Oxidative DNA Damage, Apoptosis, and Astrogliosis Induced by Acrylamide in The Cerebellar Cortex of The Growing Albino Rats and Their Mothers.

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Event Abstract Back to Event Copper impairs biliary epithelial cells and induces lipid peroxidation, protein oxidation and oxidative DNA damage in the isolated perfused rat liver Albena Alexandrova1*, Lubomir Petrov1, Margarita Kirkova1, Elina Tzvetanova1, Almira Georgieva1, Galina Nenkova1 and Stefani Vircheva1 1 Bulgarian Academy of Sciences, Institute of Physiology, Bulgaria The liver can be damaged by a wide variety of toxicants, including heavy metals. Although copper is an essential micronutrient, it can be harmful in excess. The isolated perfused rat liver model is considered to be a sensitive system for studying chemically-induced hepatotoxicity. In literature, however, there are rather scarce data about copper effects on isolated perfused rat liver. The hepatic injury, caused by copper overload is hypothesized to result from its role in the generation of reactive oxygen species (ROS). Being highly reactive ROS are able to impair all cellular constituents – lipids, proteins and DNA. The aim of the present study was to investigate whether CuSO4 (0.01, 0.03, and 0.1 mM) in perfused rat liver (for 60 min) was associated with lipid peroxidation, protein oxidation, and oxidative DNA damage, as well as with biliary epithelial cell injury. The results regarding liver viability and function (copper decreased the portal flow and bile secretion and led to liver swelling) showed a concentration-response effect. The highest tested concentration of copper caused complete blockage of portal flow and bile production within 30 min of perfusion. In confirmation of copper-induced injury, increased leakage of lactate dehydrogenase, aspartate transaminase, and alanine transaminase (indexes of hepatocellular membrane integrity) into perfusate were registered. Compared to controls, 0.01 and 0.03mM concentrations of copper increased the amount of thiobarbituric acid reacting substances, a marker of lipid peroxidation, tissue protein carbonyl groups, an index of protein oxidation, and 8-oxo-7,8-dihydro-2-deoxyguanosine, a marker of oxidative DNA damage; the biliary levels of gamma-glutamyltransferase, an index of biliary epithelial cell integrity, were also increased. The results suggest that copper toxicity in the isolated perfused rat liver could be a consequence of its ability to impair the integrity of biliary epithelial cells and is associated with the enhanced lipid peroxidation, protein oxidation, and oxidative DNA damage. Keywords: Copper, Bile, Oxidative Stress Conference: 8th Southeast European Congress on Xenobiotic Metabolism and Toxicity - XEMET 2010, Thessaloniki, Greece, 1 Oct - 5 Oct, 2010. Presentation Type: Poster Topic: Xenobiotic toxicity Citation: Alexandrova A, Petrov L, Kirkova M, Tzvetanova E, Georgieva A, Nenkova G and Vircheva S (2010). Copper impairs biliary epithelial cells and induces lipid peroxidation, protein oxidation and oxidative DNA damage in the isolated perfused rat liver. Front. Pharmacol. Conference Abstract: 8th Southeast European Congress on Xenobiotic Metabolism and Toxicity - XEMET 2010. doi: 10.3389/conf.fphar.2010.60.00099 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 28 Oct 2010; Published Online: 04 Nov 2010. * Correspondence: Dr. Albena Alexandrova, Bulgarian Academy of Sciences, Institute of Physiology, Sofia, Bulgaria, a_alexandrova_bas@yahoo.com Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Albena Alexandrova Lubomir Petrov Margarita Kirkova Elina Tzvetanova Almira Georgieva Galina Nenkova Stefani Vircheva Google Albena Alexandrova Lubomir Petrov Margarita Kirkova Elina Tzvetanova Almira Georgieva Galina Nenkova Stefani Vircheva Google Scholar Albena Alexandrova Lubomir Petrov Margarita Kirkova Elina Tzvetanova Almira Georgieva Galina Nenkova Stefani Vircheva PubMed Albena Alexandrova Lubomir Petrov Margarita Kirkova Elina Tzvetanova Almira Georgieva Galina Nenkova Stefani Vircheva Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

Studies have demonstrated that the induction of oxidative stress may be involved in brain tumor induction in rats by acrylonitrile. The present study examined whether acrylonitrile induces oxidative stress and DNA damage in rats and whether blood can serve as a valid surrogate for the biomonitoring of oxidative stress induced by acrylonitrile in the exposed population. Male Sprague-Dawley rats were treated with 0, 3, 30, 100, and 200 ppm acrylonitrile in drinking water for 28 days. One group of rats were also coadministered N-acetyl cysteine (NAC) (0.3% in diet) with acrylonitrile (200 ppm in drinking water) to examine whether antioxidant supplementation was protective against acrylonitrile-induced oxidative stress. Direct DNA strand breakage in white blood cells (WBC) and brain was measured using the alkaline comet assay. Oxidative DNA damage in WBC and brain was evaluated using formamidopyrimidine DNA glycosylase (fpg)-modified comet assay and with high-performance liquid chromatography-electrochemical detection. No significant increase in direct DNA strand breaks was observed in brain and WBC from acrylonitrile-treated rats. However, oxidative DNA damage (fpg comet and 8'hydroxyl-2-deoxyguanosine) in brain and WBC was increased in a dose-dependent manner. In addition, plasma levels of reactive oxygen species (ROS) increased in rats administered acrylonitrile. Dietary supplementation with NAC prevented acrylonitrile-induced oxidative DNA damage in brain and WBC. A slight, but significant, decrease in the GSH:GSSG ratio was seen in brain at acrylonitrile doses > 30 ppm. These results provide additional support that the mode of action for acrylonitrile-induced astrocytomas involves the induction of oxidative stress and damage. Significant associations were seen between oxidative DNA damage in WBC and brain, ROS formation in plasma, and the reported tumor incidences. Since oxidative DNA damage in brain correlated with oxidative damage in WBC, these results suggest that monitoring WBC DNA damage maybe a useful tool to assess acrylonitrile-induced oxidative stress in humans.

Effects of atorvastatin therapy on protein oxidation and oxidative DNA damage in hypercholesterolemic rabbits

A modified alkaline Comet assay for in vivo detection of oxidative DNA damage in Drosophila melanogaster

Oxidative stress and DNA damage have been proposed as mechanisms linking pesticide exposure to health effects such as cancer and neurological diseases. A pilot study of pesticide applicators and farm workers working in the fruit orchards of Oregon (i.e., apples, pears) was conducted to examine the relationship between organophosphate (OP) pesticide exposure and oxidative stress and DNA damage. Urine samples were analyzed for OP metabolites and 8-hydroxy-2′-deoxyguanosine (8-OH-dG). Lymphocytes were analyzed for oxidative DNA repair activity and DNA damage (Comet assay) and serum analyzed for lipid peroxides (i.e., malondialdehyde [MDA]). Cellular DNA damage in agricultural workers was validated using lymphocyte cell cultures. Urinary OP metabolites were significantly higher in farm workers and applicators (p < .001) when compared to controls. 8-OH-dG levels were 8.5 times and 2.3 times higher in farm workers and applicators, respectively, than in controls. Serum MDA levels were 4.9 times and 24 times higher in farm workers and applicators, respectively, than in controls. DNA damage and oxidative DNA repair were significantly greater in lymphocytes from applicators and farm workers when compared with controls. A separate field study showed that DNA damage was also significantly greater (p < .001) in buccal cells (i.e., leukocytes) collected from migrant farm workers working with fungicides in the berry crops in Oregon. Markers of oxidative stress (i.e., reactive oxygen species, reduced levels of glutathione) and oxidative DNA damage were also observed in lymphocyte cell cultures treated with an OP. The findings from these in vivo and in vitro studies indicate that pesticides induce oxidative stress and DNA damage in agricultural workers. These biomarkers may be useful for increasing our understanding of the link between pesticides and cancer.

Oxidative stress-induced DNA damage by particulate air pollution

The association between three major physiological stress systems and oxidative DNA and lipid damage

Copper impairs biliary epithelial cells and induces protein oxidation and oxidative DNA damage in the isolated perfused rat liver

Several risk factors have been identified as potential contributors to pancreatic cancer development, including environmental and lifestyle factors, such as smoking, drinking and diet, and medical conditions such as diabetes and pancreatitis, all of which generate oxidative stress and DNA damage. Oxidative stress status can be modified by environmental factors and also by an individual's unique genetic makeup. Here we examined the contribution of environment and genetics to an individual's level of oxidative stress, DNA damage and susceptibility to pancreatic cancer in a pilot study using three groups of subjects: a newly diagnosed pancreatic cancer group, a healthy genetically-unrelated control group living with the case subject, and a healthy genetically-related control group which does not reside with the subject. Oxidative stress and DNA damage was evaluated by measuring total antioxidant capacity, direct and oxidative DNA damage by Comet assay, and malondialdehyde levels. Direct DNA damage was significantly elevated in pancreatic cancer patients (age and sex adjusted mean ± standard error: 1.00±0.05) versus both healthy unrelated and related controls (0.70±0.06, p<0.001 and 0.82±0.07, p = 0.046, respectively). Analysis of 22 selected SNPs in oxidative stress and DNA damage genes revealed that CYP2A6 L160H was associated with pancreatic cancer. In addition, DNA damage was found to be associated with TNFA −308G>A and ERCC4 R415Q polymorphisms. These results suggest that measurement of DNA damage, as well as select SNPs, may provide an important screening tool to identify individuals at risk for development of pancreatic cancer.

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.

Volatile organic compounds (VOCs) are present in several working activities. This work is aimed at comparing oxidative stress and DNA damage biomarkers to specific VOCs in the occupational exposure of painters. Dose-response relationships between biomarkers of oxidative stress and of dose were studied. Unmetabolized VOCs and their urinary metabolites were analyzed. Urinary Methylhyppuric acids (MHIPPs, xylenes metabolite), Phenylglyoxylic and Mandelic acid (PGA, MA ethylbenzene metabolites), S-Benzylmercapturic acid (SBMA, toluene metabolite), and S-Phenylmercapturic acid (SPMA, benzene metabolite) were quantified at the end of work-shift. Oxidative stress was determined by: urinary excretion of 8-oxodGuo, 8-oxoGua and 8-oxoGuo and direct/oxidative DNA damage in blood by Fpg-Comet assay. Multivariate linear regression models were used to assess statistical significance of the association between dose and effect biomarkers. The regressions were studied with and without the effect of hOGG1 and XRCC1 gene polymorphisms. Statistically significant associations were found between MHIPPs and both 8-oxoGuo and oxidative DNA damage effect biomarkers measured with the Comet assay. Oxidative DNA damage results significantly associated with airborne xylenes and toluene, whilst 8-oxodGuo was significantly related to urinary xylenes and toluene. Direct DNA damage was significantly associated to SBMA. XRCC1 wild-type gene polymorphism was significantly associated with lower oxidative and total DNA damage with respect to heterozygous and mutant genotypes. The interpretation of the results requires some caution, as the different VOCs are all simultaneously present in the mixture and correlated among them.

Objective: Oxidative stress is central to Human Immunodeficiency Virus (HIV) pathogenesis. Increased oxidative stress leads to increased oxidative DNA damage in HIV infected patients. The objective of this study was to analyse oxidative DNA damage in HIV- positive pregnant women. Methods: This was a case-control study involving 100 HIV-positive women as cases and 100 HIV-negative women as controls. We used plasma levels of the oxidized base, 8-hydroxy-2-deoxyguanosine (8-OHdG), as our biomarker of oxidative DNA damage. 8-OHdG was measured with the highly sensitive 8-OHdG check enzyme-linked immunosorbent assay (ELISA) kit. Results: Increased oxidative DNA damage was observed in HIV-positive pregnant women than HIV-positive non pregnant women and controls. Conclusion: Oxidative stress-induced DNA lesions may contribute to carcinogenesis. Hence management of oxidative stress induced DNA damage is very important in HIV-positive mothers and their newborns.

Hexavalent chromium Cr (VI) causes various toxic and carcinogenic effects. The main carcinogenic effect is observed in the pulmonary system through inhalation route. Reduction of Cr (VI) to Cr (V, IV, and III) reactive intermediates within the cells by intracellular reducing agents such as glutathione is an important event leading to oxidative stress and oxidative DNA damage. This study evaluated the effects of intraperitoneal administration of Cr (VI) and GSH on total oxidant status (TOS), total antioxidant capacity (TAC), oxidative stress index, and oxidative DNA damage by evaluating the level of 8-hydroxy-2́-deoxyguanosine (8-OHdG) in Swiss-Albino mice. Seventy two mice were divided into 6 groups and treated intraperitoneally as follow: control (saline), group GSH (30mg/kg GSH) groups of Cr-20 (20mg/kg, K2Cr2O7), Cr-30 (30mg/kg K2Cr2O7), Cr-20+GSH (20mg/kg K2Cr2O7+30mg/kg GSH), Cr-30+GSH (30mg/kg K2Cr2O7+30mg/kg GSH). Total oxidant capacities of Cr-20 and Cr-30 were increased compared to control, Cr-20+GSH, and Cr-30+GSH. TOS levels in Cr-20+GSH and Cr-30+GSH were lower than in Cr-20 and Cr-30. No difference in TAC was observed among the groups. 8-Hydroxy-2́-deoxyguanosine levels were increased in groups Cr-20 and Cr-30 compared with control and groups Cr-20+GSH and Cr-30+GSH. No difference was determined in 8-OHdG levels among control, groups GSH, Cr-20+GSH and Cr-30+GSH. Results indicate that Cr (VI) given i.p. route causes increased oxidative stress and oxidative DNA damage in the blood of Swiss-Albino mice. Administration of GSH via i.p. route protects from oxidative stress and DNA damage.

Reactive oxygen species (ROS) 1The abbreviations used are: ROS, reactive oxygen species; BER, base excision repair; NER, nucleotide excision repair; AD, Alzheimer's disease; endo, endonuclease; TCR, transcription-coupled repair; 8-oxoG, 8-hydroxyguanine; 8-oxodG, 8-hydroxydeoxyguanine; TG, thymine glycol; AP, apurinic/apyrimidinc; GSR, gene-specific repair assay; XP, xeroderma pigmentosum; CS, Cockayne's syndrome; FapyG, 2,6-diamino-4hydroxyl-5-methylformamidopyrimidine. 1The abbreviations used are: ROS, reactive oxygen species; BER, base excision repair; NER, nucleotide excision repair; AD, Alzheimer's disease; endo, endonuclease; TCR, transcription-coupled repair; 8-oxoG, 8-hydroxyguanine; 8-oxodG, 8-hydroxydeoxyguanine; TG, thymine glycol; AP, apurinic/apyrimidinc; GSR, gene-specific repair assay; XP, xeroderma pigmentosum; CS, Cockayne's syndrome; FapyG, 2,6-diamino-4hydroxyl-5-methylformamidopyrimidine. are generated in cells as a by-product of cellular metabolism. ROS react with proteins, lipids, and DNA. DNA base modifications, abasic sites, deoxyribose damage, and single and double strand breaks are all induced following various forms of oxidative stress. This review will focus on DNA repair of oxidative lesions by base excision repair (BER) and nucleotide excision repair (NER). We will focus on the mammalian BER enzymes that have recently been cloned and characterized. Mitochondrial DNA repair mechanisms for oxidative damage will also be discussed. Although sugar damage and double strand breaks are critical lesions induced by ionizing radiation and bleomycin, repair of these lesions will not be discussed here (see Refs. 1Weaver D.T. Crit. Rev. Eukaryotic Gene Expression. 1996; 6: 345-375Crossref PubMed Scopus (44) Google Scholar, 2Lieber M.R. Grawunder U. Wu X. Yaneva M. Curr. Opin. Genet. Dev. 1997; 7: 99-104Crossref PubMed Scopus (127) Google Scholar, 3Povirk L.F. Mutat. Res. 1996; 355: 71-89Crossref PubMed Scopus (333) Google Scholar for recent reviews).Oxidative DNA Damage and Its ConsequencesThe endogenous attack on DNA by ROS species generates a low steady-state level of DNA adducts that have been detected in the DNA from human cells (4Dizdaroglu M. Halliwell B. Aruoma O.I. DNA and Free Radicals. Ellis Horwood, Ltd., London, United Kingdom1993: 18-39Google Scholar). Some of these base modifications are shown in Fig. 1. There are many more, and it is possible that the full spectrum of oxidative lesions in endogenous mammalian DNA exceeds 100 different types, of which 8-hydroxyguanine (8-oxoG) is one of the most abundant (5Ames B.N. Free Radical Res. Commun. 1989; 7: 121-128Crossref PubMed Scopus (627) Google Scholar).Oxidative DNA damage is thought to contribute to carcinogenesis, aging, and neurological degeneration (for reviews, see Refs. 5Ames B.N. Free Radical Res. Commun. 1989; 7: 121-128Crossref PubMed Scopus (627) Google Scholar and 6Wiseman H. Halliwell B. Biochem. J. 1996; 313: 17-29Crossref PubMed Scopus (1943) Google Scholar). Studies have shown that oxidative DNA damage accumulates in cancerous tissue. For example, higher levels of oxidative base damage were observed in lung cancer tissue compared with surrounding normal tissue (7Olinski R. Zastawny T. Budzbin J. Skokowski J. Zegarski W. Dizdaroglu M. FEBS Lett. 1992; 309: 193-198Crossref PubMed Scopus (243) Google Scholar). Another study reported a 9-fold increase in 8-oxoG, 8-hydroxyadenine, and 2,6-diamino-4-hydroxy-5-formamidopyrimidine in DNA from breast cancer tissue compared with normal tissue (8Malins D.C. Haimanot R. Cancer Res. 1991; 51: 5430-5432PubMed Google Scholar). Further, the cumulative risk of cancer increases dramatically with age in humans (9Ames B.N. Mutat. Res. 1989; 214: 41-46Crossref PubMed Scopus (290) Google Scholar), and cancer can in general terms be regarded as a degenerative disease of old age. There is evidence for the accumulation of oxidative DNA damage with age based on studies mainly measuring the increase in 8-oxoG (10Sohal R.S. Ku H.H. Agarwal S. Forster M.J. Lal H. Mech. Ageing Dev. 1994; 74: 121-133Crossref PubMed Scopus (689) Google Scholar). In Alzheimer's disease (AD), some studies have shown an accumulation of oxidative DNA damage in the brain, and a recent extensive study in cells from familial Alzheimer's disease demonstrated a deficiency in the processing of damage invoked by fluorescent light (11Parshad R.P. Sanford K.K. Price F.M. Melnick L.K. Nee L.E. Schapiro M.B. Tarone R.E. Robbins J.H. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 5146-5150Crossref PubMed Scopus (62) Google Scholar). The effects of fluorescent light exposure were inhibited by the addition of free radical scavengers, and therefore it was proposed that oxidative DNA damage was produced and responsible for the altered response seen in AD cells (11Parshad R.P. Sanford K.K. Price F.M. Melnick L.K. Nee L.E. Schapiro M.B. Tarone R.E. Robbins J.H. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 5146-5150Crossref PubMed Scopus (62) Google Scholar). AD cells also respond abnormally to ionizing radiation and simple alkylating agents, and therefore it is possible that lesions introduced by these agents such as oxidative modifications, alkylpurines, or DNA strand breaks are not repaired efficiently in AD cells (12Scudiero D.A. Polinsky R.J. Brumbach R.A. Tarone R.E. Nee L.E. Robbins J.H. Mutat. Res. 1986; 159: 125-131Crossref PubMed Scopus (45) Google Scholar).Many experimental methods have been used to expose cells to oxidative damage, all attempting to mimic endogenous processes (4Dizdaroglu M. Halliwell B. Aruoma O.I. DNA and Free Radicals. Ellis Horwood, Ltd., London, United Kingdom1993: 18-39Google Scholar, 6Wiseman H. Halliwell B. Biochem. J. 1996; 313: 17-29Crossref PubMed Scopus (1943) Google Scholar). Some studies have used hydrogen peroxide, which generates a large spectrum of lesions. Ionizing radiation also generates a wide spectrum of lesions including base damage and single and double strand breaks in DNA. Methylene blue plus visible light exposure primarily generates singlet oxygen damage, and osmium tetroxide generates primarily thymine glycols. For more discussion of this see Refs. 4Dizdaroglu M. Halliwell B. Aruoma O.I. DNA and Free Radicals. Ellis Horwood, Ltd., London, United Kingdom1993: 18-39Google Scholar and6Wiseman H. Halliwell B. Biochem. J. 1996; 313: 17-29Crossref PubMed Scopus (1943) Google Scholar. It is important to distinguish between the different types of oxidative stresses when evaluating experimental results.Technical differences in the methods used for DNA isolation may well result in differences in the analysis of the DNA adducts. A recent review compared the various methods used to detect oxidative damage in DNA (13Beckman K.B. Ames B.N. Methods Enzymol. 1996; 264: 442-453Crossref PubMed Google Scholar). One of the conclusions that emerged from the comparison was that there is a great need for methods to be more standardized and thus to provide more consistent results between different laboratories when comparing different but related techniques.One aspect that is common to many methods used to detect oxidative damage is that the DNA modifications are measured as averages in the total cellular DNA. This is of limited value since advances in recent years have shown that DNA damage processing and the biological consequences of DNA lesions vary considerably depending upon where a lesion is situated in the genome. For example, UV-induced photoproducts are processed differently whether situated in an active gene or in a non-transcribed region, and this may also be the case for oxidative lesions.The gene-specific repair assay (GSR) employs various DNA repair enzymes to detect specific lesions, and this assay has provided new insights about the heterogeneity of DNA repair in the nucleus (14Bohr V.A. Carcinogenesis. 1995; 16: 2885-2892Crossref PubMed Scopus (95) Google Scholar) and more recently about the repair mechanisms for mitochondrial DNA (see below). For example, endonuclease III (endo III) can detect oxidized pyrimidines, and the Fapy DNA glycosylase (Fpg protein) can detect oxidized purines. Endo III-sensitive sites have been assayed in the general genome (15Collins A.R. Duthie S.J. Dobson V.L. Carcinogenesis. 1993; 14: 1733-1735Crossref PubMed Scopus (761) Google Scholar), and more recently, Fpg protein has been used to detect lesions in specific genes (16Driggers W.J. LeDoux S.P. Wilson G.L. J. Biol. Chem. 1993; 268: 22042-22045Abstract Full Text PDF PubMed Google Scholar, 17Taffe B.G. Larminat F. Laval J. Croteau D.L. Anson R.M. Bohr V.A. Mutat. Res. 1996; 364: 183-192Crossref PubMed Scopus (64) Google Scholar).Base Excision Repair of Oxidative DamageBER is initiated by DNA glycosylases, a class of enzymes that recognize a specific set of modified bases such as 8-oxoG or thymine glycol (TG). Glycosylases cleave the N-glycosylic bond between the modified base and the sugar. There are two classifications of glycosylases: simple glycosylases that only cleave the N-glycosylic bond and glycosylase/AP lyase enzymes, which cleave the N-glycosylic bond and the DNA-phosphate backbone. Following the glycosylase step, AP endonucleases are required to remove the 3′-deoxyribose moiety and generate a 3′-hydroxyl group, which can be extended by a DNA polymerase. The process is completed by a DNA ligase rejoining the free DNA ends (for reviews see Refs. 18Seeberg E. Eide L. Bjoras M. Trends Biochem. Sci. 1995; 20: 391-397Abstract Full Text PDF PubMed Scopus (465) Google Scholar and19Friedberg E.C. Walker G.C. Siede W. DNA Repair and Mutagenesis. American Society for Microbiology, Wash., D. C.1995: 135-190Google Scholar).Repair of 8-oxoGThe majority of our knowledge regarding the repair of 8-oxoG has been derived from studies in Escherichia coli. 8-oxoG is considered to be a premutagenic lesion because it can mispair with adenine during DNA replication, and this mispairing results in G → T transversion mutations (20Grollman A.P. Moriya M. Trends Genet. 1993; 9: 246-249Abstract Full Text PDF PubMed Scopus (727) Google Scholar). Bacteria possess an integrated system of BER and error avoidance mechanisms to prevent damage at guanines (for a review see Ref. 20Grollman A.P. Moriya M. Trends Genet. 1993; 9: 246-249Abstract Full Text PDF PubMed Scopus (727) Google Scholar). This system is comprised of three components, an 8-oxoG glycosylase/AP lyase enzyme, called MutM or Fpg protein, an adenine DNA glycosylase, MutY, and a 8-oxodGTPase, MutT. As will be discussed, functional homologs of each of these proteins have now been identified in higher eukaryotes.Two groups have independently cloned an 8-oxoguanine glycosylase/AP lyase from yeast (yOgg1) (21van der Kemp P.A. Thomas D. Barbey R. de Oliveira R. Boiteux S. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 5197-5202Crossref PubMed Scopus (346) Google Scholar, 22Nash H.M. Bruner S.D. Schaerer O.D. Kawate T. Addona T.A. Spooner E. Lane W.S. Verdine G.L. Curr. Biol. 1996; 6: 968-980Abstract Full Text Full Text PDF PubMed Scopus (413) Google Scholar). The enzyme is a functional homolog of the Fpg protein because the yeast enzyme shares no amino acid homology with the bacterial protein. The yOgg1 cleaved DNA containing 8-oxoG opposite pyrimidines, abasic sites (21van der Kemp P.A. Thomas D. Barbey R. de Oliveira R. Boiteux S. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 5197-5202Crossref PubMed Scopus (346) Google Scholar, 22Nash H.M. Bruner S.D. Schaerer O.D. Kawate T. Addona T.A. Spooner E. Lane W.S. Verdine G.L. Curr. Biol. 1996; 6: 968-980Abstract Full Text Full Text PDF PubMed Scopus (413) Google Scholar), and 2,6-diamino-4-hydroxy-5-methylformamidopyrimidine (FapyG) (21van der Kemp P.A. Thomas D. Barbey R. de Oliveira R. Boiteux S. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 5197-5202Crossref PubMed Scopus (346) Google Scholar). Cleavage by yOgg1 was consistent with a β-elimination mechanism (21van der Kemp P.A. Thomas D. Barbey R. de Oliveira R. Boiteux S. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 5197-5202Crossref PubMed Scopus (346) Google Scholar,22Nash H.M. Bruner S.D. Schaerer O.D. Kawate T. Addona T.A. Spooner E. Lane W.S. Verdine G.L. Curr. Biol. 1996; 6: 968-980Abstract Full Text Full Text PDF PubMed Scopus (413) Google Scholar).Recently, the human and the mouse 8-oxoguanine glycosylase/AP lyase (human OGG1 or mouse Ogg1) genes have been cloned by their homology to yeast ogg1 (23Lu R. Nash H.M. Verdine G.L. Curr. Biol. 1997; 7: 397-407Abstract Full Text Full Text PDF PubMed Scopus (309) Google Scholar, 24Aburatani H. Hippo Y. Ishida T. Takashima R. Matsuba C. Kodama T. Takao M. Yasui A. Yamamoto K. Asano M. Fukasawa K. Yoshinari T. Inoue H. Ohtsuka E. Nishimura S. Cancer Res. 1997; 57: 2151-2156PubMed Google Scholar, 25Arai K. Morishita K. Shinmura K. Kohno T. Kim S. Nohmi T. Taniwaki M. Ohwada S. Yokota J. Oncogene. 1997; 14: 2857-2861Crossref PubMed Scopus (247) Google Scholar). Human OGG1 gene was localized to the short arm of chromosome 3, 3p26.2 (23Lu R. Nash H.M. Verdine G.L. Curr. Biol. 1997; 7: 397-407Abstract Full Text Full Text PDF PubMed Scopus (309) Google Scholar, 25Arai K. Morishita K. Shinmura K. Kohno T. Kim S. Nohmi T. Taniwaki M. Ohwada S. Yokota J. Oncogene. 1997; 14: 2857-2861Crossref PubMed Scopus (247) Google Scholar). Expression of the human gene in E. coli lacking mutM and mutY suppressed the spontaneous mutator phenotype of these cells (24Aburatani H. Hippo Y. Ishida T. Takashima R. Matsuba C. Kodama T. Takao M. Yasui A. Yamamoto K. Asano M. Fukasawa K. Yoshinari T. Inoue H. Ohtsuka E. Nishimura S. Cancer Res. 1997; 57: 2151-2156PubMed Google Scholar, 25Arai K. Morishita K. Shinmura K. Kohno T. Kim S. Nohmi T. Taniwaki M. Ohwada S. Yokota J. Oncogene. 1997; 14: 2857-2861Crossref PubMed Scopus (247) Google Scholar). Human OGG1 (also called MutM homolog) was shown to cleave the DNA by a β-elimination mechanism preferentially at 8-oxoG:C base pairs (23Lu R. Nash H.M. Verdine G.L. Curr. Biol. 1997; 7: 397-407Abstract Full Text Full Text PDF PubMed Scopus (309) Google Scholar, 24Aburatani H. Hippo Y. Ishida T. Takashima R. Matsuba C. Kodama T. Takao M. Yasui A. Yamamoto K. Asano M. Fukasawa K. Yoshinari T. Inoue H. Ohtsuka E. Nishimura S. Cancer Res. 1997; 57: 2151-2156PubMed Google Scholar). Several conserved domains have been identified in the yeast, mouse, and human genes including the a helix-hairpin-helix (HhH) and Gly/Pro-rich-Asp motif (GPD motif) (22Nash H.M. Bruner S.D. Schaerer O.D. Kawate T. Addona T.A. Spooner E. Lane W.S. Verdine G.L. Curr. Biol. 1996; 6: 968-980Abstract Full Text Full Text PDF PubMed Scopus (413) Google Scholar, 23Lu R. Nash H.M. Verdine G.L. Curr. Biol. 1997; 7: 397-407Abstract Full Text Full Text PDF PubMed Scopus (309) Google Scholar, 25Arai K. Morishita K. Shinmura K. Kohno T. Kim S. Nohmi T. Taniwaki M. Ohwada S. Yokota J. Oncogene. 1997; 14: 2857-2861Crossref PubMed Scopus (247) Google Scholar). In addition, Arai et al. (25Arai K. Morishita K. Shinmura K. Kohno T. Kim S. Nohmi T. Taniwaki M. Ohwada S. Yokota J. Oncogene. 1997; 14: 2857-2861Crossref PubMed Scopus (247) Google Scholar) reported that the yeast Ogg1 and human OGG1 contained a putative C2H2 zinc finger-like motif, although in the yeast sequence one of the histidines was an arginine. Alignment of the ogg1 genes with other DNA repair glycosylases suggests that these enzymes may represent a DNA repair superfamily (18Seeberg E. Eide L. Bjoras M. Trends Biochem. Sci. 1995; 20: 391-397Abstract Full Text PDF PubMed Scopus (465) Google Scholar, 22Nash H.M. Bruner S.D. Schaerer O.D. Kawate T. Addona T.A. Spooner E. Lane W.S. Verdine G.L. Curr. Biol. 1996; 6: 968-980Abstract Full Text Full Text PDF PubMed Scopus (413) Google Scholar, 23Lu R. Nash H.M. Verdine G.L. Curr. 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In human extracts, an 8-oxoG endonuclease was identified from human polymorphonuclear neutrophils, which cleaved 8-oxoG but not the ring-opened guanine adduct, FapyG (27Chung M.H. Kim H.S. Ohtsuka E. Kasai H. Yamamoto F. Nishimura S. Biochem. Biophys. Res. Commun. 1991; 178: 1472-1478Crossref PubMed Scopus (84) Google Scholar). One distinguishing feature of this enzyme was that it was magnesium-dependent. Another study identified two repair activities, an 8-oxoG glycosylase and an 8-oxoG endonuclease, from HeLa cell nuclear extracts (28Bessho T. Tano K. Kasai H. Ohtsuka E. Nishimura S. J. Biol. Chem. 1993; 268: 19416-19421Abstract Full Text PDF PubMed Google Scholar). The 8-oxoG base pairing preferences for these enzymes were similar to that of yeast Ogg1. Further experiments are required to determine whether these proteins are human OGG1 or novel enzymes.In E. coli, the MutY protein is an adenine DNA glycosylase that removes adenine when base paired with 8-oxoG. Using purified DNA polymerases, it has been demonstrated that the replicative polymerases incorporate adenine opposite 8-oxoG (29Shibutani S. Takeshita M. Grollman A.P. Nature. 1991; 349: 431-434Crossref PubMed Scopus (2024) Google Scholar). A human MutY activity has been purified from calf thymus cells (30McGoldrick J.P. Yeh Y.C. Solomon M. Essigmann J.M. Lu A.L. Mol. Cell. Biol. 1995; 15: 989-996Crossref PubMed Google Scholar). The protein removes adenine mispairs including A:G, A/8-oxoG, and A:C. The glycosylase co-purified with a AP nicking activity, which was inhibited by neutralizing MutY antibodies. Recently, the gene for a human MutY homolog was cloned (31Slupska M.M. Baikalov C. Luther W.M. Chiang J.H. Wei Y.F. Miller J.H. J. Bacteriol. 1996; 178: 3885-3892Crossref PubMed Scopus (327) Google Scholar).In cells, the deoxyribonucleotide pools are also subjected to oxidative damage. dGTP can be converted to 8-oxodGTP and incorporated into nascent DNA strands opposite adenine. 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Bjoras M. Pirovano M. Alseth I. Berdal K.G. Seeberg E. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 10735-10740Crossref PubMed Scopus (146) Google Scholar). Deletion of yeast NTG1 renders the cells sensitive to H2O2 and menadione (37Eide L. Bjoras M. Pirovano M. Alseth I. Berdal K.G. Seeberg E. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 10735-10740Crossref PubMed Scopus (146) Google Scholar).A mammalian TG glycosylase activity has been purified from extracts of calf thymus and bovine cells (38Hilbert T.P. Boorstein R.J. Kung H.C. Bolton P.H. Xing D. Cunningham R.P. Teebor G.W. Biochemistry. 1996; 35: 2505-2511Crossref PubMed Scopus (78) Google Scholar). More recently another gene for the human endonuclease III homolog was cloned (39Aspinwall R. Rothwell D.G. Roldan-Arjona T. Anselmino C. J.P. T. Proc. Natl. Acad. Sci. U. S. 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