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.

Instability of repetitive sequences is a hallmark of human cancer, and its enhancement has been linked to oxidative stress. Malondialdehyde is an endogenous product of oxidative stress that reacts with guanine to form the exocyclic adduct, pyrimido[1,2-␣]purin-10(3H)-one (M1G). We used site-specifically modified single-and double-stranded vectors to investigate the mutagenic potential of M1G in bacteria and mammalian cells. M1G induced frameshift mutations (؊1 and ؊2) when positioned in a reiterated (CpG)4 sequence but not when positioned in a nonreiterated sequence in Escherichia coli and in COS-7 cells. The frequency of frameshift mutations was highest when M1G was placed at the third G in the sequence. M1G induced base pair substitutions at comparable frequencies in both sequence contexts in COS-7 cells. These studies indicate that M1G, an endogenously generated product of oxidative stress, induces sequence-dependent frameshift mutations and base pair substitutions in bacteria and in mammalian cells. This finding suggests a potential role for the M1G lesion in the induction of mutations commonly associated with human diseases.

3-(2-Deoxy-␤-D-erythro-pentofuranosyl)pyrimido[1,2-␣]purin-10(3H)-one (M1dG) is a DNA adduct arising from the reaction of 2-deoxyguanosine with the lipid peroxidation product, malondialdehyde, or the DNA peroxidation product, base propenal. M 1dG is mutagenic in bacteria and mammalian cells and is present in the genomic DNA of healthy human beings. It is also detectable, albeit at low levels, in the urine of healthy individuals, which may make it a useful biomarker of DNA damage linked to oxidative stress. We investigated the possibility that the low urinary levels of M 1dG reflect metabolic conversion to derivatives. M 1dG was rapidly removed from plasma (t1/2 ‫؍‬ 10 min) after i.v. administration to rats. A single urinary metabolite was detected that was identified as 6-oxo-M 1dG by MS, NMR spectroscopy, and independent chemical synthesis. 6-Oxo-M1dG was generated in vitro by incubation of M 1dG with rat liver cytosols, and studies with inhibitors suggested that xanthine oxidase and aldehyde oxidase are involved in the oxidative metabolism. M1dG also was metabolized by three separate human liver cytosol preparations, indicating 6-oxo-M1dG is a likely metabolite in humans. This represents a report of the oxidative metabolism of an endogenous DNA adduct and raises the possibility that other endogenous DNA adducts are metabolized by oxidative pathways. 6-Oxo-M1dG may be a useful biomarker of endogenous DNA damage associated with inflammation, oxidative stress, and certain types of cancer chemotherapy.excretion ͉ inflammation ͉ DNA damage ͉ oxidation ͉ metabolite D NA damage from endogenous sources is believed to contribute significantly to human genetic diseases, including cancer (1, 2). A major cause of endogenous DNA damage is oxidation (3, 4). Agents such as hydroxyl radical or peroxynitrous acid oxidize nucleic acid bases or the deoxyribose backbone to form miscoding lesions or induce strand breaks (5). In addition, oxidation of other cellular constituents (i.e., lipid, protein) generates reactive derivatives that form adducts with nucleic acid bases (1). In the absence of repair, this panoply of damage results in mutations, triggers signaling to arrest cell division, or induces apoptosis.3-(2-Deoxy-␤-D-er ythro-pentofuranosyl)pyrimido[1,2-␣]purin-10(3H)-one (M 1 dG) is an adduct found at varying levels in genomic DNA of rodents and humans (6-8). It is derived from the lipid oxidation product, malondialdehyde, or the DNA oxidation product, base propenal (Scheme 1) (9-11). M 1 dG is miscoding when assayed by in vitro DNA replication and is mutagenic in bacterial and mammalian cells (12)(13)(14). It induces base pair substitutions (M 1 dG3T and M 1 dG3A) and frameshift mutations in reiterated sequences (e.g., CG n ) when shuttle vectors containing a site-specific lesion are replicated in COS-7 cells (14). Genetic and biochemical experiments indicate that M 1 dG is removed by nucleotide excision repair in both bacterial and mammalian cells (13-15).As a prerequisite for preclinical, clinical, and populationbased ...

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'‐Trihydroxyisoflavanone 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.

Template primers containing propanodeoxyguanosine (PdG) in two different sequence contexts (CPdG-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 V max /K m values for incorporation of dGMP were similar in both sequence contexts, whereas the V max /K m 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. Fulllength 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 3 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.