Carboxylic Acid Drug-Induced DNA Nicking in HEK293 Cells Expressing Human UDP_Glucuronosyltransferases : Role of Acyl Glucuronide Metabolites and Glycation Pathways

H.T. Southwood, Y.C. Degraaf, P.I. Mackenzie, J.O. Miners, Philip Burcham, B.C. Sallustio

    Research output: Contribution to journalArticle

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    Abstract

    Glucuronidation of carboxylic-acid-containing drugs can yield reactive acyl (ester-linked) glucuronide metabolites that are able to modify endogenous macromolecules. Previous research has shown that several carboxylic acid drugs are genotoxic in isolated mouse hepatocytes, and that DNA damage is prevented by the glucuronidation inhibitor, borneol. Whether these species induce comparable genetic damage in human cells is unknown. In this study, we investigated the mechanisms of clofibric acid-induced genotoxicity in HEK293 cells expressing the human UDP-glucuronosyltransferases UGT1A3, UGT1A9, or UGT2B7, and screened three other carboxylic acid drugs for UGT-dependent genotoxicity. DNA damage was detected using the alkaline version of the comet assay. HEK293 cells were incubated for 18 h with vehicle (2.5 mM NaOH), 0.1-2.5 mM clofibric acid or 0.1-1.0 mM benoxaprofen, bezafibrate, or probenecid. To identify mechanisms underlying any observed genotoxicity, we treated UGT2B7 transfectants with 10 mM aminoguanidine, 1 mM borneol, or 2 mM desferrioxamine mesylate prior to co-incubation with I mM clofibric acid for 18 h. Compared to vehicle, clofibric acid, benoxaprofen, and probenecid produced significant DNA damage in all three UGT-transfected HEK293 cell lines, detectable from the lowest concentration tested. Bezafibrate caused DNA damage only at higher concentrations (1.0 mM) in UGT2B7- and UGT1A9-, but not UGT1A3-transfected cells. No drug-induced DNA damage was detected in untransfected cells, consistent With the limited glucuronidation capacity of these cells. The glycation/glycoxidation inhibitor aminoguanidine and the glucuronidation inhibitor borneol significantly decreased clofibric-acid-mediated DNA damage in UGT2B7 transfected cells by 73.5 and 94.8%, respectively. The inhibitor of transition -metal-catalyzed oxidation, desferrioxamine mesylate, had no significant effect on DNA damage. This study demonstrates the substrate-dependent role of human UGTs in the bioactivation of carboxylic acid drugs to genotoxic acyl glucuronide metabolites that are able to damage nuclear DNA via glycation and/or glycoxidation mechanisms.
    Original languageEnglish
    Pages (from-to)1520-1527
    JournalChemical Research in Toxicology
    Volume20
    Issue number10
    DOIs
    Publication statusPublished - 2007

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    Glucuronosyltransferase
    HEK293 Cells
    Glucuronides
    Metabolites
    Carboxylic Acids
    Clofibric Acid
    DNA Damage
    Cells
    DNA
    Pharmaceutical Preparations
    Bezafibrate
    Probenecid
    Mesylates
    Deferoxamine
    Comet Assay
    Corrosion inhibitors
    Macromolecules
    Transition metals
    Hepatocytes
    Assays

    Cite this

    @article{218147c963894084b3137cd5ffd2e529,
    title = "Carboxylic Acid Drug-Induced DNA Nicking in HEK293 Cells Expressing Human UDP_Glucuronosyltransferases : Role of Acyl Glucuronide Metabolites and Glycation Pathways",
    abstract = "Glucuronidation of carboxylic-acid-containing drugs can yield reactive acyl (ester-linked) glucuronide metabolites that are able to modify endogenous macromolecules. Previous research has shown that several carboxylic acid drugs are genotoxic in isolated mouse hepatocytes, and that DNA damage is prevented by the glucuronidation inhibitor, borneol. Whether these species induce comparable genetic damage in human cells is unknown. In this study, we investigated the mechanisms of clofibric acid-induced genotoxicity in HEK293 cells expressing the human UDP-glucuronosyltransferases UGT1A3, UGT1A9, or UGT2B7, and screened three other carboxylic acid drugs for UGT-dependent genotoxicity. DNA damage was detected using the alkaline version of the comet assay. HEK293 cells were incubated for 18 h with vehicle (2.5 mM NaOH), 0.1-2.5 mM clofibric acid or 0.1-1.0 mM benoxaprofen, bezafibrate, or probenecid. To identify mechanisms underlying any observed genotoxicity, we treated UGT2B7 transfectants with 10 mM aminoguanidine, 1 mM borneol, or 2 mM desferrioxamine mesylate prior to co-incubation with I mM clofibric acid for 18 h. Compared to vehicle, clofibric acid, benoxaprofen, and probenecid produced significant DNA damage in all three UGT-transfected HEK293 cell lines, detectable from the lowest concentration tested. Bezafibrate caused DNA damage only at higher concentrations (1.0 mM) in UGT2B7- and UGT1A9-, but not UGT1A3-transfected cells. No drug-induced DNA damage was detected in untransfected cells, consistent With the limited glucuronidation capacity of these cells. The glycation/glycoxidation inhibitor aminoguanidine and the glucuronidation inhibitor borneol significantly decreased clofibric-acid-mediated DNA damage in UGT2B7 transfected cells by 73.5 and 94.8{\%}, respectively. The inhibitor of transition -metal-catalyzed oxidation, desferrioxamine mesylate, had no significant effect on DNA damage. This study demonstrates the substrate-dependent role of human UGTs in the bioactivation of carboxylic acid drugs to genotoxic acyl glucuronide metabolites that are able to damage nuclear DNA via glycation and/or glycoxidation mechanisms.",
    author = "H.T. Southwood and Y.C. Degraaf and P.I. Mackenzie and J.O. Miners and Philip Burcham and B.C. Sallustio",
    year = "2007",
    doi = "10.1021/tx700188x",
    language = "English",
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    pages = "1520--1527",
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    Carboxylic Acid Drug-Induced DNA Nicking in HEK293 Cells Expressing Human UDP_Glucuronosyltransferases : Role of Acyl Glucuronide Metabolites and Glycation Pathways. / Southwood, H.T.; Degraaf, Y.C.; Mackenzie, P.I.; Miners, J.O.; Burcham, Philip; Sallustio, B.C.

    In: Chemical Research in Toxicology, Vol. 20, No. 10, 2007, p. 1520-1527.

    Research output: Contribution to journalArticle

    TY - JOUR

    T1 - Carboxylic Acid Drug-Induced DNA Nicking in HEK293 Cells Expressing Human UDP_Glucuronosyltransferases : Role of Acyl Glucuronide Metabolites and Glycation Pathways

    AU - Southwood, H.T.

    AU - Degraaf, Y.C.

    AU - Mackenzie, P.I.

    AU - Miners, J.O.

    AU - Burcham, Philip

    AU - Sallustio, B.C.

    PY - 2007

    Y1 - 2007

    N2 - Glucuronidation of carboxylic-acid-containing drugs can yield reactive acyl (ester-linked) glucuronide metabolites that are able to modify endogenous macromolecules. Previous research has shown that several carboxylic acid drugs are genotoxic in isolated mouse hepatocytes, and that DNA damage is prevented by the glucuronidation inhibitor, borneol. Whether these species induce comparable genetic damage in human cells is unknown. In this study, we investigated the mechanisms of clofibric acid-induced genotoxicity in HEK293 cells expressing the human UDP-glucuronosyltransferases UGT1A3, UGT1A9, or UGT2B7, and screened three other carboxylic acid drugs for UGT-dependent genotoxicity. DNA damage was detected using the alkaline version of the comet assay. HEK293 cells were incubated for 18 h with vehicle (2.5 mM NaOH), 0.1-2.5 mM clofibric acid or 0.1-1.0 mM benoxaprofen, bezafibrate, or probenecid. To identify mechanisms underlying any observed genotoxicity, we treated UGT2B7 transfectants with 10 mM aminoguanidine, 1 mM borneol, or 2 mM desferrioxamine mesylate prior to co-incubation with I mM clofibric acid for 18 h. Compared to vehicle, clofibric acid, benoxaprofen, and probenecid produced significant DNA damage in all three UGT-transfected HEK293 cell lines, detectable from the lowest concentration tested. Bezafibrate caused DNA damage only at higher concentrations (1.0 mM) in UGT2B7- and UGT1A9-, but not UGT1A3-transfected cells. No drug-induced DNA damage was detected in untransfected cells, consistent With the limited glucuronidation capacity of these cells. The glycation/glycoxidation inhibitor aminoguanidine and the glucuronidation inhibitor borneol significantly decreased clofibric-acid-mediated DNA damage in UGT2B7 transfected cells by 73.5 and 94.8%, respectively. The inhibitor of transition -metal-catalyzed oxidation, desferrioxamine mesylate, had no significant effect on DNA damage. This study demonstrates the substrate-dependent role of human UGTs in the bioactivation of carboxylic acid drugs to genotoxic acyl glucuronide metabolites that are able to damage nuclear DNA via glycation and/or glycoxidation mechanisms.

    AB - Glucuronidation of carboxylic-acid-containing drugs can yield reactive acyl (ester-linked) glucuronide metabolites that are able to modify endogenous macromolecules. Previous research has shown that several carboxylic acid drugs are genotoxic in isolated mouse hepatocytes, and that DNA damage is prevented by the glucuronidation inhibitor, borneol. Whether these species induce comparable genetic damage in human cells is unknown. In this study, we investigated the mechanisms of clofibric acid-induced genotoxicity in HEK293 cells expressing the human UDP-glucuronosyltransferases UGT1A3, UGT1A9, or UGT2B7, and screened three other carboxylic acid drugs for UGT-dependent genotoxicity. DNA damage was detected using the alkaline version of the comet assay. HEK293 cells were incubated for 18 h with vehicle (2.5 mM NaOH), 0.1-2.5 mM clofibric acid or 0.1-1.0 mM benoxaprofen, bezafibrate, or probenecid. To identify mechanisms underlying any observed genotoxicity, we treated UGT2B7 transfectants with 10 mM aminoguanidine, 1 mM borneol, or 2 mM desferrioxamine mesylate prior to co-incubation with I mM clofibric acid for 18 h. Compared to vehicle, clofibric acid, benoxaprofen, and probenecid produced significant DNA damage in all three UGT-transfected HEK293 cell lines, detectable from the lowest concentration tested. Bezafibrate caused DNA damage only at higher concentrations (1.0 mM) in UGT2B7- and UGT1A9-, but not UGT1A3-transfected cells. No drug-induced DNA damage was detected in untransfected cells, consistent With the limited glucuronidation capacity of these cells. The glycation/glycoxidation inhibitor aminoguanidine and the glucuronidation inhibitor borneol significantly decreased clofibric-acid-mediated DNA damage in UGT2B7 transfected cells by 73.5 and 94.8%, respectively. The inhibitor of transition -metal-catalyzed oxidation, desferrioxamine mesylate, had no significant effect on DNA damage. This study demonstrates the substrate-dependent role of human UGTs in the bioactivation of carboxylic acid drugs to genotoxic acyl glucuronide metabolites that are able to damage nuclear DNA via glycation and/or glycoxidation mechanisms.

    U2 - 10.1021/tx700188x

    DO - 10.1021/tx700188x

    M3 - Article

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    JO - Chemical Research in Toxicology

    JF - Chemical Research in Toxicology

    SN - 0893-228X

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    ER -