Role of hepatic cytochromes P450 in bioactivation of the anticancer drug ellipticine: Studies with the hepatic NADPH:Cytochrome P450 reductase null mouse

https://doi.org/10.1016/j.taap.2007.09.017Get rights and content

Abstract

Ellipticine is an antineoplastic agent, which forms covalent DNA adducts mediated by cytochromes P450 (CYP) and peroxidases. We evaluated the role of hepatic versus extra-hepatic metabolism of ellipticine, using the HRN (Hepatic Cytochrome P450 Reductase Null) mouse model, in which cytochrome P450 oxidoreductase (POR) is deleted in hepatocytes, resulting in the loss of essentially all hepatic CYP function. HRN and wild-type (WT) mice were treated i.p. with 1 and 10 mg/kg body weight of ellipticine. Multiple ellipticine–DNA adducts detected by 32P-postlabelling were observed in organs from both mouse strains. Highest total DNA binding levels were found in liver, followed by lung, kidney, urinary bladder, colon and spleen. Ellipticine–DNA adduct levels in the liver of HRN mice were up to 65% lower relative to WT mice, confirming the importance of CYP enzymes for the activation of ellipticine in livers, recently shown in vitro with human and rat hepatic microsomes. When hepatic microsomes of both mouse strains were incubated with ellipticine, ellipticine–DNA adduct levels with WT microsomes were up to 2.9-fold higher than with those from HRN mice. The ratios of ellipticine–DNA adducts in extra-hepatic organs between HRN and WT mice of up to 4.7 suggest that these organs can activate ellipticine and that more ellipticine is available in the circulation. These results and the DNA adduct patterns found in vitro and in vivo demonstrate that both CYP1A or 3A and peroxidases participate in activation of ellipticine to reactive species forming DNA adducts in the mouse model used in this study.

Introduction

Ellipticine (5,11-dimethyl-6H-pyrido[4,3-b]carbazole, Fig. 1), an alkaloid isolated from Apocyanaceae plants, exhibit significant antitumor and anti-HIV activities (for a summary see Stiborová et al., 2001). The main reason for the interest in ellipticine and its derivatives for clinical purposes is their high efficiencies against several types of cancer, their rather limited toxic side effects, and their complete lack of haematological toxicity (Auclair, 1987). Nevertheless, ellipticine is a potent mutagen. Most ellipticine derivatives are mutagenic to Salmonella typhimurium Ames tester strains, bacteriophage T4, Neurospora crassa, and mammalian cells and induce prophage lambda in Escherichia coli (for an overview see Stiborová et al., 2001).

The prevalent mechanisms of ellipticine antitumour, mutagenic and cytotoxic activities have been suggested to be (i) intercalation into DNA (Auclair, 1987, Singh et al., 1994) and (ii) inhibition of DNA topoisomerase II activity (Auclair, 1987, Monnot et al., 1991, Fossé et al., 1992, Froelich-Ammon et al., 1995). We have demonstrated that ellipticine also covalently binds to DNA after being enzymatically activated with cytochromes P450 (CYP) or peroxidases (Stiborová et al., 2001, Stiborová et al., 2003a, Stiborová et al., 2003b, Stiborová et al., 2004, Stiborová et al., 2006a, Stiborová et al., 2007a, Stiborová et al., 2007b, Poljaková et al., 2006), suggesting a third possible mechanism of action.

Understanding which enzymes are involved in the metabolic activation of ellipticine is important in the assessment of susceptibility to this drug. Human and rat CYP1A and 3A are the predominant enzymes catalyzing oxidation of ellipticine in vitro either to metabolites that are excreted (7-hydroxy- and 9-hydroxyellipticine) or that form DNA adducts (12-hydroxy- and 13-hydroxyellipticine) (Stiborová et al., 2001, Stiborová et al., 2003a, Stiborová et al., 2003b, Stiborová et al., 2004, Stiborová et al., 2006a). Of the peroxidases, human cyclooxygenase (COX)-2, ovine COX-1, bovine lactoperoxidase (LPO), human myeloperoxidase (MPO) and horseradish peroxidase efficiently generated ellipticine-derived DNA adducts (Fig. 1) (Poljaková et al., 2006, Stiborová et al., 2007a). The same DNA adducts were also detected in cells in culture expressing enzymes activating ellipticine (CYP1A1, COX-1 and MPO), such as human breast adenocarcinoma MCF-7 cells (Bořek-Dohalská et al., 2004), leukaemia HL-60 and CCRF-CEM cells (Poljaková et al., 2007) and V79 Chinese hamster lung fibroblasts transfected with human CYP3A4, 1A1 and 1A2 (Frei et al., 2002). On the basis of these data, ellipticine might be considered a drug, whose pharmacological efficiency and/or genotoxic side effects are dependent on its activation by CYPs and peroxidases in target tissues.

However, additional factors such as route of administration, absorption and renal clearance make it difficult to extrapolate from in-vitro data to the in-vivo situation. After i.p. administration of ellipticine to rats, the highest levels of DNA adducts were found in liver, followed by kidney and lung (Stiborová et al., 2003a, Stiborová et al., 2007b). Liver is a tissue rich in CYP enzymes, while kidney and lung contain high levels of peroxidases such as COX (Eling et al., 1990, Eling and Curtis, 1992, Stiborová et al., 1991, Stiborová et al., 2005, Culp et al., 1997). Knowledge about the participation of these enzymes in ellipticine activation in individual rat tissues is, however, scarce. Although previous results indicate that CYP3A1 and 1A participate in formation of DNA adducts by ellipticine in rats in vivo (Stiborová et al., 2003a), it is unclear whether CYP or peroxidases are more important in ellipticine activation in different organs.

Gene knock-out and transgenic mice have been developed to study the role of specific enzymes in drug metabolism (Gonzalez and Kimura, 2001, Henderson and Wolf, 2003). Although CYP knock-out mouse models have yielded important data on the effect of single CYP enzymes on the metabolism of drugs and chemical carcinogens (Buters et al., 1999, Buters et al., 2002, Kimura et al., 1999, Kimura et al., 2003, Tsuneoka et al., 2003, Uno et al., 2004), the functional redundancy inevitably found in the large CYP family of isoenzymes makes it difficult to determine the role of CYPs as a whole in metabolism of xenobiotics (Henderson et al., 2006). To overcome these limitations a mouse line, HRN (Hepatic Cytochrome P450 Reductase Null), has been developed in which cytochrome P450 oxidoreductase (POR), the unique electron donor to CYPs, is deleted specifically in hepatocytes, resulting in the loss of essentially all hepatic CYP function (Henderson et al., 2003). The HRN mouse as a model has been used to establish the role of hepatic versus extra-hepatic CYP-mediated metabolism and disposition of drugs such as cyclophosphamide (Pass et al., 2005, Henderson et al., 2006). With this model we found that the carcinogen 3-nitrobenzanthrone is predominantly activated in vivo by cytosolic nitroreductases rather than microsomal POR, whereas the oxidative activation of 3-aminobenzanthrone, the main metabolite of 3-nitrobenzanthrone, is CYP-dependent, both in vitro and in vivo (Arlt et al., 2003, Arlt et al., 2004, Arlt et al., 2005, Arlt et al., 2006).

In this study, we have used the HRN mouse model to examine the hepatic CYP-dependent metabolism of ellipticine. DNA adduct formation in vivo in hepatic and extra-hepatic tissues was investigated using 32P-postlabelling. In addition, we examined ellipticine metabolism and DNA adduct formation in vitro using hepatic microsomes.

Section snippets

Animals

HRN (Porlox/lox + CreALB) mice on a C57BL/6 background used in this study were constructed as described previously (Henderson et al., 2003). Mice homozygous for loxP sites at the Por locus (Porlox/lox) were used as wild-type (WT). All procedures were carried out under the Animal (Scientific Procedures) Act (1986) in accordance with UK law, and following local ethical review.

Treatment of HRN mice and WT mice with ellipticine

Male HRN (Henderson et al., 2003) and WT mice (25–30 g; n = 3/group) were treated with a single intra-peritoneal (i.p.) dose

Hepatic microsomes of HRN mice are capable of activating ellipticine

In order to evaluate the role of the mouse hepatic POR-dependent CYP in the activation of ellipticine, we first performed in-vitro experiments. Microsomes isolated from livers of HRN and WT mice used for such experiments were characterized for POR expression by Western blotting. Hepatic POR expression was detected in WT mice, while as expected, its levels in HRN mice were very low, but still detectable, by immunostaining. Hepatic POR levels in HRN mice were estimated to be less than 1.5% of the

Discussion

Ellipticine is an anticancer agent, whose biological effects such as pharmacological efficiencies and its potential genotoxic side effects may depend on its CYP- and peroxidase mediated metabolism leading to formation of DNA adducts (Stiborová et al., 2001, Stiborová et al., 2003a, Stiborová et al., 2003b, Stiborová et al., 2004, Stiborová et al., 2007a, Frei et al., 2002, Poljaková et al., 2006, Poljaková et al., 2007). Although the physiological disposition and DNA adduct formation by this

Acknowledgments

This work was supported in part by Grant Agency of the Czech Republic, grant 203/06/0329, Ministry of Education of the Czech Republic, grants MSM0021620808 and 1M4635608802-Center of Targeted Therapeutics and by Cancer Research UK. V.M. Arlt, C.J. Henderson, C.R. Wolf and D.H. Phillips are partners of ECNIS (Environmental Cancer Risk, Nutrition and Individual Susceptibility), a network of excellence operating within the European Union 6th Framework Program, Priority 5: “Food Quality and Safety”

References (46)

  • J. Poljaková et al.

    DNA adduct formation by the anticancer drug ellipticine in human leukemia HL-60 and CCRF-CEM cells

    Cancer Lett.

    (2007)
  • I.A. Slepneva et al.

    Reversible inhibition of NADPH–cytochrome P450 reductase by alpha-lipoic acid

    Biochem. Biophys. Res. Commun.

    (1995)
  • M. Stiborová et al.

    Formation and persistence of DNA adducts of anticancer drug ellipticine in rats

    Toxicology

    (2007)
  • Y. Yasukochi et al.

    NADPH-cytochrome c (P450) reductase: spectrophotometric and stopped flow kinetic studies on the formation of reduced flavoprotein intermediates

    J. Biol. Chem.

    (1979)
  • D. Aimová et al.

    The anticancer drug ellipticine is a potent inducer of rat cytochromes P450 1A1 and 1A2, thereby modulating its own metabolism

    Drug Metab. Dispos.

    (2007)
  • V.M. Arlt et al.

    Human enzymes involved in the metabolic activation of the environmental contaminant 3-nitrobenzanthrone: evidence for reductive activation by human NADPH:cytochrome P450 reductase

    Cancer Res.

    (2003)
  • V.M. Arlt et al.

    3-Aminobenzanthrone, a human metabolite of the environmental pollutant 3-nitrobenzanthrone, forms DNA adducts after metabolic activation by human and rat liver microsomes: evidence for activation by cytochrome P450 1A1 and P450 1A2

    Chem. Res. Toxicol.

    (2004)
  • V.M. Arlt et al.

    Environmental pollutant and potent mutagen 3-nitrobenzanthrone forms DNA adducts after reduction by NAD(P)H:quinone oxidoreductase and conjugation by acetyltransferases and sulfotransferases in human hepatic cytosols

    Cancer Res.

    (2005)
  • L. Bořek-Dohalská et al.

    DNA adduct formation by the anticancer drug ellipticine and its hydroxy derivatives in human breast adenocarcinoma MCF-7 cells

    Collect. Czech. Chem. Commun.

    (2004)
  • A.R. Branfam et al.

    Characterization of metabolites of ellipticine in rat bile

    Drug Metab. Dispos.

    (1978)
  • J.T. Buters et al.

    Cytochrome P450 CYP1B1 determines susceptibility to 7,12-dimethylbenz[a]anthracene-induced lymphomas

    Proc. Natl. Acad. Sci. U. S. A.

    (1999)
  • J.T. Buters et al.

    Cytochrome P450 1B1 determines susceptibility to dibenzo[a,l]pyrene-induced tumor formation

    Chem. Res. Toxicol.

    (2002)
  • M. Chadwick et al.

    Comparative physiological disposition of ellipticine in several animal species after intravenous administration

    Drug Metab. Dispos.

    (1978)
  • Cited by (52)

    • Application of hepatic cytochrome b <inf>5</inf> /P450 reductase null (HBRN) mice to study the role of cytochrome b <inf>5</inf> in the cytochrome P450-mediated bioactivation of the anticancer drug ellipticine

      2019, Toxicology and Applied Pharmacology
      Citation Excerpt :

      HRN mice have a deletion of NADPH:cytochrome P450 oxidoreductase (POR), the predominant electron donor to P450s, specifically in their hepatocytes (Henderson et al., 2003). This deletion results in the loss of essentially all hepatic P450 activity and the mice have been used to investigate hepatic versus extra-hepatic P450 mediated metabolism of several carcinogens including ellipticine (Arlt et al., 2005; Stiborova et al., 2008; Levova et al., 2011; Arlt et al., 2012). HRN mice formed 65% lower levels of ellipticine-DNA adducts in their livers than wild-type (WT) mice, demonstrating the importance of P450 activity in the hepatic bioactivation of ellipticine (Stiborova et al., 2008).

    • The impact of chemotherapeutic drugs on the CYP1A1-catalysed metabolism of the environmental carcinogen benzo[a]pyrene: Effects in human colorectal HCT116 TP53(+/+), TP53(+/−) and TP53(−/−) cells

      2018, Toxicology
      Citation Excerpt :

      As the bioactivation of ellipticine can be catalysed by CYP enzymes including CYP1A1 (Kotrbova et al., 2011; Stiborova et al., 2012b; Stiborova et al., 2004) and based on the results that p53 function impacts on ellipticine-induced CYP1A1 expression, ellipticine-DNA adduct formation after 24 and 48 h was determined by the 32P-postlabelling method (Fig. 7). After treatment with 5 μM ellipticine the adduct pattern was qualitatively similar in TP53(+/+), TP53(+/–) and TP53(–/–) cells and consisted of one major and one minor DNA adduct (assigned spots 1 and 2; Fig. 7C) previously detected in vitro and in vivo by this method (Stiborova et al., 2008; Stiborova et al., 2012b). No DNA adducts were detected in untreated controls (data not shown).

    • Anthracyclines and ellipticines as DNA-damaging anticancer drugs: Recent advances

      2012, Pharmacology and Therapeutics
      Citation Excerpt :

      Using a 32P-postlabeling method, we have found that during ellipticine oxidation by CYPs and peroxidases, two major and several minor ellipticine-derived adducts are generated in DNA (Fig. 2) (Stiborova et al., 2003b, 2007a, 2007b, 2008; Kotrbova et al., 2006; Poljakova et al., 2006). Human and rat CYP1A, 1B1 and 3A, which are expressed at higher levels in tumors sensitive to ellipticine (i.e., breast cancer) than in peritumoral tissues (El-Rayes et al., 2003; Patterson et al., 1999), are the predominant enzymes catalyzing the oxidation of ellipticine in vitro to metabolites that are either excreted (7-hydroxy- and 9-hydroxyellipticine) or that form DNA adducts (12-hydroxy-, 13-hydroxyellipticine and ellipticine N2-oxide) (see Figs. 1 and 2A, F, G and H showing the adducts formed by ellipticine activated with CYP3A4 by 12-hydroxy-, 13-hydroxyellipticine and ellipticine N2-oxide, respectively) (Stiborova et al., 2004, 2006b, 2007a, 2008). Of the mammalian peroxidases, bovine lactoperoxidase (LPO), human myeloperoxidase (MPO), which is highly expressed in acute myeloid leukemia blasts, bovine COX-1, and human cyclooxygenase (COX)-2 efficiently generate ellipticine-derived DNA adducts (see Figs. 2B, C, D and E for adducts formed by ellipticine activated with LPO, MPO, COX-1 and COX-2, respectively) (Poljakova et al., 2006; Stiborova et al., 2007a).

    View all citing articles on Scopus
    View full text