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Toxicokinetics and biotransformation of 3-(4-methylbenzylidene)camphor in rats after oral administration
Toxicology and Applied Pharmacology 216 (2006) 331 – 338 www.elsevier.com/locate/ytaap
Toxicokinetics and biotransformation of 3-(4-methylbenzylidene)camphor in rats after oral administration Wolfgang Völkel a , Thomas Colnot b , Ute M.D. Schauer a , Thomas H. Broschard b , Wolfgang Dekant a,⁎ a
Department of Toxicology, University of Würzburg, Versbacherstrasse 9, 97078 Würzburg, Germany b Institute of Toxicology, Merck KGaA, 64271 Darmstadt, Germany Received 5 April 2006; revised 15 May 2006; accepted 18 May 2006 Available online 23 May 2006
Abstract 3-(4-Methylbenzylidene)camphor (4-MBC) is an UV-filter frequently used in sunscreens and cosmetics. Equivocal findings in some screening tests for hormonal activity initiated a discussion on a possible weak estrogenicity of 4-MBC. In this study, the toxicokinetics and biotransformation of 4-MBC were characterized in rats after oral administration. Male and female Sprague–Dawley rats (n = 3 per group) were administered single oral doses of 25 or 250 mg/kg bw of 4-MBC in corn oil. Metabolites formed were characterized and the kinetics of elimination for 4-MBC and its metabolites from blood and with urine were determined. Metabolites of 4-MBC were characterized by 1H NMR and LC-MS/MS as 3-(4carboxybenzylidene)camphor and as four isomers of 3-(4-carboxybenzylidene)hydroxycamphor containing the hydroxyl group located in the camphor ring system with 3-(4-carboxybenzylidene)-6-hydroxycamphor as the major metabolite. After oral administration of 4-MBC, only very low concentrations of 4-MBC were present in blood and the peak concentrations of 3-(4-carboxybenzylidene)camphor were approximately 500-fold above those of 4-MBC; blood concentrations of 3-(4-carboxybenzylidene)-6-hydroxycamphor were below the limit of detection. Blood concentration of 4-MBC and 3-(4-carboxybenzylidene)camphor peaked within 10 h after 4-MBC administration and then decreased with half-lives of approximately 15 h. No major differences in peak blood levels between male and female rats were seen. In urine, one isomer of 3-(4carboxybenzylidene)hydroxycamphor was the predominant metabolite [3-(4-carboxybenzylidene)-6-hydroxycamphor], the other isomers and 3-(4carboxybenzylidene)camphor were only minor metabolites excreted with urine. However, urinary excretion of 4-MBC-metabolites represents only a minor pathway of elimination for 4-MBC, since most of the applied dose was recovered in feces as 3-(4-carboxybenzylidene)camphor and, to a smaller extent, as 3-(4-carboxybenzylidene)-6-hydroxycamphor. Glucuronides of both metabolites were also present in feces, but partly decomposed during sample workup and were thus not quantified. The results show that absorbed 4-MBC undergoes extensive first-pass biotransformation in rat liver resulting in very low blood levels of the parent 4-MBC. Enterohepatic circulation of glucuronides derived from the two major 4-MBC metabolites may explain the slow excretion of 4-MBC metabolites with urine and the small percentage of the administered doses recovered in urine. © 2006 Elsevier Inc. All rights reserved. Keywords: Biotransformation; Blood levels; Endocrine; LC-MS/MS; Electrospray
Introduction Sunscreen formulations frequently contain 3-(4-methylbenzylidene)camphor (4-MBC) as an UV-filter to reduce the intensity of UV-radiation reaching the skin. Commercial sunscreen lotions contain up to 4% 4-MBC. Based on a human study, the extent of dermal absorption of 4-MBC from
⁎ Corresponding author. Fax: +49 931/201 48865. E-mail address: [email protected] (W. Dekant). 0041-008X/$ - see front matter © 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.taap.2006.05.012
sunscreen lotions in humans is estimated to be approximately 0.5% (Schauer et al., accompanying manuscript). A possible estrogenic activity of 4-MBC was suggested based on the observation that 4-MBC induced cell proliferation in MCF-7 breast cancer cells transfected with the estrogen receptor and increased uterine weight in immature Long–Evans rats exposed to doses of 119 mg/kg/day for 4 days (Schlumpf et al., 2001; Ma et al., 2003; Schlumpf et al., 2004a, 2004b). An interaction of 4-MBC with the estrogen receptor could not be consistently demonstrated in vitro (Mueller et al., 2003; Schreurs et al., 2005), but estrogenic effects of 4-MBC in in
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vivo assays for uterotrophic activity and vaginal-stratification were observed (Ashby, 2001; Tinwell et al., 2002). The equivocal or negative findings in in vitro and positive results in in vivo screening assays for estrogenicity suggest a major impact of the metabolism on the endocrine activity of 4-MBC. These considerations prompted us to investigate the metabolism and kinetic behavior of 4-MBC in rats after oral administration. These observations suggest that activation of the weak estrogen 4-MBC to a more potent estrogen may occur and that metabolite(s) may be responsible for the estrogenic effects of 4MBC in vivo. Due to the possible bioactivation of 4-MBC to a metabolite with higher estrogenic potency, toxicokinetic considerations will be important for a reasonable risk assessment for species and route-to-route extrapolations. Extent of metabolite formation and clearance is expected to be widely differing after oral administration, which was used as route of application in many studies on the toxicity of 4-MBC, compared to the dermal exposure of humans to 4-MBC from sunscreens. Material and methods Chemicals 4-MBC was supplied by Merck KGaA (Darmstadt, Germany). All other reagents and solvents were reagent grade or better and obtained from several commercial suppliers. For recording of 1H NMR-spectra, 3-(4-carboxybenzylidene)-6-hydroxycamphor was isolated from urine pools (26 ml from male and 32 ml from female rats) by liquid–liquid extraction with diethyl ether (v:v 1:2). After evaporation of the solvent, the residue was diluted 1:100 with methanol and separated by HPLC to obtain the compound in a purity >95% (Bernauer et al., 1998). 3-(4-Carboxybenzylidene)camphor was isolated from feces by Soxhlet extraction and HPLC separation to obtain the compound in a purity >95%.
Animal treatment Male and female Sprague–Dawley rats (12–13 weeks) were purchased from Harlan–Winkelmann, Borchen, Germany. The animals (3 per dose group) had free access to water and a standard diet (Altromin) and were kept under standard conditions (12 h day/night cycle, temperature 21–23 °C, humidity 45–55%). All animal experimentation was performed under permit from the appropriate authorities in the approved animal care facility of the department. Animals were transferred into metabolic cages for collection of control urine and feces for 4 days before treatment. Animals were then administered 4-MBC (25 and 250 mg/ kg body weight), dissolved in corn oil, at 10 am. To obtain a homogeneous dosing solution, 4-MBC was homogenized in corn oil during 5 min using an ultrasonic homogenizer. A dosing volume of 5 ml/kg bw of corn oil was applied. Immediately after dosing, animals were transferred to metabolic cages (one rat per cage) and urine and feces samples were collected for 94 h after dosing. Collecting vessels for urine and feces were cooled with mixtures of ice and sodium chloride. Urine samples were collected in predefined intervals, urine volume was recorded, samples were divided into aliquots, and stored at −20 °C. Feces were collected in 24 h intervals, weighted and also stored at −20 °C. Blood samples (100 μl) were collected from the tail vein at predetermined time points and plasma was immediately prepared by centrifugation (15 min at 1000 × g). Plasma samples were also stored at −20 °C. Feces were freeze-dried and 500 mg of the dry residues were extracted by Soxhlet-extraction with methanol (60 ml) for 4 h.
pump and an Agilent 1040 M series II diode array detector or an Agilent 1090 HPLC system. Gradient elution using a linear gradient from 100% water (with 0.1% trifluoroacetic acid) to 100% acetonitrile (with 0.1% trifluoroacetic acid) over 25 min, held for 5 min, with a flow rate of 1 ml/min was applied. The eluate was monitored at 300 nm. Calibration solutions were generated by addition of 4MBC to urine and blood samples from control rats. Due to the identical UVspectra, 3-(4-carboxybenzylidene)-6-hydroxycamphor and 3-(4-carboxybenzylidene)camphor were quantified based on calibration curves obtained with 4-MBC. Calibration curves were calculated from 6 data points using Microsoft Excel spreadsheets. The R2-values of the calibration curves were >0.999. Standard deviations (mean ± SD), elimination half-lives and areas under the curve were calculated using Microsoft Excel spread sheets and default setting. Polynoms given for best fit by Microsoft Excel were transferred into “Functions” (NumericalMathematics.com), AUCs were calculated from the mean of each time point. Liquid chromatography/mass spectrometry. For the recording of mass spectra of 4-MBC metabolites, urine samples were diluted with MeOH and H2O (1:2:2), vortexed and centrifuged to precipitate protein. The diluted samples (10 μl) were separated on a Nucleosil RP18 HPLC column (2.0 mm × 150 mm; 5 μm, 100 Å; Phenomenex, Aschaffenburg, Germany) using an Agilent 1100 autosampler and an Agilent 1100 HPLC-pump (Agilent, Waldbronn, Germany). Samples were separated by gradient elution with water (solvent A) and acetonitrile (solvent B) using the following conditions: 100% A, linear increase to 25% B within 5 min, then linear increase to 80% B within 20 min at a flow-rate of 200 μl/min. The HPLC system was directly coupled to a triple stage quadrupole mass spectrometer (API 3000, Applied Biosystems, Darmstadt, Germany) equipped with a TurboIon Spray source. Analytes were detected in the negativeion mode at a vaporizer temperature of 400 °C and a TurboIon Spray voltage of −4.0 kV. Spectral data were recorded with nitrogen as collision gas (CAD = 4) in the product ion scan mode or the multiple reaction monitoring (MRM) mode. Product ion scans were performed with calculated molecular weights of each metabolite and monitored from 50 amu to the expected mass units for the molecular ion. For MRM analyses, the samples were diluted 100-fold. To quantify 4-MBC in blood, 25 μl of serum samples from rats was mixed with 25 μl of cold MeOH, and the samples were centrifuged for 4 min at 15,000 × g. The supernatants were then placed on ice for 30 min and 25 μl of cold acetonitrile was added followed by centrifugation for 4 min at 15,000 × g. To quantify 4-MBC by LC-MS/MS after atmospheric pressure photoionization (APPI), 5 μl of the supernatants of the samples was injected on a Synergi Hydro-RP 80 A HPLC column (2.0 mm × 150 mm; 4 μm; Phenomenex, Aschaffenburg, Germany) using an Agilent 1100 autosampler and an Agilent 1100 HPLC-pump. The samples were separated by elution with water (solvent A) and acetonitrile (solvent B) using the following conditions: 10% A, 90% B, isocratic for 7 min, with a flow rate of 300 μl/min. Toluene was used as dopant with a flow rate of 1.5 ml/h. The HPLC system was directly coupled to the mass spectrometer equipped with an atmospheric pressure photoionization source. 4-MBC was detected in the positive ion mode at a vaporizer temperature of 400 °C. For APPI, an ion source voltage (IS) of 2000 V was applied. Spectral data were recorded with N2 as collision gas (CAD = 4) in the multiple reaction monitoring (MRM) mode with a dwell time of 200 ms for each transition monitoring the MS-MS ion-transitions given in Table 2. To confirm the presence of glucuronides of 4-MBC metabolites, samples were analyzed by LC-MS/MS monitoring the constant neutral loss of a fragment with 176 Da from the calculated molecular ions of glucuronides derived from the two identified metabolites with [M-H]− at m/z 475, respectively 459. NMR spectra were recorded with a BRUKER (Karlsruhe, Germany) 600 MHz NMR spectrometer. The dried residues obtained after metabolite isolation were dissolved in D2O containing 3-(trimethylsilyl)-2,2,3,3-d4-proprionate (TSP). 1H NMR spectra were recorded using a Bruker DPX 600 NMR operating at 600.13 MHz. Chemical shifts were normalized to TSP set at δ = 0 ppm.
Results
Instrumental analysis
Metabolite identification
HPLC with UV-detection. Blood and urine samples were separated using a 250 mm × 4.6 mm ID steel column filled with Hypersil ODS 5 μm (Bischoff, Leonberg, Germany). Separation was performed using a Agilent 1050 quaternary
To detect and identify metabolites of 4-MBC, urine and blood samples from 4-MBC-treated rats were analyzed by
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HPLC with diode array detector by LC-MS/MS and 1H NMR. The diode array detector was selected for metabolite detection since it was not expected that biotransformation results in a major modification of the chromophore present in 4-MBC. Compounds exhibiting the characteristic UV-spectra of 4-MBC present in urine and blood samples as indicated by the diode array detector were further characterized by mass spectrometry, and by 1H NMR after isolation by HPLC. In addition, LC-MS/ MS with specific acquisition conditions to detect glucuronides was used to identify the presence of glucuronides formed from metabolites of 4-MBC. In urine and feces samples collected before administration, peaks with the characteristic UV-spectra of 4-MBC or its metabolites were not present. HPLC separation of urine samples from 4-MBC-treated rats gave five peaks with the characteristic UV-spectra of 4-MBC derivatives (Fig. 1). These results suggest the presence of one major metabolite and three minor metabolites (peaks Ia–d, Fig. 1) with retention times within a range of less than 4 min. An additional metabolite (peak II, Fig. 1) with a lower polarity was present in low concentrations, and was mainly excreted by female rats. Product ion spectra of all five metabolites were recorded by LC-MS/MS. Metabolites Ia–d all exhibited identical mass spectra with molecular weights of 300 Da, m/z 299 represents (M-H)−. The major product formed by fragmentation from
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(M-H)− was an ion with m/z 255. The loss of 44 amu represents CO2, a typical fragmentation observed in the mass spectra of carboxylic acids (Fig. 2). Based on the 4-MBC-structure, the spectral data suggest an oxidation of the methyl group of the aromatic ring in 4-MBC in addition to a further hydroxylation. Analysis of metabolite II by LC-MS/MS in the product ion scan mode gave a (M-H)− ion (m/z 283) and, after collision with N2, loss of CO2 (44 amu) to give an ion m/z 239 (Fig. 3). For further structure determination, Ia and II were isolated by HPLC and analyzed by 1H NMR (Table 1). The losses of the resonances of the methyl group attached to the phenyl ring and the changes in chemical shifts of the protons on the phenyl ring in both metabolites Ia and II in relation to the chemical shifts of the phenyl ring protons in 4-MBC suggest an oxidation of the methyl group on the aromatic ring to a carboxylate in both metabolites consistent with the mass spectral data. Furthermore, the four aromatic protons of both metabolites and 4-MBC retained identical coupling constants indicating a further hydroxylation, as indicated by the mass spectra, did not occur at the phenyl ring. The three remaining methyl groups in the camphor moiety and the vinylic proton in 4-MBC and both metabolites also gave singlets with identical chemical shifts suggesting absence of modifications at these positions in the molecule (Table 1). The proton NMR-data combined with the mass spectra identified the minor urinary metabolite II
Fig. 1. HPLC separation of an urine sample from a male (A) and a female (B) rat collected 14 h after administration of 4-MBC (250 mg/kg bw). UV-spectra of major metabolites are shown in the inserts. Peaks Ia-d represents four isomers of 3-(4-carboxybenzylidene)hydroxycamphor, peak II represents 3-(4-carboxybenzylidene) camphor.
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Fig. 2. Product ion spectrum of 3-(4-carboxybenzylidene)-6-hydroxycamphor (m/z 299, M-H); characteristic fragments are m/z 255 (M·super −H–CO2) and m/z 227 (M·super −H–CO2–CO).
(Figs. 1B and 4) as 3-(4-carboxybenzylidene)camphor formed by the oxidation of the aromatic methyl group to a benzoic acid derivative of 4-MBC (Fig. 4). For metabolite Ia (Figs. 1 and 4), the chemical shift of the single proton in the camphor moiety (Hc) was similar to that of 4-MBC. Therefore, the introduced hydroxyl function is located on one of the two methylene groups in the camphor ring. As a result of the very complex coupling of the four possible diastereomers, it is difficult to localize the position of the hydroxyl group. Due to the same multiplicity (d) with coupling constants of 4.3 Hz in comparison to 4-MBC (d, 4.5 Hz), it is concluded that the hydroxylation occurred at C-6. In combination, 1H NMR and mass spectrometry data suggest that the major 4-MBC metabolite (Ia, Fig. 1) in rat urine corresponds to 3-(4-carboxybenzylidene)-6-hydroxycamphor (Fig. 4). The
three other minor metabolites represent isomers of this compound with the hydroxyl group either in another orientation on C-6 or on C-5 with two possible orientations. The presence of metabolites such as glucuronides and sulfates of the 4-MBC-metabolites was determined a using LCMS/MS method with constant neutral loss scans designed specifically to detect glucuronides and sulfates with high sensitivity. In urine and feces, the constant neutral loss scan showed that glucuronides of both 3-(4-carboxybenzylidene)6-hydroxycamphor ([M-H]− at 475 amu, major fragment at 299 amu) and 3-(4-carboxybenzylidene)camphor ([M-H]− at 459 amu, major fragment at 283 amu) were present. Most likely, both glucuronides represent acyl glucuronides. The presence of glycine conjugation products, which often are formed from reactive acyl glucuronides (Kasuya et al., 1996),
Fig. 3. Product ion spectrum of 3-(4-carboxybenzylidene)camphor (m/z 283, M−); characteristic fragment ion is m/z 239 (M−CO2).
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Table 1 H NMR chemical shifts and multiplicity of signals in 1H NMR spectra of 3-(4-carboxybenzylidene)-6-hydroxycamphor and 3-(4-carboxybenzylidene)camphor isolated as metabolites from urine or feces of 4-MBC-tretaed rats in comparison to 4-MBC (d, duplet; m, multiplet; s, singlet; q, quartet), 1H NMR-resonances of 4-MBC are given for comparison
1
4-MBC
3-(4-Carboxybenzylidene)-6-hydroxycamphor
3-(4-Carboxybenzylidene) camphor
Chemical shift (δ)
Multipl.
Assig.
Chemical shift (δ)
Multipl.
Assig.
Chemical shift (δ)
Multipl.
Assig.
0.79 1.00 1.03 1.52 1.59 1.77 2.18 2.37 3.10 7.20 7.22 7.39
s s s m m m m s d d s d
CH3 CH3 CH3 C–H C–H C–H C–H Ph–CH3 Hc Hb–Hb' Hd Ha–Ha'
0.71 0.99 1.03 – 2.78 2.82 4.30 – 3.26 7.98 7.30 7.63
s s s – m m q – d d s d
CH3 CH3 CH3 C–H C–H C–H C–H – Hc Hb–Hb′ Hd Ha–Ha′
0.72 0.92 0.96 1.52 1.59 1.81 2.20 – 3.09 7.89 7.16 7.41
s s s m m m q – d d s d
CH3 CH3 CH3 C–H C–H C–H C–H – Hc Hb–Hb′ Hd Ha–Ha′
and sulfate conjugates was not indicated by the analytical methods applied. Kinetics of 4-MBC elimination from blood and with urine Blood and urine samples collected at different time points were analyzed by HPLC for the presence of 4-MBC and its metabolites. Experimental results on the elimination of 4-MBC and its metabolites from blood and with urine are given in Figs. 5 and 6, and in Table 2. In blood samples from animals collected before 4-MBC administration, concentrations of 4-MBC and all identified metabolites were below the limit of detection. Blood samples
collected after 4-MBC administration did not contain quantifiable concentrations of 4-MBC when analyzed by HPLC with diode array detection; therefore, the concentrations of 4-MBC in these samples were determined by a more sensitive LC-MS/MS method. With this method, a dose-dependent increase in 4-MBC concentrations in blood was observed after 4-MBC administration in both male and female rats. Blood concentrations of 4-MBC were dose-dependent and peak concentrations between 35 and 60 pmol/ml were reached within 10 h (Figs. 5A, B) after administration of 250 mg/kg. The concentrations of 3(4-carboxybenzylidene)camphor in the blood samples were also dose-dependent, but were much higher as compared to those of 4-MBC and peaked between 90 and 120 nmol/ml within 8 h
Fig. 4. Biotransformation of 4-MBC to 3-(4-carboxybenzylidene)-6-hydroxycamphor and 3-(4-carboxybenzylidene)camphor.
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▪
Fig. 5. Blood concentrations of 4-MBC and its metabolite 3-(4-carboxybenzylidene)camphor, after oral administration of 25 mg/kg bw ( ) and 250 mg/kg bw (♦) to male (panels A and C) or female (panels B and D) rats.
▪
Fig. 6. Urinary excretion of 4-MBC-metabolites in male (panel A, 250 mg/kg bw 4-MBC and panel C, 25 mg/kg bw 4-MBC) and female rats (panel B, 250 mg/kg bw 4-MBC and panel D, 25 mg/kg bw 4-MBC); ♦, sum of isomers of 3-(4-carboxybenzylidene)hydroxycamphor; , 3-(4-carboxybenzylidene)camphor. Concentrations of 3-(4-carboxybenzylidene)camphor were below the limit of detection in urine samples from male rats receiving 25 mg/kg bw 4-MBC (panel C).
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Table 2 Recovery of 4-MBC and its metabolites in excreta after oral administration of doses of 25 and 250 mg/kg bw to male and female rats, data are mean ± SD from 3 male and 3 female rats ♂
♀
25 mg/kg bw
4-MBC applied Urine Feces Total
250 mg/kg bw
25 mg/kg bw
μmol
% of dose
μmol
% of dose
28 ± 1.4 0.6+0.4 n.d.
2.3 ± 1.5 n.d.
416 ± 23 36 ± 3 216 ± 107 252 ± 120
8.6 ± 2.7 52 ± 26.4 61 ± 29.1
after 4-MBC administration at 250 mg/kg. The concentration of this metabolite in blood afterwards declined following firstorder kinetics with a half-life of approximately 15 h in both male and female rats. Concentrations of 3-(4-carboxybenzylidene)-6-hydroxycamphor in the blood samples were at the limit of detection in all samples analyzed and therefore not quantified. In urine samples from 4-MBC treated rats, 3-(4-carboxybenzylidene)-6-hydroxycamphor was the predominant excretory product observed after both of the applied doses. Peak concentrations of 3-(4-carboxybenzylidene)-6-hydroxycamphor in urine were reached within 14 h in males and then slowly declined (Fig. 6). In female rats, urinary elimination of 3(4-carboxybenzylidene)-6-hydroxycamphor was slower as compared to male rats and gave a plateau from 14 h to approximately 38 h after administration; excretion of 3-(4-carboxybenzylidene) camphor was more pronounced in females as compared to males (Figs. 6A, B). In feces samples collected after administration of 4-MBC, 4-MBC and both 3-(4-carboxybenzylidene)-6-hydroxycamphor and 3-(4-carboxybenzylidene)camphor were recovered. The presence of glucuronides of both 4-MBC metabolites was also indicated by LC-MS/MS analysis of feces extracts. A direct quantitation of the glucuronides of these compounds in feces could not be performed since the glucuronides decomposed during the methanol extractions necessary for isolation from the feces matrix. Therefore, it was attempted to quantify unconjugated 3-(4-carboxybenzylidene)camphor and 3-(4-carboxybenzylidene)-6-hydroxycamphor in feces, but results obtained were not well reproducible. This likely is due to the relatively harsh conditions required for extraction and the chemical reactivity of the present acyl glucuronides (SpahnLangguth and Benet, 1992), which may bind to nucleophiles in feces matrix at the elevated temperatures required for extraction. In general, the concentration of 3-(4-carboxybenzylidene) camphor in feces was much higher than those of 3-(4carboxybenzylidene)-6-hydroxycamphor and 4-MBC. Only a minor part of the applied dose of 4-MBC was recovered in urine as metabolites, most of the dose was recovered in feces both as metabolites and as unchanged 4MBC with large variations in recovery (Table 2). Discussion The results of this study show that 4-MBC is intensively metabolized in rodents and its toxicokinetics after oral administration are complex. The identified metabolites suggest
250 mg/kg bw
μmol
% of dose
μmol
% of dose
19 ± 0.3 0.9 ± 8.7 n.d.
4.8 ± 4.7 n.d.
234 ± 7 687 28 ± 14 103 ± 44 131 ± 58
12 ± 6.2 44 ± 18.3 56 ± 24.5
the biotransformation of 4-MBC is initiated by cytochrome P450-mediated oxidation reactions. The first step in the metabolic sequence to result in the 4-MBC metabolite 3-(4carboxybenzylidene)-6-hydroxycamphor likely occurs by hydroxylation of the aromatic methyl group by cytochrome P450 (Fig. 4) (Hanioka et al., 1995). The intermediate, 3-(4hydroxymethylbenzylidene)camphor, represents the major product observed when 4-MBC is incubated with rat or human liver microsomes (Völkel et al., unpublished observation). 3-(4-Hydroxymethylbenzylidene)-6-camphor is then further oxidized, presumably by alcohol dehydrogenase and aldehyde dehydrogenase, to give 3-(4-carboxybenzylidene) camphor as described for other benzylalcohols (Bakke and Scheline, 1970). This compound may then undergo further oxidation, likely again by a cytochrome P450-catalyzed oxidation, to the final excretory product 3-(4-carboxybenzylidene)-6hydroxycamphor. Only a minor percentage of 3-(4-carboxybenzylidene)camphor is excreted with urine, the majority of this compound undergoes further biotransformation to give 3-(4carboxybenzylidene)-6-hydroxycamphor as the major urinary metabolite. This reaction seems to occur extrahepatically, since 3-(4-carboxybenzylidene)-6-hydroxycamphor is not released to blood from liver in quantifiable concentrations. Both 3-(4carboxybenzylidene)-6-hydroxycamphor and 3-(4-carboxybenzylidene)camphor are then excreted with urine due to their low molecular weight. However, the major pathway for the disposition of 3-(4carboxybenzylidene)-6-hydroxycamphor and 3-(4-carboxybenzylidene)camphor likely is represented by an efficient conjugation of both compounds to the corresponding acyl glucuronides. The glucuronides of 3-(4-carboxybenzylidene) camphor and 3-(4-carboxybenzylidene)-6-hydroxycamphor with molecular weights of 476 and 460 Da are above the molecular weight threshold for biliary elimination (approximately 400 Da in rats), are eliminated with bile and likely undergo enterohepatic circulation (Tephly and Burchell, 1990). Enterohepatic circulation may explain the comparatively slow elimination of 4-MBC metabolites with urine. The low blood levels and the low or absent estrogenicity of 4-MBC in vitro and the formation of 3-(4-carboxybenzylidene)6-hydroxycamphor, which shows a somewhat higher activity in a screening assay for estrogenicity (Müller et al., unpublished results), suggest that 4-MBC may exert its weak estrogenic effects in rats after bioactivation to 3-(4-carboxybenzylidene)-6hydroxycamphor. This observation might possibly represent a first example of a biotransformation of a hormonally inactive
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compound to a more potent estrogen and indicates that the reliance on in vitro screening systems without biotransformation capacities for xenobiotics may miss chemicals where estrogenicity only occurs after biotransformation (Degen et al., 2002). The intensive first-pass metabolism of 4-MBC and the role of biotransformation and estrogenic response also stress the importance of toxicokinetics in the risk assessment of “endocrine modulators”. Extent of biotransformation and thus formation of metabolites with either increased or decreased affinity to hormone receptors varies depending on route of application and species-specific capacities for biotransformation (see accompanying manuscript). This underscores the importance of developing complete toxicological data sets before discussing “endocrine modulation” based on in vitro activities in screening assays. For example, widely published “endocrine modulators” such as bisphenol Awith some estrogenicity in vitro are rapidly conjugated in vivo to non-estrogenic products and estrogenicity in vivo is only observed after extreme doses saturating excretory pathways or after unusual routes of application (Pottenger et al., 2000; Völkel et al., 2002). Regarding 4-MBC, the absorption of this compound from skin in the relevant human exposure scenario “application of sunscreens” seems very limited since only low concentrations of 4-MBC in blood were determined after repeated and intensive applications of sunscreens in human subjects (Lademann et al., 2005). Dermal administration may result in a less pronounced first-pass metabolism of 4-MBC and lower concentrations of the estrogenic 3-(4-carboxybenzylidene)-6-hydroxycamphor. However, studies in human subjects determining both 4-MBC and 3-(4-carboxybenzylidene)-6-hydroxycamphor in blood after sunscreen application are needed for a more conclusive hazard assessment (see accompanying manuscript). Acknowledgments This study was supported by Merck KGaA, Darmstadt, Germany. The authors thank Nataly Bittner and Heike KeimHeusler for excellent technical assistance and Dr. M. Grüne, Institut für Organische Chemie der Universität Würzburg, for the recording of 1H NMR-spectra. References Ashby, J., 2001. Confirmation of uterotrophic activity for 4-MBC in the immature rat. Environ. Health Perspect. 109, A517. Bakke, O.M., Scheline, R.R., 1970. Hydroxylation of aromatic hydrocarbons in the rat. Toxicol. Appl. Pharmacol. 16, 691–700.
Bernauer, U., Amberg, A., Scheutzow, D., Dekant, W., 1998. Biotransformation of 12C- and 2-13C-labeled methyl-tert-butyl ether, ethyl-tert-butyl ether, and tert-butyl alcohol in rats: identification of metabolites in urine by 13C nuclear magnetic resonance and gas chromatography/mass spectrometry. Chem. Res. Toxicol. 11, 651–658. Degen, G.H., Janning, P., Wittsiepe, J., Upmeier, A., Bolt, H.M., 2002. Integration of mechanistic data in the toxicological evaluation of endocrine modulators. Toxicol. Lett. 127, 225–237. Hanioka, H., Hamamura, M., Kakino, K., Ogata, H., Jinno, H., Takahashi, A., Nishimura, T., Ando, M., 1995. Dog liver microsomal P450 enzymemediated toluene biotransformation. Xenobiotica 25, 1207–1217. Kasuya, F., Igarashi, K., Fukui, M., 1996. Participation of a medium chain acylCoA synthetase in glycine conjugation of the benzoic acid derivatives with the electron-donating groups. Biochem. Pharmacol. 51, 805–809. Lademann, J., Schanzer, S., Jacobi, U., Schaefer, H., Pflucker, F., Driller, H., Beck, J., Meinke, M., Roggan, A., Sterry, W., 2005. Synergy effects between organic and inorganic UV filters in sunscreens. J. Biomed. Opt. 10, 14008. Ma, R., Cotton, B., Lichtensteiger, W., Schlumpf, M., 2003. UV filters with antagonistic action at androgen receptors in the MDA-kb2 cell transcriptional-activation assay. Toxicol. Sci. 74, 43–50. Mueller, S.O., Kling, M., Arifin Firzani, P., Mecky, A., Duranti, E., ShieldsBotella, J., Delansorne, R., Broschard, T., Kramer, P.J., 2003. Activation of estrogen receptor alpha and ERbeta by 4-methylbenzylidene-camphor in human and rat cells: comparison with phyto- and xenoestrogens. Toxicol. Lett. 142, 89–101. Pottenger, L.H., Domoradzki, J.Y., Markham, D.A., Hansen, S.C., Cagen, S.Z., Waechter Jr., J.M., 2000. The relative bioavailability and metabolism of bisphenol A in rats is dependent upon the route of administration. Toxicol. Sci. 54, 3–18. Schlumpf, M., Cotton, B., Conscience, M., Haller, V., Steinmann, B., Lichtensteiger, W., 2001. In vitro and in vivo estrogenicity of UV screens. Environ. Health Perspect. 109, 239–244. Schlumpf, M., Jarry, H., Wuttke, W., Ma, R., Lichtensteiger, W., 2004a. Estrogenic activity and estrogen receptor beta binding of the UV filter 3benzylidene camphor. Comparison with 4-methylbenzylidene camphor. Toxicology 199, 109–120. Schlumpf, M., Schmid, P., Durrer, S., Conscience, M., Maerkel, K., Henseler, M., Gruetter, M., Herzog, I., Reolon, S., Ceccatelli, R., Faass, O., Stutz, E., Jarry, H., Wuttke, W., Lichtensteiger, W., 2004b. Endocrine activity and developmental toxicity of cosmetic UV filters—an update. Toxicology 205, 113–122. Schreurs, R.H., Sonneveld, E., Jansen, J.H., Seinen, W., van der Burg, B., 2005. Interaction of polycyclic musks and UV filters with the estrogen receptor (ER), androgen receptor (AR), and progesterone receptor (PR) in reporter gene bioassays. Toxicol. Sci. 83, 264–272. Spahn-Langguth, H., Benet, L.Z., 1992. Acyl glucuronides revisited: is the clucuronidation process a toxification as well as a detoxification mechanism? Drug Metab. Rev. 24, 5–48. Tephly, T.R., Burchell, B., 1990. UDP-glucuronosyltransferases: a family of detoxifying enzymes. Trends Pharmacol. Sci. 11, 276–279. Tinwell, H., Lefevre, P.A., Moffat, G.J., Burns, A., Odum, J., Spurway, T.D., Orphanides, G., Ashby, J., 2002. Confirmation of uterotrophic activity of 3(4-methylbenzylidinecamphor) in the immature rat. Environ. Health Perspect. 110, 533–536. Völkel, W., Colnot, T., Csanady, G.A., Filser, J.G., Dekant, W., 2002. Metabolism and kinetics of bisphenol a in humans at low doses following oral administration. Chem. Res. Toxicol. 15, 1281–1287.
Toxicokinetics and biotransformation of 3-(4-methylbenzylidene)camphor in rats after oral administration Wolfgang Völkel a , Thomas Colnot b , Ute M.D. Schauer a , Thomas H. Broschard b , Wolfgang Dekant a,⁎ a
Department of Toxicology, University of Würzburg, Versbacherstrasse 9, 97078 Würzburg, Germany b Institute of Toxicology, Merck KGaA, 64271 Darmstadt, Germany Received 5 April 2006; revised 15 May 2006; accepted 18 May 2006 Available online 23 May 2006
Abstract 3-(4-Methylbenzylidene)camphor (4-MBC) is an UV-filter frequently used in sunscreens and cosmetics. Equivocal findings in some screening tests for hormonal activity initiated a discussion on a possible weak estrogenicity of 4-MBC. In this study, the toxicokinetics and biotransformation of 4-MBC were characterized in rats after oral administration. Male and female Sprague–Dawley rats (n = 3 per group) were administered single oral doses of 25 or 250 mg/kg bw of 4-MBC in corn oil. Metabolites formed were characterized and the kinetics of elimination for 4-MBC and its metabolites from blood and with urine were determined. Metabolites of 4-MBC were characterized by 1H NMR and LC-MS/MS as 3-(4carboxybenzylidene)camphor and as four isomers of 3-(4-carboxybenzylidene)hydroxycamphor containing the hydroxyl group located in the camphor ring system with 3-(4-carboxybenzylidene)-6-hydroxycamphor as the major metabolite. After oral administration of 4-MBC, only very low concentrations of 4-MBC were present in blood and the peak concentrations of 3-(4-carboxybenzylidene)camphor were approximately 500-fold above those of 4-MBC; blood concentrations of 3-(4-carboxybenzylidene)-6-hydroxycamphor were below the limit of detection. Blood concentration of 4-MBC and 3-(4-carboxybenzylidene)camphor peaked within 10 h after 4-MBC administration and then decreased with half-lives of approximately 15 h. No major differences in peak blood levels between male and female rats were seen. In urine, one isomer of 3-(4carboxybenzylidene)hydroxycamphor was the predominant metabolite [3-(4-carboxybenzylidene)-6-hydroxycamphor], the other isomers and 3-(4carboxybenzylidene)camphor were only minor metabolites excreted with urine. However, urinary excretion of 4-MBC-metabolites represents only a minor pathway of elimination for 4-MBC, since most of the applied dose was recovered in feces as 3-(4-carboxybenzylidene)camphor and, to a smaller extent, as 3-(4-carboxybenzylidene)-6-hydroxycamphor. Glucuronides of both metabolites were also present in feces, but partly decomposed during sample workup and were thus not quantified. The results show that absorbed 4-MBC undergoes extensive first-pass biotransformation in rat liver resulting in very low blood levels of the parent 4-MBC. Enterohepatic circulation of glucuronides derived from the two major 4-MBC metabolites may explain the slow excretion of 4-MBC metabolites with urine and the small percentage of the administered doses recovered in urine. © 2006 Elsevier Inc. All rights reserved. Keywords: Biotransformation; Blood levels; Endocrine; LC-MS/MS; Electrospray
Introduction Sunscreen formulations frequently contain 3-(4-methylbenzylidene)camphor (4-MBC) as an UV-filter to reduce the intensity of UV-radiation reaching the skin. Commercial sunscreen lotions contain up to 4% 4-MBC. Based on a human study, the extent of dermal absorption of 4-MBC from
⁎ Corresponding author. Fax: +49 931/201 48865. E-mail address: [email protected] (W. Dekant). 0041-008X/$ - see front matter © 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.taap.2006.05.012
sunscreen lotions in humans is estimated to be approximately 0.5% (Schauer et al., accompanying manuscript). A possible estrogenic activity of 4-MBC was suggested based on the observation that 4-MBC induced cell proliferation in MCF-7 breast cancer cells transfected with the estrogen receptor and increased uterine weight in immature Long–Evans rats exposed to doses of 119 mg/kg/day for 4 days (Schlumpf et al., 2001; Ma et al., 2003; Schlumpf et al., 2004a, 2004b). An interaction of 4-MBC with the estrogen receptor could not be consistently demonstrated in vitro (Mueller et al., 2003; Schreurs et al., 2005), but estrogenic effects of 4-MBC in in
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vivo assays for uterotrophic activity and vaginal-stratification were observed (Ashby, 2001; Tinwell et al., 2002). The equivocal or negative findings in in vitro and positive results in in vivo screening assays for estrogenicity suggest a major impact of the metabolism on the endocrine activity of 4-MBC. These considerations prompted us to investigate the metabolism and kinetic behavior of 4-MBC in rats after oral administration. These observations suggest that activation of the weak estrogen 4-MBC to a more potent estrogen may occur and that metabolite(s) may be responsible for the estrogenic effects of 4MBC in vivo. Due to the possible bioactivation of 4-MBC to a metabolite with higher estrogenic potency, toxicokinetic considerations will be important for a reasonable risk assessment for species and route-to-route extrapolations. Extent of metabolite formation and clearance is expected to be widely differing after oral administration, which was used as route of application in many studies on the toxicity of 4-MBC, compared to the dermal exposure of humans to 4-MBC from sunscreens. Material and methods Chemicals 4-MBC was supplied by Merck KGaA (Darmstadt, Germany). All other reagents and solvents were reagent grade or better and obtained from several commercial suppliers. For recording of 1H NMR-spectra, 3-(4-carboxybenzylidene)-6-hydroxycamphor was isolated from urine pools (26 ml from male and 32 ml from female rats) by liquid–liquid extraction with diethyl ether (v:v 1:2). After evaporation of the solvent, the residue was diluted 1:100 with methanol and separated by HPLC to obtain the compound in a purity >95% (Bernauer et al., 1998). 3-(4-Carboxybenzylidene)camphor was isolated from feces by Soxhlet extraction and HPLC separation to obtain the compound in a purity >95%.
Animal treatment Male and female Sprague–Dawley rats (12–13 weeks) were purchased from Harlan–Winkelmann, Borchen, Germany. The animals (3 per dose group) had free access to water and a standard diet (Altromin) and were kept under standard conditions (12 h day/night cycle, temperature 21–23 °C, humidity 45–55%). All animal experimentation was performed under permit from the appropriate authorities in the approved animal care facility of the department. Animals were transferred into metabolic cages for collection of control urine and feces for 4 days before treatment. Animals were then administered 4-MBC (25 and 250 mg/ kg body weight), dissolved in corn oil, at 10 am. To obtain a homogeneous dosing solution, 4-MBC was homogenized in corn oil during 5 min using an ultrasonic homogenizer. A dosing volume of 5 ml/kg bw of corn oil was applied. Immediately after dosing, animals were transferred to metabolic cages (one rat per cage) and urine and feces samples were collected for 94 h after dosing. Collecting vessels for urine and feces were cooled with mixtures of ice and sodium chloride. Urine samples were collected in predefined intervals, urine volume was recorded, samples were divided into aliquots, and stored at −20 °C. Feces were collected in 24 h intervals, weighted and also stored at −20 °C. Blood samples (100 μl) were collected from the tail vein at predetermined time points and plasma was immediately prepared by centrifugation (15 min at 1000 × g). Plasma samples were also stored at −20 °C. Feces were freeze-dried and 500 mg of the dry residues were extracted by Soxhlet-extraction with methanol (60 ml) for 4 h.
pump and an Agilent 1040 M series II diode array detector or an Agilent 1090 HPLC system. Gradient elution using a linear gradient from 100% water (with 0.1% trifluoroacetic acid) to 100% acetonitrile (with 0.1% trifluoroacetic acid) over 25 min, held for 5 min, with a flow rate of 1 ml/min was applied. The eluate was monitored at 300 nm. Calibration solutions were generated by addition of 4MBC to urine and blood samples from control rats. Due to the identical UVspectra, 3-(4-carboxybenzylidene)-6-hydroxycamphor and 3-(4-carboxybenzylidene)camphor were quantified based on calibration curves obtained with 4-MBC. Calibration curves were calculated from 6 data points using Microsoft Excel spreadsheets. The R2-values of the calibration curves were >0.999. Standard deviations (mean ± SD), elimination half-lives and areas under the curve were calculated using Microsoft Excel spread sheets and default setting. Polynoms given for best fit by Microsoft Excel were transferred into “Functions” (NumericalMathematics.com), AUCs were calculated from the mean of each time point. Liquid chromatography/mass spectrometry. For the recording of mass spectra of 4-MBC metabolites, urine samples were diluted with MeOH and H2O (1:2:2), vortexed and centrifuged to precipitate protein. The diluted samples (10 μl) were separated on a Nucleosil RP18 HPLC column (2.0 mm × 150 mm; 5 μm, 100 Å; Phenomenex, Aschaffenburg, Germany) using an Agilent 1100 autosampler and an Agilent 1100 HPLC-pump (Agilent, Waldbronn, Germany). Samples were separated by gradient elution with water (solvent A) and acetonitrile (solvent B) using the following conditions: 100% A, linear increase to 25% B within 5 min, then linear increase to 80% B within 20 min at a flow-rate of 200 μl/min. The HPLC system was directly coupled to a triple stage quadrupole mass spectrometer (API 3000, Applied Biosystems, Darmstadt, Germany) equipped with a TurboIon Spray source. Analytes were detected in the negativeion mode at a vaporizer temperature of 400 °C and a TurboIon Spray voltage of −4.0 kV. Spectral data were recorded with nitrogen as collision gas (CAD = 4) in the product ion scan mode or the multiple reaction monitoring (MRM) mode. Product ion scans were performed with calculated molecular weights of each metabolite and monitored from 50 amu to the expected mass units for the molecular ion. For MRM analyses, the samples were diluted 100-fold. To quantify 4-MBC in blood, 25 μl of serum samples from rats was mixed with 25 μl of cold MeOH, and the samples were centrifuged for 4 min at 15,000 × g. The supernatants were then placed on ice for 30 min and 25 μl of cold acetonitrile was added followed by centrifugation for 4 min at 15,000 × g. To quantify 4-MBC by LC-MS/MS after atmospheric pressure photoionization (APPI), 5 μl of the supernatants of the samples was injected on a Synergi Hydro-RP 80 A HPLC column (2.0 mm × 150 mm; 4 μm; Phenomenex, Aschaffenburg, Germany) using an Agilent 1100 autosampler and an Agilent 1100 HPLC-pump. The samples were separated by elution with water (solvent A) and acetonitrile (solvent B) using the following conditions: 10% A, 90% B, isocratic for 7 min, with a flow rate of 300 μl/min. Toluene was used as dopant with a flow rate of 1.5 ml/h. The HPLC system was directly coupled to the mass spectrometer equipped with an atmospheric pressure photoionization source. 4-MBC was detected in the positive ion mode at a vaporizer temperature of 400 °C. For APPI, an ion source voltage (IS) of 2000 V was applied. Spectral data were recorded with N2 as collision gas (CAD = 4) in the multiple reaction monitoring (MRM) mode with a dwell time of 200 ms for each transition monitoring the MS-MS ion-transitions given in Table 2. To confirm the presence of glucuronides of 4-MBC metabolites, samples were analyzed by LC-MS/MS monitoring the constant neutral loss of a fragment with 176 Da from the calculated molecular ions of glucuronides derived from the two identified metabolites with [M-H]− at m/z 475, respectively 459. NMR spectra were recorded with a BRUKER (Karlsruhe, Germany) 600 MHz NMR spectrometer. The dried residues obtained after metabolite isolation were dissolved in D2O containing 3-(trimethylsilyl)-2,2,3,3-d4-proprionate (TSP). 1H NMR spectra were recorded using a Bruker DPX 600 NMR operating at 600.13 MHz. Chemical shifts were normalized to TSP set at δ = 0 ppm.
Results
Instrumental analysis
Metabolite identification
HPLC with UV-detection. Blood and urine samples were separated using a 250 mm × 4.6 mm ID steel column filled with Hypersil ODS 5 μm (Bischoff, Leonberg, Germany). Separation was performed using a Agilent 1050 quaternary
To detect and identify metabolites of 4-MBC, urine and blood samples from 4-MBC-treated rats were analyzed by
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HPLC with diode array detector by LC-MS/MS and 1H NMR. The diode array detector was selected for metabolite detection since it was not expected that biotransformation results in a major modification of the chromophore present in 4-MBC. Compounds exhibiting the characteristic UV-spectra of 4-MBC present in urine and blood samples as indicated by the diode array detector were further characterized by mass spectrometry, and by 1H NMR after isolation by HPLC. In addition, LC-MS/ MS with specific acquisition conditions to detect glucuronides was used to identify the presence of glucuronides formed from metabolites of 4-MBC. In urine and feces samples collected before administration, peaks with the characteristic UV-spectra of 4-MBC or its metabolites were not present. HPLC separation of urine samples from 4-MBC-treated rats gave five peaks with the characteristic UV-spectra of 4-MBC derivatives (Fig. 1). These results suggest the presence of one major metabolite and three minor metabolites (peaks Ia–d, Fig. 1) with retention times within a range of less than 4 min. An additional metabolite (peak II, Fig. 1) with a lower polarity was present in low concentrations, and was mainly excreted by female rats. Product ion spectra of all five metabolites were recorded by LC-MS/MS. Metabolites Ia–d all exhibited identical mass spectra with molecular weights of 300 Da, m/z 299 represents (M-H)−. The major product formed by fragmentation from
333
(M-H)− was an ion with m/z 255. The loss of 44 amu represents CO2, a typical fragmentation observed in the mass spectra of carboxylic acids (Fig. 2). Based on the 4-MBC-structure, the spectral data suggest an oxidation of the methyl group of the aromatic ring in 4-MBC in addition to a further hydroxylation. Analysis of metabolite II by LC-MS/MS in the product ion scan mode gave a (M-H)− ion (m/z 283) and, after collision with N2, loss of CO2 (44 amu) to give an ion m/z 239 (Fig. 3). For further structure determination, Ia and II were isolated by HPLC and analyzed by 1H NMR (Table 1). The losses of the resonances of the methyl group attached to the phenyl ring and the changes in chemical shifts of the protons on the phenyl ring in both metabolites Ia and II in relation to the chemical shifts of the phenyl ring protons in 4-MBC suggest an oxidation of the methyl group on the aromatic ring to a carboxylate in both metabolites consistent with the mass spectral data. Furthermore, the four aromatic protons of both metabolites and 4-MBC retained identical coupling constants indicating a further hydroxylation, as indicated by the mass spectra, did not occur at the phenyl ring. The three remaining methyl groups in the camphor moiety and the vinylic proton in 4-MBC and both metabolites also gave singlets with identical chemical shifts suggesting absence of modifications at these positions in the molecule (Table 1). The proton NMR-data combined with the mass spectra identified the minor urinary metabolite II
Fig. 1. HPLC separation of an urine sample from a male (A) and a female (B) rat collected 14 h after administration of 4-MBC (250 mg/kg bw). UV-spectra of major metabolites are shown in the inserts. Peaks Ia-d represents four isomers of 3-(4-carboxybenzylidene)hydroxycamphor, peak II represents 3-(4-carboxybenzylidene) camphor.
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Fig. 2. Product ion spectrum of 3-(4-carboxybenzylidene)-6-hydroxycamphor (m/z 299, M-H); characteristic fragments are m/z 255 (M·super −H–CO2) and m/z 227 (M·super −H–CO2–CO).
(Figs. 1B and 4) as 3-(4-carboxybenzylidene)camphor formed by the oxidation of the aromatic methyl group to a benzoic acid derivative of 4-MBC (Fig. 4). For metabolite Ia (Figs. 1 and 4), the chemical shift of the single proton in the camphor moiety (Hc) was similar to that of 4-MBC. Therefore, the introduced hydroxyl function is located on one of the two methylene groups in the camphor ring. As a result of the very complex coupling of the four possible diastereomers, it is difficult to localize the position of the hydroxyl group. Due to the same multiplicity (d) with coupling constants of 4.3 Hz in comparison to 4-MBC (d, 4.5 Hz), it is concluded that the hydroxylation occurred at C-6. In combination, 1H NMR and mass spectrometry data suggest that the major 4-MBC metabolite (Ia, Fig. 1) in rat urine corresponds to 3-(4-carboxybenzylidene)-6-hydroxycamphor (Fig. 4). The
three other minor metabolites represent isomers of this compound with the hydroxyl group either in another orientation on C-6 or on C-5 with two possible orientations. The presence of metabolites such as glucuronides and sulfates of the 4-MBC-metabolites was determined a using LCMS/MS method with constant neutral loss scans designed specifically to detect glucuronides and sulfates with high sensitivity. In urine and feces, the constant neutral loss scan showed that glucuronides of both 3-(4-carboxybenzylidene)6-hydroxycamphor ([M-H]− at 475 amu, major fragment at 299 amu) and 3-(4-carboxybenzylidene)camphor ([M-H]− at 459 amu, major fragment at 283 amu) were present. Most likely, both glucuronides represent acyl glucuronides. The presence of glycine conjugation products, which often are formed from reactive acyl glucuronides (Kasuya et al., 1996),
Fig. 3. Product ion spectrum of 3-(4-carboxybenzylidene)camphor (m/z 283, M−); characteristic fragment ion is m/z 239 (M−CO2).
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Table 1 H NMR chemical shifts and multiplicity of signals in 1H NMR spectra of 3-(4-carboxybenzylidene)-6-hydroxycamphor and 3-(4-carboxybenzylidene)camphor isolated as metabolites from urine or feces of 4-MBC-tretaed rats in comparison to 4-MBC (d, duplet; m, multiplet; s, singlet; q, quartet), 1H NMR-resonances of 4-MBC are given for comparison
1
4-MBC
3-(4-Carboxybenzylidene)-6-hydroxycamphor
3-(4-Carboxybenzylidene) camphor
Chemical shift (δ)
Multipl.
Assig.
Chemical shift (δ)
Multipl.
Assig.
Chemical shift (δ)
Multipl.
Assig.
0.79 1.00 1.03 1.52 1.59 1.77 2.18 2.37 3.10 7.20 7.22 7.39
s s s m m m m s d d s d
CH3 CH3 CH3 C–H C–H C–H C–H Ph–CH3 Hc Hb–Hb' Hd Ha–Ha'
0.71 0.99 1.03 – 2.78 2.82 4.30 – 3.26 7.98 7.30 7.63
s s s – m m q – d d s d
CH3 CH3 CH3 C–H C–H C–H C–H – Hc Hb–Hb′ Hd Ha–Ha′
0.72 0.92 0.96 1.52 1.59 1.81 2.20 – 3.09 7.89 7.16 7.41
s s s m m m q – d d s d
CH3 CH3 CH3 C–H C–H C–H C–H – Hc Hb–Hb′ Hd Ha–Ha′
and sulfate conjugates was not indicated by the analytical methods applied. Kinetics of 4-MBC elimination from blood and with urine Blood and urine samples collected at different time points were analyzed by HPLC for the presence of 4-MBC and its metabolites. Experimental results on the elimination of 4-MBC and its metabolites from blood and with urine are given in Figs. 5 and 6, and in Table 2. In blood samples from animals collected before 4-MBC administration, concentrations of 4-MBC and all identified metabolites were below the limit of detection. Blood samples
collected after 4-MBC administration did not contain quantifiable concentrations of 4-MBC when analyzed by HPLC with diode array detection; therefore, the concentrations of 4-MBC in these samples were determined by a more sensitive LC-MS/MS method. With this method, a dose-dependent increase in 4-MBC concentrations in blood was observed after 4-MBC administration in both male and female rats. Blood concentrations of 4-MBC were dose-dependent and peak concentrations between 35 and 60 pmol/ml were reached within 10 h (Figs. 5A, B) after administration of 250 mg/kg. The concentrations of 3(4-carboxybenzylidene)camphor in the blood samples were also dose-dependent, but were much higher as compared to those of 4-MBC and peaked between 90 and 120 nmol/ml within 8 h
Fig. 4. Biotransformation of 4-MBC to 3-(4-carboxybenzylidene)-6-hydroxycamphor and 3-(4-carboxybenzylidene)camphor.
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▪
Fig. 5. Blood concentrations of 4-MBC and its metabolite 3-(4-carboxybenzylidene)camphor, after oral administration of 25 mg/kg bw ( ) and 250 mg/kg bw (♦) to male (panels A and C) or female (panels B and D) rats.
▪
Fig. 6. Urinary excretion of 4-MBC-metabolites in male (panel A, 250 mg/kg bw 4-MBC and panel C, 25 mg/kg bw 4-MBC) and female rats (panel B, 250 mg/kg bw 4-MBC and panel D, 25 mg/kg bw 4-MBC); ♦, sum of isomers of 3-(4-carboxybenzylidene)hydroxycamphor; , 3-(4-carboxybenzylidene)camphor. Concentrations of 3-(4-carboxybenzylidene)camphor were below the limit of detection in urine samples from male rats receiving 25 mg/kg bw 4-MBC (panel C).
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Table 2 Recovery of 4-MBC and its metabolites in excreta after oral administration of doses of 25 and 250 mg/kg bw to male and female rats, data are mean ± SD from 3 male and 3 female rats ♂
♀
25 mg/kg bw
4-MBC applied Urine Feces Total
250 mg/kg bw
25 mg/kg bw
μmol
% of dose
μmol
% of dose
28 ± 1.4 0.6+0.4 n.d.
2.3 ± 1.5 n.d.
416 ± 23 36 ± 3 216 ± 107 252 ± 120
8.6 ± 2.7 52 ± 26.4 61 ± 29.1
after 4-MBC administration at 250 mg/kg. The concentration of this metabolite in blood afterwards declined following firstorder kinetics with a half-life of approximately 15 h in both male and female rats. Concentrations of 3-(4-carboxybenzylidene)-6-hydroxycamphor in the blood samples were at the limit of detection in all samples analyzed and therefore not quantified. In urine samples from 4-MBC treated rats, 3-(4-carboxybenzylidene)-6-hydroxycamphor was the predominant excretory product observed after both of the applied doses. Peak concentrations of 3-(4-carboxybenzylidene)-6-hydroxycamphor in urine were reached within 14 h in males and then slowly declined (Fig. 6). In female rats, urinary elimination of 3(4-carboxybenzylidene)-6-hydroxycamphor was slower as compared to male rats and gave a plateau from 14 h to approximately 38 h after administration; excretion of 3-(4-carboxybenzylidene) camphor was more pronounced in females as compared to males (Figs. 6A, B). In feces samples collected after administration of 4-MBC, 4-MBC and both 3-(4-carboxybenzylidene)-6-hydroxycamphor and 3-(4-carboxybenzylidene)camphor were recovered. The presence of glucuronides of both 4-MBC metabolites was also indicated by LC-MS/MS analysis of feces extracts. A direct quantitation of the glucuronides of these compounds in feces could not be performed since the glucuronides decomposed during the methanol extractions necessary for isolation from the feces matrix. Therefore, it was attempted to quantify unconjugated 3-(4-carboxybenzylidene)camphor and 3-(4-carboxybenzylidene)-6-hydroxycamphor in feces, but results obtained were not well reproducible. This likely is due to the relatively harsh conditions required for extraction and the chemical reactivity of the present acyl glucuronides (SpahnLangguth and Benet, 1992), which may bind to nucleophiles in feces matrix at the elevated temperatures required for extraction. In general, the concentration of 3-(4-carboxybenzylidene) camphor in feces was much higher than those of 3-(4carboxybenzylidene)-6-hydroxycamphor and 4-MBC. Only a minor part of the applied dose of 4-MBC was recovered in urine as metabolites, most of the dose was recovered in feces both as metabolites and as unchanged 4MBC with large variations in recovery (Table 2). Discussion The results of this study show that 4-MBC is intensively metabolized in rodents and its toxicokinetics after oral administration are complex. The identified metabolites suggest
250 mg/kg bw
μmol
% of dose
μmol
% of dose
19 ± 0.3 0.9 ± 8.7 n.d.
4.8 ± 4.7 n.d.
234 ± 7 687 28 ± 14 103 ± 44 131 ± 58
12 ± 6.2 44 ± 18.3 56 ± 24.5
the biotransformation of 4-MBC is initiated by cytochrome P450-mediated oxidation reactions. The first step in the metabolic sequence to result in the 4-MBC metabolite 3-(4carboxybenzylidene)-6-hydroxycamphor likely occurs by hydroxylation of the aromatic methyl group by cytochrome P450 (Fig. 4) (Hanioka et al., 1995). The intermediate, 3-(4hydroxymethylbenzylidene)camphor, represents the major product observed when 4-MBC is incubated with rat or human liver microsomes (Völkel et al., unpublished observation). 3-(4-Hydroxymethylbenzylidene)-6-camphor is then further oxidized, presumably by alcohol dehydrogenase and aldehyde dehydrogenase, to give 3-(4-carboxybenzylidene) camphor as described for other benzylalcohols (Bakke and Scheline, 1970). This compound may then undergo further oxidation, likely again by a cytochrome P450-catalyzed oxidation, to the final excretory product 3-(4-carboxybenzylidene)-6hydroxycamphor. Only a minor percentage of 3-(4-carboxybenzylidene)camphor is excreted with urine, the majority of this compound undergoes further biotransformation to give 3-(4carboxybenzylidene)-6-hydroxycamphor as the major urinary metabolite. This reaction seems to occur extrahepatically, since 3-(4-carboxybenzylidene)-6-hydroxycamphor is not released to blood from liver in quantifiable concentrations. Both 3-(4carboxybenzylidene)-6-hydroxycamphor and 3-(4-carboxybenzylidene)camphor are then excreted with urine due to their low molecular weight. However, the major pathway for the disposition of 3-(4carboxybenzylidene)-6-hydroxycamphor and 3-(4-carboxybenzylidene)camphor likely is represented by an efficient conjugation of both compounds to the corresponding acyl glucuronides. The glucuronides of 3-(4-carboxybenzylidene) camphor and 3-(4-carboxybenzylidene)-6-hydroxycamphor with molecular weights of 476 and 460 Da are above the molecular weight threshold for biliary elimination (approximately 400 Da in rats), are eliminated with bile and likely undergo enterohepatic circulation (Tephly and Burchell, 1990). Enterohepatic circulation may explain the comparatively slow elimination of 4-MBC metabolites with urine. The low blood levels and the low or absent estrogenicity of 4-MBC in vitro and the formation of 3-(4-carboxybenzylidene)6-hydroxycamphor, which shows a somewhat higher activity in a screening assay for estrogenicity (Müller et al., unpublished results), suggest that 4-MBC may exert its weak estrogenic effects in rats after bioactivation to 3-(4-carboxybenzylidene)-6hydroxycamphor. This observation might possibly represent a first example of a biotransformation of a hormonally inactive
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compound to a more potent estrogen and indicates that the reliance on in vitro screening systems without biotransformation capacities for xenobiotics may miss chemicals where estrogenicity only occurs after biotransformation (Degen et al., 2002). The intensive first-pass metabolism of 4-MBC and the role of biotransformation and estrogenic response also stress the importance of toxicokinetics in the risk assessment of “endocrine modulators”. Extent of biotransformation and thus formation of metabolites with either increased or decreased affinity to hormone receptors varies depending on route of application and species-specific capacities for biotransformation (see accompanying manuscript). This underscores the importance of developing complete toxicological data sets before discussing “endocrine modulation” based on in vitro activities in screening assays. For example, widely published “endocrine modulators” such as bisphenol Awith some estrogenicity in vitro are rapidly conjugated in vivo to non-estrogenic products and estrogenicity in vivo is only observed after extreme doses saturating excretory pathways or after unusual routes of application (Pottenger et al., 2000; Völkel et al., 2002). Regarding 4-MBC, the absorption of this compound from skin in the relevant human exposure scenario “application of sunscreens” seems very limited since only low concentrations of 4-MBC in blood were determined after repeated and intensive applications of sunscreens in human subjects (Lademann et al., 2005). Dermal administration may result in a less pronounced first-pass metabolism of 4-MBC and lower concentrations of the estrogenic 3-(4-carboxybenzylidene)-6-hydroxycamphor. However, studies in human subjects determining both 4-MBC and 3-(4-carboxybenzylidene)-6-hydroxycamphor in blood after sunscreen application are needed for a more conclusive hazard assessment (see accompanying manuscript). Acknowledgments This study was supported by Merck KGaA, Darmstadt, Germany. The authors thank Nataly Bittner and Heike KeimHeusler for excellent technical assistance and Dr. M. Grüne, Institut für Organische Chemie der Universität Würzburg, for the recording of 1H NMR-spectra. References Ashby, J., 2001. Confirmation of uterotrophic activity for 4-MBC in the immature rat. Environ. Health Perspect. 109, A517. Bakke, O.M., Scheline, R.R., 1970. Hydroxylation of aromatic hydrocarbons in the rat. Toxicol. Appl. Pharmacol. 16, 691–700.
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