Abstract
The anticancer drug ellipticine exerts its genotoxic effects after metabolic activation by cytochrome P450 (CYP) enzymes. The present study has examined the role of cytochrome P450 oxidoreductase (POR) and cytochrome b5 (Cyb5), electron donors to P450 enzymes, in the CYP-mediated metabolism and disposition of ellipticine in vivo. We used Hepatic Reductase Null (HRN) and Hepatic Cytochrome b5/P450 Reductase Null (HBRN) mice. HRN mice have POR deleted specifically in hepatocytes; HBRN mice also have Cyb5 deleted in the liver. Mice were treated once with 10 mg/kg body weight ellipticine (n = 4/group) for 24 h. Ellipticine-DNA adduct levels measured by 32P-postlabelling were significantly lower in HRN and HBRN livers than in wild-type (WT) livers; however no significant difference was observed between HRN and HBRN livers. Ellipticine-DNA adduct formation in WT, HRN and HBRN livers correlated with Cyp1a and Cyp3a enzyme activities measured in hepatic microsomes in the presence of NADPH confirming the importance of P450 enzymes in the bioactivation of ellipticine in vivo. Hepatic microsomal fractions were also utilised in incubations with ellipticine and DNA in the presence of NADPH, cofactor for POR, and NADH, cofactor for Cyb5 reductase (Cyb5R), to examine ellipticine-DNA adduct formation. With NADPH adduct formation decreased as electron donors were lost which correlated with the formation of the reactive metabolites 12- and 13-hydroxy-ellipticine in hepatic microsomes. No difference in adduct formation was observed in the presence of NADH. Our study demonstrates that Cyb5 contributes to the P450-mediated bioactivation of ellipticine in vitro, but not in vivo.
Highlights
Ellipticine (5,11-dimethyl-6H-pyrido[4,5-b]carbazole) is a cytotoxic alkaloid isolated from the Apocynaceae family of plants
Ellipticine-DNA adduct formation in WT, Hepatic Reductase Null (HRN) and Hepatic Cytochrome b5/P450 Reductase Null (HBRN) livers correlated with Cyp1a and Cyp3a enzyme activities measured in hepatic microsomes in the presence of reduced nicotinamide adenine dinucleotide (NADPH) confirming the importance of P450 enzymes in the bioactivation of ellipticine in vivo
Expression of electron donor proteins (i.e. P450 oxidoreductase (POR), cytochrome b5 (Cyb5), Cyb5 reductase (Cyb5R)) associated with the mixed-function oxidase system (i.e. P450) were probed for in the hepatic microsomal fractions from WT, HRN and HBRN mice exposed to ellipticine (Fig. 2)
Summary
Ellipticine (5,11-dimethyl-6H-pyrido[4,5-b]carbazole) is a cytotoxic alkaloid isolated from the Apocynaceae family of plants. Both ellipticine and its derivatives possess anti-HIV and anti-tumour properties allowing it to be used against several cancers with limited toxic side effects and no haematological toxicity by functioning through multiple mechanisms that result in cell cycle arrest and initiation of apoptosis (Martinkova et al, 2010; Stiborova et al, 2011; Miller and McCarthy, 2012; Stiborova and Frei, 2014). Ellipticine needs to be metabolised to exert its pharmacological effects. These enzyme-catalysed reactions generate detoxication products leading to the excretion of the drug.
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