial cells and thickness of theendometrium and myometrium layers) (PC group) whencompared to all the other groups (NC TH and THC groups)

In the BPA-exposed rats (PC group) the reductions wereto 3811 1488 and 2366 in luminal epithelial cellsheight and endometrial and myometrial thickness respec-tively Interestingly consistent with the reduction of theuterine relative weight concurrent treatment of rats with BPAand Tualang honey (TH group) significantly prevented the


bbb

aaa c ddd

bbb

b

(minus30)

0

20

40

60

80

100

120

140

160

()

PC THC THNC

(244)

(497)

c

Figure 2 Levels of malondialdehyde (MDA) in all experimentalgroups Malondialdehyde level was significantly increased in BPA-exposed rats (PC group) while concurrent treatment of BPA withTualang honey (TH group) has significantly prevented the incre-ment effect of BPA Data are expressed as Mean plusmn SEM (1)aaa119875 lt 0001 versus NC (negative control) (2) b

119875 lt 005 bbb119875 lt

0001 versus PC (BPA 10mgkg) (3) c119875 lt 005 versus TH (Tualang

honey 200mgkg + BPA 10mgkg) (4) ddd119875 lt 0001 versus THC

(Tualang honey 200mgkg)

reduction in these parametersMeanwhile the values of theseparameters in the three groups were comparable to eachother

34 Morphology of the Uterus Morphological analysis of therepresentative uteri from sections of all groups is shown inFigure 4Themorphological changeswere consistentwith thehistomorphometric analysis

In the control rats (NC group) normal histologicalappearance was observed (Figures 4(A) 4(B) and 4(C))Theluminal epithelial cells were made up of tall pseudostratifiedcolumnar epithelium cylindrical in shape with well-roundednuclei and rested on a prominent basement membrane Thelamina propria was intact with healthy endometrial glandsand high cellular content in the stroma A number of mitoticfigures were visible in glandular epithelial cells The myome-trium appeared to be normal The histological appearancesof the endometria in rats treated with Tualang honey alone(THC group) were comparable to the control rats (NC group)(Figures 4(G) 4(H) and 4(I))

The uterus of BPA-exposed rats (PC group) exhibiteddisruptive changes as shown in Figures 4(D) 4(E) and 4(F)when compared to the control rats (NC group) The luminalepithelial cells were shorter the stroma had less cell pop-ulation and appeared disorganized and some cells weredistorted and had irregular-shaped nuclei Some of the nucleihad more condensed chromatin There were very little inter-stitial intracellular spaces between the stroma cells Theendometrial glands appeared unhealthy by being smaller insize and lined by shrunken and distorted epithelial cells withirregularly shaped nuclei and somepyknotic nuclei which are

less organized and reduced in number The smooth musclebundles of the myometrium which were also seen smallerand shrunkenwith the organizations of the inner circular andouter longitudinal smoothmuscle fibers lookeddisintegrated

In comparison with BPA-exposed rats (PC group) theuterine morphology of the Tualang honey (TH group) (Fig-ures 4(J) 4(K) and 4(L)) showed slightly improved surfaceepithelium but had healthy looking stromal cell populationwith larger stromal cells and relativelymore interstitial spacesbetween cells compared to the PC group The endometrialglands looked normal lined by glandular epithelium verymuch comparable to the NC group Moreover the myome-trium also appeared normal

35 ER120572 ER120573 and C3 Protein Distribution The representa-tive uterine tissue sections were stained using immunohis-tochemical technique to evaluate cell specific changes in theER120572 ER120573 and complement C3 proteins These proteins arelocalized in the nuclei of epithelial and stromal cells of theuterus

In general the staining intensity of ER120572 protein receptorexpression in all representative uterine sections was highestin the luminal and glandular epithelial cells but lower stainingintensity was found in the cells of endometrial stroma (about50 to 80) (Figure 5) Among all groups the most pro-nounced immunostaining intensity was observed in thecontrol rats (NC groups) (Figures 5(C) and 5(D)) and theTualang honey alone treated rats (THC group) (Figures 5(G)and 5(H)) As expected lower immunostaining intensity wasobserved in BPA-exposed rats (PC group) (Figures 5(E) and5(F)) and in rats with concurrent treatment with Tualanghoney (TH group) (Figures 5(I) and 5(J))

As shown in Figure 6 the immunostaining patterns andintensities of ER120573 were different from the ER120572 Comparableimmunostaining intensities were observed in rats of NCgroup (Figures 6(C) and 6(D)) THCgroup (Figures 6(G) and6 (H)) and TH group (Figures 6(I) and 6(J)) while there wasless immunostaining intensity in all compartments (namelyluminal and glandular epithelial cells and stroma) in the BPA-exposed rats (PC group)

For complement C3 protein expression the immunos-taining intensity was highest in both the control rats (NCgroup) and Tualang honey alone treated rats (THC group)(Figures 7(C) 7(D) and 7(G) 7(H) resp) There was lessimmunostaining intensity in the BPA-exposed rats (PCgroup) (Figures 7(E) and 7(F)) while there was slightly higherintensity of staining in the stroma of rats with concurrenttreatment with Tualang honey (TH group) (Figures 7(I) and7(J))

36 ER120572 ER120573 and C3 mRNA Expression The changes inmRNA expression of estrogen-related genes were measuredto support the immunohistochemistry analysis The ER120572ER120573 and complement C3 mRNA expression are shown inFigures 8(a) 8(b) and 8(c)

As shown in Figure 8(a) BPA exposure in PC group ani-mals significantly downregulated the ER120572mRNA expressionby approximately 4-fold as compared to the control rats (NC


bbb c

aaa cc ddd

bbb

a bbb

80

85

90

95

100

105(

)

(minus3811)

(minus1196)

(minus747)

PC THC THNC(a)

bbaa cc ddd

bbb

b(minus1488) (minus447)

0

20

40

60

80

100

120

140

()

PC THC THNC

(041)

(b)

bb

aa c dd

bbbb

(minus2366)

0

20

40

60

80

100

120

140

()

PC THC THNC

(003)(minus490)

(c)

Figure 3 (a) Height of luminal epithelial cells of uterus in all experimental groups BPA-induced reduction in luminal epithelial height (PCgroup) was significantly prevented by concurrent treatment with Tualang honey (TH group) Data are expressed asMeanplusmn SEM (1) a119875 lt 005and aaa119875 lt 0001 versus NC (negative control) (2) bbb

119875 lt 0001 versus PC (BPA 10mgkg) (3) c119875 lt 005 and cc

119875 lt 001 versus TH (Tualanghoney 200mgkg + BPA 10mgkg) (4) ddd

119875 lt 0001 versus THC (Tualang honey 200mgkg) (b) Thickness of endometrium of uterus in allexperimental groups BPA-induced reduction in thickness of the endometrium layer (PC group) was significantly prevented by concurrenttreatment with Tualang honey (TH group) Data are expressed as Mean plusmn SEM (1) aa

119875 lt 001 versus NC (negative control) (2) b119875 lt 005

bb119875 lt 001 and bbb

119875 lt 0001 versus PC (BPA 10mgkg) (3) c119875 lt 005 versus TH (Tualang honey 200mgkg +BPA 10mgkg) (4) ddd119875 lt 0001versus THC (Tualang honey 200mgkg) (c) Thickness of myometrium of uterus in all experimental groups BPA-induced reduction in thethickness of the myometrium layer (PC group) was significantly prevented by concurrent treatment with Tualang honey (TH group) Dataare expressed as Mean plusmn SEM (1) aa

119875 lt 001 versus NC (negative control) (2) bb119875 lt 001 versus PC (BPA 10mgkg) (3) c

119875 lt 005 versus TH(Tualang honey 200mgkg + BPA 10mgkg) (4) dd


group) However the magnitude was reduced to 3-fold byconcurrent treatment with Tualang honey (TH group) Incontrast treatmentwith Tualang honey (THCgroup) showed15-fold induction in the ER120572mRNA expression compared tothe control group

Figure 8(b) shows a dissimilar pattern of ER120573 mRNAexpression compared to ER120572 mRNA expression As com-pared to the control rats (NC group) the expression of ER120573was significantly upregulated by approximately 14-fold inBPA-exposed rats (PC group) and this was reduced to 11-foldby concurrent treatment of Tualang honey (TH group) Thisindicates that concurrent treatment with Tualang honey wasable to reduce the ER120573 mRNA expression in BPA-exposed

rats by 80 Meanwhile Tualang honey alone (THC group)had no effect on the ER120573mRNA expression with the value ofexpression comparable to the control rats (NC group)

As shown in Figure 8(c) BPA exposure (PC group)caused dramatic suppression of the complement C3 mRNAexpression by almost 90as compared to the control rats (NCgroup) Surprisingly this effect was completely reversed withconcurrent treatment with Tualang honey (TH group) Infact the C3 mRNA expression in the Tualang honey group(TH group) was 18-fold higher than the control rats (NCgroup) Tualang honey alone had no effect on the C3 mRNAexpression with the value of expression comparable to thecontrol rats (NC group)


Luminal epithelium Endometrial gland Myometrial layer

(A) (B) (C)

(a) Negative control (vehicle)


(D) (E) (F)

(b) Positive control (BPA 10mgkg)


(G) (H) (I)

(c) Tualang honey control (Tualang honey 200mgkg)


(J) (K) (L)

(d) Tualang honey (Tualang honey 200mgkg + BPA 10mgkg)

Figure 4 Representative sections of uteri from all experimental groups (HampE times40) Normal histological appearance was observed in controlgroup (NC group) ((A) (B) and (C)) Significant disruptive morphological changes were observed in BPA-exposed rats (PC group) ((D)(E) and (F)) The uterus of rat treated with Tualang honey alone (THC group) ((G) (H) and (I)) was comparable to the control group whileconcurrent treatment of rats with BPA and Tualang honey (TH group) ((J) (K) and (L)) showed partial prevention of tissues damage fromthe toxic effect of BPA


Luminal epithelium Endometrial gland

(A) (B)

(a) Negative control (vehicle) without primary antibody


(C) (D)

(b) Negative control (vehicle)


(E) (F)

(c) Positive control (BPA 10mgkg)


(H)(G)

(d) Tualang honey control (Tualang honey 200mgkg)


(J)(I)

(e) Tualang honey (Tualang honey 200mgkg + BPA 10mgkg)

Figure 5 Representative sections of uteri showing the immunohistological localization of ER120572 in all experimental groups (times40) The mostpronounced immunostaining intensity in both control ((C) (D)) (NC groups) and Tualang honey treated alone ((G) (H)) (THC groups)rats Lower immunostaining intensity in BPA-exposed rats ((E) (F)) (PC group) was observed Concurrent treatment with Tualang honeyTH group ((I) (J)) also showed similar immunostaining intensity with these BPA-exposed rats



(A) (B)



(C) (D)



(E) (F)



(G) (H)



(I) (J)


Figure 6 Representative sections of uteri showing the immunohistological localization of ER120573 in all experimental groups (times40) The mostpronounced immunostaining intensity was observed in BPA-exposed rats ((E) (F)) (PC group) Comparable immunostaining intensitieswere observed in NC ((C) (D)) THC ((G) (H)) and TH ((I) (J)) groups


(A) (B)



(C) (D)



(E) (F)



(G) (H)



(I) (J)



Figure 7 The lowest intensity of immunostaining was observed in BPA-exposed animals (PC group) ((E) (F)) However higher intensityof color in stroma was noted with concurrent treatment with Tualang honey (TH group) ((I) (J)) Compared to the PC and TH groups thehighest and comparable immunostaining intensity were observed in NC group ((C) (D)) and THC group ((G) (H))


bb d

aa c ddd

b dd

a bbb cc

PC THC THNC0

02

04

06

08

1

12

14

16

18Re

lativ

e mRN

A ex

pres

sion

(a)

bbb ccc

aaa ccc ddd

aaa bbbbbb cc

0

02

04

06

08

1

12

14

16

Relat

ive m

RNA

expr

essio

n

PC THC THNC(b)

bbb cc

aaa ccc ddd

aaa bbb ddd

bbb

0

02

04

06

08

1

12

14

16

18

2

Relat

ive m

RNA

expr

essio

n

PC THC THNC(c)

Figure 8 (a) Quantitative real-time PCR of ER120572 in all experimental groups ER120572mRNA expression was significantly downregulated in therats exposed to BPA (PC group) (4-fold) as compared to the control animals (NC group) However the decline was reversed (3-fold) byconcurrent treatment with Tualang honey (TH group) Data are expressed as Mean plusmn SEM (1) a

119875 lt 005 and aa119875 lt 001 versus NC (negative

control) (2) b119875 lt 005 bb119875 lt 001 and bbb


119875 lt 001 versus TH (Tualang honey200mgkg + BPA 10mgkg) (4) d

119875 lt 005 dd119875 lt 001 and ddd119875 lt 0001 versus THC (Tualang honey 200mgkg) (b) Quantitative real-time

PCR of ER120573 in all experimental groups Compared to the control rats (NC group) the expression of ER120573 was significantly upregulated (14-fold) in BPA-exposed rats (PC group) and this is reduced to 11-fold by concurrent treatment of Tualang honey (TH group) Data are expressedas Mean plusmn SEM (1) aaa

119875 lt 0001 versus NC (negative control) (2) bbb119875 lt 0001 versus PC (BPA 10mgkg) (3) cc

119875 lt 001 and ccc119875 lt 0001

versus TH (Tualang honey 200mgkg + BPA 10mgkg) (4) ddd119875 lt 0001 versus THC (Tualang honey 200mgkg) (c) Quantitative real-

time PCR of complement C3 in all experimental groups BPA exposure (PC group) caused a significant downregulation of the amount ofcomplement C3 mRNA (9-fold) as compared to the control rats (NC group) This effect was completely reversed by concurrent treatmentwith Tualang honey (TH group) Data are expressed as Mean plusmn SEM (1) aaa

119875 lt 0001 versus NC (negative control) (2) bbb119875 lt 0001 versus

PC (BPA 10mgkg) (3) cc119875 lt 001 and ccc

119875 lt 0001 versus TH (Tualang honey 200mgkg + BPA 10mgkg) (4) ddd119875 lt 0001 versus THC


4 Discussion

BPA as an endocrine disrupting chemical (EDC) has thecapability tomimic enhance or inhibit the activity of naturalestrogen and to disrupt the functions of estrogen nuclearhormone receptors in a diverse set of target tissues [8 1315 34] Thus exclusively defining BPA as an environmentalestrogen or selective estrogen receptor modulator (SERM) isindeed inaccurate [2]

In the last few decades a considerable amount of evidencehas been accumulated which demonstrates the fact thatyoung women may be at high risk of reproductive infertilitydue to continuous exposure to numerous BPA products intheir daily lives [35] With this concern in mind our presentstudy had investigated the disruptive effects of BPA onthe female reproductive physiology with special referenceto the morphology oxidative markers and protein andmolecular changes that occur in the rat uterus In addition


the potential protective roles of Tualang honey as a naturalproduct in reducing the disruptive effects of BPA on theabove-mentioned selected parameters were evaluated Themechanisms of the translational and transcription factors inestrogen receptors were also investigated In addition to theclassical in vivo tool (uterotrophic assay) more sensitive toolssuch as the analysis of gene transcription and protein expres-sion were adopted to investigate the molecular responses dueto BPA exposure

In toxicology study the differences in body and organweights between untreated and treated animals can be areliable indicator for the toxicity effects of the test compounds[36] In the present study the toxicology data showed that theeffects of BPA appear to be very specific to the uterus as thereduction in weight seems to affect the uterus itself withouthaving significant effects on the whole body weight Thesecurrent results are in agreement with a previous report byAshby et al [37] In contrast others have shown that BPAexposure in rodent animals was reported to be associatedwith weight gain [38ndash40]

BPA is one of the suggested EDCs that induce reactiveoxygen species (ROS) that play important roles in the pathol-ogy of female reproductive diseases such as uterine endomet-riosis [35] Furthermore lipid peroxidation (LPO) is a processof interaction between reactive oxygen species (ROS) and thecellular membrane that induces damage to cellular macro-molecules and DNA [41] Since malondialdehyde (MDA)is a secondary product of LPO it is generally accepted asan oxidative stress marker [42] The relationship betweenoxidative stress and infertility amongwomenhas been provenin several scientific studies [43 44] Epidemiological datashowed that higher levels of reactive oxygen species (ROS)and lower antioxidant levels were detected in infertile womencompared to fertile women [45 46] In the present study theuterine MDA level in BPA-exposed animals was significantlyhigher compared to the normal control animals The resultscould be due to lipid peroxidation process by BPA and thisresult correlated well with the histopathological immunohis-tochemical and gene expression analyses

Treatment with Tualang honey in BPA-exposed rats sig-nificantly reduced the uterine MDA levels This observationcould be due to the antioxidant effects of Tualang honeywhich cause reduction in the number of free radicals Thisexplanation is based on several published data that reportedTualang honey as an effective antioxidant in reducing theMDA levels in human subjects and animal models [17 47]

In addition to that striking morphologic changes ofthe uterus (disruption of the normal structure of the lumi-nal epithelium endometrium myometrium and glandularepithelial cells) associated with oxidative stress were apparentin BPA-exposed rats compared to the controls These mor-phological changes could be related to the potential directactions of BPA on the DNA itself that results in alterationof gene expression BPA-induced DNA damage has beenevidently reported in both in vivo and in vitro studies [4849] This is because BPA can be oxidized to bisphenol-o-quinone that covalently binds to deoxyguanosine to formDNA adducts Subsequently DNA adducts cause improper

and incomplete replication that leads to the formation ofatypical cells

Interestingly the disruptive effects of BPA on the uterinemorphology were partially reduced following concurrenttreatment with Tualang honey The improvement could beagain accounted for by the high natural antioxidant contentsin Tualang honey in addition to the carbohydrates aminoacids proteins organic acids vitamins and various phyto-chemicals [24 50] In the natural state cells of our body arecapable of maintaining the normal equilibrium of antioxi-dant-oxidative stress balance by neutralizing or directlydiminishing the oxidative damage bymeans of enzymatic andnonenzymatic antioxidants [51] Evidently Tualang honeycontains nonenzymatic antioxidants such as vitamins E andC with their interactive effects as lipophilic and hydrophilicfree-radical scavengers respectively [52] hence possibly exp-laining the improvement observed in treated rats

Vitamin E resides mainly in the cell membranes thusplaying an important role in the maintenance of cell mem-branes [53] Meanwhile the major function of vitamin C is asa scavenger of free radicals in the extracellular fluid deceivingradicals in the aqueous phase protecting biomembranes fromperoxidase impairment and regenerating tocopherol (vita-min E) from tocopheroxyl radicals in the membrane [54]Several studies have found that both antioxidants can miti-gate adverse pathological impacts by minimizing the geno-toxicity and cytotoxicity effects in the cells [55ndash57] Flavonols(quercetin and kaempferol) with weak estrogenic activitiesare attributed to the decrease in the intracellular reactiveoxygen species (ROS) by a mechanism that involves estrogenreceptors [58] Thus in our study the antioxidant propertiesof Tualang honey could be contributing to the protectiveeffects in cells by trapping and inactivating of free radicalsas observed in the improvement of the heterogeneous celltypes of the uterus (stromal luminal epithelium endometrialglands and smooth muscle)

Nonetheless the morphological recovery following treat-ment with Tualang honey in BPA-exposed uterus seemsto be partial This may be due to the presence of otherreactive oxygen species that could also be in play to cause thedisruptive effects Moreover even after removal of BPA theinactive detoxifying proteinsenzymes may require a certainperiod of time to recover as supported byChevallet et al [59]

Other findings in our study which are also in line withprevious studies showed that BPA can disrupt the develop-ment growth and functions of the uterus by interruptingthe regulation of mRNA expression and protein distributionof ER120572 ER120573 and complement C3 [12 13 60] Estrogenreceptor (ER) is a member of steroid receptor superfamilya ligand-activated enhancer protein that is activated by thehormone estrogen (17120573-estradiol) and is able to regulate genetranscription via estrogen responsive elements [61] Unfortu-nately it can also be activated by other compounds includingendocrine disrupting chemical such as BPA [62] The ER isencoded by two subtype genes namely ER120572 and ER120573 thatfunction as a signal transducer and a transcription factor inmodulating the expression of target genes [63] The endoge-nous estrogen (17120573-estradiol) has a lower binding affinityto ER120573 than ER120572 [34] but both receptors share similarity


in terms of transactivation via estrogen responsive element(ERE) [64] In contrast they possess dissimilar functionswithregard to their roles in transcription activation which dependvery much on the ligands and their responsive elements [65]

BPA is considered weak environmental estrogen since itsbinding affinity to ER120572 and ER120573 is estimated to be 10000-foldlower than the endogenous estrogen with a relative bindingaffinity of 6-fold higher in ER120573 compared to ER120572 [34 62] Onthe other hand in some cell types BPA exhibits estradiol-likeagonist activity via ER120573 and amixed agonistantagonist activ-ity via ER120572 [15] Both subtypes contain a ligand-dependenttransactivation domain named AF2 (Activation Function 2)for activation of transcription of target genes by the recruit-ment of transcriptional cofactors steroid receptor coactivator-1 (SRC-1) and transcriptional intermediary factor-2 (TIF-2)[66] The disruptive role of BPA in this pathway significantlyincreases the recruitment of these cofactors [67] This couldbe a possible explanation for our present data that showedsignificant upregulation in the expression of mRNA andhigher protein distribution of ER120573 in BPA-exposed animals(PC group) as compared to the control rats

In our study besides the role of lipid peroxidation theupregulation of ER120573 in BPA-exposed animals could be attri-buted to the disruptive effects in the uterus A report in 2000by Weihua et al revealed that in the uterus ER120573 acts as amodulator of ER120572-mediated gene transcription [68]The roleof ER120573 is as a transdominant repressor that inhibits the ER120572transcriptional activity [69] The inhibitory effects are due tothe ability of ER120573 to form heterodimers with ER120572 which inturn regulate the functions of estrogen receptors [70] Thusunlike ER120572 ER120573 does not have classic uterotrophic effects butrather disruptive effects on ER120572

Other studies via crystallography structure analysis andcomputer modeling studies have found that BPA has higherbinding affinity to ERR120574 to ER120572 [71 72] ERR120574 is an orphannuclear receptor that belongs to the ERR family (estrogenreceptor-related) [73] Although both ER120572 and ERR120574 recep-tors share similar sequence homology 17120573-estradiol doesnot associate to ERR120574 [74] The BPA-ERR120574 interaction wassuggested to trigger heterodimerization between ER120572 andERR120574 which results in suppression of the transcriptionalactivity of the ER120572 [71] Similarly Huppunen and Aarnisaloalso reported that the modulation of BPA as an inhibitor onER120572 signaling is also via ERR120574 [75] Such interaction could bean explanation for our present data that showed significantdownregulation of mRNA expression and lower proteindistribution of ER120572 in BPA-exposed animals (PC group)compared to the control normal animals In addition the ER120572mRNA expression was also significantly higher in Tualanghoney treated rats However the protein distribution did notdiffermuch from the normal control rats Hence these resultssuggest that Tualang honey is likely to modulate the ER120572mRNA expression at the transcriptional level but not at thetranslational level

Complement C3 is involved in innate immunity Thecrucial role of C3 is to regulate any activation of host cell dam-age by promoting phagocytosis initiating local inflammatoryresponses against pathogens and to instruct appropriateadaptive immune response towards antigens for a humoral

response [76] In 2004 Seidlova-Wuttke et al have usedquantitative RT-PCR analysis to investigate the effects of BPAon C3 mRNA transcription in rat uterus [13] According tothe authors estradiol treatment significantly upregulated theC3 mRNA expression Interestingly the effect was not mim-icked by BPA exposure where downregulation of C3 mRNAexpression was observedThis effect was also observed in ourpresent study However concurrent treatment with Tualanghoney was able to significantly increase the C3 expressionindicating some improvement in the innate immune systemInterestingly the mRNA expression in Tualang honey treatedrats was also significantly higher compared to the controlanimals This could be due to higher activation of transcrip-tional activity C3 induced by certain bioactive compoundsthat oppose the disturbance effects of BPA on the innateimmune systemHowever the protein distribution inTualanghoney treated rats did not differ much from the BPA-exposedrats Hence these results suggest that Tualang honey is likelyto modulate the C3 mRNA expression at the transcriptionallevel but not at the translational level

The improvements of protein and molecular levels ofER120572 ER120573 and C3 in BPA-exposed animals treated by Tua-lang honey could be explained by the fact that Tualanghoney contains high nutritional profile antioxidants includ-ing flavonoids specifically flavonols [58] The two main nat-urally occurring flavonols namely quercetin and kaempferolshare structural similarities with 17120573-estradiol and hencemaypotentially have weak estrogenic effects [58] Evidence fromour previous study have shown that besides the ability toscavenge oxidants and free radicals [19] biologically activeestrogen-like compounds in Tualang honey also improvedthe atrophy state of uterus and vagina [18] In addition ourprevious findings also found that the potential competitivebinding of these compounds also contributed to the improve-ment of the hypothalamic-pituitary axis function in BPA-exposed animals [77] as also supported by an earlier study[78]

The effects of lipid peroxidation of BPA on DNA damagehave also been extensively tested in in vivo and in vitro studies[79ndash81] The antigenotoxicity effects of Tualang honey werereported to occur via several mechanisms the upregulationof double stranded DNA repair enzymes [82] reduction ofcyclobutane pyrimidine dimers and 8-oxo-dG-positive cells(biomarkers of DNA damage) [83] and induction of apopto-sis [84] Similar protective mechanisms against DNA damagehave been suggested in studies on Buckwheat honey andhoney from arid regions that are also in line with the currentTualang honey findings [85 86]

5 Conclusions

In conclusion Tualang honey has potential roles in reducingBPA-induced uterine disruption possibly accounted for byits phytochemical properties In view of this Tualang honeycould be suggested as a promising natural product candidatein reducing the toxicity effects of BPA in the uterusThus fur-ther investigation is warranted to establish precise and betterunderstanding of the roles of phytochemical compounds in


Tualang honey that account for the reduction in BPA-induceduterine toxicity

Abbreviations

BPA Bisphenol AOSCC Oral squamous cell carcinomasER120572 Estrogen receptor-alphaER120573 Estrogen receptor-betaMDA MalondialdehydeTBARS Thiobarbituric Acid Reactive SubstancesPBS Phosphate buffered salineBHT Butylated hydroxytolueneRNA Ribonucleic acidcDNA Complementary deoxyribonucleic acidPCR Polymerase chain reactionNC Negative control groupPC Positive control groupTH Tualang honey + BPA groupTHC Tualang honey control

Conflict of Interests

The authors declare there is no conflict of interests

Acknowledgments

The authors would like to acknowledge University of Malayafor providing a postgraduate research grant (no PG087-2012B) Universiti Putra Malaysia and Ministry of HigherEducation Malaysia for their financial assistance to the firstauthor Siti SarahMohamadZaid Sincere thanks are due to allthe staff of the Department of Anatomy Faculty of MedicineUniversity of Malaya

References

[1] N Von Goetz M Wormuth M Scheringer and KHungerbuhler ldquoBisphenol a How the most relevant exposuresources contribute to total consumer exposurerdquo Risk Analysisvol 30 no 3 pp 473ndash487 2010

[2] Y B Wetherill B T Akingbemi J Kanno et al ldquoIn vitromolecular mechanisms of bisphenol A actionrdquo ReproductiveToxicology vol 24 no 2 pp 178ndash198 2007

[3] A Suzuki A Sugihara K Uchida et al ldquoDevelopmental effectsof perinatal exposure to bisphenol-A and diethylstilbestrol onreproductive organs in female micerdquo Reproductive Toxicologyvol 16 no 2 pp 107ndash116 2002

[4] Y J Yang Y-C Hong S-Y Oh et al ldquoBisphenol A exposureis associated with oxidative stress and inflammation in post-menopausal womenrdquo Environmental Research vol 109 no 6pp 797ndash801 2009

[5] H-W Kuo and W-H Ding ldquoTrace determination of bisphe-nol A and phytoestrogens in infant formula powders by gaschromatography-mass spectrometryrdquo Journal of Chromatogra-phy A vol 1027 no 1-2 pp 67ndash74 2004

[6] Y Sun M Wada O Al-Dirbashi N Kuroda H Nakazawaand K Nakashima ldquoHigh-performance liquid chromatographywith peroxyoxalate chemiluminescence detection of bisphenol

A migrated from polycarbonate baby bottles using 4-(45-diphenyl-1H-imidazol-2-yl)benzoyl chloride as a labelrdquo Journalof Chromatography B Biomedical Sciences and Applications vol749 no 1 pp 49ndash56 2000

[7] C Brede P Fjeldal I Skjevrak and H Herikstad ldquoIncreasedmigration levels of bisphenolA frompolycarbonate baby bottlesafter dishwashing boiling and brushingrdquo Food Additives ampContaminants vol 20 no 7 pp 684ndash689 2003

[8] J C Gould L S Leonard S C Maness et al ldquoBisphenol Ainteracts with the estrogen receptor 120572 in a distinct manner fromestradiolrdquoMolecular and Cellular Endocrinology vol 142 no 1-2 pp 203ndash214 1998

[9] M V Maffini B S Rubin C Sonnenschein and A M SotoldquoEndocrine disruptors and reproductive health the case ofbisphenol-Ardquo Molecular and Cellular Endocrinology vol 254-255 pp 179ndash186 2006

[10] CMMarkey P RWadia B S Rubin C Sonnenschein and AM Soto ldquoLong-term effects of fetal exposure to low doses of thexenoestrogen bisphenol-A in the female mouse genital tractrdquoBiology of Reproduction vol 72 no 6 pp 1344ndash1351 2005

[11] P Diel T Schulz K Smolnikar E Strunck G Vollmer andH Michna ldquoAbility of xeno- and phytoestrogens to modulateestrogen-sensitive genes in rat uterus estrogenicity profilesand uterotrophic activityrdquo Journal of Steroid Biochemistry andMolecular Biology vol 73 no 1-2 pp 1ndash10 2000

[12] G Schonfelder K Friedrich M Paul and I Chahoud ldquoDevel-opmental effects of prenatal exposure to bisphenol A on theuterus of rat offspringrdquo Neoplasia vol 6 no 5 pp 584ndash5942004

[13] D Seidlova-Wuttke H Jarry and W Wuttke ldquoPure estrogeniceffect of benzophenone-2 (BP2) but not of bisphenol a (BPA)and dibutylphtalate (DBP) in uterus vagina and bonerdquo Toxicol-ogy vol 205 no 1-2 pp 103ndash112 2004

[14] S Xiao H Diao M A Smith X Song and X Ye ldquoPreimplan-tation exposure to bisphenol A (BPA) affects embryo transportpreimplantation embryo development and uterine receptivityin micerdquo Reproductive Toxicology vol 32 no 4 pp 434ndash4412011

[15] T Kurosawa H Hiroi O Tsutsumi et al ldquoThe activity of bis-phenol A depends on both the estrogen receptor subtype andthe cell typerdquo Endocrine Journal vol 49 no 4 pp 465ndash4712002

[16] H Kabuto S Hasuike N Minagawa and T Shishibori ldquoEffectsof bisphenol A on the metabolisms of active oxygen species inmouse tissuesrdquo Environmental Research vol 93 no 1 pp 31ndash352003

[17] O O Erejuwa S A Sulaiman M S Wahab K N S Sira-judeen M S M D Salleh and S Gurtu ldquoAntioxidant pro-tection of Malaysian tualang honey in pancreas of normal andstreptozotocin-induced diabetic ratsrdquoAnnales drsquoEndocrinologievol 71 no 4 pp 291ndash296 2010

[18] S S M Zaid S A Sulaiman K N M Sirajudeen and N HOthman ldquoThe effects of Tualang honey on female reproductiveorgans tibia bone and hormonal profile in ovariectomisedratsmdashanimal model for menopauserdquo BMC Complementary andAlternative Medicine vol 10 article 82 2010

[19] S S Zaid S A Sulaiman N H Othman et al ldquoProtectiveeffects of Tualang honey on bone structure in experimentalpostmenopausal ratsrdquo Clinics vol 67 no 7 pp 779ndash784 2012


[20] M Mohamed S A Sulaiman H Jaafar and K N SalamldquoAntioxidant protective effect of honey in cigarette smoke-induced testicular damage in ratsrdquo International Journal ofMolecular Sciences vol 12 no 9 pp 5508ndash5521 2011

[21] A A Ghashm N H Othman M N Khattak N M Ismailand R Saini ldquoAntiproliferative effect of Tualang honey on oralsquamous cell carcinoma and osteosarcoma cell linesrdquo BMCComplementary and Alternative Medicine vol 10 article 492010

[22] S N S Mohamad S H Ahmad and H G Siew ldquoAntipro-liferative effect of methanolic extraction of Tualang honey onhuman keloid fibroblastsrdquo BMC Complementary and Alterna-tive Medicine vol 11 no 82 pp 1ndash8 2011

[23] S Ahmed and N H Othman ldquoReview of the medicinal effectsof tualang honey and a comparison with Manuka honeyrdquoMalaysian Journal of Medical Sciences vol 20 no 3 pp 6ndash132013

[24] M Mahaneem K N S Sirajudeen M Swamy and S YNik ldquoStudies on antioxidant properties of Tualang honeyrdquoAfrican Journal of Traditional Complementary and AlternativeMedicines vol 7 no 1 pp 59ndash63 2010

[25] M I Khalil N AlamMMoniruzzaman S A Sulaiman and SH Gan ldquohenolic acid composition and antioxidant propertiesof Malaysian honeysrdquo Journal of Food Science vol 76 no 6 ppC921ndashC928 2011

[26] S Anjum S Rahman M Kaur et al ldquoMelatonin amelioratesbisphenol A-induced biochemical toxicity in testicular mito-chondria of mouserdquo Food and Chemical Toxicology vol 49 no11 pp 2849ndash2854 2011

[27] K Okuda M Takiguchi and S Yoshihara ldquoIn vivo estrogenicpotential of 4-methyl-24-bis(4-hydroxyphenyl)pent-1-ene anactive metabolite of bisphenol A in uterus of ovariectomizedratrdquo Toxicology Letters vol 197 no 1 pp 7ndash11 2010

[28] Y Li W Zhang J Liu et al ldquoPrepubertal bisphenol A exposureinterferes with ovarian follicle development and its relevantgene expressionrdquo Reproductive Toxicology vol 44 pp 33ndash402014

[29] H H Draper andM Hadley ldquoMalondialdehyde determinationas index of lipid peroxidationrdquoMethods in Enzymology vol 186pp 421ndash431 1990

[30] K J Livak and T D Schmittgen ldquoAnalysis of relative geneexpression data using real-time quantitative PCRand the 2minusΔΔ119862TmethodrdquoMethods vol 25 no 4 pp 402ndash408 2001

[31] E Spreafico E Bettini G Pollio and A Maggi ldquoNucleotidesequence of estrogen receptor cDNA fromSprague-Dawley ratrdquoEuropean Journal of Pharmacology Molecular Pharmacologyvol 227 no 3 pp 353ndash356 1992

[32] G G J M Kuiper E Enmark M Pelto-Huikko S Nilssonand J-A Gustafsson ldquoCloning of a novel estrogen receptorexpressed in rat prostate and ovaryrdquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 93 no12 pp 5925ndash5930 1996

[33] Y Misumi M Sohda and Y Ikehara ldquoNucleotide and deducedamino acid sequence of rat complement C3rdquo Nucleic AcidsResearch vol 18 no 8 p 2178 1990

[34] G G J M Kuiper B Carlsson K Grandien et al ldquoComparisonof the ligand binding specificity and transcript tissue distribu-tion of estrogen receptors alpha and betardquo Endocrinology vol138 no 3 pp 863ndash870 1997

[35] B Yi H Kasai H-S Lee Y Kang J Y Park and M YangldquoInhibition by wheat sprout (Triticum aestivum) juice of bisphe-nol A-induced oxidative stress in young womenrdquo Mutation

ResearchGenetic Toxicology and Environmental Mutagenesisvol 724 no 1-2 pp 64ndash68 2011

[36] M JWolfsegger T Jaki BDietrich J AKunzler andK BarkerldquoA note on statistical analysis of organ weights in non-clinicaltoxicological studiesrdquo Toxicology and Applied Pharmacologyvol 240 no 1 pp 117ndash122 2009

[37] J Ashby H Tinwell P A Lefevre R Joiner and J HasemanldquoThe effect on sperm production in adult Sprague-Dawley ratsexposed by gavage to bisphenol A between postnatal days 91ndash97rdquo Toxicological Sciences vol 74 no 1 pp 129ndash138 2003

[38] C M Markey M A Coombs C Sonnenschein and A MSoto ldquoMammalian development in a changing environmentexposure to endocrine disruptors reveals the developmentalplasticity of steroid-hormone target organsrdquo Evolution andDevelopment vol 5 no 1 pp 67ndash75 2003

[39] B S Rubin M K Murray D A Damassa J C King and AM Soto ldquoPerinatal exposure to low doses of bisphenol A affectsbodyweight patterns of estrous cyclicity and plasma LH levelsrdquoEnvironmental Health Perspectives vol 109 no 7 pp 675ndash6802001

[40] K L Howdeshell A K Hotchkiss K A Thayer J G Vanden-bergh and F S Vom Saal ldquoEnvironmental toxins exposure tobisphenol A advances pubertyrdquo Nature vol 401 no 6755 pp763ndash764 1999

[41] M Guney B Oral H Demirin et al ldquoEvaluation of caspase-dependent apoptosis during methyl parathion-induced endo-metrial damage in rats ameliorating effect of vitamins E andCrdquo Environmental Toxicology and Pharmacology vol 23 no 2pp 221ndash227 2007

[42] R Aslan M R Sekeroglu M Tarakcioglu and H KoyluldquoInvestigation of malondialdehyde formation and antioxidantenzyme activity in stored bloodrdquo Haematologia vol 28 no 4pp 233ndash237 1997

[43] T Suzuki N Sugino T Fukaya et al ldquoSuperoxide dismutase innormal cycling human ovaries immunohistochemical localiza-tion and characterizationrdquo Fertility and Sterility vol 72 no 4pp 720ndash726 1999

[44] M Jozwik S Wolczynski M Jozwik and M SzamatowiczldquoOxidative stress markers in preovulatory follicular fluid inhumansrdquoMolecular Human Reproduction vol 5 no 5 pp 409ndash413 1999

[45] YWang J Goldberg R K Sharma A Agarwal and T FalconeldquoImportance of reactive oxygen species in the peritoneal fluidof women with endometriosis or idiopathic infertilityrdquo Fertilityand Sterility vol 68 no 5 pp 826ndash830 1997

[46] G Polak M Koziol-Montewka M Gogacz I Blaszkowskaand J Kotarski ldquoTotal antioxidant status of peritoneal fluid ininfertile womenrdquoEuropean Journal of Obstetrics Gynecology andReproductive Biology vol 94 no 2 pp 261ndash263 2001

[47] N Shafin Z Othman R Zakaria and N H Nik HussainldquoTualang honey supplementation reduces blood oxidative stresslevelsactivities in postmenopausal womenrdquo ISRN OxidativeMedicine vol 2014 Article ID 364836 4 pages 2014

[48] A Atkinson and D Roy ldquoIn vivo DNA adduct formation bybisphenol Ardquo Environmental and Molecular Mutagenesis vol26 no 1 pp 60ndash66 1995

[49] J S Edmonds M Nomachi M Terasaki M Morita B WSkelton and A H White ldquoThe reaction of bisphenol A 34-quinone with DNArdquo Biochemical and Biophysical ResearchCommunications vol 319 no 2 pp 556ndash561 2004

[50] R K Kishore A S Halim M S N Syazana and K N SSirajudeen ldquoTualang honey has higher phenolic content and


greater radical scavenging activity compared with other honeysourcesrdquo Nutrition Research vol 31 no 1 pp 322ndash325 2011

[51] B Oral M Guney H Demirin et al ldquoEndometrial damage andapoptosis in rats induced by dichlorvos and ameliorating effectof antioxidant vitamins E and Crdquo Reproductive Toxicology vol22 no 4 pp 783ndash790 2006

[52] S S M Zaid S Othman and N M Kassim ldquoPotentialprotective effect of Tualang honey on BPA-induced ovarian tox-icity in prepubertal ratrdquo BMC Complementary and AlternativeMedicine vol 14 article 509 12 pages 2014

[53] H W G Baker J Brindle D S Irvine and R J AitkenldquoProtective effect of antioxidants on the impairment of spermmotility by activated polymorphonuclear leukocytesrdquo Fertilityand Sterility vol 65 no 2 pp 411ndash419 1996

[54] V Kumar A Rani A K Dixit D Pratap and D BhatnagarldquoA comparative assessment of total phenolic content ferricreducing-anti-oxidative power free radical-scavenging activityvitamin C and isoflavones content in soybean with varying seedcoat colourrdquo Food Research International vol 43 no 1 pp 323ndash328 2010

[55] A S A Harabawy and Y Y I Mosleh ldquoThe role of vitaminsA C E and selenium as antioxidants against genotoxicity andcytotoxicity of cadmium copper lead and zinc on erythrocytesof Nile tilapia Oreochromis niloticusrdquo Ecotoxicology and Envi-ronmental Safety vol 104 no 1 pp 28ndash35 2014

[56] A S Y Hounkpatin R C Johnson P Guedenon et alldquoProtective effects of vitamin C on haematological parametersin intoxicated wistar rats with cadmium mercury and com-bined cadmium andmercuryrdquo International Research Journal ofBiological Sciences vol 1 no 8 pp 76ndash81 2012

[57] M Shaban El-Neweshy and Y Said El-Sayed ldquoInfluence ofvitamin C supplementation on lead-induced histopathologicalalterations in male ratsrdquo Experimental and Toxicologic Pathol-ogy vol 63 no 3 pp 221ndash227 2011

[58] A Wattel S Kamel R Mentaverri et al ldquoPotent inhibitoryeffect of naturally occurring flavonoids quercetin and kaemp-ferol on in vitro osteoclastic bone resorptionrdquo BiochemicalPharmacology vol 65 no 1 pp 35ndash42 2003

[59] M Chevallet E Wagner S Luche A Van Dorsselaer E Leize-Wagner and T Rabilloud ldquoRegeneration of peroxiredoxinsduring recovery after oxidative stress only some overoxidizedperoxiredoxins can be reduced during recovery after oxidativestressrdquo Journal of Biological Chemistry vol 278 no 39 pp37146ndash37153 2003

[60] L Vigezzi V L Bosquiazzo L Kass J G RamosMMunoz-de-Toro and E H Luque ldquoDevelopmental exposure to bisphenolA alters the differentiation and functional response of the adultrat uterus to estrogen treatmentrdquo Reproductive Toxicology vol52 no 1 pp 83ndash92 2015

[61] C M Klinge ldquoEstrogen receptor interaction with estrogenresponse elementsrdquo Nucleic Acids Research vol 29 no 14 pp2905ndash2919 2001

[62] H Hiroi O Tsutsumi M Momoeda Y Takai Y Osuga and YTaketani ldquoDifferential interactions of bisphenol A and 17beta-estradiol with estrogen receptor alpha (ERalpha) and ERbetardquoEndocrine Journal vol 46 no 6 pp 773ndash778 1999

[63] J F Couse and K S Korach ldquoEstrogen receptor null micewhat have we learned and where will they lead usrdquo EndocrineReviews vol 20 no 3 pp 358ndash417 1999

[64] P Pace J Taylor S Suntharalingam R C Coombes and S AlildquoHuman estrogen receptor 120573 binds DNA in a manner similar

to and dimerizes with estrogen receptor 120572rdquo Journal of BiologicalChemistry vol 272 no 41 pp 25832ndash25838 1997

[65] K Paech P Webb G G J M Kuiper et al ldquoDifferential ligandactivation of estrogen receptors ER120572 and ER120573 at AP1 sitesrdquoScience vol 277 no 5331 pp 1508ndash1510 1997

[66] J J VoegelM J SHeine C Zechel P Chambon andHGrone-meyer ldquoTIF2 a 160 kDa transcriptionalmediator for the ligand-dependent activation function AF-2 of nuclear receptorsrdquo TheEMBO Journal vol 15 no 14 pp 3667ndash3675 1996

[67] E J Routledge R White M G Parker and J P Sumpter ldquoDif-ferential effects of xenoestrogens on coactivator recruitmentby estrogen receptor (ER) 120572 and ER120573rdquo Journal of BiologicalChemistry vol 275 no 46 pp 35986ndash35993 2000

[68] Z Weihua S Saji S Makinen et al ldquoEstrogen receptor (ER)beta a modulator of ERalpha in the uterusrdquo Proceedings of theNational Academy of Sciences of the United States of Americavol 97 no 11 pp 5936ndash5941 2000

[69] J M Hall and D P McDonnel ldquoThe estrogen receptor 120573-isoform (ER120573) of the human estrogen receptor modulates ER120572transcriptional activity and is a key regulator of the cellularresponse to estrogens and antiestrogensrdquo Endocrinology vol140 no 12 pp 5566ndash5578 1999

[70] K Pettersson K Grandien G G J M Kuiper and J-A Gustafsson ldquoMouse estrogen receptor 120573 forms estrogenresponse element-binding heterodimers with estrogen receptor120572rdquoMolecular Endocrinology vol 11 no 10 pp 1486ndash1496 1997

[71] A Matsushima Y Kakuta T Teramoto et al ldquoStructural evi-dence for endocrine disruptor bisphenol A binding to humannuclear receptor ERR120574rdquo Journal of Biochemistry vol 142 no 4pp 517ndash524 2007

[72] T Nose and Y Shimohigashi ldquoA docking modelling rationallypredicts strong binding of bisphenol A to estrogen-relatedreceptor 120574rdquo Protein and Peptide Letters vol 15 no 3 pp 290ndash296 2008

[73] V Giguere ldquoTo ERR in the estrogen pathwayrdquo Trends inEndocrinology andMetabolism vol 13 no 5 pp 220ndash225 2002

[74] S Takayanagi T Tokunaga X Liu H Okada A Mat-sushima and Y Shimohigashi ldquoEndocrine disruptor bisphenolA strongly binds to human estrogen-related receptor gamma(ERRgamma)with high constitutive activityrdquoToxicology Lettersvol 167 no 2 pp 95ndash105 2006

[75] J Huppunen and P Aarnisalo ldquoDimerization modulates theactivity of the orphan nuclear receptor ERR120572rdquo Biochemical andBiophysical Research Communications vol 314 no 4 pp 964ndash970 2004

[76] A Sahu and J D Lambris ldquoStructure and biology of comple-ment proteinC3 a connecting link between innate and acquiredimmunityrdquo Immunological Reviews vol 180 no 1 pp 35ndash482001

[77] S S Zaid S Othman and N M Kassim ldquoPotential protectiveeffect of Tualang honey on BPA-induced ovarian toxicity in pre-pubertal ratrdquo BMC Complementary and Alternative Medicinevol 14 article 509 2014

[78] M G L Hertog P C H Hollman M B Katan and DKromhout ldquoIntake of potentially anticarcinogenic flavonoidsand their determinants in adults inThe NetherlandsrdquoNutritionand Cancer vol 20 no 1 pp 21ndash29 1993

[79] D Tiwari J Kamble S Chilgunde et al ldquoClastogenic andmuta-genic effects of bisphenol A an endocrine disruptorrdquoMutationResearch Genetic Toxicology and Environmental Mutagenesisvol 743 no 1-2 pp 83ndash90 2012


[80] T Iso TWatanabe T Iwamoto A Shimamoto and Y FuruichildquoDNA damage caused by bisphenol A and estradiol throughestrogenic activityrdquo Biological and Pharmaceutical Bulletin vol29 no 2 pp 206ndash210 2006

[81] H-J Wu C Liu W-X Duan et al ldquoMelatonin amelioratesbisphenol A-induced DNA damage in the germ cells of adultmale ratsrdquoMutation Research vol 752 no 1-2 pp 57ndash67 2013

[82] N S Yaacob and N F Ismail ldquoComparison of cytotoxicityand genotoxicity of 4-hydroxytamoxifen in combination withTualang honey in MCF-7 and MCF-10A cellsrdquo BMC Comple-mentary and Alternative Medicine vol 14 article 106 2014

[83] I Ahmad H Jimenez N S Yaacob and N Yusuf ldquoTualanghoney protects keratinocytes from ultraviolet radiation-induced inflammation and DNA damagerdquo Photochemistry andPhotobiology vol 88 no 5 pp 1198ndash1204 2012

[84] N S Yaacob A Nengsih and M N Norazmi ldquoTualanghoney promotes apoptotic cell death induced by tamoxifen inbreast cancer cell linesrdquo Evidence-Based Complementary andAlternative Medicine vol 2013 Article ID 989841 9 pages 2013

[85] J Zhou P Li N Cheng et al ldquoProtective effects of buckwheathoney onDNAdamage induced by hydroxyl radicalsrdquo Food andChemical Toxicology vol 50 no 8 pp 2766ndash2773 2012

[86] H M Habib F T Al Meqbali H Kamal U D Souka andW H Ibrahim ldquoBioactive components antioxidant and DNAdamage inhibitory activities of honeys from arid regionsrdquo FoodChemistry vol 153 pp 28ndash34 2014

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


in female rats [14] Furthermore BPA has been reportedto promote oxidative stress and inflammation in the femalereproductive tract whichmight contribute to themorpholog-ical and functional abnormalities [15]

Natural compounds with antioxidant properties havebeen extensively studied as a means to counter disease-associated oxidative stress [16] With these concerns in mindwe used a natural product with high antioxidant contentnamely Tualang honey (Agromas Malaysia) as a possiblepotential therapeutic agent to counter the deleterious effectsof BPA Previous scientific studies have claimed that Tualanghoney has the capability to ameliorate oxidative stress inrenal and pancreas of streptozotocin-induced diabetic rats[17] reverse atrophic uterus and vagina [18] and to improveosteoporotic bone [19] in postmenopausal animal model Inaddition Tualang honey has the capability to protect againstcigarette-induced damage on testis [20] and to counter theproliferation of oral squamous cell carcinomas (OSCC) [21]human osteosarcoma [21] and keloid fibroblasts [22]

Tualang honey a dark brownish honey has been reportedto containmore than 200 substances including sugars aminoacids vitamins minerals enzymes organic acids and phyto-chemicals The primary content of Tualang honey is invertedsugars that consist of fructose 296 glucose 30 sucrose06 and maltose 79 as well as a small amount of complexmixture of other saccharides (disaccharides trisaccharidesand oligosaccharides) [23] It also contains high total pheno-lic compounds (gallic syringic benzoic trans-cinnamic p-coumaric and caffeic acids) and flavonoids (catechin kaemp-ferol naringenin luteolin and apigenin) [24] It has tremen-dous antioxidant properties with high radical scavengingand antioxidant activities [25]The other compounds presentin Tualang honey are stearic acids furfural acetic acid 2-furylmethylketone palmitic acid ethyl linoleate oleic acid 2-hyroxy-2-cyclopenten-1-one hyacinthine and 5-methyl fur-fural [23]

The aims of our studywere to investigate the effects of sub-chronic administration of BPA on the uterine morphologyand function by determining the lipid peroxidation proteindistribution and mRNA expressions of estrogen receptor-120572 estrogen receptor-120573 and complement C3 Consequentlyinvestigation of the possible protective effects of Tualanghoney against BPA-induced uterine toxicity is executed

2 Material and Methods

21 Tualang Honey (Agromas Malaysia) Tualang honey waspurchased from the Federal Agricultural Marketing Author-ity (FAMA) under the Ministry of Agriculture and Agro-Based Industry Malaysia Tualang honey is wild multifloralhoney collected from Apis dorsatarsquos beehive that is built ona giant tree Koompassia excelsa (locally known as Tualangtree) in the rain forest of Kedah Malaysia At Honey Proces-singCentre inKualaNerang Kedah the honeywas processedthrough several stages (quality inspection dehydration pack-aging and labeling) In brief the honeywas filtered to removesolid particles concentrated in an oven at 40∘C and subjectedto 120574 irradiation at 25 kGy at Sterilgamma (M) Sdn Bhd

(Selangor Malaysia) The water concentration of the honeywas standardized by FAMA at 18

22 AnimalModel and Experimental Design Female Spraguerats (P21) were obtained from theAnimalHusbandry Facultyof Medicine University of Malaya All the experimentaldesign and procedures were conducted according to theNational Institutes of Health guide for the care and use ofLaboratory animals (NIH Publications number 8023 revised1978) which has been approved by the Animal Care andCommittee (ACUC) of the University ofMalayaThroughoutthe experimental period the rats were maintained underthe standard laboratory conditions (temperature 25 plusmn 2∘C50 plusmn 15 relative humidity and normal photoperiod of12 h dark and 12 h light) and supplied ad libitum with waterand commercial pellet diet (Gold Coin Feedmills Pte LtdMalaysia) The rats were placed in stainless steel cages withwood bedding and water was supplied in glass bottles tominimize additional exposures endocrine disruptors Theywere acclimatized to the laboratory environment for sevendays prior to the commencement of the experiment At 28days of age the rats were randomly divided into four groups(119899 = 8 in each group)

NC group (negative control) was administered with thevehicle (02mL of corn oil) PC group (positive control) wasadministered with BPA (10mgkg body weight suspended incorn oil) THC group (Tualang honey control) was admin-istered with Tualang honey (200mgkg body weight 30minbefore administration of corn oil) TH group (Tualang honey+ BPA) was administered with Tualang honey (200mgkgbodyweight 30min before administration of BPA at 10mgkgbody weight) The procedure was performed in the morning(between 0900 and 1000 AM) once daily by oral gavage(to mimic the most likely route of human exposure) for sixconsecutive weeks Throughout the administration perioddaily body weight was recorded After the last treatment therats were sacrificed during their diestrous phase

Once the rat was sacrificed the wet weight of the wholeuterus was recorded for uterotrophic response evaluationThe left horn of the uterus was immediately fixed in 10buffered formalin for histopathological analysis One half ofthe right horn of uterus was kept in phosphate buffer formalondialdehyde (MDA) determination while the other halfwas kept in RNAlater for mRNA extraction and subsequentlystored in minus80∘C freezer until further analysisThe dose selec-tion of BPA at 10mgg body weight was based on previousstudies where BPA at this dose was reported to induce disrup-tion on themorphological and biochemical parameters in thereproductive system [3 26ndash28] As for Tualang honey it wasfreshly prepared daily to avoid oxidation of the antioxidantscontentThe dose of Tualang honey at 200mgkg bodyweightwas based on previous study that showed positive biologicaleffects on female reproductive organs and the dose was equalto one tablespoonwhich is routinely taken by an adult human[18]

23 Histopathological Evaluation Theuteri were fixed in 10buffered formalin for 24 hours prior to further processingfor histopathological examination The uteri were trimmed




















260280while the







3 Results








weight)



(120583m)












0

20

40

60

80

100

120

140

()

173088 1655

THNC PC THC(a)

bbbb

0

20

40

60

80

100

120

()

PC THC THNC

aaa c dd(minus2539)


(b)













bbb

aaa c ddd

bbb

b

(minus30)

0

20

40

60

80

100

120

140

160

()

PC THC THNC

(244)

(497)

c


119875 lt 005 bbb119875 lt

















bbb c

aaa cc ddd

bbb

a bbb

80

85

90

95

100

105(

)

(minus3811)

(minus1196)

(minus747)

PC THC THNC(a)

bbaa cc ddd

bbb


0

20

40

60

80

100

120

140

()

PC THC THNC

(041)

(b)

bb

aa c dd

bbbb

(minus2366)

0

20

40

60

80

100

120

140

()

PC THC THNC

(003)(minus490)

(c)

















(A) (B) (C)



(D) (E) (F)



(G) (H) (I)



(J) (K) (L)





(A) (B)



(C) (D)



(E) (F)



(H)(G)



(J)(I)





(A) (B)



(C) (D)



(E) (F)



(G) (H)



(I) (J)




(A) (B)



(C) (D)



(E) (F)



(G) (H)



(I) (J)





bb d

aa c ddd

b dd

a bbb cc

PC THC THNC0

02

04

06

08

1

12

14

16

18Re

lativ

e mRN

A ex

pres

sion

(a)

bbb ccc

aaa ccc ddd

aaa bbbbbb cc

0

02

04

06

08

1

12

14

16

Relat

ive m

RNA

expr

essio

n

PC THC THNC(b)

bbb cc

aaa ccc ddd

aaa bbb ddd

bbb

0

02

04

06

08

1

12

14

16

18

2

Relat

ive m

RNA

expr

essio

n

PC THC THNC(c)









119875 lt 001 and ccc119875 lt 0001







4 Discussion























5 Conclusions




Abbreviations




Acknowledgments


References



































































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of



































260280while the







3 Results








weight)



(120583m)












0

20

40

60

80

100

120

140

()

173088 1655

THNC PC THC(a)

bbbb

0

20

40

60

80

100

120

()

PC THC THNC

aaa c dd(minus2539)


(b)













bbb

aaa c ddd

bbb

b

(minus30)

0

20

40

60

80

100

120

140

160

()

PC THC THNC

(244)

(497)

c


119875 lt 005 bbb119875 lt

















bbb c

aaa cc ddd

bbb

a bbb

80

85

90

95

100

105(

)

(minus3811)

(minus1196)

(minus747)

PC THC THNC(a)

bbaa cc ddd

bbb


0

20

40

60

80

100

120

140

()

PC THC THNC

(041)

(b)

bb

aa c dd

bbbb

(minus2366)

0

20

40

60

80

100

120

140

()

PC THC THNC

(003)(minus490)

(c)

















(A) (B) (C)



(D) (E) (F)



(G) (H) (I)



(J) (K) (L)





(A) (B)



(C) (D)



(E) (F)



(H)(G)



(J)(I)





(A) (B)



(C) (D)



(E) (F)



(G) (H)



(I) (J)




(A) (B)



(C) (D)



(E) (F)



(G) (H)



(I) (J)





bb d

aa c ddd

b dd

a bbb cc

PC THC THNC0

02

04

06

08

1

12

14

16

18Re

lativ

e mRN

A ex

pres

sion

(a)

bbb ccc

aaa ccc ddd

aaa bbbbbb cc

0

02

04

06

08

1

12

14

16

Relat

ive m

RNA

expr

essio

n

PC THC THNC(b)

bbb cc

aaa ccc ddd

aaa bbb ddd

bbb

0

02

04

06

08

1

12

14

16

18

2

Relat

ive m

RNA

expr

essio

n

PC THC THNC(c)









119875 lt 001 and ccc119875 lt 0001







4 Discussion























5 Conclusions




Abbreviations




Acknowledgments


References



































































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of























260280while the







3 Results








weight)



(120583m)












0

20

40

60

80

100

120

140

()

173088 1655

THNC PC THC(a)

bbbb

0

20

40

60

80

100

120

()

PC THC THNC

aaa c dd(minus2539)


(b)













bbb

aaa c ddd

bbb

b

(minus30)

0

20

40

60

80

100

120

140

160

()

PC THC THNC

(244)

(497)

c


119875 lt 005 bbb119875 lt

















bbb c

aaa cc ddd

bbb

a bbb

80

85

90

95

100

105(

)

(minus3811)

(minus1196)

(minus747)

PC THC THNC(a)

bbaa cc ddd

bbb


0

20

40

60

80

100

120

140

()

PC THC THNC

(041)

(b)

bb

aa c dd

bbbb

(minus2366)

0

20

40

60

80

100

120

140

()

PC THC THNC

(003)(minus490)

(c)

















(A) (B) (C)



(D) (E) (F)



(G) (H) (I)



(J) (K) (L)





(A) (B)



(C) (D)



(E) (F)



(H)(G)



(J)(I)





(A) (B)



(C) (D)



(E) (F)



(G) (H)



(I) (J)




(A) (B)



(C) (D)



(E) (F)



(G) (H)



(I) (J)





bb d

aa c ddd

b dd

a bbb cc

PC THC THNC0

02

04

06

08

1

12

14

16

18Re

lativ

e mRN

A ex

pres

sion

(a)

bbb ccc

aaa ccc ddd

aaa bbbbbb cc

0

02

04

06

08

1

12

14

16

Relat

ive m

RNA

expr

essio

n

PC THC THNC(b)

bbb cc

aaa ccc ddd

aaa bbb ddd

bbb

0

02

04

06

08

1

12

14

16

18

2

Relat

ive m

RNA

expr

essio

n

PC THC THNC(c)









119875 lt 001 and ccc119875 lt 0001







4 Discussion























5 Conclusions




Abbreviations




Acknowledgments


References



































































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of




















weight)



(120583m)












0

20

40

60

80

100

120

140

()

173088 1655

THNC PC THC(a)

bbbb

0

20

40

60

80

100

120

()

PC THC THNC

aaa c dd(minus2539)


(b)













bbb

aaa c ddd

bbb

b

(minus30)

0

20

40

60

80

100

120

140

160

()

PC THC THNC

(244)

(497)

c


119875 lt 005 bbb119875 lt

















bbb c

aaa cc ddd

bbb

a bbb

80

85

90

95

100

105(

)

(minus3811)

(minus1196)

(minus747)

PC THC THNC(a)

bbaa cc ddd

bbb


0

20

40

60

80

100

120

140

()

PC THC THNC

(041)

(b)

bb

aa c dd

bbbb

(minus2366)

0

20

40

60

80

100

120

140

()

PC THC THNC

(003)(minus490)

(c)

















(A) (B) (C)



(D) (E) (F)



(G) (H) (I)



(J) (K) (L)





(A) (B)



(C) (D)



(E) (F)



(H)(G)



(J)(I)





(A) (B)



(C) (D)



(E) (F)



(G) (H)



(I) (J)




(A) (B)



(C) (D)



(E) (F)



(G) (H)



(I) (J)





bb d

aa c ddd

b dd

a bbb cc

PC THC THNC0

02

04

06

08

1

12

14

16

18Re

lativ

e mRN

A ex

pres

sion

(a)

bbb ccc

aaa ccc ddd

aaa bbbbbb cc

0

02

04

06

08

1

12

14

16

Relat

ive m

RNA

expr

essio

n

PC THC THNC(b)

bbb cc

aaa ccc ddd

aaa bbb ddd

bbb

0

02

04

06

08

1

12

14

16

18

2

Relat

ive m

RNA

expr

essio

n

PC THC THNC(c)









119875 lt 001 and ccc119875 lt 0001







4 Discussion























5 Conclusions




Abbreviations




Acknowledgments


References



































































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















bbb

aaa c ddd

bbb

b

(minus30)

0

20

40

60

80

100

120

140

160

()

PC THC THNC

(244)

(497)

c


119875 lt 005 bbb119875 lt

















bbb c

aaa cc ddd

bbb

a bbb

80

85

90

95

100

105(

)

(minus3811)

(minus1196)

(minus747)

PC THC THNC(a)

bbaa cc ddd

bbb


0

20

40

60

80

100

120

140

()

PC THC THNC

(041)

(b)

bb

aa c dd

bbbb

(minus2366)

0

20

40

60

80

100

120

140

()

PC THC THNC

(003)(minus490)

(c)

















(A) (B) (C)



(D) (E) (F)



(G) (H) (I)



(J) (K) (L)





(A) (B)



(C) (D)



(E) (F)



(H)(G)



(J)(I)





(A) (B)



(C) (D)



(E) (F)



(G) (H)



(I) (J)




(A) (B)



(C) (D)



(E) (F)



(G) (H)



(I) (J)





bb d

aa c ddd

b dd

a bbb cc

PC THC THNC0

02

04

06

08

1

12

14

16

18Re

lativ

e mRN

A ex

pres

sion

(a)

bbb ccc

aaa ccc ddd

aaa bbbbbb cc

0

02

04

06

08

1

12

14

16

Relat

ive m

RNA

expr

essio

n

PC THC THNC(b)

bbb cc

aaa ccc ddd

aaa bbb ddd

bbb

0

02

04

06

08

1

12

14

16

18

2

Relat

ive m

RNA

expr

essio

n

PC THC THNC(c)









119875 lt 001 and ccc119875 lt 0001







4 Discussion























5 Conclusions




Abbreviations




Acknowledgments


References



































































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















bbb c

aaa cc ddd

bbb

a bbb

80

85

90

95

100

105(

)

(minus3811)

(minus1196)

(minus747)

PC THC THNC(a)

bbaa cc ddd

bbb


0

20

40

60

80

100

120

140

()

PC THC THNC

(041)

(b)

bb

aa c dd

bbbb

(minus2366)

0

20

40

60

80

100

120

140

()

PC THC THNC

(003)(minus490)

(c)

















(A) (B) (C)



(D) (E) (F)



(G) (H) (I)



(J) (K) (L)





(A) (B)



(C) (D)



(E) (F)



(H)(G)



(J)(I)





(A) (B)



(C) (D)



(E) (F)



(G) (H)



(I) (J)




(A) (B)



(C) (D)



(E) (F)



(G) (H)



(I) (J)





bb d

aa c ddd

b dd

a bbb cc

PC THC THNC0

02

04

06

08

1

12

14

16

18Re

lativ

e mRN

A ex

pres

sion

(a)

bbb ccc

aaa ccc ddd

aaa bbbbbb cc

0

02

04

06

08

1

12

14

16

Relat

ive m

RNA

expr

essio

n

PC THC THNC(b)

bbb cc

aaa ccc ddd

aaa bbb ddd

bbb

0

02

04

06

08

1

12

14

16

18

2

Relat

ive m

RNA

expr

essio

n

PC THC THNC(c)









119875 lt 001 and ccc119875 lt 0001







4 Discussion























5 Conclusions




Abbreviations




Acknowledgments


References



































































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


















(A) (B) (C)



(D) (E) (F)



(G) (H) (I)



(J) (K) (L)





(A) (B)



(C) (D)



(E) (F)



(H)(G)



(J)(I)





(A) (B)



(C) (D)



(E) (F)



(G) (H)



(I) (J)




(A) (B)



(C) (D)



(E) (F)



(G) (H)



(I) (J)





bb d

aa c ddd

b dd

a bbb cc

PC THC THNC0

02

04

06

08

1

12

14

16

18Re

lativ

e mRN

A ex

pres

sion

(a)

bbb ccc

aaa ccc ddd

aaa bbbbbb cc

0

02

04

06

08

1

12

14

16

Relat

ive m

RNA

expr

essio

n

PC THC THNC(b)

bbb cc

aaa ccc ddd

aaa bbb ddd

bbb

0

02

04

06

08

1

12

14

16

18

2

Relat

ive m

RNA

expr

essio

n

PC THC THNC(c)









119875 lt 001 and ccc119875 lt 0001







4 Discussion























5 Conclusions




Abbreviations




Acknowledgments


References



































































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


















(A) (B)



(C) (D)



(E) (F)



(H)(G)



(J)(I)





(A) (B)



(C) (D)



(E) (F)



(G) (H)



(I) (J)




(A) (B)



(C) (D)



(E) (F)



(G) (H)



(I) (J)





bb d

aa c ddd

b dd

a bbb cc

PC THC THNC0

02

04

06

08

1

12

14

16

18Re

lativ

e mRN

A ex

pres

sion

(a)

bbb ccc

aaa ccc ddd

aaa bbbbbb cc

0

02

04

06

08

1

12

14

16

Relat

ive m

RNA

expr

essio

n

PC THC THNC(b)

bbb cc

aaa ccc ddd

aaa bbb ddd

bbb

0

02

04

06

08

1

12

14

16

18

2

Relat

ive m

RNA

expr

essio

n

PC THC THNC(c)









119875 lt 001 and ccc119875 lt 0001







4 Discussion























5 Conclusions




Abbreviations




Acknowledgments


References



































































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


















(A) (B)



(C) (D)



(E) (F)



(G) (H)



(I) (J)




(A) (B)



(C) (D)



(E) (F)



(G) (H)



(I) (J)





bb d

aa c ddd

b dd

a bbb cc

PC THC THNC0

02

04

06

08

1

12

14

16

18Re

lativ

e mRN

A ex

pres

sion

(a)

bbb ccc

aaa ccc ddd

aaa bbbbbb cc

0

02

04

06

08

1

12

14

16

Relat

ive m

RNA

expr

essio

n

PC THC THNC(b)

bbb cc

aaa ccc ddd

aaa bbb ddd

bbb

0

02

04

06

08

1

12

14

16

18

2

Relat

ive m

RNA

expr

essio

n

PC THC THNC(c)









119875 lt 001 and ccc119875 lt 0001







4 Discussion























5 Conclusions




Abbreviations




Acknowledgments


References



































































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















(A) (B)



(C) (D)



(E) (F)



(G) (H)



(I) (J)





bb d

aa c ddd

b dd

a bbb cc

PC THC THNC0

02

04

06

08

1

12

14

16

18Re

lativ

e mRN

A ex

pres

sion

(a)

bbb ccc

aaa ccc ddd

aaa bbbbbb cc

0

02

04

06

08

1

12

14

16

Relat

ive m

RNA

expr

essio

n

PC THC THNC(b)

bbb cc

aaa ccc ddd

aaa bbb ddd

bbb

0

02

04

06

08

1

12

14

16

18

2

Relat

ive m

RNA

expr

essio

n

PC THC THNC(c)









119875 lt 001 and ccc119875 lt 0001







4 Discussion























5 Conclusions




Abbreviations




Acknowledgments


References



































































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















bb d

aa c ddd

b dd

a bbb cc

PC THC THNC0

02

04

06

08

1

12

14

16

18Re

lativ

e mRN

A ex

pres

sion

(a)

bbb ccc

aaa ccc ddd

aaa bbbbbb cc

0

02

04

06

08

1

12

14

16

Relat

ive m

RNA

expr

essio

n

PC THC THNC(b)

bbb cc

aaa ccc ddd

aaa bbb ddd

bbb

0

02

04

06

08

1

12

14

16

18

2

Relat

ive m

RNA

expr

essio

n

PC THC THNC(c)









119875 lt 001 and ccc119875 lt 0001







4 Discussion























5 Conclusions




Abbreviations




Acknowledgments


References



































































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of




































5 Conclusions




Abbreviations




Acknowledgments


References



































































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

























5 Conclusions




Abbreviations




Acknowledgments


References



































































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


















Abbreviations




Acknowledgments


References



































































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of






























































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of





























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of





























of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of





















of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















Documents

Research Article Tualang Honey Protects against BPA ... · PDF fileEvidence-Based Complementaryand AlternativeMedicine accordingly,dehydratedthroughagradedseriesofincreasing concentration