Genotoxicity of acyl glucuronide metabolites formed from clofibric acid and gemfibrozil: a novel role for phase-II-mediated bioactivation in the hepatocarcinogenicity of the parent aglycones?

Glucuronides formed from carboxylate-containing xenobiotics are more chemically reactive than most Phase II conjugates. However, while they have been shown to form protein adducts, their reactions with DNA have received little attention. We thus used the M13 forward mutational assay to assess the genotoxicity of acyl glucuronides formed from two widely used fibrate hypolipidemics, clofibric acid and gemfibrozil. Single-stranded M13mp19 bacteriophage DNA was incubated in pH 7.4 buffer for 16 h in the presence of 0, 1, 2.5, and 5 mM concentrations of each glucuronide as well as the respective aglycones. The modified DNA was then transfected into SOS-induced competent Escherichia coli JM105 cells and the transfection efficiency was determined after phage growth overnight at 37 degrees C. Significantly, both acyl glucuronides, but not the aglycones, caused a concentration-dependent decrease in the transfection efficiency of the DNA, with a greater than 80% decrease in phage survival produced by the 5 mM concentrations of the glucuronides. No increase in lacZa mutations accompanied the loss of phage survival. We propose that these genotoxic effects involve reactions with nucleophilic centers in DNA via a Schiff base mechanism that is analogous to the glycosylation of DNA by endogenous sugars. Since strand nicking is known to accompany such damage, we also analyzed glucuronide-treated pSP189 plasmids for strand breakages via agarose gel electrophoresis. Both clofibric acid and gemfibrozil glucuronides produced significant concentration-related strand nicking and exhibited over 10-fold greater reactivity than the endogenous glycosylating agent, glucose 6-phosphate. On the basis of these findings, the possibility that this novel bioactivation route participates in the carcinogenicity of the fibrate hypolipidemics deserves investigation.

[1]  P. Burcham,et al.  Role of G-->T transversions in the mutagenicity of alkylperoxyl radicals: induction of alkali-labile sites in bacteriophage M13mp19. , 1997, Chemical Research in Toxicology.

[2]  B. Sallustio,et al.  Interaction of human serum albumin with the electrophilic metabolite 1-O-gemfibrozil-beta-D-glucuronide. , 1997, Drug metabolism and disposition: the biological fate of chemicals.

[3]  M. Bailey,et al.  Chemical and immunochemical comparison of protein adduct formation of four carboxylate drugs in rat liver and plasma. , 1996, Chemical research in toxicology.

[4]  B. Sallustio,et al.  Reactivity of gemfibrozil 1-o-beta-acyl glucuronide. Pharmacokinetics of covalently bound gemfibrozil-protein adducts in rats. , 1995, Drug Metabolism And Disposition.

[5]  R. Bucala,et al.  Identification of N2-(1-carboxyethyl)guanine (CEG) as a guanine advanced glycosylation end product. , 1995, Biochemistry.

[6]  C. Fenselau,et al.  Characterization and formation of the glutathione conjugate of clofibric acid. , 1995, Drug metabolism and disposition: the biological fate of chemicals.

[7]  T G Myers,et al.  Metabolic activation and immunochemical localization of liver protein adducts of the nonsteroidal anti-inflammatory drug diclofenac. , 1994, Chemical research in toxicology.

[8]  S Kettle,et al.  Mechanistically-based Human Hazard Assessment of Peroxisome Proliferator-induced Hepatocarcinogenesis , 1994, Human & experimental toxicology.

[9]  L. Benet,et al.  Irreversible binding of tolmetin to macromolecules via its glucuronide: binding to blood constituents, tissue homogenates and subcellular fractions in vitro. , 1994, Xenobiotica; the fate of foreign compounds in biological systems.

[10]  A. Burlingame,et al.  Evidence for covalent binding of acyl glucuronides to serum albumin via an imine mechanism as revealed by tandem mass spectrometry. , 1993, Proceedings of the National Academy of Sciences of the United States of America.

[11]  U. Boelsterli,et al.  Diclofenac covalent protein binding is dependent on acyl glucuronide formation and is inversely related to P450-mediated acute cell injury in cultured rat hepatocytes. , 1993, Toxicology and applied pharmacology.

[12]  P. Smith,et al.  Nonenzymic glycation of albumin by acyl glucuronides in vitro. Comparison of reactions with reducing sugars. , 1992, Biochemical pharmacology.

[13]  G. Mulder.  Glucuronidation and its role in regulation of biological activity of drugs. , 1992, Annual review of pharmacology and toxicology.

[14]  L. Benet,et al.  Acyl glucuronides revisited: is the glucuronidation process a toxification as well as a detoxification mechanism? , 1992, Drug metabolism reviews.

[15]  K. Knights,et al.  In vivo covalent binding of clofibric acid to human plasma proteins and rat liver proteins. , 1991, Biochemical pharmacology.

[16]  A. Cerami,et al.  The formation of reactive intermediate(s) of glucose 6-phosphate and lysine capable of rapidly reacting with DNA. , 1987, Mutation research.

[17]  C. Fenselau,et al.  Reaction of 1-O-acyl glucuronides with 4-(p-nitrobenzyl)pyridine. , 1986, Drug metabolism and disposition: the biological fate of chemicals.

[18]  R. Bucala,et al.  Modification of DNA by glucose 6-phosphate induces DNA rearrangements in an Escherichia coli plasmid. , 1985, Proceedings of the National Academy of Sciences of the United States of America.

[19]  T. Kunkel.  Mutational specificity of depurination. , 1984, Proceedings of the National Academy of Sciences of the United States of America.

[20]  R. Bucala,et al.  Modification of DNA by reducing sugars: a possible mechanism for nucleic acid aging and age-related dysfunction in gene expression. , 1984, Proceedings of the National Academy of Sciences of the United States of America.

[21]  J. Sambrook,et al.  Molecular Cloning: A Laboratory Manual , 2001 .

[22]  C. Schlatter,et al.  Lack of covalent binding to rat liver DNA of the hypolipidemic drugs clofibrate and fenofibrate. , 1981, Toxicology letters.