8-oxodG Lesions Research Articles

Antiestrogens and the Formation of DNA Damage in Rats: A Comparison

Tamoxifen (TAM) has been used as an agent for the treatment and prevention of breast cancer. However, long-term treatment of TAM in women increases the risk of developing endometrial cancer. The secondary cancer may be due to the genotoxicity of TAM. To find safer alternatives, four selective estrogen receptor modulators (SERMs), 4-hydroxytamoxifen (4-OHTAM), toremifene (TOR), raloxifene (RAL), and ICI 182,780, were administered to rats with an equimolar dose of TAM [54 micromol/kg (20 mg/kg)/day, p.o. for 7 days]. To evaluate the genotoxicity of each SERM, the presence of bulky DNA adducts was determined by (32)P-postlabeling/polyacrylamide gel electrophoresis and (32)P-postlabeling/high-performance liquid chromatography. The formation of 7,8-dihydro-8-oxodeoxyguanosine (8-oxodG) was analyzed as a marker of typical oxidative damage, using liquid chromatography electrospray tandem mass spectrometry. Among the SERMs, bulky DNA adducts were detected in the livers of rats treated with TAM; the total amount of TAM-DNA adducts was 26.1 adducts/10(7) nucleotides. However, with a detection limit of approximately 2 adducts/10(9) nucleotides, no bulky DNA adducts were observed with 4-OHTAM, TOR, RAL, or ICI 182,780. In addition, no significant increase of hepatic 8-oxodG lesions was detected in rats treated with any of the antiestrogens. Therefore, TOR, RAL, and ICI 182,780 are likely to be less genotoxic than TAM.

Effet protecteur des acides gras contre le stress oxydatif : Implication en physiopathologie vasculaire

Oxidative stress play a major role in the genesis and the evolution of vascular pathologies. Recent data suggest that redox mechanisms are involved in the signaling network of vascular cells and the physiology of the vascular wall. Experimental cell models allow to analyse the biological effect of pro-oxidants, and the regulation exerted by antioxidants and cytoprotective agents.Fatty acids play a complex role in atherosclerosis, since they exhibit both anti and proatherogenic properties, as function of their concentration and insaturation level. Beside their role in cell metabolism and mitochondrial b-oxidation, fatty acids activate various signaling pathways, and could modulate intracellular oxidative stress via an uncoupling effect on mitochondria. We report here that fatty acids protect against mitochondrial oxidative stress by activating cellular glutathione peroxidase (GPX) activity. This effect is independent of the chain length and unsaturation level. EGF receptor is activated by fatty acids and involved in their protective effect against mitochondrial oxidative stress and GPX activation, and similar protective results are observed with EGF. Oleic acid and EGF protect DNA against the formation of 8oxodG lesions occurring subsequently to oxidative stress. Taken together, this new ‘antioxidant’ and cytoprotective property of fatty acids and EGF could participate to the stabilization of atherosclerotic plaque, and the more general anti-atherogenic properties of oleic acid.

Two clustered 8-oxo-7,8-dihydroguanine (8-oxodG) lesions increase the point mutation frequency of 8-oxodG, but do not result in double strand breaks or deletions in Escherichia coli.

Multiply damaged sites (MDSs) are generated in DNA by ionizing radiation. In vitro studies predict that base excision repair in cells will convert MDSs to lethal double strand breaks (DSBs) when two opposing base damages are situated >/=2 bp apart. If the lesions are situated immediately 5' or 3' to each other, repair is predicted to occur sequentially due to inhibition of the DNA glycosylase by a single strand break repair intermediate. In this study, we examined how the distance between two opposing lesions alters the mutation frequency of an 8-oxodG in an MDS, and whether repair generates DSBs and deletions in bacteria. The 8-oxodG mutation frequency declined in MutY-deficient bacteria when the opposing 8-oxodG was 6 bp away, and was similar to a single 8-oxodG when the lesions were separated by 14 bp. However, the number of deletions detected for the MDSs was equivalent to the undamaged sequence. Using a separate assay, MDSs consisting of two 8-oxodG or an 8-oxodG opposite a uracil were not converted to DSBs in the absence of DNA replication in wild-type and transcription-coupled repair-deficient bacteria. This is the first study showing that DSB-repair intermediates and deletions are not formed during repair of clustered 8-oxodGs in cells.

Structural Basis for the Recognition of the FapydG Lesion (2,6-Diamino-4-hydroxy-5-formamidopyrimidine) by Formamidopyrimidine-DNA Glycosylase

Formamidopyrimidine-DNA glycosylase (Fpg) is a DNA repair enzyme that excises oxidized purines such as 7,8-dihydro-8-oxoguanine (8-oxoG) and 2,6-diamino-4-hydroxy-5-formamidopyrimidine (FapyG) from damaged DNA. Here, we report the crystal structure of the Fpg protein from Lactococcus lactis (LlFpg) bound to a carbocyclic FapydG (cFapydG)-containing DNA. The structure reveals that Fpg stabilizes the cFapydG nucleoside into an extrahelical conformation inside its substrate binding pocket. In contrast to the recognition of the 8-oxodG lesion, which is bound with the glycosidic bond in a syn conformation, the cFapydG lesion displays in the complex an anti conformation. Furthermore, Fpg establishes interactions with all the functional groups of the FapyG base lesion, which can be classified in two categories: (i) those specifying a purine-derived lesion (here a guanine) involved in the Watson-Crick face recognition of the lesion and probably contributing to an optimal orientation of the pyrimidine ring moiety in the binding pocket and (ii) those specifying the imidazole ring-opened moiety of FapyG and probably participating also in the rotameric selection of the FapydG nucleobase. These interactions involve strictly conserved Fpg residues and structural water molecules mediated interactions. The significant differences between the Fpg recognition modes of 8-oxodG and FapydG provide new insights into the Fpg substrate specificity.

Urinary 8-oxo-2'-deoxyguanosine: redox regulation of DNA repair in vivo?

DNA is susceptible to damage by reactive oxygen species (ROS). ROS are produced during normal and pathophysiological processes in addition to ionizing radiation, environmental mutagens, and carcinogens. 8-oxo-2'-deoxyguanosine (8-oxodG) is probably one of the most abundant DNA lesion formed during oxidative stress. This potentially mutagenic lesion causes G --> T transversions and is therefore an important candidate lesion for repair, particularly in mammalian cells. Several pathways exist for the removal, or repair, of this lesion from mammalian DNA. The most established is via the base excision repair enzyme, human 8-oxoguanine glycosylase (hOgg1), which acts in combination with the human apurinic endonuclease (hApe). The latter is known to respond to regulation by redox reactions and may act in combination with hOgg1. We discuss evidence in this review article concerning alternative pathways in humans, such as nucleotide excision repair (NER), which could possibly remove the 8-oxodG lesion. We also propose that redox-active components of the diet, such as vitamin C, may promote such repair, affecting NER specifically.

Anti-8-oxo-2′-deoxyguanosine Phage Antibodies: Isolation, Characterization, and Relationship to Disease States

We have used human single chain Fv (scFv) phage display antibody libraries to isolate recombinant antibodies against the DNA adduct 8-oxo-2′-deoxyguanosine (8-oxodG). One of these scFvs (175G) bound to several 8-oxodG-containing oligonucleotides whilst demonstrating no cross-reactivity with G-containing control oligonucleotides, and bound to 8-oxodG lesions introduced into DNA by treatment with methylene blue and white light. In addition, 175G inhibited the cleavage of an 8-oxodG-containing oligonucleotide by the Escherichia coli enzyme formamidopyrimidine-DNA glycosylase (Fpg). The nucleotide sequence of the 175G VH gene segment was 98% homologous to the published VH sequence of a human hybridoma derived from a patient with systemic lupus erythematosus (SLE). Sera from two SLE patients bound to damaged DNA, and this binding could be inhibited by 175G. The use of human scFv phage display libraries has thus produced a unique reagent with specificity for 8-oxodG, which may have a role in damage detection and quantitation and in modifying DNA repair activity. 175G also offers support to the hypothesis that SLE might be associated with oxidative damage to DNA.

Gamma-Ray footprinting and fluorescence polarization anisotropy of a 30-mer synthetic DNA fragment with one 2'-deoxy-7-hydro-8- oxoguanosine lesion.

The influence of the oxidative lesion 2'-deoxy-7-hydro-8-oxoguanosine (8-oxodG) on some conformational properties of DNA has been studied. Four 30-mer duplexes of the form [5'-GATCCTCTAGAGTC[G(*) or G]ACCTGCAGGCATGCA-3']:[3'-CTAGGAGATCTCAG[C or A]TGGACGTCCGTACGT-5'], in which G(*) is the 8-oxodG lesion, were synthesized in order to compare the effect of the GA mismatch and of the damaged G(*)C and G(*)A forms with the normal GC. Spectroscopic measurements performed by means of UV denaturation and circular dichroism experiments do not show gross changes of stability and overall structure in the damaged and mismatched samples. The control DNA and the samples containing GA mismatch show very similar gamma-rays cutting patterns, indicating that the introduction of the GA mismatch does not perturb the phosphate backbone geometry. In the samples containing the 8-oxodG there are some variations of the cleavage pattern near G(*) which are extended for almost one helical turn. Some differences are observed between G(*)C and G(*)A duplexes. In particular, in the G(*)C sample the reduced accessibility to OH radicals at the G15 site, observed in the control, spreads on the intrastrand adjacent bases and in the G(*)A sample a shift of the minimum is observed. The hydrodynamic radius R(h) derived by fluorescence polarization anisotropy decay exhibits a constant value of 11.4 +/- 0.2 A between 5 and 40 degrees C, in all the samples. The torsional constant alpha of each oligomer decreases when the temperature is raised and the alpha values of the damaged samples are higher than those of the normal ones.

Efficient in vitro repair of 7-hydro-8-oxodeoxyguanosine by human cell extracts: involvement of multiple pathways.

To investigate the repair of oxidative damage in DNA, we have established an in vitro assay utilizing human lymphoblastoid whole cell extracts and plasmid DNA damaged by exposure to methylene blue and visible light. This treatment has been shown to produce predominantly 7-hydro-8-oxodeoxyguanosine (8-oxodG) in double-stranded DNA at low levels of modification. DNA containing 1. 6 lesions per plasmid is substrate for efficient repair synthesis by cell extracts. The incorporation of dGMP is 2.7 +/- 0.5 times greater than the incorporation of dCMP, indicating an average repair patch of 3-4 nucleotides. Damage-specific nicking occurs within 15 min, while resynthesis is slower. The incorporation of dGMP increases linearly, while the incorporation of dCMP exhibits a distinct lag. Extracts from xeroderma pigmentosum (XP) complementation groups A and B exhibit 25 and 40%, respectively, of the incorporation of dCMP compared with normal extracts, but extracts from an XP-D cell line exhibit twice the activity. These data suggest that the efficient repair of 8-oxodG lesions observed in human cell extracts involves more than one pathway of base excision repair.

Leading versus lagging strand mutagenesis induced by 7,8-dihydro-8-oxo-2′-deoxyguanosine in Escherichia coli

We have previously shown that a single N-2-acetylaminofluorene (AAF) adduct bound to the C-8 position of a guanine residue located within plasmids containing the unidirectional ColE1 origin of replication induces a 20-fold higher mutation frequency when the adduct is located in the lagging strand as compared to the leading strand. In this study, single 7,8-dihydro-8-oxo-2′-deoxyguanosine (8-oxodG) lesions have been intro duced in the leading and lagging strand orientation within the same sequence context as for the AAF adducts. The induced frequency of guanine to thymine transversions has been measured, using a specific PCR-based quantitative assay, in strains deficient in the repair of the oxidative lesion. The potential involvement of the UvrABC excision repair system in the removal of 8-oxodG has also been investigated and ruled out. Concerning the mutation frequency asymmetry, in contrast to AAF adducts, 8-oxodG adducts induce the same mutation frequency, irrespective of their location in the leading or lagging strands. This striking difference between 8-oxodG and dGuo-C8-AAF adducts is discussed in terms of their differential capacity to block DNA replication.

Finding new mutator strains of Escherichia coli — a review

Mutations in the lacZ gene were constructed that allowed the detection of mutators that caused specific transversion mutations. This allowed the detection of two new mutator genes, mutt and mutM, which are involved in the repair of the 8-oxodguanine (8oxodG) lesion.

Site-specific mutagenesis using a gapped duplex vector: A study of translesion synthesis past 8-oxodeoxyguanosine in E. coli

We have constructed a gapped plasmid vector in which a single defined lesion is introduced, site-specifically, within a single-strand region. Efficiency of translesional synthesis is determined by the number of colonies recovered following transformation of E. coli. The nucleotide sequence of progeny plasmids in the gapped region of the vector reflects incorporation of bases opposite and near the lesion. The analysis detects non-mutagenic as well as mutagenic events. This system was used to establish the mutagenic potential of 2'-deoxy-7,8-dihydro-8-oxoguanosine (8-oxodG), a lesion produced by the action of active oxygen species on DNA. The presence of 8-oxodG did not affect the number of transformants recovered. Most transformants (greater than 99%) contained G:C pairs at the site of the lesion; however, a limited number of targeted G----T transversions were observed in the presence and absence of SOS induction. Base substitutions neighboring the lesion, reported for an in vitro system, were not observed. We conclude that the 8-oxodG lesion in DNA is weakly mutagenic in E. coli.