Ellipticine oxidation and DNA adduct formation in human hepatocytes is catalyzed by human cytochromes P450 and enhanced by cytochrome b5

Toxicology 302 (2012) 233–241

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Ellipticine oxidation and DNA adduct formation in human hepatocytes is catalyzed by human cytochromes P450 and enhanced by cytochrome b5 b ˇ Marie Stiborová a,∗ , Jitka Poljaková a , Eva Martínková a , Jitka Ulrichová b , Vilím Simánek , b,1 c Zdenˇek Dvoˇrák , Eva Frei a

Department of Biochemistry, Faculty of Science, Charles University, Albertov 2030, 128 40 Prague 2, Czech Republic Department of Medical Chemistry and Biochemistry, Faculty of Medicine and Dentistry, Palacky University, Olomouc, Czech Republic c Division of Preventive Oncology, National Center for Tumour Diseases, German Cancer Research Center (DKFZ), Heidelberg, Germany b

a r t i c l e

i n f o

Article history: Received 1 July 2012 Received in revised form 5 August 2012 Accepted 6 August 2012 Available online 16 August 2012 Keywords: Ellipticine Oxidation Human cytochromes P450 Hepatic microsomes Hepatocytes ellipticine–DNA adduct formation

a b s t r a c t Ellipticine is an antineoplastic agent considered a pro-drug, the pharmacological and genotoxic effects of which are dependent on cytochrome P450 (CYP)- and/or peroxidase-mediated activation to covalent DNA adducts. We investigated whether ellipticine–DNA adducts are formed in human hepatic microsomes and human hepatocytes. We then identified which human CYPs oxidize ellipticine to metabolites forming DNA adducts and the effect of cytochrome b5 on this oxidation. 13-Hydroxyellipticine, the metabolite forming the major ellipticine–DNA adduct, was generated mainly by CYP3A4 and 1A1, followed by CYP2D6 > 2C19 > 1B1 > 1A2 > 2E1 and >2C9. Cytochrome b5 increased formation of this metabolite by human CYPs, predominantly by CYP1A1, 3A4, 1A2 and 2C19. Formation of 12-hydroxyellipticine is generated mainly by CYP2C19, followed by CYP2C9 > 3A4 > 2D6 > 2E1 and >2A6. Other CYPs were less active (CYP2C8 and 2B6) or did not oxidize ellipticine to this metabolite (CYP1A1, 1A2 and 1B1). CYP2D6 was the most efficient enzyme generating ellipticine N2 -oxide. CYP3A4 and 1A1 in the presence of cytochrome b5 are mainly responsible for bioactivation of ellipticine to DNA adduct 1 (formed by ellipticine-13-ylium from 13-hydroxyellipticine), while 12-hydroxyellipticine generated during the CYP2C19-mediated ellipticine oxidation is the predominant metabolite forming ellipticine-12-ylium that generates ellipticine–DNA adduct 2. These ellipticine–DNA adducts were also generated by human hepatic microsomes and in primary human hepatocytes exposed to ellipticine. Ellipticine is toxic to these hepatocytes, decreasing their viability; the IC50 value of ellipticine in these cells was 0.7 ␮M. In liver CYP3A4 is the predominant ellipticine activating CYP species, which is expected to result in efficient metabolism after oral ingestion of ellipticine in humans. © 2012 Published by Elsevier Ireland Ltd.

1. Introduction Ellipticine (5,11-dimethyl-6H-pyrido[4,3-b]carbazole) and its derivatives are efficient anticancer compounds that function through multiple mechanisms leading to cell cycle arrest and initiation of apoptosis [for a summary see (Auclair, 1987; Stiborová

Abbreviations: CYP, cytochrome P450; DMSO, dimethyl sulfoxide; HPLC, high performance liquid chromatography; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyl tetrazoliumbromide; NADP+ , nicotinamidadeninedinucleotide phosphate; NADPH, nicotinamidadeninedinucleotide phosphate (reduced); PEI-cellulose, polyethylenimine-cellulose; r.t., retention time; RAL, relative adduct labelling; SEM, standard error medium; TLC, thin-layer chromatography. ∗ Corresponding author. Tel.: +420 221951285; fax: +420 221951283. E-mail address: [email protected] (M. Stiborová). 1 Present address: Department of Cell Biology and Genetics, Faculty of Science, Palacky University, Slechtitelu 11, 783 71 Olomouc, Czech Republic. 0300-483X/$ – see front matter © 2012 Published by Elsevier Ireland Ltd. http://dx.doi.org/10.1016/j.tox.2012.08.004

et al., 2001, 2006b, 2011; Garbett and Graves, 2004; Kizek et al., 2012)]. Ellipticine was found (i) to arrest cell cycle progression due to modulation of levels of cyclinB1 and Cdc2, and phosphorylation of Cdc2 in human mammary adenocarcinoma MCF-7 cells (Kuo et al., 2005a,b), (ii) to initiate apoptosis due to formation of toxic free radicals, stimulation of the Fas/Fas ligand system and modulation of proteins of the Bcl-2 family in several tumor cell lines, and (iii) to induce the mitochondria-dependent apoptotic processes (Garbett and Graves, 2004; Kuo et al., 2005a,b, 2006; Stiborová et al., 2011; Kizek et al., 2012). The predominant mechanisms of biological effects of ellipticine were suggested to be (i) intercalation into DNA (Auclair, 1987; Singh et al., 1994; Chu and Hsu, 1992; Garbett and Graves, 2004) and (ii) inhibition of topoisomerase II (Auclair, 1987; Monnot et al., 1991; Froelich-Ammon et al., 1995). In addition, we showed that this antitumor agent forms covalent DNA adducts after its enzymatic activation with cytochromes P450 (CYP) and peroxidases (Stiborová et al., 2001, 2004, 2006b,

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Fig. 1. Scheme of ellipticine metabolism by CYPs and peroxidases showing the identified metabolites and those proposed to form DNA adducts. The compounds shown in brackets were not detected under the experimental conditions and/or not structurally characterized. The CYP enzymes predominantly oxidizing ellipticine shown in the figure were identified in this work and/or in our previous studies (Stiborová et al., 2004, 2012; Kotrbová et al., 2011).

2007a,b, 2010b, 2011, 2012; Kotrbová et al., 2011; Kizek et al., 2012), which suggests an additional DNA-damaging effect of ellipticine. The CYP enzymes oxidize ellipticine to up to five metabolites, 9-hydroxy-, 12-hydroxy-, 13-hydroxy-, 7-hydroxyellipticine and ellipticine N2 -oxide (Fig. 1), found previously to be formed by human, rat and rabbit hepatic microsomes (Stiborová et al., 2004, 2006a). Of the CYP enzymes investigated, human CYP3A4 and rat CYP3A1 seem to be the most active enzymes oxidizing ellipticine to 13-hydroxy- and 12-hydroxyellipticine, these reactive metabolites dissociate to ellipticine-13-ylium and ellipticine-12ylium which bind to DNA (Stiborová et al., 2004, 2007a,b, 2010b, 2011, 2012), while the CYP1A isoforms preferentially form the other ellipticine metabolites, 9-hydroxy- and 7-hydroxyellipticine, which are the detoxication products. In intact organs, in cells and in isolated microsomal subcellular systems cytochrome b5 , which is a component of CYP-dependent enzymatic systems, might

influence reactions catalyzed by CYPs, thereby modulating their contributions to the oxidation of different substrates (Yamazaki et al., 1997, 2001; Schenkman and Jansson, 2003; Zhang et al., 2005, 2007). Recently we have found that in reconstituted systems cytochrome b5 alters the ratio of ellipticine metabolites formed by CYP1A1, 1A2 and 3A4 (Kotrbová et al., 2011; Stiborová et al., 2012). While the amounts of the detoxication metabolites (7-hydroxyand 9-hydroxyellipticine) were either decreased or not changed with added cytochrome b5 , 12-hydroxy-, 13-hydroxyellipticine and ellipticine N2 -oxide increased considerably. These changes in amounts of metabolites resulted in increased formation of covalent ellipticine–DNA adducts (Kotrbová et al., 2011; Stiborová et al., 2012). Since orally administered drugs which are taken up in the intestine first pass through the liver, the most active organ in the metabolism of foreign compounds, we investigated ellipticine metabolism in this organ in three different settings. We looked at

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the influence of cytochrome b5 upon ellipticine oxidation by human CYP enzymes expressed in liver. We also looked at ellipticine activation in isolated human liver microsomes to compare the influence of an intact balanced expression of CYP and other components of the CYP-mediated enzyme systems in a physiological membrane and finally the formation of ellipticine DNA adducts in these systems and in primary cultures of human hepatocytes. In these intact cells other enzymes such as peroxidases and conjugating enzymes are active which compete with CYP for ellipticine or might scavenge active metabolites reducing the yield in DNA adducts.

of the Czech Republic. Human liver samples used in this study were obtained from two patients: LH 20, a woman, aged 67 and LH 21, a woman aged 61. We do not use steatic or cirrhotic liver, and two donors used had negative virology on human immunodeficiency virus (HIV), hepatic C virus (HCV), and cytomegalovirus (CMV). The other medical records of the patients were not accessible to us. Hepatocytes were isolated as previously described (Pichard-Garcia et al., 2002; Vrzal et al., 2009). Following their isolation, cells were plated on collagen-coated culture dishes (BD Biosciences, Le Pont de Claix, France) at a density of 1.4 × 105 cells/cm2 . Cultures were maintained at 37 ◦ C and 5% CO2 in a humidified incubator. Culture medium was as described previously (Isom et al., 1985) enriched for plating with 2% fetal calf serum (v/v). Medium was exchanged for a serum-free medium the next day and the culture was allowed to stabilize for an additional 48–72 h prior to treatment.

2. Materials and methods

2.4. Treatment of primary cultures of human hepatocytes

2.1. Chemicals

Following the stabilization period, primary cultures of human hepatocytes were treated for 48 h with 1.0 and 5 ␮M ellipticine or with vehicle for control (DMSO; 0.1%, v/v). Hepatocytes were harvested, and DNA was isolated from the cells by phenol/chloroform extraction essentially as reported previously (Frei et al., 2002; Poljaková et al., 2009, 2011; Stiborová et al., 2010a). Cell viability assay. The MTT assay was used as an indicator of cell viability (Frei et al., 2002; Poljaková et al., 2009, 2011; Stiborová et al., 2010a). Cells were seeded on 96-well dishes at a density of 2 × 104 cells/well using culture medium enriched with fetal calf serum (2%, v/v). The medium was exchanged for a serum-free medium the next day and the culture was allowed to stabilize for an additional 48–72 h prior to treatment. The cells were treated for 48 h with ellipticine (0.05, 0.1, 1.0 or 5.0 ␮M). In parallel, cultures were treated with vehicle (DMSO, 0.1%, v/v) or 1% (v/v) Triton X-100 to assess the minimal and maximal cell damage, respectively.

NADP+ , NADPH, ellipticine, d-glucose 6-phosphate, d-glucose 6-phosphate dehydrogenase, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazoliumbromide (MTT) and calf thymus DNA were obtained from Sigma Chemical Co. (St. Louis, MO, USA); 9-hydroxyellipticine (5,11-dimethyl-9-hydroxy-6H-pyrido[4,3-b]carbazole) was from Calbiochem (San Diego, CA, USA). All these and other chemicals from commercial sources used in the experiments were reagent grade or better. 7-Hydroxyellipticine and the N2 -oxide of ellipticine were synthesized as described (Stiborová et al., 2004) by J. Kuˇcka (Charles University, Prague, Czech Republic); their purity was >99.5% as estimated by high-performance liquid chromatography (HPLC). Enzymatically prepared 12-hydroxy- and 13-hydroxyellipticine were obtained from multiple HPLC runs of ethyl acetate extracts of incubations of ellipticine with human and/or rat hepatic microsomes as described (Stiborová et al., 2004). Enzymes and chemicals for the 32 P-postlabeling assay were obtained from sources described previously (Stiborová et al., 2001, 2004, 2007a,b). SupersomesTM are microsomes isolated from insect cells transfected with Baculovirus constructs containing cDNA of human CYPs and expressing NADPH:CYP reductase with or without cytochrome b5 and were obtained from Gentest corp. (Woburn, MA, USA). Microsomes from livers of eight human donors who died after traffic accidents were isolated and characterized as described (Stiborová et al., 2004). 2.2. Microsomal- and CYP-incubations Incubations used to study the ellipticine metabolism contained in a final volume of 500 ␮l: 50 mM potassium phosphate buffer (pH 7.4), 1 mM NADP+ , 10 mM dglucose 6-phosphate, 1 U/ml d-glucose 6-phosphate dehydrogenase, 10 mM MgCl2 , microsomes containing 0.1 ␮M CYP and 5 ␮M ellipticine dissolved in 5 ␮l methanol. Incubations, in which the efficiencies of CYPs in SupersomesTM were tested, were the same except that 50 nM CYP without or with cytochrome b5 (1:3) were used. In the case of human CYP1A1 and 1A2 in SupersomesTM these were reconstituted with rabbit hepatic cytochrome b5 at a ratio of 1:3; CYP:cytochrome b5 ; as described in our former studies (Kotrbová et al., 2011; Stiborová et al., 2012), other human CYPs contained cytochrome b5 that was co-expressed with CYPs and NADPH:CYP reductase in insect cells used for preparation of SupersomesTM . In the control incubation, ellipticine was omitted from the incubation mixture. After incubation (37 ◦ C, 20 min) the reaction was stopped by adding 100 ␮l of 2 M NaOH. 5 ␮l of 1 mM phenacetine in methanol was added as an internal standard and the ellipticine metabolites were extracted with ethyl acetate. The extracts were evaporated and dissolved in 50 ␮l of methanol. The ellipticine metabolites were separated by HPLC (Stiborová et al., 2004, 2006a, 2010b, 2011, 2012; Klejdus et al., 2005; Kotrbová et al., 2011). The column used was a 5 ␮m Ultrasphere ODS (Beckman, 4.6 × 250 mm) preceded by a C-18 guard column, the eluent was 64% methanol plus 36% of 5 mM heptane sulfonic acid containing 32 mM acetic acid in water with flow rate of 0.8 ml/min, detection was at 296 nm. Five ellipticine metabolites with the retention times (r.t.) of 6.3, 6.9, 7.8, 8.5 and 11.2 min, were separated, and identified as 9-hydroxy-, 12-hydroxy-, 13hydroxy, 7-hydroxyellipticine and N2 -oxide of ellipticine, respectively. Recoveries of ellipticine metabolites were around 95%. Incubations in which ellipticine–DNA adducts were analyzed contained in a final volume of 750 ␮l: 50 mM potassium phosphate buffer (pH 7.4), 1 mM NADPH, microsomes containing 0.4 ␮M CYP or 0.1 ␮M Supersomal CYP, 100 ␮M ellipticine dissolved in 7.5 ␮l methanol and 1 mg calf thymus DNA. Incubations in which 13-hydroxyellipticine and 12-hydroxyellpticine, collected from multiple HPLC runs were used instead of ellipticine, contained in a final volume of 500 ␮l: 50 mM potassium phosphate buffer (pH 7.4) and 10 ␮M 13-hydroxyellipticine or 12-hydroxyellipticine dissolved in 5 ␮l of methanol and 1 mg DNA. After 60 min (37 ◦ C), incubations were extracted with ethyl acetate. DNA was isolated by phenol/chloroform extraction (Stiborová et al., 2001, 2004, 2007a,b, 2012) and used for evaluation of DNA adduct formation by the 32 P-postlabeling assay (see below). 2.3. Primary cultures of human hepatocytes Hepatocytes were prepared from lobectomy segments, resected from adult patients for medical reasons unrelated to our research program. Tissue acquisition protocol was in accordance with the requirements issued by local ethic commission

2.5. 32 P-postlabeling analysis and HPLC analysis of 32 P-labeled 3 ,5 -deoxyribonucleoside bisphosphate adducts The 32 P-postlabeling of nucleotides using nuclease P1 enrichment procedure, found previously to be appropriate to detect and quantify ellipticine-derived DNA adducts formed in vitro (Stiborová et al., 2001, 2003a,b, 2004, 2007a) and in vivo (Stiborová et al., 2003a, 2007b, 2008, 2011), was employed in the experiments. The TLC and HPLC analyzes were done as reported recently (Stiborová et al., 2001, 2004, 2007a,b, 2010b, 2011, 2012). 2.6. Statistical analyses The correlation coefficients were based on a sample size of 8 for human hepatic microsomes and 11 for human CYPs and calculated using version 6.12 Statistical Analysis System software. All Ps are two-tailed and considered significant at the 0.05 level.

3. Results and discussion 3.1. The effect of cytochrome b5 on oxidation of ellipticine and formation of ellipticine–DNA adducts by human recombinant cytochromes P450 in SupersomesTM To characterize the effect of cytochrome b5 on oxidation of ellipticine by human CYP enzymes, several human CYPs expressed in SupersomesTM in the absence or the presence of this protein were used (Fig. 2). Cytochrome b5 was either co-expressed in SupersomesTM , or reconstituted into SupersomesTM expressing a CYP. All CYPs in SupersomesTM in the absence or the presence of cytochrome b5 were active with their typical substrates (data not shown). The present study was focused on the effects of cytochrome b5 on the formation of those ellipticine metabolites which are responsible for covalent DNA adduct formation; 13-hydroxyelipticine and 12-hydroxyellipticine, which dissociate (hydrolyze) to ellipticine-13-ylium and ellipticine12-ylium which bind to DNA (Stiborová et al., 2004, 2007a, 2011, 2012), and ellipticine N2 -oxide that spontaneously rearranges to 12-hydroxyellipticine (Fig. 1, Stiborová et al., 2004, 2007a). which forms the major 13-Hydroxyellipticine, ellipticine–deoxyguanosine adduct in DNA (dG-adduct 1 in Fig. 1), was generated mainly by human CYP3A4 and 1A1, followed by CYP2D6*1 (site mutation at [374]Val), CYP2C19, 1B1, 1A2, 2E1 and 2C9*1 (site mutation at [144]Arg) even in the absence of cytochrome b5 . Cytochrome b5 present in the enzymatic system

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Fig. 2. Ellipticine metabolism by human CYPs in SupersomesTM without (A) and with cytochrome b5 (B) determined by HPLC with UV detection. 50 nM CYP and 5 ␮M ellipticine were used in all experiments, cyt b5 co-expressed in SupersomesTM or added in the ratio CYP:cyt b5 1:3. Values are averages and SEM of triplicate incubations.

increased formation of this metabolite by most human CYPs tested, predominantly by CYP1A1, 3A4 and 1A2, but decreased its generation by CYP1B1 (Fig. 2). Several human CYPs without cytochrome b5 oxidized ellipticine to 12-hydroxyellipticine to a lower extent than to 13-hydroxyellipticine. In the presence of cytochrome b5 , however, formation of 12-hydroxyellipticine catalyzed particularly by CYP2C19, 2C9*1 increased considerably, to levels even higher than 13-hydroxyellipticine, but CYP1A1, 1A2 and 1B1 still did not oxidize ellipticine to this metabolite (Fig. 2). Human recombinant CYP2D6*1, either without or with cytochrome b5 , was the most efficient enzyme generating ellipticine N2 -oxide. This ellipticine metabolite was also formed by most of the other CYP enzymes, but to a much lower extent (levels < 0.1 pmol min−1 nmol CYP−1 ), while CYP2C9 and 1B1 were

without activity to form this metabolite, CYP1B1 even in the presence of cytochrome b5 (Fig. 2). Except of CYP2A6, efficiencies of all CYP enzymes forming ellipticine N2 -oxide were elevated by adding cytochrome b5 . Using the same Supersomal systems we showed that all human CYPs in the absence or the presence of cytochrome b5 activate ellipticine to species binding to DNA. Utilizing the nuclease P1 version of the 32 P-postlabeling, two major DNA adducts (see adduct spots 1 and 2 in Fig. 3A and B formed by human CYP3A4), found previously to be formed with deoxyguanosine in DNA from 13-hydroxyellipticine (spot 1 in Fig. 3E) and 12hydroxyellipticine (spot 2 in Fig. 3F) (Stiborová et al., 2004, 2007a,b, 2012), were generated (Fig. 4). In all cases cytochrome b5 present in the incubations enhanced the formation of both adducts.

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Fig. 3. Autoradiographic profile of 32 P-labeled DNA adducts generated in calf thymus DNA by ellipticine after its activation with CYP3A4 without (A): and with cytochrome b5 (CYP3A4:cytochrome b5 1:3) (B): of 32 P-labeled digests of DNA of human hepatocytes LH 21 exposed to 5 ␮M ellipticine (C): or to DMSO (control exposition) (D): from calf thymus DNA reacted with 13-hydroxyellipticine (E): or 12-hydroxyelipticine (F). Analyses were performed by the nuclease P1 version of the 32 P-postlabeling assay. Adduct spots 1, 2, 6 and 7 correspond to the ellipticine-derived DNA adducts. Besides adduct 2 formed by 12-hydroxyellipticine, another strong adduct (spot X in panel F), which was not found in any other activation systems or in vivo (Stiborová et al., 2001, 2003a,b, 2004, 2007a,b, 2008) was generated.

Fig. 4. Ellipticine–DNA adduct formation catalyzed by human CYPs in SupersomesTM without (A) and with cytochrome b5 (B) determined by 32 P-postlabeling assay. 0.1 ␮M CYP and 100 ␮M ellipticine were used in all experiments; cyt b5 co-expressed in SupersomesTM or added in the ratio CYP:cyt b5 1:3. Values are averages and SEM of triplicate incubations.

All data are presented as means of duplicate experiments. a pmol ellipticine metabolites/min/nmol CYP. b Relative adduct labeling (RAL)/107 nucleotides per nmol CYP. c n.d., not detectable (less than 0.001 nmol ellipticine metabolites/min/nmol CYP). d Arithmetic means for eight hepatic microsomes (1–8). In the case of ellipticine–DNA adducts, the results shown were calculated from data published in our previous study (Stiborová et al., 2004). Values in parentheses are standard deviations, representing the interindividual variability.

6.35 3.91 6.33 19.1 12.2 9.68 3.39 4.96 8.24 (4.95)

Ellipticine–DNA adduct 1b Table 2 CYP-dependent amounts of ellipticine metabolites and DNA adducts formed by ellipticine in human hepatic microsomes.

Of the CYPs tested in this study, human CYP3A4 and 1A1 in the presence of cytochrome b5 were most efficient in generating adduct 1 (Fig. 4), which corresponds to the highest levels of 13-hydroxyellipticine formed by these enzymes (compare Fig. 2). Human CYP2C19, CYP2D6*1 and 2C9 were also very efficient enzymes generating this DNA adduct (Fig. 4). The ellipticine–DNA adduct 2 (spot 2 in Fig. 3) was predominantly formed by ellipticine activated with CYP2C19, CYP2D6*1, 3A4, 1A1, 2E1 and 2C9*1, while other CYPs were less active (Fig. 4). Surprisingly, relatively high levels of the adduct 2 were generated by CYP1B1, even though this CYP did not form 12-hydroxyellipticine nor ellipticine N2 -oxide which lead to ellipticine–DNA adduct 2 (compare Figs. 2 and 4). This unexpected phenomenon remains to be explained. The CYP-catalyzed formation of 13-hydroxyellipticine correlated with CYP-mediated generation of the DNA adduct 1 with a high correlation coefficient of 0.847 (P = 0.001) (Table 1). These and previous results confirm that 13-hydroxyellipticine is responsible for formation of ellipticine–DNA adduct 1 and indicate that CYP3A4 and 1A1 in the presence of cytochrome b5 are the major enzymes responsible for bioactivation of ellipticine to this adduct. In the case of the ellipticine–DNA adduct 2, a correlation tendency close to statistical significance (P = 0.059) was found between the CYP-mediated formation of this adduct and 12hydroxyellipticine, a precursor of ellipticine-12-yllium binding to DNA (Stiborová et al., 2007a, 2012) (0.689, P = 0.059). This finding suggests that CYP enzymes forming 12-hydroxyellipticine, namely CYP2C19, 2C9*1, 3A4 and 2D6*1 predominantly participate in formation of adduct 2. It should be, however, noted that situation with the adduct 2 formation is more complicated than that with the DNA adduct 1. Recently, we have found that not only the amounts of 12hydroxyellipticine formed in the reaction mixture determine DNA adduct 2 levels, but that the rate of 12-hydroxyellipticine hydrolysis to ellipticine-12-ylium dictate levels of this DNA adduct (Stiborová et al., 2012). The results of this former work demonstrated that the dissociation (hydrolysis) of 13-hydroxyellipticine to ellipticine13-ylium was preferred over that of 12-hydroxyellipticine to ellipticine-12-ylium (Stiborová et al., 2012). In addition, the adduct 2 might be generated not only directly from 12-hydroxyellipticine, but also from ellipticine N2 -oxide, which spontaneously rearranges to 12-hydroxyellipticine (Stiborová et al., 2004, 2007a). However, no correlation was found between levels of adduct 2 and ellipticine N2 -oxide. All these phenomena might explain why the correlation coefficient between the CYP-mediated formation of the adduct 2 and amounts of 12-hydroxyellipticine formed by CYPs, was not statistically significant. Nevertheless, the results of this study demonstrate that besides human CYP2C19, 2C9*1, 3A4 and 2D6*1 forming 12-hydroxyellipticine at high amounts, human CYP2D6*1 in the presence of cytochrome b5 , also participates in generation of ellipticine–DNA adduct 2, because of its high efficiency to form ellipticine N2 -oxide generating through 12hydroxyellipticine ellipticine-12-ylium.

N2 -oxide of ellipticinea

Values of correlation coefficient (r) and significance (P) were calculated from data shown in Figs. 2B and 4B, n = 11. *** P < 0.001.

0.022 n.d.c 0.023 0.070 0.009 0.020 0.007 0.009 0.023 (0.020)

0.059 0.109 0.159

0.011 0.019 0.029 0.041 0.029 0.039 n.d.c 0.068 0.029 (0.019)

P

0.689 0.510 0.481

0.013 0.027 0.035 0.054 0.040 0.074 0.023 0.091 0.045 (0.025)

r

0.338 0.001 0.914

1 2 3 4 5 6 7 8 Average valued

P

0.393 0.847*** 0.040

7-OH-ellipticinea

r

12-OH-ellipticine 13-OH-ellipticine Ellipticine N2 -oxide

13-OH-ellipticinea

Correlation data

Adduct 2

12-OH-ellipticinea

Adduct 1

9-OH-ellipticinea

Ellipticine metabolite

0.511 0.313 0.408 2.318 1.008 0.528 0.337 0.419 0.730 (0.634)

Human CYPs

0.108 0.447 0.195 0.468 0.298 0.410 0.298 0.302 0.316 (0.116)

Ellipticine–DNA adduct 2b

Table 1 Correlation coefficients (r) between levels of ellipticine metabolites formed by human CYP enzymes in the presence of cytochrome b5 and those of ellipticine–DNA adducts formed by the same enzyme systems.

0.47 0.14 0.25 1.10 0.27 0.08 0.013 0.07 0.299 (0.332)

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Human hepatic microsomal samples

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M. Stiborová et al. / Toxicology 302 (2012) 233–241 Table 3 Correlation coefficients (r) between levels of ellipticine metabolites and ellipticine–DNA adducts formed by human hepatic microsomes. Human hepatic microsomes Ellipticine metabolite

Adduct 1

Correlation data

r

P

r

P

12-OH-ellipticine 13-OH-ellipticine ellipticine N2 -oxide

0.414 0.949*** 0.815

0.307 0.0003 0.025

0.193 0.912** 0.937**

0.647 0.002 0.002

Adduct 2

Values of correlation coefficient (r) and significance (P) were calculated from data shown in Table 2 and those published in our previous study (Stiborová et al., 2004), n = 8. ** P < 0.01. *** P < 0.001.

3.2. Oxidation of ellipticine and formation of ellipticine–DNA adducts by human hepatic microsomes Recently, we have found that microsomal samples isolated from livers of several human donors were also capable of oxidizing ellipticine to the metabolites described above and to ellipticine-derived DNA adducts (Stiborová et al., 2004). These subcellular fractions represent natural enzymatic system consisting of CYPs, their reductase (NADPH:CYP reductase), cytochrome b5 and its reductase (NADH:cytochrome b5 reductase) located in the membrane of endoplasmic reticulum, comprising the important part of the xenobiotic metabolizing system of human liver. In the present study, we again analyzed ellipticine oxidation by human hepatic microsomes, using the same conditions as were employed with human recombinant CYPs. Under these conditions, 13-hydroxy- and 12hydroxyellipticine were the metabolites formed at the highest levels (Table 2), followed by 9-hydroxy-, 7-hydroxyellipticine and ellipticine N2 -oxide. As shown in our former study (Stiborová et al., 2004), 13-hydroxyellipticine and ellipticine N2 -oxide were formed mainly by CYP3A4 in human hepatic microsomes. Because of low levels of CYP2D6 in human liver microsomes (∼2.5%, Rendic and Di Carlo, 1997), even though this enzyme is most effective on formation of ellipticine N2 -oxide in vitro (compare Fig. 2), the impact of CYP2D6 in formation of this metabolite in human liver microsomes was low; the only impact of CYP3A4, highly expressed in human liver (∼30% of the liver complement, Rendic and Di Carlo, 1997) on the ellipticine N2 -oxide formation, could be observed (Stiborová et al., 2004). Because the active metabolites of ellipticine, 13hydroxyellipticine and 12-hydroxyellitpticine, are formed by human hepatic microsomes at high levels, both major DNA adducts were generated in human hepatic microsomal samples (Table 2). The formation of 13-hydroxyellipticine in human hepatic microsomes was highly correlated with levels of DNA adduct 1 formed in the same microsomes (r = 0.949, P = 0.0003, Table 3). These findings are consistent with the results found in experiments utilizing human recombinant CYPs; 13-hydroxyellipticine, formed predominantly by CYP3A4, the major CYP expressed in human liver, generates the DNA adduct 1. Surprisingly, a lower but still significant correlation was also found between formation of 13-hydroxyellipticine and ellipticine–DNA adduct 2 (r = 0.912, P = 0.002, Table 3). This phenomenon cannot be explained by the chemistry of the reactions. One possible explanation might be the fact that levels of the adduct 1 also correlated with amounts of adduct 2 (r = 0.827, P = 0.011), thereby contributing to a degree of correlation between formation of 13-hydroxyellipticine and the levels of DNA adduct 2. The formation of this adduct 2 in DNA highly significantly correlated with amounts of ellipticine N2 -oxide produced by human hepatic microsomal samples (r = 0.937, P = 0.002, Table 3).

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Even though cross-correlation between formation of the adducts 1 and 2 in human microsomes might influence the interpretation of the results found here, they nevertheless indicate that ellipticine N2 -oxide generated during the CYP3A4-mediated oxidation of ellipticine in human liver microsomes is the predominant metabolite forming finally, through the Polonowski rearrangement to 12-hydroxyelliptiicne, ellipticine-12-ylium that generates the ellipticine–DNA adduct 2. 3.3. DNA adduct formation by ellipticine in primary cultures of human hepatocytes To confirm our in vitro data obtained in incubations with subcellular hepatic fractions we isolated viable hepatocytes from two human donors and incubated these with two concentrations of ellipticine for 48 h. We also determined the cytotoxicity of ellipticine in primary cultures of human hepatocytes. For the latter experiment hepatocytes LH 21 were treated with increasing concentrations of ellipticine (0.05, 0.1, 1.0 or 5.0 ␮M) for 48 h. Primary human hepatocytes were sensitive to ellipticine, the IC50 value was 0.7 ␮M. The IC50 values in these primary cells are one order of magnitude higher than those determined previously in V79 cells transfected with human CYPs where IC50 values between 0.25 and 0.40 ␮M ellipticine were determined (Frei et al., 2002). The higher value found here in hepatocytes might reflect these cells’ capacity to detoxify xenobiotics and repair DNA adducts. The DNA adduct levels were indeed much lower than in V79 cells transfected with human CYP3A4, the most expressed liver CYP, with which RAL of 33.4 adducts/107 normal nucleotides were seen at 1 ␮M ellipticine after 48 h (Frei et al., 2002), while in hepatocytes these were only 0.24–0.47 (Table 4). In addition to the two major ellipticine–DNA adducts formed in vitro two additional adducts were visible (spots 6 and 7 in Fig. 3C), which were previously found in DNA of several organs including the livers of rats and mice treated with ellipticine (Stiborová et al., 2007b, 2008). These findings indicated similarities between ellipticine activation in human hepatocytes in vitro and this process in other in vitro systems (human CYPs, hepatic microsomes) as well as livers of experimental animals in vivo and demonstrate that human hepatocytes might be a suitable model mimicking the fate of ellipticine in the human liver. DNA from controls (human hepatocytes treated with DMSO) was devoid of adduct spots in the region of interest (Fig. 3D). Cochromatographic analyses of individual spots on HPLC confirmed that adducts 1 and 2 are formed from ellipticine metabolites, 13hydroxy- and 12-hydroxyellipticine, respectively (data not shown). These results demonstrated that human hepatocytes contain CYP enzymes forming these metabolites. Indeed, the CYP3A4 enzyme forming 13-hydroxy-, 12-hydroxyellipticine and ellipticine N2 oxide (Fig. 2) is highly expressed in human livers (hepatocytes), accounting ∼30% of the liver CYP complement (Rendic and Di Carlo, 1997). Levels of ellipticine–DNA adducts found in human hepatocytes are shown in Table 4.

Table 4 Amounts of ellipticine–DNA adducts formed in human hepatocytes. Total ellipticine–DNA adducts (RAL/107 normal nucleotides)

No ellipticine (DMSO) 1 ␮M ellipticine 5 ␮M ellipticine

LH 20

LH 21

n.d.a 0.47 ± 0.08b 2.12 ± 0.25b

n.d.a 0.24 ± 0.06b 1.29 ± 0.19b

a Not detectable (detection limit of DNA adducts is 1010 adducts/normal nucleotides). b Averages and SEM of three parallel 32 P-postlabeling analyses.

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4. Conclusions and perspectives The results of this work demonstrate that oxidation of ellipticine to its active metabolite 13-hydroxyellipticine by pure human CYP enzymes and those present in human hepatic microsomes correlates with formation of the major DNA adduct by these systems. They also indicate importance of cytochrome b5 on these CYPcatalyzed processes, whereby the levels of ellipticine–DNA adduct 1 catalyzed mainly by CYP3A4 and 1A1 and those of ellipticine–DNA adduct 2 predominantly by CYP2C19, 2C9 or 2D6 were higher with cytochrome b5 . The results also show for the first time that ellipticine is toxic to human hepatocytes and forms the same DNA adducts in these cells that were formed by pure CYPs and human hepatic microsomes and in vivo in livers of experimental animals. These data are important to assess the effects upon liver of orally administered ellipticine in a first pass. The study forms the basis to further predict the susceptibility of human cancers to ellipticine and suggests this alkaloid for treatment in combination with CYP gene transfer increasing the anticancer potential of this prodrug. As we found in this and our former studies (Stiborová et al., 2004, 2007a, 2012; Kotrbová et al., 2011), two of the ellipticine metabolites formed by oxidation with CYPs in combination with cytochrome b5 , 13hydroxyellipticine and 12-hydroxyellipticine, are reactive enough to decompose spontaneously to the carbenium ions forming DNA adducts that are predominantly responsible for killing cancer cells. Among them leukemias, lymphosarcomas, lung carcinoma, human non-small-cell-lung-cancer, hepatocellular carcinomas, glioblastomas, osteosarcoma, breast adenocarcinoma and neuroblastomas were very sensitive to ellipticine treatment [for a summary see (Stiborová et al., 2011)]. Both these ellipticine metabolites are, therefore, excellent candidates for tumor cell-specific targeting to these cancers that include the use of systemic delivery of these metabolites. Hence, we suggest these two ellipticine derivatives for potential clinical usage. Furthermore, preparation of appropriate conjugates that would deliver these ellipticine derivatives to cancer cells is one of the major challenges in research of our laboratory to prepare suitable ellipticine derivatives for clinical usage. In addition, because the CYP activation enzymes, when expressed in tumor cells confer the ability to metabolize ellipticine into more potent cytotoxins, ellipticine itself should be a suitable candidate for CYP-gene-directed enzyme-prodrug therapy (Ma and Waxman, 2007; Lu et al., 2009), which has the potential to provide efficient activation of ellipticine in target tumor tissue. Conflict of interest The authors state that they have no conflict of interest. Acknowledgements This work was supported in part by Grant Agency of the Czech Republic, Grant P301/10/0356, Charles University in Prague, Grant UNCE204025/2012 and by the Institutional Support of Palacky University in Olomouc. References Auclair, C., 1987. Multimodal action of antitumor agents on DNA. The ellipticine series. Arch. Biochem. Biophys. 259, 1–14. Chu, Y., Hsu, M.T., 1992. Ellipticine increases the superhelical density of intracellular SV40 DNA by intercalation. Nucleic Acids Res. 20, 4033–4038. Frei, E., Bieler, C.A., Arlt, V.M., Wiessler, M., Stiborová, M., 2002. Covalent binding of the anticancer drug ellipticine to DNA in V79 cells transfected with human cytochrome P450 enzymes. Biochem. Pharmacol. 64, 289–295. Froelich-Ammon, S.J., Patchan, M.W., Osheroff, N., Thompson, R.B., 1995. Topoisomerase II binds to ellipticine in the absence or presence of DNA. Characterization

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