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Endocrinology, doi:10.1210/en.2006-1280
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Endocrinology Vol. 148, No. 6 2835-2844
Copyright © 2007 by The Endocrine Society

The Ultraviolet Filter Benzophenone 2 Interferes with the Thyroid Hormone Axis in Rats and Is a Potent in Vitro Inhibitor of Human Recombinant Thyroid Peroxidase

Cornelia Schmutzler, Anja Bacinski, Inka Gotthardt, Katrin Huhne, Petra Ambrugger, Holger Klammer, Christiane Schlecht, Cuong Hoang-Vu, Annette Grüters, Wolfgang Wuttke, Hubertus Jarry and Josef Köhrle

Institut für Experimentelle Endokrinologie (C.Schm., A.B., I.G., K.H., J.K.), Institut für Experimentelle Pädiatrische Endokrinologie (P.A., A.G.), Charité Universitätsmedizin Berlin, D-10117 Berlin, Germany; Klinische und Experimentelle Endokrinologie (H.K., C.Schl., W.W., H.J.), Universitäts-Frauenklinik Göttingen, D-37075 Göttingen, Germany; and Experimentelle und Chirurgische Onkologie (C.H.-V.), Martin-Luther-Universität Halle/Wittenberg, D-06120 Halle/Saale, Germany

Address all correspondence and requests for reprints to: Cornelia Schmutzler, Institut für Experimentelle Endokrinologie, Charité Universitätsmedizin Berlin, Charitéplatz 1, D-10117 Berlin, Germany. E-mail: cornelia.schmutzler{at}charite.de.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Endocrine disrupting chemicals (EDCs), either plant constituents or contaminants deriving from industrial products, may interfere with the thyroid hormone (TH) axis. Here, we examined whether selected EDCs inhibit the key reactions of TH biosynthesis catalyzed by thyroid peroxidase (TPO). We used a novel in vitro assay based on human recombinant TPO (hrTPO) stably transfected into the human follicular thyroid carcinoma cell line FTC-238. F21388 (synthetic flavonoid), bisphenol A (building block for polycarbonates), and the UV filter benzophenone 2 (BP2) inhibited hrTPO. BP2 is contained in numerous cosmetics of daily use and may be in regular contact with human skin. Half-maximal inhibition in the guaiacol assay occurred at 450 nmol/liter BP2, a concentration 20- and 200-fold lower than those required in case of the TPO-inhibiting antithyroid drugs methimazole and propylthiouracil, respectively. BP2 at 300 nmol/liter combined with the TPO substrate H2O2 (10 µmol/liter) inactivated hrTPO; this was, however, prevented by micromolar amounts of iodide. BP2 did not inhibit iodide uptake into FRTL-5 cells. In BP2-treated rats (333 and 1000 mg/kg body weight), serum total T4 was significantly decreased and serum thyrotropin was significantly increased. TPO activities in the thyroids of treated animals were unchanged, a finding also described for methimazole and propylthiouracil. Thus, EDCs, most potently BP2, may disturb TH homeostasis by inhibiting or inactivating TPO, effects that are even more pronounced in the absence of iodide. This new challenge for endocrine regulation must be considered in the context of a still prevailing iodide deficiency in many parts of the world.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THYROID PEROXIDASE (TPO) is a heme protein localized in the apical cytoplasmic membrane of thyroid epithelial cells, facing the colloid-filled, extracellular lumen of the follicle. TPO plays a key role in thyroid hormone (TH) biosynthesis (1, 2). Therefore, apart from representing the "microsomal antigen" in autoimmune thyroiditis, TPO, if mutated, is of pathophysiological importance in dyshormonogenic forms of congenital hypothyroidism. Furthermore, TPO so far is the only target of antithyroid drugs with therapeutic relevance in the treatment of hyperthyroidism (3). Conversely, this susceptibility to inhibition may promote goiter development, especially in the context of iodine deficiency.

Endocrine disrupting chemicals (EDCs) are either derived as pollutants from industrial products (plasticizers, flame retardants, pesticides, etc.) or consumed as vegetable or fruit constituents and as high dose plant extracts (4). Both involuntary exposure to contaminating EDCs and voluntary intake of EDCs, e.g. as "phytoestrogens" for hormone replacement, are constantly increasing. Especially, certain UV filters are suspected as EDCs. These substances are produced in high amounts, i.e. several hundreds of tons per year, and their main use is in sun lotions for skin protection against sunburn, photoaging, and skin cancer. This applies to, for example, 4-methylbenzylidene-camphor (4-MBC), octyl-methoxycinnamate (OMC), and benzophenone 3 (BP3). Concentrations of up to 10% (wt/wt) of UV filter, depending on the light protection factor, are allowed in commercial preparations. Another UV filter, benzophenone 2 (BP2) is no longer permitted to be used in sun lotions within, for example, the European Union. However, it is still contained in plastic materials or many cosmetics to prevent their UV-induced damage. BP2 concentrations in these preparations are much lower than the concentrations allowed for UV filters in sun protection products, but because the substance is found in a vast variety of products (5, 6), it may be in contact with human skin virtually every day.

Several EDCs have been extensively characterized concerning their interference with development and regulation of the reproductive system (7), but it is increasingly accepted that the TH endocrine axis may be a target for endocrine disruption, too. Polyhalogenated phenolic compounds (polychlorinated biphenyls, polybrominated diphenylethers, etc.), probably because of their structural resemblance to TH (8), may cause disturbance of TH homeostasis, hypothyroidism, thyroid hyperplasia and neoplasia (9, 10), and developmental defects of the central nervous system (11, 12) in experimental animals and humans. Approximately 50 yr ago, it was reported that feeding with soybean milk caused goiter in infants, although adequate iodine supply could prevent or revert this condition (for review, see Ref. 13). This is probably attributable to the soy isoflavone genistein, which interacts with nuclear receptors for estrogen and other ligands (14, 15) but also inhibits TPO in vitro and in vivo (16, 17). Additional possible targets for interference by EDCs with the complex regulatory network of TH metabolism and action are iodide uptake by the sodium iodide symporter (NIS) (18), type I 5'-deiodinase (5'DI) (19, 20, 21, 22), the TH transport protein transthyretin (TTR) (23, 24, 25), and TH receptor (TR) (26, 27, 28, 29). However, data on thyroid-related effects of EDCs are still comparatively scarce.

To further elucidate possible adverse effects of EDCs on the TH axis, we here examined whether they interfere with TH production by inhibiting the central biosynthetic steps catalyzed by TPO. We used in vitro assays based on human recombinant TPO (hrTPO) and concentrated on a selection of UV filters for which, so far, primarily estrogenic effects had been described (30, 31), but an antithyroid action was to be suspected according to our previous results (22, 32). These compounds, 4-MBC, OMC, BP2, and BP3, were compared with other substances with known antithyroid properties: the goitrogen genistein (13), F21388, a synthetic flavonoid that displaces T4 from its binding to TTR and inhibits 5'DI (23, 33), bisphenol A (BPA), which interacts with TR{alpha} (28), as well as the two TPO-inhibiting antithyroid drugs propylthiouracil (PTU) and methimazole (MMI) (3). We show that some of these substances indeed may interfere with TH biosynthesis by inhibiting TPO and that the degree of this inhibition is dependent on the iodide concentration available. Furthermore and most importantly, we demonstrate that the UV filter BP2 is probably one of the most potent inhibitors of TPO described so far.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Construction of the cell line FTC-238/TPO
A cDNA coding for human TPO was ligated into the eukaryotic high copy number vector pcDNA3 (Invitrogen, Karlsruhe, Germany). Thereafter, the human follicular thyroid carcinoma cell line FTC-238 (kindly provided by P. Goretzki, Lukaskrankenhaus, Neuss, Germany), which does not express TPO endogenously, was stably transfected with this construct using Lipofectamine plus (Invitrogen) according to the instructions of the manufacturer. Three days after transfection, cells were incubated with selection medium containing 700 µg/ml G418 (PAA Laboratories, Coelbe, Germany). Twelve single clones were isolated, seeded, and tested for TPO activity in the guaiacol assay. The clone with the highest activity, referred to as FTC-238/TPO, was chosen for additional experiments. Cells were grown in Iscove’s basal medium with 10% (vol/vol) fetal bovine serum, 700 µg/ml G418 (Calbiochem, Bad Soden, Germany), and penicillin/streptomycin (100 U/ml and 100 µg/ml, respectively). Media and solutions for cell culture were obtained from Biochrom (Berlin, Germany).

Tissue samples
Human tissue samples were obtained from patients undergoing surgery for goiter at the Klinik für Allgemein-, Viszeral- und Gefaßchirurgie, Martin-Luther-Universität Halle/Wittenberg. The study has been approved by the local University Ethical Committee, and all patients have given their written consent.

Chemicals
Compounds tested were as follows: genistein (plant isoflavone), 4-MBC, OMC, BP2 and BP3 (UV filters), BPA (building block for polycarbonates), and F21388 (synthetic flavonoid). Chemical structures are shown in Fig. 1Go. They were purchased at the highest purity available from Sigma (Munich, Germany), Roth (Karlsruhe, Germany), BASF (Ludwigshafen, Germany), Riedel-de Haen (Seelze, Germany), or Merck (Darmstadt, Germany). F21388 was synthesized and kindly provided by P. Schreier (Institute for Food Chemistry, University of Würzburg, Würzburg, Germany). The chemicals were dissolved at 1 mol/liter or 100 mmol/liter in dimethylsulfoxide (DMSO) and kept at –20 C in the dark. In the assay mixes, they were used at concentrations from 1 nmol/liter up to 600 µmol/liter final concentration added to the assay mixtures in 10 µl of DMSO. Although DMSO did not alter TPO activities at the concentration used (1%), control mixes routinely contained 10 µl of DMSO as a solvent control.


Figure 1
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  		FIG. 1. Chemical structures of substrates, inhibitors, and EDCs used in TPO assays. 1) guaiacol; 2) tyrosine (for comparison); 3) BP3; 4) BP2; 5) BPA; 6) genistein; 7) 4-MBC; 8) OMC; 9) F21388; 10) PTU; and 11) MMI.

 
TPO assay
FTC-238/TPO cells were grown in basal Iscove’s medium with 10% (vol/vol) fetal bovine serum and stimulated with hematin (1 µg/ml; Sigma) for 2 d. Afterwards, cells were harvested, lysed by ultrasonification (10 pulses of 0.6 sec at 200 W), and centrifuged for 1 h at 105 x g and 4 C. The precipitate was incubated for 24 h at 4 C in 1% (wt/vol) digitonin and centrifuged again as described. The supernatant was used for TPO assays.

For the preparation of rat TPO (17), single thyroid lobes were homogenized in 1 ml of a buffer containing 5 mmol/liter potassium phosphate (pH 7.4), 200 mmol/liter sucrose, 500 U/ml catalase, and 1 mmol/liter EDTA. The homogenate was centrifuged at 600 rpm for 10 min at 4 C, the precipitate was discarded, and the supernatant was centrifuged at 100,000 x g for 45 min at 4 C. The supernatant was discarded, and the precipitate was resuspended in 100 µl of 200 mmol/liter potassium phosphate buffer (pH 7.4) and stored at –80 C. Protein concentrations were determined by a modified Bradford assay (Bio-Rad, Munich, Germany).

All TPO measurements were repeated at least three times. The guaiacol oxidation assay (34) was performed in a mixture containing 100 µg extract protein, 50 mmol/liter potassium phosphate buffer (pH 7.4), 40 mmol/liter guaiacol, and 220 µmol/liter H2O2. The increase in the concentration of the oxidation product 3,3'-dimethoxy-4,4'-biphenochinone was measured at a wavelength of 470 nm (molar extinction coefficient {epsilon}: 26,600 liter x mol–1 x cm–1). In the iodine oxidation assay (35), 20 µg of membrane protein was used in a mixture containing 50 mmol/liter potassium phosphate buffer (pH 7.4), 50 mmol/liter iodide, and 0.25 mmol/liter H2O2. Accumulation of the I3– ion was detected photometrically at a wavelength of 353 nm ({epsilon}: 22,900 liter x mol–1 x cm–1). TPO activities are given as micromole H2O2 reduced per minute and per milligram membrane extract protein. For the TPO inactivation assay (16), TPO-containing extract (325 µg) was preincubated with EDCs (100 nmol/liter to 10 µmol/liter) and/or H2O2 (1–10 µmol/liter) in 100 mmol/liter potassium phosphate buffer (pH 7.4) at a final volume of 600 µl. The incubation was started with H2O2, and aliquots were removed after 0 (i.e. before starting), 2.5, 5, 7.5, and 10 min. The remaining TPO activity was then determined in a conventional guaiacol assay using 100 µl of the preincubation mixture.

Iodide uptake assay
The rat thyroid cell line FRTL-5 was grown in Coon’s modified Ham’s F12 medium with 5% fetal calf serum and a mix of six hormones and growth factors (6H medium) (36). Cells were passaged every 7 d, and medium was changed every 3–4 d. For iodide uptake assays, cells were seeded into 24-well plates. Cells were then grown to confluence and iodide uptake was tested in the presence of EDCs to detect direct interference with the function of NIS. Alternatively, cells were grown for 5 d in the presence of EDCs or 1 µmol/liter all trans-retinoic acid in serum-free medium, and iodide uptake was measured in the absence of additives to elucidate possible effects on NIS expression. Incubation with DMSO (solvent control) did not result in any difference.

For the assay, cells were washed with 0.5 ml Hank’s buffered salt solution (HBSS) [in mmol/liter: 137 NaCl, 5.4 KCl, 1.3 CaCl2, 0.4 MgSO4, 0.5 MgCl2, 0.4 Na2HPO4, 0.44 KH2PO4, 5.55 glucose, and 10 HEPES (pH 7.3)] and overlaid with HBSS containing 10 µmol/liter NaI and carrier-free [125I]Na to give a specific activity of 10–20 mCi/mmol. NaClO4 (10 µmol/liter) was added to part of the reactions to control for specific uptake. After 30 min at 37 C in a humid atmosphere, the radioactive medium was aspirated, cells were washed with ice-cold HBSS, and radioactive iodide was extracted with 1 ml of ethanol at –20 C and counted in an LKB Wallac 1277 Gammamaster counter (GE Healthcare, Little Chalfont, Buckinghamshire, UK). Protein concentrations in cell homogenates were determined by a modified Bradford protein assay (Bio-Rad), and results were calculated as the amount of iodide accumulated per microgram of protein.

Animals and treatments
Adult 2-month-old female Sprague Dawley rats were purchased from Winkelmann (Borchen, Germany). Animals were bilaterally ovariectomized and kept under standard conditions (soy-free rat diet containing 2.1 mg/kg iodide, water available ad libitum, illumination from 0600 to 1800 h, room temperature at 23 C, and relative humidity of 55%). At 14 d after ovariectomy, animals (12 per group) were treated orally via gavage once per day with either olive oil (control), 600 µg/kg body weight (BW), estradiol valerate (E2) or with 10, 33, 100, 333, and 1000 mg/kg BW BP2. The applied volume was 1 ml. The treatment was conducted between 0530 and 0630 h. At day 5, 3–4 h after the last application, animals were decapitated under deep CO2 anesthesia, and blood was collected from the trunk. Thyroid glands were removed and snap frozen. Samples were stored at –70 C until workup for enzyme assays or immunological analysis. The study has been approved by the local University Ethical Committee.

Measurement of serum hormone levels
Total T4 and total T3 were determined by RIA using the DSL-3200 and DSL-3100 kits, respectively, according to the instructions of the manufacturer (Diagnostic Systems Laboratories, Webster, TX). TSH was determined by RIA as described previously (37, 38).

5'DI assay
Livers of nine to 12 animals per treatment group were analyzed for 5'DI activity. Pulverized liver samples (~50 mg) were homogenized in a buffer containing 250 mmol/liter glucose, 20 mmol/liter HEPES, 1 mmol/liter EDTA, and 1 mmol/liter dithiothreitol by ultrasonification (20 pulses of 0.6 sec at 200 W) and centrifuged at 10,000 x g. Supernatants were decanted. Precipitates were resuspended in homogenization buffer by another course of ultrasonification (10 pulses of 0.6 sec at 200 W) and used for the assay. The specific activity of 5'DI was determined measuring the release of 125I– from 125I-labeled reverse T3 in the presence of 100 nmol/liter unlabeled reverse T3. The incubation volume was 100 µl, and the incubation time was 1 h at 37 C. Samples were measured in triplicates using 20 µg protein. 5'DI activity was expressed as picomoles of 125I– released per milligram of protein per minute.

Statistics
Statistical analysis was performed using the SYSTAT program (SPSS, Chicago, IL). Data were analyzed by ANOVA or Kruskal-Wallis test with the different treatments as grouping factors. When the main effect was significant (P < 0.05), treatment groups were individually compared with the control group by Student’s t test or Mann-Whitney U test. P < 0.05 was considered significant. IC50 values were determined using the GraphPad Prism program (GraphPad Software, San Diego, CA).


   Results
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 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Functional characterization of hrTPO extracts
Three membrane protein extracts containing hrTPO prepared from FTC-238/TPO cells were used in these series of experiments. The total yields of protein and the specific activities for the different batches (calculated as the mean of all control reactions performed with the respective extract in a guaiacol assay) are summarized in Table 1Go. The various hrTPO extracts were characterized by determining their sensitivity to the classical TPO inhibitors MMI and PTU as well as to BP2 and compared with TPO extracts prepared from four different human goiter samples. Figure 2Go shows the respective dose-response curves for TPO inhibition in the guaiacol assay, and the corresponding IC50 values are listed in Table 1Go. They were in the same order of magnitude in the recombinant cellular and the human thyroid tissue extracts.


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  		TABLE 1. Properties of several TPO-containing membrane extract preparations

 

Figure 2
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  		FIG. 2. Comparison of the inhibition of TPO activity in membrane protein extracts prepared from FTC-238/TPO cells and human goiters. Guaiacol oxidation assays were performed with varying concentrations of inhibitors as indicated or with DMSO as a solvent control. Dose-response curves were used to determine IC50 values for MMI (A), PTU (B), and BP2 (C).

 
Several compounds suspected as EDCs, especially BP2, inhibit human TPO
The compounds displayed in Fig. 1Go were tested for their ability to interfere with hrTPO activity (Fig. 3Go). Genistein, a well-characterized inhibitor of rat TPO and a goitrogen in humans (13, 16, 17), was used as a control. This compound reduced hrTPO activity with IC50 values of 61.1 and 2.06 µmol/liter in the guaiacol and in the iodide oxidation assay, respectively. 4-MBC, OMC, and BP3 had no effect (data not shown), whereas half-maximal inhibition of TPO by BPA and F21388 occurred at 174 and 37.5 µmol/liter in the guaiacol assay. Interestingly, IC50 values for the UV filter BP2 were 0.45 µmol/liter in the guaiacol assay and 0.37 µmol/liter in the iodide oxidation assay. These values are markedly lower than those observed for genistein, MMI (9.2 and 8.0 µmol/liter), and PTU (125 and 13.5 µmol/liter). Inhibition by nanomolar concentrations of BP2 was also observed in the case of rat TPO (data not shown).


Figure 3
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  		FIG. 3. Inhibition of human TPO activity by EDCs. Shown is the dose response for TPO inhibition in the guaiacol oxidation assay by genistein, F21388, BPA, and BP2.

 
hrTPO is inactivated by H2O2 in combination with low concentrations of BP2
hrTPO-containing extract was preincubated for different time intervals without guaiacol but with increasing concentrations of H2O2 and/or EDCs. Aliquots of this preincubation mixture were then measured in a conventional guaiacol assay to determine the remaining TPO activity. Figure 4AGo shows a rapid inactivation of hrTPO by H2O2 and genistein, which was already maximal after 2.5 min. Accordingly, all of the following inactivation assays were preincubated for 2.5 min.


Figure 4
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  		FIG. 4. Inactivation of human TPO by EDC. A, Membrane extracts containing hrTPO were preincubated in the presence of genistein and H2O2 at the specified concentrations. Aliquots were taken after different time intervals as indicated and tested for remaining enzyme activity in the guaiacol oxidation assay. {circ}, Untreated control; {square}, 10 µmol/liter H2O2; {blacktriangleup}, 10 µmol/liter H2O2 plus 100 nmol/liter genistein; {diamondsuit}, 10 µmol/liter H2O2 plus 1 µmol/liter genistein; and {blacksquare}, 10 µmol/liter H2O2 plus 10 µmol/liter genistein. B, Dose-dependent inactivation of TPO by H2O2 after an incubation time of 2.5 min. C, Dose-dependent inactivation of TPO by increasing concentrations of EDCs in the presence of constant amounts of H2O2 (10 µmol/liter). D, Dose-dependent inactivation of TPO by increasing concentrations of H2O2 in the presence of constant amounts of EDCs.

 
Preincubation with H2O2 alone lead to a dose-dependent decrease in the remaining TPO activity, reaching about 50% of the control value at 10 µmol/liter (Fig. 4BGo), whereas 10 µmol/liter genistein, 10 µmol/liter F21388, or 0.3 µmol/liter BP2 alone had no inactivating effect (Fig. 4Go, C and D). However, when EDCs and H2O2 were added in combination, the remaining TPO activity was lower than with H2O2 alone. In the presence of the highest amount of H2O2, 10 µmol/liter, inactivation became more marked also with the low EDC concentrations (Fig. 4CGo). If H2O2 was lower than 10 µmol/liter, inactivation was comparatively "mild" even with the highest levels of EDCs (Fig. 4DGo). Again, BP2 was active at the lowest, i.e. nanomolar, concentrations.

Inactivation of hrTPO by H2O2 and BP2 is prevented by iodide
When iodide was added to the preincubation mix, inactivation of hrTPO by H2O2 and by the combination of H2O2 and BP2 was prevented (Fig. 5Go, A and B). The extent of this protection was, however, dependent on the concentrations of the inactivating compounds, because the efficiency decreased with rising H2O2 and BP2 concentrations. Conversely, increasing concentrations of the protecting iodide caused higher remaining TPO activities (Fig. 5CGo). Iodide at 1 mmol/liter present in the preincubation mix was able to restore about 70% of the TPO activity measured in the untreated control reaction. To achieve an optimal rescue effect, iodide had to be present throughout the preincubation; addition of iodide for 2 or 5 min before the following guaiacol assay, but after the preincubation, resulted in a less efficient rescue of TPO activity (Fig. 5CGo). Similar protective effects of iodide were obtained in the case of hrTPO inactivation by genistein and F21388 (data not shown).


Figure 5
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  		FIG. 5. Inactivation of TPO is prevented by iodide. Inactivation assays were performed under addition of increasing concentrations of iodide to the preincubation mixtures. A and B, Prevention by iodide (KI) of the TPO inactivation caused by preincubation with H2O2, BP2, and a combination of both. C, The protective effect of iodide is dependent on its concentration in the reaction mixture and the time point of iodide addition to the reaction mixture; if iodide is added only after preincubation and immediately before the guaiacol assay (for 2 and 5 min), remaining TPO activity is lower.

 
BP2 alters serum hormone levels in rats
To study acute interference of BP2 with thyroid function in vivo, female ovariectomized rats were treated for 5 d with different amounts of BP2. There was a dose-dependent decrease in serum total T4 levels, with significant alterations at 333 and 1000 mg/kg BW (Fig. 6AGo). The small decrease in total T3 levels was not significant (Fig. 6BGo). TSH levels were significantly increased at 333 and 1000 mg/kg BW BP2 (Fig. 6CGo). BP2 did not interfere with the determination of any of the three hormones. TPO activities in the thyroid glands of the same BP2-treated rats were measured ex vivo, but they did not show any significant dose-dependent changes (Fig. 6DGo). In the livers of animals treated with 1000 mg/kg BW BP2, 5'DI activity was significantly decreased, whereas an increase was observed at 33 mg/kg (Fig. 7AGo).


Figure 6
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  		FIG. 6. Serum hormone levels and thyroidal TPO activity in BP2-treated rats. Female ovariectomized rats (n = 11–12) were treated with olive oil (control), 600 µg/kg BW E2, or 10, 33, 100, 333, and 1000 µg/kg BW BP2. A, TSH; B, total T4; C, total T3; D, TPO activity.

 

Figure 7
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  		FIG. 7. BP2 effects on 5'DI activity in rat liver and on iodide uptake in FRTL-5 rat thyroid cells. A, Female ovariectomized rats were treated with olive oil (control), 600 µg/kg BW E2, or 10, 33, 100, 333, and 1000 µg/kg BW BP2. Livers (n = 9–12) were used for a deiodinase assay. B, FRTL-5 cells were incubated with 100 nmol/liter to 100 µmol/liter BP2 for 5 d, and iodide uptake was determined. Retinoic acid (RA) at 1 µmol/liter was used as a positive control, and NaClO4 was included to test for specific uptake.

 
BP2 does not inhibit iodide uptake in FRTL-5 cells
We also determined whether BP2 interferes with NIS by testing iodide uptake in vitro. If FRTL-5 cells were preincubated with 100 nmol/liter to 100 µmol/liter BP2 for 1, 3, or 5 d, no change in iodide uptake was detectable (Fig. 7BGo, shown are the values for 5 d). In contrast, addition of 1 µmol/liter retinoic acid markedly reduced iodide accumulation (to 17% after 5 d), which is attributable to a down-regulation of NIS expression (39). Direct addition of BP2 during the uptake assay did not interfere with NIS, whereas addition of 10 µmol/liter NaClO4 reduced iodide accumulation to 3%. A cytotoxic effect of BP2 on FRTL-5 cells was only observed at the highest concentration of 100 µmol/liter (data not shown).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Our previous experiments performed in rats had shown that administration of the UV filters BP2, 4-MBC, and OMC or the presence of soy in the animals’ chow altered thyroid gland weight as well as TSH and/or TH levels (22, 30, 32). Because TPO is a well-known target of the endocrine-disrupting activity of the soy isoflavone genistein (16, 17), we tested whether TPO is also inhibited by UV filters and by other representative substances with endocrine-disrupting activity against components of the TH axis.

For this purpose, a functional enzyme assay system monitoring effects on human TPO was established. We succeeded in the repeated preparation of diverse TPO-containing membrane protein extracts with highly comparable properties such as yield, specific TPO activity, and sensitivity to inhibition by antithyroid drugs. IC50 values for PTU and MMI were approximately 100 and 10 µmol/liter, respectively, which is in agreement with published data (1). This assay system will also provide a suitable platform for the systematic in vitro screening of potential antithyroid drugs in the forthcoming REACH program (for Registration, Evaluation, and Authorization of Chemicals) of the European Union regulating the evaluation and authorization of industrial and environmental chemicals.

Human goiter samples were considered undesirable as a routine supply for TPO isolation because of ethical considerations; in addition, interindividual differences in TPO expression and differences in the clinical and therapeutic history of the thyroid tissues used might result in inconsistencies in the quality and stability of the extract preparations. Therefore, the cell line FTC-238/TPO, which was stably transfected with an expression clone coding for hrTPO, was used as a source for human TPO. TPO-containing extracts prepared from several human goiter samples displayed similar characteristics as those prepared from FTC-238/TPO cells. However, specific activities and IC50 values for inhibitors showed a slightly broader range of variation. We conclude that the use of cell culture-derived hrTPO extracts allows working with a system accurately simulating the properties of native human TPO while ensuring at the same time a greater homogeneity in the TPO activity measurements compared with goiter tissue-derived TPO extracts. This permits reproducible determination of EDC effects on TPO in a "humanized" system with relevance for human health.

We were, above all, interested in the effects of some UV filters, namely OMC, 4-MBC, BP2, and BP3. Among these compounds, only BP2 was a potent inhibitor of hrTPO. The IC50 values of BP2 in the guaiacol oxidation assay were about 20- and 200-fold lower, respectively, than those of the TPO inhibitors MMI and PTU, which are established antithyroid drugs used for the therapy of hyperthyroidism. Different IC50 values were determined for BP2 in the guaiacol vs. the iodide oxidation assay. This was also the case for genistein, MMI, and PTU (data not shown). Because different mechanisms and intermediates are involved in the reactions measured by the two assays, this is not surprising. For MMI and PTU, this was already described, and the relative inhibitory potency of the two compounds may even vary with species (1).

In our experiments, genistein was used as a positive control and inhibited hrTPO with IC50 values of 61.6 and 2.06 µmol/liter in the guaiacol and iodide oxidation assays, respectively. Thus, hrTPO exhibited a similar sensitivity for genistein as reported previously for porcine, bovine, rat, and human goiter TPO (17). BPA and the synthetic flavonoid F21388 compete with TH for their binding to TR (28) and TTR (33), respectively. As phenol derivatives, they also inhibited TPO with IC50 values in the same range as that of genistein. Thus, these chemicals demonstrate that a single compound may interact with more than one target in an endocrine axis. Whether they act as TPO inhibitors in vivo remains to be established.

TPO catalyzes all steps in the formation of TH (1): the oxidation of iodide, the iodination of selected tyrosyl side chains of thyroglobulin, and the covalent coupling of monoiodotyrosyl and diiodotyrosyl residues to give thyroglobulin-bound T3 and T4. By reaction with its essential cosubstrate H2O2, TPO is transformed into "compound I," a FeIV-containing porphyrin {pi}-cation, which is regenerated to the native, FeIII-harboring form by the oxidation of iodide. In the absence of iodide, the {pi}-cation radical isomerizes irreversibly to an inactive protein radical form of TPO. Thus, incubation of TPO with H2O2 in the absence of iodide results in inactivation of TPO (2). Furthermore, the protein radical is the form of TPO that reacts with genistein during suicide inactivation of TPO by this flavonoid (16).

We tested whether this inactivation also occurs in our system by first preincubating hrTPO in the absence of an oxidizable substrate (i.e. iodide or guaiacol) with increasing concentrations of H2O2. This resulted in a dose-dependent inactivation of TPO, measured as a reduction of the remaining TPO activity in a succeeding guaiacol test. The remaining TPO activity was even lower when the preincubation was performed with H2O2 and genistein together, although genistein alone had no inactivating effect. Similar results have been reported by Chang and Doerge (17) who observed a reduction of the remaining enzyme activity by 62% after a 5 min preincubation with 10 µmol/liter genistein and 100 µmol/liter H2O2 using a commercial preparation of human TPO.

We then examined whether BP2 can inactivate hrTPO. Similar to genistein, BP2 alone had no such effect. However, the combination of H2O2 and BP2 did inactivate the enzyme. Thus, BP2 is dependent on H2O2 to exert its inhibitory action, but, when H2O2 is present, BP2 enhances its effect. This reactivity probably depends on the presence of two resorcinol groups in the BP2 molecule. Resorcinol as well as several plant flavonoids that contain a resorcinol moiety in their structure were shown to be inhibitors of TPO (40, 41). For example, genistein serves as an alternative substrate for iodination in its resorcinol ring or, in the absence of sufficient iodide, covalently binds to TPO and causes suicide inactivation (16). It is plausible that BP2 acts in a comparable manner.

However, if adequate concentrations of iodide were present in the incubation mixture, TPO inhibition by BP2 was prevented. Similar findings have been reported for genistein (16). The efficiency of this prevention was clearly dependent on the concentration of iodide during the inactivation reaction. This is probably attributable to the above-mentioned ability of iodide to regenerate the native FeIII-harboring form of TPO and thereby prevent the {pi}-radical form of TPO compound I from irreversible isomerization to the inactive protein radical form (2). Moreover, suicide inactivation is prevented because this mechanism requires the protein radical form of TPO (16). These findings are also of physiological relevance, because they suggest that adverse effects of EDCs may add to and aggravate the outcome of an inadequate iodide supply.

In vivo administration of BP2 revealed rapid interference with the thyroid axis. Already after 5 d of exposure, T4 was significantly reduced whereas TSH was significantly increased at the two highest BP2 concentrations. This indicates the relevance of our observations in vitro for a possible disruption of endocrine regulation in vivo.

TPO activity in the thyroid glands of the treated animals was not yet significantly altered. However, this does not imply that there is no inhibitory effect on TPO by circulating BP2 in vivo. Similar observations were reported by Davidson et al. (42) and Taurog and Dorris (43). In one of their experiments, PTU and MMI were injected into rats, whereas control animals received saline. After 15 min, an injection of 125I– followed. After 30 min more, thyroid glands were isolated and homogenized. The thyroids of the animals were removed and used for either measuring the accumulated radioactivity or preparation of TPO. PTU- and MMI-treated rats had incorporated into their thyroid homogenate proteins less than 1% of the amount of 125I– that was measured in the thyroid extracts of the controls, indicating a reduced TPO activity in the drug-treated intact animals. However, in vitro TPO activity in MMI- and PTU-treated animals was not diminished. This was interpreted as a consequence of the iodide that was present in vivo and prevented irreversible inactivation of TPO as discussed above. In vivo, TPO activity is reduced nevertheless as a result of reaction with the alternative substrates PTU and MMI. A similar scenario may be imagined for BP2.

In contrast to our findings, Chang and Doerge (17) reported that TPO activity in the thyroid glands of rats treated with genistein was reduced; enzyme activity was indirectly proportional to the content of genistein that had accumulated in their thyroid glands. Interestingly, however, these animals did not show signs of hypothyroidism, although they experienced long-term treatment in this case starting in utero and ending at postnatal day 190. However, the authors also discuss that iodide supply is an important factor that influences the outcome of such an in vitro experiment, because iodide can reverse soy-induced goiter in rats and also in humans (13), and they state that their rats received normal amounts of iodide. Our rats were also fed an iodide-sufficient diet. Thus, the marked reduction in T4 levels might be attributable to the low IC50 of BP2 that probably makes it a more efficient inhibitor than genistein. Alternatively, BP2 might induce additional processes that lower serum levels of T4 (e.g. liver metabolism).

Finally, histological examination of the thyroids of BP2-treated rats did not yet reveal any significant changes either (data not shown). Thus, profound changes in thyroid morphology as well as protein levels might require longer exposure to this UV filter.

In the liver of the treated animals, we found a significant reduction of 5'DI activity at the highest BP2 concentration of 1000 mg/kg BW. Preliminary RT-PCR data indicate a more pronounced down-regulation of 5'DI mRNA in the liver of the BP2-treated rats, probably preceding the down-regulation of 5'DI enzyme activity. RT-PCR data also indicate an induction of sulfotransferase and uridine 5'-diphosphate-glucuronyltransferase mRNAs (Huhne, K., and C. Schmutzler, unpublished data). It is an open question whether BP2 also induces the corresponding enzyme activities; this will have to be tested in additional experiments. In contrast, we found neither binding of BP2 to TTR in vitro nor a T3-agonistic or -antagonistic action in a TH-dependent transcriptional reporter assay (Radovic, B., and P. J. Hofmann, unpublished data). We also tested whether BP2 interferes with iodide uptake in FRTL-5 cells. However, we did not observe any effect of this UV screen on NIS activity, neither via direct inhibition nor by down-regulation of NIS expression.

It is still intensively debated whether UV filters can pass the skin and reach concentrations in the serum that are high enough to have adverse effects on endocrine regulation. Among others, three recent studies addressed this problem. After submerging female immature hairless (hr/hr) rats in olive oil containing 5 or 7.5% 4-MBC, uterine weight was increased (44). In the contribution by Gonzalez et al. (45), 25 healthy volunteers applied a commercially available sun protection solution containing 4% BP3 two times a day for 5 d evenly over their whole body. Each volunteer received 2 mg/cm2 body surface area, resulting in total amounts of BP3 applied varying from 10.4 to 18.8 g. During the time of application, all urine was collected, and BP3 was determined by HPLC. A total of 1.2–8.7% of the applied amounts of BP3 were detected in the urine, suggesting dermal resorption of the compound. In the work of Janjua et al. (46), a specially prepared basic cream formulation containing 10% (wt/wt) of each 4-MBC, OMC, and BP3, i.e. the concentrations allowed for sunscreen formulations in Europe, was applied by healthy volunteers. Afterwards, 4-MBC was present in the serum at a concentration of 79 nmol/liter, which is a level showing already significant effects on iodide uptake in the rat thyroid cell line FRTL-5 (Huhne, K., and C. Schmutzler, unpublished data). BP2 was not examined, but the closely related compound BP3 was found at concentrations of up to 1.31 µmol/liter. Suppose that a similar value is achieved by BP2; this would be well above its IC50 values determined for the inhibition of TPO. In this context, it should be mentioned that BP3 is metabolized in rats to give 2,4,-dihydroxy-benzophenone as one of the major metabolites, which also contains the resorcinol moiety found in BP2 (47).

Altogether, it must be assumed that several UV filters penetrate the skin, distribute to tissues such as liver, kidney, spleen, heart, muscle, testes, and fat in rats (30, 48), and appear in the serum at concentrations that cause inhibition of processes involved in TH biosynthesis in vitro, namely iodide uptake and organification. Whether UV screens leaking from plastic material and daily life articles present any risk attributable to human exposure remains to be clarified.

In summary, using a "humanized" in vitro test system, we present evidence that several suspected EDCs inhibit hrTPO and, in some cases, also synergize with H2O2 in the inactivation of this enzyme. The most potent compound in these experiments was BP2, which is contained in many cosmetics and may be in contact with human skin practically every day. BP2 displayed IC50 values for inhibition much lower than those of PTU and MMI. Rats treated with BP2 for only 5 d experienced the classical hormonal alterations characteristic for a beginning hypothyroid state. This indicates a strong antithyroid potential of this UV filter. If BP2 can, analogous to the closely related compound BP3, penetrate human skin, this could well have consequences for human health. Because the ability of the tested EDCs to inactivate TPO was clearly dependent on the availability of iodide during the incubation, an additional point of concern in this context is the still prevailing iodide deficiency in wide parts of the world, including developed countries (49, 50). EDCs such as BP2 may add to and aggravate adverse effects by an inadequate iodide supply, especially in risk groups such as children, adolescents, and lactating women who usually have a higher iodide demand. This may represent a challenge to endocrine regulation that requires more consideration in the future.


   Acknowledgments
 
We acknowledge the excellent technical assistance of Nicole Abdallah, Rosemarie Großklaus, and Benjamin Minow.


   Footnotes
 
First Published Online March 22, 2007

Abbreviations: BP2, Benzophenone 2; BP3, benzophenone 3; BPA, bisphenol A; BW, body weight; 5'DI, type I 5'-deiodinase; DMSO; dimethylsulfoxide; E2, estradiol valerate; EDC, endocrine disrupting chemical; HBSS, Hank’s buffered salt solution; hr, human recombinant; 4-MBC, 4-methylbenzylidene-camphor; MMI, methimazole; NIS, sodium iodide symporter; OMC, octyl-methoxycinnamate; PTU, propylthiouracil; TH, thyroid hormone; TPO, thyroid peroxidase; TR, thyroid hormone receptor; TTR, transthyretin.

This work was supported by European Union Grant EKV1-CT-2002-00128 Multi-Organic Risk Assessment of Selected Endocrine Disrupters (www.eurisked.org) and by a Ph.D. student fellowship of the Charité awarded to I.G.

C.Schm., A.B., K.H., P.A., H.K., C.Schl., C.H.-V., A.G., H.J. have nothing to disclose. I.G. received lecture fees from Bionorica (Neumarkt, Germany). W.W. consults for and received lecture fees from Bionorica. J.K. received lecture fees from Biosyn Arzneimittel (Fellbach, Germany).

Received September 18, 2006.

Accepted for publication March 9, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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