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M Dizdaroglu

Publications and source records attributed to M Dizdaroglu.

At least 55 records · Page 3Linked to original sources
PubMed1996

Novel activities of human uracil DNA N-glycosylase for cytosine-derived products of oxidative DNA damage.

Uracil DNA N-glycosylase is a repair enzyme that releases uracil from DNA. A major function of this enzyme is presumably to protect the genome from pre-mutagenic uracil resulting from deamination of cytosine in DNA. Here, we report that human uracil DNA N-glycosylase also recognizes three uracil derivatives that are generated as major products of cytosine in DNA by hydroxyl radical attack or other oxidative processes. DNA substrates were prepared by gamma-irradiation of DNA in aerated aqueous solution and incubated with human uracil DNA N-glycosylase, heat-inactivated enzyme or buffer. Ethanol-precipitated DNA and supernatant fractions were then separated. Supernatant fractions after derivatization, and pellets after hydrolysis and derivatization were analyzed by gas chromatography/isotope-dilution mass spectrometry. The results demonstrated that human uracil DNA N-glycosylase excised isodialuric acid, 5-hydroxyuracil and alloxan from DNA with apparent K(m) values of approximately 530, 450 and 660 nM, respectively. The excision of these uracil analogues is consistent with the recently described mechanism for recognition of uracil by human uracil DNA N-glycosylase [Mol,C.D., Arval,A.S., Slupphaug,G., Kavil,B., Alseth,I., Krokan,H.E. and Tainer,J.A. (1995) Cell, 80, 869-878]. Nine other pyrimidine- and purine-derived products that were identified in DNA samples were not substrates for the enzyme. The results indicate that human uracil DNA N-glycosylase may have a function in the repair of oxidative DNA damage.

Animals↗
PubMed1996

A novel DNA N-glycosylase activity of E. coli T4 endonuclease V that excises 4,6-diamino-5-formamidopyrimidine from DNA, a UV-radiation- and hydroxyl radical-induced product of adenine.

We report on a novel activity of T4 endonuclease V. This enzyme is well known to be specific for the excision of pyrimidine dimers from UV-irradiated DNA. In this work, we show that T4 endonuclease V excises 4,6-diamino-5-formamidopyrimidine from DNA. 4,6-Diamino-5-formamidopyrimidine is formed as a product of adenine in DNA upon action of hydroxyl radicals and upon UV-irradiation. DNA substrates were prepared by UV-or gamma-irradiation of DNA in aqueous solution. DNA substrates were incubated either with active T4 endonuclease V or with heat-inactivated T4 endonuclease V or without the enzyme. After incubation, DNA was precipitated and supernatant fractions were separated. Supernatant fractions after derivatization, and pellets after hydrolysis and derivatization were analyzed by gas chromatography/isotope-dilution mass spectrometry. The results provide evidence for the excision of 4,6-diamino-5-formamidopyrimidine by T4 endonuclease V from both gamma-and UV-irradiated DNA. Kinetics of excision were also determined. Fifteen other pyrimidine- and purine-derived base lesions that were identified in DNA samples were not substrates for this enzyme. It was concluded that, in addition to its well known activity for pyrimidine photodimers, T4 endonuclease V possesses an N-glycosylase activity for a major UV-radiation- and hydroxyl radical-induced monomeric product in DNA.

Adenine↗
PubMed1995

Treatment of Wistar rats with a renal carcinogen, ferric nitrilotriacetate, causes DNA-protein cross-linking between thymine and tyrosine in their renal chromatin.

Ferric nitrilotriacetate (Fe-NTA) induces renal proximal tubular damage associated with lipid peroxidation and oxidative DNA base modifications that finally leads to a high incidence of renal adenocarcinoma in rodents. In the present study, we report on the in vivo formation of DNA-protein cross-links (DPCs) involving thymine and tyrosine in the renal chromatin of Wistar rats treated with single or repeated i.p. administration of Fe-NTA. Analyses of chromatin samples by gas chromatography/mass spectrometry revealed a significant increase in the amount of 3-[(1,3-dihydro-2,4-dioxopyrimidin-5-yl)-methyl]-L-tyrosine (Thy-Tyr cross-link) 24 and 48 hr after the administration of Fe-NTA. At 19th day of Fe-NTA treatment, the amount of Thy-Tyr cross-link decreased to the control level, indicating the presence of cellular repair activity. Thy-Tyr cross-link may play a role in the genetic alteration of this renal carcinogenesis model, since mitoses for regeneration of renal proximal tubules were closely associated with the increase in DPCs.

Adenocarcinoma↗
PubMed1995

A novel activity of E. coli uracil DNA N-glycosylase excision of isodialuric acid (5,6-dihydroxyuracil), a major product of oxidative DNA damage, from DNA.

We describe a novel activity of E. coli uracil DNA N-glycosylase (UNG) that excises isodialuric acid from DNA. Isodialuric acid is formed in DNA as a major oxidative product of cytosine. DNA substrates, which were prepared by gamma-irradiation, were incubated with UNG. Following precipitation of DNA, analyses of pellets and supernatant fractions by gas chromatography/mass spectrometry showed an efficient excision of isodialuric acid from DNA by UNG. None of the other 15 identified DNA base lesions was excised. The excision of isodialuric acid indicates that the non-aromaticity of a substrate may not be a limiting factor for UNG.

Cytosine↗
PubMed1995

Monomeric base damage products from adenine, guanine, and thymine induced by exposure of DNA to ultraviolet radiation.

The formation of monomeric products in DNA upon exposure to UV radiation was investigated. Three novel products were identified in DNA in aqueous solution upon exposure to UV radiation at 254 nm in a dose range from 100 to 10,000 J/m2. These were 4,6-diamino-5-formamidopyrimidine, 2,6-diamino-4-hydroxy-5-formamidopyrimidine, and 5-hydroxy-5,6-dihydrothymine. These three products are known to be substrates for base excision repair enzymes involved in the reversal of oxidative DNA damage. The dependence of the yields of formamidopyrimidines on UV radiation dose was nonlinear, whereas the yield of 5-hydroxy-5,6-dihydrothymine was increased linearly in the entire dose range. Of these products, 4,6-diamino-5-formamidopyrimidine was the only compound produced in appreciable amounts at 310 nm. At the highest dose used, the formation of other pyrimidine- and purine-derived products was also observed. Their amounts, however, were increased above control levels up to 2-fold only. The hydroxyl radical scavenger dimethyl sulfoxide had no effect on product yields excluding the involvement of hydroxyl radical in product formation. 4,6-Diamino-5-formamidopyrimidine and 2,6-diamino-4-hydroxy-5-formamidopyrimidine may be produced by hydration of adenine and guanine, respectively, across the N(7)-C(8) double bond by mechanisms similar to those proposed previously for well-known formation of pyrimidine hydrates with the hydroxyl group located at C(6). Formation of 5-hydroxy-5,6-dihydrothymine indicates that hydration of thymine with the hydroxyl group located at C(5) of the pyrimidine ring also occurs.(ABSTRACT TRUNCATED AT 250 WORDS)

DNA↗
PubMed1995

DNA base modifications and antioxidant enzyme activities in human benign prostatic hyperplasia.

The authors have studied DNA base damage and activities of antioxidant enzymes in human benign prostatic hyperplasia (BPH) tissues and surrounding disease-free tissues removed from prostate glands of 15 patients. In these tissues, endogenous levels of various typical hydroxyl radical-induced products of DNA bases and activities of catalase and superoxide dismutase were measured. The majority of patients had higher levels of DNA base lesions and lower activities of enzymes in BPH tissues than in normal prostate tissues. When activities of both enzymes were lower in BPH tissues than in normal tissues, the increases in the amounts of DNA base lesions over control levels were most prominent. In the case of similar enzyme activities in both BPH and normal tissues, no changes in levels of DNA base lesions were observed. These results suggest a possible association between antioxidant enzyme activities and levels of DNA base lesions in BPH tissues. Some of the identified DNA lesions are known to be premutagenic and may play a role in carcinogenesis. Although a possible link between BPH and prostate cancer is controversial, BPH patients with both decreased antioxidant enzyme activities and increased levels of DNA lesions may be at risk of developing prostate cancer.

Antioxidants↗
PubMed1995

DNA base modifications and membrane damage in cultured mammalian cells treated with iron ions.

We investigated DNA base damage in mammalian cells exposed to exogenous iron ions in culture. Murine hybridoma cells were treated with Fe(II) ions at concentrations of 10 microM, 100 microM, and 1 mM. Chromatin was isolated from treated and control cells and analyzed by gas chromatography/mass spectrometry for DNA base damage. Ten modified DNA bases were identified in both Fe(II)-treated and control cells. The quantification of modified bases was achieved by isotope-dilution mass spectrometry. In Fe(II)-treated cells, the amounts of modified bases were increased significantly above the background levels found in control cells. Dimethyl sulfoxide at concentrations up to 1 M in the culture medium did not significantly inhibit the formation of modified DNA bases. A mathematical simulation used to evaluate the plausibility of DNA damage upon Fe(II) treatment predicted a dose-dependent response, which agreed with the experimental results. In addition, Fe(II) treatment of cells increased the cell membrane permeability and caused production of lipid peroxides. The nature of DNA base lesions suggests the involvement of the hydroxyl radical in their formation. The failure of dimethyl sulfoxide to inhibit their formation indicates a site-specific mechanism for DNA damage with involvement of DNA-bound metal ions. Fe(II) treatment of cells may increase the intracellular iron ion concentration and/or cause oxidative stress releasing metal ions from their storage sites with subsequent binding to DNA. Identified DNA base lesions may be promutagenic and play a role in pathologic processes associated with iron ions.

Animals↗
PubMed1995

Formation of DNA-protein cross-links in cultured mammalian cells upon treatment with iron ions.

Formation of DNA-protein crosslinks (DPCs) in mammalian cells upon treatment with iron or copper ions was investigated. Cultured murine hybridoma cells were treated with Fe(II) or Cu(II) ions by addition to the culture medium at various concentrations. Subsequently, chromatin samples were isolated from treated and control cells. Analyses of chromatin samples by gas chromatography/mass spectrometry after hydrolysis and derivatization revealed a significant increase over the background amount of 3-[(1,3-dihydrio-2,4-dioxopyrimidin-5-yl)-methyl]- L-tyrosine (Thy-Tyr crosslink) in cells treated with Fe(II) ions in the concentration range of 0.01 to 1 mM. In contrast, Cu(II) ions at the same concentrations did not produce this DPC in cells. No DNA base damage was observed in cells treated with Cu(II) ions, either. Preincubation of cells with ascorbic acid or coincubation with dimethyl sulfoxide did not significantly alleviate the Fe(II) ion-mediated formation of DPCs. In addition, a modified fluorometric analysis of DNA unwinding assay was used to detect DPCs formed in cells. Fe(II) ions caused significant formation of DPCs, but Cu(II) ions did not. The nature of the Fe(II)-mediated DPCs suggests the involvement of the hydroxyl radical in their formation. The Thy-Tyr crosslink may contribute to pathological processes associated with free radical reactions.

Animals↗
PubMed1994

DNA base modifications in renal chromatin of Wistar rats treated with a renal carcinogen, ferric nitrilotriacetate.

Ferric nitrilotriacetate (Fe-NTA) causes renal proximal tubular necrosis, a consequence of iron ion-mediated free-radical-associated damage, that finally leads to a high incidence of renal adenocarcinoma in male rats and mice. We have investigated the levels of typical hydroxyl radical-induced DNA base modifications in renal chromatin of male Wistar rats treated with a single or repeated administrations of Fe-NTA. Five pyrimidine-derived and 5 purine-derived modified DNA bases were identified and quantified by gas chromatography/mass spectrometry with selected-ion monitoring. The modified bases were 5-hydroxy-5-methylhydantoin, 5-(hydroxymethyl)uracil, 5-hydroxycytosine, thymine glycol, 5,6-dihydroxyuracil, 4,6-diamino-5-formamidopyrimidine, 8-hydroxyadenine, xanthine, 2-hydroxyadenine and 8-hydroxyguanine. The amounts of most of these compounds were significantly increased over control levels in renal chromatin of Fe-NTA-treated rats as measured 3 and 24 hr after treatment. Elevated levels of modified bases were accompanied by proximal tubular necrosis. On the 19th day, however, accumulation of modified DNA bases was not observed. Morphologically, scattered karyomegalic cells were seen in the proximal tubules, but necrosis was rarely found. Some of the identified DNA base lesions are known to be promutagenic, although others have not been investigated. Presence of modified DNA bases concomitant with necrosis and regeneration of the renal proximal tubules may be a critical step in Fe-NTA-induced carcinogenesis.

Animals↗
PubMed1994

tert.-butyl hydroperoxide-mediated DNA base damage in cultured mammalian cells.

tert.-Butyl hydroperoxide has been utilized to study the effect of oxidative stress on living cells; however, its effect on DNA bases in cells has not been characterized. In the present work, we have investigated DNA base damage in mammalian cells exposed to this organic hydroperoxide. SP2/0 derived murine hybridoma cells were treated with 4 concentrations of tert.-butyl hydroperoxide for varying periods of time. Chromatin was isolated from treated and control cells and subsequently analyzed by gas chromatography-mass spectrometry with selected-ion monitoring for DNA base damage. Quantification of damaged DNA bases was achieved by isotope-dilution mass spectrometry. The amounts of 8 products were significantly higher than control levels in cells treated with tert.-butyl hydroperoxide at a concentration range of 0.01-0.1 mM. At concentrations from 1.0 to 10 mM, product formation was inhibited and the amounts of products were similar to those in control cells. The bimodal nature of the dose-response may be qualitatively analogous to previous reports of bimodal killing of E. coli bacteria by hydrogen peroxide. The nature of the identified DNA base lesions suggests the involvement of the hydroxyl radical in their formation. tert.-Butyl hydroperoxide is known to produce the tert.-butoxyl radical in reactions with metal ions. However, it is unlikely that the tert.-butoxyl radical produces these DNA lesions. It is suggested that DNA base damage arises from tert.-butyl hydroperoxide-mediated oxidative stress in cells, resulting in formation of hydroxyl radicals in close proximity to DNA. The inhibition of product formation at high concentrations of tert.-butyl hydroperoxide may be explained by the scavenging of tert.-butoxyl radical by tert.-butyl hydroperoxide resulting in inhibition of oxidative stress. The plausibility of the scavenging mechanism was evaluated with a mathematical simulation of the dose-response for DNA damage in solutions containing hydrogen peroxide. The simulation model predicted a bimodal dose-response which agreed qualitatively with the results in this study and with other in vivo and in vitro studies reported in the literature.

Animals↗
PubMed1994

Oxidative DNA base damage and antioxidant enzyme activities in human lung cancer.

We have investigated levels of antioxidant enzymes and free radical-induced DNA base modifications in human cancerous lung tissues and in their cancer-free surrounding tissues. Various DNA base lesions in chromatin of lung tissues were measured by gas chromatography-mass spectrometry. Activities of superoxide dismutase, catalase and glutathione peroxidase were also measured in lung tissues. Higher levels of DNA lesions were observed in cancerous tissues than in cancer-free surrounding tissues. Antioxidant enzyme levels were lower in cancerous tissues. The results indicate an association between decreased activities of antioxidant enzymes and increased levels of DNA lesions in cancerous tissues. Higher levels of DNA lesions suggest that free radical reactions may be increased in malignant tumor cells.

Antioxidants↗
PubMed1994

Ionizing radiation causes greater DNA base damage in radiation-sensitive mutant M10 cells than in parent mouse lymphoma L5178Y cells.

DNA base damage in radiation-sensitive mutant M10 cells and parent mouse lymphoma L5178Y cells was studied. Cells were exposed to ionizing radiation in the dose range of 48 to 400 Gy. Chromatin was isolated from cells and analyzed by gas chromatography-mass spectrometry. Ten DNA base products were identified and quantified. A dose-dependent formation of the products was observed. The yields of products in M10 cells were up to threefold greater than in L5178Y cells. Of the products measured, formamidopyrimidines had the highest difference in their yields between the two cell lines. The greater initial DNA base damage in M10 cells may play a role in their hypersensitivity to ionizing radiation.

Animals↗
PubMed1993

beta-D-glucosyl-hydroxymethyluracil: a novel modified base present in the DNA of the parasitic protozoan T. brucei.

We have previously shown that the DNA of the unicellular eukaryote T. brucei contains about 0.1% of a novel modified base, called J. The presence of J correlates with a DNA modification associated with the silencing of telomeric expression sites for the variant surface antigens of trypanosomes. Here we show that J is 5-((beta-D-glucopyranosyloxy)-methyl)-uracil (shortened to beta-D-glucosyl-hydroxymethyluracil), a base not previously found in DNA. We discuss putative pathways for the introduction of this base modification at specific positions in the DNA and the possible contribution of this modification to repression of surface antigen gene expression.

Animals↗
PubMed1993

Substrate specificity of the Escherichia coli endonuclease III: excision of thymine- and cytosine-derived lesions in DNA produced by radiation-generated free radicals.

The excision of modified bases from DNA by Escherichia coli endonuclease III was investigated. Modified bases were produced in DNA by exposure of dilute buffered solutions of DNA to ionizing radiation under oxic or anoxic conditions. The technique of gas chromatography/mass spectrometry (GC/MS) was used to identify and quantify 16 pyrimidine- and purine-derived DNA lesions. DNA substrates were incubated either with the native enzyme or with the heat-inactivated enzyme. Subsequently, DNA was precipitated. Pellets were analyzed by GC/MS after hydrolysis and derivatization. Supernatant fractions were analyzed after derivatization without hydrolysis. The results provided unequivocal evidence for the excision by E. coli endonuclease III of a number of thymine- and cytosine-derived lesions from DNA. These were 5,6-dihydrothymine, 5-hydroxy-5-methylhydantoin, thymine glycol, 5-hydroxy-6-hydrothymine, 5,6-dihydrouracil, alloxan, uracil glycol, and 5-hydroxy-6-hydrouracil. None of the purine-derived lesions was excised by endonuclease III. The present work extends the substrate specificity of E. coli endonuclease III to another thymine-derived and four cytosine-derived lesions. It is the first investigation of the substrate specificity of this repair enzyme in the context of a large number of pyrimidine- and purine-derived lesions in DNA.

Animals↗
PubMed1993

Modification of DNA bases in chromatin of intact target human cells by activated human polymorphonuclear leukocytes.

We investigated whether phorbol-12-acetate-13-myristate (PMA)-activated human polymorphonuclear leukocytes (PMNs) induce base modifications in target cell DNA in vivo. Human PMNs produced 9.4 +/- 0.8 (SD) nmol of H2O2/10(6) cells during 50 min of exposure to 2 micrograms/ml PMA and 13.7 +/- 2.8 nmol/10(6) cells during exposure to PMA plus 5 mM NaN3. Neither nonstimulated PMNs, nor PMA alone, nor NaN3 alone induced base modifications in chromatin-associated DNA of human Ad293 cells above control levels, when assayed by gas chromatography/mass spectrometry with selected-ion monitoring. However, a 60-min exposure to 1.7 +/- 0.4 x 10(6) PMNs/ml in the presence of 2 micrograms/ml PMA induced a 2-3-fold increase in the level of all modified bases detected by gas chromatography/mass spectrometry with selected-ion monitoring. The guanine-derived products 8-hydroxyguanine and 2,6-diamino-4-hydroxy-5-formamidopyrimidine, and the adenine-derived product 4,6-diamino-5-formamidopyrimidine were induced to the highest levels among those bases detected. These data demonstrate that exposure to activated PMNs causes DNA base modifications in target cells in vivo typical of those induced by hydroxyl radical attack. The induction of potentially promutagenic modified bases may contribute to the mutagenicity of activated PMNs.

Cell Line↗
PubMed1993

Quantitative determination of oxidative base damage in DNA by stable isotope-dilution mass spectrometry.

For understanding of the role of oxidative DNA damage in biological processes such as mutagenesis and carcinogenesis, it is essential to identify and quantify this type of DNA damage in cells. This can be achieved by gas chromatography/mass spectrometry. The present study describes the quantification of modified bases in DNA by isotope-dilution mass spectrometry with the use of stable isotope-labeled analogues as internal standards. A number of isotopically labeled DNA bases were synthesized. The mass spectra of their trimethylsilyl derivatives were recorded. Calibration plots were obtained for known quantities of modified bases and their isotope-labeled analogues. Quantification of various modified DNA bases by isotope-dilution mass spectrometry was demonstrated in isolated chromatin exposed to ionizing radiation. The results indicate that gas chromatography/stable isotope-dilution mass spectrometry is an ideally suited technique for selective and sensitive quantification of modified bases in DNA.

Chromatin↗
PubMed1993

DNA base damage generated in vivo in hepatic chromatin of mice upon whole body gamma-irradiation.

DNA base lesions in hepatic chromatin formed upon whole-body irradiation of mice were studied. After gamma-irradiating (20-470 Gy) and killing animals, chromatin was isolated from their livers and analysed by GC-MS. Five pyrimidine- and five purine-derived DNA lesions were identified and quantified. These were 5-hydroxy-5-methylhydantoin, 5-hydroxycytosine, 5-(hydroxymethyl)uracil, 4,6-diamino-5-formamidopyrimidine, 7,8-dihydro-8-oxoadenine, 2-hydroxyadenine, 2,6-diamino-4-hydroxy-5-formamidopyrimidine, 7,8-dihydro-8-oxoguanine, thymine glycol and 5,6-dihydroxyuracil. Except for the latter two, the amounts of these compounds were increased significantly over control levels in the dose range of 100-470 Gy. Above 200 Gy, a deviation from linearity was observed, although the yields were increased in most cases up to 470 Gy. The modified bases that were identified are typically produced by hydroxyl radical attack on the DNA bases. This may indicate a role for hydroxyl radicals in their formation in vivo. These lesions may play a role in the biological consequences of ionizing radiation such as mutagenesis and carcinogenesis.

Animals↗