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At least 901 records · Page 50Linked to original sources
PubMed2001

Formaldehyde-mediated modification of natural deoxyguanosine with amines: one-pot cyclization as a molecular model for genotoxicity.

Stable cross-linked adducts, 3-(2-deoxy-beta-D-ribofuranosyl)-7-phenyl-5,6,7,8-tetrahydro[1,3,5]triazino[1,2-a]purin-10(3H)-one and 7-butyl-3-(2-deoxy-beta-D-ribofuranosyl)-5,6,7,8-tetrahydro[1,3,5]triazino[1,2-a]purin-10(3H)-one, that formed chemically from natural deoxyguanosine and aniline or buthyl amine in the presence of formaldehyde were identified. This reaction appears to be a general reaction of deoxyguanosine and primary amines, and it may be a model of DNA modification with carcinogenic aromatic amines without metabolic activation, if formaldehyde is present.

Amines↗
PubMed2002

Dissociation of DNA damage and mitochondrial injury caused by hydrogen peroxide in SV-40 transformed lung epithelial cells.

BACKGROUND: Since lung epithelial cells are constantly being exposed to reactive oxygen intermediates (ROIs), the alveolar surface is a major site of oxidative stress, and each cell type may respond differently to oxidative stress. We compared the extent of oxidative DNA damage with that of mitochondrial injury in lung epithelial cells at the single cell level. RESULT: DNA damage and mitochondrial injury were measured after oxidative stress in the SV-40 transformed lung epithelial cell line challenged with hydrogen peroxide (H2O2). Single cell analysis of DNA damage was determined by assessing the number of 8-oxo-2-deoxyguanosine (8-oxo-dG) positive cells, a marker of DNA modification, and the length of a comet tail. Mitochondrial membrane potential, DeltaPsim, was determined using JC-1. A 1 h pulse of H2O2 induced small amounts of apoptosis (3%). 8-oxo-dG-positive cells and the length of the comet tail increased within 1 h of exposure to H2O2. The number of cells with reduced DeltaPsim increased after the addition of H2O2 in a concentration-dependent manner. In spite of a continual loss of DeltaPsim, DNA fragmentation was reduced 2 h after exposure to H2O2. CONCLUSION: The data suggest that SV-40 transformed lung epithelial cells are resistant to oxidative stress, showing that DNA damage can be dissociated from mitochondrial injury.

Journal Article↗
PubMed1993

Detoxication products of the carcinogenic azodye Sudan I (solvent yellow 14) bind to nucleic acids after activation by peroxidase.

The C-hydroxyderivatives of the carcinogenic dye Sudan I, 1-phenylazo-2,6-dihydroxynaphthalene and 1-(4-hydroxyphenylazo)-2-hydroxynaphthalene, which are considered to be detoxication products of this dye bind to DNA or tRNA after oxidation into active metabolites by peroxidase and H2O2 in vitro. The 32P-postlabeling analysis of DNA modified by active metabolites of both Sudan I derivatives provides evidence that the covalent binding to DNA is the principal type of DNA modification. Since the urinary bladder is rich in peroxidases, the participation of these enzymes in activation of detoxicating products of Sudan I may be involved in the initiation of Sudan I-carcinogenesis in this organ.

Animals↗
PubMed2005

Occurrence of restriction-modification systems in ruminal butyrate-producing bacteria.

Thirty-five strains of ruminal bacteria belonging to the former Butyrivibrio fibrisolvens species were screened for the presence of site-specific restriction endonuclease and modification methyltransferase activities. Seven strains possessed endonuclease activities detectable in crude cell extracts. The recognition sequences and optimal reaction conditions for seven of them were determined. Five enzymes were found to be isoschizomers of type II endonucleases (EcoRV, NsiI, AseI (2x) and SauI), one was type IIS (FokI) and two remained unknown. The optimal reaction buffer was found to be a low ionic strength buffer and all enzymes possessed sufficient activity at 39 degrees C. The presence of DNA modification among all strains was also determined. Most of the methylation activities correlated with restriction activities, yet some strains possessed unaccompanied modification methyltransferases.

Journal Article↗
PubMed1983

The correlation between benzo[a]pyrene-induced mutagenicity and DNA adduct formation in Salmonella typhimurium TA100.

In an attempt to stabilize the dose response in the Salmonella typhimurium test (STT), the use of DNA-bound products from BP was evaluated as a measure of the biologically effective dose. In addition to the previously documented interlaboratory variation, we observed a 3-fold difference in the dose response of TA100 to BP even when the assay was repeated with the same experimental conditions. When overall BP-DNA adduct formation was related to the level of His+ revertants, a series of responses emerged with two predominating. In the first type of response around 70 revertants per plate were generated for every BP molecule bound per 10(6) nucleotides of cellular DNA. The second response gave about 1400 revertants per plate for one BP bound in every 10(6) nucleotides. Several intermediates curves were also detected. The variation in the mutational response to binding levels occurred regardless of the source of S9 or the growth stage of the cells. These experiments indicate that there was no constant level of DNA damage that would lead to a specified number of revertants of TA100 by BP and that DNA modification was not solely responsible for mutagenic potency. It is possible that an induction of an error-prone repair function of the muc gene carried by the plasmid pKM101 in TA100 may be affecting the relationship between the measured adduct level and reversion frequency.

Benzopyrenes↗
PubMed1985

Detection of DNA adducts in N-acetoxy-2-acetylaminofluorene-treated human fibroblasts by means of immunofluorescence microscopy and quantitative immunoautoradiography.

An immunohistochemical procedure was developed for the detection of adducts in DNA of cultured cells exposed to N-acetoxy-2-acetylaminofluorene (N-AcO-AAF) with the use of antibodies raised in rabbits against N-(guanosin-8-yl)-2-acetyl-aminofluorene (Guo-8-AAF) conjugated to bovine serum albumin. Binding of these antibodies to the nuclear DNA was visualized by means of immunofluorescence microscopy with fluorescein-conjugated anti-rabbit-Ig antibodies, or by means of immunoautoradiography with 125I-labeled protein A. The dose-response curve obtained when the number of autoradiographic grains developed over the nuclei was plotted as a function of the concentration of N-AcO-AAF used to treat the cells, indicated that the extent of specific antibody-binding is determined by the amount of adducts in the cells. DNA modification levels allowing for 20% survival of the cells could be detected with the immunofluorescence technique, while cells exposed to concentrations of N-AcO-AAF resulting in 60% survival were still positive with the immunoautoradiographic method.

2-Acetylaminofluorene↗
PubMed2005

N-methylpurine DNA glycosylase overexpression increases alkylation sensitivity by rapidly removing non-toxic 7-methylguanine adducts.

Previous studies indicate that overexpression of N-methylpurine DNA glycosylase (MPG) dramatically sensitizes cells to alkylating agent-induced cytotoxicity. We recently demonstrated that this sensitivity is preceded by an increased production of AP sites and strand breaks, confirming that overexpression of MPG disrupts normal base excision repair and causes cell death through overproduction of toxic repair intermediates. Here we establish through site-directed mutagenesis that MPG-induced sensitivity to alkylation is dependent on enzyme glycosylase activity. However, in contrast to the sensitivity seen to heterogeneous alkylating agents, MPG overexpression generates no cellular sensitivity to MeOSO2(CH2)2-lexitropsin, an alkylator which exclusively induces 3-meA lesions. Indeed, MPG overexpression has been shown to increase the toxicity of alkylating agents that produce 7-meG adducts, and here we demonstrate that MPG-overexpressing cells have dramatically increased removal of 7-meG from their DNA. These data suggest that the mechanism of MPG-induced cytotoxicity involves the conversion of non-toxic 7-meG lesions into highly toxic repair intermediates. This study establishes a mechanism by which a benign DNA modification can be made toxic through the overexpression of an otherwise well-tolerated gene product, and the application of this principle could lead to improved chemotherapeutic strategies that reduce the peripheral toxicity of alkylating agents.

Alkylating Agents↗
PubMed2004

Effects of vitamin E depletion/repletion on biomarkers of oxidative stress in healthy aging.

The effects on ex vivo LDL resistance to oxidation and biomarkers of in vivo oxidative stress in response to 3-month dietary vitamin E restriction to 25% of recommended intake and 2-month unrestricted dietary intake and supplementation with 800 IU/d were studied in 100 healthy, nonsmoking 20-75-year-old volunteers. Significant changes in vitamin E status were associated with decreases and increases, respectively, in LDL resistance to oxidation in the depletion and supplementation period and with decreases in lipid peroxidation and oxidative DNA modification in the supplementation period. Healthy aging was not associated with enhanced susceptibility to oxidation in the depletion period.

Adult↗
PubMed2001

A common mechanism for ATP hydrolysis in ABC transporter and helicase superfamilies.

ABC (ATP-binding cassette) transporters and helicases are large superfamilies of seemingly unrelated proteins, whose functions depend on the energy provided by ATP hydrolysis. Comparison of the 3D structures of their nucleotide-binding domains reveals that, besides two well-characterized ATP-binding signatures, the folds of their nucleotide-binding sites are similar. Furthermore, there are striking similarities in the positioning of residues thought to be important for ATP binding or hydrolysis. Interestingly, structures have recently been obtained for two ABC proteins that are not involved in transport activities, but that have a function related to DNA modification. These ABC proteins, which contain a nucleotide-binding site akin to those of typical ABC transporters, might constitute the missing link between the two superfamilies.

ATP-Binding Cassette Transporters↗
PubMed1989

Interactions between eukaryotic DNA topoisomerase I and a specific binding sequence.

The interaction between eukaryotic DNA topoisomerase I and a high affinity binding sequence was investigated. Quantitative footprint analysis demonstrated that the substrate preference results from strong specific binding of topoisomerase I to the sequence. The specificity was conferred by a tight noncovalent association between the enzyme and its target DNA, whereas the transient formation of a covalently bound enzyme.nicked DNA intermediate contributed insignificantly to the overall affinity. Topoisomerase I protected both strands over a 20-base pair region in which the cleavage site was centrally located. DNA modification interference analysis revealed a 16-base pair interference region on the scissile strand. Essential bases were confined to the 5' side of the cleavage site. The 6-base pair interference region observed on the complementary strand did not contain essential bases.

Animals↗
PubMed1998

Intraperitoneal cisplatin with regional hyperthermia in advanced ovarian cancer: pharmacokinetics and cisplatin-DNA adduct formation in patients and ovarian cancer cell lines.

The purpose of this study was to investigate the influence of hyperthermia on cisplatin pharmacokinetics and DNA adduct formation. The latter was investigated both in tumour cell lines in vitro and in tumour cells and buccal cells from cancer patients. The patients had advanced ovarian carcinoma and were entered into a phase I study for cytoreductive surgery followed by hyperthermia in combination with intraperitoneal cisplatin administration. The cisplatin-DNA modifications in vivo and in vitro were studied by an immunocytochemical method with the polyclonal antiserum NKI-A59. The patient samples for pharmacokinetic determinations were analysed by flameless atomic absorption spectrometry. In vitro, the combination of hyperthermia and cisplatin enhanced cell killing compared with either treatment alone, such that the cisplatin-resistant ovarian cell line A2780/DDP became almost as sensitive as the parent A2780 cell line (resistance factor reduced from 30 to 2 at the IC50). In addition, increased cisplatin-DNA adducts were observed in the resistant cell line after the combined treatment compared with cisplatin alone. A good correlation was found between nuclear staining density and surviving fraction for all groups, indicating that the DNA adducts generated are an important determinant of toxicity and that the mechanism by which hyperthermia enhances kill is by increasing adduct levels. In the patients, the ratio of drug concentration in the peritoneal perfusate compared with that in plasma was found to be approximately 15, indicating a favourable pharmacokinetic ratio. Cisplatin-DNA adduct formation in tumour cells from patients was higher than in buccal cells, reflecting this higher drug exposure, i.e. local plus systemic versus systemic only. In addition, the tumour cells but not buccal cells were exposed to hyperthermia. The higher number of tumour adducts also suggests that a favourable therapeutic ratio could be achieved. Platinum-DNA adduct formation was found to decrease with distance from the surface of the tumour nodules. However, at a distance of 3-5 mm, the nuclear staining density levels were still measurable and higher than in buccal cells. In conclusion, the combined pharmacokinetic and adduct data in patients support the advantages of the intraperitoneal route for drug administration, and the addition of heat.

Adult↗
PubMed1984

Immunoglobulin gene expression and DNA methylation in murine pre-B cell lines.

DNA modification accompanying immunoglobulin gene expression was examined in various Abelson murine leukemia virus (A-MuLV)-transformed cell lines, which were able to differentiate from the mu- to mu+ stage or to undergo an isotype switch during in vitro culture. The C mu genes were relatively demethylated in the A-MuLV-transformed cell lines examined irrespective of whether or not the C mu genes were expressed. Normal IgM-bearing B cells, as well as a T cell line, also showed a similar DNA methylation pattern and the C mu genes were relatively demethylated. In one of the mu+ clones, however, the expressed C mu gene was heavily methylated. The DNA methylation pattern did not change and remained hypermethylated before and after gamma 2b expression in the two cell lines which underwent class switch to gamma 2b during in vitro culture, suggesting that expression of the gamma 2b gene was not accompanied by demethylation of the C gamma 2b gene. Taken together, these results indicate that DNA demethylation within and around the CH gene may not be necessary for its expression.

Animals↗
PubMed1997

A prediction of the amino acids and structures involved in DNA recognition by type I DNA restriction and modification enzymes.

The S subunits of type I DNA restriction/modification enzymes are responsible for recognising the DNA target sequence for the enzyme. They contain two domains of approximately 150 amino acids, each of which is responsible for recognising one half of the bipartite asymmetric target. In the absence of any known tertiary structure for type I enzymes or recognisable DNA recognition motifs in the highly variable amino acid sequences of the S subunits, it has previously not been possible to predict which amino acids are responsible for sequence recognition. Using a combination of sequence alignment and secondary structure prediction methods to analyse the sequences of S subunits, we predict that all of the 51 known target recognition domains (TRDs) have the same tertiary structure. Furthermore, this structure is similar to the structure of the TRD of the C5-cytosine methyltransferase, Hha I, which recognises its DNA target via interactions with two short polypeptide loops and a beta strand. Our results predict the location of these sequence recognition structures within the TRDs of all type I S subunits.

Amino Acid Sequence↗
PubMed1978

Application of alkaline sucrose gradient sedimentation to the study of DNA damage and its repair in mammalian cells treated with methylmethanesulfonate and 4-nitroquinoline-1-oxide.

KB cells and L cells were treated with methylmethanesulfonate (MMS) or 4-nitroquinoline-1-oxide (4 NQO) and the resulting damage to DNA and its repair were examined by sedimentation in an alkaline sucrose gradient. The sedimentation profiles obtained were found to be the resultant of a complex interrelationship between drug dosage, duration of the lysis period and the repair capacity of the cells. A systematic study of these variables was made which led to a plausible and useful interpretation of the sedimentation profiles. Both drugs produce two kinds of DNA modifications which show up as a single-strand breaks but affect the sedimentation profile in characteristic ways. One of these modifications which is quite alkali-labile can be studied using a 30-min lysis period. The other modification is less alkali-labile and can be studied using a long lysis period. Both KB cells and L cells can repair the former type of damage but only KB cells can repair the latter type of damage.

4-Nitroquinoline-1-oxide↗
PubMed1986

The mom gene of bacteriophage Mu: the mechanism of methylation-dependent expression.

Transcription of the DNA modification gene (mom) of bacteriophage Mu requires methylation of three GATC sites upstream of the mom promoter by the Escherichia coli deoxyadenosine methylation function (Dam). The three sites map within a 40-bp segment termed region I. Small deletions, inversions, duplications and specific point mutations have been introduced in region I. Their effect on mom expression has been studied in dam+ and dam strains. Dam-dependent expression of the mom gene requires a specific arrangement of the three GATC sites and the presence of the methylated base in at least two of the three sites. We show that mom specific modification is regulated by a host protein. The Mom function is expressed in dam strains if they are defective in one component of the methylation-instructed mismatch correction system, mutH. We suggest that the product of mutH functions as a transcriptional repressor by binding to region I.

Base Sequence↗
PubMed1999

Analysis of oxidative DNA damage and HPRT mutations in humans after hyperbaric oxygen treatment.

DNA damage induced by reactive oxygen species (ROS) seems to play an important role in the induction of mutations and cancer. We have recently shown that hyperbaric oxygen (HBO) treatment of volunteers (i.e., exposure to 100% oxygen at a pressure of 2.5 ATA) induces DNA damage detected in leukocytes with the comet assay. Using formamidopyrimidine-DNA glycosylase (FPG protein) we provided indirect evidence for the induction of oxidative DNA base damage. We now comparatively evaluated FPG-sensitive sites with the comet assay and 7,8-dihydro-8-oxo-deoxyguanosine (8-OHdG) with HPLC analysis after a single HBO. As 8-OHguanine (8-OHgua) is one of the major DNA modifications induced by ROS and a pre-mutagenic lesion, we looked for HBO-induced mutations at the HPRT locus with the T cell cloning test. We also determined the genotypes for glutathione transferases (GST) and tested a possible influence of the GSTM1 and GSTT1 genotypes on the sensitivity of subjects against HBO-induced genotoxicity. Our results indicate that despite a clear induction of FPG-sensitive sites no increased levels of 8-OHdG and no induction of HPRT mutations was detected in lymphocytes after HBO. Furthermore, the DNA effects in the comet assay and the mutant frequencies in the HPRT test seem to be unrelated to the GST genotypes of the test subjects.

8-Hydroxy-2'-Deoxyguanosine↗
PubMed1988

New bacteriophages active on strains of Hyphomicrobium.

Fifty-five lytic bacteriophages isolated from water and soil samples were active on many strains of the genus Hyphomicrobium. The optimal isolation procedure was an adsorption method in which samples from a habitat similar to that of the respective host bacterium were used as the phage inoculum. According to the morphology and nucleic acid type these bacteriophages belonged to different families: Myoviridae (type A1: five phages); Styloviridae (type B1: 33 phages; type B2: eight phages) and Podoviridae (type C1: nine phages). The Styloviridae (type B1) appeared in two morphological variants (tails flexible or rigid). All phages investigated were specific for the genus Hyphomicrobium and were unable to lyse members of other genera of hyphal, budding bacteria (e.g. Hyphomonas, Pedomicrobium, genus D, genus T). The host specificity of 42 phages was tested with 156 Hyphomicrobium strains: 122 strains were lysed by at least one of these phages, but 34 Hyphomicrobium strains were not susceptible. Morphotype B1 phages with identical morphology could be distinguished according to their host-range properties on prophage-containing Hyphomicrobium strains. With regard to differences in morphology and host range, 25 phages were selected for more detailed investigations. From these phages DNA was isolated; the melting transition midpoints (Tm) ranged from 67 to 93 degrees C. The upper and higher values suggested the presence of DNA modifications. Six different adsorption patterns could be distinguished among the Hyphomicrobium phages. Preferred attachment sites were the proximal pole of the mother cell, the hyphal tip, the distal pole of the bud, and the distal pole of the swarmer cell.

Bacteria↗
PubMed1984

DNA methylation in 5-aza-2'-deoxycytidine-resistant variants of C3H 10T1/2 C18 cells.

A cell line (T17) was derived from C3H 10T1/2 C18 cells after 17 treatments with increasing concentrations of 5-aza-2'-deoxycytidine. The T17 cell line was very resistant to the cytotoxic effects of 5-aza-2'-deoxycytidine, and the 50% lethal dose for 5-aza-2'-deoxycytidine was ca. 3 microM, which was 30-fold greater than that of the parental C3H 10T1/2 C18 cells. Increased drug resistance was not due to a failure of the T17 cell line to incorporate 5-aza-2'-deoxycytidine into DNA. The cells were also slightly cross-resistant to 5-azacytidine. The percentage of cytosines modified to 5-methylcytosine in T17 cells was 0.7%, a 78% decrease from the level of 3.22% in C3H 10T1/2 C18 cells. The DNA cytosine methylation levels in several clones isolated from the treated lines were on the order of 0.7%, and clones with methylation levels lower than 0.45% were not obtained even after further drug treatments. These highly decreased methylation levels appeared to be unstable, and DNA modification increased as the cells divided in the absence of further drug treatment. The results suggest that it may not be possible to derive mouse cells with vanishingly low levels of 5-methylcytosine and that considerable de novo methylation can occur in cultured lines.

Animals↗