Effect of hepatic cytochrome P450 (P450) oxidoreductase deficiency on 2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine-DNA Adduct Formation in P450 reductase conditional null mice

Volker M. Arlt, Rajinder Singh, Marie Stiborova, Goncalo Gamboa da Costa, Eva Frei, James D. Evans, Peter B. Farmer, C. Roland Wolf, Colin J. Henderson, David H. Phillips

    Research output: Contribution to journalArticle

    11 Citations (Scopus)

    Abstract

    2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), formed during the cooking of foods, induces colon cancer in rodents. PhIP is metabolically activated by cytochromes P450 (P450s). To evaluate the role of hepatic P450s in the bioactivation of PhIP, we used Reductase Conditional Null (RCN) mice, in which cytochrome P450 oxidoreductase (POR), the unique electron donor to P450s, can be specifically deleted in hepatocytes by pretreatment with 3-methylcholanthrene (3-MC), resulting in the loss of essentially all hepatic P450 function. RCN mice were treated orally with 50 mg/kg b.wt. PhIP daily for 5 days, with and without 3-MC pretreatment. PhIP-DNA adducts (i.e., N-(deoxyguanosin-8-yl)-2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine [dG-C8-PhIP]), measured by liquid chromatography-tandem mass spectrometry, were highest in colon (1362 adducts/10(8) deoxynucleosides), whereas adduct levels in liver were similar to 3.5-fold lower. Whereas no differences in PhIP-DNA adduct levels were found in livers with active POR versus inactivated POR, adduct levels were on average similar to 2-fold lower in extrahepatic tissues of mice lacking hepatic POR. Hepatic microsomes from RCN mice with or without 3-MC pretreatment were also incubated with PhIP and DNA in vitro. PhIP-DNA adduct formation was similar to 8-fold lower with hepatic microsomes from POR-inactivated mice than with those with active POR. Most of the hepatic microsomal activation of PhIP in vitro was attributable to CYP1A. Our results show that PhIP-DNA adduct formation in colon involves hepatic N-oxidation, circulation of activated metabolites via the bloodstream to extrahepatic tissues, and further activation, resulting in the formation of dG-C8-PhIP. Besides hepatic P450s, PhIP may be metabolically activated mainly by a non-P450 pathway in liver.

    Original languageEnglish
    Pages (from-to)2169-2173
    Number of pages5
    JournalDrug Metabolism and Disposition
    Volume39
    Issue number12
    DOIs
    Publication statusPublished - Dec 2011

    Cite this

    Arlt, Volker M. ; Singh, Rajinder ; Stiborova, Marie ; da Costa, Goncalo Gamboa ; Frei, Eva ; Evans, James D. ; Farmer, Peter B. ; Wolf, C. Roland ; Henderson, Colin J. ; Phillips, David H. / Effect of hepatic cytochrome P450 (P450) oxidoreductase deficiency on 2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine-DNA Adduct Formation in P450 reductase conditional null mice. In: Drug Metabolism and Disposition. 2011 ; Vol. 39, No. 12. pp. 2169-2173.
    @article{cf996470891a4bcdb1aba3c66a9c69c0,
    title = "Effect of hepatic cytochrome P450 (P450) oxidoreductase deficiency on 2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine-DNA Adduct Formation in P450 reductase conditional null mice",
    abstract = "2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), formed during the cooking of foods, induces colon cancer in rodents. PhIP is metabolically activated by cytochromes P450 (P450s). To evaluate the role of hepatic P450s in the bioactivation of PhIP, we used Reductase Conditional Null (RCN) mice, in which cytochrome P450 oxidoreductase (POR), the unique electron donor to P450s, can be specifically deleted in hepatocytes by pretreatment with 3-methylcholanthrene (3-MC), resulting in the loss of essentially all hepatic P450 function. RCN mice were treated orally with 50 mg/kg b.wt. PhIP daily for 5 days, with and without 3-MC pretreatment. PhIP-DNA adducts (i.e., N-(deoxyguanosin-8-yl)-2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine [dG-C8-PhIP]), measured by liquid chromatography-tandem mass spectrometry, were highest in colon (1362 adducts/10(8) deoxynucleosides), whereas adduct levels in liver were similar to 3.5-fold lower. Whereas no differences in PhIP-DNA adduct levels were found in livers with active POR versus inactivated POR, adduct levels were on average similar to 2-fold lower in extrahepatic tissues of mice lacking hepatic POR. Hepatic microsomes from RCN mice with or without 3-MC pretreatment were also incubated with PhIP and DNA in vitro. PhIP-DNA adduct formation was similar to 8-fold lower with hepatic microsomes from POR-inactivated mice than with those with active POR. Most of the hepatic microsomal activation of PhIP in vitro was attributable to CYP1A. Our results show that PhIP-DNA adduct formation in colon involves hepatic N-oxidation, circulation of activated metabolites via the bloodstream to extrahepatic tissues, and further activation, resulting in the formation of dG-C8-PhIP. Besides hepatic P450s, PhIP may be metabolically activated mainly by a non-P450 pathway in liver.",
    author = "Arlt, {Volker M.} and Rajinder Singh and Marie Stiborova and {da Costa}, {Goncalo Gamboa} and Eva Frei and Evans, {James D.} and Farmer, {Peter B.} and Wolf, {C. Roland} and Henderson, {Colin J.} and Phillips, {David H.}",
    year = "2011",
    month = "12",
    doi = "10.1124/dmd.111.041343",
    language = "English",
    volume = "39",
    pages = "2169--2173",
    journal = "Drug Metabolism and Disposition",
    issn = "0090-9556",
    publisher = "American Society for Pharmacology and Experimental Therapeutics",
    number = "12",

    }

    Effect of hepatic cytochrome P450 (P450) oxidoreductase deficiency on 2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine-DNA Adduct Formation in P450 reductase conditional null mice. / Arlt, Volker M.; Singh, Rajinder; Stiborova, Marie; da Costa, Goncalo Gamboa; Frei, Eva; Evans, James D.; Farmer, Peter B.; Wolf, C. Roland; Henderson, Colin J.; Phillips, David H.

    In: Drug Metabolism and Disposition, Vol. 39, No. 12, 12.2011, p. 2169-2173.

    Research output: Contribution to journalArticle

    TY - JOUR

    T1 - Effect of hepatic cytochrome P450 (P450) oxidoreductase deficiency on 2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine-DNA Adduct Formation in P450 reductase conditional null mice

    AU - Arlt, Volker M.

    AU - Singh, Rajinder

    AU - Stiborova, Marie

    AU - da Costa, Goncalo Gamboa

    AU - Frei, Eva

    AU - Evans, James D.

    AU - Farmer, Peter B.

    AU - Wolf, C. Roland

    AU - Henderson, Colin J.

    AU - Phillips, David H.

    PY - 2011/12

    Y1 - 2011/12

    N2 - 2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), formed during the cooking of foods, induces colon cancer in rodents. PhIP is metabolically activated by cytochromes P450 (P450s). To evaluate the role of hepatic P450s in the bioactivation of PhIP, we used Reductase Conditional Null (RCN) mice, in which cytochrome P450 oxidoreductase (POR), the unique electron donor to P450s, can be specifically deleted in hepatocytes by pretreatment with 3-methylcholanthrene (3-MC), resulting in the loss of essentially all hepatic P450 function. RCN mice were treated orally with 50 mg/kg b.wt. PhIP daily for 5 days, with and without 3-MC pretreatment. PhIP-DNA adducts (i.e., N-(deoxyguanosin-8-yl)-2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine [dG-C8-PhIP]), measured by liquid chromatography-tandem mass spectrometry, were highest in colon (1362 adducts/10(8) deoxynucleosides), whereas adduct levels in liver were similar to 3.5-fold lower. Whereas no differences in PhIP-DNA adduct levels were found in livers with active POR versus inactivated POR, adduct levels were on average similar to 2-fold lower in extrahepatic tissues of mice lacking hepatic POR. Hepatic microsomes from RCN mice with or without 3-MC pretreatment were also incubated with PhIP and DNA in vitro. PhIP-DNA adduct formation was similar to 8-fold lower with hepatic microsomes from POR-inactivated mice than with those with active POR. Most of the hepatic microsomal activation of PhIP in vitro was attributable to CYP1A. Our results show that PhIP-DNA adduct formation in colon involves hepatic N-oxidation, circulation of activated metabolites via the bloodstream to extrahepatic tissues, and further activation, resulting in the formation of dG-C8-PhIP. Besides hepatic P450s, PhIP may be metabolically activated mainly by a non-P450 pathway in liver.

    AB - 2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), formed during the cooking of foods, induces colon cancer in rodents. PhIP is metabolically activated by cytochromes P450 (P450s). To evaluate the role of hepatic P450s in the bioactivation of PhIP, we used Reductase Conditional Null (RCN) mice, in which cytochrome P450 oxidoreductase (POR), the unique electron donor to P450s, can be specifically deleted in hepatocytes by pretreatment with 3-methylcholanthrene (3-MC), resulting in the loss of essentially all hepatic P450 function. RCN mice were treated orally with 50 mg/kg b.wt. PhIP daily for 5 days, with and without 3-MC pretreatment. PhIP-DNA adducts (i.e., N-(deoxyguanosin-8-yl)-2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine [dG-C8-PhIP]), measured by liquid chromatography-tandem mass spectrometry, were highest in colon (1362 adducts/10(8) deoxynucleosides), whereas adduct levels in liver were similar to 3.5-fold lower. Whereas no differences in PhIP-DNA adduct levels were found in livers with active POR versus inactivated POR, adduct levels were on average similar to 2-fold lower in extrahepatic tissues of mice lacking hepatic POR. Hepatic microsomes from RCN mice with or without 3-MC pretreatment were also incubated with PhIP and DNA in vitro. PhIP-DNA adduct formation was similar to 8-fold lower with hepatic microsomes from POR-inactivated mice than with those with active POR. Most of the hepatic microsomal activation of PhIP in vitro was attributable to CYP1A. Our results show that PhIP-DNA adduct formation in colon involves hepatic N-oxidation, circulation of activated metabolites via the bloodstream to extrahepatic tissues, and further activation, resulting in the formation of dG-C8-PhIP. Besides hepatic P450s, PhIP may be metabolically activated mainly by a non-P450 pathway in liver.

    U2 - 10.1124/dmd.111.041343

    DO - 10.1124/dmd.111.041343

    M3 - Article

    C2 - 21940903

    VL - 39

    SP - 2169

    EP - 2173

    JO - Drug Metabolism and Disposition

    JF - Drug Metabolism and Disposition

    SN - 0090-9556

    IS - 12

    ER -