Study on inter-ethnic human differences in bioactivation and detoxification of estragole using physiologically based kinetic modeling

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1 Arch Toxicol (2017) 91: DOI /s x TOXICOKINETICS AND METABOLISM Study on inter-ethnic humn differences in bioctivtion nd detoxifiction of estrgole using physiologiclly bsed kinetic modeling Ji Ning 1 Jochem Louisse 1 Bert Spenkelink 1 Sebstin Wesseling 1 Ivonne M. C. M. Rietjens 1 Received: 24 October 2016 / Accepted: 21 Februry 2017 / Published online: 29 Mrch 2017 The Author(s) This rticle is n open ccess publiction Abstrct Considering the rpid developments in food sfety in the pst decde in Chin, it is of importnce to obtin insight into wht extent sfety nd risk ssessments of chemicls performed for the Cucsin popultion pply to the Chinese popultion. The im of the present study ws to determine physiologiclly bsed kinetic (PBK) modeling-bsed predictions for differences between Chinese nd Cucsins in terms of metbolic bioctivtion nd detoxifiction of the food-borne genotoxic crcinogen estrgole. The PBK models were defined bsed on kinetic constnts for heptic metbolism derived from in vitro incubtions using liver frctions of the two ethnic groups, nd used to evlute the inter-ethnic differences in metbolic ctivtion nd detoxifiction of estrgole. The models predicted tht t relistic dietry intke levels, only 0.02% of the dose ws converted to the ultimte crcinogenic metbolite 1 -sulfooxyestrgole in Chinese subjects, wheres this mounted to 0.09% of the dose in Cucsin subjects. Detoxifiction of 1 -hydroxyestrgole, minly vi conversion to 1 -oxoestrgole, ws similr within the two ethnic groups. The 4.5-fold vrition in formtion of the ultimte crcinogenic metbolite of estrgole ccompnied by similr rtes of detoxifiction my indicte lower risk of estrgole for the Chinese popultion t similr levels of exposure. The study provides proof of principle for how PBK modeling cn identify differences in ethnic sensitivity Electronic supplementry mteril The online version of this rticle (doi: /s x) contins supplementry mteril, which is vilble to uthorized users. * Ji Ning ji.ning@wur.nl 1 Division of Toxicology, Wgeningen University, Stippeneng 4, 6708 WE Wgeningen, The Netherlnds nd provide more refined risk ssessment for specific ethnic group for compound of concern. Keywords Inter-ethnic difference Chinese Cucsin Physiologiclly bsed kinetic modeling Estrgole Introduction Recently, the dose-dependent bioctivtion nd detoxifiction of estrgole in different species, including humn, hs been studied by physiologiclly bsed kinetic (PBK) nd dynmic (PBD) modeling (Punt et l. 2008, 2009, 2016). The models defined for the humn popultion were specific for Cucsins, since the prmeters used to describe the kinetics were derived using smples from relevnt tissues from Cucsin origin. Considering the rpid developments in food sfety in the pst decde in Chin, it is of importnce to obtin insight into to wht extent sfety nd risk ssessments of chemicls performed for the Cucsin popultion would lso pply to the Chinese popultion. Given the fct tht rce diversity might result in the vribility of dose-response reltionships ffecting the sfety nd efficcy of chemicl exposures (Mlinowski et l. 2008), the bsence of knowledge in this field implies tht hrmoniztion in legisltion on chemicls between regultory bodies of Europe, USA nd Asi is hmpered. Severl studies hve shown ethnic differences in cytochrome P450 enzymes. Significnt differences in rections ctlyzed by CYP1A2, CYP2C9, CYP2C19, nd CYP2E1 in liver microsomes hve been observed between Chinese nd Cucsin smples (Yng et l. 2012). Brter et l. (2013) developed PBK models nd used them for in vitro to in vivo extrpoltion to predict the P450-medited phrmcokinetics of selected drugs in the Chinese popultion. The results showed tht Vol.:( )

2 3094 Arch Toxicol (2017) 91: the predicted clernces for phencetin, tolbutmide, desiprmine, omeprzole, lprzolm (intrvenous), lprzolm (orl), midzolm (intrvenous), nd midzolm (orl) in Chinese subjects were predicted to be 36, 25, 43, 51, 21, 22, 24, nd 17% lower, respectively, thn in Cucsin subjects, nd the experimentlly observed clernces in treted Chinese individuls were 28, 2, 42, 75, 20, 21, 19, nd 62% lower, respectively, thn in Cucsin volunteers (Brter et l. 2013). Thus, inter-ethnic differences in metbolism nd metbolic bioctivtion nd detoxifiction my occur. However, systemtic ttempts to predict the kinetic processes nd relted toxicity in different popultions using physiologiclly bsed kinetic (PBK) modeling re lcking. The im of the present study ws to determine PBK modeling-bsed predictions for differences between Chinese nd Cucsins in terms of metbolic bioctivtion nd detoxifiction of the food-borne genotoxic crcinogen estrgole (Fig. 1). Estrgole (1-llyl-4-methoxybenzene) is n lkenylbenzene tht is nturlly present in vriety of herbs nd spices, such s fennel, bsil, nd trrgon (Smith et l. 2002). Consumption of herbs, spices, nd their essentil oils nd food products contining these is n importnt route of exposure to estrgole. The Flvor nd Extrct Mnufcturers Assocition (FEMA) estimted the dily intke of estrgole to be less thn 0.01 mg/kg bw/dy bsed on the nnul production volume dt of estrgole for use in flvorings (Smith et l. 2002). Estrgole is known to be genotoxic nd crcinogenic in rodents t high-dose levels (Drinkwter et l. 1976; Miller et l. 1983). Bioctivtion of estrgole to DNA rective ultimte crcinogen proceeds by cytochrome P450 medited conversion to 1 -hydroxyestrgole nd subsequent conversion of 1 -hydroxyestrgole to the ultimte crcinogen 1 -sulfooxyestrgole by sulfotrnsferses (SULTs) (Fig. 1). Detoxifiction of 1 -hydroxyestrgole proceeds by glucuronidtion to 1 -hydroxyestrgole glucuronide nd oxidtion to 1 -oxoestrgole (Fig. 1). Given the vriety of biotrnsformtion enzymes involved in estrgole Fig. 1 Metbolic pthwys of estrgole

3 Arch Toxicol (2017) 91: bioctivtion nd detoxifiction, estrgole ws selected s n dequte model compound to study ethnic differences in bioctivtion nd detoxifiction. To define the PBK models for the Chinese nd Cucsin popultions, kinetic constnts for the vrious biotrnsformtion rections of estrgole were quntified using in vitro incubtions with relevnt tissue frctions of the two ethnic groups. The outcomes predicted by the PBK model for the Chinese popultion were compred to those predicted by the PBK model for Cucsins to evlute the inter-ethnic differences in metbolic ctivtion nd detoxifiction of estrgole nd to demonstrte the potentil of PBK modeling to study such inter-ethnic vribility. Mterils nd methods Chemicls nd biologicl mterils Estrgole (1-llyl-4-methoxybenzene), dimethylsulfoxide (DMSO), lmethicin, uridine 5 -diphosphoglucuronic cid (UDPGA), 3 -phosphodenosine-5 -phosphosulfte (PAPS), phencetin, cetminophen, coumrin, 7-hydroxycoumrin, 7-hydroxycoumrin sulfte, glucose-6-phosphte dehydrogense, nd reduced L-glutthione (GSH) were purchsed from Sigm Aldrich (Steinheim, Germny). Potssium dihydrogen phosphte, dipotssium hydrogen phosphte trihydrte, hydrochloric cid (37%), trifluorocetic cid (TFA), nd mgnesium chloride were purchsed from VWR Interntionl (Drmstdt, Germny). Reduced nicotinmide denine dinucleotide phosphte (NADPH), nicotinmide denine dinucleotide phosphte nicotinmide (NADP + ), denine dinucleotide (NAD + ), nd glucose-6-phosphte were obtined from Roche Dignostics (Mnnheim, Germny). Acetonitrile (UPLC/MS grde) ws obtined from Biosolve BV (Vlkenswrd, Netherlnds). 1 -Hydroxyestrgole, 4-llylphenol, estrgole- 2,3 -oxide, 3 -hydroxynethole, nd 1 -oxoestrgole were synthesized s previously described by Punt et l. (2007, 2008). Chinese liver microsomes nd Chinese S9, mde from 40 donors, were purchsed from PrimeTox (Wuhn, Chin). Cucsin liver microsomes were purchsed from BD Gentest (Woburn, MA, USA), nd Cucsin liver S9 ws purchsed from Corning (Amsterdm, Netherlnds). All microsomes nd S9 were of pooled mixed gender. In vitro incubtions Assessment of metbolic cpbilities of CYP nd SULT enzymes in Chinese nd Cucsin liver smples The qulity of the Chinese nd Cucsin liver microsomes ws checked by mesuring the ctivity of CYP1A2 nd 3095 CYP2A6 by the method of Yng et l. (2012). The qulity of Chinese nd Cucsin liver S9 smples ws checked by mesuring the SULT ctivity bsed on the method provided by Wng et l. (2006). The detils of the methods for the microsoml nd S9 in vitro incubtions nd UPLC nlysis for ssessing the metbolic cpbilities of CYP nd SULT enzymes cn be found in the supporting mterils 1. Microsoml metbolism of estrgole The kinetic constnts for the microsoml conversion of estrgole were determined s previously described by Punt et l. (2009). Briefly, mixed gender Chinese or Cucsin liver microsomes were incubted with estrgole in the presence of NADPH. The incubtion mixtures contined (finl concentrtions) 3 mm NADPH nd 1 mg/ml microsoml protein in 0.2 M Tris HCl (ph 7.4). Incubtions were performed for 10 min t substrte concentrtions rnging from 25 to 1000 μm, fter which the rection ws terminted by dding 25 μl ice-cold cetonitrile. Blnk incubtions were performed in the bsence of the cofctor NADPH. All incubtions were performed in triplicte. In the supporting mterils 2, detiled informtion cn be found (Tble S1 cn be used to keep trck of detiled informtion). Glucuronidtion of 1 hydroxyestrgole Pooled mixed gender Chinese or Cucsin liver microsomes were incubted with 1 -hydroxyestrgole in the presence of UDPGA. As previously described by Punt et l. (2009), the incubtion mixtures contined (finl concentrtions) 10 mm UDPGA, nd 1 mg/ml microsoml protein in 0.2 M Tris HCl (ph 7.4) with 10 mm MgCl 2. Incubtions were crried out for 6 h, nd the rection ws terminted by dding 25 μl ice-cold cetonitrile. Blnk incubtions were performed in the bsence of the cofctor UDPGA. All incubtions were performed in triplicte. In the supporting mterils 2, detiled informtion cn be found (Tble S1 cn be used to keep trck of detiled informtion). Oxidtion of 1 hydroxyestrgole Mixed gender Chinese or Cucsin liver S9 ws incubted with 1 -hydroxyestrgole in the presence of NAD + nd GSH, the ltter dded to trp the trnsient 1 -oxoestrgole. Formtion of the 1 -oxoestrgole dducts with GSH forming GS-1 -oxoestrgole reflects the formtion of 1 -oxoestrgole (Punt et l. 2009). The incubtions hd finl volume of 100 μl, contining (finl concentrtions) 3 mm NAD +, 2 mm GSH, nd 1 mg/ml liver S9 in 0.2 M Tris HCl (ph 7.4), s described previously by Punt et l. (2016). The rections were terminted fter 10 min by the

4 3096 Arch Toxicol (2017) 91: ddition of 25 μl ice-cold cetonitrile. Blnk incubtions were performed without cofctor NAD +. All incubtions were performed in triplicte. In the supporting mterils 2, detiled informtion cn be found (Tble S1 cn be used to keep trck of detiled informtion). Sulftion of 1 hydroxyestrgole The formtion of 1 -sulfooxyestrgole ws determined by incubting 0.2 mg/ml pooled mixed gender Chinese or Cucsin liver S9 in the presence of 0.2 mm PAPS s cofctor nd 10 mm GSH s trpping gent for the rective 1 -sulfooxyestrgole in 0.1 M potssium phosphte (ph 8.0). The incubtions were crried out for 2 h, nd the rections were terminted by dding 25 μl ice-cold cetonitrile. The blnk smples were performed without cofctor. All incubtions were performed in triplicte. In the supporting mterils 2, detiled informtion cn be found (Tble S1 cn be used to keep trck of detiled informtion). UPLC nlysis UPLC nlysis of estrgole metbolites Before UPLC nlysis, ll smples were centrifuged for 5 min t 16,000 g to precipitte microsoml proteins. Superntnt of ech smple ws nlyzed on UPLC using BEH C18 (1.7 μm, mm) column with gurd column nd diode rry detector (Acquity, Wters). The grdient for nlysis of metbolites of estrgole cn be found in supporting mterils 2. Tble S1 cn be used to keep trck of detiled informtion. Identifiction of microsoml metbolites of estrgole, including 4-llylphenol, estrgole- 2,3 -diol, 1 -hydroxyestrgole, nd 3 -hydroxynethole, nd M5 ws chieved by comprison of the UV spectr nd retention times of formed metbolites to those of synthesized reference compounds identified previously (Punt et l. 2007, 2008). Formtion of 4-llylphenol, estrgole- 2,3 -diol, nd 1 -hydroxyestrgole ws quntified by compring the pek res to those of the corresponding reference stndrd curves t wvelength 225 nm (Aghrhimi nd LeBel 1995; Drinkwter et l. 1976; Iyer et l. 2003; Luo et l. 1992). Becuse the UV spectrum of M5 is similr to tht of estrgole-2,3 -diol, quntifiction of M5 could be chieved by comprison of the pek re to the clibrtion curve of estrgole-2,3 -diol t 225 nm. 3 -Hydroxynethole ws quntified by comprison of the pek res of the metbolite in the chromtogrms obtined t wvelength of 206 nm to the clibrtion curve of the synthesized reference compound (Aghrhimi nd LeBel 1995; Drinkwter et l. 1976; Iyer et l. 2003; Luo et l. 1992). The mounts of formed microsoml estrgole metbolites were corrected for the mounts detected in the blnk incubtions performed without the respective cofctor NADPH. UPLC nlysis of 1 hydroxyestrgole metbolites Before UPLC nlysis, ll smples were centrifuged for 5 min t 16, 000 g to precipitte microsoml or cytosolic proteins. Superntnt of ech smple ws nlyzed on UPLC using BEH C18 (1.7 μm, mm) column with gurd column nd diode rry detector (Acquity, Wters). Secondry metbolism of 1 -hydroxyestrgole includes glucuronidtion, oxidtion, nd sulftion. The grdient of nlysis of the metbolites of 1 -hydroxyestrgole cn be found in supporting mterils 2. Tble S1 cn be used to trck more detiled informtion. Identifiction of 1 -hydroxyestrgole glucuronide ws chieved by the fct tht it ws the only metbolite formed nd by LC-MS nlysis s reported previously (Punt et l. 2008). Both 1 -hydroxyestrgole glucuronide nd 1 -hydroxyestrgole hve the sme UV spectrum nd the sme extinction coefficient t wvelength of 225 nm. Thus, the quntifiction of 1 -hydroxyestrgole glucuronide could be chieved by compring the pek re to the clibrtion curve of 1 -hydroxyestrgole t wvelength 225 nm (Punt et l. 2009). The mounts of 1 -hydroxyestrgole glucuronide formed were corrected for the mounts detected in the blnk incubtions performed without the respective cofctor UDPGA. Identifiction of the GS-1 -oxoestrgole which reflects the formtion of 1 -oxoestrgole ws done by compring the UV spectr nd retention time of the formed GSH dduct to those of GS-1 -oxoestrgole identified s described previously (Punt et l. 2009). Quntifiction of the GSH conjugte of 1 -oxoestrgole ws chieved by compring the pek re to the clibrtion curve of GS-1 -oxoestrgole t wvelength of 280 nm prepred s described previously by Punt et l. (2009, 2016). Briefly, the clibrtion curve of GS-1 -oxoestrgole ws prepred by incubting 40 μm 1 -oxoestrgole with rnge of GSH concentrtions. The rections were incubted for 4 h fter which mximl formtion of GS-1 -oxoestrgole ws previously shown to be reched (Punt et l. 2009). The mounts of 1 -oxoestrgole formed were corrected for the mounts detected in the blnk incubtions performed without the respective cofctor NAD +. Since the UV spectrum of 3 -hydroxynethole is similr to tht of the GSH dduct of 1 -sulfooxyestrgole, quntifiction of 1 -sulfooxyestrgole could be chieved by compring the pek re of the GSH dduct of 1 -sulfooxyestrgole to the clibrtion curve of 3 -hydroxynethole t wvelength 260 nm s described for the quntifiction of 1 -sulfooxysfrole nd 1 -sulfooxyelemicin (Mrtti et l. 2012; Vn den Berg et l. 2012). The mount of

5 Arch Toxicol (2017) 91: sulfooxyestrgole GSH dduct formed ws corrected for the mount detected in the blnk smples performed without the respective cofctor PAPS. Kinetic nlysis The dt for the formtion of estrgole nd 1 -hydroxyestrgole metbolites with incresing substrte concentrtion [S] were fitted to the stndrd Michelis Menten eqution: υ = V mx (1 +(K m [S]). The pprent mximum velocity (V mx ) nd the pprent Michelis Menten constnt (K m ) were determined by fitting the dt to this eqution using GrphPd Prism version 5.0 (GrphPd softwre, Sn Diego Cliforni USA). PBK model structure The PBK models developed in this study were bsed on the PBK models previously defined by Punt et l. (2008). The models hve six comprtments, including blood, ft, rpidly perfused tissue, slowly perfused tissue, liver, nd GI trct tht re mutully connected through the systemic circultion. A schemtic digrm of the PBK models for estrgole kinetics is presented in Fig. 2, nd the code of the models cn be found in the supporting mterils 6. Tble 1 summrizes the physiologicl prmeters for Cucsin nd Chinese subjects, respectively, which were derived from the literture (Brown et l. 1997; NHFPC 2007, 2014b). Bsed on the method described by DeJongh et l. (1997; Punt et l. 2016), prtition coefficients were estimted bsed on the log K ow. The log K ow vlues for estrgole nd 1 -hydroxyestrgole were estimted by ChemBio 3D 2010 (CmbrigeSoft, USA). Model equtions were coded nd numericlly integrted 3097 in Berkely Mdonn (Mcey nd Oster, UC Berkeley, CA, USA) using the Rosenbrock s lgorithm for stiff systems. Estrgole ws ssumed to directly enter from the gstrointestinl trct vi the portl vein into the liver with Tble 1 Prmeters used in the physiologiclly bsed kinetic model for estrgole in Chinese nd Cucsin popultions s obtined from the literture Model prmeters Chinese,c Cucsin b,c Physiologicl prmeters Body weight (kg) Percentge of body weight Liver Ft Rpidly perfused Slowly perfused Blood Crdic output (l/hr/kg bw 0.74 ) Percentge of crdic output Liver Ft Rpidly perfused Slowly perfused Tissue: blood prtition coefficients Estrgole Liver Ft Rpidly perfused Slowly perfused Hydroxyestrgole Liver NHFPC (2007); NHFPC (2014b) b Brown et l. (1997) c DeJongh et l. (1997) Fig. 2 Schemtic digrm of the PBK model for estrgole in humn, including the nme of kinetic prmeters for metbolites of estrgole nd 1 -hydroxyestrgole s used in the mss blnce equtions of the PBK model

6 3098 Arch Toxicol (2017) 91: n bsorption rte constnt (K ) of 1.0 h 1 s first-order process. This vlue ws used bsed on the fct tht complete nd fst bsorption of estrgole from the GI trct hs been observed (Anthony et l. 1987). As reported by Punt et l. (2009), conversion of estrgole minly occurs in the liver nd not in other orgns. In ddition, since the liver is known to be the mjor trget orgn for estrgole-induced tumor formtion in rt nd mice, the PBK model focused on metbolism of estrgole nd 1 -hydroxyestrgole in the liver. 1 -Hydroxyestrgole, 2,3 -estrgole oxide, 3 -hydroxynethole, 4-llylphenol, nd n unidentified minor metbolite referred to s M5 were formed in incubtions with liver microsomes from both popultions (see Results section), nd conversions of estrgole into these metbolites were included in the liver comprtment of the models. In both models, descriptions of the conversions of 1 -hydroxyestrgole into its further metbolites were included, but the conversions of 2,3 -estrgole oxide, 3 -hydroxynethole, 4-llylphenol, nd M5 to their further metbolites were not included, since these re ssumed to not influence the formtion of the ultimte crcinogenic metbolite 1 -sulfooxyestrgole. The mss blnce equtions for microsoml conversion of estrgole when using Chinese or Cucsin liver microsomes were described s follows: dal E dt = duptke E dt + QL (CA E CL E PL E ) V mx, L_AP (CL E PL E ) (K m, L_AP + CL E PL E ) V mx, L_HE (CL E PL E ) (K m, L_HE + CL E PL E ) V mx, L_EE (CL E PL E ) (K m, L_EE + CL E PL E ) V mx, L_HA (CL E PL E ) (K m, L_HA + CL E PL E ) V mx, L_M5 (CL E PL E ) (K m, L_M5 + CL E PL E ) duptke E dt = dagi E dt = K AGI E, AGI E (0) = orl dose CL E = AL E VL where AL E is the mount of estrgole in the liver tissue (μmol). Uptke E is the mount of estrgole tken up from the GI trct (μmol), QL is the blood flow rte to nd from the liver tissue (L/h), CA E is the estrgole concentrtion in the rteril blood (μmol/l), nd CL E is the estrgole concentrtion in the liver tissue. PL E is the liver/blood prtition coefficient, V mx, L_M nd K m, L_M re the mximum rte of formtion nd Michelis Menten constnt for the metbolites 4-llylphenol (AP), 1 -hydroxyestrgole (HE), estrgole-2,3 -oxide (EE), 3 -hydroxynethole (HA) nd metbolite 5 (M5) in the liver tissue, AGI E (μmol) is the mount of estrgole in the GI trct, nd VL is the volume of the liver. The mss blnce eqution for the metbolism of 1 -hydroxyestrgole in the liver ws s follows: dal HE dt = V mx, L_HE (CL E PL E ) (K m, L_HE + CL E PL E ) CL HE = AL HE /VL where AL HE is the mount of 1 -hydroxyestrgole in the liver tissue (μmol), CL HE is the 1 -hydroxyestrgole concentrtion in the liver tissue (μmol/l), PL HE is the liver/blood prtition coefficient of 1 -hydroxyestrgole, V mx, L_M, nd K m, L_M re the mximum rte nd the Michelis Menten constnt for the formtion of 1 -hydroxyestrgole glucuronide (HEG), 1 -oxoestrgole (OE) nd 1 -sulfooxyestrgole (HES) in the liver tissue. The kinetic constnts for metbolites formed were determined in vitro in the present study. V mx vlues expressed s nmol/min/(mg microsoml or S9 protein) were scled to the V mx per μmol/h/(g liver) using mirosoml nd S9 protein yields of 35 nd 143 mg/g liver, respectively, s previously described by Punt et l. (2009), Al-Subeihi et l. (2012), Mrtti et l. (2012), nd Vn den Berg et l. (2012). Currently, no dt re vilble specificlly for the liver microsoml nd S9 protein relevnt for the Chinese tissue smples. Therefore, the vlue of liver microsoml protein nd liver S9 protein vilble for the Cucsins ws used for the Chinese popultion. Sensitivity nlysis V mx, L_HEG (CL HE /PL HE ) (K m, L_HEG + CL HE /PL HE ) V mx, L_OE (CL HE /PL HE ) (K m, L_OE + CL HE /PL HE ) V mx, L_HES (CL HE /PL HE ) (K m, L_HES + CL HE /PL HE ) To identify which prmeters hve the gretest impct on the model predictions on the formtion of 1 -hydroxyestrgole nd 1 -sulfooxyestrgole, sensitivity nlysis ws performed. Normlized sensitivity coefficients (SC) were determined using the following eqution: SC = ( C C ) ( P P ) (P C) where C is the initil vlue of the model output; C is the modified model output resulting from 5% increse of the prmeter vlue; P is the initil prmeter vlue; nd P is the modified prmeter vlue (Evns nd Andersen 2000). A 5% increse in prmeter vlues ws chosen to nlyze the effect of chnge in prmeter vlues on formtion of 1 -hydroxyestrgole nd 1 -sulfooxyestrgole t dose 0.01, 5, nd 150 mg/kg bw/dy for 24 h exposure, representing respectively relistic dily intke (Smith et l. 2002), n intke tht my result from supplement use (Vn Den Berg et l. 2011) nd dose level known to cuse liver tumors in rodent biossys (Drinkwter et l. 1976; Miller et l. 1983). Ech prmeter ws nlyzed individully, while other prmeters were kept s their initil vlue.

7 Arch Toxicol (2017) 91: Comprison of the PBK model bsed predictions for bioctivtion nd detoxifiction of estrgole in the Chinese nd Cucsin popultion The PBK model-bsed predictions for the formtion of metbolites of estrgole nd 1 -hydroxyestrgole in the Chinese popultion were compred with the predicted formtion of these metbolites in the Cucsin popultion. Model predictions were mde for period of 24 h fter exposure. Results Metbolic cpbilities of CYP nd SULT enzymes The results of metbolic cpbilities of CYP nd SULT enzymes cn be found in supporting mterils 3. The qulity of the Chinese tissue smples used to determine the vrious kinetic prmeters ppered to be in line with wht hs been reported before (Yng et l. 2012; Wng et l. 2006). Microsoml conversion of estrgole The microsoml conversion of estrgole ws determined by incubting estrgole with Chinese or Cucsin liver microsomes in the presence of NADPH. Figure S1 in supporting mterils 4 presents representtive chromtogrm of n incubtion with Chinese liver microsomes showing the formtion of estrgole-2,3 -diol (Rt = 2.9 min), 1 -hydroxyestrgole (Rt = 4.7 min), 3 -hydroxynethole (Rt = 4.9 min), M5 (Rt = 5 min) nd 4-llylphenol (Rt = 6.8 min). Since the metbolite referred to s M5 ws only formed in reltively smll mount in incubtions with liver microsomes from both ethnic groups, its identifiction ws not deemed essentil. Due to the presence of epoxide hydrolse in the humn liver microsomes, the formtion of estrgole-2,3 -diol reflects formtion of 3099 estrgole-2,3 -oxide (Guenthner nd Luo 2001; Luo nd Guenthner 1996; Luo et l. 1992). The estrgole concentrtion-dependent rtes of formtion of estrgole-2,3 -oxide, 1 -hydroxyestrgole, 3 -hydroxynethole, M5, nd 4-llylphenol by Chinese or Cucsin liver microsomes is shown in Fig. 3. The kinetic constnts for these metbolic conversions of estrgole nd the ctlytic efficiency, clculted s V mx /K m, obtined from these dt re summrized in Tble 2. The results showed tht estrgole-2,3 -oxide ws the most bundnt metbolite formed in incubtions with liver microsomes from both popultions, followed by 1 -hydroxyestrgole. Anlysis of ctlytic efficiencies for the formtion of the microsoml estrgole metbolites reveled tht the ctlytic efficiency for formtion of 1 -hydroxyestrgole ws the highest, followed by the formtion of estrgole- 2,3 -oxide, 4-llylphenol, 3 -hydroxynethole nd M5 in both popultions. Thus, formtion of 1 -hydroxyestrgole ws the mjor microsoml metbolic pthwy of estrgole, wheres formtion of M5 ws the lest importnt route of estrgole metbolism. The ctlytic efficiency for the formtion of 1 -hydroxyestrgole ws higher thn tht for the other microsoml metbolites becuse of reltively low K m. Comprison of the ctlytic efficiencies obtined for the two ethnic groups reveled tht the ctlytic efficiencies for the formtion of 4-llylphenol, estrgole-2,3 -oxide, 1 -hydroxyestrgole, 3 -hydroxynethole, nd M5 in incubtions with the Chinese smples were 1.8-, 4.9-, 2.4-, 3.2-, nd 2.9-fold lower, respectively, thn those in incubtions with Cucsin smples. The reltively low ctlytic efficiencies for the Chinese smples were minly due to reltively low V mx vlues, which were 5.0-, 4.4-, 3.4-, 3.1-, nd 6.5-fold lower thn the V mx for O-demethyltion, epoxidtion, 1 -hydroxyltion, 3 -hydroxyltion, nd the rection for the formtion of M5 in Cucsin smples, respectively. The pprent K m vlues for these rections by Chinese nd Cucsin smples were similr. Fig. 3 Concentrtion-dependent rte of metbolic conversion of estrgole in incubtions with Chinese () or Cucsin (b) liver microsomes. Dt points represent men vlues ± SEM of three individul experiments for ech metbolite, including estrgole-2,3 - oxide (closed upwrd tringle), 1 -hydroxyestrgole (closed circle), 3 -hydroxynethole (closed squre), 4-llylphenol (closed downwrd tringle), nd M5 (closed dimond) Rte of formtion (nmol min -1 mg microsoml protein -1 ) Chinese b Rte of formtion (nmol min -1 mg microsoml protein -1 ) Substrte concentrtion ( µ M) Substrte concentrtion ( µ M) Cucsin

8 3100 Arch Toxicol (2017) 91: Glucuronidtion of 1 hydroxyestrgole Kinetic constnts for the formtion of 1 -hydroxyestrgole glucuronide were determined by incubtions with Chinese or Cucsin liver microsomes in the presence of UDPGA nd 1 -hydroxyestrgole. 1 -Hydroxyestrgole glucuronide eluted t 2.6 min nd ws identified by LC-MS s previously described (Punt et l. 2008). The metbolite ws quntified using the clibrtion curve of 1 -hydroxyestrgole on the bsis of the similrity in their UV spectr ssuming similr molr extinction coefficient. Figure 4 shows the 1 -hydroxyestrgole concentrtiondependent rte of formtion of 1 -hydroxyestrgole glucuronide by liver microsomes nd kinetic constnts derived from these plots re displyed in Tble 2. The pprent K m nd V mx for formtion of 1 -hydroxyestrgole glucuronide by Chinese smples were 4656 µm nd 1.63 nmol/min/(mg microsoml protein), respectively, wheres K m nd V mx for formtion of 1 -hydroxyestrgole glucuronide by Cucsin smples were 4607 µm nd 4.29 nmol/min/(mg microsoml protein), respectively. These vlues result in ctlytic efficiency tht is 2.7-fold lower for Chinese thn for Cucsin subjects. Oxidtion of 1 hydroxyestrgole 1 -Oxoestrgole ws formed in incubtions with 1 -hydroxy estrgole nd Chinese or Cucsin liver S9 using NAD + s cofctor nd GSH to trp 1 -oxoestrgole forming GS-1 -oxoestrgole. Chromtogrphic nlysis reveled tht GS-1 -oxoestrgole eluted t 2.6 min bsed on its previous identifiction (Punt et l. 2009). The rte of oxidtion of 1 -hydroxyestrgole with incresing concentrtion of 1 -hydroxyestrgole is shown in Fig. 4b nd Tble 2 displys the kinetic constnts for formtion of 1 -oxoestrgole derived from these dt. The pprent K m for formtion of 1 -oxoestrgole by Chinese smples ws 403 µm, nd the V mx ws 1.82 nmol/min/(mg S9 protein). For Cucsin smples, the pprent K m nd the V mx were 521 µm nd 2.8 nmol/min/(mg S9 protein), respectively. The ctlytic efficiencies for formtion of 1 -oxoestrgole by both ethnic groups were similr. Sulftion of 1 hydroxyestrgole Formtion of the ultimte crcinogenic metbolite 1 -sulfooxyestrgole upon sulftion of 1 -hydroxyestrgole ws observed in incubtions with Chinese s well s Cucsin liver S9 in the presence of PAPS nd GSH. GSH ws used to trp the trnsient 1 -sulfooxyestrgole forming the GSH dduct of 1 -sulfooxyestrgole. Chromtogrphic nlysis reveled pek t 1.4 min, identified s the GSH Tble 2 Kinetic constnts (verge ± SEM) for metbolic conversion of estrgole nd 1 -hydroxyestrgole in incubtions with Chinese or Cucsin liver tissue frctions Ethnic group Chinese Cucsin Metbolite K m (µm) V mx (nmol /min/ mg microsoml or S9 protein) Ctlytic efficiency (µl/ min/mg microsoml or S9 protein) K m (µm) V mx (nmol /min/ mg microsoml or S9 protein) Ctlytic efficiency (µl/min/mg microsoml or S9 protein) Conversion of estrgole 4-Allylphenol 115 ± ± ± ± Estrgole-2,3-161 ± ± ± ± oxide 1 -Hydroxyestrgole 49 ± ± ± ± Hydroxynethole 450 ± ± ± ± M5 618 ± ± ± ± Conversion of 1 -hydroxyestrgole 1 -Hydroxyestrgole 4656 ± ± ± ± glucuronide 1 -Oxoestrgole 403 ± ± ± ± Sulfooxyestrgole 694 ± ± ± ± V mx /K m 1000

9 Arch Toxicol (2017) 91: Fig. 4 Concentrtion-dependent rte of metbolic conversion of 1 -hydroxyestrgole to () 1 -hydroxyestrgole glucuronide, (b) 1 -oxoestrgole, nd (c) 1 -sulfooxyestrgole in incubtions with Chinese or Cucsin liver tissue frctions. Dt points represent men vlues ± SEM of three individul experiments for ech metbolite Rte of formtion (nmol min -1 mg microsoml protein -1 ) Chinese Rte of formtion (nmol min -1 mg microsoml protein -1 ) Cucsin b Rte of formtion (nmol min -1 mg S9 protein -1 ) Substrte concentrtion ( µ M) Rte of formtion (nmol min -1 mg S9 protein -1 ) Substrte concentrtion ( µ M) c Rte of formtion (nmol min -1 mg S9 protein -1 ) Substrte concentrtion ( µ M) Substrte concentrtion ( µ M) Rte of formtion (nmol min -1 mg S9 protein -1 ) Substrte concentrtion ( µ M) Substrte concentrtion ( µ M) dduct of 1 -sulfooxyestrgole. This identifiction ws bsed on the fct tht the pek ws bsent in the incubtions with PAPS nd S9 in the bsence of GSH (chromtogrm not shown), nd the previous identifiction of this (Punt et l. 2007) nd similr lkenylbenzene 1 -sulfooxy dducts by LC-MS (Al-Mlhmeh et l. 2017; Al-Subeihi et l. 2012; Aljlouni et l. 2016; Mrtti et l. 2012). Figure 4c presents the 1 -hydroxyestrgole concentrtion-dependent rte of formtion of 1 -sulfooxyestrgole in incubtions with Chinese or Cucsin liver S9 s determined by quntifiction of the GSH dduct of 1 -sulfooxyestrgole. Tble 2 presents summry of the kinetic constnts for formtion of 1 -sulfooxyestrgole in the two ethnic groups derived from these dt. The ctlytic efficiency for formtion of 1 -sulfooxyestrgole in incubtion with Chinese smples ws fivefold lower thn those in incubtions with Cucsin smples. Comprison of the kinetic constnts for conversion of estrgole nd 1 hydroxyestrgole by Chinese nd Cucsin liver tissue frctions Tble 3 displys the scled kinetic prmeters for conversion of estrgole nd 1 -hydroxyestrgole by Chinese or Cucsin liver tissue frctions. In this tble, V mx vlues tht were obtined in vitro expressed s nmol/min/ (mg microsoml protein or S9) (Tble 2) were scled to whole liver nd expressed in µmol/h/(g tissue). A scled ctlytic efficiency (V mx in vivo/k m ) for formtion of the different metbolites of estrgole nd 1 -hydroxyestrgole could be clculted. The ctlytic efficiencies thus obtined revel tht formtion of the proximte crcinogenic metbolite of estrgole, 1 -hydroxyestrgole, ws 2.5-fold lower for Chinese liver microsomes s compred to Cucsin liver micorosomes. This difference in ctlytic efficiency ws minly due to the low V mx in Chinese

10 3102 Arch Toxicol (2017) 91: liver microsomes, since the vlue of V mx in Chinese liver incubtions ws 3.5-fold lower thn in Cucsin liver incubtions. Epoxidtion of estrgole turned out to be the min route of microsoml metbolism of estrgole in both ethnic groups, lthough the ctlytic efficiency for formtion of estrgole-2,3 -oxide ws fivefold lower for the Chinese popultion. This lower ctlytic efficiency ws minly due to fourfold lower V mx for Chinese liver smples. Regrding the metbolic rections of 1 -hydroxyestrgole, formtion of 1 -oxoestrgole ws the min detoxifiction pthwy for this proximte crcinogenic metbolite in both popultions. The ctlytic efficiencies for formtion of 1 -oxoestrgole in both popultions were similr. However, the ctlytic efficiency for formtion 1 -hydroxyestrgole glucuronide, nother detoxifiction route of 1 -hydroxyestrgole ws found to be threefold lower in incubtions with Chinese thn with Cucsin liver microsomes, resulting from threefold lower V mx. Sulftion of 1 -hydroxyestrgole in both popultions, reflecting bioctivtion to rective ultimte crcinogen, ws found to be the lest efficient pthwy for 1 -hydroxyestrgole metbolism for both ethnic groups. For Chinese smples, the scled ctlytic efficiency for this bioctivtion rection ws 4.5-fold lower thn for the Cucsin smples. Comprison of the PBK model bsed predictions for bioctivtion nd detoxifiction of estrgole in Chinese nd Cucsins Comprison of the overll consequences of the observed differences in kinetic constnts for ll the individul bioctivtion nd detoxifiction rections of estrgole between the two ethnic groups requires integrtion of ll dt in PBK model. In the present study, these PBK models were defined bsed on the PBK model for estrgole in humn previously developed (Punt et l. 2009), using the popultion specific physiologicl nd kinetic prmeters defined in the present study. The PBK model for estrgole for Cucsins ws evluted previously by compring the predicted formtion of 4-llylphenol nd 1 -hydroxyestrgole glucuronide to the observed dose levels of these metbolites in vivo (Punt et l. 2009). The performnce of the PBK model for estrgole for the Chinese popultion could not be evluted ginst Chinese in vivo dt becuse quntittive dt on the excretion of different metbolites of estrgole in Chinese subjects fter orl exposure to estrgole re not vilble. Evlution of the model could only be mde by compring the predicted inter-ethnic differences in formtion of 1 -hydroxyestrgole to reported ethnic differences in heptic drug-metbolizing enzymes tht minly ctlyze the formtion of metbolites of Tble 3 Scled kinetic constnts (verge ± SEM) for metbolic conversion of estrgole nd 1 -hydroxyestrgole from incubtions with Chinese or Cucsin liver tissue frctions Ethnic Chinese Cucsin Metbolite K m (µm) Scled V mx (µmol/h/g liver) Ctlytic efficiency b (µl/h/g liver) K m (µm) Scled V mx (µmol/h/g liver) Ctlytic efficiency b (µl/h/g liver) Conversion of estrgole 4-Allylphenol 115 ± ± Estrgole-2,3-161 ± ± oxide 1 -Hydroxyestrgole 49 ± ± Hydroxynethole 450 ± ± M5 618 ± ± Conversion of 1 -hydroxyestrgole 1 -Hydroxyestrgole 4656 ± ± glucuronide 1 -Oxoestrgole 403 ± ± Sulfooxyestrgole 694 ± ± Scled V mx in vivo expressed s µmol/h/(g liver), clculted from the in vitro V mx bsed on microsoml protein yield of 35 mg/(g tissue) nd n S9 protein yield of 143 mg/(g tissue) b Ctlytic efficiency expressed s µl/h/(g liver) is the rtio of scled V mx nd K m

11 Arch Toxicol (2017) 91: estrgole to 1 -hydroxyestrgole. CYP1A2 nd CYP2A6 predominntly ctlyze formtion of 1 -hydroxyestrgole with CYP2C19, CYP2D6, nd CYP2E1 contributing to some extent t high concentrtions of estrgole (Jeurissen et l. 2007). The PBK model-bsed predicted formtion of 1 -hydroxyestrgole t n orl dose of 0.01 mg/kg bw, relistic dietry intke level (Smith et l. 2002), in Chinese liver, mounted to 43% of the dose, which ws similr to the reltive mount of 1 -hydroxyestrgole predicted to be formed in the Cucsin liver, which ws 47% of the dose s shown in Fig. 6. Although the ctlytic efficiency in formtion of 1 -hydroxyestrgole in Chinese subjects is two to threefold lower thn tht of those in Cucsin incubtions, the predicted overll formtion of 1 -hydroxyestrgole over 24 h in both ethnic groups is similr. Therefore, lthough the ctlytic efficiency for estrgole 1 -hydroxyltion in the Chinese subjects is lower, when considering time for conversion of 24 h the overll % of the dose converted to 1 -hydroxyestrgole still ppers to be similr. The prediction of similr overll formtion of 1 -hydroxyestrgole in both ethnic groups is in line with the experimentl observtion tht pek plsm concentrtions chieved t h nd pprent orl clernce vlues within 7 h of dministrtion for phencetin, mrker for CYP1A2 ctivity, did lso not differ between Chinese nd Cucsin subjects (Brtoli et l. 1996). At high-dose level of 300 mg/kg bw/dy estrgole, the predicted formtion of 1 -hydroxyestrgole in the liver of Chinese subjects ws twofold lower (13 versus 25% of the dose) thn the predicted formtion in the liver of Cucsin subjects. This would be in line with the fct tht t higher estrgole concentrtions, CYP2C19 might become involved in estrgole 1 -hydroxyltion nd the fct tht the frequency of poor metbolizers (PM) for CYP2C19 in the Chinese popultions ws 14.6% compred to 3.3% in Swedish popultion (Bertilsson et l. 1992). Altogether, the PBK models obtined were considered dequte for further predictions of inter-ethnic differences in estrgole metbolism, lso becuse of the previous evlution of the model for humn s reported in the literture (Punt et l. 2009). Figure 5 shows the PBK model-bsed predictions for the time-dependent formtion of 1 -hydroxyestrgole nd 1 -hydroxyestrgole metbolites in both ethnic groups t dose level of 0.01 mg/kg bw/dy of estrgole. Although the ctlytic efficiency in formtion of 1 -hydroxyestrgole in incubtions with liver smples from Chinese subjects is two to threefold lower thn tht in incubtions with Cucsin smples, the predicted C mx nd 24 h AUC of 1 -hydroxyestrgole in both the groups re similr. The predicted formtion of the different metbolites mounted to 0.02 nmol/ (g liver) for 1 -hydroxyestrgole glucuronide, 1.2 nmol/ (g liver) for 1 -oxoestrgole nd nmol/(g liver) for 1 -sulfooxyestrgole in Chinese subjects, corresponding 3103 to 0.8, 42, nd 0.02% of the dose, respectively (Fig. 5). For the Cucsins, these vlues mounted to 0.05 nmol/ (g liver) for 1 -hydroxyestrgole glucuronide, 1.2 nmol/ (g liver) for 1 -oxoestrgole nd nmol/(g liver) for 1 -sulfooxyestrgole, corresponding to 2, 45 nd 0.09% of the dose, respectively (Fig. 5). Figure 6 presents the PBK model-bsed predictions for the dose-dependent formtion of 1 -hydroxyestrgole nd metbolites of 1 -hydroxyestrgole in both ethnic groups. Differences on predicted formtion of the vrious estrgole metbolites in both ethnic groups cn be considered following estrgole exposure t 0.01, 5, nd 150 mg/kg bw/ dy, representing, respectively, relistic dily intke (Smith et l. 2002), n intke tht my result from supplement use (Vn Den Berg et l. 2011) nd dose level known to cuse liver tumors in rodent biossys (Drinkwter et l. 1976; Miller et l. 1983). Figure 6 shows tht the ethnic differences in predicted formtion of estrgole metbolites in both ethnic groups t dose of 0.01 mg/kg bw/dy re similr to the sitution for these metbolites formed in both ethnic groups t dose of 5 mg/kg bw/dy estrgole. When the dose level increses up to 150 mg/kg bw/dy, the ethnic difference in predicted metbolites of estrgole increses. Figure 6 displys the predicted formtion of 1 -hydroxyestrgole in both ethnic groups. At dose level of 0.01 mg/kg bw/dy of estrgole, no ethnic difference in the formtion of 1 -hydroxyestrgole is observed. When the dose level increses up to 150 mg/kg bw/dy, the reltive formtion of 1 -hydroxyestrgole in both ethnic subjects decresed due to the sturtion of this metbolic route. However, the formtion of 1 -hydroxyestrgole predicted for Chinese liver ws 1.5-fold lower thn for Cucsin liver. At reltively high concentrtions of estrgole, CYP2C19 might strt to be involved in 1 -hydroxyestrgole formtion nd genetic polymorphism between Chinese nd Cucsins hs been reported with higher frequency of slow metbolizers in the Chinese popultion (Bertilsson et l. 1992) which my provide n explntion for the observed effect. Figure 6b presents the predicted formtion of 1 -oxoestrgole in both ethnic groups. Oxidtion of 1 -hydroxyestrgole represents the min metbolic route for 1 -hydroxyestrgole in both ethnic groups t ll three dose levels of estrgole. The predicted formtion of 1 -oxoestrgole ws similr t 0.01 mg/kg bw/dy of estrgole, 41.2% of the dose for Chinese, nd 45% of the dose for Cucsins. At 5 mg/kg bw/dy of estrgole, 39% of dose ws converted to 1 -oxoestrgole in Chinese nd 44% of the dose in Cucsins. When dose of 150 mg/ kg bw/dy estrgole ws pplied, only 19% of the dose ws converted to 1 -oxoestrgole for Chinese nd 28% for Cucsins. The ethnic difference in formtion of this metbolite ws incresed from onefold t 0.01 mg/kg bw/ dy to 1.5-fold t 150 mg/kg bw/dy estrgole. Figure 6c

12 3104 Arch Toxicol (2017) 91: Fig. 5 PBK model-bsed predictions for the timedependent concentrtion of 1 -hydroxyestrgole in liver (), nd overll formtion of 1 -oxoestrgole (b), 1 -hydroxyestrgole glucuronide (c), nd 1 -sulfooxyestrgole (d) in Chinese (dshed line) nd Cucsin (continuous line) liver following exposure to 0.01 mg/ kg bw estrgole Concentrtion (nmol/g liver) b Time (h) c Formtion (nmol/g liver) d Time (h) Formtion (nmol/g liver) Formtion (nmol/g liver) Time (h) Time (h) Fig. 6 PBK model-bsed predictions for the dose-dependent formtion of () 1 -hydroxyestrgole, (b) 1 -oxoestrgole, (c) 1 -hydroxyestrgole glucuronide, nd (d) 1 -sulfooxyestrgole in Chinese (dshed line) nd Cucsin (continuous line) liver 1'-Hydroxyestrgole formtion (mmol/kg bw) c 1'-Hydroxyestrgole glucuronide formtion (mmol/kg bw) Dose estrgole (mg/kg bw) Dose estrgole (mg/kg bw) b 10 3 d '-Oxoestrgole formtion (mmol/kg bw) '-Sulfooxyestrgole formtion (mmol/kg bw) Dose estrgole (mg/kg bw) Dose estrgole (mg/kg bw) shows tht the PBK model-bsed predicted formtion of 1 -hydroxyestrgole glucuronide in the Chinese liver ws 2.4-fold lower thn the levels predicted to be formed in Cucsins t dose level of 0.01 mg/kg bw/dy of estrgole. At dose level of 5 mg/kg bw/dy of estrgole, there ws 2.6-fold difference in formtion of 1 -hydroxyestrgole glucuronide between the ethnic subjects, nd t dose level of 150 mg/kg bw/dy of estrgole, the difference incresed to 3.4-fold. Figure 6d revels tht the PBK model-bsed predicted formtion of 1 -sulfooxestrgole ws 4.5-fold lower for Chinese thn Cucsin liver t 0.01 mg/kg bw/dy of estrgole. The ethnic difference in formtion of 1 -sulfooxyestrgole ws 5.1-fold t 5 mg/ kg bw/dy nd 6.6-fold t 150 mg/kg bw/dy of estrgole pointing t lower levels of bioctivtion to the ultimte crcinogenic metbolite for the Chinese popultion.

13 Arch Toxicol (2017) 91: Sensitivity nlysis A sensitivity nlysis ws performed to determine model prmeters tht re ble to influence the formtion of 1 -hydroxyestrgole nd 1 -sulfooxyestrgole in both ethnic groups to the lrgest extent. Sensitivity coefficients were clculted for ll model prmeters t dose of 0.01, 5, nd 150 mg/kg bw/dy estrgole, but only the prmeters tht ppered to hve sensitivity coefficient higher thn 0.1 re presented in Fig. S2 in supporting mterils 5. In Chinese nd Cucsin liver t 0.01 mg/kg bw of estrgole (Fig. S2), formtion of 1 -hydroxyestrgole is predominntly influenced by the kinetic constnt for 1 -hydroxyltion, nd to minor extent by the kinetic constnts for formtion of estrgole-2,3 -oxide. Focusing on inter-ethnic differences, it cn be concluded tht prmeters, including liver volume, blood flow to ft nd microsome protein yield of liver, hve higher effect on the formtion of 1 -hydroxyestrgole in Chinese liver compred to Cucsin liver. Formtion of 1 -sulfooxyestrgole minly depends on the kinetic constnts for its formtion from 1 -hydroxyestrgole, the formtion of 1 -hydroxyestrgole itself, nd the kinetic constnts for formtion of 1 -oxoestrgole, which is n importnt competing metbolic pthwy to sulftion in both ethnic groups. At 5 mg/ kg bw/dy of estrgole (Fig. S2b), the results of the sensitivity nlysis of both ethnic groups re similr to the sitution t dose of 0.01 mg/kg bw/dy. When the dose increses to 150 mg/kg bw/dy (Fig. S2c), besides the influentil prmeters t dose of 0.01 nd 5 mg/kg bw/ dy of estrgole, the prmeters relted to ft tissue, such s volume of ft nd ft/blood prtition coefficient, lso hve influence on the formtion of 1 -hydroxyestrgole nd 1 -sulfooxyestrgole in Chinese liver. For the Cucsin group, the results of the sensitivity nlysis t 150 mg/kg bw/dy re similr to those t 0.01 or 5 mg/kg bw/dy of estrgole. Discussion In the present study, bioctivtion nd detoxifiction of estrgole in Chinese nd Cucsin popultions upon orl exposure to estrgole were exmined using PBK modeling. The structure of the PBK models ws bsed on the model developed nd vlidted previously for estrgole in Cucsins (Punt et l. 2009). The prmeter vlues for the Chinese PBK model were derived from literture or from incubtion experiments using humn liver frctions. To gurntee dequte experimentl comprison, the prmeters for heptic metbolism in the Cucsin model were redefined in the present study s well. The kinetic constnts of estrgole metbolism obtined from incubtion with 3105 Cucsin liver frctions nd PBK model predictions of estrgole in Cucsins in this study were similr to results reported in the study by Punt et l. (2009). Since the ctul kinetic dt on plsm or urinry estrgole metbolite levels were not vilble for the Chinese popultion, the performnce of the model ws evluted by compring the predicted inter-ethnic differences in formtion of 1 -hydroxyestrgole to reported ethnic differences in heptic drug-metbolizing enzymes CYP1A2 tht minly ctlyze the formtion of metbolites of estrgole to 1 -hydroxyestrgole. Furthermore, the PBK model for the Chinese popultion ws bsed on the model for estrgole defined nd vlidted previously (Punt et l. 2009). In ddition, the qulity of the Chinese tissue smples used to determine the vrious kinetic prmeters ppered to be in line with wht hs been reported before (Yng et l. 2012; Wng et l. 2006). Considering these fcts, it cn be concluded tht the developed PBK model for the Chinese popultion is expected to dequtely describe the in vivo levels of metbolites upon orl exposure. The ethnic difference in bioctivtion of estrgole ws evluted by predicting the formtion of 1 -sulfooxyestrgole t doses level of 0.01 nd 5 mg/kg bw/dy of estrgole. The PBK models predicted tht following n exposure of 0.01 mg/kg bw/dy estrgole, only 0.02% of the estrgole dose ws predicted to be converted to 1 -sulfooxyestrgole in the Chinese popultion, wheres 0.09% of the dose ws converted to 1 -sulfooxyestrgole in Cucsins, which ws 4.5-fold higher. At 5 mg/kg bw/dy of estrgole, worst cse scenrio intke for people using plnt food supplements contining estrgole (Vn Den Berg et l. 2011), the difference in the predicted formtion of 1 -sulfooxyestrgole between the two ethnic groups ws incresed to fivefold. This four to fivefold difference in the level of bioctivtion of estrgole between the two ethnic groups minly origintes from the difference in SULT medited conversion of 1 -hydroxyestrgole. The humn SULTs involved in this sulftion rection hve not been identified to dte, but sulftion of the 1 -hydroxy metbolite of the relted lkenylbenzene methyleugenol in the liver hs been shown to be ctlyzed minly by SULT1A1 nd to smll extent by SULT1E1 (Herrmnn et l. 2012). It hs lso been shown tht exon 7 of the SULT1A1 gene my contin G to A trnsition t codon 213 (rs ), which induces n Arg to His mino cid substitution (Rftoginis et l. 1997). This SULT1A1 Arg213His (rs ) polymorphism is reported to be ssocited with incresed risk of vrious cncer types (Xio et l. 2014). In the ethnic subgroup nlysis of Xio et l., they found tht the genotype distributions of the SNP site re different for different ethnic groups. Occurrence of the His llele in Asins (9.58%) ws reported to be significntly lower thn in Cucsins (35.2%). This difference of the