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Reproductive Toxicology 24 (2007) 259–264

Bisphenol-A and chlorinated derivatives in adipose tissue of women

M.F. Fernandez a,∗, J.P. Arrebola a, J. Taoufiki b, A. Navalon b,O. Ballesteros b, R. Pulgar a, J.L. Vilchez b, N. Olea a

a Laboratory of Medical Investigations, San Cecilio University Hospital,University of Granada, 18071 Granada, Spain

b Research Group of Analytical Chemistry and Life Sciences, Department of Analytical Chemistry,University of Granada, 18071 Granada, Spain

Received 1 March 2007; received in revised form 25 May 2007; accepted 11 June 2007Available online 26 June 2007

bstract

Bisphenol-A (BPA) and chlorinated derivatives (ClxBPA) were investigated in adipose tissue of women in Southeast Spain. BPA was above limitf detection (LOD) in 11 out of 20 samples (55%). Among ClxBPA, Cl2BPA was the most frequent (80%) and abundant, constituting 94.6% ofotal chlorinated BPA in adipose tissue. Mean ± S.D. of BPA, monochloro-BPA (ClBPA), dichloro-BPA (Cl2BPA), and trichloro-BPA (Cl3BPA)ere 5.83 ± 3.48, 3.05 ± 0.28, 9.21 ± 9.26, and 0.74 ± 0.15 ng/g of adipose tissue, respectively. No tetrachloro-BPA (Cl4BPA) was found aboveOD. There are no published data on BPA in human adipose tissue or on Cl BPA in adipose tissue or blood, limiting comparisons. BPA levels

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ere similar (w/w) to findings in blood (w/v) in other populations and below levels reported in placenta tissue (w/w). Because of the estrogenimicking effects of BPA and its ClxBPA, further research is needed to explore their combined effects on human health and trends in human

xposure.2007 Published by Elsevier Inc.

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eywords: Bisphenol-A; Bisphenol-A chlorinated derivatives; Adipose tissue;

. Introduction

In 2005, the world-wide production of bisphenol A [2,2-is(4-hydroxyphenyl)propane, BPA] was 3,200,000 tonnes/year1]. In Europe, four companies manufacture a total amount of00,000 tonnes/year of BPA at six production sites, with one fac-ory in Southern Spain producing more than 250,000 tonnes/year2,3].

Bisphenol-A is used as an intermediate for binding, plasticiz-ng, or hardening of plastics, paints/lacquers, binding materials,nd filling-in materials. BPA is also a substrate for the produc-ion of polycarbonate resins (71%) and epoxy resins (27%).urthermore, BPA is used as an additive for flame-retardants,rake fluids, and thermal papers. An historical growth in BPA

onsumption of 6.9–7% per year is driven primarily by heavyemand for polycarbonate resins [4,5]. Automotive applicationsccount for about 20% of total polycarbonate consumption, with

∗ Corresponding author. Tel.: +34 958 24 2864; fax: +34 958 24 9953.E-mail address: marieta@ugr.es (M.F. Fernandez).

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890-6238/$ – see front matter © 2007 Published by Elsevier Inc.oi:10.1016/j.reprotox.2007.06.007

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esins used in place of traditional materials such as metal andlass. Uses in glazing and sheet form, e.g., in construction andransportation, make up a further 20% of consumption. Opti-al media, including audio compact discs (CDs), CD-ROMs,ecordable CDs, and digital versatile disks (DVDs) account forrapidly growing proportion (currently 15–20%) of the poly-

arbonate market [6].This massive production implies the continuous emission

f BPA during its manufacture and from utilization of BPA-ontaining products [4,6]. Emissions during BPA productionave been estimated at around 2 tonnes/year to surface watersnd 1 ton to air. The most important emissions result from itsse in the manufacture of phenoplast cast resins (43 tonneso water in Europe), thermal paper (151 tonnes to water inurope) and PVC (25 tonnes to water in Europe). In sum-ary, total emissions in Europe are 2.1 tonnes to air, 199 tonnes

o water and 30 tonnes to soil. Emissions from products-in-

se are estimated at 160 kg from polycarbonates and <1 kgrom epoxy resins used in can lining. Additionally, losses fromVC articles in use are 20 tonnes to air and 30 tonnes to water4].

2 ctive Toxicology 24 (2007) 259–264

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Table 1Age and body mass index (BMI) corresponding to the 20 human adipose tissuesamples from Southeast Spain

Age (years) BMI (kg/m2)

1 69 36.02 81 19.13 70 30.44 58 22.95 76 30.56 62 37.07 57 32.48 53 31.19 49 23.810 74 25.311 62 33.612 69 27.013 70 31.314 24 21.315 50 28.116 43 >6517 74 26.918 41 59.819 50 30.820 62 24.8

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The release of BPA into the environment and its use in aariety of high-volume consumer products means that there areany possible routes of exposure for individuals in the general

opulation [7,8]: (i) direct and indirect environmental exposureue to its release during production, use, and disposal; (ii) con-act with or inhalation of non-food-contact consumer products;nd (iii) exposure via leaching into food.

In the environment, BPA has been reported in leachates fromaste landfills [9–12], with BPA levels ranging from 1.3 to7,200 ng/ml and a mean of 269 ng/ml [11]. Landfill effluents aremajor source of BPA contamination in the aquatic environmentecause BPA is moderately soluble (120–300 mg/l at pH 7) andas a low volatility [13]. BPA can also migrate from PVC hoseso water at room temperature and at neutral pH [14], makinghese plastic-to-water migrations a significant source of humanxposure via drinking water. Moreover, BPA that migrates toap water may react with residual chlorine in treated drinkingater. Hence, BPA chlorinated derivatives are by-products of the

eaction between BPA and free chlorine [15,16] with a complexiodegradation [17] and contributing to the inadvertent exposuref humans to bisphenols.

Despite its significant production, environmental release andidespread utilization, possibly higher than that of any other

ingle man-made chemical, BPA has not been subjected to anynvironmental legislative control. Non-food products are stillot regarded as significant sources of BPA exposure and themportance of environmental BPA exposure for the general pop-lation has yet to be established [18]. Thus, exposure fromental composites and sealants [19,20] remains controversial21].

Food is acknowledged to be the main source of exposureo bisphenols [22] as a consequence of BPA migration fromood containers. Epoxy resins produced with BPA are used asacquers to coat metal can surfaces in contact with foods and bev-rages, while polycarbonate plastics are used in food and drinkontainers. Many studies have reported BPA migration froman surface coatings or plastics into foods and food-simulatingiquids, especially at high temperatures and with repeated usef plastic products [23,24], and from wine vats [25], cannedrinks [26], and baby bottles [27]. In 2002, the European Foodafety Agency (EFSA) set a Specific Migration Limit (SML)f 3 mg bisphenol-A per kg food (3 ppm) for the protection ofU consumers. A recent re-evaluation of BPA [28] confirmed aon-observed-adverse-effect level (NOAEL) of 5 mg/(kg (bodyeight)/day) and established a maximum total daily intake (TDI)f 0.05 mg/kg body weight. Unfortunately, this report did notake into consideration newly discovered sources of food expo-ure to BPA, e.g., from recycled paper and cardboard containers29].

The present study forms part of a wider investigation intohe exposure of women in Southern Spain to environmentalhemicals. Data have already been published on their expo-ure to organochlorine pesticides [30], polybrominated diphenyl

thers (PBDE) and biphenyls (PBB) [31], and polychlorinatediphenyls (PCB) [32]. The present objective was to charac-erize BPA exposure by quantifying BPA and its chlorinatederivatives in the adipose tissue of these women.

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. Materials and methods

.1. Subjects

Twenty human adipose tissue samples were collected from women in theourse of surgical treatment of malignant and benign diseases at the San Cecilioniversity Hospital of Granada (Spain). The mean age was 59.7 years, rang-

ng from 24 to 81 years (Table 1). Body mass index (BMI) values, estimatedrom reported height and weight (kg/m2), ranged from 19.1 to 65. After subjectsigned their consent to participation, they were interviewed face-to-face by arained person using a structured questionnaire on their sociodemographic char-cteristics, reproductive history, and life-style factors. No subject reported anynown occupational exposure to BPA. The study was approved by the Ethicsommittee of our institution.

.2. Sample collection and storage

Adipose tissues were placed into a glass vial on ice, coded, and frozen to70 ◦C, always within 30 min of being excised, and samples were stored at the

ame temperature at the Laboratory of Medical Investigations until their dispatcho laboratory for analysis.

.3. Reagents and standards

All reagents were of analytical grade unless otherwise specified. Water18.2 M�/cm) was purified with a Milli-Q plus system (Millipore, Bedford,SA). Methanol, hexane, ethanol, ethyl acetate, diethyl ether, dichloromethane,

nhydrous sodium sulfate, o-phosphoric acid, and sodium hydroxide were sup-lied by Panreac (Barcelona, Spain). All solvents and reagents were tested tonsure they were free of contamination from compounds. Bisphenol-F (BPF),PA, and tetrachlorobisphenol A (Cl4BPA) were supplied by Sigma–Aldrich

Madrid, Spain). Monochloro-, dichloro-, and trichloro-bisphenol A (ClBPA,l2BPA, and Cl3BPA) were synthesized at the Department of Analyticalhemistry of our university (Fig. 1). Stock standard solutions (100 mg/l) ofach chemical compound were prepared in n-hexane and stored in dark glassottles at 4 ◦C until use, remaining stable for at least three months. These

M.F. Fernandez et al. / Reproductive Toxicology 24 (2007) 259–264 261

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Fig. 1. Structural formula of bisphenol-A and chlorinated derivatives.

olutions were used to spike the adipose tissue samples. The mixture of,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA) and trimethylchloro silane

TMCS) (99:1, v/v), supplied by Supelco (Bellefonte, PA, USA), was used asilylation reagent.

.4. Sample preparation, extraction, and derivatization

Two hundred milligrams of adipose tissue were homogenized with 6 ml n-exane. Then, 2 ml of acetonitrile were added to the n-hexane solution. Afterhaking for 3 min, the aqueous phase was separated and dried with a gentle streamf nitrogen. Prior to the extraction, adipose tissue samples were spiked withPF as internal standard. SPE cartridges (silica-based bonded C18 cartridgesccuBONDII ODS-C18, Agilent Technologies, Waldbron, Germany) were con-itioned with 3 ml of diethylether, 3 ml of methanol, and 3 ml of deionized watern an SPE manifold at a rate of 1–2 ml/min.

Sample extracts were resuspended using 15 ml of deionized water and passedhrough SPE cartridges at a flow rate of 1–2 ml/min. Then, cartridges were driednder vacuum for 20 min. BPA and its chlorinated metabolites were eluted fromorbents with 3 ml of a mixture of diethyl ether/methanol (9:1 v/v) at a flowate of 1 ml/min. Finally, eluents were evaporated to dryness under a stream ofitrogen; 120 �l of ethyl acetate and 30 �l of BSTFA/TMCS (1:1, v/v) weredded to the reaction vial in order to resuspend the residue and carry out theerivatization. Next, vials were closed and heated at 60 ◦C for 30 min. Once theerivatization process was completed, 2 �l of the reaction mixture was injectednto the gas chromatography–mass spectrometry (GC–MS) system.

.5. Apparatus: gas chromatographic–mass spectrometricnalysis

GC–MS analysis was performed using a 6890 Agilent (Agilent Technolo-ies, Wilmington, USA) gas chromatograph with a 7683 series injector andquadruple mass filter 5976 network mass selective detector (MSD) follow-

ng a previously published method [33]. In brief, a ZB-5 MS Zebron capillaryolumn (30 m × 0.25 mm i.d.; 0.25 �m film thickness) from Phenomenex wassed. The MSD was operated in full-scan mode from m/z 50 to 550 for qualita-ive determinations and in selected ion-monitoring (SIM) mode for quantitativeeterminations. The mass spectrometer was calibrated every day, using perflu-

rotributylamine (PFTBA) as calibration standard. HPCHEM chromatographyoftware was used for data acquisition and integration. The injector port of theC was set at 250 ◦C. The silicated samples were automatically injected using

he splitless-injection mode. The transfer line of the GC to the MS was set at70 ◦C, and the electron impact (EI) ion source of the MS was set at 250 ◦C.

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ig. 2. Chromatogram of a standard mixture of bisphenol-A, bisphenol A chlo-inated derivatives, and bisphenol F in a spiked adipose tissue sample.

he ionization energy was 70 eV. The GC oven temperature program was asollows: the initial oven temperature was set at 120 ◦C, held for 2 min, and thenhe temperature was increased to 230 ◦C via ramp of 30 ◦C/min and maintainedt 230 ◦C for 2 min, and from 230 up to 270 ◦C via ramp of 40 ◦C/min and main-ained at 270 ◦C for 6 min. The carrier gas was high-purity helium (99.999%)ith a constant flow of 1 ml/min. A solvent delay time of 4 min was used torotect the ion multiplier of the MS instrument from saturation. SIM mode wassed to carry out measurements. SPE was carried out on a Supelco vacuum man-fold for 12 columns connected to a Supelco vacuum tank and a vacuum pump.ig. 2 shows a chromatogram of a standard mixture of bisphenol-A, bisphenol-Ahlorinated derivatives, and bisphenol-F in a spiked adipose tissue sample.

.6. Analytical performance

Calibration graphs for samples treated according to the above procedure wereonstructed using SIM mode. Linearity of the calibration graphs was testedccording to the Analytical Methods Committee [34]; the lack-of-fit test waspplied to two replicates and two injections of each standard. The results for thentercept (a), slope (b), correlation coefficient (R2), and probability level of theack-of-fit test and for the linearity, precision, accuracy, sensitivity, and selec-ivity of the overall assay have been presented elsewhere [33,35,36]. Recoveriesf tested compounds were 95–105% in all cases. The limit of detection (LOD)as 0.5 ng/ml for BPA, ClBPA and Cl2BPA, and 2.7 and 3 ng/ml for Cl3BPA

nd Cl4BPA, respectively [33].

.7. Quality control

The methodology was validated using recovery studies with spiked samples.ecoveries of BPA and its chlorinated derivatives ranged from 91.3 to 100.4%,ith mean values of 96.4% (BPA), 94.4% (ClBPA), 97.3%, (Cl2BPA), 96.7%

Cl3BPA), and 94.9% (Cl4BPA).BPF was selected as internal standard because this compound was efficiently

xtracted from adipose tissue (mean recovery: 98.4%) and did not elute with anyf the evaluated chemicals.

. Results

Table 1 shows the age and BMI of the women in the study.wo previous studies by our group [30,37] reported a high BMI

n our series of women (mean of 29.1 and 28.9 kg/m2, respec-ively). These BMI data are consistent with the finding by theuropean Prospective Investigation into Cancer and Nutrition

EPIC) in 2002 that 92% of Granada women in the same ageange were overweight [38]. Lipid content ranged from 61 to

9%, with a mean value of around 80%.

BPA and three of the chlorinated derivatives were found atoncentrations above the LOD in at least one sample (Fig. 3).l2BPA was the most frequent (16/20 samples, 80%), followed

262 M.F. Fernandez et al. / Reproductive

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ig. 3. Individual concentrations (ng/g of adipose tissue) of bisphenol-A andhlorinated derivatives in adipose tissue. (�) BPA, ( ) ClBPA, (�) Cl2BPA, ( )l3BPA.

y BPA (55%), ClBPA (15%), and Cl3BPA (10%). Cl4BPA waslways below the LOD. Table 2 shows means, medians, andercentiles of BPA and chlorinated derivatives in samples withresence of these chemicals above the LOD. BPA concentrationsanged from 1.80 to 12.01 ng/g of adipose tissue. Interestingly,he sample with the highest concentration of BPA also showedhe highest concentration of Cl2BPA (41.77 ng/g of adipose tis-ue). No correlation was found between BPA and chlorinatederivatives or among chlorinated BPA derivatives.

The mean ± S.D. of BPA in all samples was 3.16 ± 4.11 ng/gf adipose tissue. In mono-, di-, and trichloro-BPA derivatives,he mean concentrations were 0.35 ± 1.00, 7.84 ± 9.68, and.04 ± 0.15 ng/g of adipose tissue, respectively. Age did emerges a determinant of exposure, as findings for other persistent andat soluble compounds. The Spearman correlation coefficientr) was significant between age and the concentration of any

hlorinated derivative or of their sum total (BPA/age, r = 0.538,= 0.014; total chlorinated

∑BPA/age, r = 0.529, p = 0.016).

or instance, one of the oldest women in the series (76 yearsld, sample 16) showed the highest BPA and Cl2BPA lev-

able 2oncentrations of BPA (ng/g) and chlorinated derivatives in adipose tissue

amples

n (%) >LOD

Mean (±S.D.) Median 5th 25th 75th 95th

PA 11/20 (55) 5.83 (3.48) 4.79 2.07 3.54 7.12 11.88l-BPA 3/20 (15) 3.05 (0.28) 3.14 2.82 2.94 3.21 3.26l2-BPA 16/20 (80) 9.21 (9.26) 7.77 2.61 5.62 8.83 21.49l3-BPA 2/20 (10) 0.74 (0.15) 0.74 0.66 0.69 0.80 0.84l4-BPA 0/20 (0) <LOD <LOD – – – –

BPA 16/20 (80) 9.00 (9.22) 8.12 0 1.93 9.60 28.25

(%), number of subjects (percentage of detection); LOD, Limit of detection.

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Toxicology 24 (2007) 259–264

ls. Moreover, body fat content had a negative effect on theresence or concentration of these chemicals, but Spearman cor-elation coefficients were not significant between the BMI andhe concentration of BPA or of total chlorinated BPA (BPA/BMI= −0.213, p = 0.368; total chlorinated

∑BPA/BMI r = −0.262,

= 0.264). All bisphenols were below the LOD in the samplerom the most obese woman in this series (sample 16, BMI > 65).

. Discussion

There are few data on BPA human exposure, thereforehis study was undertaken to determine BPA and its chlori-ated derivates in adipose tissue from adult women. We usedpreviously developed sensitive GC/MS method that excludes

alse-positive results due to BPA leaching into samples fromeaction solvents or chromatographer components [33,35,36].

BPA was present in 11 out of the 20 (55%) adipose tissueamples, with a mean value of 3.16 ± 4.11 ng/g of adipose tis-ue. To our best knowledge, this is the first report on BPA indipose tissue in humans, precluding comparisons with lev-ls in other populations. However, because BPA is a lipophilicompound, levels can be compared with results of similar ana-ytical techniques in other fatty fluids or tissues. Thus, lowerevels of BPA were found in the breast milk of all 23 healthyomen investigated by Sun et al. [39], who reported a rangef 0.28–0.97 ng/ml and a mean concentration of 0.61 ng/ml. Aower mean concentration (1.9 ng/ml) in breast milk samplesas also described by Ye et al. [40], who found free BPA in0% and total BPA (including free BPA and BPA conjugates) in0% of samples. Similar levels of BPA (3.41 ± 0.01 ng/ml, range–7 ng/ml) to present findings in adipose tissue were recentlyeported in human calostrum [41], which is less fatty than matureilk.BPA levels were found to be higher in tissue samples from

lacentas, which have a modestly higher fat content versuslood, than in serum samples from the same women, with aean value of 11.2 ng/g of tissue and a maximum of 104.9 ng/g

42] versus a mean of 4.4 ± 0.64 ng/ml of serum. Serum BPAevels in these pregnant women appeared to be high, contrastingith findings of 1.49 ± 0.11 and 0.64 ± 0.10 ng/ml, respectively

n a group of Japanese men and women [43]. Some studies haveeported blood BPA concentrations in individuals with no inten-ional exposure that ranged from below 1 to 19 ng/ml [44–46],lthough these wide differences may be related to the differentethodologies used for BPA quantification.Evidence of fat accumulation of BPA was demonstrated by

unez et al. [47], who investigated the distribution of BPA in dif-erent target tissues after repeated BPA doses (4 or 5 mg/day for5 days). BPA was detected in all tissue samples from the treatednimals but preferentially accumulated in brown adipose tissue,hich plays an important role in diet-induced thermogenesis and

nergy balance.The accumulation of Cl2BPA in the adipose tissue of these

omen can more readily be understood than that of BPA, sincehere is much more information about organohalogenated com-ounds in fat tissue [30–32]. However, the lack of previousata on chlorinated bisphenols in humans hinders compar-

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sons. The main concern about the accumulation of chlorinatedPAs in fatty tissue derives from their demonstrated estro-enicity in various in vitro and in vivo models [48,49]. Allhe chlorinated bisphenols investigated here, except Cl4BPA,nduced the proliferation of MCF-7 breast cancer cells in cul-ure at concentrations of ≥1 mM [48]. In ovariectomized rats,l2BPA mimicked BPA by increasing uterine wet weight and

howed a stronger effect than BPA in increasing the num-er of BrdU-positive cells in the uterine endometrium [49].nother organohalogenated BPA derivative, fluorine-containingisphenol-A (bisphenol-AF), is also estrogenic for MCF-7reast cancer cells, promoting cell proliferation and increas-ng the synthesis and secretion of cell-type specific proteins50,51]. These organohalogenated compounds have been col-ected together with organochlorine pesticides in adipose tissuextracts from breast cancer patients [48,52] and may contributeo the combined estrogenicity of the fat extract and, therefore, tohe risk associated with the total effective xenoestrogen burden37,53].

Data presented here should be interpreted with care. The sam-le size is small, hampering detection of differences betweenubgroups (e.g., groups defined by age or residence), and theeries does not reflect the composition of the general Spanishopulation. Nevertheless, our findings are of interest, since BPAs a widespread chemical present in products in everyday use,nd there is inadequate information on the degree of humanxposure. Determination of the tissue distribution, metabolism,nd excretion of these biologically active contaminants is a pre-equisite of risk assessment, since these are essential parametersor establishing the in vivo activity and biological significance ofnvironmental data. The effects of bisphenols on adipose tissue,highly active metabolic and endocrine organ, appear worthy

f special attention [54].

cknowledgments

We thank Richard Davies for editorial assistance. Thisesearch was supported by grants from the Spanish Ministry ofealth (FIS 02/1314 and FIS C03/081) and the EU Commission

EDEN QLK4-CT-2002-00603 and CASCADE Food-CT-2003-06319).

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