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Evaluation of Interindividual Human Variation in Bioactivation and DNA Adduct Formation of Estragole in Liver Predicted by Physiologically Based Kinetic/Dynamic and Monte Carlo Modeling Ans Punt,*,†,∥ Alicia Paini,†,‡,∥ Albertus Spenkelink,† Gabriele Scholz,‡ Benoit Schilter,‡ Peter J. van Bladeren,†,§ and Ivonne M. C. M. Rietjens† †

Division of Toxicology, Wageningen University, Tuinlaan 5, 6703 HE Wageningen, The Netherlands Nestlé Research Center, P.O. Box 44, 1000 Lausanne 26, Switzerland § Nestec S.A, Avenue Nestlé 55, 1800 Vevey, Switzerland ‡

S Supporting Information *

ABSTRACT: Estragole is a known hepatocarcinogen in rodents at high doses following metabolic conversion to the DNA-reactive metabolite 1′-sulfooxyestragole. The aim of the present study was to model possible levels of DNA adduct formation in (individual) humans upon exposure to estragole. This was done by extending a previously defined PBK model for estragole in humans to include (i) new data on interindividual variation in the kinetics for the major PBK model parameters influencing the formation of 1′-sulfooxyestragole, (ii) an equation describing the relationship between 1′-sulfooxyestragole and DNA adduct formation, (iii) Monte Carlo modeling to simulate interindividual human variation in DNA adduct formation in the population, and (iv) a comparison of the predictions made to human data on DNA adduct formation for the related alkenylbenzene methyleugenol. Adequate model predictions could be made, with the predicted DNA adduct levels at the estimated daily intake of estragole of 0.01 mg/kg bw ranging between 1.6 and 8.8 adducts in 108 nucleotides (nts) (50th and 99th percentiles, respectively). This is somewhat lower than values reported in the literature for the related alkenylbenzene methyleugenol in surgical human liver samples. The predicted levels seem to be below DNA adduct levels that are linked with tumor formation by alkenylbenzenes in rodents, which were estimated to amount to 188−500 adducts per 108 nts at the BMD10 values of estragole and methyleugenol. Although this does not seem to point to a significant health concern for human dietary exposure, drawing firm conclusions may have to await further validation of the model’s predictions.

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INTRODUCTION

as the addition of estragole as a pure compound to individual food categories is no longer allowed in Europe.3 Estragole is known to induce hepatocarcinomas in rodents at high dose levels.4 Metabolic conversion of estragole into a genotoxic metabolite that forms DNA adducts is involved in the formation of liver tumors.5 Bioactivation of estragole (Figure 1) proceeds by cytochrome P450-catalyzed hydroxylation of estragole to 1′-hydroxyestragole, followed by sulfotransferase (SULT)-mediated conversion of 1′-hydroxyestragole to 1′sulfooxyestragole.6 This metabolite is unstable in aqueous environment and reacts, via a putative carbocation, with nucleophilic molecules including glutathione, proteins, RNA, and DNA.6−9 The involvement of 1′-sulfooxyestragole in the induction of liver tumors is supported by observations that the formation of hepatocellular carcinomas in B6C3F1 mice is significantly reduced by coexposure with the SULT inhibitor pentachlorophenol.9 Important detoxification pathways of estragole include O-demethylation to 4-allylphenol, 3′-hydro-

Estragole is an alkenylbenzene that is naturally present in various herbs and spices; thus, it is traditionally present in the human diet. Daily intake estimates are available from the EU Scientific Committee of Food (SCF) and the U.S. Flavor and Extract Manufacturers Association (FEMA).1,2 The SCF has estimated an average daily intake of estragole of 4.3 mg per day, corresponding to ∼0.07 mg/kg bw/day for a 60 kg person. This estimation is based on a relative conservative method using theoretical maximum use levels of estragole (including addition as a pure compound) in 28 food categories and consumption data for these food categories based on 7 day dietary records of adult individuals. Using a different method, a lower average per capita daily intake of estragole was estimated by the Expert Panel of the FEMA. This estimation was performed using production volume data of herbs, essential oils, and flavor substances containing estragole in the United States. The FEMA estimated the daily per capita intake to be less than 0.01 mg/kg bw day. At present, the intake estimate of the SCF might be an overestimation of the daily exposure to estragole, © XXXX American Chemical Society

Received: December 2, 2015

A

DOI: 10.1021/acs.chemrestox.5b00493 Chem. Res. Toxicol. XXXX, XXX, XXX−XXX

Article

Chemical Research in Toxicology

Several in vitro and rodent studies with estragole have revealed that the major adduct formed from the reactive metabolite 1′-sulfooxyestragole is N2-(trans-isoestragol-3′-yl)deoxyguanosine (E-3′-N2-dGuo).1,6 However, adducts with 2′deoxyadenosine have also been reported to be formed in livers of male F344 and F344 gpt delta rats exposed to estragole,14 albeit to a lesser extent than adducts formed with 2′deoxyguanosine, at dose levels lower than 22 mg/kg bw.15 Therefore, at low realistic levels of exposure, the E-3′-N2-dGuo adduct can be considered the most relevant adduct that is formed upon estragole exposure. This is supported by observations for the related alkenylbenzene methyleugenol, showing that the analogous 2′-deoxyguanosine adduct is the most predominant adduct that can be found in human surgical liver samples, occurring at 64 ± 40 times higher levels than N6(trans-methylisoeugenol-3′-yl)-2′-deoxyadenosine adducts.16 The mutagenic potential of estragole DNA adducts has not been established so far. Estragole, as well as the related alkenylbenzene methyleugenol, generally give negative results in standard mutagenicity tests due to the absence of the sulfonation pathway required for their bioactivation.17−21 However, a recent study by Herrmann et al.22 revealed that different isomeric hydroxylated metabolites of methyleugenol are mutagenic in an Ames test using Salmonella typhimirum TA100 strains expressing different sulfotransferase enzymes. This supports the mutagenic potential of sulfate conjugates of 1′-hydroxy alkenylbenzenes. Considering that the formation of 1′-sulfooxyestragole and subsequently formed DNA adducts play an important role in estragole-induced carcinogenicity, knowledge of the levels of DNA adduct formation in human livers at relevant dietary intake levels would be valuable to facilitate risk assessment for low-dose estragole intake. In previous work, we developed a physiologically based kinetic (PBK) model that predicts 1′sulfooxyestragole formation in humans.11,23 Using this model as a basis, the aim of the present study was to predict E-3′-N2-

Figure 1. Estragole metabolism.

xylation to 3′-hydroxyanethole, and epoxidation to estragole2′,3′-oxide (Figure 1).1,10,11 Even though this latter metabolite has been observed to form DNA adducts in vitro, these adducts have not been observed in vivo due to rapid detoxification of estragole-2′,3′-oxide by epoxide hydrolases and/or glutathioneS-transferases.12 Therefore, this metabolite is not considered to contribute to the mode of action of estragole-induced carcinogenicity. Detoxification of the proximate carcinogenic metabolite 1′-hydroxyestragole can proceed by its oxidation to 1′-oxoestragole6,11 or by conjugation to form 1′-hydroxyestragole glucuronide.13

Table 1. Information on Individual Human Donors from Which S9 Fractions Were Taken, Including Code of Supplier, Supplier, Age, Gender, Race, Cause of Death, and Social History codea

supplier

age

genderb

race

cause of deathb

H0420 H0236 H0120 F0962 H0280 H0346 H0441 H0311 H0428 H0354 H0133 H0393 H0422 H0322 H0251 H0438 H0442 M0962 H0432

Xenotech Xenotech Xenotech Celsis Xenotech Xenotech Xenotech Xenotech Xenotech Xenotech Xenotech Xenotech Xenotech Xenotech Xenotech Xenotech Xenotech Celsis Xenotech

42 17 57 44 36 3 63 21 57 0 17 30 69 1 42 56 49 72 60

M M F F F M M M F F M F M M F M M M M

Caucasian Asian Caucasian Caucasian Caucasian Caucasian Caucasian Hispanic Caucasian Caucasian Caucasian Caucasian Caucasian Hispanic Caucasian Caucasian Caucasian Caucasian African-American

Anoxia CVA CVA CVA Anoxia CVA CVA MVA HT CVA HT Anoxia CVA Anoxia Anoxia HT HT CVA CVA

social historyb Smoker: Smoker: Smoker: Smoker: Smoker: Smoker: Smoker: Smoker: Smoker: Smoker: Smoker: Smoker: Smoker: Smoker: Smoker: Smoker: Smoker: Smoker: Smoker:

Y; EtOH: Y N; EtOH: N N; EtOH: Y Y; EtOH: Y Y; EtOH: N N; EtOH: N N; EtOH: Y Y; EtOH: Y Y; EtOH: N N; EtOH: N Y; EtOH: Y N; EtOH: Y N; EtOH: N N; EtOH: N Y; EtOH: Y Y; EtOH: Y N; EtOH: Y N; EtOH: N N; EtOH: N

a

Code = S9 fraction code of supplier. bH, human; F, female; M, male; CVA, cerebrovascular accident; HT, head trauma; MVA, motor vehicle accident ; EtOH, ethanol consumption; Y, yes; N, no. B


Article


ESI probe (Applied Biosystem, Foster City, CA). The gradient was made with ultrapure water containing 0.1% (v/v) formic acid and 100% acetonitrile. The flow rate was set at 0.3 mL/min, a linear gradient was applied, and the mass spectrometric analysis was performed with settings reported in Paini et al.26 Quantification of the DNA adduct was carried out using selected-ion detection in the multiple reaction-monitoring mode (MRM), recording the following characteristic transitions: for E-3′-N2-dGuo: 414 m/z → 298 m/z (CE = 18 eV), 414 m/z → 164 m/z (CE = 37 eV), and 414 m/z → 147 m/ z (CE = 40 eV); for (15N5) E-3′-N2-dGuo: 419 m/z → 303 m/z (CE = 18 eV) and 419 m/z → 169 m/z (CE = 37 eV). Data analysis of the calibration series and sample sequence was performed using Analyst software (Applied Biosystem) and integrated using an Excel spreadsheet developed internally (Nestlé Research Center), complying with EU guidelines 2002/657/EC concerning the performance of analytical methods and the interpretation of results (EU guidelines 2002/657/EC). The apparent kinetic parameters (Km and Vmax) for the PAPS-dependent formation of the E-3′-N2-dGuo adducts were determined by fitting the data to the standard Michaelis−Menten equation using GraphPad Prism, version 5 (GraphPad Software, San Diego, CA). When substrate inhibition of the sulfotransferase was observed at the highest concentration(s), these concentrations were not taken into account when fitting the Michaelis−Menten curves to the data. Substrate inhibition occurred only at concentrations higher than 500 μM, which is above the physiologically relevant 1′hydroxyestragole concentrations predicted to be reached in the liver at a dose of 0.01 mg/kg bw (i.e., ∼0.5 μM11). The apparent kinetic constants obtained were therefore considered to adequately capture metabolic conversion at physiologically relevant concentrations. Determination of Apparent Kinetic Constants for NAD+Dependent Conversion of 1′-Hydroxyestragole to 1′-GSOxoestragole, Reflecting Oxidation of 1′-Hydroxyestragole. Apparent kinetic constants for the oxidation of 1′-hydroxyestragole by human liver S9 homogenates, as a major detoxification route of 1′hydroxyestragole, were performed as previously described by Punt et al.23 by indirect trapping of the reactive 1′-oxoestragole. The incubations (200 μL final volume in 0.1 M Tris-HCl, pH 7.4) were performed in triplicate in the presence of different concentrations of the substrate 1′-hydroxyestragole (added to a final concentration of 25−1000 μM from 100× concentrated stock solutions in DMSO) dissolved in 0.2 M Tris-HCl (pH 7.4), with NAD+ (3 mM final concentration) as cofactor, GSH (2 mM final concentration) as a trapping agent for reactive 1′-oxoestragole, and 1 mg of S9 protein/mL final concentration). The reactions were started after a 1 min preincubation of the samples at 37 °C by adding the substrate 1′hydroxyestragole. All incubations were carried out for 10 min at 37 °C. The reactions were terminated by adding 50 μL of ice-cold acetonitrile, after which the samples were centrifuged for 5 min at 16 000g to precipitate the S9 proteins. Under these conditions, the formation of the 1′-oxoestragole metabolite was linear with time and with the S9 protein concentration for all human S9 fractions (data not shown). The supernatant was collected and analyzed by ultraperformance liquid chromatography with a diode array detector (UPLC-DAD). To this end, 3.5 μL of the samples was injected into an Acquity BEH C18 1.7 μm, 2.1 × 50 mm column, with a guard column (Acquity, Waters). The flow was 0.6 mL/min, and the gradient started with 100% of 0.1% acetic acid in nanopure water and 0% acetonitrile. After 1.2 min, the amount of acetonitrile was increased to 25% and kept at 25% for 0.2 min; then, the acetonitrile was increased to 100% and kept at 100% for 30 s, after which starting conditions were reset. Under these conditions, the glutathione adduct of 1′-oxoestragole eluted at 0.97 min. Blanks, without the addition of NAD+, were performed for each individual human S9 batch. The amount of 1′oxoestragole formed in the different samples was corrected for the average amount formed in blank incubations. The apparent kinetic parameters (Km and Vmax) for the oxidation reaction were determined by fitting the data to the standard Michaelis−Menten equation using GraphPad Prism. PBK/D Modeling. The PBK model previously developed for estragole in humans11,23 was used in the present study to integrate the

dGuo adduct formation in (individual) humans at realistic dietary exposure levels by (i) including new data on interindividual variation in the major parameters influencing the formation of 1′-sulfooxyestragole, (ii) defining an equation describing the relationship between 1′-sulfooxyestragole and DNA adduct formation, (iii) performing Monte Carlo simulations to simulate interindividual human variation in estragole DNA adduct formation in the population, and (iv) comparing the predictions made to human data on DNA adduct formation in human liver by the related alkenylbenzene methyleugenol. The results obtained provide an estimate of DNA adduct levels to be expected in human liver at low dietary exposure levels of estragole and can be evaluated with respect to their biological relevance by comparing the levels with DNA adduct levels that have been associated with tumor formation by alkenylbenzenes in rodents.

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MATERIALS AND METHODS

Chemicals and Biological Materials. Two individual human liver S9 homogenates were purchased from Celsis (Brussels, Belgium). Seventeen individual human liver S9 homogenates and pooled male Sprague−Dawley rat liver S9 fractions were purchased from Xenotech (Lenexa, KS). The characteristics of each human liver S9 homogenate donor are reported in Table 1. 2′-Deoxyguanosine was purchased from Sigma (Basel, Switzerland). 1,2,3,7,9-15N5-2′-Deoxyguanosine (15N5dG) was obtained from Cambridge Isotope Laboratories (Cambridge, MA). Glutathione (GSH) and 3′-phosphoadenosine-5′-phosphosulfate (PAPS, 60−70%) were purchased from Sigma-Aldrich (Schnelldorf, Germany). Acetonitrile, formic acid, hydrochloric acid (HCl), and tris(hydroxymethyl)-aminomethane (Tris) were purchased from VWR (Darmstadt, Germany). Nicotinamide adenine dinucleotide (NAD+) was purchased from Roche Diagnostics (Mannheim, Germany). The methods used for the synthesis of 1′-hydroxyestragole, E-3′-N2-dGuo, and (15N5) E-3′-N2-dGuo were described previously.24 Determination of Apparent Kinetic Constants for PAPSDependent Conversion 1′-Hydroxyestragole to E-3′-N2-dGuo, Reflecting Sulfonation of 1′-Hydroxyestragole. Apparent kinetic constants for the conversion of 1′-hydroxyestragole to its sulfate conjugate were determined as described previously based on an indirect method, namely, trapping the transient 1′-sulfooxyestragole with 2′-deoxyguanosine.25 Briefly, incubations with S9 fractions (1 mg of S9 protein/mL final concentration) were performed in triplicate in the presence of different concentrations of the substrate 1′hydroxyestragole at final concentrations of 10−2000 μM (added from 100× concentrated stock solutions in DMSO) using PAPS (1 mM final concentration) as cofactor and 2′-deoxyguanosine (1 mM final concentration) as a trapping agent of transient 1′-sulfooxyestragole. The final volume of the incubations was 200 μL. The reactions were started after a 1 min preincubation at 37 °C by adding PAPS, and all incubations were carried out for 1 h at 37 °C. The reactions were terminated by adding 50 μL of ice-cold acetonitrile, after which the samples were centrifuged for 5 min at 16 000g to precipitate the S9 proteins. Under these conditions, the formation of the metabolite was linear with time and with the S9 protein concentration for all human S9 fractions (data not shown). The supernatant was collected, speed vacuum-dried, reconstituted in 100 μL of water, and stored at −80 °C until analysis by LC-MS. Blanks were made without the addition of PAPS, and the amount of E-3′-N2-dGuo formed in the samples was corrected for the average amount formed in these blank incubations. Analysis of E-3′-N2-dGuo Adducts. Formation of E-3′-N2-dGuo in the different incubations was quantified by LC-MS/MS, performed as described previously,24 based on a method by Punt et al.25 Briefly, 50 μL of each sample was spiked with 10 μL of (0.012 nmol) internal standard (as reported by Paini et al.24), of which 10 μL was then injected on an Agilent Zorbax Extend C18 column, 2.1 × 50 mm, 3.5 μm, 80 Å (Basel, Switzerland), with a Zorbax guard column. LC-MS/ MS analysis was performed on a PerkinElmer 200 series HPLC system (PerkinElmer, Waltham, MA) coupled to an API 3000 system with an C


Article


Table 2. Apparent Kinetic Constants for PAPS-Dependent Conversion of 1′-Hydroxyestragole as a Representation of Sulfonation of 1′-Hydroxyestragole (to SHE) and Oxidation of 1′-Hydroxyestragole (to OE) by Individual Human Liver S9 Fractions as Well as PBK-Model-Predicted Formation of 1′-Sulfooxyestragole in the Liver of Each Individual NAD+-dependent conversion

PAPS-dependent conversion codeb H0420 H0236 H0120 F0962 H0280 H0346 H0441 H0311 H0428 H0354 H0133 H0393 H0442 H0322 H0251 H0438 H0422 M0962 H0432 mean (μx) SD CVxf μwg σwh

Vmax(SHE) (pmol/min/mg S9)c

Km(SHE) (μM)c

CE (μL/min/mg S9)d

Vmax(OE) (nmol/min/mg S9)c

6.3 ± 0.4 20.5 ± 2.4 23.2 ± 2.6 25.1 ± 0.5 31.4 ± 1.2 27.6 ± 0.4 31.8 ± 6.3 38.4 ± 2.1 48.0 ± 2.7 6.9 ± 0.7 43.4 ± 2.3 83.1 ± 3.1 59.8 ± 5.6 31.0 ± 0.5 84.1 ± 6.9 128.6 ± 6.5 123.0 ± 7.3 63.9 ± 6.5 55.8 ± 1.4 49.0

165 ± 24 378 ± 88 342 ± 127 303 ± 16 219 ± 24 343 ± 13 281 ± 157 216 ± 28 186 ± 33 111 ± 48 164 ± 29 249 ± 34 237 ± 67 133 ± 8 253 ± 61 304 ± 43 357 ± 55 143 ± 61 54 ± 6 239

0.04 0.05 0.07 0.08 0.14 0.08 0.11 0.18 0.26 0.06 0.26 0.31 0.25 0.23 0.33 0.42 0.34 0.45 1.03 0.25

2.0 3.1 3.3 3.1 3.2 3.6 3.2 3.7 4.9 1.4 3.0 3.3 2.5 2.5 5.5 2.2 2.5 3.1 3.9 3.2

34.8 0.7 3.7 0.6

94 0.4 5.4 0.4

0.23 0.9 n.a.a n.a.

1.0 0.3 1.1 0.3

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.1 0.6 1.5 0.6 0.2 0.8 0.6 0.2 0.6 0.1 1.3 0.4 0.2 0.3 3.2 0.4 0.2 0.8 0.1

predicted

Km(OE) (μM)c

CE (μL/min/mg S9)d

formation of 1′-sulfooxyestragole (nmol/g liver)e

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

4.84 4.64 5.74 6.43 9.99 4.85 5.73 9.38 9.87 1.98 8.52 9.07 5.56 4.56 6.12 6.44 4.95 4.95 7.22 6.36

0.01 0.02 0.02 0.02 0.02 0.03 0.03 0.03 0.04 0.05 0.05 0.06 0.07 0.08 0.08 0.10 0.11 0.12 0.21 0.06

2.14 0.3 n.a. n.a.

0.05 0.8 n.a. n.a.

421 672 581 488 325 751 556 398 498 716 358 364 447 541 894 345 499 627 534 527 153 0.3 6.2 0.3

45 354 382 225 25 222 200 87 72 87 101 51 89 106 567 131 68 243 21

a n.a., not applicable. bCode of the individual human liver S9 fractions. cMean ± standard error of the fit. dCatalytic efficiency (Vmax/Km). ePBKmodel predicted formation of 1′-sulfooxyestragole in 24 h at 0.01 mg/kg bw estragole. fCoefficients of variation (SD/μx). gμw = ln[μx/√(1 + CV2x)]. hσw = √ln(1 + CV2x).

apparent kinetic constants obtained from the incubations with 19 individual human liver S9 samples (resulting in 19 separate models). With the models obtained, predictions were made on the level of formation of 1′-sulfooxyestragole in human livers expressed as nanomoles per gram of liver at different estragole doses. Subsequently, these levels of 1′-sulfooxyestragole were converted to DNA adduct formation, assuming a linear relationship between 1′-sulfooxyestragole formation and DNA adduct formation at different oral doses. The proportion of 1′-sulfooxyestragole converted to E-3′-N2-dGuo adducts was derived from previously published in vivo data on DNA adduct formation in rats after a single oral exposure26 and a previously defined PBK model for estragole in rats that allows 1′-sulfooxyestragole formation in rat liver to be predicted27,24 (see the Results section). By assuming that the chemical reaction of 1′-sulfooxyestragole to generate DNA adducts will be the same for humans as it is for rats, the derived correlation from the rat data allowed the development of a PBK/D model in humans that predicts DNA adduct formation in humans. An overview of all model parameters of the human models and for the rat model is provided as Supporting Information (Table S1). It should be noted that for the PBK model for estragole in rat the apparent kinetic constants for the PAPS-dependent conversion of 1′hydroxyestragole to E-3′-N2-dGuo by rat liver S9, which were used in the current model to reflect sulfonation of 1′-hydroxyestragole, were obtained within the experimental range of the present study to exclude effects of experimental variation on the relationship between 1′sulfooxyestragole and E-3′-N2-dGuo formation (see Supporting Information Table S1 for the apparent kinetic constants obtained). In addition, the fact that both sulfonation and oxidation of 1′hydroxyestragole have been measured indirectly by trapping the respective metabolites and the fact that no pure cofactors were used for the incubations have no influence on the model predictions

because the prediction of DNA adduct formation in humans is based on the relative difference in the predicted formation of 1′sulfooxyestragole between rats and humans, which were measured in the same way and do not rely on the absolute predicted formation of 1′-sulfooxyestragole in the liver. The models were coded and numerically integrated in Berkeley Madonna 8.0.1 (Macey and Oster, UC Berkeley, CA), using Rosenbrock’s algorithm for stiff systems. Monte Carlo Simulations. Monte Carlo simulations were performed to simulate E-3′-N2-dGuo adduct formation for a larger population based on the data on interindividual human variation obtained with the 19 individual human tissue fractions. For the Monte Carlo analysis, a total of 10 000 simulations were performed, where in each simulation the Vmax and Km values for both formation of 1′sulfooxyestragole and oxidation of 1′-hydroxyestragole were randomly taken from a log-normal distribution. Each value was varied independently because no correlation was observed between the apparent Km and Vmax values within each reaction, nor was it observed between the measured sulfonation and oxidation reactions. The mean μw and standard deviation σw describing these log-normal distributions of the Km and Vmax values were derived from the 19 individual human subjects of the present study using the following equation μw = ln(μx /√1 + CV2 x) and σ 2 w = ln(1 + CV2 x) where μx is the mean and CVx is the coefficient of variation of the nonlog-normal-transformed kinetic constants as observed for the 19 individual human subjects of the present study (see the Results section). The distributions were truncated at ±3 SD by removing individuals with kinetic constants that were 3× the SD higher or lower than the geometric mean values from the Monte Carlo simulation, D


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Chemical Research in Toxicology resulting in a final population of 9896 individuals. The Berkeley Madonna code of the PBK/D model used to perform the Monte Carlo simulations is provided as Supporting Information (code S1). It should be noted that no variation was introduced in the physiological parameters and partition coefficients since the predicted formation of 1′-sulfooxyestragole was previously observed to be relatively insensitive to changes in these parameters.11 Only the liver volume may affect the predicted levels of 1′-sulfooxyestragole to some extent (normalized sensitivity coefficient of 0.5). However, because of the low level of variability in human liver volume (coefficient of variation of 5%),28 variation in liver volume did not affect the model outcome to a significant extent and was therefore not taken into account in the final model. Of the different metabolic parameters, interindividual human variation in the enzymes involved in the formation 1′-hydroxyestragole was previously observed to affect 1′sulfooxyestragole formation only to a very limited extent (sensitivity coefficients range from 0.05 to 0.2),11 and interindividual human variation in this metabolic route was, therefore, not taken into account in the present study. Furthermore, formation of 1′-sulfooxyestragole in humans was previously shown to be relatively insensitive to changes in other metabolic pathways, including epoxidation, O-demethylation, and 3′-hydroxylation of estragole and glucuronidation of 1′hydroxyestragole (sensitivity coefficients < 0.1).11 Glucuronidation of 1′-hydroxyestragole has also been observed to be only a minor metabolic route in humans in vivo.29,30 Interindividual human variation in these metabolic pathways was therefore not taken into account. The output of the Monte Carlo simulations was analyzed statistically to calculate the different percentiles of the model outcome distributions using IBM SPSS statistics, version 21 (IBM Corporatio, Armonk, NY).

was predicted for individual H0420, corresponding to 0.01 nmol/g liver (Table 2). The low observed formation of 1′sulfooxyestragole in this individual was due to the relatively low apparent Vmax for the sulfonation of 1′-hydroxyestragole as compared with that for the other individuals. The highest level of formation of 1′-sulfooxyestragole was predicted for individual H0432, corresponding to 0.21 nmol/g liver, due to a relatively low apparent Km for the sulfonation of 1′hydroxyestragole as compared with that for the other individuals. Overall, the differences in the formation of 1′sulfooxyestragole were 21-fold among the 19 individuals. On the basis of the apparent kinetic constants for the sulfonation and oxidation reactions obtained with the tissue fractions from 19 individuals, Monte Carlo simulations were performed to simulate the formation of 1′-sulfooxyestragole in a larger population. Parameters used to calculate the mean μw and the standard deviation σw describing the log-normal distribution of each Vmax and Km for the Monte Carlo simulations are shown in Table 2. Statistical analysis of the outcomes of the Monte Carlo simulations showed that the 50th, 90th, and 99th percentiles for the amounts of 1′sulfooxyestragole that are formed at an estragole dose of 0.01 mg/kg bw correspond to 0.05, 0.13, and 0.27 nmol/g liver. Thus, the differences between the 50th and 90th percentiles and the 50th and 99th percentiles were 2.6- and 5.4-fold, respectively. Derivation of an Equation Describing E-3′-N2-dGuo Adduct Formation as a Function of 1′-Sulfooxyestragole Levels. Figure 2 presents the level of E-3′-N2-dGuo

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RESULTS Effect of Interindividual Human Variation in Sulfonation and Oxidation of 1′-Hydroxyestragole on the Predicted Formation of 1′-Sulfooxyestragole. Table 2 shows the apparent kinetic constants, Vmax and Km, for PAPSand NAD+-dependent conversion of 1′-hydroxyestragole for 19 human individuals, derived experimentally by incubating liver S9 homogenates with the respective cofactors and increasing concentrations of 1′-hydroxyestragole. The apparent Vmax and Km values for the sulfonation reaction by the 19 human individuals ranged between 6.3 and 128.6 pmol/min/mg protein and between 54 and 378 μM, respectively. Apparent Vmax and Km values for the oxidation reaction for the 19 human individuals ranged between 1.4 and 5.5 nmol/min/mg protein and between 325 and 894 μM, respectively. The catalytic efficiency, calculated as the Vmax/Km ratio, was quantified for the individual S9 fractions, for both sulfonation and oxidation (Table 2). These catalytic efficiencies ranged between 0.04 and 1.03 μL/min/mg protein for PAPS-dependent conversion of 1′-hydroxyestragole to E-3′-N2-dGuo and between 1.98 and 9.99 μL/min/mg protein for NAD+-dependent conversion of 1′-hydroxyestragole. The average catalytic efficiency for the oxidation reaction was 25-fold higher than that for the sulfonation reaction, indicating that oxidation of 1′-hydroxyestragole is preferred over sulfonation, which is in agreement with previous observations using pooled human liver fractions.23 No correlation was observed between the deduced catalytic efficiencies for the sulfonation and oxidation reactions and the gender, age, smoking status, or alcohol consumption of the person. Using a previously defined human PBK model11 and the individual Vmax and Km values for the 19 individuals, the formation of 1′-sulfooxyestragole at an oral estimated daily intake of 0.01 mg/kg bw of estragole1 was modeled for each individual. The lowest level of formation of 1′-sulfooxyestragole

Figure 2. Formation of E-3′-N2-dGuo adducts in 108 nts in vivo in male Sprague−Dawley rats versus the levels of 1′-sulfooxyestragole (nmol/g liver) predicted by the rat PBK model.

adducts per 108 nucleotides (nts) detected in the liver of rats exposed to estragole by oral gavage at doses of 5, 30, 75, 150, and 300 mg/kg bw26 plotted against the level of 1′sulfooxyestragole (nmol/g liver) predicted to be formed in rat liver at the same doses using the PBK model for estragole in rat.24,27 On the basis of these data, an equation describing the relationship between the PBK-model-based predicted levels of 1′-sulfooxyestragole formation and DNA adduct formation in the liver over 24 h was derived as follows: E‐3′‐N 2‐dGuo adducts (no. adducts in 108 nts) = 32.6 × concentration of 1′‐sulfooxyestragole (nmol/g liver)

The equation indicates how much of the 1′-sulfooxyestragole reacts with DNA, with the remaining 1′-sulfooxyestragole most probably reacting with other nucleophilic cellular macroE


Article


Figure 3. Number of E-3′-N2-dGuo DNA adducts at a dose of 0.01 mg/kg bw as predicted by the human PBK models (A) for the 19 individuals and (B) results from the Monte Carlo simulation. The frequency distribution of E-3′-N2-dGuo DNA adduct formation for the general population at an estragole (ES) dose of 0.01 mg/kg bw (black bars) was predicted and compared with distribution of the in vivo observed methyleugenol (ME) DNA adduct levels in surgical human liver samples from (unknown) dietary exposure (gray bars).

Table 3. DNA Adduct Levels Estimated at the BMD10 for Different Alkenylbenzenes reference alkenylbenzene

slope (increase in DNA adduct formation, no. adducts/108 nts, per mg/kg bw increase in dosea)

ref

BMD10 (mg/kg bw)b

estimated DNA adduct levels at the lowest BMD10 (no. adducts/108 nts)

methyleugenol

25 15 40

Williams et al.34 Herrmann et al.33 Paini et al.26

20−88 20−88 5−8

500 300 188

estragole

Values represent the slopes obtained by linear fitting of in vivo observed DNA adduct formation with increasing dose levels, as provided in the Supporting Information (Figures S1−S3). bBMD10 values obtained from van den Berg et al.31 Note that for estragole BMD10 could be estimated only based on extrapolation of available carcinogenicity data at dose levels higher than the presumed BMD10, which may have resulted in a less certain estimate of the BMD10. a

dGuo adducts per 108 nts, and the 90th and 99th percentiles were 4.3 and 8.8 adducts per 108 nts. There is considerable overlap in the predicted distribution for estragole and the in vivo observed distribution for methyleugenol, with the median DNA adduct formation for methyleugenol being 13 adducts per 108 nts.16 Comparison of the Predicted DNA Adduct Formation in Humans to DNA Adduct Levels Related with Alkenylbenzene-Induced Tumor Formation in Rodents. The biological relevance of the predicted DNA adduct levels in humans at physiologically relevant exposures may be evaluated by comparing the predicted DNA adduct levels with DNA adduct levels that occur at dose levels of alkenylbenzene that have induced liver tumors in rodents. On the basis of available data for estragole and its related alkenylbenzene methyleugenol, dose levels that induce a 10% increase in tumor incidence upon lifetime exposure (BMD10) have been estimated to amount to approximately 5−8 mg/kg bw for estragole and 20−88 mg/ kg bw for methyleugenol.31 On the basis of a method previously described by Paini et al.32 for various carcinogens, the number of adducts that can be expected to occur at the respective BMD10 of each compound can be calculated from available data on DNA adduct formation in rodents. Recently published data on DNA adduct formation by methyleugenol and estragole in rodents26,33,34 allows similar predictions to be performed for estragole and methyleugenol. A summary of these latter studies that were compatible with a linear increase in observed DNA adduct levels with increasing doses is provided as Supporting Information (Figures S1−S3). Table 3 provides the slope obtained from the different curves by linear fitting, allowing the DNA adduct levels at the BMD10 to be estimated for the respective alkenylbenzenes. Overall, these results reveal that for estragole and methyleugenol the DNA adduct levels expected to occur at dose levels that induce 10%

molecules. Accordingly, the overall percentage of 1′-sulfooxyestragole that is converted to E-3′-N2-dGuo adducts at the different dose levels corresponds to 0.39, 0.18, 0.19, 0.25, and 0.16%, resulting in an average value of 0.23 ± 0.09% of the amount of 1′-sulfooxyestragole that is formed or to 0.0003 ± 0.0001% of the estragole dose. Since the equation defined is based on in vivo data for DNA adduct formation, the values implicitly represent the result from processes including DNA adduct formation and repair. Evaluation of Interindividual Human Variation in DNA Adduct Formation and Comparison of the Predictions Made to Human Data on DNA Adducts Levels. Using the equation defined above based on the rat data and assuming that the relationship between 1′-sufooxyestragole formation and DNA adduct levels detected would be comparable for rats and humans, the PBK/D model for human was used to predict the dose-dependent formation of DNA adducts in humans. With the models obtained, the amount of E-3′-N2-dGuo adducts predicted to be formed at the estimated daily intake of 0.01 mg/kg bw day ranged between 0.4 and 7 adducts per 108 nts (Figure 3A) in the 19 human individuals. In addition, DNA adduct formation was predicted for a larger population using Monte Carlo simulations. Figure 3B shows the results of this simulation, including a comparison of the predicted E-3′-N2dGuo DNA adduct levels to literature reported levels of analogous guanosine adducts that have been observed for the related alkenylbenzene methyleugenol in livers of human subjects that had to undergo liver surgery.16 The fact that intake levels of estragole and methyleugenol from the use of herbs, essential oils, and flavor substances containing estragole are estimated to be similar for estragole and methyleugenol1 supports this comparison. The median predicted amount of guanosine adducts formed for estragole, at the estimated average human daily intake of 0.01 mg/kg bw, was 1.6 E-3′-N2F


Article


Vmax and Km is considered to represent an average level of variation.39 The results obtained with the Monte Carlo simulation on the variability in the formation of 1′-sulfooxyestragole can potentially be used to derive a so-called chemical-specific adjustment factor (CSAF), which can be used to evaluate the appropriateness of the default factor of 3.16 generally assumed to reflect interindividual variation in kinetics within the human population.23,40−42 On the basis of the guidelines of the International Program on Chemical Safety (IPCS, 2005), CSAFs can be calculated as the ratio between given percentiles (such as 90th, 95th, 97.5th, or 99th) and the median for the whole population. Using the 90th percentile to represent a sensitive individual and the 50th percentile to represent the average individual, the CSAF can be calculated to amount to 2.6 based on the results of the present study. This predicted CSAF is comparable to the default factor of 3.16 for kinetic variability and suggests that the default uncertainty factor adequately protects 90% of the population. Using the 99th percentile to represent a sensitive individual, the CSAF amounts to 5.4, indicating that protecting 99% of the population would require a higher uncertainty factor than the default value of 3.16. Predicted liver levels of 1′-sulfooxyestragole were converted to E-3′-N2-dGuo adduct levels using an equation defined based on observed levels of E-3′-N2-dGuo adducts in the livers of estragole-exposed rats and PBK-predicted formation of 1′sulfooxyestragole in rat liver. By including this equation, dosedependent levels of DNA adduct formation in humans can be predicted at relevant exposure levels of estragole. The predicted DNA adduct formation at this dose level, corresponding to 1.6 adducts per 108 nts (50th percentile) to 8.8 adducts per 108 nts (99th percentile), appeared to be slightly lower as the literature reported DNA adduct levels measured from 30 individual humans for the related alkenylbenzene methyleugenol, corresponding to 13 adducts per 108 nts.16 This result suggests that adequate predictions could be made based on the developed method. Still, some uncertainties remain. For example, in the present study, DNA adduct levels in the liver of humans are predicted based on data of DNA adduct levels in rats, without taking possible differences in DNA repair between rats and humans into account and assuming that the chemical reaction of 1′-sulfooxyestragole to generate DNA adducts will be the same for humans as it is for rats. In addition, a linear relationship between 1′-sulfooxyestragole formation and DNA adduct formation at different oral doses was assumed, as reported previously.24 Moreover, the predicted level of DNA adduct formation in humans could be validated only against available data for the related alkenylbenzene methyleugenol. Although this comparison is supported by the comparable intake estimates for estragole and methyleugenol (i.e., 0.01 mg/ kg bw/day for both compounds1) and the similar level of bioactivation that has been estimated for the two compounds in humans (i.e., varying less than 5-fold11,27,43−45), ultimate validation of the model’s predictions will rely on in vivo human data for estragole. The biological relevance of the predicted DNA adduct levels in humans at physiologically relevant exposure levels was evaluated against DNA adduct levels that occur at dose levels of alkenylbenzenes that have induced liver tumors in rodents. On the basis of data for both estragole and methyleugenol, DNA adduct levels at dose levels that induce a 10% tumor incidence in rodents were estimated to range between 188 and 500

tumor incidence in rats can be estimated to range between 188 and 500 adducts per 108 nts (Table 3; range is based on the lowest estimates of the BMD10 values). The predicted DNA adduct formation in humans at a dose of 0.01 mg/kg bw, ranging from 1.6 adducts per 108 nts (50th percentile) to 8.8 adducts per 108 nts (99th percentile), is 21−330 times below these levels.

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DISCUSSION The objective of the present study was to obtain insight into possible levels of DNA adduct formation in human liver upon exposure to estragole at realistic human dietary intake levels while taking into account variation in the major metabolic reactions that influence the formation of carcinogenic metabolite 1′-sulfooxyestragole. Sensitivity analysis previously reported for the human PBK model showed that the formation of 1′-sulfooxyestragole depends mainly on the kinetic constants for the conversion of 1′-hydroxyestragole to 1′-oxoestragole and the kinetic constants for the conversion of 1′hydroxyestragole to 1′-sulfooxyestragole itself.11 By determining the Vmax and Km values for these metabolic reactions experimentally for a set of liver S9 fractions from 19 human individuals, interindividual human variation in the formation of 1′-sulfooxyestragole could be modeled for these 19 individuals as well as for a larger population, the latter using a Monte Carlo simulation. To this end, the variation in the sulfonation and oxidation reactions observed for the 19 individuals was assumed to represent the level of variation in the population as a whole. Additional insight into the level of variability in sulfonation and oxidation of 1′-hydroxyestragole that could occur in the population may be obtained by characterizing the enzymes involved in these reactions. In the case of estragole, the human sulfotransferase enzymes involved in the sulfonation of 1′hydroxyestragole have not been identified so far. No data are available on the relative expression of different SULT isoforms in the individual human liver samples used in the present study. Sulfonation of the 1′-hydroxy metabolite of the related alkenylbenzene methyleugenol in the liver has been observed to be primarily catalyzed by SULT1A1 and to a lesser extent by SULT1E1.22 The reported coefficients of variation for the catalytic activities of SULT1A1 and SULT1E1, as determined with enzyme-selective substrates in human liver tissue fractions, amount to 34 and 47%, respectively.35 The level of variation in sulfonation was observed to be higher in the present study, with an overall coefficient of variation of 90% for the catalytic efficiency. The differences in the sulfonation of 1′-hydroxyestragole were not related to the gender, age, smoking status, or alcohol consumption of the person. This finding is supported by the literature, which reveals mainly polymorphic differences for SULT1A1 and SULT1E1 expression.36 In addition, SULT1A1 and SULT1E1 are not differently expressed in children.37 In the case of the oxidation reaction, 17βhydroxysteroid dehydrogenase type 2 (17β-HSD2) was previously suggested to be involved based on the subcellular localization of the reaction, which occurred mainly in incubations with microsomes, and based on enzyme specificity, with NAD+ being the preferred cofactor.11 Audet-Walsh et al.38 reported that genetic polymorphisms occur in the gene encoding 17β-HSD2, which could lead to reduced 17β-HSD2 expression or function. The impact of such polymorphisms in 17β-HSD2 on its activity remains to be elucidated. The current variation in the oxidation of 1′-hydroxyestragole of 30% in both G



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adducts per 108 nts. These levels are higher than previously calculated for methyleugenol by Paini et al.,32 amounting to 34−40 adducts per 108 nts. However, as this previous estimation for methyleugenol was derived based on data that has been published in a conference abstract that does not contain details on how the data were derived, no explanation can be given for the difference. The obtained range of 188 and 500 adducts per 108 nts at the BMDL10 are in the same range as previously reported by Phillips et al.,7 490 adducts per 108 nts (i.e., 15 pmol/mg DNA), to be linked to statistically significant tumor formation by estragole and related alkenylbenzenes in newborn mice. Furthermore, these levels are also in the same range as DNA adduct levels that have been historically linked to tumor formation in rodents upon exposure to various hepatocarcinogens, amounting to 50−5500 adducts per 108 nucleosides at the TD50 (daily dose that induces 50% tumor response in long-term studies)46 or 10−10 000 adducts per 108 nucleosides at the BMD10.32 For estragole, the predicted level of DNA adduct formation in humans at a dose of 0.01 mg/kg bw estragole of 1.6−8.8 adducts per 108 nts (at the 50th and 99th percentiles, respectively) is below the 188 and 500 adducts per 108 nts that are linked to 10% tumor incidence in rodents, which may suggest that risk related to daily estragole exposure is limited, although care should be taken when making such a comparison as DNA adduct formation does not equate to tumor formation. It is worth noting that higher dose levels from the addition of estragole, which has been estimated to result in an average daily intake of 0.07 mg/kg, would lead to higher levels of DNA adduct formation of 11 to 63 adducts per 108 nts (at the 50th and 99th percentiles, respectively) that may be considered too close to the DNA adduct levels that are associated with liver tumors. A similar comparison was made by Herrmann et al.,16 who concluded that DNA adduct levels observed in human liver samples by the related compound methyleugenol, ranging from 13 to 37 adducts per 108 nts in human liver samples, might be too close to the 50−5500 adducts per 108 nucleosides at the TD50. Whether DNA adduct levels from estragole are indeed lower than those for methyleugenol remains to be elucidated, as the observed differences in the present study may not necessarily be due to differences in the level of bioactivation of the two compounds and the reactivity of their 1′sulfooxymetabolites but may also be due to uncertainty in the model’s predictions and/or to the exposure estimate of estragole that was used. Taken together, it can be concluded that at an estimated daily intake of 0.01 mg/kg bw/day estragole results in E-3′-N2dGuo adduct levels in humans predicted to be between 1.6 adducts per 108 nts (50th percentile) and 8.8 adducts per 108 nts (99th percentile). These adduct levels seem to be below DNA adduct levels that have been associated with tumor formation by alkenylbenzenes in rodents, which were estimated in the present study to amount to 188−500 adducts per 108 nts at the BMD10 values of estragole and methyleugenol. These data may suggest a limited concern for estragole carcinogenicity at average daily intake levels, although more firm conclusions may have to await validation of the model’s predictions against in vivo human data for estragole.

Article

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.chemrestox.5b00493. PBK model parameters that were used in the rat and human model, the PBK model code for the human model, and an analysis of DNA adduct formation observed in rodents exposed to different alkenylbenzenes (PDF)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Tel: + 0031 317 484936. Fax: + 0031 317 484931. Author Contributions ∥

Ans Punt and Alicia Paini contributed equally to this work.

Funding

This work was financially supported by Nestlé Research Center, Lausanne, Switzerland. Notes

The authors declare the following competing financial interest(s): Dr. Rietjens is a member of the Expert Panel of the Flavor and Extract Manufacturers Association (FEMA).

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ABBREVIATIONS nts, nucleotides; PAPS, 3′-phosphoadenosine-5′-phosphosulfate; PBK/D, physiologically based kinetic/dynamic; GST, glutathione S-transferase; GSH, reduced glutathione; NAD+, oxidized β-nicotinamide adenine dinucleotide phosphate; NADPH, reduced β-nicotinamide adenine dinucleotide phosphate; dGUO, 2′-deoxyguanosine; UPLC, ultra-performance liquid chromatography

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REFERENCES

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