piperidines and 8-hydroxyguanine

It has been reported that oxidative stress and chronic inflammation may be involved in the pathogenesis of polycystic ovary syndrome (PCOS). 8-oxoguanine DNA glycosylase (OGG1) is the main glycosylase that catalyzes the excision of DNA oxidation products. In this study, we investigated the role and potential mechanisms of OGG1 in the development of PCOS. We first analyzed OGG1 levels in serum and follicular fluid (FF) of PCOS patients, and significantly elevated OGG1 levels were noted in PCOS patients. We similarly observed a significant upregulation of OGG1 expression levels in ovarian tissue of the dehydroepiandrosterone (DHEA)-induced PCOS rat model. In addition, increased apoptosis and increased production of reactive oxygen species (ROS) were observed after the addition of OGG1-specific inhibitor (TH5487) in human granulosa-like tumor cell line (KGN) cells following a concentration gradient, along with a significant decrease in mRNA levels of inflammatory factors such as CXCL2, IL-6, MCP1, IL-1β, and IL-18. Significant decreases in protein phosphorylation levels of P65 and IκBα were also observed in cells. In addition, we found a significant positive correlation between OGG1 and IL-6 expression levels in human and DHEA-induced PCOS rat models. In conclusion, our results suggest that OGG1 might be involved in the pathogenesis of PCOS by regulating the secretion of IL-6 through NF-κB signaling pathway, and there might be a balance between the inhibition of oxidative stress and the promotion of chronic inflammation by OGG1 on KGN cells.

Topics: Animals; Benzimidazoles; Dehydroepiandrosterone; DNA Glycosylases; Female; Guanine; Humans; Inflammation; Interleukin-6; NF-kappa B; Piperidines; Polycystic Ovary Syndrome; Rats; Signal Transduction

DNA damage caused by reactive oxygen species may result in genetic mutations or cell death. Base excision repair (BER) is the major pathway that repairs DNA oxidative damage in order to maintain genomic integrity. In mammals, eleven DNA glycosylases have been reported to initiate BER, where each recognizes a few related DNA substrate lesions with some degree of overlapping specificity. 7,8-dihydro-8-oxoguanine (8-oxoG), one of the most abundant DNA oxidative lesions, is recognized and excised mainly by 8-oxoguanine DNA glycosylase 1 (OGG1). Further oxidation of 8-oxoG generates hydantoin lesions, which are recognized by NEIL glycosylases. Here, we demonstrate that NEIL1, and to a lesser extent NEIL2, can potentially function as backup BER enzymes for OGG1 upon pharmacological inhibition or depletion of OGG1. NEIL1 recruitment kinetics and chromatin binding after DNA damage induction increase in cells treated with OGG1 inhibitor TH5487 in a dose-dependent manner, whereas NEIL2 accumulation at DNA damage sites is prolonged following OGG1 inhibition. Furthermore, depletion of OGG1 results in increased retention of NEIL1 and NEIL2 at damaged chromatin. Importantly, oxidatively stressed NEIL1- or NEIL2-depleted cells show excessive genomic 8-oxoG lesions accumulation upon OGG1 inhibition, suggesting a prospective compensatory role for NEIL1 and NEIL2. Our study thus exemplifies possible backup mechanisms within the base excision repair pathway.

Topics: Benzimidazoles; Cell Line; DNA; DNA Damage; DNA Glycosylases; DNA Repair; DNA-(Apurinic or Apyrimidinic Site) Lyase; Guanine; Humans; Kinetics; Mutation; Oxidative Stress; Piperidines; Prospective Studies; Reactive Oxygen Species

In Escherichia coli, endonuclease III (endo III) repairs the oxidation products of 8-OHGua. However, the corresponding repair enzymes in eukaryotes have not been identified. Here we report that 8-hydroxyguanine (8-OHGua) is highly sensitive to further oxidation. We also show that Ntg2, a functional homolog of endo III in Saccharomyces cerevisiae, is capable of nicking the irradiated duplex DNA containing 8-OHGua. Moreover, Ntg2 formed a stable complex with the DNA upon incubation with NaBH(4). In contrast, Ntg1, another functional homolog of endo III, showed no such activities. These findings indicate that Ntg2 is, at least in part, responsible for repairing the oxidation products of 8-OHGua in eukaryotic cells.

Topics: Borohydrides; Deoxyadenosines; Deoxycytidine; Deoxyguanosine; DNA; DNA-(Apurinic or Apyrimidinic Site) Lyase; DNA-Formamidopyrimidine Glycosylase; Dose-Response Relationship, Radiation; Escherichia coli Proteins; Gamma Rays; Guanine; Hydrogen Peroxide; Hydroxyl Radical; Iron; Macromolecular Substances; N-Glycosyl Hydrolases; Oxidation-Reduction; Piperidines; Protein Binding; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; Substrate Specificity; Thymidine

Synthetic oligodeoxynucleotides containing secondary oxidative lesions at guanine nucleobases have been prepared by the site-specific oxidation by ONOO(-) of oligomers containing 8-oxoguanine (8-oxo-G). The oligomers have been tested for their stability to the standard hot piperidine treatment that is commonly used to uncover oxidized DNA lesions. While DNA containing oxaluric acid and oxazolone was cleaved at the site of modification under hot piperidine conditions, the corresponding cyanuric acid and 8-oxo-G lesions were resistant to piperidine. The recognition of the oxidative lesions by formamidopyrimidine glycosylase (Fpg enzyme) was examined in double-stranded versions of the synthetic oligodeoxynucleotides. Fpg efficiently excised 8-oxo-G and oxaluric acid and to some extent oxazolone, but not cyanuric acid. These data suggest that some DNA lesions formed via ONOO(-) exposures (cyanuric acid) are not repaired by Fpg and are not uncovered by assays based on piperidine cleavage at the site of lesion. Our results indicate that cryptic secondary and tertiary oxidation products arising from 8-oxo-G may contribute to the overall mutational spectra arising from oxidative stress.

Topics: Chromatography, High Pressure Liquid; DNA; DNA Repair; DNA-Formamidopyrimidine Glycosylase; Guanine; N-Glycosyl Hydrolases; Nitrates; Oligonucleotides; Oxidants; Oxidation-Reduction; Piperidines; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization

Two kinds of double-stranded oligonucleotides containing a single 8-oxo-7,8-dihydroguanine were labeled with (32)P at their 5' ends and exposed to gamma rays in the frozen aqueous state at 77 K, where both direct and quasi-direct effects of ionizing radiation predominate. Analysis of the oligonucleotides with 20% denaturing polyacrylamide gel electrophoresis revealed no difference in the immediate induction of strand breaks between oligonucleotides containing 8-oxo-7,8-dihydroguanine and their corresponding oligonucleotides with normal guanine, but piperidine-sensitive damage was induced more frequently in the former than in the latter. Sequence analysis of irradiated oligonucleotides showed that not only 8-oxo-7,8-dihydroguanine but also its neighboring bases and the cytosine residue that is paired to it became piperidine-sensitive in both oligonucleotides. These results suggest that 8-oxo-7,8-dihydroguanine, its neighboring bases and the opposite cytosine are candidates for radiation damage hot spots.

Topics: Cold Temperature; DNA Damage; Electrophoresis, Polyacrylamide Gel; Gamma Rays; Guanine; Humans; Oligonucleotides; Piperidines; Water

Several studies have shown that ionizing radiation generates a wide spectrum of lesions to DNA including base modifications, abasic sites, strand breaks, crosslinks and tandem base damage. One example of tandem base damage induced by @OH radical inX-irradiated DNA oligomers is N -(2-deoxy-beta-d- erythro -pentofuranosyl)-formylamine/8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodGuo). In order to investigate the biological significance of such a tandem lesion, both 8-oxo-7,8-dihydroguanine and formylamine were introduced into synthetic oligonucleotides at vicinal positions using the solid phase phosphoramidite method. For this purpose, a new convenient method of synthesis of 8-oxodGuo was developed. The purity and integrity of the modified synthetic DNA fragments were assessed using different complementary techniques including HPLC, polyacrylamide gel electrophoresis, electrospray and MALDI-TOF mass spectrometry. The piperidine test applied to the double modified base-containing oligonucleotides revealed the high alkaline lability of formylamine in DNA. In addition, various enzymatic experiments aimed at determining biochemical features of such multiply damaged sites were carried out using the synthetic substrates. The pro-cessing of the vicinal lesions by nuclease P1, snake venom phosphodiesterase, calf spleen phospho-diesterase and repair enzymes including Escherichia coli endonuclease (endo) III and Fapy-glycosylase was studied and is reported.

Topics: Alkaline Phosphatase; Animals; Base Pairing; Cattle; Deoxyribonuclease (Pyrimidine Dimer); DNA Damage; DNA-Formamidopyrimidine Glycosylase; Endodeoxyribonucleases; Escherichia coli Proteins; Exonucleases; Guanine; N-Glycosyl Hydrolases; Oligodeoxyribonucleotides; Phosphodiesterase I; Phosphoric Diester Hydrolases; Piperidines; Single-Strand Specific DNA and RNA Endonucleases; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization

The frequency of oxidative base damage, such as 8-hydroxyguanine (8-OH-Gua), was determined at the nucleotide level of resolution using the ligation-mediated PCR technique. Administration of a renal carcinogen, ferric nitrilotriacetate (Fe-NTA), is known to induce oxidative stress and subsequent formation of 8-OH-Gua in the rat kidney. Whole genomic DNA was isolated from the rat kidney after or without Fe-NTA treatment and then cleaved with hot piperidine. In order to assess the frequency of 8-OH-Gua formation, we chose three genes, the tumor suppressor gene p53, the heat shock protein 70 (HSP70-1) gene and the Na,K-ATPase alpha1 subunit gene. No alteration in the cleavage profile was observed in the p53 and HSP70 genes after Fe-NTA treatment. In the case of the p53 gene, a low incidence of point mutations has been observed in this carcinogenesis system. On the other hand, time-dependent alterations, corresponding to the time course of overall 8-OH-Gua formation and repair, were detected in the promoter region of the Na,K-ATPase alpha1 subunit gene. GpG and GpGpG in specific regions seem to be hotspots for the formation of 8-OH-Gua. These results were confirmed by formamidopyrimidine-DNA glycosylase-dependent DNA cleavage patterns. Thus, oxidative base damage, such as 8-OH-Gua, was not distributed uniformly along the whole genome, but seemed to be restricted to particular genes and regions.

Topics: Animals; Base Sequence; Carcinogens; DNA; DNA Damage; Ferric Compounds; Guanine; HSP70 Heat-Shock Proteins; Kidney; Male; Molecular Sequence Data; Nitrilotriacetic Acid; Piperidines; Protozoan Proteins; Rats; Rats, Wistar; Sodium-Potassium-Exchanging ATPase; Tumor Suppressor Protein p53

The two findings indicate that 8-hydroxyguanine(8-OH-Gua) is a hot piperidine-sensitive lesion in DNA. These are cleavages of DNA containing 8-OH-Gua at the site of this residue and decomposition of 8-hydroxydeoxyguanosine when the DNA and nucleoside were treated in 1 M piperidine for 30 min at 90 degrees C. However, no cleavage was observed in DNA containing 8-hydroxyadenine or O6-methylguanine. 8-OH-Gua was found to be different from apurinic sites that are also alkali-labile lesions since the former was more resistant to alkali treatment. This property of 8-OH-Gua can be used as a check for the incorporation of this base into DNA after the synthesis of DNA containing 8-OH-Gua at a specific position or possibly can be one of the markers for the identification of 8-OH-Gua formed in DNA exposed to reactive oxygen species.

Topics: Base Sequence; DNA; DNA Damage; Guanine; Hot Temperature; Molecular Sequence Data; Oligodeoxyribonucleotides; Piperidines; Spectrophotometry, Ultraviolet