Characterization of Xenobiotic Metabolizing Enzymes of A

Characterization of xenobiotic metabolizing enzymes of a reconstructed human epidermal model from adult hair follicles Daniel Bacqueville, Carine Jacques, Laure Duprat, Emilien L. Jamin, Beatrice Guiraud, Elisabeth Perdu, Sandrine Bessou-Touya, Daniel Zalko, Hélène Duplan

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Daniel Bacqueville, Carine Jacques, Laure Duprat, Emilien L. Jamin, Beatrice Guiraud, et al.. Characterization of xenobiotic metabolizing enzymes of a reconstructed human epidermal model from adult hair follicles. Toxicology and Applied Pharmacology, Elsevier, 2017, 329, pp.190-201. 10.1016/j.taap.2017.05.040. hal-01608588

HAL Id: hal-01608588 https://hal.archives-ouvertes.fr/hal-01608588 Submitted on 26 May 2020

HAL is a multi-disciplinary open access L’archive ouverte pluridisciplinaire HAL, est archive for the deposit and dissemination of sci- destinée au dépôt et à la diffusion de documents entific research documents, whether they are pub- scientifiques de niveau recherche, publiés ou non, lished or not. The documents may come from émanant des établissements d’enseignement et de teaching and research institutions in France or recherche français ou étrangers, des laboratoires abroad, or from public or private research centers. publics ou privés. Version postprint Bessou-Touya, Zalko, D.,Duplan(2017). Characterization ofxenobiotic metabolizing enzymesof Bacqueville, D.(Auteur decorrespondance), Jacques, Duprat,Jamin, E.L.,Guiraud, Perdu,E., a reconstructed human epidermal model fromadult hair follicles. Toxicology and Applied journal pertain. be it manuscript a This 10.1016/j.taap.2017.05.040 captured epidermal Duplan Jamin, Please Accepted date: Revised date: Received date: T Reference: DOI: PII: T Jamin, Daniel reconstructed humanepidermalmodelfromadulthairfollicles Characterization Accepted Manuscript o appearin: ouya, DanielZalko,HélèneDuplan is service discovered published is Pharmacology, 329, 190-201. DOI : 10.1016/j.taap.2017.05.040 cite Beatrice a Bacqueville, , Beatrice Characterization PDF as to model this will af our in which filiation file article under Guiraud, customers its from Guiraud, of of Comment citer cedocument: final Carine could go an xenobiotic as: adult 30 May2017 10 May2017 7 March2017 T YT doi: S0041-008X(17)30258-2 for oxicology andAppliedPharmacology copyediting, unedited form. Elisabeth Daniel AAP 13975 of 10.1016/j.taap.2017.05.040 all af Elisabeth we hair Jacques, xenobiotic fect authors.

Please are follicles. Bacqueville, the manuscript metabolizing Perdu, providing typesetting, content, Laure note Perdu, metabolizing Please Sandrine The that Duprat, that this Sandrine and Carine address during check enzymes and has early all Bessou-T review Emilien enzymes Jacques, legal been if the for Bessou- version appropriate. production the of disclaimers accepted of ouya, L. corresponding Laure of the a of a resulting Daniel reconstructed the for Duprat, process Ytaap(2017), that manuscript. publication. Zalko, proof apply author Emilien errors Hélène human before to may doi: was The the As L. Version postprint Bessou-Touya, Zalko, D.,Duplan(2017). Characterization ofxenobiotic metabolizing enzymesof Bacqueville, D.(Auteur decorrespondance), Jacques, Duprat,Jamin, E.L.,Guiraud, Perdu,E., a reconstructed human epidermal model fromadult hair follicles. Toxicology and Applied Manuscript correspon  UPS, Toulouse, France. 2 Département Pharmacologie, 1 affiliations:Complete Perdu DanielBacqueville adulthairfollicles Characterizationofxenobiotic metabolizing enzymes of a reconstructed humanepidermal model from E Fax: Tel.:+33 534506427 DanielBacqueville *

Toxalim (Research ToxalimCentre (Research in Food Toxicology), UniversitéToulouse,de INRA, ENVT,INP Pierre Fabre Dermo - Equalcontribution C mail address: orrespo

+33 2 , , SandrineBessou Pharmacology, 329, 190-201. DOI : 10.1016/j.taap.2017.05.040 534503424 nding author:

daniel

1,*

ACCEPTED MANUSCRIPT , -  . cosmétique, , bacqueville

Carine Jacques

dence ACCEPTED MANUSCRIPT - Comment citer cedocument: Touya Centre R&D Pierre Fabre, 3avenue Hubert Curien,Toulouse, France.

1 ,

@pierre DanielZalko ServicePharmacologie Division 2

1 ,  , - Laure Duprat fabre.com 2 , Hélène Duplan

1 , Emilien L. Jamin 1

et Pharmacocinétique et Cutané, 2 , Beatrice Guiraud 1 - , Purpan, Elisabeth

Pharmacology, 329, 190-201. DOI : 10.1016/j.taap.2017.05.040

responsiveto AhRligands

within 24 h after topicalapplication. reconstructed a a comprehensivecharacterization of - ethoxycoumarin, testosterone, benzo(a)py The presence of functional CYPs, UGTsand SULTs was confirmedbyincubating the

ACCEPTED metabolism byMANUSCRIPTthe XMEsandthe on residence timewithin themodel.

were - Functional XME . activities also were demonstrated using systemicor topical RHE modelexpress ACCEPTED MANUSCRIPT the Comment citer cedocument: human

observed for alcohol/aldehyde dehydrogenasesand (carboxyl) CYP inducers,CYP the cutaneous metabolism and potential toxicity ofinnovative dermo

epiderm s ,

evident at thegene andenzyme activity level Cytochromes is, I was was made

XMEs,thehighest levelswere 3 es - hairfollicle, cutaneous metabolism, gene expression, methylcholanthrene (

a a numberof

The extentof metabolism was dependent on ORS xenobiotic metabolizing enzymes ( in P450( - a a reconstructed skinepidermal model derived RHE rene and rene 3 Phase I Phase CYPs ) . The - RHE model. )

- 3 and consistentlyreported todetected be in ORS MC - MC II , ofallwhich were -

) and RHE model XMEs, GSTs Thehighest  -

and transferases, naphthoflavone someof .

XMEs Phase II Phase XME

whichmay be esterases. The esterases. I Phase rapidly ) gene based onbased

saturable XME

profile ( β

XME - NF)

levels

- is q

oprto ad eeomn (ED; ue ro sheath root outer (OECD); Development and Cooperation

PCR); PCR); spermine (ORS

- H) prostaglandinRHE); - ctlrnfrs 2 (A2) N (NAA20); 20 acetyltransferase - deethylase (EROD); flavinglutathionedeethylase(EROD); (FMO);monooxidase catechol ectrometry (HRMS);ectrometry

N1 - acetyltransferase1 (SAT1); sulfotransferase (SULT); - - - chain APCI);benzo(a)pyrene (B(a)P); O - methyltransferase - - fatty noeoie ytae PG2; el time real (PTGS2); synthase endoperoxide - acid hydroxysteroid(17 - o lgs 1 AS1; 3 (ACSL1); 1 ligase CoA ( - COMT ctl rnfrs (NAT); transferase acetyl ); - P450 cytochrome  hydrocarbon aryl  -

naphthoflavone dehydrogenase - - 7 ESI); derived - -

bio ORS outersheath root assays. assays. ORS the rapidandnon Introduction ® -

RHE model Pharmacology, 329, 190-201. DOI : 10.1016/j.taap.2017.05.040 Full

derivedkeratinocytes affords a numberofadvantages over otherRHE models Inthis study, we aimedto characterize as precisely as possible the metabolic capabilities of -

hcns skinmodelThickness andEpiDerm™

the function (Bacquevilleal., et 2015) 004; Jacques Jacques et 004;al.,2010 that

ACCEPTED MANUSCRIPT and

was developedin orderto establish a cost - could be customized according to specific assay needs Tothis end,we derivedreco ,

- ACCEPTED MANUSCRIPT moreover, invasive Comment citer cedocument: al

activitiesofseveral I Phase in engineered the same thesame way as nstructed humanepidermal samplecol the lternativeto human skinfor Jäckh al., et 2011 , firstinvestigated

repair The select . , Oesch , a fromadult undifferentiated keratinocytes from Here, Here, o ), anda ), ORS

lection, theability toselect a particular donor ionof andinduction ofapoptosis

previousreports

- urintention et et al. RHE model s s compoundspenetrate the skin, theymay be subject to

adult donors, as opposed to neonatal donors that are - , solublemetabolites orbioactivate them to reactive

that , 2007, ) the .

and

support overall

and II was was ( was was shown be to - ORS

effective andreproducible xenobiotic metabolizing enzymes (XMEs)

us 2 studying of theeffects

basal to extend 014 their ing - RHE) model ). Therefore, ). inorderto help RHE models applicability tothis purpose XMEgene expression profile andinvestigating ) the theuse ofthe ORS

(Guiraud et al.,2014)

a a good

is , a a three

such as suchas andrepro This UV UV radiation(e.g. ORS ,

in in vitro including the - (gender,age, dimensional ORS SkinEthic,

cells - sunscreen RHE ducible

- . human s

RHE ofhair

The

used by of of

ase I ase belowthe model) andtopical (added the tou

- we we confirmedinduction effect RHE Pharmacology, 329, 190-201. DOI : 10.1016/j.taap.2017.05.040 oxido of

and B(a)P

timepoint a model XMEgene does not necessarily indicate thatthe - eutv capacity reductiveofthemodel a II - n established diagnostic substrate for activities.CYP

, and

hee ( threne sequentialpathways ( ( ACCEPTED MANUSCRIPT Haag Haag et al.,2012 vanEijl . using s The twoendpoints

3 (24 ACCEPTED MANUSCRIPT - Comment citer cedocument: exposureto theskin istopical MC 3 RT

h) - et et al MC . . 7 . . Additional timepoints - qPCR - ., 2012., ) EC represents represents EC a chemicalwith a ,XMEon gene )

; therefore, ; we selected t analysis

s (De (De Kanter et al.,1999) )

by incubating controlandinducer nt testosterone represents an idea (gene expressionand

metabolism ( OECD,2004b

The test chemicals 7 were of Thisisalso of importance sincethe metabolic capacity of

. knownAhR

morethan Testosterone isalso specified inthe Organization for expression topical pper surface ppersurface of themodel) -

RHE model, we compl (48 and(48 h) added72were for functional metabolic itimportantis to show, thatRHE models can be offour test chemicalswi treatment with a test compound (Hewitt et )

todescribe methodsassessing drug after after systemic (addedto theculture medium ligands, namely 0

estosterone Phase I Phase . functional corresponding Theskin knownis to ablebe to

simple metaboliteprofile Thissubstrate can alsobe

and 7 - lsubstrate toconfirm the ( ethoxycoumarin (7 OECD, 2004a - metabolism) as treated treated e 0

mented thismentedby a 

Phase Phase B(a)P XMEfunctionalis application - model naphthoflavone ( th varyingth skin

II is ofan

) XME genes a a knowngenotoxi comparedwere modelswith the and a .

T endogenous - involving metabolic EC), investigating he  In . NF .

al., Since

)

c ,

ethoxy[3 -

dihydrodiol, benzo(a)pyrene test test chemicalmetabolism) Chemicals andMaterials methods Pharmacology, 329, 190-201. DOI : 10.1016/j.taap.2017.05.040 s - which , especially, tohelp interpret negative outcomes in genotoxicity assays usin - 14 ( andthat th lrc (StQuentinAldrich Fallavier, France) had a purity greater than 98.5%. Conney et al., 1994 C] - ™ - isimportant tounderstand thebalance ofmetabolism ofsuch genotoxi a coumarin, [U

r specificactivities of2,2.074, - , toxicity 7 24h

t ACCEPTED MANUSCRIPT - 8 is

, e theinducibility e ofXMEs in ORS t (Brea, United States). alongside after - ACCEPTED MANUSCRIPT model 9 Comment citer cedocument: relieson - , hydroxybenzo(a)pyrene, 3 t - 10 topicalapplication - . .

14 - Thesuitability ofthemodel forcompound metabolism assays was 3 tetrahydrotetrol respon ) - C] - . 1,6

MC its inductionits effects. B(a)P - CYP450 a - bisphenolA,[5,6 dione

is ds

a a directgenotoxin

has been to CYP1 induction ,

benzo(a)pyrene - Unlabeled7 depend

, benz,

2 (Götzal., et 2012), suggesting itinduces its own

characterizationof theXMEs present inthe and 3 also - M - o(a)pyrene - e 2.03GBq/mmol, respectively, purchasedwere from 14 ethylcholanthrene hydroxybenzo(a)pyrene shownto induce EROD activities intheepidermal nt bioactivation, into

C] b C] (PCR)

- - EC and - . RHEs RHEs and therefore it enzo(a)pyrene[ 3,6 B(a)P ,

and functional activities - is metabolizedis B(a)P in, benzo(a)pyrenedione, - cis

- is detoxifiedalso byGSTs, SULTs 7,8 , 3 - - - dihydrodiol [ MC 3 H] was was purchased H] from 14

and testosterone,purchased

C 7 , ultimate DNA reactive via - B(a)P], B(a)P], [4,7 -

hydroxybenzo(a)pyrene was P benz , hase hase I

gskin epidermal

- ofinterest to 7,8 ( c compound c EROD EROD activity

and o(a)pyrene -

in, 8 dione, - 14 novel ORS II C]

pathways - -

- in cis - - .

- ol healthy

- 3,17 Preparation humanofreconstituted epidermis pluckedhairfollicles explanted were - Pharmacology, 329, 190-201. DOI : 10.1016/j.taap.2017.05.040 France) France) was used for 3 -  in, 4 dione, - Caucasian ORS - ol Le - -

g 17 to lutamine obtained were from Gibco - Canal, France). Ultrapure water from a Milli

- - the RHE tissues andthestructure of thetissues at thetime of incubation (histology). ACCEPTED MANUSCRIPT 5 one, - androsten - 

Aldrich. protocol - - volunteerswhoha ketotestosterone,1 hydroxytestosterone, 7 ACCEPTED MANUSCRIPT Comment citer cedocument:  - androstan were were

- HPLC HPLC mobilephase 16 describedby Guiraud et al.

 used compounds.as reference - ol using - -

3 3,17  - ol d given theirinformed consent. T ORS - - - dione dehydrotestosterone,4 17

on a a on

-  Chemical e CarcinogenStandardsReference Repository M)an

n, 5 one, keratinocytesisolated hairfromfollicles obtainedfrom - epitestosterone , hydroxytestosterone microporous membrane of a cellcultureinsert (Costar, preparation - TM Aldrich (StQuentin Fallavier, France): 2 d sodium hydroxide, from Sigma  (Cergy Pontoise, France). The antibiotics - androstan

( 2014 - system (Millipore, Saint QuentinQ en s . Dulbecco’s . Modified EagleMedium )

. , androsterone,, epiandrosterone, 5 - - usedin the culture media) were Figure1 androstene 3,17 , 11 - dione and5  - A hydroxytestosterone, 16 he t

providesoverviewan of the - 3,17 issue - in, 4 dione,  s

- were engineeredwere - androstan Aldrich; - androsten  - - 3,17   - - -

- in -

2 providesan overview ofthe cultureofthe RHE tissuesand the incubation conditions (systemic

before ORS cellharvesting and long

3 r - reached , the , MC EROD EROD activity and - treated treated with mitotic humandermal fibroblasts

in acetone acetone into thesurface of the model (topicalapplication). Thecontrol treated tissues ORS

ACCEPTED MANUSCRIPT Follicles

confluence, at which 2 xenobiotic

first strandfirst kitandAppliedan Biosystems2720 ThermalCycler. The I Phase and cells were cellst were ACCEPTED MANUSCRIPT Comment citer cedocument: 25

were submergedwere inthemedium after 6 daysand then cultured for a further were  and ORS M - 1000spectrophotometer. Then,1µg RNA was reverse transcripted into qPCR treatment andthe XMEgeneexpression profiles measured were using - 

toform mitotic humandermal fibroblasts used accordingto thesupplier’s instructions Biosciences/Qiagen). (SA hen plated a at density of0.3 x10 NF inDMSO to themedium (systemicapplication) orof 100 - RHE

- / q metabolismassays, respectively) contained DMEM/Ham’s F12 PCR). ThePCR). RT models(1.12cm a a functional timethecultures

em storage term test test compoun as as describedpreviously

stratumcorneum 2 and

Profiler™ PCR Arrays humandrug metabolis 2

were liftedwere totheair culturedwith1ml supplemented DMEM . . The growthmedium (0.5ml and1 ml per d Supplementary .

- free free DNAse I set (Qiagen), and

6 sub

cells/cm After . ree ree times a week. - ( lethally treated withmitomycin insert as as isolatedfromthe Table1 2

on cellon culture inserts, picture) 2 – - mediuminterface 3days, .

. After After 16. days of

the

F or tissue ORS - in - m 2

for1 h at 37°C, after which hod

SYBR GreenMastermix containing HotStart DNA Taq Polymerase enzymeand other components T - -

RHE model RHE methodbyusing the web Induction ofethoxyresorufin O Measurement of functional XME activities previously Finally, t Pharmacology, 329, 190-201. DOI : 10.1016/j.taap.2017.05.040 models(0.33cm B ). The ). controltissues were -

pcfc amplificationspecific and dyesreference was mixed with andcDNA amplification was - and µM33dic od higherfoldthansystemically applied doses were placedwere 24in a concentration 2 he epidermis was lyzedbyusing

describedby 3

ACCEPTED MANUSCRIPTmis(n=3 - MC  NF ACCEPTED MANUSCRIPT

hot start at 95°C,followed by 40 cycles of 95°C for andsec 15 60°Cfor 1 min. performed Comment citer cedocument: or 2  (25

- culturedwith0.5ml supplemented DMEM medium) were 8) depending8) the xenobiotic treatments. NF in acetone tothesurface of the model(topical application, 10 n o s -

fluorescence umarol fr e based RT Harri  ofthe xcitation M) after after runningcheck tosingle amplicons. Assays for 5housekeeping - or wellplatecontaining 500 EROD µLb s s et al.

inducers 3 - - eshlyprepared inculture medium. The 2 treated treated with deethylase deethylase (EROD) activity MC

wavelength Profiler™ PCR Array Data Analysis Software. A C

( was was measured 2002

(10

andthe - changes of changes geneexpression obtainedwere fromdifferent )  0.5M such that,, a on M)

DMSO (systemicapplication) or acetone (topical of measurement of EROD activity w inDMSO to themedium (systemic application) or

530andnm emission NaOH andNaOH totalproteins assayed were with a

using . . After incubation and PBS washing, the ctionsystem (Bio a multiplate reader Mithras  g/cm

ts were analyzedwere ts according to the 2

basis,topical dosesare uffer containing 3.4µM wavelengthof incubation was - Rad laboratories).Rad The treated treated with ere LB940

based on the 590n T

 valueof carried l/cm m.

non 2 ) -

nmoles/cm min 10% l /m

n consistedofin, A: ammonium (20 acetate and -

RHE min 62.5% , each , Test compound Test metabolism

mm,(5 ORS were storedwere at toobtain themass balance Pharmacology, 329, 190-201. DOI : 10.1016/j.taap.2017.05.040

inpmole/mg/min. Experiments performedwere from

models( B,15 , B(a) 2

, For 3 assayed

- , France). France). The, scintillation cocktailused forFlo as as of test compoundin acetone (10 RHE

µm) ZorbaxSb

previously described Jacques by et al

P P and B,65 min18% - MC,

ACCEPTED modelsMANUSCRIPT . intriplicate (n=9) 12 radiolabelled

min100% – 3

the HPLC system for B(a)P, 7 ACCEPTED MANUSCRIPT - cm 20°Cuntil theanalysis. incubationsAll carried were out in triplicate Comment citer cedocument: MC

B,28 2 were were

culturedwith to reach toreach the requireddose RHE 4 -

C18column (Agilent, Interchim, Montluçon, France) protected by a Briefly, ). min 18% dissolved B, 70B,

. 7

- Metabolitea extracts extracts andwashing solutions - EC andtestosterone

EC,

min100%

testosterone, B(a) consistedofAgilent an 1100(

B,33 1 ORS in

mlsupplemented DMEM medium) were

4 tima 

mM,pH3.5): ml l/cm

- min28% RHE nalyses

)

ofsolvable

B,71 inorder tomeasure EROD activity 2 ) to the surface tothesurface of ) the model(topical application). - .

i e radioactivityline detection usin surfaces, tissue cultureinserts and wells were (2010a .

the Culturemedia were min0% was B, P P and physicochemical propertiesof

- 40 acetonitrile(95:5; v/v) and acetonitrile.B: one detection onewas Ultima FlowM (Packard

performed

performed byradio overnight and epidermis reconstructed from

min 28%

B, 75B, 3 , - band MC was was quantified byscintillation

min0% were were , France) France) associated with a

(PerkinElmer, France).

adjustedwith unlabeled 2014

as as previously described B,45 collected at 24 )

for B. min40% - HPLC HPLC andmass

3 7 H - EC,B(a) - g a Flo a g incubatedwith , which was whichwas , 3 - MC andrelated

B,50 the

h - 0 (3 , 48,h 3batch one A500one

P P and min 0% compound

min 50% batch

7 by -

EC, and

1 e

s to B, ) .

au,

- ESI) oran sourceAPCI inthepositive mode(PI

optimized and and Phase I Phase expressiongene Basal of XMEs Results ase to the ase source. ion Pharmacology, 329, 190-201. DOI : 10.1016/j.taap.2017.05.040 to capillary temperature 150°C the variety of genes expressed as well as theirwellgenesvarietyexpressed astheas of “ 36 weak the Ct P 0°C.Optimal sourceAPCI conditions - hase hase MC andmetabolitesits quantifiedwere integratingby from reference frommetabolitesreference of

sprayvoltage XMEs and discussion ”

(which are to ACCEPTED MANUSCRIPT I XMEs after systemic application ofDMSO was very similar tothat in “ . good

of10 ACCEPTED MANUSCRIPT Comment citer cedocument: ture 30 ” of3

a a panelof  expression between were these values - also 2.5 l/cm Since reference Sincemetabolitesreference of3

- MCmetabolites 0°C

kV, sheathkV, gas (N2) shownin Supplementary Table1) 2

acetone, suggesting that neither , Corona, discharge4

gene targets for TheOrbitrap mass

benzo(a)pyrenewhich display similar structures, and were w ere ere performed using an LTQ

- were obtainedwere from3 APCI). A APCI). post columnsplit diverted 25% of the 30 P hase hase

a µ analyzer u , sheath gas A,(N ,auxiliary gas (N Thelevels ofexpression assignedwere levels of expression lowerlevels wereglobally of I - and MC,operating

. II was was used a at resolution of whereby;

the

the ofarea theradio metabolism in solventthenor method of - MC analysis is 2 ) 2 ) 60 shownin Figure2. “ ESIsource parameters - 1 no expressionno Orbitrap 0

au,auxiliary gas (N

au ORS

and were set were as - - - XLmass capillary RHE ORS ” 60 was was models - RHEs ,000. Basal 2 )

expression playan - terminal esterase L3, esterase terminal a protease), HSD17B10(hydroxysteroid 17 Pharmacology, 329, 190-201. DOI : 10.1016/j.taap.2017.05.040

inthe basement epidermis andinvolved inestrogen formation/metabolism (Hikima and

important expressed

ofthis XMEis important sincethis enzyme involved is inthe detoxification viahydrolysis -

many ACCEPTED RHE tissues. MANUSCRIPTIn addition tothegood expression of dehydrogenases, UCHL3 (ubiquitin - 1 RHE tissues. Theepoxid - role ). ). Int CYP phaseI XMEs CYP

ACCEPTED MANUSCRIPT - reactive epo at low(DMSO treated) and good (acetone treated) levels in ORS Comment citer cedocument: RHE models.For example, ADH1B was absent intheRHE modelsa inthehydrolysis large ofa number of structurally diversedrugs erestingly, onlyoneof theseven alcohol dehydrogenases (ADH) measured for - RHE models.CES1 is themajor hydrolasein human liver organ:thereference liver, ADH3 transwellinsert,which couldthe affect overall XMEprofile by . Sincethe . genepanel xides(Benhamou et al.,1998). Carboxylesterase 1and 2 (CES1/2) t oxidativet stress (Hu et al.,2010). TheNQO1 gene was alsowell

inour - MCand B(a)P especially,, aldehyde dehydrogenases (ALDH), forwhich e hydrolase e gene, EPHX1, preferentiallyis expressed in the stu dy . Although .

, viahydrolysis, andplays an important rolein used <30)orstrong (Ct - The et Theet al.,2009). NQO1 a iscytoplasmic was was developedtheby our skin

was was notincluded inth - The et Theet al.,2009).Likewise, this model includes a fibroblast - 

dehydrogenase 10 <25)(Figure2A supplier for - ; whereas, whereas, ; RHE t . They . is

genepanel

and nd this ) , issues.

were were

adulthuman liver (Edwards et al.,1998)) present inmeasureable levels (weak orgood expression). These absent orpresent onlylow at levels inORS the CES3 was not ,expressed inORS - The al., et 200

CYP2B6; ho . mone or more donors

In previous studies,a . c

). ACCEPTED MANUSCRIPT ompared to other CYPs

itreportedis thatthe expressionofthis XME is differently intheepidermis and

majorhepaticphase No ACCEPTED MANUSCRIPT Comment citer cedocument: e e expression tableXMEs thatnot were expressed dium andthe presence of fibroblasts which inhibitorysecrete factors (Luu w , Wiegand9, ever, these be presentbe inthe epidermis orwhole ofskin nativeskin (Luu

human whereas, skin; the expression of CES3 is lacking (Huet al.,20 CYP3A5, CYP4B1, CYP4F2,

llof these genes, except CYP4F11, (Luu

of FMOgenes inRHE models

et et al.,2014). While it were were - I The et Theet al.,2009, Wi XMEs, innative human skin all reportedto be

andOe - RHE models.

namely , havebeen shown CYP4B1 andCYP26B1 - - The et Theet al.,2009, Huet al.,2010). RHE modelsand sch

CYP1A2,CYP2C9/18/19 and CYP3A4 were

et et al. i s expected that s FMO2and FMO3 would be CYP4F3, inORS expressed e

gand et al.,2014, Hu al., et 2010)

,2007 (Luu -

ha - RHE models toexpress ). Bothof these ). isoforms are also The et Theet al.,2009 thedermis, FMO1,FMO4 and CYP4F11,CYP7B1, were were s

at at verylow levels only few ofa the

previously been genes foundto be were were

FMO1,3,and 4 mRNA

were CYP1B1,were were CYP1A1,were also reportedto be - , Hu et Huet ,al.,2010, RHE tissues. This CYP1B1 - expressed in

reportedto be Theet al., 42 in CYP26B1 native target CYP

,and

(which - The 10 ). ).

. targeted undetectable.were CYP2D6 Phase Phase . In our. study, it Pharmacology, 329, 190-201. DOI : 10.1016/j.taap.2017.05.040 notall at GSTisoforms alsowere present at goodlevelsandhave shownalsoto be present innative II n l expressed GSTtly was GSTP1 is

XMEs were allwere expres

notably, UGT1A10,,which is repor intheskin ACCEPTED MANUSCRIPT reported to a be majordetected CYP at the mRNA levelinnative human skin (Oesch et

intopically applied acetone models (Supplemen Luu ctivity ACCEPTED MANUSCRIPT

Comment citer cedocument: was was - The et Theet al.,2009). 10 at at the gene level - thanin the ). specificsuch thatNAT1 ispreferentially expressed foundtobe expressed II

intheORS SULT2B1,SULT1A3, sed ingood XMEs expressed at significant levelsinRHE tissues (Figure 2B). To further check this, additional s Allthe inhuman keratinocytes (Saeki et 2002) al ORS

liver - UGT isoforms RHE tissues measured here andin native from skin one donor - RHE models inthe AllGSTsplay importantan role levelsin ( Hu al., et 2010

(Ct =20.5) VB VB radiation(Black et al.,2008). ORS

only at verylow levels inDMSO other ORS ted to be affected tedto affected beby the complexity oftheskin - SULT1E1

. RHE modelsnotbut thefetal form of this CYP, foundbe to CYP3A4 expression appears to donorbe - donors(Wiegand et al., , whichis, known tobe themajor RHE tissues;however, other . , The expressionofN Kawakubo et al.,1990) and tudiesusing CYP2D6

expressed ay Table1) tary t hiosulfatesulfurtransferase

andhuman skin (Oesch et al., inthedetoxification of inourmodel belongdo to andhas a higher . . This - acetyl transferases 2014). Other - . In keeping. with treated treated control

mayreflect the - sulfurtransferase selective cytosolic and As As withnative GST - thickness isoform

The

 NF and Induction ofPhase leve Pharmacology, 329, 190-201. DOI : 10.1016/j.taap.2017.05.040  of3 NF (133NF of l

andthestatistical analysis 3 .

- -  MC MC NF and expressionofCYP1A1 5 )

and NAA20 (N(alpha)

ACCEPTED MANUSCRIPT induction ofCYP1A1 and CYP1B1 orsystemic application ( - Figure3 od andfold) xpression . A biologically A relevantinduction ACCEPTED MANUSCRIPT 3 Phase II Phase Comment citer cedocument: - MC on ofonspermidine and spermine, and involvedis intheregulation ofthe I ) XME XME expression . . DespiteCYP1A1 beinge

3 are are ligandsforAhR and of - - MC XME foldby phase more

(25 (catechol

genes ACSL1were (

for control andinducer I than2 insolvent treated tissues 0

 Luu - - XMEs acetyltransferase acetyltransferase 20) NF andNF 11.9 fold) ofβ , suggesting, its presence isimportant in metabolismskin - The et Theet al.,2009) - -

foldandwhen the inducedexpressed level was greater O - to NF in - thisenzyme m clearlymeasurable ORS ethyltransferase), isshown in Figure3 - AhR od byfold expressionin - xpressed at verylow levels response was called when a gene was RHE ORS Long

regulates a numberofPhase

; however, ;we andothers (Huet al.,20 is - models . - 3 treated models are shownin

RHE The . involved in - -

MC chain expression ), which was most duelikely th to was was confirmedin ourstudies. ORS SAT1 levels withthat24 h -

fatty .

- RHE

. Theresponse . (spermineN1 the metabolism of -

was was a selective acid ofCOMT inskinhas models - CoA ligase1,CoA

it was readily after after treated treated with I - topical

was was much XMEs, and

. 10 e )

- ,ALDH3A1 or fold, P<0.05). Pharmacology, 329, 190-201. DOI : 10.1016/j.taap.2017.05.040

lly applied. and moreconsistent across donors topical application

 additional genes inducedwere eitherby treated treated topically andsystemically with NF (25 relevant after the systemicapplication of , ORS

ACCEPTED MANUSCRIPT h24after the start ofinduction -

RHE and NQO1  M)). Thiscould ALDH3A1 ACCEPTED MANUSCRIPT Comment citer cedocument: models

topical

ORS of affinity by both .

likely be treated treated topically orsystemically withvehicle control solvents(DMSO ORS Inaddition, the induction response was higher using - was was significantly induced(P<0.001)

the RHE application of

inducer

of - systemicapplication of RHE tissues with 3 -

dueto difference inthebioavailability of theinducers such MC

- s when applied topically insystemically treated offvalue considered to biologicallybe relevant(Ct was

(Figure3) with forAhR 3 -  MC   theCYP1A1/2 andCYP1B1 selective substrate, NF or NF orNF NF or3 

(despitethelower dose of caused a significant

; however,; theinduction was onlyconsidered to NF measurable in (P (P 3 3 values: - - since fold(ALDH1A3 andCY4F12) - - MC MC MC  NF and . . Theextent ofinduction inducers by

theinduced level was below Ct a of (Figure4) (but

0.323 ORS (even thoughthe by>2 never ORS PTGS2by topical - and 0. and RHE

- . - induction ofthis gene

RHE fold bothbyinducers) EROD EROD activity was tissues than whenthey 245 following the 3 tissues onefrom or , respectively, - MC theoretical 3 -

(10 MC ly

applied 3 by  than M) ,namely,  ) dose NF and/or 

(2.64 NF ; - , -

2014 also onetype of orsystemic Induction ofPhase Metabolism ofMetabolism substratesmodel after topical application this ) considered as as measured after 24h), we have expressed thedose ) Pharmacology, 329, 190-201. DOI : 10.1016/j.taap.2017.05.040

and thereforewere appliedtopically.

two phase notsignificant fromcontrol treated acterized the Phase 6/7 correlated with 2

andSULT1A1; however, thiswas effect considered notbe to biologically  8 and 10),/8 NF Phase fromthestudies presented here andfrom previous rep application -

ACCEPTED MANUSCRIPT models treated are shown in tobe (3.8 II II - XMEs thatdown were ACCEPTED MANUSCRIPT

4.6

XMEgene Comment citer cedocument: a

and

suitable functional XMEactivities by incubating them with four test compounds - II

fold)than of β XME XME expression theextent ofinduction was alsorelatively low I of . and The lowerinduction ofUGTs - phase I NF

timepoint s and/or

II that is shownisin Figure

aft XMEgeneexpression II was er XMEsin II

topicalapplication of100 nmoles/cm

XMEs.

inducedby sinceall ofthe 4model substrates foundwere tobe -

ORS regulatedby more than 2 T Supplementary Table1 The incubations included three timepoints (24,48 and 72

he responses were - ORS RHE Allfour test chemicals are known to 5 -  RHEs models . NF and TheCt values -

dependent in compared to

ORS withthat h24 .

higher - MC - RHE orts( .

effects usingeffects h24incubations. - Unlike Phase ,namely UGTs fold .

and thestatistical analysis

after after systemicapplication of tissues, we then

Jacques Jacques et al.,2010 CYPs has CYPs alsobeen reported

( by topicallyby applied 2 after after topicalapplication -

to 2

3 5 - MC - fold I

XMEs,there relevant

(2.6 ) penetrate

comparedto

investigated - 2.9 a and ba

because because - fold) 3 - MC for was

It of ,

A1 (Figure2A). 7 ngly, the production ofHC - and CYP2E1 Pharmacology, 329, 190-201. DOI : 10.1016/j.taap.2017.05.040 Ethoxycoumarin the expressed - EC, indicatingEC, radioactivity s HC ORS he 4he test chemicals that was present as the parent chemicalinsignificant amountsinthe

(Figure6A) (forall mediasamples into the medium (HC) (HC) of7

- The glucuronidation of HC isreported to

ACCEPTED MANUSCRIPTglucuronide expressed - RHE -

EC tohydroxycoumarin was likely toCYP1B1,be which was expressed (and a minor amountvia 1 inthe ( A ACCEPTED MANUSCRIPT Shimada . Comment citer cedocument:

- Duringthecourse of the h72incubation, t ) EC The , model

, applied (7 which we attributedtothe shortresidence timeof7 suggestingthattheenzymes inv sulfonation that ORS producedin - re re was a at

EC) and

, with, goodlevelsin (an observationmadealso thischemical does not require metabolism inorder to pass through the et al.,199 (SupplementaryFigure - - s RHE

that was HC ulfate

anddosesof themajor metabolite being HC dose –

of HC. models sulfate andHC recombinanthuman liver microsomes 7 - ), dependent recovered inthemedium ranged between 60%and 82% ORS ),with a subsequent conjugation oftheintermediate

ORS

CYP1A2 ( in acetone treated control models De De Kanter et al.,1999 - - g RHE

7 luc - - RHE EC

uronide linearproduction of three all metabolites models

and 1 over72 h) models. )

. previously be mediatedbe by UGT1A6 (Lampe et al., olved were not were olved saturated

CYP1B1

reached reached a plateau after 48h , the ,responsibleCYP for There were There were he productionhe of HC was maximal at andof this, 54

- ) ) g (Jacques (Jacques et al.,2010 .

luc mediated The - s uronide ulf se ate

- six three metabolites were EC inEC the RHE models, are are

andHC O

(Figure2B SULT isoforms - (Figure 73% was was non73% - producedvia deethylation , evenat thehighest - the g

6) b luc ) ) . )

. uronide which Since at at good This was

over1 to - to

of .

O - liver Testosterone testosterone (peak testosteroneIII) - RHE tissues,7 deethylated Pharmacology, 329, 190-201. DOI : 10.1016/j.taap.2017.05.040 hydroxy

that . microsomes This was Thiswas alsoo thisobservation was notspecific to - 

ACCEPTED MANUSCRIPT (peak 4.dione metabolite was detected in themedium, whichmay duebe to its

 the faster thefaster rate of its - ACCEPTED MANUSCRIPT andfresh humanskin explants hydroxytestosterone(peak producedI, by CYP2A6 and2A13 (Bogaards et al., Comment citer cedocument: bserved

(Figure7B)  epiandrosterone(peakXII), androstenedione (peak XI), - s hydroxytestosterone(peak II, produced CYP1A1/2,by CYP3A4/5/7

IV in

 (Jacques (Jacques et al.,2 , V’ andVIII

ex ex vivo - andtime dependence reductase reductase ( . conjugation

analysis.Two other hydroxylated metabolites producedwere One ofthemajor metabolites formedin theORS

humanskin our ), Liuand Yamauchi, 2008)) 4 urther

- 0 ORS androsten metabolitesoftestosterone thanreduced in 14) were alsowere 14)produced by the (Jacques (Jacques et al.,2 o the to

andpig ear skin (Jacques et al, 2014) - metabolism was possible. RHE either (Figure7A) HC - 16α model. - hydroxyl glucuronide - Possiblereasons for the ol - 0 3,17 . 14 Considering thelower CYP , producing manymetabolites in An interesting finding was that ated ). Th ). - . dione

andHC or e e majormetabolites

reduced metabolites (peak s

low ORS Comparatively less -  sulfate

6 VI) VI) and16α rate of - - tes RHE - RHE models relative - t androstan . osterone

models -

andtime Benzo(a) Pharmacology, 329, 190-201. DOI : 10.1016/j.taap.2017.05.040

ofthis lipophilic compound suggesting, - - s metabolized testosterone dependent radioactivity . (forall mediasamples

pyrene (B previously

ACCEPTED) MANUSCRIPT ) . T ain andreduction lationpathways totestosterone metabolism he that the ACCEPTED MANUSCRIPT

Comment citer cedocument: (Fig

percentage of ( applied a ure ) observedusing pig a skin P) rate rate

andSupplementary Figure1 that was of metabolism

because because itsmetabolism ismore complex (Fig

anddosesof testosterone ,indicating thatthis ishighly

reduced metabolites ure recovered inthemedium ranged between 62%and 78% 7

andSupplementary Figure1

dependenton its (including bolism decreased after decreased , suggesting, that these metabolites are metabolized t higherdoses skin .

The production of metabolitesall was

modelmolecule ( hydroxylated metabolites) models J also acques acques et al.,2010 B) over 72h) and of this, only metabolism . decreased decreased

48 W . T

(400 hiletheincrease in hydroxy h following testosterone he hairfollicle cellswhichfrom hydrox –

B) residedin the

800nmoles/skin over time such thatonly the than7 .

T a ylation he could be dueto ).

Likewise, inthe percentage of - EC and because andEC because the

( andenzyme from25% after pathways ORS 2 - explant 4 - % was was % RHE both the - ,

sulfate conjugatessulfateof B(a)P metaboliteproduced to be ableidentifyto themby lth

8A uced. uh thesyntheough by CYP1B1,by CYP1A1 and microsomal epoxidehydrolase Pharmacology, 329, 190-201. DOI : 10.1016/j.taap.2017.05.040 ). ). Bycontrast, expressed at the gene level solventincontrol RHE tissues ex ex vivo by , only 1 ,

over 72h ORS II metabolites ofproducedB(a)P inORS ( 

ACCEPTEDpigskin (Jacques et al., 2010a). B( MANUSCRIPT NF and3 - - ithUGT1A10, 1A7and 1A8, andSULT1A3 and1E1 presentwere inORS ) 3% RHE P . . B , the , majority ofbeingB(a)P metabolized inthefirst h24( ACCEPTED MANUSCRIPT I t of theradioactivity Comment citer cedocument: i sulfation and glucuronidation c standards c etween 9% and 21% of theapplied dose w metabolites of models - - MC;therefore, thismayCYP contribute metabolismB(a)P to once it mediated metabolism ofB( - hydroxide(Figure8B), which consistentis with themetabolites tohydroxide, diol, catecholand dione metabolites

forsome metabolites not were avail

B(a)P

present intheculture medium

was was dose

a)P phase a)P

pathways saturatedwere at doses higherthan1 Of Of these XMEs,only - - mass spectrometryand a)P, a)P, at leastin the few first hours of the dependent and overlinear thedose rangetested RHE models are the mono I metabolites are conjugated viaUGTs s as as recovered

(EPHX) . . T is herefore able an AhRligand withsimilar was was foundto correspond to

there was enough, ofeach CYP1B1 ( Hvastkovs et al.,2007) , these isoformsare NMR. in SupplementaryFigure mediasamplesfor all - a

glucuronide and (Figure8B) ) and )

B(a)P and EPHX

are are

was - mainly - RHE

. The .

we has

likely

re and

conjugation of

at at the genelevel after topical application Figure (see 5B) 3

activities.l - Pharmacology, 329, 190-201. DOI : 10.1016/j.taap.2017.05.040 trans Methylcholanthrene ( - hydroxyl functionsnorconjugation positions,

(Meinl et al.,2013). RHE models(Figure 2B);which sugge to -

hydroxy  1 - or hydrolyzed 1,2 - g) keto “ - i notdidallow NMR identification reduced ACCEPTED MANUSCRIPTdihydroxy T 3 re re involved inthe detoxification of - - he s he 3 MC - are are subsequentlyconjugat - 3 MC ACCEPTED MANUSCRIPT - onthehydroxide Comment citer cedocument: MC tandard syntheticmetabolites of

metabolitesor ” (Stoming et al.,1977) 3 , andcholanthrene, (Myers et al.,1989, Myers andFlesher, 1990)

- metabolites -

MC C and MC (withSULT1C3 and SULT2A1playing a minor role) Interestingly, noneof these SULT 3 3

- H to DNA toDNA reactive metabolitesinvolves CYP1 XMEs(Guengerich, - MC MC X1,which was expressed in these tissues.

with 11,12 ) and dosesandof

(Figure

that conjugated - hydroxyl ORS -

the genelevel of these XMEs does correlatenot withthe dihydroxy

1 ed tosulfates ed andglucuronides. - - 9A ) hydroxy RHE

sts either sts and number a of multiple T . ) - SULT1B1:is primarily responsibleforthe . 3 3

Detoxification viaepoxide hydrolases in the - - herefore, MC MC models 3 - - 11,12 anddenoted these metabolites MC - 3 3

- - over 72 h) and ofoverthis,h)72and 2 only that (withSULT1A2and SULT2A1 playing a MC MC . Interestingly, . we foundthat3 - . dihydro and these conjugates readilywere

anotherisoform was responsible

we we were notwere available and2 isoforms presentwere at the gene level were unablewere to - - MC

hydroxy -

yrxltd metaboliteshydroxylated (ClaytonandClayton, 1981

As As with - Thesulfated conjugates 3 - MC determinethe andthelow amount andSULT1A1 is

theother 3

, which are whichare further , either as - 4% was presentwas4% - , MC induced epoxides

forthe exact ORS in ,

MC en

UGT data showeddata thatt and ) reported as UGTenzymes involved inthe 3

phase - metabolized Conclusions 1A7 were present at the genelevelpresentwereORS 1A7thein at the Pharmacology, 329, 190-201. DOI : 10.1016/j.taap.2017.05.040 h SLs novd n h frain f h slae cnuae ae SULT1B1, SULT1C3, are conjugates sulfated the of formation the in involved SULTs the ,which notdo havethe same XMEprofile I metabolites are conjugated viaUGTs ( by ACCEPTED MANUSCRIPTand1A8 3

enabling the measurement of - he ORS MC ACCEPTED MANUSCRIPT bothof which impactlocal ,skin andsystemic toxicity Comment citer cedocument: idctn ta ti chemica this indicating that , exposure . The use . oftheORS ofthehair follicle a isnew concept UGT - RHE model 1A10 were induced after topicalinduced 1A10wereapplication after 3 of In

scenarios andis RHE modelsand to characterize them as a suitablesurrogate for contrast could

Meinl Such models - is a a isnovel alternativeapproach RHE models2B).(FigureRHE represent a to t l, 03. Interes 2013). al., et

- B(a)P MCmetabolism intheliterature.

potential metabolic activation to toxic metabolites or, more predictive 1A5, s

complementary are importantin thesafety assessment of topically as as skin(van Eijlal., et 2012) andcannot be h frain f hydroxylated formationthe of - be to enough models long RHE the in resided l RHE modelsRHE2B)(Figure 1A7,1A6 igy nn o tee UT isoforms SULT these of tingly, none

, 1A1 , than

Of the UGTs,UGT the Of modelto a number a of different assay Although several commercial ) ( ) usingextrapolations from withextensive xenobiotic Gramsal, 2000et Thismodel .

the commercially

. Interestin. - MC but they have nothave theybut MC that 3

allowsfor - MC 1A gly, the genethegly, refl 5 , Finelal, et metabolites

, (Figure6) UGT ects ects skin liver 1 A6 A6

ishigher a bioavailability totheepidermal XMEs).Our study demonstrate thatthe ORS : CYP1B1, CYP2C18,e:CYP2D6, CYP2S1,CYP3A5, CYP4B1, CYP4F3,CYP7B1, CYP26B1 and

withothe - Pharmacology, 329, 190-201. DOI : 10.1016/j.taap.2017.05.040 RHE model a has XME profile that consistentis with nativehuman skin, supportedtheby investigatewhether XME

profiles morewere sensitivethanother endpoints such thatthereproducibility ofdata ACCEPTED MANUSCRIPTreports,r the levels ofII Phase ansportationconditions e.g. the duration oftransport our tolaboratory andseasonal vanEijl et al., ni ACCEPTED MANUSCRIPT mportant aspect aspect mportantof thismodel that is iti Comment citer cedocument: . These . topical application enzymesfunctionality 2012),t

were

also present at thegene levelintheORS here here are a numberof that CYPs are consistentlyreportedto be

XMEs presentgenerallywere higher than those of Phase of test compounds

dueto t

andfor environmental pollutants ). ). bioavailabilityoften is assumedto be ow incomparisonow with reductases, esterases The hi ransport effects. ransporteffects. s generated ghest gene ghestexpression levelofP couldemployedbe insuchassays.

withinlaboratory our and Since topical application is - RHE mode w , - RHE e e also l

and hase hase

the model isresponsive prototypical to inducers, CYP such that CYP1A1 and CYP1B1 are correspondto functional of innovative

and theprediction ofthesystemic exposure toparent

ACCEPTEDautoinduction MANUSCRIPT - RHE model ACCEPTED MANUSCRIPT - Comment citer cedocument: MC. TheMC. metabolitesproduced after incubation withtestosterone also indicates -

and25 ain with bationinducers.CYP Functional XMEactivities also were active

during the incubation period tested vity level, whichofissignificance because gene expression  0 was was shownto express a numberof important XMEs,which -

- ingredients od respectively).fold, reductase, the presence of which isreported to pivotalbe inthe

XMEactivities ( t toconfirm ntwhether XMEs can alteredbe b  NF and .

3 - MC Hewitt et al.,2013) TheORS . Theinduction ofCYP1A1 and CYP1B1 was ndtheon residence timewithin the - mediated metabolism of7 - RHE models are intended foruse in .

(LiuandYamauchi, 2008). and/ the . ormetabolites af  cutaneous - andNF

to5 - fold) as thatfor 3 y the y test - metabolism and MC -

also inducedalso EC, ter ter topical levelsdo could

The be

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StephenD, Bratton MackenzieS, PI, Radominska 2):56 – 3 ACCEPTED MANUSCRIPT Comment citer cedocument: - 387. methylcholanthrene

- P 3 doi: 10.1016/j.mrfmmm.2008.07.002.63.

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30 and30strong expression (  NF (A) (A) NF and topical application of3 er er treatment with25µM s /mg/min. Data were /mg/min. Data obtainedwere from 3 donors intriplicate - - 2 tailedStudent’s t od Symbolsfold. inred represent genes that inducedwere on)in themedium. Thedifferent levels ofexpression

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Pharmacology, 329, 190-201. DOI : 10.1016/j.taap.2017.05.040 - Dose Dose Effect of systemic applicatio

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- hydroxytestosterone; II Peak =6 3,17 ACCEPTED MANUSCRIPT Comment citer cedocument: 2 - - ) forthegoodness of fit 0.94,were 0.98and 0.86 for theHC, HC RHE models.Thegene profilesmeasured were 24 hafter treatment with25 dione; Peak VI Peak =4 dione; - EC. Thedoses applied 1, were 2 and 4 nmoles/cm - 4 SEM SEM - androstene of n

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ACCEPTED) andsulfated ( MANUSCRIPT) andsulfated ( donors,mean± - hydroxy ACCEPTED MANUSCRIPT Comment citer cedocument: - hydroxy - MC. Thedoses applied 1, were 2 and 4 nmoles/cm - 3MC - - VI =2 SEM hydroxide.The correlation coefficients(R SEM - 3MC metabolites, respectively.ed hydroxy metabolites.) Th metabolites.) The metabolites ofshownB(a)P inthe -

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