Mutagenesis by Acrolein-Derived Propanodeoxyguanosine Adducts in Human Cells

Abstract

Acrolein, which is widely spread in the environment and is produced by lipid peroxidation in cells, reacts with DNA to form two exocyclic 1,N²-propanodeoxyguanosine (PdG) adducts. To establish their relative contribution to the acrolein mutagenicity, the genotoxic properties of α-OH-PdG and γ-OH-PdG together with their model DNA adduct, PdG, were studied in human cells. DNA adducts were incorporated site-specifically into a SV40/BK virus origin-based shuttle vector and replicated in xeroderma pigmentosum complementation group A (XPA) cells. Analysis of progeny plasmid revealed that α-OH-PdG and PdG strongly block DNA synthesis and that both adducts induced base substitutions with G → T transversions predominating. Primer extension studies, catalyzed by the 3‘→5‘ exonuclease-deficient Klenow fragment of Escherichia coli pol I, revealed limited extension from the 3‘ primer termini opposite these two adducts. In contrast, γ-OH-PdG did not strongly block DNA synthesis or miscode in XPA cells. Primer extension from a dC terminus opposite γ-OH-PdG was much more efficient than that opposite α-OH-PdG or PdG. These results indicate that the minor α-OH-PdG adduct is more genotoxic than the major γ-OH-PdG. Furthermore, experiments using a HeLa whole cell extract indicate that all three DNA adducts are not efficiently removed from DNA by base excision repair.