Formamido Pyrimidine Research Articles

Genome Profiling for Aflatoxin B1 Resistance in Saccharomyces cerevisiae Reveals a Role for the CSM2/SHU Complex in Tolerance of Aflatoxin B1-Associated DNA Damage

Exposure to the mycotoxin aflatoxin B1 (AFB1) strongly correlates with hepatocellular carcinoma (HCC). P450 enzymes convert AFB1 into a highly reactive epoxide that forms unstable 8,9-dihydro-8-(N7-guanyl)-9-hydroxyaflatoxin B1 (AFB1-N7-Gua) DNA adducts, which convert to stable mutagenic AFB1 formamidopyrimidine (FAPY) DNA adducts. In CYP1A2-expressing budding yeast, AFB1 is a weak mutagen but a potent recombinagen. However, few genes have been identified that confer AFB1 resistance. Here, we profiled the yeast genome for AFB1 resistance. We introduced the human CYP1A2 into ∼90% of the diploid deletion library, and pooled samples from CYP1A2-expressing libraries and the original library were exposed to 50 μM AFB1 for 20 hs. By using next generation sequencing (NGS) to count molecular barcodes, we initially identified 86 genes from the CYP1A2-expressing libraries, of which 79 were confirmed to confer AFB1 resistance. While functionally diverse genes, including those that function in proteolysis, actin reorganization, and tRNA modification, were identified, those that function in postreplication DNA repair and encode proteins that bind to DNA damage were over-represented, compared to the yeast genome, at large. DNA metabolism genes also included those functioning in checkpoint recovery and replication fork maintenance, emphasizing the potency of the mycotoxin to trigger replication stress. Among genes involved in postreplication repair, we observed that CSM2, a member of the CSM2(SHU) complex, functioned in AFB1-associated sister chromatid recombination while suppressing AFB1-associated mutations. These studies thus broaden the number of AFB1 resistance genes and have elucidated a mechanism of error-free bypass of AFB1-associated DNA adducts.

In-vivo assesment of the genotoxic and oxidative stress effects of particulate matter on Echinogammarus veneris

Seven types of atmospheric dusts (road dust, soil dust, brake dust, desert dust, pellet ash and coke and certified material NIST1648a – urban dust) have been tested for their genotoxicity on specimens of Echinogammarus veneris, a small aquatic amphipod. Experiments were carried out in vivo, by exposing the animals for 24 h to water containing 25 mg/L of dust. Each dust has been chemically analyzed for ions, elemental carbon, organic carbon and for the soluble and insoluble fractions of elements. Non-specific damages to DNA have been evaluated by the comet test, while oxidative damages have been estimated by coupling the comet test with formamido pyrimidine DNA glycosylase reaction. The animal tissues have been acid digested and analyzed for their elemental content to evaluate the bioaccumulation. All the considered dusts have caused a significant non-specific DNA damage, while the oxidative stress was shown only by dust types containing high concentration of elements. Furthermore, the oxidative damage has shown a positive correlation with the total bio-accumulated elemental concentration. For all the dust samples, the correlation with bio-accumulation in the tissues was more satisfactory for the insoluble fraction than for the soluble fraction of elements. Elements contained in solid particles seem then to be the main responsible bioaccumulation and for the oxidative stress.

Exposure to runoff from coal-tar-sealed pavement induces genotoxicity and impairment of DNA repair capacity in the RTL-W1 fish liver cell line

Coal-tar-based (CTB) sealcoat, frequently applied to parking lots and driveways in North America, contains elevated concentrations of polycyclic aromatic hydrocarbons (PAHs) and related compounds. The RTL-W1 fish liver cell line was used to investigate two endpoints (genotoxicity and DNA-repair-capacity impairment) associated with exposure to runoff from asphalt pavement with CTB sealcoat or with an asphalt-based sealcoat hypothesized to contain about 7% CTB sealcoat (AS-blend). Genotoxic potential was assessed by the Formamido pyrimidine glycosylase (Fpg)-modified comet assay for 1:10 and 1:100 dilutions of runoff samples collected from 5h to 36d following sealcoat application. DNA-repair capacity was assessed by the base excision repair comet assay for 1:10 dilution of samples collected 26h and 36d following application. Both assays were run with and without co-exposure to ultraviolet-A radiation (UVA). With co-exposure to UVA, genotoxic effects were significant for both dilutions of CTB runoff for three of four sample times, and for some samples of AS-blend runoff. Base excision repair was significantly impaired for CTB runoff both with and without UVA exposure, and for AS-blend runoff only in the absence of UVA. This study is the first to investigate the effects of exposure to the complex mixture of chemicals in coal tar on DNA repair capacity. The results indicate that co-exposure to runoff from CT-sealcoated pavement and UVA as much as a month after sealcoat application has the potential to cause genotoxicity and impair DNA repair capacity.

Fluorogenic DNA ligase and base excision repair enzyme assays using substrates labeled with single fluorophores

Continuing our work on fluorogenic substrates labeled with single fluorophores for nucleic acid modifying enzymes, here we describe the development of such substrates for DNA ligases and some base excision repair enzymes. These substrates are hairpin-type synthetic DNA molecules with a single fluorophore located on a base close to the 3′ ends, an arrangement that results in strong fluorescence quenching. When such substrates are subjected to an enzymatic reaction, the position of the dyes relative to that end of the molecules is altered, resulting in significant fluorescence intensity changes. The ligase substrates described here were 5′ phosphorylated and either blunt-ended or carrying short, self-complementary single-stranded 5′ extensions. The ligation reactions resulted in the covalent joining of the ends of the molecules, decreasing the quenching effect of the terminal bases on the dyes. To generate fluorogenic substrates for the base excision repair enzymes formamido–pyrimidine–DNA glycosylase (FPG), human 8-oxo-G DNA glycosylase/AP lyase (hOGG1), endonuclease IV (EndoIV), and apurinic/apyrimidinic endonuclease (APE1), we introduced abasic sites or a modified nucleotide, 8-oxo-dG, at such positions that their enzymatic excision would result in the release of a short fluorescent fragment. This was also accompanied by strong fluorescence increases. Overall fluorescence changes ranged from approximately 4-fold (ligase reactions) to more than 20-fold (base excision repair reactions).

Induction and repair of DNA damage measured by the comet assay in human T lymphocytes separated by immunomagnetic cell sorting

The comet assay is widely used in human biomonitoring to measure DNA damage in whole blood or isolated peripheral blood mononuclear cells (PBMC) as a marker of exposure to genotoxic agents. Cytogenetic assays with phytohemagglutinin (PHA)-stimulated cultured T lymphocytes are also frequently performed in human biomonitoring. Cytogenetic effects (micronuclei, chromosome aberrations, sister chromatid exchanges) may be induced in vivo but also occur ex vivo during the cultivation of lymphocytes as a consequence of DNA damage present in lymphocytes at the time of sampling. To better understand whether DNA damage measured by the comet assay in PBMC is representative for DNA damage in T cells, we comparatively investigated DNA damage and its repair in PBMC and T cells obtained by immunomagnetic cell sorting. PBMC cultures and T cell cultures were exposed to mutagens with different modes of genotoxic action and DNA damage was measured by the comet assay after the end of a 2h exposure and after 18h post-incubation. The mutagens tested were methyl methanesulfonate (MMS), (±)-anti-B[a]P-7,8-dihydrodiol-9,10-epoxide (BPDE), 4-nitroquinoline-1-oxide (4NQO), styrene oxide and potassium bromate. MMS and potassium bromate were also tested by the modified comet assay with formamido pyrimidine glycosylase (FPG) protein. The results indicate that the mutagens tested induce DNA damage in PBMC and T cells in the same range of concentrations and removal of induced DNA lesions occurs to a comparable extent. Based on these results, we conclude that the comet assay with PBMC is suited to predict DNA damage and its removal in T cells.

Assessment of RTG-W1, RTL-W1, and PLHC-1 fish cell lines for genotoxicity testing of environmental pollutants by means of a Fpg-modified comet assay

In a context of growing awareness of aquatic pollution impacts, there is an increasing need to develop methods for hazard and risk assessment of pollutants. For this purpose, in vitro models such as fish cell lines warrant to be evaluated as possible alternative to in vivo fish testing, and new toxicity endpoints such as genotoxicity deserve to be considered. This study assesses the interest of the formamido pyrimidine glycosylase (Fpg)-modified comet assay applied to three fish cell lines (RTL-W1, RTG-W1, and PLHC-1) regarding the sensitivity of the system for measuring genotoxicity of various classes of pollutants. Cytochrome P450-dependent EROD activity has also been measured to evaluate the importance of the biotransformation capacity of the cell lines in genotoxicity assessment. For all cell lines and chemicals tested, a concentration dependent genotoxic effect was observed with a 10- to 1000-fold increased sensitivity when using the Fpg-protocol compared to the standard comet assay. Such a modified assay led in particular to improve the detection threshold of oxidative and alkylating DNA damages following exposure at environmentally relevant contaminant concentrations and could partly compensate for the lower sensitivity of cell lines versus whole organism testing often cited as a limit of in vitro testing.

Vitamin A and C compounds permitted in supplements differ in their abilities to affect cell viability, DNA and the DNA nucleoside deoxyguanosine

In accordance with the European Parliament and Council's directive, vitamin A and C supplements can include any of four (vitamin A) or five (vitamin C) specified compounds. This study focuses on these compounds and compares their abilities to affect the DNA and viability of cells in culture, but also their potencies to chemically oxidise the DNA nucleoside deoxyguanosine (dG). To study the vitamins' strict chemical oxidation potencies, dG was exposed to vitamin solution and the amount of the oxidation product 8'-hydroxydeoxyguanosine (8-oxodG) formed was estimated using a high-performance liquid chromatography system with electrochemical and ultraviolet detection. The vitamin's ability to cause DNA damage to promyelocytic leukaemia cells (HL-60), as detected by strand breaks, alkaline labile sites and formamido pyrimidine DNA glycosylase (FPG)-sensitive sites was, after vitamin exposure, measured using the comet assay and cytotoxicity was estimated using trypan blue staining. The results highlight that vitamin A and C compounds found in supplements do have different properties, chemically as well as in a cellular system. Among the vitamin C compounds, ascorbic acid, sodium ascorbate and calcium ascorbate stood out causing both oxidation to dG and cytotoxicity to cells. The vitamin A compounds retinol, retinyl acetate and retinal (a breakdown product found in vivo) caused oxidation of dG, while retinal was the only compound causing cytotoxicity, giving rise to an almost complete cell death. β-carotene caused, as the only vitamin compound, a small increase in FPG-sensitive sites. It is concluded that even though the compounds are found under the same name (vitamin A or C), they do have different properties linked to oxidation, cytotoxicity and DNA damage.

New insights on the barrel medic MtOGG1 and MtFPG functions in relation to oxidative stress response in planta and during seed imbibition

In plants, 8-oxoguanine DNA glycosylase/lyase (OGG1) and formamidopyrimidine-DNA glycosylase (FPG) play similar roles within the base excision repair (BER) pathway involved in the removal of oxidized bases, e.g. 7,8-dihydro-8-oxoguanine (8-oxo-dG) and formamidopyrimidine (FAPy) lesions. To date, it is not clear why plants have retained both the OGG1 and FPG functions. In the present work, we have investigated the possible roles played in planta by MtOGG1 and MtFPG genes from Medicago truncatula Gaertn. (barrel medic). Bioinformatic investigation revealed the presence of putative mitochondrial and nuclear localization signals in the MtOGG1 and MtFPG amino acid sequences, respectively, thus suggesting for different subcellular fates. The expression profiles of both genes were evaluated by Quantitative Real-Time PCR (QRT-PCR) in barrel medic plantlets grown in vitro under oxidative stress conditions induced by copper (CuCl(2), 0.05, 0.1 and 0.2 mM) and polyethylene glycol (PEG6000, 50, 100 and 150 g L(-1)). The MtOGG1 and MtFPG genes were up-regulated in response to stress agents, at different levels, depending on treatment and tissue. As for copper, MtOGG1 showed significant up-regulation (up to 1.2- and 1.7-fold) only in roots while the MtFPG mRNA significantly increased (up to 1.3- and 2.8-fold, respectively) in roots and aerial parts. In response to PEG, the MtOGG1 expression was significantly enhanced in aerial parts (up to 1.3-fold) while the MtFPG showed significant (1.2-fold) up-regulation in roots. The expression profiles of MtOGG1 and MtFPG genes were also evaluated during seed imbibition, a physiological process which is characterized by Reactive Oxygen Species (ROS) accumulation and requires active DNA repair.

Antioxidant activities of major thyme ingredients and lack of (oxidative) DNA damage in V79 Chinese hamster lung fibroblast cells at low levels of carvacrol and thymol

The leafy parts of thyme and its essential oil have been used in foods for the flavour, aroma and preservation and also in folk medicines. In the present study the genotoxicity of thymol and carvacrol was examined using comet assay. In V79 Chinese hamster lung fibroblast cells treated with 1, 5, 25 μM thymol and carvacrol, only 25 μM thymol caused some clastogenic DNA damage. For detection of oxidative DNA damage, the comet assay with formamido pyrimidine glycosylase (Fpg) protein was used: When V79 cells were treated with 1, 5, 25 μM thymol and carvacrol and post-treated with Fpg enzyme, no significant increase of Fpg-sensitive sites was observed at all concentrations studied. Reactive oxygen species (ROS) generation decreased slightly in the presence of thymol (1–100 μM) and carvacrol (5 μM) between 1 and 4 h, yet increased at the highest 100 μM concentration of carvacrol after 24 h. Thymol and carvacrol displayed a concentration dependent antioxidant capacity, whilst γ-terpinene which lacks a phenolic group did not show any antioxidant capacity in the trolox equivalent antioxidant capacity (TEAC) assay. The results of this study indicate a lack of clastogenic activity for thymol and carvacrol at biologically relevant concentrations, and a moderate antioxidant activity in vitro.

Glycaemic status in relation to oxidative stress and inflammation in well-controlled type 2 diabetes subjects

The aim of the present observational study was to investigate the relationships between glycaemic status and levels of oxidative stress and inflammation in well-controlled type 2 diabetes subjects. Metabolic variables (weight, BMI, waist circumference (waist), blood glucose, glycated Hb (HbA(1c)), insulin, blood lipids), biomarkers of oxidative stress (8-iso-PGF(2alpha), malondialdehyde, 8-oxo-7,8-dihydro-2'-deoxyguanosine, formamido pyrimidine glycosylase-sites, frequency of micronucleated erythrocytes, nitrotyrosine) and inflammatory markers (high sensitivity C-reactive protein (hsCRP), IL-6, cyclo-oxygenase-catalyzed PGF(2alpha)-metabolite) were measured. Fifty-six patients (thirty women and twenty-six men, age 62.3 (SD 7.0) years, HbA(1c) 6.1 (SD 0.9) %, BMI 28.3 (SD 3.8) kg/m(2), waist 99.6 (SD 11.1) cm) were included in the study. HbA(1c) (r 0.29, P=0.03) and blood glucose (r 0.33, P=0.01) correlated positively with 8-iso-PGF(2alpha). Positive correlations were also observed between HbA(1c) and nitrotyrosine (r 0.42, P=0.01), waist and hsCRP (r 0.37, P=0.005), hsCRP and IL-6 (r 0.61, P<0.0001) and between PGF(2alpha)-metabolite and 8-iso-PGF(2alpha) (r 0.27, P=0.048). The present study indicates that glycaemic status is associated with oxidative stress even in subjects with well-controlled type 2 diabetes. Furthermore, inflammation was more related to abdominal obesity than to glycaemic control. A large number of biomarkers of oxidative stress and inflammation were investigated, but only a few associations were found between the markers. This could be due to the fact that none of these biomarkers biosynthesises via similar pathways or simultaneously owing to their diverse nature and origin.

Permethrin induces lymphocyte DNA lesions at both Endo III and Fpg sites and changes in monocyte respiratory burst in rats

Pyrethroids are widely used insecticides of low acute toxicity in mammals but the consequences of long-term exposure are of concern. Their insecticidal action is related to neurotoxicity and, in addition, there are indications of mammalian immuno-toxicity. In this work the effect of 60 days permethrin (150 mg kg(-1) body weight/day) exposure on two types of leukocytes (monocytes and lymphocytes) in adolescent rats was investigated. In particular, the monocyte respiratory burst response was first investigated, followed by studies on the degree and type of lymphocyte DNA damage induced by permethrin at this stage of life. Permethrin treatment reduces the monocyte respiratory burst response to phorbol myristate acetate, thereby decreasing superoxide anion (65%) and hydrogen peroxide (37%) production. Moreover an increase [correction made here after initial online publication] in monocyte plasma membrane fluidity in the hydrophilic-hydrophobic interface of the lipid bilayer was measured. Data obtained from the comet assay show that permethrin induces lymphocyte DNA lesions at both formamido pyrimidine glycosylase (Fpg) and endonuclease III (Endo III) sites in adolescent rats. Our results indicate the key role of permethrin in oxidative stress whose consequences lead to biochemical and functional changes. The reduced phagocyte respiratory burst induced by permethrin treatment and the type of DNA damage measured could represent new relevant aspects of pyrethroid toxicity which should be considered for human health.

DNA single- and double-strand breaks by alkaline- and immuno-comet assay in lymphocytes of workers exposed to styrene

Occupational exposure to styrene was studied in 34 workers employed in the production of fiberglass-reinforced plastic sheets and compared to 29 unexposed healthy controls. We evaluated genotoxic effects induced by occupational styrene exposure in lymphocytes by alkaline version of the comet assay to detect single-strand breaks (SSBs), DNA oxidation products (formamido pyrimidine glycosilase (Fpg)- and endonuclease (Endo III)-sensitive sites) and DNA repair kinetics studies, as well as the neutral version of comet assay for DNA double-strand breaks (DSBs). An innovative aspect of this study was the use of immuno-comet assay, a new technique that recognizes DSBs with specific antibody by DAPI/FITC method. The battery of parameters included markers of external and internal exposure. Exposed workers showed significant high levels of SSBs ( p < 0.0001) and DSBs ( p < 0.0001) in neutral- and immuno-comet assay. A drastic decrease in DNA repair activity as compared to controls was observed (180 min vs. 35 min). Styrene workplace concentration significantly correlated with alkaline comet parameters (TM, p = 0.013; TI, p = 0.008), in negative with TL ( p = 0.022), and with DNA-base oxidation (TM Endo III, p = 0.048 and TI Endo III, p = 0.028). There was a significant negative correlation between urinary metabolites (MA + PGA) and TM Endo III ( p = 0.032) and TI Endo III ( p = 0.017).

The Formamidopyrimidine Derivative of 7-(2-Oxoethyl)-2′-deoxyguanosine

Vinyl chloride induces hepatic angiosarcomas, which are otherwise rare malignancies. The biochemical basis involves the formation of the epoxide, which reacts with DNA to give approximately 98% of the 7-(2-oxoethyl) adduct (4) of dGuo plus small amounts of the etheno derivatives of dGuo, dCyd, and dAdo. The carcinogenicity is generally ascribed to the etheno adducts, not 4, because 4 has been shown to disappear from cells rapidly and to have negligible mutagenicity, which argues against its biological importance, whereas etheno adducts are both persistent and mutagenic. It has also been shown that apurinic sites derived from 4 are unlikely to be crucial lesions. A confounding factor with regard to the etheno hypothesis is that etheno adducts arise in unexposed cells by reactions of various lipid peroxidation products. The present study explores the possibility that a major contributor to the carcinogenicity of vinyl chloride may be formamidopyrimidine (FAPy) 12, N-[2-amino-6-[(2-deoxy-beta-D-erythro-pentofuranosyl)amino]-3,4-dihydro-4-oxo-5-pyrimidinyl]-N-(2-oxoethyl)-formamide, which can arise by ring opening of 4, although its formation has not been observed until the present study. N7 adduct 4 undergoes deglycosylation to give 7-(2-oxoethyl)-Gua (13) in acid and imidazolium ring-opening to 12 in base. At pH 7.4, both processes occur with the formation of 12 representing approximately 10% of the product mixture. FAPy 12 spontaneously cyclizes to 22, which upon mild acid treatment yields the deglycosylation product 2-amino-3,4,7,8-tetrahydro-7-hydroxy-4-oxopteridine-5(6H)-carbaldehyde (14). The structure of 14 has been established by NMR and mass spectroscopy and by independent synthesis. Reaction of the epoxide of crotonaldehyde with dGuo failed to give either 13 or 14, indicating that both compounds are unique products of the reactions of dGuo with the epoxides of vinyl monomers. Although FAPy 12 was found to be unstable, carbinolamine 22 arising from cyclization of 12 may be an important contributor to the carcinogenicity of vinyl chloride.

Oxidative DNA damage and augmentation of poly(ADP-ribose) polymerase/nuclear factor-kappa B signaling in patients with Type 2 diabetes and microangiopathy

Although oxidative stress and the subsequent DNA damage is one of the obligatory signals for poly(ADP-ribose) polymerase (PARP) activation and nuclear factor-kappa B (NFκB) alterations, these molecular aspects have not been collectively examined in epidemiological and clinical settings. Therefore, this study attempts to assess the oxidative DNA damage and its downstream effector signals in peripheral blood lymphocytes from Type 2 diabetes subjects without and with microangiopathy along with age-matched non-diabetic subjects. The basal DNA damage, lipid peroxidation and protein carbonyl content were significantly (p<0.05) higher in patients with and without microangiopathy compared to control subjects. Formamido Pyrimidine Glycosylase (FPG)-sensitive DNA strand breaks which represents reliable indicator of oxidative DNA damage were also significantly (p<0.001) higher in diabetic patients with (19.41±2.5) and without microangiopathy (16.53±2.0) compared to control subjects (1.38±0.85). Oxidative DNA damage was significantly correlated to poor glycemic control. PARP mRNA expression and PARP activity were significantly (p<0.05) increased in cells from diabetic patients with (0.31±0.03 densitometry units; 0.22±0.02PARPunits/mgprotein, respectively) and without (0.35±0.02; 0.42±0.05) microangiopathy compared to control (0.19±0.02; 0.11±0.02) subjects. Diabetic subjects with and without microangiopathy exhibited a significantly (p<0.05) higher (80%) NFκB binding activity compared to control subjects. In diabetic patients, FPG-sensitive DNA strand breaks correlated positively with PARP gene expression, PARP activity and NFκB binding activity. This study provides a comprehensive molecular evidence for increased oxidative stress and genomic instability in Type 2 diabetic subjects even prior to vascular pathology and hence reveals a window of opportunity for early therapeutic intervention.

Unraveling the Aflatoxin−FAPY Conundrum: Structural Basis for Differential Replicative Processing of Isomeric Forms of the Formamidopyrimidine-Type DNA Adduct of Aflatoxin B1

Aflatoxin B1 (AFB) epoxide forms an unstable N7 guanine adduct in DNA. The adduct undergoes base-catalyzed ring opening to give a highly persistent formamidopyrimidine (FAPY) adduct which exists as a mixture of forms. Acid hydrolysis of the FAPY adduct gives the FAPY base which exists in two separable but interconvertible forms that have been assigned by various workers as functional, positional, or conformational isomers. Recently, this structural question became important when one of the two major FAPY species in DNA was found to be potently mutagenic and the other a block to replication [Smela, M. E.; Hamm, M. L.; Henderson, P. T.; Harris, C. M.; Harris, T. M.; Essigmann, J. M. Proc. Natl. Acad. Sci. U.S.A. 2002, 99, 6655-6660]. NMR studies carried out on the AFB-FAPY bases and deoxynucleoside 3',5'-dibutyrates now establish that the separable FAPY bases and nucleosides are diastereomeric N5 formyl derivatives involving axial asymmetry around the congested pyrimidine C5-N5 bond. Anomerization of the protected beta-deoxyriboside was not observed, but in the absence of acyl protection, both anomerization and furanosyl --> pyranosyl ring expansion occurred. In oligodeoxynucleotides, two equilibrating FAPY species, separable by HPLC, are assigned as anomers. The form normally present in duplex DNA is the mutagenic species. It has previously been assigned as the beta anomer by NMR (Mao, H.; Deng, Z. W.; Wang, F.; Harris, T. M.; Stone, M. P. Biochemistry 1998, 37, 4374-4387). In single-stranded environments the dominant species is the beta anomer; it is a block to replication.

Sensitivity to oxidative damage and DNA repair capacity of Japanese skin

Sensitivity to oxidative damage and DNA repair capacity of Japanese skin

Sensitivity of the FPG protein towards alkylation damage in the comet assay

The comet assay (single cell gel electrophoresis) is widely used for the evaluation of DNA-damaging effects in genotoxicity testing and population monitoring. In its standard version at pH >13, DNA double strand breaks (DSB), DNA single strand breaks (SSB) and alkali-labile sites (ALS) lead to increased DNA migration. At reduced pH (12.5–12.1) the expression of ALS as SSB can be eliminated and the effect of SSB only can be identified. Specific endonucleases have been used to characterize specific classes of DNA damage. The formamido pyrimidine glycosylase (FPG) protein has been used to assess oxidative DNA base damage because it detects 8-OH guanine and other oxidatively damaged purines. Here, we show that the FPG protein also detects alkylation damage with high sensitivity in the comet assay. Human whole blood, isolated lymphocytes and V79 cells were treated with alkylating agents and post-incubated with FPG. FPG strongly enhanced MMS- and EMS-induced DNA damage but had no significant effect on ENU-induced DNA damage, indicating that the amount of N-7 guanine alkylation is responsible for the observed effect. Reducing the pH during alkali unwinding and electrophoresis to 12.5 to avoid the contribution of ALS to the comet assay effects, strongly decreased the sensitivity of the comet assay with and without FPG treatment and prevented DNA migration. We conclude that enhanced DNA effects in the comet assay by FPG after exposure to genotoxins with unknown mode of action should not directly be regarded as evidence for the presence of oxidative damage. Furthermore, reducing the pH leads to a considerable loss in sensitivity and should not be used in biomonitoring and other applications which require a sensitive protocol.

Modulation of exogenous and endogenous levels of thioredoxin in human skin fibroblasts prevents DNA damaging effect of ultraviolet A radiation

Thioredoxin (Trx) plays important biological roles both intra- and extracellularly via thiol redox control. We have previously demonstrated that Trx exhibited protective effects against UVA cytotoxicity in human skin fibroblasts. As an extension of the latter investigation, the present work is aimed at assessing ability of Trx to maintain genomic integrity in human skin fibroblasts upon exposure to UVA radiation. Indeed, UVA (320–380 nm) is mutagenic and induces genomic damage to skin cells. The alkaline comet assay was used in association with DNA repair enzyme including formamido pyrimidine glycosylase (Fpg) and endonuclease III (endo III) to estimate the amount of modified bases together with the level of strand breaks and alkali-labile sites. The HPLC-EC assay was applied to assess 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodGuo) levels and to permit the calibration of comet assay as previously described. We reported that overexpression of human Trx (transient transfection) as well as exogenous human recombinant Trx added to the culture medium, decreased the level of DNA damage in UVA irradiated cells. Interestingly, transfection appeared to prevent UVA-induced 8-oxodGuo (3.06 au per Joules.cm −2 compared to 4.94 au per Joules.cm −2 for nontransfected cells). Moreover, Trx accumulates into nuclei in transfected cells. This finding supports the notion that Trx is important for the maintenance of the integrity of genetic information. This work demonstrated that under conditions of UVA oxidative stress, Trx prevented the UVA-induced DNA damage.

Escherichia coli mutM suppresses illegitimate recombination induced by oxidative stress.

DNA damage by oxidative stress is one of the causes of mutagenesis. However, whether or not DNA damage induces illegitimate recombination has not been determined. To study the effect of oxidative stress on illegitimate recombination, we examined the frequency of lambdabio transducing phage in the presence of hydrogen peroxide and found that this reagent enhances illegitimate recombination. To clarify the types of illegitimate recombination, we examined the effect of mutations in mutM and related genes on the process. The frequency of lambdabio transducing phage was 5- to 12-fold higher in the mutM mutant than in the wild type, while the frequency in the mutY and mutT mutants was comparable to that of the wild type. Because 7,8-dihydro-8-oxoguanine (8-oxoG) and formamido pyrimidine (Fapy) lesions can be removed from DNA by MutM protein, these lesions are thought to induce illegitimate recombination. Analysis of recombination junctions showed that the recombination at Hotspot I accounts for 22 or 4% of total lambdabio transducing phages in the wild type or in the mutM mutant, respectively. The preferential increase of recombination at nonhotspot sites with hydrogen peroxide in the mutM mutant was discussed on the basis of a new model, in which 8-oxoG and/or Fapy residues may introduce double-strand breaks into DNA.

Mass Spectral Identification and Positional Mapping of Aflatoxin B 1–Guanine Adducts in Oligonucleotides

The biological consequences of a carcinogen–DNA adduct are defined by the structure of the lesion and its position within the genome. Electrospray ionization ion trap mass spectrometry (ESI-ITMS) is shown here to be a sensitive and rapid approach capable of defining both of these parameters. Three isomeric oligonucleotides of the sequence 5′-CCGGAGGCC modified by the potent human carcinogen aflatoxin B 1 (AFB 1) at different guanines were analyzed by ESI-ITMS. All three samples possessed the same molecular ion confirming the presence of an intact aflatoxin moiety in each oligonucleotide. In addition, each sample displayed a characteristic fragmentation pattern that permitted unambiguous identification of the site of modification within the sequence. Furthermore, an AFB 1-modified oligonucleotide was converted under alkaline conditions to its more stable formamidopyrimidine (FAPY) derivative. Analysis of this sample revealed the presence of a molecular ion corresponding to the presence of the FAPY adduct and a distinctive fragmentation pattern that paralleled the known chemical stability of the FAPY metabolite. This approach should be of general use in the determination of not only the nature and site of covalent modifications, but also the chemical stability of DNA adducts.