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.
Sallustio
B C
BC
Department of Clinical Pharmacology, Queen Elizabeth Hospital, Woodville, South Australia.
Harkin
L A
LA
Mann
M C
MC
Krivickas
S J
SJ
Burcham
P C
PC
D002995:Clofibric Acid / Q000378:metabolism / Q000633:toxicity*; D004277:DNA, Single-Stranded / Q000187:drug effects; D004926:Escherichia coli / Q000235:genetics; D015248:Gemfibrozil / Q000378:metabolism / Q000633:toxicity*; D005965:Glucuronates / Q000378:metabolism / Q000633:toxicity*; D009152:Mutagenicity Tests; D009153:Mutagens / Q000378:metabolism / Q000633:toxicity*; D010957:Plasmids / Q000235:genetics; D013014:SOS Response, Genetics
D004277:DNA, Single-Stranded; D005965:Glucuronates; D009153:Mutagens; D002995:Clofibric Acid; D015248:Gemfibrozil