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At least 973 records · Page 54Linked to original sources
PubMed2000

Classical conditioning of oxidative DNA damage in rats.

This study investigated whether the formation of 8-hydroxydeoxyguanosine (8-OH-dG), a known oxidative DNA modification relevant to carcinogenicity, can be classically conditioned to a novel taste in order to clarify the possible role of the central nervous system (CNS) or psychological stress on cancer initiation via a classical conditioning mechanism. Male Wistar rats underwent one or two conditioned taste aversion (CTA) experiments in which ferric nitrilotriacetate (Fe-NTA), which has renal toxicity and can induce renal cell carcinoma, served as a visceral unconditioned stimulus (US), and a saccharin solution (SAC) was used as a conditioned stimulus (CS). The 8-OH-dG levels in the group conditioned with the combination of SAC and Fe-NTA significantly increased as compared to those of the uncombined groups by two repeats of the conditioning procedure (P=0.013). The rats that showed a painful response at the Fe-NTA administration had significantly higher values of 8-OH-dG than those without pain (P=0. 003). These results not only provide the first evidence regarding classical conditioning of oxidative DNA damage using the CTA procedure, but also suggest the involvement of the CNS and psychological stress in the pathogenesis of cancer via oxidative DNA damage.

8-Hydroxy-2'-Deoxyguanosine↗
PubMed2006

Cytosine methylation and DNA repair.

Cytosine methylation is a common form of post-replicative DNA modification seen in both bacteria and eukaryotes. Modified cytosines have long been known to act as hotspots for mutations due to the high rate of spontaneous deamination of this base to thymine, resulting in a G/T mismatch. This will be fixed as a C-->T transition after replication if not repaired by the base excision repair (BER) pathway or specific repair enzymes dedicated to this purpose. This hypermutability has led to depletion of the target dinucleotide CpG outside of special CpG islands in mammals, which are normally unmethylated. We review the importance of C-->T transitions at non-island CpGs in human disease: When these occur in the germline, they are a common cause of inherited diseases such as epidermolysis bullosa and mucopolysaccharidosis, while in the soma they are frequently found in the genes for tumor suppressors such as p53 and the retinoblastoma protein, causing cancer. We also examine the specific repair enzymes involved, namely the endonuclease Vsr in Escherichia coli and two members of the uracil DNA glycosylase (UDG) superfamily in mammals, TDG and MBD4. Repair brings its own problems, since it will require remethylation of the replacement cytosine, presumably coupling repair to methylation by either the maintenance methylase Dnmt1 or a de novo enzyme such as Dnmt3a. Uncoupling of methylation from repair may be one way to remove methylation from DNA. We also look at the possible role of specific cytosine deaminases such as Aid and Apobec in accelerating deamination of methylcytosine and consequent DNA demethylation.

5-Methylcytosine↗
PubMed2005

The analysis of X-chromosome inactivation-related gene expression from single mouse embryo with sex-determination.

Chromatin remodeling by histone and DNA modification is important for the initiation of X-chromosome inactivation (XCI). In this study, a thorough transcriptional analysis of five XCI-related genes was performed by single cell reverse-transcribed PCR. An analysis of the XCI-related gene (Xist, Tsix, SUV39H1, SET7, and DNMT1) expression was performed to investigate the initiation process of XCI from early mouse single embryo (1-cell, 2-cell, 4-cell, 8-cell, and blastocyst). Detection of the expression of Xist and Tsix from single 2-cell embryo was feasible, although the expression of those genes was very low in single 1-cell embryo. Transcription of those genes may be activated from single 2-cell embryo. After determining the sex of single embryo by Y-chromosome-specific Zfy expression, we found that Tsix could be detected from both male and female single embryos, but it was only possible to detect Xist from female single embryo. XCI chromatin-remodeling genes, such as histone H3 methylation enzymes (SUV39H1 and SET7) and DNA methylation enzyme (DNMT1), were expressed during all early phases of embryogenesis. The expression of those genes in single embryo was not dependent on sex. Our study illustrated that the expression of these chromatin-remodeling genes, SUV39H1, DNMT1, and SET7, may be originated from germ cells, which were not dependent on zygotic activation of Xist from female single embryo.

Animals↗
PubMed1998

Germ cell mutagenesis in Drosophila: multiple endpoint analysis.

Genotoxic carcinogens, able to damage DNA by alkylation reactions, represent a very diverse class of agents which are capable of producing a wide range of DNA modifications. The mechanisms leading to genetic changes as a result of exposure to alkylating agents (AAs) have been studied in male germ cells of Drosophila using a structure-activity relationship approach (SAR). The analytical tools available concern both genetic and molecular assays. The genetic tests enable to quantify excision repair and clastogenic potency of the AA after treatment of post-meiotic male germ cells and to determine the degree of germ-cell specificity, i.e., the mutagenic effectiveness in post- versus premeiotic cell stages. For a selected group of alkylating agents the molecular spectra have been studied in post-meiotic cell stages. On the basis of these descriptors clear SAR's between genotoxic activity in germ cells and physico-chemical parameters (s-values and O6/N7-alkylguanine adducts) and carcinogenic potency in rodents became apparent, resulting in five distinct classes of alkylating agents so far. These classes are: 1) SN2-type monofunctional AAs, 2) SN1-type monofunctional AAs, 3) polyfunctional AAs, 4) agents able to form etheno-DNA adducts, and 5) aflatoxin B1 (AFB1) a bulky-adduct forming agent. The recent finding that the molecular data obtained with Drosophila and data of the specific locus tests in male mice show remarkable similarities for most genotoxic agents supports the view that Drosophila is a useful model system for the study of transgenerational damage.

Alkylating Agents↗
PubMed1991

Formation of interaction products of carboplatin with DNA in vitro and in cancer patients.

Binding of the cytostatic drug carboplatin to DNA was studied in solution, in RIF-1 and CHO cell lines and in human buccal cells after in vitro or in situ drug exposure. Results were compared with DNA adduction by cisplatin. The rate of binding in solution, determined by atomic absorption spectroscopy, was 35 times lower for carboplatin than for cisplatin. Adduct formation in cells in vitro was determined in a quantitative immunostaining assay. Staining intensities after carboplatin treatment were at least 29 times lower than after an equimolar dose of cisplatin. For RIF-1 and CHO cells, maximum levels of carboplatin-induced DNA modification were obtained 24 h after treatment; these levels correlated with cell killing. Adduct-specific staining in buccal cells from two carboplatin-treated patients increased 5-7 fold between 0 and 14 h after infusion, reaching a maximum at 10-14 h. This strongly contrasts with buccal cells from a cisplatin-treated patient, in which the adduct-specific staining signal increased by only 23% between 0 and 6 h after infusion, and then declined. This difference in the rate of adduct formation in vivo is consistent with the in vitro data.

Animals↗
PubMed2003

Genetic and cytological characterization of the RecA-homologous proteins Rad51 and Dmc1 of Schizosaccharomyces pombe.

The Schizosaccharomyces pombe rad51(+) and dmc1(+) genes code for homologues of the Escherichia coli recombination protein RecA. Deletion of rad51(+) causes slow growth, retardation of cell division and a decrease in viability. rad51Delta cells have a defect in mating-type switching. The DNA modification at the mating-type locus required for mating-type switching contributes to slow growth in the rad51 mutant. Cell mating is reduced in crosses homozygous for rad51Delta. Ectopic expression of the dmc1(+) gene allowed us to demonstrate that the reduction in meiotic recombination in dmc1 mutants is not caused by a disturbance of rad24 expression from the dmc1- rad24 bicistronic RNA. We describe the functional defects of terminally epitope-tagged Dmc1 and Rad51 and discuss it in terms of protein interaction. Presumptive Rad51 and Dmc1 foci were detected on spreads of meiotic chromatin.

Adenosine Triphosphatases↗
PubMed1993

DNA adducts in model systems and humans.

The etiology of chemically induced cancer is thought to involve the covalent binding of carcinogens to DNA (adducts) leading to mutations in oncogenes or tumor suppressor genes, and ultimately to tumors. Thus, the DNA-carcinogen adduct has been used as a measurable biochemical endpoint in laboratory studies designed to assess carcinogen exposure, carcinogen metabolism, mutagenesis, and tumorigenesis. Unfortunately, the significance of adducts in the etiology of human cancer is still unclear. This is partially due to the difficulty detecting adducts at carcinogen exposures relevant to humans, which are often orders of magnitude lower than animal model exposures. The relationship between adducts and higher biological effects is also not known at low doses. We have been assessing the DNA damage caused by exposure to heterocyclic amine carcinogens in the diet. Using the technique of 32P-postlabeling in combination with accelerator mass spectrometry, we have determined that DNA adduction in rodents decreases linearly with decreasing dose from the high doses used in typical cancer bioassays to the low doses relevant to human exposures. For a given tissue, adduct levels are correlated with dose, but the level of DNA modification by carcinogens is tissue-specific and does not completely correlate with tumor site. This lack of correlation may be due to differences in adduct formation and repair rates among tissues. Comparison of carcinogen metabolism routes between rodents and humans also indicates that species differences could influence the amount and type of damage resulting from exposure to these carcinogens. The use of model systems to study dosimetry, species differences in adduction, and role of adducts in mutation will ultimately lead to a better understanding of the significance of adducts in human disease. This should eventually allow the use of adducts as biomarkers for estimating carcinogen exposure and individual susceptibility.

Amines↗
PubMed1994

Thyroid hormone receptor monomer, homodimer, and heterodimer (with retinoid-X receptor) contact different nucleotide sequences in thyroid hormone response elements.

Thyroid hormone receptors (TRs) bind as monomers, homodimers, and heterodimers with nuclear proteins such as retinoid-X receptors (RXRs) to thyroid hormone response elements (TREs). However, it is not known which nucleotides each TR complex contacts in a particular TRE. To identify the precise contact sites on a synthetic DR4 (TRE half-sites arranged as a direct repeat with a four-nucleotide spacer) and a chick lysoenzyme TRE, F2 (half-sites arranged as an inverted palindrome with a six-nucleotide spacer), for various TR complexes, mobility shift assays, and dimethylsulfate and KMnO4 DNA modification interference assays were employed. First, TR alpha monomer bound to the downstream half-site of these TREs, whereas TR alpha homodimer bound to both half-sites. TR alpha/RXR alpha heterodimer also bound to both half-sites, but "preferred" to contact the down-stream half-site. Second, the specific flanking and spacing sequences of DR4 influenced the contact sites and the binding of TR alpha monomer and homodimer, but not TR alpha/RXR alpha heterodimer. Finally, cotransfection studies, using reporter plasmids containing DR4 or F2 in both orientations with respect to the basal promoter, provide evidence that preferential contact with the down-stream half-site by TR/RXR heterodimer may be important for maximal transcriptional activation.

Animals↗
PubMed1996

Induction of 8-hydroxy-2'-deoxyguanosine in CHO-K1 cells exposed to phenyl-hydroquinone, a metabolite of ortho-phenylphenol.

The induction of 8-hydroxy-2'-deoxyguanosine (8-OHdG), an index of oxidative DNA modification, was investigated in CHO-K1 cells exposed to phenyl-hydroquinone (PHQ), a major metabolite of ortho-phenylphenol (OPP), an antimicrobial. Addition of PHQ at a concentration of 50 microM to CHO cell suspensions (10(6) cells/ml) induced slight elevation of intracellular 8-OHdG levels. Pretreatment of CHO cells with 3-amino-1,2,4-triazole (AT, 20 mM) enhanced PHQ-induced 8-OHdG formation which was accompanied by cell death. Pretreatment of CHO-K1 cells with AT (20 mM) and deferoxamine (DeFe, 20 mM) inhibited the formation of 8-OHdG as well as cell death caused by PHQ. Neither AT nor DeFe affected cell viability or the formation of 8-OHdG in untreated CHO cells during the incubation period. The loss of cellular glutathione induced by the addition of PHQ alone was enhanced by the pretreatment of CHO cells with AT or AT plus DeFe. When PHQ was added to AT-pretreated cell suspensions, the concentration of PHQ decreased with time. This decrease was accompanied by the formation of phenyl-benzoquinone (PBQ). These results suggest that the reactive oxygen species derived from autoxidation of PHQ which converts to PBQ via phenyl-semiquinone elicit DNA damage in CHO cells, especially when the activity of cellular catalase is inhibited.

8-Hydroxy-2'-Deoxyguanosine↗
PubMedDate not supplied

Energy restriction and oxidative DNA damage in humans.

The cancer-preventive effect of energy restriction in rodents has been related to a decrease in oxidative damage to DNA. We have investigated the effect of energy restriction on the rate of oxidative DNA modification estimated from the urinary excretion of the repair product, 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG), in healthy, normal weight men. Before and after 10 weeks on a diet containing 80 (n = 16) or 100% (n = 8) of the estimated weight-maintaining energy, resting metabolic rate (RMR) was measured and 24-h urine was collected for 8-oxodG determination by HPLC. During the study, the weight loss was 10 and 2.5% of the initial weight, mostly in terms of fat, and the RMR decreased by 13 and 8% in the energy-restricted and control groups, respectively. With the use of t tests there was no significant difference within or between groups with respect to 8-oxodG excretion. However, if RMR was included as a covariate in multifactorial ANOVA, an average relative 17% (2-31%; 95% confidence interval) increase in 8-oxodG excretion in the energy-restricted group was significantly different from the corresponding value of the control group (P < 0.02). In the energy-restricted group the change in 8-oxodG excretion was correlated closely with the decrease in RMR (r = 0.63; P = 0.013). In the present study, 20% energy restriction for 10 weeks did not reduce oxidative DNA damage; we question a beneficial effect on cancer risk in normal weight humans.(ABSTRACT TRUNCATED AT 250 WORDS)

8-Hydroxy-2'-Deoxyguanosine↗
PubMed1996

Sequence-specific DNA binding of the phage Mu C protein: footprinting analysis reveals altered DNA conformation upon protein binding.

The mom gene of bacteriophage Mu, which codes for a DNA modification function, is regulated in a complex manner at both transcriptional and translational levels. The phage-encoded C protein functions as an activator of mom transcription. The mom promoter has features of an activator-dependent weak promoter, and the C binding site is located upstream and overlapping the -35 region and includes the palindromic sequence TTAT(N)6ATAA. The interactions of this activator protein at its binding site in Pmom has been investigated using four different chemical footprinting reagents. The protein footprint spans a region of 18 to 25 bp, depending on the nature of the chemical reagent used. Dimethylsulfate protection experiments revealed the base-specific interactions. The protected guanines are separated by 15 bp and are located beyond the interrupted palindromic sequence. A tripartite footprint was observed with hydroxyl radical, generated by Fe(II)-EDTA, which shows the binding of the protein to one face of the helix. The extent of protection conferred by the bound protein, however, is not uniform, suggesting that the interaction is asymmetric. The chemical nuclease 1,10-phenanthroline-copper, a minor groove specific ligand, shows hyper-reactivity upon protein binding in the top strand nucleotide triplet CAC, again confirming the protein-induced alterations in DNA conformation. Gel exclusion chromatography and chemical crosslinking experiment with the purified protein suggest that this mode of interaction is accomplished by a dimeric protein. This observation is supported by electrophoretic mobility shift assay using heterodimer of pure C protein and staphylococcal protein A-C fusion. The deletion analysis implicates a role for the carboxyl-terminal region of the protein in DNA binding.

Base Sequence↗
PubMed2001

Significant differences in the cellular and molecular reactions of rat and hamster lung after quartz exposure.

Exposure of rats to high doses of quartz and other insoluble isometric particles can produce lung tumors. In contrast, after exposure of such particles in hamsters no tumor outcome has been observed. Recent studies have demonstrated that the tumorigenic effect of particles is closely linked to the induction of inflammatory processes and the subsequent formation and persistence of mutagenic oxidative DNA-modifications. Species-specific differences in sensitivity to particles should therefore be reflected in the molecular reaction of the lung cells. We exposed rats and hamsters to two different doses of quartz (0.3 mg, 1.2 mg/100 g body weight) by intratracheal instillation and characterized the dose-related pattern of pulmonary inflammation (neutrophil recruitment, TNF), toxicity (protein content, surfactant phospholipids), antioxidant defence (glutathione content), mutagenicity (8-oxoguanine, p53) and proliferation. Our results clearly demonstrate a significantly higher response of the rat to quartz exposure for all determined molecular and cellular parameters. Therefore the examination of these parameters in humans would contribute to the evaluation of the relevance of rats or hamsters as models to predict particle-induced human lung cancer risk.

Animals↗
PubMed1999

Methylation, gene expression and the chromatin connection in cancer (review)

The mechanisms by which cells regulate gene expression are often altered in tumors. Modulating aspects of the components responsible for the nuclear packaging of DNA is one means by which the cell can control transcription, either by packaging the DNA such that access to specific sites of transcription is blocked or by modifying the DNA itself to prevent transcription factor binding. One such DNA modification is the methylation of cytosines. In addition, histone acetylation status has been linked recently through a large number of studies to the regulation of gene expression. Expressed genes are located in highly acetylated chromatin. The acetylation status of nucleosomes (the basic packaging unit of chromatin), is regulated by a group of enzymes, histone acetyltransferases (HATs), and histone deacetylases (HDACs). These two elements, methylation and histone acetylation have also been linked together, whereby methylation is used to direct gene repression through a histone deacetylase complex. Methylation, HATs, and HDACs have been shown to be altered in tumors. We present an overview of the current knowledge surrounding these elements in cancer, and in the final sections describe the likelihood of alterations of the histone modifying apparatus in hepatocellular carcinoma.

Animals↗
PubMed1980

The ral gene of phage lambda. III. Interference with E. coli ATP dependent functions.

The ral gene of phage lambda has previously been shown to counteract host controlled restriction and to enhance DNA modification in Escherichia coli (Zabeau et al., 1980). The studies presented in this paper demonstrate that although ral plays only a minor role in the lytic development of phage lambda, it counteracts different E. coli functions, which, like the E. coli restriction system, are ATP dependent. First, ral was found to specifically decrease the efficiency of recombination mediated by the RecBC pathway. Secondly, we observed that E. coli strains in which ral is constitutively expressed, exhibit phenotypes analogous to those of strains carrying mutations in the transcription termination factor rho. In addition, in rho deficient strains general recombination and host controlled restriction are both reduced. These findings strongly suggest that ral might be a second anti-termination function, which in contrast to the lambda N gene product directly antagonizes rho.

Adenosine Triphosphate↗
PubMed1984

Methylation dependent expression of the mom gene of bacteriophage Mu: deletions downstream from the methylation sites affect expression.

The expression of the DNA modification gene (mom) of bacteriophage Mu requires the cellular deoxyadenosine methylase (dam) and a transactivation factor from the phage. By hypothesis, the transcription of mom is activated by methylation of three GATC sequences upstream from the mom gene. We have introduced small deletions at a fourth GATC site located about 140 base pairs downstream from the primary methylation region. Some of the deletions severely affect the mom gene expression. We propose from this analysis that (1) some important elements, possibly the promoter, concerned with the expression of mom are located between nucleotides 840 and 880 from the right end of Mu and (2) the mom protein starts with the codon GTG located at position 810. We favor the hypothesis that methylation turns off transcription upstream, thereby allowing the main mom promoter to function.

Base Sequence↗
PubMed1988

A simple method for locating methylated bases in DNA, as applied to detect asymmetric methylation by M.FokIA.

Class-IIS restriction enzymes, which cut the DNA outside their recognition sequence, could be used for locating the bases methylated by a DNA-modification methylase. This is possible because methylation of the class-IIS cut sites does not interfere with the cleavage. The method consists of (i) selection of a nucleotide sequence with appropriate overlap between the methylase recognition site and the class-IIS enzyme cut site, (ii) methylation using S-adenosylmethionine as [3H]methyl donor, (iii) cleavage of the methylated sequence with the class-IIS enzyme, (iv) separation of the cleavage products and identification of the 3H-labelled fragment. Using this simple and straightforward method, we have shown that M.FokIA is an adenine methylase and methylates asymmetrically one strand of the FokI recognition site, resulting in the (Formula: see text) sequence. In addition, it was observed that another class-IIS restriction enzyme, SfaNI, is completely inhibited by methylation of its recognition site, (Formula: see text), by M.FokIA.

Base Sequence↗
PubMed2004

Comparison of the mutagenic activity of the benzene metabolites, hydroquinone and para-benzoquinone in the supF forward mutation assay: a role for minor DNA adducts formed from hydroquinone in benzene mutagenicity.

Benzene, a ubiquitous environmental pollutant and occupational hazardous chemical, is a recognised human leukaemogen and rodent carcinogen. The mechanism by which benzene exerts its carcinogenic effects is to date unknown but it is considered that mutations induced by benzene-DNA adducts may play a role. The benzene metabolite, para-benzoquinone (p-BQ) following reaction in vitro with DNA, forms four major adducts, which include two adducts on 2'-deoxyguanosine 3'-monophosphate (dGp). Reaction of DNA with the benzene metabolite hydroquinone (HQ) results in only one major DNA adduct, which corresponds to one of the dGp adducts formed following reaction with p-BQ. The mutagenicity of the adducts formed from these two benzene metabolites was investigated using the supF forward mutation assay. Metabolite-treated plasmid (pSP189) containing the supF gene was replicated in human Ad293 cells before being screened in indicator bacteria. Treatment with 5-20 mM p-BQ gave a 12 to 40-fold increase in mutation rate compared to 5-20 mM HQ treatment, a result reflected in the level of DNA modification observed (8 to 26-fold increase compared to HQ treatment). Treatment with p-BQ gave equal numbers of GC --> TA transversions and GC --> AT transitions, whereas treatment with HQ gave predominantly GC-->AT transitions. The spectra of mutations achieved for the two individual treatments were shown to be significantly different (P = 0.004). A combination of both treatments also resulted in a high level of GC --> AT transitions and a synergistic increase in the number of multiple mutations, which again predominated as GC --> AT transitions. Sites of mutational hotspots were observed for both individual treatments and one mutational hotspot was observed in the multiple mutations for the combined treatment. These results suggest that the dGp adducts formed from benzene metabolite treatment may play an important role in the mutagenicity and myelotoxicity of benzene.

Base Sequence↗
PubMed1988

Subunit structure of a yeast site-specific endodeoxyribonuclease, endo SceI. A study using monoclonal antibodies.

Endo SceI is a eucaryotic site-specific endoDNase of 120 kDa that causes double-stranded scission at well-defined sites, but is distinguishable from procaryotic restriction endonucleases by its mode of sequence recognition and lack of related specific DNA modification. In purified preparations of endoSceI, only two polypeptide species of 75 kDa (75-kDa peptide) and 50 kDa (50-kDa peptide) are detected in apparently equal amounts. We prepared mouse monoclonal IgGs that bound specifically to the 75-kDa peptide (but not the 50-kDa peptide) without inhibiting the endoSceI activity. Immunoprecipitation experiments with these IgGs revealed that the 75-kDa peptide and the 50-kDa peptide are physically associated with each other and with the endonucleolytic activity. Full endoSceI activity was recovered by mixing the purified 75-kDa peptide and the partially purified 50-kDa peptide, each of which exhibited little or no endonuclease activity alone. These observations indicate that endoSceI consists of two non-identical subunits of 75 kDa and 50 kDa, and that both subunits are required for full enzyme activity.

Antibodies, Monoclonal↗