8-hydroxyguanine and potassium-bromate

Repair of some oxidized purines such as 8-oxo-7,8-dihydroguanine (8-oxoG) is inefficient in human cells in comparison to repair of other major endogenous lesions (e.g. uracil, abasic sites or oxidized pyrimidines). This is due to the poor catalytic properties of hOGG1, the major DNA glycosylase involved in 8-oxoG removal. The formamidopyrimidine DNA glycosylase (FPG) protein from E. coli is endowed with a potent 8-oxoG glycolytic activity coupled with a beta,delta-AP lyase. In this study, we have expressed FPG fused to the enhanced green fluorescent protein (EGFP) in human bladder cells to accelerate the repair of oxidative DNA damage. Cells expressing the fusion protein EGFP-FPG repaired 8-oxoG and AP sites at accelerated rates, in particular via the single-nucleotide insertion base excision repair (BER) pathway and were resistant to mutagenicity of the oxidizing carcinogen potassium bromate. FPG may stably protect human cells from some harmful effects of oxidative DNA damage.

Topics: Bromates; Carcinogens; Cell Culture Techniques; DNA Damage; DNA Repair; DNA-Formamidopyrimidine Glycosylase; Escherichia coli Proteins; Fibroblasts; Green Fluorescent Proteins; Guanine; Humans; Oxidative Stress; Reactive Oxygen Species; Urinary Bladder; Urinary Bladder Neoplasms

Exposure of mammalian cells to bromate (BrO3-) generates oxidative DNA modifications, in particular 7,8-dihydro-8-oxo-guanine (8-oxoG). The damaging mechanism is quite unique, since glutathione, which is protective against most oxidants and alkylating agents, mediates a metabolic activation, while bromate itself does not react directly with DNA. Neither enzymes nor transition metals are required as catalysts in the activation. The ultimate DNA damaging species has not yet been established, but experiments under cell-free conditions suggest that neither molecular bromine nor reactive oxygen species such as superoxide, hydrogen peroxide or singlet oxygen are involved. Rather bromine radicals (Br*) or oxides (BrO*, BrO2*) might be responsible. Compared to hypochlorite (ClO-), bromate is much less cytotoxic, probably because the former halite efficiently reacts with proteins and other vitally important cellular constituents. In consequence, oxidative DNA damage and the induction of mutations and micronuclei is easily detectable at non-cytotoxic concentrations of bromate, while DNA damage by hypochlorite is observed only at cytotoxic concentrations and follows a non-linear (hockey-stick-like) dose response.

Topics: Animals; Bromates; Cell Line, Tumor; Cell Survival; Cricetinae; Cricetulus; DNA Damage; Dose-Response Relationship, Drug; Guanine; Mice; Micronuclei, Chromosome-Defective; Micronucleus Tests; Mutagens; Reactive Oxygen Species

In order to assess the effect of potassium bromate (KBrO3) on the induction of tumor formation, a 1-year carcinogenesis study was performed using Ogg1 knockout mice (Ogg1(-/-)) and wild-type mice (Ogg1(+/+)). The mice were chronically exposed to KBrO3 by putting it in the drinking water for 29 weeks, at 2 g/l for the first 18 weeks, and then at 1 g/l for another 11 weeks. After termination of treatment the mice were kept for an additional 23 weeks. The amount of 8-hydroxydeoxyguanosine (8-OH-dG) in kidney DNA after 29 weeks of KBrO3 exposure reached 500 8-OH-dG/10(6) dG, almost 250-fold that of untreated wild-type mice. During the course of study the mice appeared normal, although a decrease of body weight gain in both Ogg1(-/-) and Ogg1(+/+) mice exposed to KBrO3, and some kidney malfunction in KBrO3 treated Ogg1(-/-) mice was observed. Surprisingly, when Ogg1(-/-) and Ogg1(+/+) mice were sacrificed at 52 weeks, no tumor formation could be found in kidney or other organs such as lung, liver, spleen, thymus, stomach and intestine. Microscopic examination also showed the absence of precancerous foci in all tissues of both Ogg1(-/-) and Ogg1(+/+) mice. A possible explanation is presented to reconcile these results with those of others which showed an increased incidence of tumor formation in untreated Ogg1(-/-) mice.

Topics: Administration, Oral; Animals; Bromates; Carcinogenicity Tests; DNA Damage; DNA Glycosylases; Guanine; Kidney; Mice; Mice, Knockout; Neoplasms

The European Standards Committee on Oxidative DNA Damage (ESCODD) recommended the use of the lesion-specific repair enzyme, formamidopyrimidine DNA-glycosylase (FPG) in the comet assay to detect oxidative DNA damage. In the present study, FPG was compared with endonuclease III (ENDOIII) and human 8-hydroxyguanine DNA-glycosylase (hOGG1) for the ability to modify the sensitivity of the comet assay. Mouse lymphoma L5178Y cells were treated with dimethyl sulphoxide (DMSO) as a standard solvent or reference agents known to induce oxidative damage (gamma irradiation and potassium bromate) or alkylation (methyl methanesulfonate, MMS; ethylnitrosurea, ENU). Using DMSO even up to toxic concentrations, no increase in breaks was seen with FPG, ENDOIII or hOGG1. With gamma irradiation (1-10 Gy), dose-related increases in breaks were seen with all three enzymes. FPG and hOGG1 gave similar increases in breaks after potassium bromate treatment between 0.25 and 2.5 mmol/l, but ENDOIII showed an increase only at the highest concentration, 2.5 mmol/l. Following MMS treatment (5-23 micromol/l), FPG induced a dramatic increase in breaks compared with control levels and ENDOIII also showed a significant but smaller increase; in marked contrast, hOGG1 gave no increase. With ENU (0.5-2.0 mmol/l), increases in breaks were seen with FPG and ENDOIII at 1 and 2 mmol/l but, again, no increase was observed with hOGG1. These data indicate that all three endonucleases recognize oxidative DNA damage and, in addition, FPG and ENDOIII also recognize alkylation damage. Therefore, caution should be taken when using FPG and ENDOIII in the comet assay with an agent that has an unknown mode of action since any additional strand breaks induced by either enzyme cannot necessarily be ascribed to oxidative damage. The use of hOGG1 in the modified comet assay offers a useful alternative to FPG and is apparently more specific for 8-oxoguanine and methyl-fapy-guanine.

Topics: Animals; Bromates; Comet Assay; Deoxyribonuclease (Pyrimidine Dimer); Dimethyl Sulfoxide; DNA Damage; DNA Glycosylases; DNA Repair Enzymes; DNA-Formamidopyrimidine Glycosylase; Escherichia coli Proteins; Ethylnitrosourea; Gamma Rays; Guanine; Humans; Leukemia L5178; Methyl Methanesulfonate; Mice; Oxidation-Reduction; Sensitivity and Specificity

Xeroderma pigmentosum (XP) C is involved in the recognition of a variety of bulky DNA-distorting lesions in nucleotide excision repair. Here, we show that XPC plays an unexpected and multifaceted role in cell protection from oxidative DNA damage. XP-C primary keratinocytes and fibroblasts are hypersensitive to the killing effects of DNA-oxidizing agents and this effect is reverted by expression of wild-type XPC. Upon oxidant exposure, XP-C primary keratinocytes and fibroblasts accumulate 8,5'-cyclopurine 2'-deoxynucleosides in their DNA, indicating that XPC is involved in their removal. In the absence of XPC, a decrease in the repair rate of 8-hydroxyguanine (8-OH-Gua) is also observed. We demonstrate that XPC-HR23B complex acts as cofactor in base excision repair of 8-OH-Gua, by stimulating the activity of its specific DNA glycosylase OGG1. In vitro experiments suggest that the mechanism involved is a combination of increased loading and turnover of OGG1 by XPC-HR23B complex. The accumulation of endogenous oxidative DNA damage might contribute to increased skin cancer risk and account for internal cancers reported for XP-C patients.

Topics: Bromates; Cells, Cultured; DNA Damage; DNA Glycosylases; DNA Repair; DNA Repair Enzymes; DNA-Binding Proteins; Guanine; Humans; Keratinocytes; Oxidants; Skin Neoplasms; X-Rays; Xeroderma Pigmentosum

Public drinking water treated with chemical disinfectants contains a complex mixture of disinfection by-products (DBPs) for which the relative toxicity of the mixtures needs to be characterized to accurately assess risk. Potassium bromate (KBrO(3)) is a by-product from ozonation of high-bromide surface water for production of drinking water and is a rodent carcinogen that produces thyroid, mesothelial, and renal tumors. The proposed mechanism of KBrO(3) renal carcinogenesis involves the formation of 8-oxoguanine (8-oxoG), a promutagenic base lesion in DNA typically removed through base excision repair (BER). In this study, male Long-Evans rats were exposed via drinking water to carcinogenic concentrations of KBrO(3) (0.4 g/L), 3-chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone (0.07 g/L), chloroform (1.8 g/L), bromodichloromethane (0.7 g/L), or a mixture of all these chemicals at the same concentrations for 3 weeks. Half of one kidney was processed for microscopic examination, and the remaining kidney was frozen for isolation of genomic DNA. Levels of 8-oxoG were measured using HPLC with electrochemical detection in DNA samples incubated with formamidopyrimidine-DNA glycosylase. Aldehydic lesions (e.g. abasic sites) in DNA samples were quantitated using an aldehyde-reactive probe slot-blot assay. Treatment with KBrO(3) produced a measurable increase of 8-oxoG in the kidney, and this effect was greater than that produced by treatment with the DBP mixture. No other single chemical treatment caused measurable increases of 8-oxoG. The mixture effect on the amount of 8-oxoG observed in this study suggests an interaction between chemicals that reduced the generation of oxidative DNA damage. No increases in abasic sites were observed with treatment, but a decrease was apparent in the rats treated with the DBP mixture. These data are consistent with previous studies where chronic exposure to this chemical mixture in drinking water resulted in a less than additive carcinogenic response in Tsc2 mutant Long-Evans rats.

Topics: Animals; Bromates; Chloroform; Disinfectants; DNA; DNA Damage; Furans; Guanine; Histocytochemistry; Kidney Neoplasms; Male; Oxidative Stress; Rats; Rats, Long-Evans; Rats, Mutant Strains; Trihalomethanes; Water Purification; Water Supply

It has been previously reported that the elevated accumulation of repair incision intermediates in cells from patients with combined characteristics of xeroderma pigmentosum complementation group D (XP-D) and Cockayne syndrome (CS) XP-D/CS fibroblasts following UV irradiation is caused by an "uncontrolled" incision of undamaged genomic DNA induced by UV-DNA-lesions which apparently are not removed. This could be an explanation for the extreme sensitivity of these cells to UV light. In the present study, we confirm the immediate DNA breakage following UV irradiation also for CS group B (CS-B) fibroblasts by DNA migration in the "comet assay" and extend these findings to other lesions such as 8-oxodeoxyguanosine (8-oxodG), selectively induced by KBrO3 treatment. In contrast, X-ray exposure does not induce differential DNA breakage. This indicates that additional lesions other than the UV-induced photoproducts (cyclobutane pyrimidine dimers, CPD, and 6-pyrimidine-4-pyrimidone products, 6-4 PP), such as 8-oxodG, specifically induced by KBrO3, are likely to trigger "uncontrolled" DNA breakage in the undamaged genomic DNA in the CS-B fibroblasts, thus accounting for some of the clinical features of these patients.

Topics: Bromates; Chromosomal Instability; Cockayne Syndrome; Comet Assay; DNA; DNA Damage; DNA Repair; Fibroblasts; Guanine; Humans; Oxidation-Reduction; Photochemistry; Pyrimidine Dimers; Radiation Tolerance; Transcription, Genetic; Ultraviolet Rays; Xeroderma Pigmentosum

The Mmh/Ogg1 gene product maintains the integrity of the genome by removing the damaged base 8-hydroxyguanine (8-OH-G), one of the major DNA lesions generated by reactive oxygen species. Using Ogg1-deficient mice, we sought to establish if cells having high amounts of 8-OH-G have the ability to proliferate and whether the mutation frequency increases after proliferation in vivo. When KBrO(3), a known renal carcinogen, at a dose of 2 grams/liter was administered to Ogg1 mutant mice for 12 weeks, the amount of 8-OH-G in liver DNA from treated Ogg1(-/-) mice increased 26.1 times that of treated Ogg1(+/+) mice. The accumulated 8-OH-G did not decrease 4 weeks after cessation of KBrO(3) treatment. Partial hepatectomy was performed on Ogg1(+/-) and Ogg1(-/-) mice after being treated with KBrO(3) for 12 weeks. The remnant liver from Ogg1(-/-) mice treated with KBrO(3) regenerated to the same extent as nontreated Ogg1(+/-) mice. In addition, 8-OH-G was not repaired during cell proliferation by partial hepatectomy, indicating that there is no replication coupled repair of preexisting 8-OH-G. The mutation frequency after the regeneration of liver from treated Ogg1(-/-) mice showed a 3.5-fold increase compared with before regeneration. This represents a mutation frequency 6.2 times that of normal levels. The proliferation of cells having accumulated amounts of 8-OH-G caused mainly GC-->TA transversions. These results showed that inactivation of the Ogg1 gene leads to a higher risk of cancer because cells with accumulated 8-OH-G still retain the ability to proliferate, leading to an increase in the mutation frequency.

Topics: Animals; Bromates; Carcinogens; Cell Division; DNA; DNA Damage; DNA Repair; DNA-Formamidopyrimidine Glycosylase; Female; Guanine; Hepatectomy; Liver; Liver Regeneration; Male; Mice; Mice, Inbred C57BL; Mice, Transgenic; Mutation; N-Glycosyl Hydrolases

8-Hydroxyguanine (8-OH-G) is a major pre-mutagenic lesion generated from reactive oxygen species. The Mmh/Ogg1 gene product plays a major role in maintaining genetic integrity by removing 8-OH-G by way of the base excision repair pathway. To investigate how oxidative stress influences the formation of 8-OH-G in Ogg1 mutant mice, a known oxidative agent, potassium bromate (KBrO(3)), was administered at a dose of 2 g/l in the drinking water to Ogg1(+/+), Ogg1(+/-) and Ogg1(-/-) mice for 12 weeks. Apurinic (AP) site lyase activity, measured by the excision of 8-OH-G from synthetic oligonucleotides, remained unchanged in kidney cell extracts isolated from Ogg1 mutant mice when the mice were pre-treated by KBrO(3). The levels of 8-OH-G in kidney DNA tremendously increased in a time-dependent manner following exposure of Ogg1(-/-) mice to KBrO(3). Of particular note, the amount of 8-OH-G in kidney DNA from Ogg1(-/-) mice treated with KBrO(3) was approximately 70 times that of KBrO(3)-treated Ogg1(+/+) mice. The accumulated 8-OH-G did not decrease 4 weeks after discontinuing treatment with KBrO(3). KBrO(3) treatment for 12 weeks gave rise to increased mutation frequencies at the transgenic gpt gene in Ogg1(+/+) mice kidney. Absence of the Ogg1 gene further enhanced the mutation frequency. Sequence data obtained from gpt mutants showed that the accumulated 8-OH-G caused mainly GC-->TA transversion and deletion. Other mutations including GC-->AT transition also showed a tendency to increase. These results indicate that 8-OH-G, produced by chronic exposure to exogenous oxidative stress agents, is not repaired to any significant extent within the overall genome of Ogg1(-/-) mice kidney.

Topics: Animals; Bromates; Carbon-Oxygen Lyases; Chromatography, High Pressure Liquid; DNA; DNA Damage; DNA Mutational Analysis; DNA-(Apurinic or Apyrimidinic Site) Lyase; DNA-Formamidopyrimidine Glycosylase; Genotype; Guanine; Kidney; Mice; Mice, Inbred C57BL; Mice, Transgenic; Mutation; N-Glycosyl Hydrolases; Oligonucleotides; Oxidants; Oxidative Stress; Time Factors

The oxidative DNA damage induced by the renal carcinogen potassium bromate (KBrO3) in cultured mammalian cells and in a cell-free system was characterized by means of various repair endonucleases. Under cell-free conditions, no modifications were induced by KBrO3 alone, but extensive DNA damage was observed in the presence of glutathione (GSH). The DNA damage was found to consist mostly of base modifications sensitive to Fpg protein (formamidopyrimidine-DNA glycosylase). HPLC analysis demonstrated that many of the modifications were 7,8-dihydro-8-oxoguanine(8-hydroxyguanine) residues. Single-strand breaks, sites of base loss (AP sites) and base modifications sensitive to endonuclease III (5,6-dihydropyrimidine derivatives) were formed in only low amounts. This 'damage profile' and experiments with various scavengers (catalase, superoxide dismutase, deferoxamine, azide, tert-butanol) and D2O as solvent excluded the involvement of hydroxyl radicals and single oxygen in the damage production, but were consistent with a radical mechanism involving bromine radicals. In L1210 mouse leukemia cells and LLC-PK1 porcine kidney cells, KBrO3 alone gave rise to a DNA damage profile similar to that observed after treatment of cell-free DNA with KBrO3 plus GSH, i.e. base modifications sensitive to Fpg protein were formed in high excess of all other lesions quantified. In LLC-PK1 cells (derived from the target organ of KBrO3-induced carcinogenesis) the level of DNA damage was twice that in the L1210 cells. DNA damage was partially prevented by depletion of intracellular GSH with diethylmaleate, indicating that GSH played an activating role in the cells similar to that seen under cell-free conditions. The Fpg-sensitive base modifications induced by KBrO3 were repaired with only moderate efficiency (38 +/- 10% of the lesions were still present after 18 h in full medium) under conditions that did not influence cell proliferation.

Topics: Animals; Bromates; Bromine; Carcinogens; Cattle; Cell-Free System; Cells, Cultured; DNA Damage; DNA, Viral; Free Radical Scavengers; Free Radicals; Glutathione; Guanine; Hydroxyl Radical; Kidney; Leukemia L1210; Mice; Oxidation-Reduction; Reactive Oxygen Species