Application of hepatic cytochrome b5/P450 reductase null (HBRN) mice to study the role of cytochrome b5 in the cytochrome P450-mediated bioactivation of the anticancer drug ellipticine

Lindsay Reed, Radek Indra, Iveta Mrizova, Michaela Moserova, Heinz H. Schmeiser, C. Roland Wolf, Colin J. Henderson, Marie Stiborova, David H. Phillips, Volker M. Arlt (Lead / Corresponding author)

Research output: Contribution to journalArticle

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.
LanguageEnglish
Pages64-74
Number of pages11
JournalToxicology and Applied Pharmacology
Volume366
Early online date25 Jan 2019
DOIs
Publication statusE-pub ahead of print - 25 Jan 2019

Fingerprint

ellipticine
Cytochromes b5
NADPH-Ferrihemoprotein Reductase
Cytochrome P-450 Enzyme System
Oxidoreductases
Liver
Pharmaceutical Preparations
DNA Adducts
NADP
NAD
Microsomes

Keywords

  • Cytochrome P450
  • cytochrome b5
  • Mouse models
  • Metabolism
  • DNA Adducts

Cite this

Reed, Lindsay ; Indra, Radek ; Mrizova, Iveta ; Moserova, Michaela ; Schmeiser, Heinz H. ; Wolf, C. Roland ; Henderson, Colin J. ; Stiborova, Marie ; Phillips, David H. ; Arlt, Volker M. / Application of hepatic cytochrome b5/P450 reductase null (HBRN) mice to study the role of cytochrome b5 in the cytochrome P450-mediated bioactivation of the anticancer drug ellipticine. In: Toxicology and Applied Pharmacology. 2019 ; Vol. 366. pp. 64-74.
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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.",
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note = "Lindsay Reed is supported by a King's College London Health Faculty PhD Studentship funded by the Medical Research Council (Grant 1524896). Work at King's College London (Grant C313/A14329) and the University of Dundee (Grant C4639/A10822) were supported by Cancer Research UK. Work at King's College London was supported by the Wellcome Trust (Grants 101126/Z/13/Z and 101126/B/13/Z). Work at Charles University is supported by the Grant Agency of Czech Republic (grant 17-12816S).",
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Application of hepatic cytochrome b5/P450 reductase null (HBRN) mice to study the role of cytochrome b5 in the cytochrome P450-mediated bioactivation of the anticancer drug ellipticine. / Reed, Lindsay ; Indra, Radek; Mrizova, Iveta; Moserova, Michaela; Schmeiser, Heinz H.; Wolf, C. Roland; Henderson, Colin J.; Stiborova, Marie; Phillips, David H.; Arlt, Volker M. (Lead / Corresponding author).

In: Toxicology and Applied Pharmacology, Vol. 366, 01.03.2019, p. 64-74.

Research output: Contribution to journalArticle

TY - JOUR

T1 - Application of hepatic cytochrome b5/P450 reductase null (HBRN) mice to study the role of cytochrome b5 in the cytochrome P450-mediated bioactivation of the anticancer drug ellipticine

AU - Reed, Lindsay

AU - Indra, Radek

AU - Mrizova, Iveta

AU - Moserova, Michaela

AU - Schmeiser, Heinz H.

AU - Wolf, C. Roland

AU - Henderson, Colin J.

AU - Stiborova, Marie

AU - Phillips, David H.

AU - Arlt, Volker M.

N1 - Lindsay Reed is supported by a King's College London Health Faculty PhD Studentship funded by the Medical Research Council (Grant 1524896). Work at King's College London (Grant C313/A14329) and the University of Dundee (Grant C4639/A10822) were supported by Cancer Research UK. Work at King's College London was supported by the Wellcome Trust (Grants 101126/Z/13/Z and 101126/B/13/Z). Work at Charles University is supported by the Grant Agency of Czech Republic (grant 17-12816S).

PY - 2019/1/25

Y1 - 2019/1/25

N2 - 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.

AB - 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.

KW - Cytochrome P450

KW - cytochrome b5

KW - Mouse models

KW - Metabolism

KW - DNA Adducts

U2 - 10.1016/j.taap.2019.01.020

DO - 10.1016/j.taap.2019.01.020

M3 - Article

VL - 366

SP - 64

EP - 74

JO - Toxicology and Applied Pharmacology

T2 - Toxicology and Applied Pharmacology

JF - Toxicology and Applied Pharmacology

SN - 0041-008X

ER -