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At least 793 records · Page 44Linked to original sources
PubMed2005

Genotoxicity of the benzene metabolites para-benzoquinone and hydroquinone.

Our interest in benzene-DNA adduct formation and their consequence has led us to develop a number of sensitive methods for their analysis. A HPLC method for the analysis of 32P-postlabelled benzene-DNA adducts was developed and used to detect adducts formed from the reaction of DNA or individual deoxynucleotides with the metabolites para-benzoquinone (p-BQ) and hydroquinone (HQ). Reaction of DNA with BQ yielded four adducts, the major product being a deoxycytidine adduct. HQ formed a single detectable deoxyguanosine DNA adduct, which was a minor product of the reaction of DNA with p-BQ. The supF forward mutation assay was used to assess the mutagenicity of p-BQ and HQ after transfection of treated plasmid in the human kidney cell line, Ad293. Single base substitution mutations at GC base pairs (bp) predominated for each treatment. However, when the mutation spectra achieved for each treatment were compared they were shown to be significantly different (p=0.004). These results may suggest either a possible role for the minor benzene-deoxyguanosine adducts in benzene genotoxicity or that HQ is causing DNA modification via a different mechanism, such as oxidative damage.

Benzene↗
PubMed1999

DNA methylation represses transcription in vivo.

DNA in somatic tissue is characterized by a bimodal pattern of methylation, which is established in the animal through a series of developmental events. In the mouse blastula, most DNA is unmethylated, but after implantation a wave of de novo methylation modifies most of the genome, excluding the majority of CpG islands, which are mainly associated with housekeeping genes. This genomic methylation pattern is broadly maintained during the life of the organism by maintenance methylation, and generally correlates with gene expression. Experiments both in vitro and in vivo indicate that methylation inhibits transcription. It has not yet been possible, however, to determine the role of DNA methylation on specific sequences during normal development. Cis-acting regulatory elements and trans-acting factors appear to be involved in both stage- and tissue-specific demethylation processes. Sp1-like elements have a key role in protecting the CpG island of Aprt (encoding adenine phosphoribosyltransferase) from de novo methylation, and when these elements are specifically mutated, the Aprt CpG island becomes methylated in transgenic mice. We have now characterized an embryo-specific element from the CpG island sequence upstream of Aprt that can protect itself from de novo methylation in transgenic mice as well as reduce methylation of flanking sequences. We placed this element on a removable cassette adjacent to a human HBB (encoding beta-globin) reporter and generated a transgene whose methylation pattern can be switched in vivo. Analysis of globin transcription in this system showed that methylation in cis inhibits gene expression in a variety of tissues, indicating that DNA modification may serve as a global genomic repressor.

Adenine Phosphoribosyltransferase↗
PubMed2006

Recruitment of the de novo DNA methyltransferase Dnmt3a by Kaposi's sarcoma-associated herpesvirus LANA.

The Kaposi's sarcoma-associated herpesvirus LANA protein is expressed in all Kaposi's sarcoma-associated herpesvirus-infected cells, including the tumor cells of endemic and AIDS-associated Kaposi sarcoma, primary effusion lymphoma, and Castleman disease. LANA modulates cell gene expression, but the mechanisms of LANA-mediated transcriptional reprogramming are poorly understood. LANA-repressed cell genes were identified by using retroviral-transduced telomerase-immortalized microvascular endothelial cells. Transciptional repression of targeted genes was relieved by treatment with the methyltransferase inhibitor 5-aza-2'-deoxycytidine, suggesting a role for DNA methylation in repression. We found that LANA coprecipitated with DNA methyltransferases (Dnmts) and recruited endogenous DNA methyltransferase activity from the cell extract. LANA preferentially relocalized Dnmt3a from the nuclear matrix into the chromatin fraction. Further, LANA associated with repressed cellular promoters, recruited Dnmt3a to DNA, and facilitated de novo promoter methylation of a down-regulated gene, cadherin 13 (H-cadherin). The data provide an example of promoter-specific epigenetic DNA modification through viral protein recruitment of de novo Dnmt activity.

Antigens, Viral↗
PubMed1983

Potential for genetic damage from multivitamin solutions exposed to phototherapy illumination.

The ability of standard phototherapy illumination to produce damage in intracellular DNA is well established. In this study, the addition of a dilute solution (1:6400) of a clinically-used multivitamin concentrate to human KB cells was found to enhance the generation of single-strand DNA breaks produced by broad-spectrum fluorescent light. The effect of the exogenous photosensitizing agent (multivitamins) was blocked by the enzyme catalase; thus, the photoproduct responsible for the DNA modification was hydrogen peroxide, an extremely reactive molecule capable of damaging a variety of biologic macromolecules.

Catalase↗
PubMed1989

[DNA damage by oxygen radicals and carcinogenesis].

Oxygen radicals are produced by ionizing radiation and many other environmental carcinogens. They are also produced in cells endogenously by the oxygen metabolism. Therefore it appears to be important to study DNA damage by oxygen radicals and its relation with mutagenesis and carcinogenesis. During a study on DNA damage caused in vitro by heated carbohydrates, which were being used as a model of cooked foods, a new type of DNA modification was discovered, the formation 8-hydroxyguanine (8-OH-Gua). Various oxygen radical forming carcinogenic agents (radiation, cigarette smoke components, asbestos + H2O2 etc.) have been found to be effective in the formation of 8-OH-Gua in DNA in vitro. The formation of 8-OH-Gua was also observed in cellular DNA in vivo after mice or cultured cells were irradiated by ionizing radiation. The 8-OH-Gua residue in DNA may cause mutagenesis and carcinogenesis, since 8-OH-Gua in DNA induces misreading during DNA synthesis in vitro. Among the various types of DNA damage induced by oxygen radicals, 8-OH-Gua can be most readily measured at high sensitivity by high-pressure liquid chromatography coupled to an electrochemical detector. Recently, we found that oral administration of several chemical carcinogens, which are known to produce oxygen radicals, induce 8-OH-Gua in rat target organ DNA. Therefore, 8-OH-Gua can be used as a marker for monitoring oxidative DNA damage as a means of evaluating the carcinogenic potency of various oxygen radical forming agents. We are also measuring 8-OH-Gua levels in human lymphocyte DNA in order to investigate the relation between oxidative DNA damage and the incidence of cancer.

Animals↗
PubMed2004

HhaI DNA methyltransferase uses the protruding Gln237 for active flipping of its target cytosine.

Access to a nucleotide by its rotation out of the DNA helix (base flipping) is used by numerous DNA modification and repair enzymes. Despite extensive studies of the paradigm HhaI methyltransferase, initial events leading to base flipping remained elusive. Here we demonstrate that the replacement of the target C:G pair with the 2-aminopurine:T pair in the DNA or shortening of the side chain of Gln237 in the protein severely perturb base flipping, but retain specific DNA binding. Kinetic analyses and molecular modeling suggest that a steric interaction between the protruding side chain of Gln237 and the target cytosine in B-DNA reduces the energy barrier for flipping by 3 kcal/mol. Subsequent stabilization of an open state by further 4 kcal/mol is achieved through specific hydrogen bonding of the side chain to the orphan guanine. Gln237 thus plays a key role in actively opening the target C:G pair by a "push-and-bind" mechanism.

2-Aminopurine↗
PubMed1989

Synthesis of heterocyclic N-acetoxyarylamines and their reactivity with DNA.

2-Acetoxyamino-5-phenylpyridine and 2-acetoxyamino-3-methyl-5-phenylpyridine, being proposed ultimate carcinogens of the heterocyclic aromatic amines 2-amino-5-phenylpyridine (APP) and 2-amino-3-methyl-5-phenylpyridine (AMPP), respectively, were synthesized, crystallized and characterized. Using the 32P-postlabelling technique, we show that the total amount of adducts found in DNA after reaction with these N-acetoxyarylamines is at least 30- and 450-fold higher than in DNA reacted with equimolar amounts of the proposed proximate carcinogens 2-hydroxyamino-5-phenylpyridine and 2-hydroxyamino-3-methyl-5-phenylpyridine, respectively. These results support a postulated activation mechanism, in which N-acetoxyarylamines are the ultimate reactive species responsible for DNA modification by carcinogenic aromatic amines in vivo. The possibility to obtain the reactive 0-acetyl derivatives of APP and AMPP in crystalline form makes them unique model compounds for studies on the interaction of ultimate carcinogens of aromatic amines with DNA.

Amines↗
PubMed2000

DNA damage formation, DNA repair, and survival after exposure of DNA repair-proficient and nucleotide excision repair-deficient human lymphoblasts to UVA1 and UVB.

PURPOSE: The comet assay has been used to visualize DNA damage in single cells after exposure to UV light. These comets are commonly thought to reflect transient, repair-induced DNA breaks. The goal of the work presented here was to further characterize the nature of UV-induced comets and to further elucidate DNA damage formation by different wavelengths of ultraviolet light. MATERIALS AND METHODS: Detailed dose-response and time-course experiments with comet formation were carried out with normal and nucleotide excision repair (NER)-deficient xeroderma pigmentosum (XP) lymphoblasts. Irradiation was carried out with low, intermediate, or high doses of UVA1 or UVB, comet formation was observed, cell survival and viability were determined, and UV-induced apoptosis was measured. RESULTS: All responses were dose-dependent. With the intermediate dose of UVA1, a pronounced comet formation was observed without subsequent growth inhibition. Raising levels of porphyrins, which act as photosensitizers, by preincubation with 5-amino-levulinic acid increased comet formation with UVA1, but not with UVB. UVA1-sensitivity and comet formation in XP cells was not significantly different from the normal cells. With UVB no comet formation was seen without subsequent apoptotic cell death. XP cells exhibited the known UVB-hypersensitivity, but their comet formation was not significantly different from that of normal cells. CONCLUSIONS: The findings are compatible with the hypothesis that UV-induced comets represent transient repair-induced DNA breaks. Both, the NER of dimers and the base excision repair of oxidative DNA modifications are thought to contribute to comet formation.

Apoptosis↗
PubMed2006

DNA damage by bromate: mechanism and consequences.

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

Animals↗
PubMed2001

Nuclear matrix in developing rat spermatogenic cells.

The nonchromatin structure or nuclear matrix in developing spermatogenic cells of the rat was studied using a biochemical fractionation in concert with resinless section electron microscopy. Observations demonstrated that the nuclear matrix of spermatogenic cells consisted of a three-dimensional network of filaments of variable thicknesses. In spermatogonia and spermatocytes the nuclear matrix consisted of relatively thin filaments, while that of round spermatids consisted of a thicker interconnecting network of filament. In elongating spermatids, the interior of the nuclear matrix consisted of a network of dense filaments bounded by a peripheral lamina. The protein composition of the nuclear matrix in spermatogenic cells was examined by high-resolution two-dimensional gel electrophoresis and correlated with morphological changes characteristic of each stage. The results showed that the proteins of nuclear matrix changed in a cell stage-specific manner. These stage-specific changes corresponded to the major transitions of chromatin structure and function during spermatogenesis. Furthermore, immunocytochemical and immunoblotting analysis of DNA topoisomerase II (topo II) revealed that this enzyme exhibited stage-specific variations and was associated with the nuclear matrix. These results suggest that the nuclear matrix in spermatogenic cells may be involved in mediating DNA modifications and maintaining nuclear organization during spermatogenesis. Mol. Reprod. Dev. 59:314-321, 2001.

Animals↗
PubMed1988

DNA hypomethylation in ethionine-induced rat preneoplastic hepatocyte nodules.

DNA from hepatocyte nodules induced in rats with dietary DL-ethionine and from the surrounding non-nodular liver contained less 5-methyldeoxycytidine per deoxycytidine when compared with that from normal adult liver. The degree of apparent hypomethylation, 37% in nodules and 20% in the surrounding liver, decreased somewhat (29% and 16% respectively) at 2 weeks after terminating the exposure to ethionine. Nodules and surrounding liver, like normal liver, responded to partial hepatectomy with a decrease in the 5-methyldeoxycytidine level at 24 hrs and a return to the level at the time of partial hepatectomy by 38 hrs. These findings indicate the need for careful control of cell proliferation in comparing the levels of a post-replicative DNA modification, methylation, in proliferating and non-proliferating cell populations. These findings also suggest that a portion of the hypomethylation in preneoplastic nodules may be due to a bona fide decrease in the level of cytosine methylation in the parental strand of DNA. This hypomethylation could be one basis for the altered gene expression in hepatocyte nodules, possible precursors for liver cancer.

Animals↗
PubMedDate not supplied

Pyrrolo[1,4]benzodiazepine antitumor antibiotics: relationship of DNA alkylation and sequence specificity to the biological activity of natural and synthetic compounds.

The DNA alkylation and sequence specificity of a group of natural and synthetic pyrrolo-[1,4]benzodiazepines [P(1,4)Bs] were evaluated by using an exonuclease III stop assay, and the results were compared with in vitro and in vivo biological potency and antitumor activity. The P(1,4)B antibiotics are potent antitumor agents produced by various Actinomycetes, which are believed to mediate their cytotoxic effects by covalent bonding through N-2 of guanine in the minor groove of DNA. In this article we describe the results of a sensitive DNA alkylation assay using exonuclease III which permits both estimation of the extent of DNA modification as well as location of the precise guanines to which the drugs are covalently bound. Using this assay, we have evaluated a series of natural and synthetic compounds of the P(1,4)B class for their ability to bind to DNA and also determined their DNA sequence preference. The compounds included in this study are P(1,4)Bs carrying different substituents in the aromatic ring, having varying degrees of saturation in the five-membered ring, or differing in the stereochemistry at C-11a. These same compounds were evaluated for in vitro cytotoxic activity against B16 melanoma cells, for potency in vivo in B6D2F1 mice (LD50), and for antitumor activity (ILSmax) against P388 leukemia cells. A good correlation was found between extent of DNA alkylation and in vitro and in vivo potency. Furthermore, on the basis of electronic and steric considerations, it was possible to rationalize why those compounds that showed negligible biological activity were unable to bond covalently to DNA. Last, we have determined that the degree of saturation in the five-membered ring of the P(1,4)Bs has a significant effect on the DNA bonding reactivity and biological activity of this class of compounds.

Alkylation↗
PubMed2006

Kinetics of O(6)-methyl-2'-deoxyguanosine repair by O(6)-alkylguanine DNA alkyltransferase within K-ras gene-derived DNA sequences.

O(6)-Methyl-2'-deoxyguanosine (O(6)-Me-dG) is a potent mutagenic DNA adduct that can be induced by a variety of methylating agents, including tobacco-specific nitrosamine, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK). O(6)-Me-dG is directly repaired by the specialized DNA repair protein, O(6)-alkylguanine DNA alkyltransferase (AGT), which transfers the O(6)-alkyl group from the modified guanine to a cysteine thiol within the active site of the protein. Previous investigations suggested that AGT repair of O(6)-alkylguanines may be sequence-dependent as a result of flanking nucleobase effects on DNA conformation and energetics. In the present work, a novel high-performance/pressure liquid chromatography-electrospray ionization tandem mass spectrometry (HPLC-ESI+-MS/MS)-based approach was developed to analyze the kinetics of AGT-mediated repair of O(6)-Me-dG adducts placed at different sites within the double-stranded DNA sequence representing codons 8-17 of the K-ras protooncogene, 5'-G1TA G2TT G3G4A G5CT G6G7T G8G9C G10TA G11G12C AAG13 AG14T-3', where G5, G6, G7, G8, G9, G10, or G11 was replaced with O(6)-Me-dG. The second guanine of K-ras codon 12 (G7 in our numbering system) is a major mutational hotspot for G --> A transitions observed in lung tumors of smokers and in neoplasms induced in laboratory animals by exposure to methylating agents. O(6)-Me-dG-containing duplexes were incubated with human recombinant AGT protein, and the reactions were quenched at specific times. Following acid hydrolysis to release purines, isotope dilution HPLC-ESI-MS/MS was used to determine the amounts of O(6)-Me-G remaining in DNA. The relative extent of demethylation for O(6)-Me-dG adducts located at G5, G6, G7, G8, G9, G10, or G11 following a 10 s incubation with AGT showed little variation as a function of sequence position. Furthermore, the second-order rate constants for the repair of O(6)-Me-dG adducts located at the first and second positions of the K-ras codon 12 (5'-G6G7T-3') were similar (1.4 x 10(7) M(-1) s(-1) vs 7.4 x 10(6) M(-1) s(-1), respectively), suggesting that O(6)-Me-dG repair by AGT is not the determining factor for K-ras codon 12 mutagenesis following exposure to methylating agents. The new HPLC-ESI-MS/MS assay developed in this work is a valuable tool which will be used to further explore the role of local sequence environment and endogenous DNA modifications in shaping mutational spectra of NNK and other methylating agents.

Base Sequence↗
PubMed1994

Both physiological and pharmacological levels of melatonin reduce DNA adduct formation induced by the carcinogen safrole.

Hepatic DNA adduct formation induced by the chemical carcinogen, safrole, was suppressed by both endogenous pineal melatonin release and by the exogenous administration of melatonin to rats. DNA damage after administration of of melatonin to rats. DNA damage after administration of 100 mg/kg safrole (i.p.) was measured by the P1 enhanced 32P-postlabeling analysis method. The RAL (relative adduct labeling) x 10(7) of carcinogen modified DNA in the liver of untreated controls and in safrole treated animals killed during the day, at night, after pinealectomy and pinealectomy plus melatonin injection (0.15 mg/kg x 4 or a total of 0.6 mg/kg) was 0, 12.6 +/- 0.75, 10.9 +/- 0.72, 13.6 +/- 1.12 and 5.7 +/- 0.53 respectively. For the same groups of animals, circulating melatonin levels at the termination of the study were 31 +/- 3, 29 +/- 2, 276 +/- 31, 24 +/- 1 and 13,950 +/- 1016 pg/ml serum respectively. The higher the melatonin concentration in the serum the lower was DNA adduct formation in the rat liver. Thus, high nocturnal levels of melatonin were protective against safrole-induced DNA damage. These findings indicate that the functional pineal gland plays an important role in oncostatic actions of carcinogens such as safrole. At physiological levels, melatonin seemed to prevent especially the formation of what was referred to as the N1 DNA adduct. Melatonin's ability to suppress DNA adduct formation may relate to its inhibitory effect on a mixed function oxidase, cytochrome p-450, and on the recently identified hydroxyl radical scavenging capacity of the indole. The oncostatic action of melatonin is also suggested by its nuclear accumulation and DNA stabilization characteristics. At pharmacological levels melatonin is extremely potent in preventing DNA modification induced by the chemical carcinogen, safrole.

Animals↗
PubMed1987

Modifications of DNA-gyrase and of permeability in a norfloxacin-resistant clinical isolate of Providencia stuartii.

We obtained a clinical isolate of Providencia stuartii showing a high level of resistance to norfloxacin and to other 4-quinolones, whose target is the enzyme DNA-gyrase. This strain showed resistance also to beta-lactam and aminoglycoside antibiotics. In order to detect modification of DNA-gyrase, we performed supercoiling assays in vitro in presence of norfloxacin and ciprofloxacin. Furthermore, outer membrane proteins, which are involved in permeability mechanisms, were analyzed on SDS-polyacrylamide gels. Results showed that both modifications in DNA-gyrase and changes in outer membrane proteins can be held responsible for resistance to 4-quinolones; moreover, these modification are probably supported by a third mechanism of resistance.

Bacterial Outer Membrane Proteins↗
PubMed1999

Cytotoxicity and mutagenicity of UVB assessed using cultured rat fibroblast.

A retroviral vector carrying both positive (neo) and negative (herpes simplex virus thymidine kinase or HSV-tk) selection markers was constructed as a substrate for mutational assay in mammalian cells. Using a population of rat fibroblast cells carrying a single copy per cell of retroviral DNA randomly integrated in their chromosomes, we examined the cytotoxic and mutagenic activities of ultraviolet light (UV) at four wavelengths (254, 290, 300, and 320 nm). The action spectra for these activities are similar to some of the previously reported spectra for photochemical DNA modifications, erythema, cell killing, and mouse skin carcinogenesis, except at 290 and 320 nm. At 290 nm, no significant mutagenicity was observed. At 320 nm, both cytotoxic and mutagenic activities were 10 times higher than the values expected from the absorption spectrum for DNA and the action spectrum for bacterial inactivation and mutagenesis. Structural comparison of some of the HSV-tk mutants obtained after irradiation with 300 and 320 nm UV revealed partially different patterns of mutation specificity, suggesting the involvement of multiple molecular mechanisms in the genotoxicity associated with this range of UV.

Animals↗
PubMedDate not supplied

Site-specific photomodification of DNA by porphyrin-oligonucleotide conjugates synthesized via a solid phase H-phosphonate approach.

meso-Tris(4-pyridyl)[[(omega-hydroxyhexamethylene)carbamoyl]phenyl ] porphyrin was converted to its H-phosphonate derivative and conjugated using solid phase synthesis with the 5'-hydroxyl group of deoxyribonucleotides d(TCTTCCCA) and d(T)12. These conjugates were transformed into their (N-methylpyridiniumyl)porphyrin analogs in the reaction with methyl iodide. A 532 nm laser beam was utilized to photoactivate both types of the conjugates in the presence of the target 22-mer and 16-mer oligonucleotides. Photoactivation of porphyrin-oligonucleotide conjugates resulted in site-specific DNA modification characterized by a main reaction site size of approximately 5 bases.

Chromatography, High Pressure Liquid↗
PubMed1995

Recognition of DNA interstrand cross-links of cis-diamminedichloroplatinum(II) and its trans isomer by DNA-binding proteins.

Recognition and processing by cellular proteins of DNA modified by platinum complexes have been suggested to be relevant to the mechanism of their antitumor activity. Platinum complexes form on DNA various mono- and bifunctional adducts. It has already been described by other authors that intrastrand cross-links formed on DNA by antitumor cis-diamminedichloroplatinum(II) (cisplatin) between neighboring purine residues are recognized by several DNA-binding proteins. In contrast, these proteins do not recognize the intrastrand cross-links formed on DNA by cisplatin or its clinically ineffective trans isomer (transplatin) between nonadjacent base residues. An eventuality heretofore not addressed is that DNA interstrand cross-links (ICLs) of platinum compounds may be recognized by and bound to DNA-binding proteins. DNA probes of 110 base pairs (bp) were constructed containing five equally spaced ICLs of cisplatin or transplatin. These ICLs were formed at specific sites at which these adducts are preferentially formed in natural DNA. Gel electrophoresis mobility shift and competition assays with these probes were used to investigate the specific recognition and binding of the calf thymus HMG1 protein to the DNA ICLs of both platinum isomers. The ICL of antitumor cisplatin was recognized by and bound to the HMG1 protein with a similar affinity as the 1,2-intrastrand d(GpG) cross-link of this drug. The protein binding to the ICL is selective for the DNA modification by cisplatin, but not by chemotherapeutically inactive transplatin.(ABSTRACT TRUNCATED AT 250 WORDS)

Antineoplastic Agents↗