Arch Toxicol

DOI 10.1007/s00204-008-0317-7

TOXICOKINETICS AND METABOLISM

Pharmacologic proWling of human and rat cytochrome P450 1A1 and 1A2 induction and competition

Walter M. A. Westerink · Joe C. R. Stevenson · Willem G. E. J. Schoonen

Received: 8 January 2008 / Accepted: 6 May 2008© Springer-Verlag 2008

Abstract Strong activation of the AhR can lead to vari-ous toxic eVects such as (non-genotoxic) carcinogenicity.Moreover, drug–drug interactions by non- or competitiveinhibition of CYP1A1 and 1A2 may cause adverse sideeVects. Normally the majority of toxicity studies are per-formed in rats, while for the prediction of human toxicityhuman AhR activation and CYP1A competition should bestudied. The present study focused on the deselection ofstrong AhR activators and/or CYP1A inducers and (non-)competitive inhibitors in the early phase of drug develop-ment, as well as on species diVerences between humans andrats. Induction studies were performed in the human HepG2and rat H4IIE cell lines. A set of 119 compounds, includingknown AhR ligands were tested. CYP1A induction wasobserved for 24 compounds. In H4IIE cells, more com-pounds showed induction and most EC50 values werebelow those of HepG2 cells. Species speciWc CYP1Ainduction in H4IIE and HepG2 cells was obtained for eightand three compounds, respectively. The same compoundsexcept four in-house NCEs were used to study diVerencesbetween CYP1A1 and 1A2 competition in human and ratsupersomes. Of the 115 compounds 46 showed CYP1A1competition. Competition was human and rat speciWc for 12and 10 compounds, respectively. CYP1A2 competition wasobserved for 37 compounds of which 14 and 3 compoundsshowed human and rat speciWc inhibition, respectively. Inconclusion, for several compounds species diVerencesbetween CYP1A induction and competition in human and

rat were found. Therefore, parallel screening in both spe-cies might be a very useful strategy.

Keywords Aryl hydrocarbon receptor · CYP1A · Induction · Competition · HepG2 · H4IIE

Introduction

In drug therapy, cytochrome P450 (CYP) 1A inductionmay lead to undesirable drug–drug interactions and toxicside eVects. The regulation of CYP1A is mainly aryl hydro-carbon receptor mediated (AhR). The AhR was discoveredas the receptor that binds the environmental contaminantTCDD (Bertazzi et al. 1998). The AhR is a basic helix-loop-helix protein belonging to the Per-ARNT-Sim (PAS)family (Hahn 2002) and is located in the cytoplasm in aninactive complex with Hsp90 and p23. Experiments inHsp90 deWcient yeast show that Hsp90 is needed for geneinduction by the AhR (Carver et al. 1994; Whitelaw et al.1995). Hsp90 is thought to protect AhR from degradationand to stabilize the high-aYnity ligand-binding conforma-tion of the AhR (Pongratz et al. 1992a, b). Binding of aligand to the AhR leads to activation of the receptor andsubsequently in translocation to the nucleus. In the nucleusAhR releases its partner Hsp90 and forms a heterodimerwith the AhR nuclear translocator (ARNT) protein. Furtheractivation of this heterodimer by phosphorylation and/ordephosphorylation is required for DNA binding (Chen andTukey 1996; Long et al. 1998).

Strong activation of the AhR by TCDD results in toxiceVects like a wasting syndrome, thymic involution, endo-crine disorders, and very important teratogenicity and (non-genotoxic) carcinogenicity. Mortality studies involvingoccupational exposure to TCDD have demonstrated an

W. M. A. Westerink (&) · J. C. R. Stevenson · W. G. E. J. SchoonenDepartment of Pharmacology, NV Organon, a part of Schering-Plough Corporation, Molenstraat 110, 5340 BH Oss, The Netherlandse-mail: walter.westerink@organon.com

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increased risk for a lot of cancers in humans (Fingerhutet al. 1991; Manz et al. 1991; Zober et al. 1990).

Important for this non-genotoxic carcinogenic eVectmight be the change in expression patterns of several fac-tors that are involved in cellular growth and diVerentiation.Another eVect contributing to carcinogenicity might be theimpairment of the p53 response (Paajarvi et al. 2005; Rayand Swanson 2004).

Studies in AhR-null mice have been used to investigatethe role of the AhR in mediating the toxic eVects. The stud-ies revealed that these mice were resistant to TCDDinduced lesions, strongly suggesting that the toxic eVectsare mediated by the AhR (Fernandez-Salguero et al. 1996).Furthermore, skin tumors that appear after topical applica-tion of the AhR agonist benzo[a]pyrene (B[a]P) are notpresent in AhR-null mice (Shimizu et al. 2000). Besidesactivating the AhR, B[a]P is metabolized by CYP1A1 to agenotoxic metabolite (Jack and Brookes 1981). The meta-bolic activation of procarcinogens to reactive metabolitesby CYP1A1 and 1A2 was also shown for other AhR ago-nists by using Aroclor pretreated rat liver S9-mixture formetabolic activation in the Ames test. CYP1A1 and/or 1A2activates PAHs, nitrosamines and aryl amines into reactivemetabolites that induce mutations (Hecht 1998). Inductionof CYP1A enzyme activity is often used for the detection ofAhR ligands as good speciWc CYP1A substrates are avail-able.

Besides metabolic activation into genotoxic com-pounds, drug-drug interactions caused by non- or compet-itive-inhibition of CYP1A1 and 1A2 can also lead toadverse side eVects. A lot of drugs are metabolized byCYP1A2 and therefore drug-drug interactions caused byCYP1A2 inhibition are known. CYP1A2 constitutesabout 13% of the total hepatic CYP content and is one ofthe clinically most relevant CYP isoenzymes. Togetherwith CYP2C9, 2C19, 2D6, and 3A4 it performs the mainpart of drug metabolism. Examples of drugs metabolizedby CYP1A2 are the antidepressants amitriptyline HCl,clomipramine HCl, and desipramine HCl (Danie et al.2001; Wu et al. 1998; Yoshimoto et al. 1995). The levelsof CYP1A1 in the human liver are low, however, thisenzyme is highly inducible in the liver and extra hepatictissues. Induction of CYP1A1 by AhR agonists in humanprecision-cut liver slices and primary human hepatocyteshas been shown in several studies (Drahushuk et al. 1998;Olinga et al. 2008; Pushparajah et al. 2008; Dvorak et al.2008). Little is known about drug–drug interactions inwhich CYP1A1 is involved. However, some of the drug–drug interactions caused by ketoconazole might be theresult of a lower CYP1A1 enzyme activity caused by inhi-bition or competition in the intestine (Paine et al. 1999).Furthermore synergistic embryotoxicity of AhR agonistswith CYP1A1 inhibitors has been shown (Wassenberg

and Di Giulio 2004). This was probably caused by a pro-longed activation of the AhR.

Because of the adverse side eVects it might be very use-ful to screen for and deselect candidate drugs that are strongAhR activators and/or strong CYP1A1 and 1A2 inhibitors/competitors in the early phase of drug development.

Induction of CYP1A enzyme activity was measured withthe Xuorogenic substrate 3-Cyano-7-ethoxycoumarin(CEC) and luminogenic P450-glo substrate Luciferin-CEE(Luc-CEE). The latter substrate is converted by CYP1A1into luciferin, which in turn reacts with luciferase to pro-duce light that is directly proportional to the activity ofCYP1A1.

Because species diVerences have been describedbetween the CYP1A inducing properties of AhR agonists inrats and humans (Zeiger et al. 2001), the CEC and theP450-glo 1A1 induction assays were performed in thehuman HepG2 and the rat H4IIE cell line.

CYP1A1 and 1A2 inhibition was measured in a 384 wellhigh-throughput assay using CYP1A1 and 1A2 expressingsupersomes and CEC as Xuorogenic substrate. With theseassays competition was measured between the Xuorogenicsubstrate CEC and the tested compounds for CYP1A1 and1A2. Whether compounds are non- or competitiveCYP1A1/1A2 inhibitors cannot be determined with theseassays. Moreover, in order to study species diVerences,competition experiments were performed by using bothhuman and rat supersomes. A total of approximately 119compounds were tested in the CYP1A induction and com-petition assays. These compounds included narcotic analge-sics, hypnotics, vasodilators, speciWc cellular energyblockers, cellular proliferation inhibitors, ion channelblockers, estrogens, antiestrogens, androgens, progesta-gens, PCBs, and others.

Materials and methods

Materials

All compounds and reagents were of analytical grade. Poly-chlorinated biphenyls (PCBs) including 2,3’,4,4’-tetrachlo-robiphenyl (PCB 77), 2,3,3’,4,4’-pentachlorobiphenyl(PCB 105), 2,3,4,4’,5 pentachlorobiphenyl (PCB 114),2,3’,4,4’,5’-pentachlorobiphenyl (PCB 118), 2,3’,4,4’,5’-pentachlorobiphenyl (PCB 123), 3,3’,4,4’,5’-pentachloro-biphenyl (PCB 126), 2,3,3’,4,4’,5-hexachlorobiphenyl(PCB 156), 2,3,3’,4,4’,5’-hexachlorobiphenyl (PCB 157),2,3’,4,4’,5,5’-hexachlorobiphenyl (PCB 167), 3,3’,4,4’,5,5’-hexachlorobiphenyl (PCB 169), 2,3,3’,4,4’,5,5’-hepta-chlorobiphenyl (PCB 189), and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) were purchased from Promochem (Wesel,Germany). All other compounds were from Sigma–Aldrich

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(St. Louis, USA). Luciferin-CEE (Luc-CEE) was fromPromega (Madison, USA). CEC and supersomes express-ing human and rat CYP1A1 and 1A2 were from BD Biosci-ences (San Jose, USA). Trypsin and Dulbecco’s ModiWedEagles medium, Nutrient Mixture F-12 (DMEM/HAM F12medium in a ratio of 1:1) without phenol red was fromInvitrogen (Kalsruhe, Germany), deWned supplementedbovine calf serum (dBCS) from Hyclone (Utah, USA) andwhite 96 and 384 well culture plates from Perkin-Elmer(Groningen, The Netherlands).

Cell culture

HepG2 and H4IIE cells were obtained from the Americantype culture collection (Rockville, MD, USA). Cells werecultured in Dulbecco’s modiWed eagles medium and nutri-ent mixture F-12 mixed in a ratio of 1:1 with 10% dBCSand 1% penicillin–streptomycin (10,000 U/ml, Invitrogen).Cultures were maintained in a humidiWed atmosphere with5% CO2 at 37°C and medium was refreshed every 3 or4 days with subculturing.

Preparation of compound solutions

Stock solutions of 0.1 M were dissolved in 100% dimethylsulfoxide (DMSO). The stock solution of TCDD had a con-centration of 3.16 £ 10¡4 M. From the stock, q10 dilutionswere prepared in DMSO.

Test plate preparation induction assays

HepG2 and H4IIE cells were trypsinized, counted andseeded in 96 well plates. HepG2 and H4IIE cells wereresuspended in culture medium to a Wnal concentration of3 £ 104 and 2 £ 104 cells/well, respectively. The 96 wellmicrotiter plates were incubated for 24 h in a humidiWedatmosphere at 37°C under 5% CO2. Following the incuba-tion, 11 serial culture medium dilutions of the compoundsor a control sample were added as 10 �l fractions to HepG2and H4IIE cells leading to a Wnal volume of 200 �l. Thehighest tested concentration for the compounds was10¡4 M. An exception was TCDD for which the highesttested concentration was 3.16 £ 10¡7 M. The Wnal concen-tration of DMSO in the assays was 0.1%.

CEC induction assay

The induction of CYP1A activity in HepG2 and H4IIE cellswas measured with the Xuorogenic substrate CEC. CECreacts with both CYP1A1 and 1A2. After incubation ofcells with compounds for 24 h, cells were washed twicewith PBS. Next 100 �l of 40 �M CEC dissolved in culturemedium without dBCS was added. After 30 min the Xuo-

rescent signal was read on the Victor II (Perkin-Elmer) bymeans of excitation at 409 nm and emission measurementat 460 nm.

P450-Glo CYP1A1 induction assay

The induction of CYP1A1 activity in HepG2 and H4IIEcells was measured with the luminogenic substrate Luc-CEE. After incubation of cells with compounds for 24 h,HepG2 and H4IIE cells were washed twice with PBS andthe CYP1A1 induction was assessed with Luc-CEE. To theplates 50 �l Luc-CEE (30 �M) was added. Subsequently,50 �l luciferin detection reagent was added. Plates wereshaken for 10 min and the luminescence signal was mea-sured with a TopCountNT luminometer (Perkin-Elmer).

Cytochrome P450 1A1 and 1A2 competition assays

CYP1A1 and 1A2 competition assays were carried out byusing supersomes. Compounds were tested at concentra-tions of 10¡7–10¡4 M with q10-fold dilution steps in a 384well plate in 0.1% DMSO. The highest concentrationTCDD in the assay was 3.16 £ 10¡7 M. The cofactor solu-tion was prepared in 25 mM phosphate buVer (pH 7.4) andcontained 2.6 mM NADP+, 6.6 mM glucose-6-phosphate,6.6 mM MgCl2 and 0.8 U/ml glucose-6-phosphate dehy-drogenase. This solution was prewarmed at 37°C. Fiveminutes before the start of the reaction human or ratCYP1A1 or 1A2 supersomes were added to the cofactorsolution leading to a concentration of 5 pmol/ml super-somes in the Wnal reaction. To the 384 well plates, contain-ing 10 �l compound dilution or DMSO control, 10 �l of asubstrate solution containing 20 �M CEC in 325 mM phos-phate buVer was added. The plates were covered with a lidand shaken for 20 min. Then plates were pre-warmed at37°C in an incubator and 20 �l of an enzyme/cofactor solu-tion was added leading to a Wnal volume of 40 �l/well.Thereafter the plate was put in the Victor II reader, shakenfor 20 s and pre-incubated for 2 min at 37°C. Next the Xuo-rescence was measured (t = 0 min). The excitation wave-length was set at 409 nm and the emission was measured at460 nm. Then the plate was incubated for 30 min at 37°C.Thereafter the Xuorescence was measured (t = 30 min). ThediVerence in Xuorescence between the measurement at 0and 30 min was used for calculation of CYP competition.The increase in Xuorescence of the solvent control (0.1%DMSO) during these 30 min was set at 100% activity. Forcalculation of the IC50 values of competitors, the data werelogit transformed. The software program XlWt (version 4.1,ID business solutions limited) was used for the calculationof the best line Wtting the experimental data. From this linethe IC50 was determined. The eYcacy was deWned in per-centage by means of the total inhibition of the Xuorescence

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increase at the highest tested compound concentration. Thisconcentration was 10¡4 M, with exception for TCDD forwhich the highest concentration tested was 3.16 £ 10¡7 M.

Statistical analysis

Each experiment was performed in duplicate in three inde-pendent experiments. Data analysis was performed byusing a Student’s t test. This indicated that an inductionabove control level of 20 and 50% for the CEC and P450-glo assay, respectively, was statistical signiWcant. For theassessment of this threshold 5 independent experimentswere performed with 3-methylcholanthrene, indirubin, andindigo (data not shown). Since compounds with an induc-tion factor equal to or greater than 2-fold are deWned asinducers this is based on statistically signiWcant diVerences.Likewise Wve independent competition assays were carriedout with furafylline and ketoconazole. The Student’s t testshowed that an inhibition of 20% was statistically signiW-cant. Therefore the IC50 values are based on statisticallysigniWcant diVerences.

Results

Cytochrome P450 1A induction measured with CEC

HepG2 and H4IIE cells were exposed for 24 h to a dose-range of 119 diVerent compounds. Thereafter the CYP1Aactivity was measured with CEC. Exposure to 95 of thesecompounds did not aVect CYP1A activity (Table 1). How-ever, 24 compounds caused an induction of the CYP1Aactivity in either HepG2, H4IIE, or both cell lines. Thesecompounds are listed in Table 2 that shows also the EC50values and induction factors (ratio of treated cells:0.1%DMSO control). Dose response curves are presented inFig. 1.

The results clearly show that there was a diVerencebetween CYP1A induction in the human HepG2 and ratH4IIE cell line. More compounds caused CYP1A inductionin H4IIE cells and most EC50 values were lower than inHepG2 cells. TCDD was as expected the most potentCYP1A inducer in both H4IIE and HepG2 cells. The EC50value was 1.35 £ 10¡9 M in H4IIE cells and 36-timeshigher in HepG2 cells. Furthermore benzo[a]pyrene(B[a]P), �-naphthoXavone (BNF), dihydroergotamine mes-ylate (DHE), 2,4-dinitrophenol, ellipticin, Xutamide,indigo, indirubin, 3-methylcholanthrene (3MC,) Org C,Org D, PCB 77, PCB 105, PCB 114, PCB 118, PCB 123,PCB 126, PCB 156, PCB 157, and PCB 167 induced theCYP1A activity in the H4IIE cell line. Of these 20 com-pounds B[a]P, BNF, DHE, indigo, indirubin, 3MC, Org C,

PCB 77, PCB 114, PCB 123, PCB 126, and PCB 167 alsoinduced the CYP1A activity in HepG2 cells. Menadione,Org A, and Org B were HepG2 speciWc CYP1A inducers.Moreover, Org C was more potent in HepG2 than in H4IIEcells.

Cytochrome P450 1A1 induction measured with Luciferin-CEE

Luc-CEE was used to measure the CYP1A1 induction inHepG2 and H4IIE cells. The same compounds except thefour organic compounds were tested. Results were compa-rable with the results measured with CEC. The same com-pounds with exception of 2,4-dinitrophenol causedCYP1A1 induction in either HepG2, H4IIE, or both celllines (Table 2). Although not statistically signiWcant, 2,4-dinitrophenol showed a tendency to induction (1.8-fold) inH4IIE cells. The EC50 values of the CEC and Luc-CEEassay were almost similar. Nevertheless, the induction fac-tors were for most compounds higher in the Luc-CEEassay.

Cytochrome P450 1A1 and 1A2 competition

CYP1A1 and 1A2 competition in human and rat super-somes was measured for 115 compound, 4 compoundswere skipped from analysis i.e. Org A, B, C, and D. InFig. 2 the dose related competition for human and ratCYP1A1 and 1A2 is demonstrated for a representative setof 8 compounds, i.e. B[a]P, ellipticine, furafylline, indigo,indirubin, ketoconazole, nitrofurantoin, and 4-NQO. TheeYcacies (EFF) and IC50 values are shown in Table 3.DiVerences were observed between CYP1A1 and 1A2competition in human and rat supersomes. Of the 115tested compounds, 36 compounds inhibited humanCYP1A1 activity for 50% or more. Human competition forCYP1A1 was found speciWc for 12 compounds, being �-naphthoXavone, cisplatin, dehydroergotamine mesylate,dopamine HCl, 17�-estradiol, 4-hydroxytamoxifen, nosca-pine HCl, papaverine, quinidine, quinidine sulfate, reser-pine and RU 58668.. The remaining 24 compounds alsoinhibited rat CYP1A1. Furthermore, rat speciWc competi-tion for CYP1A1 was found for ten compounds.

Similar diVerences were observed for CYP1A2. Compe-tition was found for 37 compounds of which 14 and 3 com-pounds showed speciWc competition for human and ratCYP1A2, respectively.

A x:y plot for the IC50 values of human vs. rat competi-tion for both CYP1A1 and 1A2 is shown in Fig. 3. Thenumbers in the Wgure represent the compounds in Table 3.The solid line in Fig. 3 is the line of identity (x = y). Theoutlier hydralazine HCl is not shown in the CYP1A1 plot.

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As can be seen in Fig. 3 there are besides the human andrat speciWc CYP1A1 competitors, compounds that arealmost equally potent in both species (compounds justaround the line of identity). However, the potency is formost compounds higher in human supersomes as comparedto rat supersomes (compounds above the line of identity).The same is true for CYP1A2 as almost all competitorshave a lower IC50 value in human supersomes as comparedto rat supersomes.

Discussion

The present study focused on CYP1A induction, (non-)competitive inhibition and species diVerences between

humans and rats. Strong AhR activation leads to a series oftoxic eVects and consequently it might be useful for thepharmaceutical industry to screen for and deselect NCEsthat are strong CYP1A inducers (AhR activators) in theearly phase of drug development.

Primary cultures of hepatocytes can be used for CYP1Ainduction studies, however, donor variation and a highnumber of compounds in the early developmental phasemake the use of cell lines preferable. Two such cell linesmight be the human and rat hepatoma cell lines HepG2 andH4IIE. Previously we demonstrated that the eVects of AhRagonists in HepG2 on Cytochrome P450 enzymes are simi-lar to the eVects observed in primary human hepatocytes(Westerink and Schoonen 2007). In line with this it wasreported that HepG2 cells are a better model reXecting

Table 1 Compounds that do not activate CYP1A activity in the human HepG2 and rat H4IIE cell lines

Acetylsalicylic acid Ferrous sulfate Orphenadrine citrate

�-NaphthoXavone Fluorouracil Papaverine

2-amino-3-methyl-3H-imidazo-[4,5-f]-quinoline

Furafylline PCB 169

Aminophylline Gentamicin A PCB 189

Amiodarone HCl Hexachlorobutadiene Paracetamol

Antazoline mesylate 2,5-Hexanedione Perhexiline

Atropine sulfate Hydralazine HCl Perphenazine

Bishydroxycoumarin (Dicumarol) Hydrochlorothiazide Phenobarbital sodium

Bromobenzene Hydroxychloroquinone sulfate Phentolamine mesylate

Carbon tetrachloride (CCl4) 4-Hydroxytamoxifen Quinidine

Chlorpromazine HCl ICI 164.384 (anti-estrogen) Quinidine sulfate

Cis-Platin Imipramine HCl Raloxifen

CITCO Indomethacin Reserpine

Clozapine Iodoacetate Rifampicin

Colchicine Iproniazid HCl Rotenone

Cyclophosphamide Isoprenaline HCl RU 1881, Methyltrienolone

Cytarabine Ketoconazole RU 58668 (anti-estrogen)

Dacarbazine Labetalol Salicylamide

Dantrolene sodium L-DOPA Strychnine nitrate

Dehydroepiandrosterone (DHEA) Levonorgestrel Sulfamoxole

Dexamethasone Medroxyprogesterone acetate Sulphaphenazole

Diclofenac Melphalan Tacrine

Diethyldithio-carbamic acid Methadone HCl Tamoxifen

Diethylstilbestrol Methampyrone (Dipyrone) Tertiair-butyl-hydroperoxide

Digoxin Methotrexate Testosterone

2,7-DinitroXuorene 7�-methyltestosterone Tetracycline HCl

Dopamine HCl 17�-Methyltestosterone Tolcapone

Doxorubicin Naphazoline nitrate Tularik 901317

Erythromycin N-ethylmaleimide Uramustine

Estradiol-17� Nitrofurantoin

Ethacrinic acid Nitropyrene

Ethinylestradiol-17� 4-Nitrosoquinoline-1-oxide (4-NQO)

Ethionine Noscapine HCl

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CYP1A induction in human hepatocytes than hepatocytesfrom Rhesus monkeys and Sprague–Dawley rats (Silk-worth et al. 2005). Others found that the H4IIE cell line is agood model to study CYP1A induction reXecting eVects inprimary rat hepatocytes (Zeiger et al. 2001). Thus it seemsthat HepG2 and H4IIE cells are good cell lines to studyhuman and rat CYP1A induction, respectively.

For induction studies the Xuorogenic substrate CEC wasused. Fluorescence detection of the deethylation of CECwas reported to be 50 up to 100 times more sensitive thanthat of ethoxyresoruWn, primarily because of the faster turn-over rate of CEC (White 1988). CEC is not speciWc for aCYP1A isoform, it reacts with both human and ratCYP1A1 and 1A2 with a preference for CYP1A1 (Stresseret al. 2002). Besides CEC, the luminogenic substrate Luc-CEE was used for measuring CYP1A induction. The resultswere quite similar to results observed with CEC. Therefore

Luc-CEE is a good alternative for the detection of CYP1Ainducers. An advance of this substrate might be it is speci-Wcity for human and rat CYP1A1 (Promega). There is alsoa CYP1A2 speciWc luminogenic substrate (Luciferin-BE)available. However, in the present study this substrate wasnot used because it reacts only with human CYP1A2 andshows no reactivity for rat CYP1A2 (data not shown).

Of the 119 compounds that were tested 24 compoundswere able to induce the CYP1A activity in either humanHepG2, rat H4IIE, or both cell lines. Pronounced diVer-ences were observed between induction in the cells ofhuman and rat origin. Zeiger et al. (2001) reported similardiVerences. In their study HepG2 and H4IIE cells were alsoexposed to the dioxin-like PCBs 77, 105, 114, 118, 123,126, 156, 157, 167, 169, and 189. In H4IIE cells, theyfound in concordance with the present study induction afterexposure to PCB 77, 105, 114, 118, 123, 126, 156, 157, and

Table 2 Cytochrome P450 1A induction measured with CEC in human HepG2 and rat H4IIE cells

Compounds indicated in bold and italic show rat and human speciWc CYP1A activation, respectively. The EC50 values and induction factors (IF)are shown

B[a]P benzo[a]pyrene; BNF �-naphthoXavone; DHE dehydroergotamine mesylate; 3MC 3-methylcholanthrene; PCB polychlorobiphenyl; TCDD2,3,7,8-tetrachlorodibenzo-p-dioxin

Compound HepG2 H4IIE

CEC Luc-CEE CEC Luc-CEE

EC50(M) IF EC50(M) IF EC50(M) IF EC50(M) IF

B[a]P 3.73 £ 10¡6 10.2 2.00 £ 10¡6 2.8 2.08 £ 10¡8 16.7 8.00 £ 10¡8 33.2

BNF 1.22 £ 10¡5 20.3 3.16 £ 10¡6 9.2 2.95 £ 10¡8 24.6 3.16 £ 10¡8 52.9

DHE 1.21 £ 10¡5 7.9 6.09 £ 10¡6 11 3.35 £ 10¡6 2.43 1.00 £ 10¡5 6.5

Dinitrophenol >1.00 £ 10¡4 >1.00 £ 10¡4 2.59 £ 10¡6 3.79 >1.00 £ 10¡4

Ellipticin >1.00 £ 10¡4 >1.00 £ 10¡4 1.00 £ 10¡8 4.6 1.00 £ 10¡8 5.8

Flutamide >1.00 £ 10¡4 >1.00 £ 10¡4 7.89 £ 10¡7 13.1 5.00 £ 10¡7 25.7

Indigo 5.79 £ 10¡6 49.7 3.85 £ 10¡6 116 8.52 £ 10¡9 25.3 3.16 £ 10¡8 63.0

Indirubin 1.73 £ 10¡7 93 1.27 £ 10¡7 368 4.91 £ 10¡8 29 2.00 £ 10¡7 68.3

Menadione 1.29 £ 10¡5 16.9 1.00 £ 10¡5 20.4 >1.00 £ 10¡4 >1.00 £ 10¡4

3MC 6.48 £ 10¡7 62.3 5.22 £ 10¡7 222 4.45 £ 10¡8 24.4 3.16 £ 10¡8 69.5

Org A 2.00 £ 10¡6 9.94 ND >1.00 £ 10¡4 ND

Org B 2.00 £ 10¡6 14.0 ND >1.00 £ 10¡4 ND

Org C 2.00 £ 10¡6 22.6 ND 3.16 £ 10¡5 2.34 ND

Org D >1.00 £ 10¡4 ND 2.00 £ 10¡7 9.50 ND

PCB 77 8.27 £ 10¡6 7.9 2.56 £ 10¡6 25.5 4.35 £ 10¡8 17.5 7.00 £ 10¡8 25.5

PCB 105 >1.00 £ 10¡4 >1.00 £ 10¡4 >1.00 £ 10¡4 11.5 >1.00 £ 10¡4 9.6

PCB 114 >1.00 £ 10¡4 32.4 >1.00 £ 10¡4 29.8 1.30 £ 10¡6 13.6 2.35 £ 10¡6 15.6

PCB 118 >1.00 £ 10¡4 >1.00 £ 10¡4 >1.00 £ 10¡4 8.9 >1.00 £ 10¡4 13.6

PCB 123 >1.00 £ 10¡4 2.2 >1.00 £ 10¡4 3.5 >1.00 £ 10¡4 2.4 >1.00 £ 10¡4 3.5

PCB 126 1.65 £ 10¡6 50.6 2.55 £ 10¡6 65.4 1.00 £ 10¡9 21.6 2.00 £ 10¡9 23.6

PCB 156 >1.00 £ 10¡4 >1.00 £ 10¡4 5.37 £ 10¡6 16.0 3.09 £ 10¡6 24

PCB 157 >1.00 £ 10¡4 >1.00 £ 10¡4 8.15 £ 10¡6 19.7 7.56 £ 10¡6 40.6

PCB 167 >1.00 £ 10¡4 52.6 >1.00 £ 10¡4 45.5 3.55 £ 10¡8 15.3 3.02 £ 10¡8 30.5

TCDD 4.88 £ 10¡8 61.8 4.00 £ 10¡8 250 1.35 £ 10¡9 22.3 1.25 £ 10¡9 78.5

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no induction after exposure to PCB 189. However, in con-tradiction we did not Wnd induction with PCB 169 in H4IIEcells. Induction in HepG2 cells was found for less com-pounds at higher doses. In both studies induction was mea-sured after exposure to PCB 77, 114, and 126. Incontradiction with Zeiger et al. (2001) we found induction

by PCB 123 and 167. The results suggest that the rat cellline H4IIE is more sensitive to detect CYP1A inducers thanthe HepG2 cell line.

The high sensitivity of rat for CYP1A inducers was alsoreported by Silkworth et al. (2005), who found CYP1Ainduction at much lower concentrations after exposure to

Fig. 1 Induction of CYP1A activity measured with CEC in humanHepG2 and rat H4IIE cells after 24 h exposure to the compounds. Ofthe 119 compounds, 13 induced CYP1A activity in both HepG2 andH4IIE cells (a, b), 8 showed H4IIE speciWc induction (c, d), and 3compounds showed HepG2 speciWc induction (e, f). Results are pre-

sented as the mean of three independent measurements. The SD is notshown as it interferes with the reading of the marker spots. However,SD is < 5% for all data points. Abbreviations: BNF �-naphthoXavone;DHE dehydroergotamine mesylate; 3MC 3-methylcholanthrene;TCDD 2,3,7,8-tetrachlorodibenzo-p-dioxin

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PCB 126 and Aroclor 1254 in rat than human cells. A studyby Aluru et al. (2005) also revealed species diVerences.While in humans �-naphthoXavone (ANF) is an AhR antag-onist in rainbow trout hepatocytes it was a partial agonist.

Although mono-ortho PCBs such as PCB 114, PCB 126,and PCB 167 are considered as weak AhR agonists (Peterset al. 2006), they showed a high potency in the presentstudy. Peters et al. (2006) showed that after puriWcation ofmono-ortho PCBs with active charcoal, mono-ortho, PCBsshow only a low potency in an AhR-EGFP reporter assay inmouse Hepa1c1c7 and rat H4IIE cells. The purity of thePCBs in the present study was 99%. Therefore we do notexpect large eVects of impurities on CYP1A induction,however we cannot rule out that contaminations with AhRagonists of high activity had an eVect on the activity.

The importance of deselecting CYP1A inducers wasstressed by Org D. This rat speciWc CYP1A inducer causednon-genotoxic carcinogenicity in in vivo rat studies(unpublished in-house data).

We observed that Org A and B were HepG2 speciWcCYP1A inducers and Org C was much more potent in theHepG2 cell line. Menadione was also active in HepG2 cellsand not in H4IIE cells. Apparently there are CYP1A induc-ers that are more potent in humans than rats. In line withthis, in human, but not in mouse and in rat, the anti-ulcerdrug omeprazole has been reported to induce CYP1A2

(Diaz et al. 1990; Lu and Li 2001; Roymans et al. 2005).Overall these observations make screening in cell linesfrom both rat and human a useful strategy.

Recently Sonneveld et al. (2007) showed that glucocorti-coids enhance the induction of CYP1A1 and other AhR tar-get genes in rat H4IIE cells but not in human cells. In thepresent study we did not observe an eVect of dexametha-sone in H4IIE cells also not when charcoal treated serumwas used (data not shown).

There are also other methods available for screeningCYP1A inducers and/or AhR activators. Two often usedmethods are the CALUX bioassay (Murk et al. 1996) andthe use of 101L cells (Postlind et al. 1993). In the CALUXbioassay rat H4IIE and mouse Hepa1c1c7 cells containinga luciferase gene under control of dioxin responsive ele-ments are used. A disadvantage of this assay is that rodentcells are used which not always reXect eVects in humans.The 101L cell line is a stable cell line derived from HepG2cells which contain a human CYP1A1 luciferase reporter.An advantage of this system might be that substrate inhibi-tion of CYP1A1 does not play a role. However, in our assaywe included a washing step after compound incubationwhich reduced substrate inhibition of CEC.

Ellipticine showed CYP1A induction in the H4IIE cellline. In HepG2 cells induction was just below two fold anddirectly after the increase in activity the signal dropped

Fig. 2 CYP1A1 and 1A2 competition in supersomes from human andrat by benzo[a]pyrene (B[a]P), ellipticine, furafylline, indigo, indiru-bin, ketoconazole, nitrofurantoin, and 4-nitrosoquinolineoxide-1-ox-ide (4-NQO). The activity of the 0.1% DMSO control was set at 100%.

Results are presented as the mean of three independent measurements.The SD is not shown as it interferes with the reading of the markerspots. However, SD is < 5% for all data points. Abbreviations: B[a]Pbenzo[a]pyrene; 4-NQO 4-nitrosoquinolineoxide-1-oxide

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Table 3 Compounds that show CYP1A1 and 1A2 competition in human and rat supersomes

No. Compound Human Rat

CYP1A1 inhibition CYP1A2 inhibition CYP1A1 inhibition CYP1A2 inhibition

EFF (%) IC50 (M) EFF (%) IC50 (M) EFF (%) IC50 (M) EFF (%) IC50 (M)

1 �-NaphthoXavone 111 3.47 £ 10¡6104 2.43 £ 10¡6

101 1.00 £ 10¡598 3.60E-06

2 2-Amino-quinoline 67 5.75 £ 10¡5 95 2.64 £ 10¡6 53 4.71 £ 10¡5 56 3.11E-04

3 B[a]P 33 >1.00 £ 10¡4 61 1.50 £ 10¡6 23 >1.00 £ 10¡4 51 2.30 £ 10¡5

4 BNF 91 2.01 £ 10¡6 83 8.07 £ 10¡7 11 >1.00 £ 10¡4 108 3.80E-06

5 Chlorpromazine HCl 74 1.81 £ 10¡5 72 8.56 £ 10¡6 98 2.50 £ 10¡6 31 >1.00 £ 10¡4

6 Cisplatin 72 3.02 £ 10¡5 82 1.29 £ 10¡5 43 >1.00 £ 10¡4 93 1.96 £ 10¡5

7 Clozapine ¡3 >1.00 £ 10¡4 52 1.00 £ 10¡4 107 1.88 £ 10¡6 47 1.15E-04

8 Dantrolene sodium 91 1.34 £ 10¡5 60 7.74 £ 10¡5 75 3.00 £ 10¡5 39 >1.00 £ 10¡4

9 DETC 37 >1.00 £ 10¡4 71 5.73 £ 10¡5 12 >1.00 £ 10¡4 87 3.00 £ 10¡5

10 DHE 66 4.25 £ 10¡5 ¡6 >1.00 £ 10¡4 19 >1.00 £ 10¡4 ¡29 >1.00 £ 10¡4

11 2,7-DinitroXuorene 64 6.61 £ 10¡5 86 3.85 £ 10¡7 53 2.75 £ 10¡5 61 2.62 £ 10¡5

12 DNP 69 8.01 £ 10¡6 81 4.20 £ 10¡6 49 2.27 £ 10¡5 51 5.82 £ 10¡5

13 Dopamine HCl 84 3.13 £ 10¡5 8 >1.00 £ 10¡4 27 >1.00 £ 10¡4 2 >1.00 £ 10¡4

14 Doxorubicin 73 3.80 £ 10¡5 67 4.29 £ 10¡5 90 6.16 £ 10¡6 69 4.89 £ 10¡5

15 Ellipticine 87 1.08 £ 10¡6 98 4.71 £ 10¡8 104 1.42 £ 10¡6 100 3.23 £ 10¡7

16 Estradiol-17� 73 2.75 £ 10¡5 30 >1.00 £ 10¡4 4 >1.00 £ 10¡4 ¡2 >1.00 £ 10¡4

17 Ethacrinic acid 14 >1.00 £ 10¡4 20 >1.00 £ 10¡4 53 7.01 £ 10¡5 9 >1.00 £ 10¡4

18 Flutamide 30 >1.00 £ 10¡4 71 8.97 £ 10¡6 ¡16 >1.00 £ 10¡4 9 >1.00 £ 10¡4

19 Furafylline 43 >1.00 £ 10¡4 100 8.01 £ 10¡7 77 3.01 £ 10¡5 56 3.68 £ 10¡5

20 Hexachlorobutadiene 83 1.75 £ 10¡6 5 >1.00 £ 10¡4 59 1.89 £ 10¡5 5 >1.00 £ 10¡4

21 Hydralazine HCl 69 5.99 £ 10¡5 58 3.35 £ 10¡5 66 5.80 £ 10¡8 55 3.05 £ 10¡5

22 4-Hydroxytamoxifen 81 3.34 £ 10¡5 ¡14 >1.00 £ 10¡4 6 >1.00 £ 10¡4 17 >1.00 £ 10¡4

23 Indigo 71 2.83 £ 10¡5 43 >1.00 £ 10¡4 53 7.00 £ 10¡5 36 >1.00 £ 10¡4

24 Indirubin 65 8.90 £ 10¡6 11 >1.00 £ 10¡4 99 3.53 £ 10¡6 46 >1.00 £ 10¡4

25 Isoprenaline HCl 10 >1.00 £ 10¡4 64 9.43 £ 10¡5 7 >1.00 £ 10¡4 14 >1.00 £ 10¡4

26 Ketoconazole 100 2.00 £ 10¡7 45 >1.00 £ 10¡4 100 3.00 £ 10¡6 ¡14 >1.00 £ 10¡4

27 L-DOPA 18 >1.00 £ 10¡4 ¡62 >1.00 £ 10¡4 56 1.31 £ 10¡4 112 1.14 £ 10¡5

28 Melphalan 66 4.68 £ 10¡5 53 7.41 £ 10¡5 63 6.84 £ 10¡5 59 7.51 £ 10¡5

29 Menadione 85 1.02 £ 10¡5 102 2.43 £ 10¡7 80 1.52 £ 10¡5 93 2.88 £ 10¡7

30 Methotrexate 18 >1.00 £ 10¡4 88 2.70 £ 10¡5 4 >1.00 £ 10¡4 28 >1.00 £ 10¡4

31 3-Methylcholanthrene 25 >1.00 £ 10¡4 74 4.79 £ 10¡6 8 >1.00 £ 10¡4 57 1.10 £ 10¡5

32 7�-Methyltestosterone 64 5.47 £ 10¡5 6 >1.00 £ 10¡4 116 1.74 £ 10¡7 10 >1.00 £ 10¡4

33 Nitrofurantoin 103 1.11 £ 10¡5 84 2.01 £ 10¡5 55 3.48 £ 10¡4 2 >1.00 £ 10¡4

34 Nitropyrene 83 1.63 £ 10¡6 92 1.00 £ 10¡7 99 3.58 £ 10¡7 108 1.00 £ 10¡7

35 4-NQO 41 >1.00 £ 10¡4 86 2.41 £ 10¡5 19 >1.00 £ 10¡4 27 >1.00 £ 10¡4

36 Noscapine HCl 65 4.04 £ 10¡5 1 >1.00 £ 10¡4 12 >1.00 £ 10¡4 5 >1.00 £ 10¡4

37 Orphenadrine citrate 33 >1.00 £ 10¡4 40 >1.00 £ 10¡4 15 >1.00 £ 10¡4 53 1.03 £ 10¡4

38 Papaverine 92 9.15 £ 10¡6 71 3.06 £ 10¡5 38 >1.00 £ 10¡4 18 >1.00 £ 10¡4

39 PCB 105 11 >1.00 £ 10¡4 42 7.59 £ 10¡5 57 5.18 £ 10¡5 22 >1.00 £ 10¡4

40 PCB 114 ¡8 >1.00 £ 10¡4 69 2.21 £ 10¡5 95 1.19 £ 10¡5 34 >1.00 £ 10¡4

41 PCB 118 22 >1.00 £ 10¡4 54 2.54 £ 10¡5 86 2.96 £ 10¡6 24 >1.00 £ 10¡4

42 PCB 123 13 >1.00 £ 10¡4 54 3.60 £ 10¡5 5 >1.00 £ 10¡4 8 >1.00 £ 10¡4

43 PCB 156 11 >1.00 £ 10¡4 39 >1.00 £ 10¡4 101 6.42 £ 10¡6 5 >1.00 £ 10¡4

44 PCB 167 28 >1.00 £ 10¡4 53 3.22 £ 10¡5 72 4.32 £ 10¡5 45 >1.00 £ 10¡4

45 Paracetamol 5 >1.00 £ 10¡4 ¡4 >1.00 £ 10¡4 62 1.32 £ 10¡4 57 5.10 £ 10¡5

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Table 3 continued

The eYcacy (EFF) and IC50 values are shown. Values are marked in bold when EFF > 50% (P < 0.05)

B[a]P benzo[a]pyrene; BNF �-naphthoXavone; DETC diethyldithio-carbamic acid; DNP 2,4-dinitrophenol; 3MC 3-methylcholanthrene; 4-NQO4-nitrosoquinolineoxide-1-oxide

No. Compound Human Rat

CYP1A1 inhibition CYP1A2 inhibition CYP1A1 inhibition CYP1A2 inhibition

EFF (%) IC50 (M) EFF (%) IC50 (M) EFF (%) IC50 (M) EFF (%) IC50 (M)

46 Perhexiline 49 >1.00 £ 10¡4 65 6.39 £ 10¡5 22 >1.00 £ 10¡4 16 >1.00 £ 10¡4

47 Perphenazine 101 4.52 £ 10¡6 1 >1.00 £ 10¡4 82 1.02 £ 10¡5 1 >1.00 £ 10¡4

48 Quinidine 76 1.87 £ 10¡5 ¡2 >1.00 £ 10¡4 27 >1.00 £ 10¡4 30 >1.00 £ 10¡4

49 Quinidine sulfate 89 1.10 £ 10¡5 24 >1.00 £ 10¡4 20 >1.00 £ 10¡4 5 >1.00 £ 10¡4

50 Raloxifen 100 3.80 £ 10¡6 43 >1.00 £ 10¡4 80 1.56 £ 10¡5 23 >1.00 £ 10¡4

51 Reserpine 59 1.73 £ 10¡5 47 >1.00 £ 10¡4 23 >1.00 £ 10¡4 ¡5 >1.00 £ 10¡4

52 Rifampicin 74 2.08 £ 10¡5 90 6.22 £ 10¡6 60 4.18 £ 10¡5 58 4.68 £ 10¡5

53 RU 58668 102 5.70 £ 10¡5 2 >1.00 £ 10¡4 ¡15 >1.00 £ 10¡4 39 >1.00 £ 10¡4

54 Tacrine 115 6.32 £ 10¡7 108 7.79 £ 10¡7 131 2.01 £ 10¡6 50 1.30E-04

55 Testosterone 64 7.47 £ 10¡5 ¡6 >1.00 £ 10¡4 45 2.40 £ 10¡4 ¡4 >1.00 £ 10¡4

56 Tolcapone 91 2.53 £ 10¡5 92 9.15 £ 10¡6 110 2.17 £ 10¡5 92 2.11 £ 10¡5

Fig. 3 Comparison of the IC50 values of human versus rat for CYP1A1 and 1A2 competition. The numbers represent the com-pounds in Table 3. The solid line is the line of identity (x = y)

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sharply again. The problem with the detection of ellipticinein the HepG2 cell line was cytotoxicity. At the concentra-tion where this compound starts to activate the AhR itshows also a strong cytotoxic eVect (Schoonen et al. 2005a,b). All other compounds in the present study were tested forcytotoxicity by using the glutathione depletion and calcein-AM assay (Schoonen et al. 2005a, b). These resultsrevealed (data not shown) that compounds not showingCYP1A induction in HepG2 cells but showing CYP1Ainduction in H4IIE cells were not missed in HepG2 cellsbecause of cytotoxicity.

CYP1A induction can easily be used in the early devel-opmental phase to detect compounds that might showdioxin-like toxicity. However, care should be taken to dese-lect such compounds directly as CYP1A activation does notnecessarily mean dioxin-like toxicity. There are for exam-ple marketed therapeutics like omeprazole, leXunomide,Xutamide, and nimodipine which are safely used but havebeen proven to be AhR agonists (Hu et al. 2007). Further-more, AhR agonists like indirubin and meisoindigo havebeen shown to be eVective in the treatment of chronic mye-logenous leukemia (Xiao et al. 2002). Hu et al. (2007) alsodemonstrated that induction of CYP1A1 is a non-speciWcmarker of direct AhR aYnity. In the present study we mea-sured CYP1A induction and not AhR activation. Thereforealso compounds are detected that alter the CYP1A expres-sion by pathways in addition to those mediated by the AhR.Induction of CYP1A has been reported after oxidativestress (Delescluse et al. 2001; Hazinski et al. 1995). Thesecompounds do not have the side eVects reported for someAhR activators, however, an adverse side eVect of thesecompounds might be the eVect of increased CYP1A activ-ity on the eYcacy of the compound itself or the inductionmight result in drug–drug interactions.

The potentially toxic eVect of CYP1A inducers can beconWrmed by using in vitro or in vivo transcriptional proWl-ing. By studying the change of a broad set of genes dioxin-like toxicity might be predicted more precisely. Conse-quently a compound might be deselected or selected.

Thus summarized, CYP1A induction can be used as aprescreening tool to detect compounds that might showdioxin-like toxicity. However, further studies are needed toconWrm this dioxin-like toxicity. When during drug devel-opment equally potent compounds without CYP1A induc-tion are available, selection of these compounds might bepreferred to avoid possible safety problems.

Besides species diVerences between CYP1A1 induction,species diVerences between the AhR receptor of human andrat could lead to diVerentially regulated gene expression.Recently it has been shown by Flaveny et al. (2008) thatdiVerences between the transactivation domains of thehuman and mouse AhR results in diVerential recruitment ofco-activators. It is likely that this leads to a divergent regu-

lation of target genes. DiVerences in the recruitment of co-activators between the human and rat AhR receptor havenot been studied yet, however, similar diVerences as foundbetween human and mouse might exist.

CYP1A1 and 1A2 competition assays were performedby using human and rat supersomes. The same set of com-pounds with exception of four in-house NCEs was tested.Like for induction species diVerences were observed. Ofthe 115 compounds 46 showed CYP1A1 competition.Competition was human and rat speciWc for 12 and 10 com-pounds, respectively. CYP1A2 competition was observedfor 37 compounds of which 14 and 3 compounds showedhuman and rat speciWc inhibition, respectively. The similar-ity between the amino acid sequence of human and ratCYP1A1 is 79%, and of human and rat CYP1A2 73%. ThediVerence in amino acid sequences might account for thediVerences in competition. Other studies also reported spe-cies diVerences between CYP1A metabolism in humansand rats. Shinkyo et al. (2003) studied the metabolism ofTCDD and other polychlorinated dibenzo-p-dioxins inhuman and rat microsomes and found signiWcant speciesdiVerences. Bogaards et al. (2000) compared the CYPactivities towards marker CYP substrates for human, rat,rabbit, dog, and micropig microsomes. They found that innone of the tested species metabolism was similar to CYPmetabolism in man. With respect to CYP1A human metab-olism was most similar to mouse, followed by rabbit,micropig, rat and dog.

In summary, we used a medium/high-throughput assayin a 96 well format for detecting CYP1A inducers in thehuman HepG2 and rat H4IIE cell line. Moreover, CYP1A1and 1A2 competition assays were performed by usinghuman and rat supersomes in a 384 well high throughputassay. The induction and competition assays revealed forseveral compounds a real species diVerence betweenhuman and rat. Therefore, parallel screening in both speciesmight be a very useful strategy.

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