Article pubs.acs.org/est
Characteristic Profiles of Benzonphenone‑3 and its Derivatives in Urine of Children and Adults from the United States and China Lei Wang†,‡ and Kurunthachalam Kannan*,† †
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 12201-0509, United States ‡ 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 S Supporting Information *
ABSTRACT: Widespread exposure of humans to benzophenone-3 (BP-3) is a concern due to this compound’s potential to disrupt endocrine function. BP-3 can be metabolized by phase I and phase II reactions of the human cytochrome P450 system. Urinary measurements of BP-3 have been used as a biomarker of exposure. Nevertheless, metabolic transformation pathway and the transformation products of BP-3 in humans are still less known. In this study, 166 urine samples collected from children and adults in the U.S. and China were analyzed for free and total forms (free plus conjugated) of BP-3 as well as four of its metabolic derivatives, 4-OH−BP, 2,4-diOH−BP, 2,2′,4,4′-tetraOH−BP, and 2,2′-diOH−4-MeO−BP, using high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS). BP-3 was found in almost all urine samples from the U.S. and China. Concentrations of BP-3 in urine from children (GM: 9.97 ng/mL) and adults (15.7 ng/mL) in the U.S. were significantly higher than those in children (0.622 ng/mL) and adults (0.977) from China. A significant positive relationship was found between the concentrations of urinary BP-3 and its derivatives. The profiles of BP derivatives in urine suggested that demethylation was a major route of BP-3 metabolism. The percentage of the free form of BP-3 in urine was used in the determination of efficacy of phase II metabolism among the different population groups studied. A significantly lower percentage of the free form of BP-3 was found in urine from the U.S. population than in the Chinese population.
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of BP-3.6,14−16 High urinary concentrations of 2,4-diOH-BP were associated with increased odds of developing endometriosis in the U.S. women.17 The conjugation of reactive metabolites with glucuronide or sulfate in the phase II metabolism is expected to reduce the toxicity of many xenobiotics.16,18,19 Nevertheless, little is known about metabolic transformation products and transformation pathways of BP-3 in humans. In this study, concentrations of free (i.e., aglycone) and total forms (i.e., free plus conjugated) of BP-3, as well as total concentrations of four metabolic derivatives of BP-3, 4-OH− BP, 2,4-diOH−BP, 2,2′,4,4′-tetraOH−BP, and 2,2′-diOH−4MeO−BP, were determined in 166 urine samples collected from children and adults in the U.S. and China. Relative percentages of the free form of BP-3 in total concentrations and hydroxylation (phase I reaction) of BP-3 and its derivatives were determined. The objectives of this study were to (i) determine the concentrations and profiles of BP-3 and its
INTRODUCTION Benzophenone-3 (BP-3; i.e., 2-hydroxy-4-methoxy benzophenone; 2-OH-4-MeO-BP) is a commonly used sunscreen agent (UV filter) in a variety of cosmetic products.1 The U.S. Food and Drug Administration (FDA) has set the maximum allowable BP-3 level in sunscreen products at 6%. Approximately 10% of the dermally applied dose of BP-3 can reach a human’s internal circulation.2 The 2003−2004 National Health and Nutrition Examination Survey (NHANES) conducted by the U.S. Centers for Disease Control and Prevention (CDC) showed that urine from 97% of U.S. residents contained measurable levels of BP-3.3 The acute toxicities of BP-3 were reported to be low, but in vivo and in vitro studies have shown that this compound possesses estrogenic4−6 and antiandrogenic activities.7,8 After dermal or dietary exposure to BP-3 in laboratory animals, this compound is metabolized by phase I and phase II reactions9,10 (Figure 1), which result in conjugation and urinary excretion. Occurrence of free and conjugated forms of BP-3 and its derivatives has been reported in urine.11−13 Some metabolic products of BP-3 are more toxic than BP-3 itself. For example, the estrogenic activities of BP-3 metabolites, 2,4-diOH-BP, 2,2′,4,4′-tetraOH−BP, and 4-OH−BP, were higher than those © 2013 American Chemical Society
Received: Revised: Accepted: Published: 12532
July 24, 2013 September 23, 2013 September 27, 2013 September 27, 2013 dx.doi.org/10.1021/es4032908 | Environ. Sci. Technol. 2013, 47, 12532−12538
Environmental Science & Technology
Article
Figure 1. Metabolic pathways of BP-3 in the phase I and phase II reactions.
derivatives in urine from children and adults in the U.S. and China; and (ii) examine the metabolic transformation pattern of BP-3 in humans in the U.S. and China.
State Department of Health (NYSDOH) for the analysis of urine. Sample Preparation. The urine samples were analyzed for BP-3 and four of its derivatives by liquid−liquid extraction (LLE), as described earlier, with some modifications.20 Briefly, 50 microliters of 13C12−BP-3 (100 ng/mL) were spiked into 500 microliters of urine taken in a 15 mL PP tube, followed by the addition of 300 μL of 1 M ammonium acetate buffer. The mixture was extracted three times with 3 mL of ethyl acetate each time by shaking in an oscillator shaker for 60 min (Eberbach Corp., Ann Arbor, MI) and then centrifuged at 4500g for 5 min (Eppendorf 5804, Hamburg, Germany). The supernatant was combined, washed with 1 mL of Milli-Q water, and then concentrated to near-dryness under a gentle nitrogen stream. Finally, 0.5 mL of methanol was added and vortex mixed for analysis by HPLC-MS/MS. Ethyl acetate extractable fraction, without enzymatic digestion, was considered to represent the “free” form (i.e., aglycone/unconjugated) of BP-3. For the determination of “total” (i.e., free plus conjugated) concentrations of BP-3 and its derivatives, urine samples were enzymatically deconjugated by the addition of 300 μL of 1 M ammonium acetate, which contained 22 units of β-glucuronidase (prepared by spiking 50 μL of β-glucuronidase into 100 mL of 1 M ammonium acetate solution) prior to extraction. After incubation at 37 °C for 12 h, the digested samples were extracted by LLE, as described above. Chemical Analysis. Separation and detection of target analytes were accomplished with an Agilent 1100 Series HPLC (Agilent Technologies Inc., Santa Clara, CA), interfaced with an Applied Biosystems API 3200 electrospray triple quadrupole mass spectrometer (ESI-MS/MS; Applied Biosystems, Foster City, CA). Ten microliters of the extract were injected onto an analytical column (Betasil C18, 100 × 2.1 mm column; Thermo Electron Corporation, Waltham, MA), which was connected to
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MATERIALS AND METHODS Chemicals. BP-3 (98%), 4-OH−BP (98%), 2,4-diOH−BP (99%), 2,2′,4,4′-tetraOH−BP (97%), and 2,2′-diOH−4-MeO− BP (98%) were purchased from Sigma-Aldrich (St. Louis, MO). The physicochemical properties of the target chemicals are presented in Table S1 (Supporting Information (SI)).13Cisotopically labeled 2-OH-4-MeO-BP (13C12−BP-3) (99%) was purchased from Cambridge Isotope Laboratories (Andover, MA). β-Glucuronidase from Helix pomatia (145700 units/mL β-glucuronidase and 887 units/mL sulfatase) was purchased from Sigma-Aldrich. Milli-Q water was prepared using an ultrapure water system (Barnstead International, Dubuque, IA). The stock solutions of target analytes and internal standards were prepared at 1 mg/mL in methanol and stored at −20 °C. Sample Collection. Spot urine samples from U.S. children aged 3−10 yrs (15 males and 23 females) were collected in 2012. Urine samples from U.S. adults aged 15−66 yrs (20 males and 10 females) were collected in Albany, New York, during May-July, 2011. Urine samples from Chinese children aged 9−10 yrs (38 males and 32 females) were collected in Tianjin, China, during March and April 2012. Urine from Chinese adults (15 males and 11 females) was collected in Shanghai during August and September 2010, with the majority of adult volunteers aged 22−30 yrs, except for two participants (one 58-yr-old male and one 56-yr-old female). Detailed information regarding adult participants is provided in SI Table S2. All spot urine samples were collected in polypropylene (PP) tubes and stored at −80 °C until analysis. Institutional Review Board approvals were obtained from the New York 12533
dx.doi.org/10.1021/es4032908 | Environ. Sci. Technol. 2013, 47, 12532−12538
Environmental Science & Technology
Article
Table 1. Urinary Concentrations (ng/mL) and Detection Rates (DR %) of Benzophenone Derivatives (BPs) in Children and Adults in the U.S. and China BP derivatives in total form BP-3 in total forma
BP-3 in free form
4-OH-BP
2,4-diOH-BP
2,2′,4,4′-tetraOH-BP
2,2′-diOH-4-MeO-BP
U.S. Children (n = 38) GMb 9.97 median 8.34 min-max
