Toxicology in Vitro 24 (2010) 425–429

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Toxicology in Vitro

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Effects of dinuclear copper(II) complexes with 6-(benzylamino)purinederivatives on AhR and PXR dependent expression of cytochromes P450 CYP1A2and CYP3A4 genes in primary cultures of human hepatocytes

Zdenek Dvorák a,*, Radim Vrzal a, Pavel Štarha b, Alena Klanicová b, Zdenek Trávnícek b

a Department of Cell Biology and Genetics, Faculty of Science, Palacky University, Šlechtitelu 11, 783 71 Olomouc, Czech Republicb Department of Inorganic Chemistry, Faculty of Science, Palacky University, Tr. 17. listopadu 12, 771 46 Olomouc, Czech Republic

a r t i c l e i n f o

Article history:Received 23 July 2009Accepted 20 October 2009Available online 23 October 2009

Keywords:Human hepatocytesCytochrome P450Dinuclear copper(II) complexesGene induction

0887-2333/$ - see front matter � 2009 Elsevier Ltd. Adoi:10.1016/j.tiv.2009.10.012

Abbreviations: AhR, Aryl hydrocarbon receptor; Creceptor; CYP, Cytochrome P450; DRE, Dioxin-responhepatoma cells; L1, 6-[(2-methoxybenzyl)amino]purinzyl)amino]purine; L3, 6-[(4-methoxybenzyl)amino]pzyl)amino]purine; L5, 6-[(4-fluorobenzyl)amino]purinTCDD, 2,3,7,8- tetrachlorodibenzo-p-dioxin; XRE, Xen

* Corresponding author. Tel.: +420 58 5634903; faxE-mail address: moulin@email.cz (Z. Dvorák).

a b s t r a c t

A series of dinuclear copper(II) complexes of the compositions [Cu2(l-Ln)2(l-Cl)2Cl2] (1, 2), [Cu2-(l-Ln)4Cl2]Cl2�2H2O (3, 4) and [Cu2(l-Ln)4(ClO4)2](ClO4)2�xSolv (5, 6; xSolv = 4MeOH for 5 and 2EtOHfor 6), involving 6-(benzylamino)purine derivatives (Ln), have been evaluated with the aim to determinetheir possible drug interactions and their capability to induce the expression of major drug-metabolizingcytochromes P450. The above-mentioned complexes have been chosen based on the fact that substantialboth in vitro (cytotoxicity, SOD-mimic) and in vivo (antidiabetic) biological activity has been found forthem. As models, primary cultures of human hepatocytes and human hepatoma cells HepG2 transientlytransfected with a plasmid containing dioxin-responsive element fused to the luciferase reporter gene(DRE-LUC) have been chosen. It has been found that the tested complexes 1–6 did not significantly inducethe expression of CYP1A2 and CYP3A4 mRNAs in the concentration range of 0.1–10.0 lM, in three differ-ent primary human hepatocyte cultures after 24 h of the treatment. On the other hand, the model induc-ers, i.e. 2,3,7,8- tetrachlorodibenzo-p-dioxin (TCDD) and rifampicin, significantly increased the levels ofCYP1A2 and CYP3A4 mRNAs in all cultures. In addition, compounds 1–6 did not transactivate DRE-LUCin transiently transfected HepG2, while TCDD strongly induced luciferase activity after 24 h of incubation.Based on the obtained results, it may be concluded that the studied dinuclear copper(II) complexes 1–6possess very low toxicological potential to cause drug interactions in terms of transcriptional activationof the major human cytochromes P450.

� 2009 Elsevier Ltd. All rights reserved.

1. Introduction

A series of 21 dinuclear copper(II) complexes involving benzyl-substituted derivatives of 6-(benzylamino)purine, (Ln), of generalcompositions [Cu2(l-Ln)4(ClO4)2](ClO4)2, [Cu2(l-Ln)4Cl2]Cl2,[Cu2(l-Ln)2(l-Cl)2Cl2], [Cu2(l-Ln)2(l-Cl)2(Ln)2Cl2], [Cu2(l-Cl)2Cl2

(Ln)2] and ½Cu2ðl-L�n Þ2ðl-ClÞ2ðH2OÞ2� were recently prepared andfully characterized by various physical methods. Moreover, theywere tested for their in vitro cytotoxicity (5 human cancer celllines), in vitro superoxide dismutase-mimic (SOD-mimic) activity,and in vivo cryoprotective effect against alloxan-induced diabetes

ll rights reserved.

AR, Constitutive androstanesive element; HepG2, Humane; L2, 6-[(2,3-dimethoxyben-urine; L4, 6-[(2-chloroben-

e; PXR, Pregnane X receptor;obiotic responsive element.: +420 58 5634905.

(Trávnícek et al., 2001; Malon et al., 2001, 2002; Klanicová et al.,submitted for publication; Štarha et al., 2009). The above-men-tioned complexes demonstrated significant both in vitro andin vivo biological activity, e.g. the IC50 value of 0.253 lM for[Cu2(l-L1)2(l-Cl)2Cl2] (1), where L1 = 6-[(2-methoxybenzyl)-amino]purine, exceeded in vitro SOD-mimic activity of the nativebovine Cu-, Zn-SOD enzyme (IC50 = 0.480 lM) (Štarha et al.,2009). In addition, the complexes [Cu2(l-L2)2(l-Cl)2Cl2] (2) and[Cu2(l-L3)4Cl2]Cl2�2H2O (3) {L2 = 6-[(2,3-dimethoxybenzyl)amino]-purine and L3 = 6-[(4-methoxybenzyl)amino]purine} successfullyprotected the mice against the alloxan toxic effect (Štarha et al.,2009). The best results of in vitro cytotoxic activity were obtainedfor the [Cu2(l-L4)2(l-Cl)2(L4)2Cl2]�2H2O complex {L4 = 6-[(2-chlo-robenzyl)amino]purine}, with IC50 = 8–31 lM (Malon et al., 2001;Trávnícek et al., 2001).

Due to the mentioned positive biological properties of the dis-cussed copper(II) complexes, we decided to study their possibledrug interactions. Namely, dinuclear copper(II) complexes havingthe compositions of [Cu2(l-L1)2(l-Cl)2Cl2] (1), [Cu2(l-L2)2(l-Cl)2

426 Z. Dvorák et al. / Toxicology in Vitro 24 (2010) 425–429

Cl2] (2), [Cu2(l-L3)4Cl2]Cl2�2H2O (3), [Cu2(l-L1)4Cl2]Cl2�2H2O (4),[Cu2(l-L4)4(ClO4)2](ClO4)2�4MeOH (5) and [Cu2(l-L5)4(ClO4)2]-(ClO4)2�2EtOH (6) were chosen for this study (Fig. 1); L5 = 6-[(4-fluorobenzyl)amino]purine.

A substantial number of pharmacokinetic drug interactions re-sults from the induction of the gene expression of cytochromeP450 (CYP) enzymes (Maurel 1996; Pavek and Dvorák, 2008). Themolecular mechanism of cytochrome P450 induction involves theactivation of several xenoreceptors, including the pregnane Xreceptor (PXR, NR1I2), the constitutive androstane receptor (CAR,NR1I3) and the aryl hydrocarbon receptor (AhR) (Willson and Klie-wer, 2002; Denison and Nagy, 2003; Pascussi et al., 2008). Theexpression of the CYP1 gene family (1A1, 1A2, 1B1) is transcrip-tionally regulated by AhR while the CYP2 (2A6, 2B6, 2C8/9) andCYP3 (3A4) families are regulated by the PXR and CAR receptors(Denison and Nagy, 2003). Therefore, new drugs are tested for theircapability to activate xenoreceptors. Surprisingly, there are stillseveral drugs used in the clinic, which are well known P450 induc-ers, the examples being omeprazole, carbamazepine, phenytoine,valproic acid or barbiturates (Pascussi et al., 2005; Gerbal-Chaloinet al., 2006; Cerveny et al., 2007).

As can be concluded from the above-mentioned facts, the com-plexes 1–6 have been chosen for the study in connection withpromising results which have been obtained during their bothin vitro and in vivo biological activity tests. In the present study,we have determined the effects of dinuclear copper(II) complexes1–6 on the expression of CYP1A2 and CYP3A4 mRNAs, as the rep-resentative target genes for the AhR and PXR/CAR receptors,respectively. We used the in vitro model of primary cultures of hu-man hepatocytes, which is the most respected model for such astudy (Maurel, 1996; Ferrini et al., 1997). We found that the testedcompounds did not induce CYP1A2 and CYP3A4 mRNA expressionin primary human hepatocyte cultures prepared from three differ-ent liver tissue donors. Additionally, we showed that the testedcompounds did not transactivate AhR, as revealed by gene reporterassays using HepG2 cells transiently transfected with a dioxin-responsive element fused to the luciferase reporter gene (DRE-LUC). In summary, the obtained data are promising for possible

Fig. 1. Proposed structures of the tested dinuclear copper(II) complexes (1–6).

therapeutic use of copper(II) complexes 1–6, in terms of their ef-fects on cytochrome P450 induction.

2. Materials and methods

2.1. Materials

Collagen-coated culture dishes were from BD Biosciences (LePont de Claix, France). 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD)was from Ultra Scientific (RI, USA). LightCycler FastStart DNAMasterPLUS SYBR Green I was from Roche Diagnostic Corporation(Meylan, France). Oligonucleotide primers used in RT-PCR reac-tions were from Invitrogen. Rifampicin (RIF) and dimethyl sulfox-ide (DMSO) were from Sigma–Aldrich (St. Quentin Fallavier,France). FuGENE 6 transfection reagent was obtained from Roche(Mannheim, Germany). The syntheses and characterizations ofthe copper(II) complexes [Cu2(l-L1)2(l-Cl)2Cl2] (1), [Cu2(l-L2)2

(l-Cl)2Cl2] (2), [Cu2(l-L3)4Cl2]Cl2�2H2O (3), [Cu2(l-L1)4Cl2]Cl2 �2H2O (4), [Cu2(l-L4)4(ClO4)2](ClO4)2�4MeOH (5) and [Cu2(l-L5)4-(ClO4)2](ClO4)2�2EtOH (6) were published elsewhere (Klanicováet al., submitted for publication; Štarha et al., 2009). All otherchemicals were of the highest quality commercially available.

2.2. Primary cultures of human hepatocytes

Hepatocytes were prepared from lobectomy segments, resectedfrom multiorgan donors. The tissue acquisition protocol was inaccordance with the requirements issued by a local ethical commis-sion in the Czech Republic. Human liver samples used in this studywere obtained from three patients: LH 28 (male, 48 years), LH 29(male, 74 years) and LH 31 (male, 28 years). Hepatocytes were iso-lated as previously described (Pichard-Garcia et al., 2002). Follow-ing the isolation, the cells were plated on collagen-coated culturedishes at the density of 1.4 � 105 cells/cm2. The culture mediumwas, as described previously (Isom et al., 1985), enriched for platingwith 2% fetal calf serum (v/v). The medium was exchanged for a ser-um-free medium the day after and the culture was allowed to sta-bilize for an additional 48 h. Thereafter, the cells were treated for24 h with 1, 2 and 3 (0.1 and 1 lM), 4 (1 and 5 lM), 5 and 6 (1and 10 lM), rifampicin (10 lM), 2,3,7,8-tetrachlorodibenzo-p-di-oxin (5 nM) and/or vehicle (DMSO; 0.1% v/v). The cultures weremaintained at 37 �C and 5% CO2 in a humidified incubator.

2.3. mRNA determination and quantitative reverse transcriptasepolymerase chain reaction (qRT-PCR)

Total RNA was isolated using TRIZOL Reagent (Invitrogen).cDNA was synthesized from 200 ng of total RNA using M-MLV Re-verse Transcriptase (Invitrogen) at 37 �C for 50 min in the presenceof random hexamers (Amersham Biosciences). One tenth was usedfor qRT-PCR amplification using the Light Cycler apparatus (RocheDiagnostic Corporation, Meylan, France). The levels of CYP1A2,CYP3A4 and GAPDH mRNAs were determined as we describedelsewhere (Pavek et al., 2007; Dvorák et al., 2008). All the experi-ments and measurements were performed in duplicates. The datawere normalized to glyceraldehyde-3-phosphate dehydrogenase(GAPDH) as a housekeeping gene and further processed by the del-ta–delta method assuming Pfapfl coefficient to be 2.

2.4. Gene reporter assays

2.4.1. HepG2 cellsHuman hepatoma cells HepG2 (ECACC No. 85011430) were cul-

tured in Dulbecco‘s modified Eagle‘s medium (DMEM) supple-mented with 10% FCS, 100 U/ml streptomycin, 100 lg/ml

Z. Dvorák et al. / Toxicology in Vitro 24 (2010) 425–429 427

penicillin, 4 mM L-glutamine, 1% non-essential amino acids, and1 mM sodium pyruvate. The cells were maintained at 37 �C and5% CO2 in a humidified incubator.

2.4.2. PlasmidspDRE-luc plasmid containing two inverted repeats of the XRE

(xenobiotic responsive element) of mouse cyp1a1 upstream ofthe thymidine kinase promoter and the luciferase reporter gene(Daujat et al., 1996; Backlund et al., 1997) was kindly providedby Dr. L. Poellinger (Karolinska Institute, Stockholm, Sweden).

2.4.3. Transient transfection and luciferase gene reporter assaysHepG2 cells were transiently transfected by lipofection

(FuGENE 6) with 300 ng/well of the reporter pDRE-luc in 24-wellplates, as we described elsewhere (Dvorák et al., 2008). After 16 hof stabilization, the cells were treated for 24 h with increasing con-centrations of 1–6 in the presence or absence of TCDD (5 nM). Afterthe treatments, the cells were lysed and luciferase activity wasmeasured and standardized per milligram of protein. Dose–

1.0 2.9 0.8 1.7 4.0 2.8 2.3 1.9 1.7 3.5 3.1 5.5 1.3

114.6

0

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DMSO

TCDD 5nM

(1) 1 µM

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µM

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µM

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µM

(6) 10

µM

(6) 1 µM

1.0 3.3 1.6 2.0 1.3 5.7 2.9 7.9 3.8 12.4 4.0 3.4 2.3

539.3

0100200300400500600700800

DMSO

TCDD5 nM

(1) 1 µM

(1) 0.1µM

(2) 1 µM

(2) 0.1

µM

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(3) 0.1µM

(4) 5

µM

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µM

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(5) 1 µM

(6) 10

µM

(6) 1 µM

1.0 1.0 0.5 0.7 0.9 1.2 0.7 2.3 1.2 2.4 1.4 0.8 0.7

124.5

020406080

100120140160

DMSO

TCDD5 nM

(1) 1 µM

(1) 0.1µM

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µM

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µM

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µM

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µM

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fold

ofco

ntro

lfo

ldof

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rol

fold

ofco

ntro

l

CYP1A2 mRNA

Human hepatocytes - culture LH 28

Human hepatocytes - culture LH 29

Human hepatocytes - culture LH 31

*

*

*

*

*

**

*

*

Fig. 2. Effects of the dinuclear copper(II) complexes (1–6) on CYP1A2 and CYP3A4 mRNtreated for 24 h with 1 (0.1 and 1 lM), 2 (0.1 and 1 lM), 3 (0.1 and 1 lM), 4 (1 and 5 lM),dioxin (5 nM) and/or vehicle (DMSO; 0.1% v/v). RT-PCR analyses of CYP1A2 (Panel A.)measurements and are expressed as fold induction over DMSO-treated cells. The data wetreated cells (p < 0.05).

response analyses using non-linear regression were performed tocalculate the half maximal effective concentration (EC50) and thehalf maximal inhibitory concentration (IC50) of the testedcompounds.

3. Results

3.1. Effects of copper(II) complexes 1–6 on CYP1A2 and CYP3A4 mRNAexpressions in primary cultures of human hepatocytes

We used primary cultures of human hepatocytes prepared fromthree different patients, as indicated in detail in the Section 2.Hepatocytes were treated for 24 h with 1 (0.1 and 1 lM), 2 (0.1and 1 lM), 3 (0.1 and 1 lM), 4 (1 and 5 lM), 5 (1 and 10 lM), 6(1 and 10 lM) and/or vehicle (DMSO; 0.1% v/v). In parallel, hepato-cytes were challenged with RIF (10 lM) and TCDD (5 nM) as themodel inducers of the CYP1A2 and CYP3A4 genes, respectively.

The expression of CYP1A2 mRNA was strongly induced by TCDDin all hepatocyte cultures; the magnitude of the induction varied

1.0 1.5 0.5 0.6 1.6 0.7 2.1 1.1 0.5 0.6 1.0 0.8 0.4

49.4

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RIF 10 µM

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µM

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1 µM

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µM

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µM

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(6) 1µM

1.0 0.8 1.2 1.1 0.8 3.4 1.0 0.5 0.8 1.1 1.2 3.2 1.2

78.5

0

20

40

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120

DMSO

RIF 10 µM

(1) 1

µM

(1) 0.

1 µM

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µM

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1 µM

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µM

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1 µM

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µM

(4) 1 µM

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µM

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(6) 1

µM

1.0 1.0 1.1 1.71.0 0.5 0.9 0.5 0.9

12.3

1.11.1 1.20.8

02468

10121416

DMSO

RIF 10 µM

(1) 1

µM

(1)0.1

µM

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µM

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1 µM

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µM

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1 µM

(4) 5

µM

(4) 1

µM

(5) 10

µM

(5) 1

µM

(6) 10

µM

(6) 1

µM

fold

ofco

ntro

lfo

ldof

cont

rol

fold

ofco

ntro

l

CYP3A4 mRNA

Human hepatocytes - culture LH 28

Human hepatocytes - culture LH 29

Human hepatocytes - culture LH 31

*

*

*

A expression in primary cultures of human hepatocytes. Human hepatocytes were5 (1 and 10 lM), 6 (1 and 10 lM), rifampicin (10 lM), 2,3,7,8- tetrachlorodibenzo-p-and CYP3A4 (Panel B.) mRNAs are shown. The data are mean ± SD from duplicatere normalized to GAPDH mRNA levels. * - value is significantly different from DMSO-

Table 1Effects of the dinuclear copper(II) complexes (1–6) on activation of pDRE-luc reporter plasmid in HepG2 cells. HepG2 cells were transiently transfected by lipofection (FuGENE 6)with 300 ng/per well of pDRE-luc plasmid. After 16 h of stabilization, the cells were treated for 24 h with the tested complexes (using concentration range indicated in the table)in the presence or absence of 2,3,7,8- tetrachlorodibenzo-p-dioxin (TCDD; 5 nM). After the treatments, the cells were lysed and luciferase activity was measured and standardizedper milligram of protein. The data are mean ± SD from triplicate measurements and are expressed as fold induction over DMSO-treated cells. The presented IC50 and EC50 valuesare the means from five independent transfection experiments.

Compounda Concentration range EC50 IC50

TCDD 10.0 pM–10.0 nM 2 nM n.a.[Cu2(l-L1)2(l-Cl)2Cl2] (1) 0.1 nM–1.0 lM >1.0 lM >1.0 lM[Cu2(l-L2)2(l-Cl)2Cl2] (2) 0.1 nM–1.0 lM >1.0 lM >1.0 lM[Cu2(l-L3)4Cl2]Cl2�2H2O (3) 0.1 nM–1.0 lM >1.0 lM >1.0 lM[Cu2(l-L1)4Cl2]Cl2�2H2O (4) 1.0 nM–5.0 lM >5.0 lM >5.0 lM[Cu2(l-L4)4(ClO4)2](ClO4)2�4MeOH (5) 2.0 nM–10.0 lM >10.0 lM 8.5 ± 3.2 lM[Cu2(l-L5)4(ClO4)2](ClO4)2�2EtOH (6) 1.0 nM–10.0 lM >10.0 lM 6.5 ± 2.1 lM

n.a. not applicable.a L1, 6-[(2-methoxybenzyl)amino]purine; L2, 6-[(2,3-dimethoxybenzyl)amino]purine; L3, 6-[(4-methoxybenzyl)amino]purine; L4, 6-[(2-chlorobenzyl)amino]purine; L5 = 6-

[(4-fluorobenzyl)amino]purine.

428 Z. Dvorák et al. / Toxicology in Vitro 24 (2010) 425–429

between the cultures and fold induction was 114.6 � (LH 28),539.3 � (LH 29) and 124.5 � (LH 31) (Fig. 2A). We observed a sig-nificant increase (p < 0.05) of CYP1A2 mRNA by compound 5(10 lM) in all three hepatocyte cultures, by 6 (10 lM) in culturesLH 28 and LH 29, and by 4 (10 lM) in culture LH 29 (Fig. 2A).The complexes 1, 2 and 3 did not significantly induce CYP1A2mRNA in any hepatocyte culture and in any concentration.

Rifampicin induced CYP3A4 mRNA in all hepatocyte cultureswith a various potency and fold induction was 49.4 � (LH 28),78.5 � (LH 29) and 12.3 � (LH 31) (Fig. 2B). The expression ofCYP3A4 mRNA was not induced by any of the tested complexesin any hepatocyte culture, implying no drug-interaction potentialof the complexes in terms of the PXR/CAR activation and CYP3A4induction.

3.2. Effects of copper(II) complexes 1–6 on transactivation of DRE-LUCin HepG2 cells

Since some of the tested complexes slightly, but significantly in-creased the expression of CYP1A2 mRNA in human hepatocytes,we tested whether this could be due to the activation of AhR andconsequently to transcriptional mechanism. For this purpose, wetreated HepG2 cells transiently transfected with pDRE-luc plasmidcontaining two inverted repeats of the XRE (xenobiotic responsiveelement) of mouse cyp1a1 upstream of the thymidine kinase pro-moter and the luciferase reporter gene for 24 h with tested com-plexes (using the concentration range indicated in Table 1) in thepresence or absence of TCDD (5 nM). A half maximal effective con-centration (EC50) for model TCDD as a model AhR activator was2 nM, while EC50 for all the tested complexes was not calculated,since it largely exceeded the solubility in aqueous milieu (Table1). Complexes 1, 2, 3 and 4 had no effect on DRE-LUC activity in-duced by TCDD within the tested concentration range (limited bythe solubility in aqueous milieu). Compounds 5 and 6 slightly de-creased TCDD-inducible DRE-LUC activity with a half maximalinhibitory concentration (IC50) of 8.5 ± 3.2, and 6.5 ± 2.1 lM,respectively (Table 1). Since the concentration range for thesetwo compounds spanned between 2 nM and 10 lM, the calculatedIC50 are not likely to be of a biological significance. In summary, thetested complexes do not influence AhR transcriptional activity asrevealed by gene reporter assays.

4. Discussion

In this paper, we demonstrate that recently synthesized dinu-clear copper(II) complexes (1–6) involving benzyl-substitutedderivatives of 6-(benzylamino)purine with promising selectivein vitro cytotoxicity, in vitro SOD-mimic activity and in vivo cryo-protective activity do not exert a potential for drug interactions,

in terms of major drug-metabolizing cytochrome P450 induction.This is supported by the findings that the tested complexes donot induce CYP1A2 and CYP3A4 mRNAs in three different primarycultures of human hepatocytes. In addition, the complexes wereineffective in gene reporter assays using HepG2 cells transientlytransfected with DRE-LUC plasmid.

The large number of pharmacokinetic drug interactions occursvia ligand(drug)-dependent activation of xenoreceptors (PXR,CAR, AhR) that transcriptionally up-regulates cytochromes P450and consequently increased enzyme activities lead to alterationsin a drug metabolic profile (Pascussi et al., 2008). Therefore, newlydeveloped drugs are imperatively screened for their capability totransactivate xenoreceptors and to induce cytochromes P450. Incontrast, there are no legislative requirements to test ex-postdrugs, introduced to the market before the discovery of xenorecep-tors. Indeed, the interactions of old drugs with xenoreceptors arereported incidentally and randomly. For instance, we have recentlydescribed activation of PXR and CYP3A4 induction by valproic acid(anticonvulsive) (Cerveny et al., 2007) and azoles antimycotics(Švecová et al., 2008), up-regulation of CYP2A6 by dexamethasone(corticoid) (Onica et al., 2008) or CYP1A2, CYP2B6, CYP2C9 andCYP3A4 down-regulation by colchicine (antimitotic) (Dvoráket al., 2003; Vrzal et al., 2008). The most respected in vitro modelfor studying P450 induction by drugs are the primary cultures ofhuman hepatocytes (Maurel, 1996), which we also used in thepresent study. The disadvantage of this model is the scarcity of hu-man biological material; however, human hepatocyte cultures arealso available commercially either as classical primary cells or inthe form of immortalized hepatocyte lines (Kenny et al., 2008).Alternative models are cancer cell lines transiently transfectedwith an appropriate reporter system to assess xenoreceptors trans-activation; e.g. in this study we used HepG2 cells transfected withDRE-LUC. Several laboratories and companies developed and arestill developing stably transfected cell lines allowing high through-put screening (Besselink et al., 2004; Guillouzo et al., 2007). The re-cent achievements in the field comprise the use of transgenic miceharbouring humanized xenoreceptors (Gonzalez, 2007).

In conclusion, we show in the current paper that the dinuclearcopper(II) complexes (1–6) with 6-(benzylamino)purine deriva-tives, as potential therapeutics with selective in vitro cytotoxicand SOD-mimic activity and in vivo cryoprotective activity, are safefor humans in terms of possible induction of drug-metabolizingcytochromes P450.

Acknowledgements

Financial support from the Grant Agency of the Czech Republic(Grants No. 303/07/0128 and 305/08/P089) and the Ministry of

Z. Dvorák et al. / Toxicology in Vitro 24 (2010) 425–429 429

Education, Youth and Sports of the Czech Republic(Grant No. MSM6198959218) is gratefully acknowledged.

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