Small-molecule activation of OGG1 increases oxidative DNA damage repair by gaining a new function.

Oxidative DNA damage is recognized by 8-oxoguanine (8-oxoG) DNA glycosylase 1 (OGG1), which excises 8-oxoG, leaving a substrate for apurinic endonuclease 1 (APE1) and initiating repair. Here, we describe a small molecule (TH10785) that interacts with the phenylalanine-319 and glycine-42 amino acids of OGG1, increases the enzyme activity 10-fold, and generates a previously undescribed ß,δ-lyase enzymatic function. TH10785 controls the catalytic activity mediated by a nitrogen base within its molecular structure. In cells, TH10785 increases OGG1 recruitment to and repair of oxidative DNA damage. This alters the repair process, which no longer requires APE1 but instead is dependent on polynucleotide kinase phosphatase (PNKP1) activity. The increased repair of oxidative DNA lesions with a small molecule may have therapeutic applications in various diseases and aging.

Chinese bayberry (Myrica rubra) phenolics mitigated protein glycoxidation and formation of advanced glycation end-products: A mechanistic investigation.

Protein glycation and formation of advanced glycation end-products (AGEs) impose threats to the human health. This study firstly investigated the inhibition of Chinese bayberry (Myrica rubra) phenolics on AGEs formation through mechanistic analysis. Four common Chinese bayberry cultivars were selected to prepare phenolic-rich extracts (CBEs) and characterized for phenolic composition, and their anti-AGE properties were evaluated in multiple in vitro systems. Total sixteen phenolics were quantified in CBEs by UPLC-ESI-MS/MS. CBEs reduced total and specific fluorescent AGEs formation in various simulating models, and protected the protein from structural modification, oxidation, and cross-linking. Mechanistic analysis unveiled that scavenging of free radicals, inactivation of transition metals, interaction with protein to form complexes, and trapping of reactive α-dicarbonyls to form adducts underlain the mechanisms of the anti-glycative actions of CBEs. Chinese bayberry fruits, especially the cultivars Biqi and Wuzi, may be a promising dietary strategy to mitigate AGEs load in the human body.

NEIL1 and NEIL2 Are Recruited as Potential Backup for OGG1 upon OGG1 Depletion or Inhibition by TH5487.

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.

Effects of airborne toxicants on pulmonary function and mitochondrial DNA damage in rodent lungs.

Inhalation of airborne toxicants such as cigarette smoke and ozone is a shared health risk among the world's populations. The use of toxic herbicides like paraquat (PQ) is restricted by many countries, yet in the developing world PQ has demonstrable ill effects. The present study examined changes in pulmonary function, mitochondrial DNA (mtDNA) integrity and markers of DNA repair induced by acute or repeated exposure of PQ to rats. Similar to cigarette smoke and ozone, PQ promotes oxidative stress, and the impact of PQ on mtDNA was compared with that obtained with these agents. Tracheal instillation (i.t.) of PQ (0.01-0.075 mg/kg) dose dependently increased Penh (dyspnoea) by 48 h while body weight and temperature declined. Lung wet weight and the wet/dry weight ratio rose; for the latter, by as much as 52%. At low doses (0.02 and 0.03 mg/kg), PQ increased Penh by about 7.5-fold at 72 h. It quickly waned to near baseline levels. The lung wet/dry weight ratio remained elevated 7 days after administration coincident with marked inflammatory cell infiltrate. Repeated administration of PQ (1 per week for 8 weeks) resulted in a similar rise in Penh on the first instillation, but the magnitude of this response was markedly attenuated upon subsequent exposures. Pulmonary [lactate] and catalase activity, [8-oxodG] and histone fragmentation (cell death) were significantly increased. Repeated PQ instillation downregulated the expression of the mitochondrial-encoded genes, mtATP8, mtNd2 and mtcyB and nuclear ones for the DNA glycosylases, Ogg1, Neil1, Neil2 and Neil3. Ogg1 protein content decreased after acute and repeated PQ administration. mtDNA damage or changes in mtDNA copy number were evident in lungs of PQ-, cigarette smoke- and ozone-exposed animals. Taken together, these data indicate that loss of pulmonary function and inflammation are coupled to the loss of mtDNA integrity and DNA repair capability following exposure to airborne toxicants.

No oxidative stress or DNA damage in peripheral blood mononuclear cells after exposure to particles from urban street air in overweight elderly.

Exposure to traffic-related particulate matter (PM) has been associated with increased risk of lung disease, cancer and cardiovascular disease especially in elderly and overweight subjects. The proposed mechanisms involve intracellular production of reactive oxygen species (ROS), inflammation and oxidation-induced DNA damage studied mainly in young normal-weight subjects. We performed a controlled cross-over, randomised, single-blinded, repeated-measure study where 60 healthy subjects (25 males and 35 females) with age 55-83 years and body mass index above 25 kg/m(2) were exposed for 5h to either particle-filtered or sham-filtered air from a busy street with number of concentrations and PM2.5 levels of 1800/cm(3) versus 23 000/cm(3) and 3 µg/m(3) versus 24 µg/m(3), respectively. Peripheral blood mononuclear cells (PBMCs) were collected and assayed for production of ROS with and without ex vivo exposure to nanosized carbon black as well as expression of genes related to inflammation (chemokine (C-C motif) ligand 2, interleukin-8 and tumour necrosis factor), oxidative stress response (heme oxygenase (decycling)-1) and DNA repair (oxoguanine DNA glycosylase). DNA strand breaks and oxidised purines were assayed by the alkaline comet assay. No statistically significant differences were found for any biomarker immediately after exposure to PM from urban street air although strand breaks and oxidised purines combined were significantly associated with the particle number concentration during exposure. In conclusion, 5h of controlled exposure to PM from urban traffic did not change the gene expression related to inflammation, oxidative stress or DNA repair, ROS production or oxidatively damaged DNA in PBMCs from elderly overweight human subjects.

Micronucleus, alkaline, and human 8-oxoguanine glycosylase 1 modified comet assays evaluation of glass-ionomer cements - in vitro.

The purpose of this study was to evaluate the genotoxic potential of components leached from two conventional self-curing glass-ionomer cements (Fuji IX and Ketac Molar), and light-curing, resin modified glass-ionomer cements (Vitrebond, Fuji II LC). Evaluation was performed on human lymphocytes using alkaline and hOGG1 modified comet, and micronucleus assays. Each material, polymerised and unpolymerised, was eluted in extracellular saline (1 cm2 mL-1) for 1 h, 1 day, and 5 days. Cultures were treated with eluates using final dilutions of 10(-2), 10(-3), and 10(-4). Alkaline comet assay did not detect changes in DNA migration of treated cells regardless of the ionomer tested, polymerisation state, and elution duration. Glass ionomers failed to significantly influence micronucleus frequency. No oxidative DNA damage in treated lymphocytes was observed using hOGG1 modified comet assay. Obtained results indicate high biocompatibility of all tested materials used in the study under experimental conditions.

[Effect of hOGG1 gene on the oxidative damage of mitochondrial DNA induced by hexavalent chromium].

OBJECTIVE: To explore the effect of hOGG1 gene on the repair capability of oxidative damage in mitochondrial DNA induced by hexavalent chromium (Cr (VI)). METHODS: After incubating with Cr(VI) at the concentrations of 2, 8, 32 micromol/L for 24 hours, mitochondria from L-02 hepatocytes were collected. Cellular ROS and hOGG1 mRNA and mitochondrial 8-OHdG and hOGG1 protein in L-02 hepatocytes were examined by Fluorometric Assay Kit, RT-qPCR, 8-OHdG ELISA Kit and western blot respectively. RESULTS: Compared with the control group, the levels of cellular ROS and mitochondrial 8-OHdG in 8 and 32 micromol/L Cr(VI) treated groups were significantly increased (P < 0.05). The levels of cellular hOGG1 mRNA and mitochondrial hOGG1 protein in 2 micromol/L Cr (VI) treated group were increased significantly (P < 0.05), but decreased in 32 micromol/l. group (P < 0.05). CONCLUSION: Overproduction of cellular ROS could be induced by Cr (VI) and could result in genomic DNA oxidative damage, and the lower expression of cellular hOGG1 gene could decrease the repair capability of mitochondrial DNA. Therefore, the cellular hOGG1 gene exerted important effect in the repair of mitochondrial DNA oxidative damage induced by Cr( VI).

The Fanconi anemia pathway promotes DNA glycosylase-dependent excision of interstrand DNA crosslinks.

Fanconi anemia (FA) is a recessive cancer prone syndrome featuring bone marrow failure and hypersensitivity to DNA interstrand crosslinks (ICLs) and, to a milder extension, to ionizing radiation and oxidative stress. Recently, we reported that human oxidative DNA glycosylase, NEIL1 excises with high efficiency the unhooked crosslinked oligomer within three-stranded DNA repair intermediate induced by photoactivated psoralen exposure. Complete reconstitution of repair of the ICL within a three-stranded DNA structure shows that it is processed in the short-patch base excision repair (BER) pathway. To examine whether the DNA damage hypersensitivity in FA cells follows impaired BER activities, we measured DNA glycosylase and AP endonuclease activities in cell-free extracts from wild-type, FA, and FA-corrected cells. We showed that immortalized lymphoid cells of FA complementation Groups A, C, and D and from control cells from normal donors contain similar BER activities. Intriguingly, the cellular level of NEIL1 protein strongly depends on the intact FA pathway suggesting that the hypersensitivity of FA cells to ICLs may, at least in part, arise from downregulation or degradation of NEIL1. Consistent with this result, plasmid-based expression of the FLAG-tagged NEIL1 protein partially complements the hypersensitivity FA cells to the crosslinking agents exposures, suggesting that NEIL1 specifically complements impaired capability of FA cells to repair ICLs and oxidative DNA damage. These findings shed light to how the FA pathway may regulate DNA repair proteins and bring explanation for the long-time disputed problem of the oxidative stress sensitive phenotype of FA cells.

Inactivation by oxidation and recruitment into stress granules of hOGG1 but not APE1 in human cells exposed to sub-lethal concentrations of cadmium.

The induction of mutations in mammalian cells exposed to cadmium has been associated with the oxidative stress triggered by the metal. There is increasing evidence that the mutagenic potential of Cd is not restricted to the induction of DNA lesions. Cd has been shown to inactivate several DNA repair enzymes. Here we show that exposure of human cells to sub-lethal concentrations of Cd leads to a time- and concentration-dependent decrease in hOGG1 activity, the major DNA glycosylase activity responsible for the initiation of the base excision repair (BER) of 8-oxoguanine, an abundant and mutagenic form of oxidized guanine. Although there is a slight effect on the level of hOGG1 transcripts, we show that the inhibition of the 8-oxoguanine DNA glycosylase activity is mainly associated with an oxidation of the hOGG1 protein and its disappearance from the soluble fraction of total cell extracts. Confocal microscopy analyses show that in cells exposed to Cd hOGG1-GFP is recruited to discrete structures in the cytoplasm. These structures were identified as stress granules. Removal of Cd from the medium allows the recovery of the DNA glycosylase activity and the presence of hOGG1 in a soluble form. In contrast to hOGG1, we show here that exposure to Cd does not affect the activity of the second enzyme of the pathway, the major AP endonuclease APE1.

Can low level exposure to ochratoxin-A cause parkinsonism?

Mycotoxins are fungal metabolites with pharmacological activities that have been utilized in the production of antibiotics, growth promoters, and other classes of drugs. Some mycotoxins have been developed as biological and chemical warfare agents. Bombs and ballistic missiles loaded with aflatoxin were stockpiled and may have been deployed by Iraq during the first Gulf War. In light of the excess incidence of amyotrophic lateral sclerosis (ALS) in veterans from Operation Desert Storm, the potential for delayed neurotoxic effects of low doses of mycotoxins should not be overlooked. Ochratoxin-A (OTA) is a common mycotoxin with complex mechanisms of action, similar to that of the aflatoxins. Acute administration of OTA at non-lethal doses (10% of the LD(50)) have been shown to increase oxidative DNA damage in brain up to 72 h, with peak effects noted at 24 h in midbrain (MB), caudate/putamen (CP) and hippocampus (HP). Levels of dopamine (DA) and its metabolites in the striatum (e.g., CP) were shown to be decreased in a dose-dependent manner. The present study focused on the effects of chronic low dose OTA exposure on regional brain oxidative stress and striatal DA metabolism. Continuous administration of low doses of OTA with implanted subcutaneous Alzet minipumps caused a small but significant decrease in striatal DA levels and an upregulation of anti-oxidative systems and DNA repair. It is possible that low dose exposure to OTA will result in an earlier onset of parkinsonism when normal age-dependent decline in striatal DA levels are superimposed on the mycotoxin-induced lesion.

In vivo mutagenicity and initiation following oxidative DNA lesion in the kidneys of rats given potassium bromate.

To clarify the role of 8-OHdG formation as a starting point for carcinogenesis, we examined the dose-dependence and time-course of changes of OGG1 mRNA expression, 8-OHdG levels and in vivo mutations in the kidneys of gpt delta rats given KBrO3 in their drinking water for 13 weeks. There were no remarkable changes in OGG1 mRNA in spite of some increments being statistically significant. Increases of 8-OHdG occurred after 1 week at 500 p.p.m. and after 13 weeks at 250 p.p.m. Elevation of Spi- mutant frequency, suggestive of deletion mutations, occurred after 9 weeks at 500 p.p.m. In a two-stage experiment, F344 rats were given KBrO3 for 13 weeks then, after a 2-week recovery, treated with 1% NTA in the diet for 39 weeks. The incidence and multiplicity of renal preneoplastic lesions in rats given KBrO3 at 500 p.p.m. followed by NTA treatment were significantly higher than in rats treated with NTA alone. Results suggest that a certain period of time might be required for 8-OHdG to cause permanent mutations. The two-step experiment shows that cells exposed to the alteration of the intranuclear status by oxidative stress including 8-OHdG formation might be able to form tumors with appropriate promotion.

A new protein superfamily includes two novel 3-methyladenine DNA glycosylases from Bacillus cereus, AlkC and AlkD.

Soil bacteria are heavily exposed to environmental methylating agents such as methylchloride and may have special requirements for repair of alkylation damage on DNA. We have used functional complementation of an Escherichia coli tag alkA mutant to screen for 3-methyladenine DNA glycosylase genes in genomic libraries of the soil bacterium Bacillus cereus. Three genes were recovered: alkC, alkD and alkE. The amino acid sequence of AlkE is homologous to the E. coli AlkA sequence. AlkC and AlkD represent novel proteins without sequence similarity to any protein of known function. However, iterative and indirect sequence similarity searches revealed that AlkC and AlkD are distant homologues of each other within a new protein superfamily that is ubiquitous in the prokaryotic kingdom. Homologues of AlkC and AlkD were also identified in the amoebas Entamoeba histolytica and Dictyostelium discoideum, but no other eukaryotic counterparts of the superfamily were found. The alkC and alkD genes were expressed in E. coli and the proteins were purified to homogeneity. Both proteins were found to be specific for removal of N-alkylated bases, and showed no activity on oxidized or deaminated base lesions in DNA. B. cereus AlkC and AlkD thus define novel families of alkylbase DNA glycosylases within a new protein superfamily.

Low dose DDT inhibition of hepatocarcinogenesis initiated by diethylnitrosamine in male rats: possible mechanisms.

Previously we reported a tendency for reduction of the development of glutathione-S-transferase placental form (GST-P) positive foci, recognized as preneoplastic changes in rat liver, by a low dose of 1,1-bis(p-chlorophenyl)-2,2,2-trichloroethane (DDT), which belongs to the same group of hepatic cytochrome P-450 inducers as phenobarbital and is itself a non-genotoxic hepatocarcinogen. In order to clarify the biological significance of this phenomenon, we investigated the reproducibility and changes in other parameters using an initiation-promotion model in which male F344 rats were treated with DDT at doses of 0, 0.005, 0.5, 500 ppm in the diet for 11 or 43 weeks after initiation of hepatocarcinogenesis with N-diethylnitrosamine (DEN). When 500 ppm DDT was applied, the formation of GST-P positive foci and tumor were markedly elevated. In contrast, induction of GST-P positive foci and liver tumors tended to be inhibited at a dose of 0.005 ppm, correlating with protein levels of cytochrome P450 2B1 and 3A2 (CYP2B1 and 3A2) and generation of 8-hydroxy-2'-deoxyguanosine (8-OHdG), a marker of oxidative DNA damage. mRNA levels for 8-oxoguanine glycosylase 1 (OGG1), an 8-OHdG repair enzyme, connexin 32 (Cx32), a major component of Gap junctions, and hepatic nuclear factor 1alpha (HNF-1alpha), a Cx32 regulator, were inversely correlated with GST-P positive foci and tumor formation. These results indicate that low dose DDT may indeed exhibit inhibitory effects on chemically initiated-rat hepatocarcinogenicity, in contrast to the promotion observed with high doses, and that this is related to changes in metabolizing enzymes, cell communication, and DNA damage and its repair.

Suppression of uracil-DNA glycosylase induces neuronal apoptosis.

A chronic imbalance in DNA precursors, caused by one-carbon metabolism impairment, can result in a deficiency of DNA repair and increased DNA damage. Although indirect evidence suggests that DNA damage plays a role in neuronal apoptosis and in the pathogenesis of neurodegenerative disorders, the underlying mechanisms are poorly understood. In particular, very little is known about the role of base excision repair of misincorporated uracil in neuronal survival. To test the hypothesis that repair of DNA damage associated with uracil misincorporation is critical for neuronal survival, we employed an antisense (AS) oligonucleotide directed against uracil-DNA glycosylase encoded by the UNG gene to deplete UNG in cultured rat hippocampal neurons. AS, but not a scrambled control oligonucleotide, induced apoptosis, which was associated with DNA damage analyzed by comet assay and up-regulation of p53. UNG mRNA and protein levels were decreased within 30 min and were undetectable within 6-9 h of exposure to the UNG AS oligonucleotide. Whereas UNG expression is significantly higher in proliferating as compared with nonproliferating cells, such as neurons, the levels of UNG mRNA were increased in brains of cystathionine beta-synthase knockout mice, a model for hyperhomocysteinemia, suggesting that one-carbon metabolism impairment and uracil misincorporation can induce the up-regulation of UNG expression.

Dietary elevated sucrose modulation of diesel-induced genotoxicity in the colon and liver of Big Blue rats.

Earlier studies have indicated that sucrose possesses either co-carcinogenic or tumor-promoter effects in colon carcinogenesis induced by genotoxic carcinogens. In this study we investigated the role of sucrose on diesel exhaust particle (DEP)-induced genotoxicity in the colonic mucosa and liver. Big Blue rats were fed with DEP (0.8 ppm in feed) and/or sucrose (3.45% or 6.85% w/w in feed) for 3 weeks. DEP increased both DNA strand breaks and DNA adducts in colon. Interestingly, sucrose also increased the level of bulky DNA adducts in colon. DEP and sucrose had no effect on DNA strand-breaks and DNA adducts in liver. DEP and sucrose treatment did not have any effect on mutation frequency in colon and liver. Oxidative DNA damage detected as 8-oxodG (8-oxo-7,8-dihydro-2'-deoxyguanosine) and endonuclease III or formamidopyrimidine DNA glycosylase sensitive sites was unaltered in colon and liver. The mRNA expression levels of the DNA repair enzymes N-methylpurine DNA glycosylase ( MPG), 8-oxoguanine DNA glycosylase ( OGG1) and ERCC1 (part of the nucleotide excision repair complex) measured by reverse transcription-polymerase chain reaction were increased in liver by DEP feeding. In colon, expression was unaffected by DEP or sucrose feeding. Among biomarkers of oxidative stress, including vitamin C, malondialdehyde and protein oxidations (gamma-glutamyl semialdehyde and 2-amino adipic semialdehyde) in plasma and liver, only malondialdehyde was increased in plasma by sucrose/DEP feeding. In conclusion, sucrose feeding did not increase DEP-induced DNA damage in colon or liver.