Evaluation of the mutagenic potential of the principal DNA adduct of acrolein.

Acrolein is produced extensively in the environment by incomplete combustion of organic materials, and it arises endogenously in humans as a metabolic by-product. Acrolein reacts with DNA at guanine residues to form the exocyclic adduct, 8-hydroxypropanodeoxyguanosine (HOPdG). Acrolein is mutagenic, and a correlation exists between HOPdG levels in Salmonella typhimurium treated with acrolein and a resultant increase in mutation frequency. Site-specifically modified oligonucleotides were used to explore the mutagenic potential of HOPdG in Escherichia coli strains that were either wild-type for repair or deficient in nucleotide excision repair or base excision repair. Oligonucleotides modified with HOPdG were inserted into double-stranded bacteriophage vectors using the gapped-duplex method or into single-stranded bacteriophage vectors and transformed into SOS-induced E. coli strains. Progeny phage were analyzed by oligonucleotide hybridization to establish the mutation frequency and the spectrum of mutations produced by HOPdG. The correct base, dCMP, was incorporated opposite HOPdG in all circumstances tested. In contrast, in vitro lesion bypass studies showed that HOPdG causes misincorporation opposite the modified base and is a block to replication. The combination of these studies showed that HOPdG is not miscoding in vivo at the level of sensitivity of these site-specific mutagenesis assays.

Pyrimido[1,2-alpha]purin-10(3H)-one: a reactive electrophile in the genome.

Malondialdehyde and base propenal react with deoxyguanosine residues in DNA to form an exocyclic adduct, pyrimido[1, 2-alpha]purin-10(3H)-one (1), that has been detected at high levels in genomic DNA of healthy humans. Previous studies have shown that tris(hydroxymethyl)aminomethane adds to 1 at elevated pH, forming an enaminoimine (2), but it is uncertain whether 1 reacts directly or hydrolyzes under basic conditions to N(2)-(3-oxo-1-propenyl)deoxyguanosine (3) prior to amine addition. We report that 1 reacts at neutral pH with hydroxylamines to form oximes. The rate of reaction of 1 with hydroxylamines at pH 7 is at least 150 times faster than the rate of hydrolysis of 1 to 3. Thus, 1 is directly reactive to nucleophiles. These observations indicate that 1 is an electrophile in the human genome that may react with cellular nucleophiles to form novel cross-linked adducts.

Replication of template-primers containing propanodeoxyguanosine by DNA polymerase beta. Induction of base pair substitution and frameshift mutations by template slippage and deoxynucleoside triphosphate stabilization.

Propanodeoxyguanosine (PdG) is a model for several unstable exocyclic adducts formed by reaction of DNA with bifunctional carbonyl compounds generated by lipid peroxidation. The effect of PdG on DNA synthesis by human DNA polymerase beta was evaluated using template-primers containing PdG at defined sites. DNA synthesis was conducted in vitro and the products were analyzed by polyacrylamide gel electrophoresis and DNA sequencing. The extent of PdG bypass was low and the products comprised a mixture of base pair substitutions and deletions. Sequence analysis of all of the products indicated that the deoxynucleoside monophosphate incorporated "opposite" PdG was complementary to the base 5' to PdG in the template strand. These findings are very similar to recent results of Efrati et al. (Efrati, E., Tocco, G., Eritja, R., Wilson, S. H., and Goodman, M. F. (1997) J. Biol. Chem. 272, 2559-2569) obtained in DNA replication of template-primers containing abasic sites and suggest that PdG is a non-informational lesion when acted upon by polymerase (pol) beta. In addition to base pair substitutions and one- or two-base deletions, a four-base deletion was observed and the mechanism of its formation was probed by site-specific mutagenesis. The results indicated that this deletion occurred by one-base insertion followed by slippage to form a four-base loop followed by extension. All of the observations on pol beta replication of PdG-containing template-primers are consistent with a mechanism of lesion bypass that involves template slippage and dNTP stabilization followed by deoxynucleoside monophosphate incorporation and extension. This mechanism of PdG bypass is completely different than that previously determined for the Klenow fragment of DNA polymerase I and is consistent with recent structural models for DNA synthesis by pol beta.

Sequence-dependent induction of base pair substitutions and frameshifts by propanodeoxyguanosine during in vitro DNA replication.

Template primers containing propanodeoxyguanosine (PdG) in two different sequence contexts (C-PdG-C and T-PdG-T) were replicated by the Klenow fragment of DNA polymerase I. The presence of PdG in the template strand reduced the extent of in vitro DNA synthesis 10(3) - 10(4)-fold compared with unmodified template primers. Partial blockade was observed 1 base 3' to the adduct and opposite the adduct. Purines were preferentially incorporated opposite the adduct; the Vmax/Kmvalues for incorporation of dGMP were similar in both sequence contexts, whereas the Vmax/Km for dAMP incorporation increased 4.7-fold when the base pair 3' to PdG was changed from C:G to T:A. Oligonucleotides containing 1- and 2-base deletions were major products of replication in both sequence contexts. Full-length products were observed with templates containing T-PdG-T but not C-PdG-C. The major full-length product resulted from incorporation of dAMP residues opposite PdG. Kinetic analysis revealed that the major factor contributing to the selective incorporation of dAMP in full-length products was preferential extension of template primers containing PdG:dA termini rather than preferential incorporation of dAMP opposite PdG. The observation of PdG --> T mutations in the T-PdG-T context but not the C-PdG-C context during in vitro DNA replication parallels findings of in vivo experiments that base pair substitutions are induced by PdG in the former sequence context but not the latter.

Reaction mechanism of oxidative rearrangement of flavanone in isoflavone biosynthesis.

Microsomes that were prepared from elicitor-treated Pueraria lobata cell cultures catalyzed the conversion of liquiritigenin, a flavanone, into daidzein, an isoflavone. The reaction was resolved into two steps. 2, 7, 4'-Trihydroxyisoflavonone was formed as a major product when liquiritigenin was incubated with carefully washed microsomes in the presence of NADPH. The structure of 2, 7, 4'-trihydroxyisoflavanone was confirmed by mass and 1H NMR spectroscopies. The enzyme responsible for this rearrangement reaction is a cytochrome P-450-dependent monooxygenase. Upon treatment with a soluble enzyme fraction 2, 7, 4'-trihydroxyisoflavone yielded daidzein quantitatively. The incorporation of 18O from 18O2 into the 2-hydroxy group of 2, 7, 4'-trihydroxyisoflavanone was demonstrated by the shift of molecular ion in its mass spectrum. Based on these observations a new reaction mechanism, hydroxylation associated with 1,2-migration, is proposed for the oxidative rearrangement reaction catalyzed by the cytochrome P-450 enzyme of Pueraria lobata.