Widespread Occurrence and Accumulation of Bisphenol A Diglycidyl

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Widespread Occurrence and Accumulation of Bisphenol A Diglycidyl Ether (BADGE), Bisphenol F Diglycidyl Ether (BFDGE) and their Derivatives in Human Blood and Adipose Fat Lei Wang, Jingchuan Xue, and Kurunthachalam Kannan Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.5b00096 • Publication Date (Web): 13 Feb 2015 Downloaded from http://pubs.acs.org on February 18, 2015

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Environmental Science & Technology

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Widespread Occurrence and Accumulation of Bisphenol A Diglycidyl Ether

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(BADGE), Bisphenol F Diglycidyl Ether (BFDGE) and their Derivatives in

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Human Blood and Adipose Fat

4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20

Lei Wang†, Jinchuan Xue‡, and Kurunthachalam Kannan‡,¶,*

†

Tianjin Key Laboratory of Environmental Remediation and Pollution Control / Ministry of Education Key Laboratory of Pollution Processes and Environmental Criteria, Nankai University, Tianjin 300071, China

‡

Wadsworth Center, New York State Department of Health, and Department of Environmental Health Sciences, School of Public Health, State University of New York at Albany, Empire State Plaza, P.O. Box 509, Albany, New York 12210-0509, United States

¶

Biochemistry Department, Faculty of Science and Experimental Biochemistry Unit, King Fahd Medical Research Center, King Abdulaziz University, Jeddah 21589, Saudi Arabia

21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36

*Corresponding author: K. Kannan Wadsworth Center Empire State Plaza, P.O. Box 509 Albany, NY 12201-0509 Tel: 1-518-474-0015 Fax: 1-518-473-2895 E-mail: [email protected]

For submission to: Environmental Science & Technology

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TOC

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ABSTRACT

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Despite the widespread use of bisphenol A diglycidyl ether (BADGE) and bisphenol F diglycidyl

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ether (BFDGE) in various consumer products, studies on human exposure to these compounds

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are scarce. In this study, BADGE, BFDGE, and seven of their derivatives were determined in

45

human adipose fat and blood plasma samples collected from New York City, NY, USA.

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Bisphenol A bis (2,3-dihydroxypropyl) ether [BADGE▪2H2O] was the major BADGE derivative

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found in 60% of the adipose samples and 70% of the plasma samples analyzed. High

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concentrations and detection frequencies of BFDGE were found in both adipose and plasma

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samples. BFDGE concentrations in adipose fat ranged from 19.1 to 4500 ng/g wet weight. A

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significant correlation between BADGE or BFDGE and their derivatives in adipose and plasma 2 ACS Paragon Plus Environment

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samples suggested hydration of these reactive compounds in humans. A significant positive

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correlation existed between BADGEs (i.e., the sum of BADGE and its five derivatives) and

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BFDGEs in adipose samples, which suggested similar exposure sources and pathways for these

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compounds in humans. Bisphenol A (BPA) also was analyzed in adipose fat and plasma, and its

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concentrations were positively correlated with those of BADGEs, which confirmed co-exposure

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of BADGEs and BPA in humans.

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INTRODUCTION

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Bisphenol A diglycidyl ether (BADGE) and bisphenol F diglycidyl ether (BFDGE) are widely

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used in the protective coatings of food and beverage cans and in paints and adhesives (1, 2).

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These compounds also are used as monomers in the production of epoxy-based polymers and as

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additives for the elimination of surplus hydrochloric acid in polyvinyl chloride (PVC) production

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(1). These are high production-volume chemicals. For instance, the annual global production of

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BADGE was estimated at 957 thousand metric tons in 2003 (2).

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BADGE is a reactive molecule and has been reported to bind to nucleic acids (3). In vitro

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studies have shown mutagenic and teratogenic effects of BADGE and its derivatives

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(collectively referred to as BADGEs in this study) (4, 5). Bisphenol A bis (2,3-dihydroxypropyl)

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ether [BADGE▪2H2O], the stable hydrated product of BADGE, was reported to have endocrine

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disrupting potentials even greater than those of bisphenol A (BPA) (6). The antagonistic activity

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of a chlorohydrin derivative of BADGE toward androgen has been reported (7). In addition,

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genotoxicity and cytotoxicity as well as developmental and reproductive toxicity of BADGEs

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have been reported in laboratory animals (5, 8-10). A reduction in a follicle-stimulating hormone

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in male workers exposed to BADGE was reported (11). The European Commission for food



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contact applications has set a migration limit for ten derivatives of BADGE from food cans at 1

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mg/kg (12).

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BADGE and its hydrated as well as chlorinated derivatives are present in many canned

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foods at concentrations on the order of several micrograms to milligrams per kilogram (13-17).

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Studies on human exposure to BADGEs, however, are scarce. Limited human biomonitoring

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studies conducted in our laboratory have shown that BADGE▪2H2O and BADGE are widespread

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in urine specimens from Greece, China, India, and the USA (18-20). Considering the moderately

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high lipophilicity (i.e., Kow; see Table 1) of BADGE and its derivatives, there is a potential for

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these chemicals’ accumulation in adipose tissue of humans. Because BADGE has been reported

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to elicit adipogenic gene expression in stromal stem cells (21), it is relevant to assess

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accumulation in human adipose fat. Nevertheless, no earlier studies have reported the occurrence

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of BADGEs in human fat.

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BFDGE is also used as a coating material and has been detected in canned foods (22).

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Cytotoxic, genotoxic, mutagenic, and endocrinal effects of BFDGEs have been reported in in

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vitro studies (7, 10, 23, 24). For instance, BFDGE was shown to elicit cytotoxic effects on

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human colorectal adenocarcinoma cell line (CACO-2), by inducing morphological changes, cell

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detachment from substratum, inhibition of cell proliferation and F-actin depolymerization (10).

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BFDGE was also reported to induce mutagenic effects in prokaryotic cell bioassays, and to

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increase frequency of sister chromatid exchanges and micronuclei in human peripheral blood

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lymphocytes (23). In addition, chlorohydrin derivative of BADGE was reported to be a strong

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antagonist for androgen receptor (7). However, no earlier studies have reported the occurrence of

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BFDGEs in human tissues.


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In this study, concentrations of six BADGEs, i.e., BADGE, BADGE▪H2O, BADGE▪2H2O,

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bisphenol A (3-chloro-2-hydroxypropyl) (2,3-dihydroxypropyl) ether [BADGE▪HCl▪H2O],

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bisphenol A

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(3-chloro-2-hydroxypropyl) glycidyl ether [BADGE▪HCl], and three BFDGEs, i.e., BFDGE,

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bisphenol F bis (2,3-dihydroxypropyl) ether (BFDGE▪2H2O), and bisphenol F bis

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(2-chloro-1-propanol) ether (BFDGE▪2HCl), were determined in 20 adipose fat tissue and 20

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blood plasma samples collected from New York City. BPA also was analyzed in these samples

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to examine the association of this compound with BADGE/BFDGE. The objectives of this study

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were to (i) determine the occurrence, distribution, and bioaccumulation potentials of

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BADGEs/BFDGEs in human tissues; and (ii) elucidate the correlation between various

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BADGE/BFDGE derivatives and BPA in human tissues.

bis

(3-chloro-2-hydroxypropyl)

ether

[BADGE▪2HCl],

bisphenol A

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MATERIALS AND METHODS

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Chemicals. Analytical standards of BADGE (≥95%) and its derivatives, BADGE·H2O (≥95%),

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BADGE·2H2O (≥97%), BADGE·HCl·H2O (≥95%), BADGE·HCl (≥90%), BADGE·2HCl

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(≥97%), BFDGE·2H2O (≥95%), and BFDGE·2HCl (≥90%), as well as BPA (>99%) and

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β-glucuronidase from Helix pomatia (145700 units/mL β-glucuronidase and 887 units/mL

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sulfatase), were purchased from Sigma-Aldrich (St. Louis, MO). BFDGE (98%) was purchased

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from

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2-hydroxy-4-methoxybenzophenone (13C12-BP-3) (99%) and

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from Cambridge Isotope Laboratories (Andover, MA). D6-BADGE (99%) was purchased from

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Toronto Research Chemicals (North York, Ontario, Canada). The molecular structures and

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selected physicochemical properties of the target compounds are shown in Figure S1 (Supporting

LGC

standards

(Teddington,

Middlesex,

UK). 13

13

C-isotopically

labeled

C12-BPA (99%) were purchased



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Information) and Table 1, respectively. Milli-Q water was prepared using an ultrapure water

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system (Barnstead International, Dubuque, IA). The stock solutions of target analytes and

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internal standards were prepared at 1 mg/mL in methanol and stored at -20° C.

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Sample Collection. A total of 20 human adipose fat samples were originally collected in

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2003–2004 from donors who underwent liposuction in New York City. The samples were stored

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in solvent-cleaned I-Chem jars at -20° C, as recommended by the National Human Monitoring

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Program (25). The samples were from 15 females (age: 18–58 years) and 5 males (age: 32–51

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years). Additional demographic information of the donors is shown in Table S1 (Supporting

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Information). Further details on the sample collection have been provided elsewhere (26).

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Plasma samples (n = 20) were collected in 2002–2003 from adult male donors (age: 24–58 years)

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in New York City. Further details of the plasma samples are shown in Table S1. Institutional

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Review Board approvals were obtained from the New York State Department of Health for the

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analysis of samples.

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Sample Preparation. For adipose fat tissue samples, 200 to 300 milligrams of sample were

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accurately weighed and spiked with 50 µL of methanol containing 13C12-BPA, D6-BADGE, and

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13

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quantification of BADGE, BFDGE, BADGE·H2O, and BADGE·HCl (analytes with epoxy

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moiety) (Table S2; Supporting Information) (27), whereas

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BADGE·2H2O, BADGE·HCl·H2O, BADGE·2HCl, BFDGE·2H2O, and BFDGE·2HCl (analytes

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without epoxy moiety).

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equilibration for 30 min at room temperature, 5 mL of acetone were added into the sample. The

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mixture was homogenized in a mortar, and the extract was then transferred to a 15 mL

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polypropylene (PP) tube by washing with 2 mL of methanol. The combined extracts were

C12-BP-3 (200 ng/mL each). D6-BADGE was used as the internal standard (IS) for the

13

13

C12-BP-3 was used as the IS for

C12-BPA was used as the IS in the quantification of BPA. After


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concentrated to ~2 mL under a gentle nitrogen stream (to remove acetone). Then an ultra-low

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temperature (-20° C) incubation was used to separate the lipid from the solvent. After storage at

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-20° C for 15 min, the extracts were centrifuged immediately at 4500 g for 5 min (Eppendorf

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centrifuge 5804, Hamburg, Germany) for the separation of organic solvent and solidified lipid.

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The supernatant was transferred and concentrated to 0.5 mL under a gentle nitrogen stream and

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analyzed

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(HPLC-MS/MS).

by

high-performance

liquid

chromatography-tandem

mass

spectrometry

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Plasma samples were extracted using a liquid-liquid extraction (LLE) method. Five hundred

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microliters of plasma samples were transferred into a 15-mL PP tube, and 50 µL of methanol

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containing 13C12-BPA, D6-BADGE and 13C12-BP-3 (200 ng/mL each) were spiked. The samples

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were then extracted three times each with 3 mL of ethyl acetate. The mixture was shaken in an

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oscillator shaker for 60 min (Eberbach Corp., Ann Arbor, MI) and then centrifuged at 4500 g for

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5 min (Eppendorf). The extracts were combined and washed with 1 mL of Milli-Q water. The

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supernatant was transferred into a glass tube and concentrated to near-dryness under a gentle

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nitrogen stream. Finally, 0.5 mL of methanol was added and vortex mixed for analysis by

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HPLC-MS/MS.

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Instrumental Analysis. Chromatographic separation and detection of analytes were

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accomplished by the use of an Agilent 1100 Series HPLC (Agilent Technologies Inc., Santa

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Clara, CA) interfaced with an Applied Biosystems API 2000 electrospray triple quadrupole mass

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spectrometer (ESI-MS/MS; Applied Biosystems, Foster City, CA). Ten microliters of the sample

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were injected onto an analytical column (Betasil® C18, 100 x 2.1 mm column; Thermo Electron

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Corporation, Waltham, MA), which was connected to a Javelin guard column (Betasil® C18, 20

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x 2.1 mm column). The mobile phase comprised 100% methanol (A) and 10% methanol in 7 ACS Paragon Plus Environment


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Milli-Q water that contained 2 mM of ammonium acetate (B). Two different gradient elutions at a

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flow rate of 200 µL/min were used for the analysis of BADGEs/BFDGEs and BPA (Table S3;

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Supporting Information). The MS/MS was operated in the multiple reaction monitoring (MRM),

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positive and negative ionization modes for the analysis of BADGEs/BFDGEs and BPA,

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respectively, and the parameters were optimized by infusion of individual analytes (Table S4;

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Supporting Information). Select MRM chromatograms of BFDGE in standard solution and in

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human specimens are shown in Figure S2 (Supporting Information).

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Quality Assurance and Quality Control (QA/QC). Solvent-cleaned glass jars were used to

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store adipose fat samples. Contamination that arose from laboratory materials and solvents was

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evaluated by the analysis of procedural blanks with every batch of samples. Recoveries of native

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compounds spiked into procedural blanks ranged from 81% to 105%. Quantification of BADGEs

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and BFDGEs was performed by an isotope-dilution method based on the responses of

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D6-BADGE (for BADGE, BFDGE, BADGE▪H2O, and BADGE▪HCl),

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BADGE▪2H2O, BADGE▪2HCl, BADGE▪HCl▪H2O, BFDGE▪2H2O, and BADGE▪2HCl), and

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13

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BADGEs, 3 BFDGEs, and BPA were spiked along with 13C12-BP-3, 13C12-BPA, and D6-BADGE

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(10 ng each) and passed through the entire analytical procedure. In spiked matrices, recoveries of

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all target compounds ranged from 98% to 125% for adipose tissue and from 94% to 107% for

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plasma (corrected for by the recoveries of D6-BADGE or

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recoveries of target chemicals through the analytical procedure are presented in Table S2.

13

C12-BP-3 (for

C12-BPA (for BPA). Four adipose and four plasma samples were selected randomly; 6

13

C12-BP-3 or

13

C12-BPA). The

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Duplicate analysis of selected samples showed a coefficient of variation of

Widespread Occurrence and Accumulation of Bisphenol A Diglycidyl

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