Biomedical subjects

M Dizdaroglu

Publications and source records attributed to M Dizdaroglu.

At least 109 records · Page 6Linked to original sources

PubMed1987

Ionizing-radiation-induced damage in the DNA of cultured human cells. Identification of 8,5-cyclo-2-deoxyguanosine.

Epstein-Barr-virus-transformed peripheral-blood B-lymphocytes were gamma-irradiated at 0 degree C at doses from 10 to 100 Gy. The cells were immediately lysed and the DNA was isolated. Subsequently, the DNA was hydrolysed to 2'-deoxyribonucleosides with a mixture of DNAase I, venom and spleen exonucleases and alkaline phosphatase. The hydrolysate was dried, trimethylsilylated and analysed by capillary gas chromatography-mass spectrometry with selected-ion monitoring. The (5'R)- and (5'S)-diastereomers of 8,5'-cyclo-2'-deoxyguanosine were observed in a ratio of 1:3, and their formation was dose-dependent. It was possible to detect and characterize one such lesion in approx. 4 X 10(4) guanine nucleotide subunits of DNA.

B-Lymphocytes↗

PubMed1986

Characterization of free radical-induced damage to DNA by the combined use of enzymatic hydrolysis and gas chromatography-mass spectrometry.

The use of gas chromatography-mass spectrometry (GC-MS) for characterization of free radical-induced base damage to DNA is presented. Damage introduced to DNA by reactive oxygen species such as hydroxyl radicals appears to play an important role in mutagenesis, carcinogenesis and aging. Elucidation of the chemical nature of such DNA lesions is necessary for the assessment of their biological consequences and enzymatic repair. DNA exposed to radiation-generated hydroxyl radicals in aqueous solution was hydrolyzed to 2'-deoxyribonucleosides with a mixture of DNase I, venom and spleen exonucleases and alkaline phosphatase. The hydrolysate was subsequently trimethylsilylated and analyzed by GC-MS. A large number of DNA lesions were separated and identified. Mass spectra obtained were interpreted on the basis of the typical fragmentation pathways of trimethylsilylated nucleosides. The use of GC-MS with selected-ion monitoring facilitated the detection of these lesions at the very low quantities and radiation doses (below 10 Gray) that might be relevant to those in biological systems.

Animals↗

PubMed1986

Free-radical-induced formation of an 8,5'-cyclo-2'-deoxyguanosine moiety in deoxyribonucleic acid.

Isolation and identification of a novel .OH-induced product, namely an 8,5'-cyclo-2'-deoxyguanosine moiety, in DNA and 2'-deoxyguanosine are described. .OH radicals were generated in dilute aqueous solutions by gamma-irradiation. Analyses of 2'-deoxyguanosine and enzymic hydrolysates of DNA by gas chromatography-mass spectrometry (g.c.-m.s.) after trimethylsilylation showed the presence of 8,5-cyclo-2'-deoxyguanosine on the basis of its fragment ions. This product was isolated by h.p.l.c. Its u.v. and n.m.r. spectra taken were in agreement with the structure suggested by its mass spectrum. Exact masses of the typical ions from the mass spectrum of the trimethylsilyl derivative of this product were measured by high-resolution m.s. The values found were in excellent agreement with the theoretical mass derived from the suggested fragmentation patterns. Both (5'R)- and (5'S)-epimers of 8,5'-cyclo-2'-deoxyguanosine were observed. These two diastereomers were separated from each other by g.c. as well as by h.p.l.c. The assignment of the epimers was accomplished on the basis of the n.m.r. data. The formation of 8,5'-cyclo-2'-deoxyguanosine was suppressed by the presence of O2 in the solutions. The use of g.c.-m.s. with the selected-ion monitoring technique facilitated the detection of 8,5'-cyclo-2'-deoxyguanosine in DNA at radiation doses as low as 1 Gy. Its mechanism of formation probably involves hydrogen atom abstraction by .OH radicals from the C-5' of the 2'-deoxyguanosine moiety followed by intramolecular cyclization with the formation of a covalent bond between the C-5' and C-8 and subsequent oxidation of the resulting N-7-centred radical.

Animals↗

PubMed1986

Formation of cytosine glycol and 5,6-dihydroxycytosine in deoxyribonucleic acid on treatment with osmium tetroxide.

OsO4 selectively forms thymine glycol lesions in DNA. In the past, OsO4-treated DNA has been used as a substrate in studies of DNA repair utilizing base-excision repair enzymes such as DNA glycosylases. There is, however, no information available on the chemical identity of other OsO4-induced base lesions in DNA. A complete knowledge of such DNA lesions may be of importance for repair studies. Using a methodology developed recently for characterization of oxidative base damage in DNA, we provide evidence for the formation of cytosine glycol and 5,6-dihydroxycytosine moieties, in addition to thymine glycol, in DNA on treatment with OsO4. For this purpose, samples of OsO4-treated DNA were hydrolysed with formic acid, then trimethylsilylated and analysed by capillary gas chromatography-mass spectrometry. In addition to thymine glycol, 5-hydroxyuracil (isobarbituric acid), 5-hydroxycytosine and 5,6-dihydroxyuracil (isodialuric acid or dialuric acid) were identified in OsO4-treated DNA. It is suggested that 5-hydroxyuracil was formed by formic acid-induced deamination and dehydration of cytosine glycol, which was the actual oxidation product of the cytosine moiety in DNA. 5-Hydroxycytosine obviously resulted from dehydration of cytosine glycol, and 5,6-dihydroxyuracil from deamination of 5,6-dihydroxycytosine. This scheme was supported by the presence of 5-hydroxyuracil, uracil glycol and 5,6-dihydroxyuracil in OsO4-treated cytosine. Treatment of OsO4-treated cytosine with formic acid caused the complete conversion of uracil glycol into 5-hydroxyuracil. The implications of these findings relative to studies of DNA repair are discussed.

Barbiturates↗

PubMed1986

Characterization of free radical-induced base damage in DNA at biologically relevant levels.

DNA damage induced by oxygen radicals, e.g., hydroxyl radicals generated in living cells either by cellular metabolism or external agents such as ionizing radiations, appears to play an important role in mutagenesis, carcinogenesis, and aging. Elucidation of the chemical nature of such DNA lesions at biologically significant quantities is required for the assessment of their biological consequences and repair. For this purpose, a sensitive method using gas chromatography-mass spectrometry with the selected-ion-monitoring technique (GC-MS/SIM) was developed in the present work. DNA was exposed to hydroxyl radicals and hydrogen atoms produced by ionizing radiation in N2O-saturated aqueous solution. DNA samples were subsequently hydrolyzed with formic acid, trimethylsilylated, and analyzed by GC-MS/SIM. Characteristic ions from previously known mass spectra of DNA base products as their trimethylsilyl derivatives were recorded and the area counts of each ion were integrated. From these acquired data, a partial mass spectrum of each product was generated and then compared with those of authentic materials. This technique permitted the detection and characterization of a large number of free radical-induced based products of DNA, i.e., 5,6-dihydrothymine, 5-hydroxy-5,6-dihydrothymine, 5-hydroxymethyluracil, 5-hydroxyuracil, 5-hydroxycytosine, thymine glycol, 4,6-diamino-5-formamidopyrimidine, 8-hydroxyadenine, 2,6-diamino-4-hydroxy-5-formamidopyrimidine, and 8-hydroxyguanine, simultaneously in a single sample after radiation doses from 0.1 to 10 Gy. Detectable amounts of the base products were found to be as low as approximately 10 fmol per injection.(ABSTRACT TRUNCATED AT 250 WORDS)

DNA↗

PubMed1986

Free radical-induced cross-linking of polydeoxythymidylic acid in deoxygenated aqueous solution.

Radiation-generated hydroxyl radicals and hydrogen atoms were shown to induce the cross-linking of polydeoxythymidylic acid (mol X wt approximately 170 000) in N2O-saturated acqueous solution. The irradiated samples were hydrolysed with formic acid and then analysed by high performance liquid chromatography. Products were isolated and subsequently characterized by capillary gas chromatography-mass spectrometry. The presence of previously described monomeric thymine products was also shown. Yields were determined and mechanisms of formation were described for the products.

Chromatography, High Pressure Liquid↗

PubMed1985

Formation of an 8-hydroxyguanine moiety in deoxyribonucleic acid on gamma-irradiation in aqueous solution.

Isolation and characterization of a novel radiation-induced product, i.e., the 8-hydroxyguanine residue, produced in deoxyribonucleic acid (DNA), 2'-deoxyguanosine, and 2'-deoxyguanosine 5'-monophosphate by gamma-irradiation in aqueous solution, are described. For this purpose, gamma-irradiated DNA was first hydrolyzed with a mixture of four enzymes, i.e., DNase I, spleen and snake venom exonucleases, and alkaline phosphatase. Analysis of the resulting mixture by capillary gas chromatography-mass spectrometry after trimethylsilylation revealed the presence of a product, which was identified as 8-hydroxy-2'-deoxyguanosine on the basis of the typical fragment ions of its trimethylsilyl (Me3Si) derivative. This product was then isolated by using reversed-phase high-performance liquid chromatography. The UV and proton nuclear magnetic resonance spectra taken from the isolated product confirmed the structure suggested by the mass spectrum of its Me3Si derivative. In addition, the accurate molecular mass of the Me3Si derivative of the isolated product was determined by MS. The obtained value agreed with the theoretical molecular mass within 1 millimass unit. The yield of 8-hydroxyguanine was also measured. Its mechanism of formation is believed to involve OH radical addition to the C-8 position of guanine followed by oxidation of the radical adduct.

Animals↗

PubMed1985

Application of capillary gas chromatography-mass spectrometry to chemical characterization of radiation-induced base damage of DNA: implications for assessing DNA repair processes.

The application of capillary gas chromatography-mass spectrometry (GC-MS) to the chemical characterization of radiation-induced base products of calf thymus DNA is presented. Samples of calf thymus DNA irradiated in N2O-saturated aqueous solution were hydrolyzed with HCOOH, trimethylsilylated, and subjected to GC-MS analysis using a fused-silica capillary column. Hydrolysis conditions suitable for the simultaneous analysis of the radiation-induced products of all four DNA bases in a single run were determined. The trimethylsilyl derivatives of these products had excellent GC properties and easily interpretable mass spectra; an intense molecular ion (M+.) and a characteristic (M-CH3)+ ion were observed. The complementary use of t-butyldimethylsilyl derivatives was also demonstrated. These derivatives provided an intense characteristic (M-57)+ ion, which appeared as either the base peak or the second most intense ion in the spectra. All mass spectra obtained are discussed. Because of the excellent resolving power of capillary GC and the accurate high-sensitivity identification by MS, the capillary GC-MS is suggested as a very suitable technique for identification of altered bases removed from DNA by base excision-repair enzymes such as DNA glycosylases and, thus, as very useful for an understanding of the base excision-repair of DNA.

Animals↗

PubMed1985

Formation of radiation-induced cross-links between thymine and tyrosine: possible model for cross-linking of DNA and proteins by ionizing radiation.

A model for radiation-induced cross-linking of DNA and proteins has been developed. It is based on initial formation of free radicals on a DNA base, i.e., thymine, and on an amino acid, i.e., tyrosine. It was demonstrated that interaction of these radicals is highly favored as measured by their kinetics and the cross-linked products. The gas chromatography-mass spectrometry methodology used for the identification of the thymine-tyrosine cross-links is suggested as an experimental approach in the measurements of biological cross-links.

Chromatography, Gas↗

PubMed1985

Radiation-induced crosslinks between thymine and phenylalanine.

OH radicals generated by ionizing radiation in aqueous solutions of thymine (T) and phenylalanine (Phe) induce crosslinking between thymine and phenylalanine. The crosslinked products were isolated and characterized by capillary gas chromatography-mass spectrometry. They are formed via OH radical adducts to thymine and phenylalanine and the reaction between dissimilar radicals is greatly favoured (T-Phe:Phe-Phe:T-T = 0.46:0.14:0.05). The reaction mechanism presented may serve as a model for radiation or any free radical-induced crosslinks between DNA and proteins.

Hydroxides↗

PubMed1984

The use of capillary gas chromatography-mass spectrometry for identification of radiation-induced DNA base damage and DNA base-amino acid cross-links.

Application of capillary gas chromatography-mass spectrometry (GC-MS) to isolation and identification of radiation-induced DNA base damage including DNA base-amino acid crosslinks is reported. gamma-Irradiated samples of thymine (thy), thymidine (dT), thymidine-5'-monophosphate (pdT), cytosine(cyt),2'-deoxycytidine (dC), 2'-deoxycytidine-5'-monophosphate (pdC), and mixtures of thy, dT and pdT with tyrosine were used as model systems. Trimethylsilylation was used as the derivatization method. Samples containing nucleosides and nucleotides were first subjected to hydrolysis with formic acid or hydrochloric acid to remove sugar or sugar-phosphate moieties, then trimethylsilylated and analyzed by GC-MS. Trimethylsilyl derivatives of radiation-induced monomeric and dimeric products of the model systems mentioned above were shown to have excellent GC properties and easily interpretable mass spectra. The presence of the molecular ion (M+.) and a characteristic (M-CH3)+ ion in the mass spectra facilitated structural elucidation. The complementary use of tert.-butyldimethylsilyl derivatives was also demonstrated. These derivatives provided an intense characteristic (M-57)+ ion in their mass spectra, which may be used to corroborate the molecular weight and to monitor the corresponding compounds in a complex mixture by means of selected-ion monitoring. All gas chromatograms and mass spectra obtained are discussed in detail.

Amino Acids↗

PubMed1984

Enzymatic digestibility of peptides cross-linked by ionizing radiation.

p6gestibility by proteolytic enzymes of peptides cross-linked by ionizing radiation was investigated. Small peptides of alanine and phenylalanine were chosen as model compounds and aminopeptidases and carboxypeptidases were used as proteolytic enzymes. Peptides exposed to gamma-radiation in aqueous solution were analysed by high-performance liquid chromatography before and after hydrolysis by aminopeptidase M, leucine aminopeptidase, carboxypeptidase A and carboxypeptidase Y. The results obtained clearly demonstrate the different actions of these enzymes on cross-linked aliphatic and aromatic peptides. Peptide bonds of cross-linked dipeptides of alanine were completely resistant to enzymatic hydrolysis whereas the enzymes, except for carboxypeptidase Y, cleaved all peptide bonds of cross-linked peptides of phenylalanine. The actions of the enzymes on these particular compounds are discussed in detail.

Aminopeptidases↗

PubMed1984

OH radical-induced crosslinks of methionine peptides.

Reactions of radiation-generated OH radicals with methionine (Met) and its homopeptides, L-Met-L-Met and tri-L-Met, were investigated through reaction products. Samples of irradiated Met and HCl-hydrolysates of its irradiated homopeptides were trimethylsilylated and analysed by capillary gas chromatography-mass spectrometry. Mass spectra taken revealed the formation of three products, 2-amino-4-(methyldithio)butanoic acid, 4,4'-thiobis(2-aminobutanoic acid) and homocystine. G values of these products were determined to be 0.10, 0.16 and 0.30, respectively.

Cobalt Radioisotopes↗

PubMed1984

Radiation-induced formation of thymine-thymine crosslinks.

The formation of dimers upon gamma-irradiation of thymine, thymidine and thymidine-5'-monophosphate in N2O-saturated aqueous solution is reported. A method using capillary gas chromatography-mass spectrometry was developed for their separation and identification. Quantitative measurements reveal that about 50 per cent of OH adduct radicals of thymine undergo dimerization.

Cobalt Radioisotopes↗

PubMed1984

OH radical-induced products of tyrosine peptides.

Reactions of radiation-generated OH radicals with tyrosine and its homopeptides, i.e. L-Tyr-L-Tyr and L-Tyr-L-Tyr-L-Tyr, in N2O saturated solutions were shown to give crosslinks between the peptide chains with high yields. High-performance liquid chromatography, capillary gas chromatography and mass spectrometry were used for isolation and identification of the monomeric and dimeric products. Evidence is presented for the crosslinking to occur through C-C and C-O-C bonds. To the best of our knowledge, the formation of the ether type of crosslink is demonstrated for the first time. Mechanisms of product formation are also discussed, which involve radicals that were described in previous pulse radiolysis studies.

Chromatography, Gas↗

PubMed1984

Radiation-induced crosslinking of cytosine.

Formation of dimers upon gamma irradiation of cytosine, 2'-deoxycytidine, and 2'-deoxycytidine-5'-monophosphate in N2O-saturated aqueous solutions was found to be a major process. Quantitative measurements revealed that more than 50% of OH adduct radicals of cytosine undergo dimerization. Derivatized dimers and monomeric products were separated and tentatively identified by combined capillary gas chromatography-mass spectrometry.

Cobalt Radioisotopes↗

PubMed1983

Comparison of reversed-phase and weak anion-exchange high-performance liquid chromatographic methods for peptide separations.

Weak anion-exchange and reversed-phase high-performance liquid chromatographic methods for peptide separations were compared using a tryptic digest of "rat small myelin basic protein". In these experiments, a number of tryptic peptides that were not resolved on the reversed-phase column could be separated on the weak anion-exchange column, and in other instances, as might be expected, reversed-phase chromatography provided better resolution of certain peptides than did the weak anion-exchange method. The results obtained strongly suggest that the combined use of these two methods of separation, which utilize different selectivities, can provide an excellent improvement in resolving power for a number of peptide separations.

Amino Acid Sequence↗

PubMed1983

Identification of some OH radical-induced products of lysozyme.

OH radical reactions with lysozyme in gamma-irradiated N2O saturated aqueous solutions caused formation of allo-threonine, alpha-amino-n-butyric acid, o- and m- tyrosines, and 2- and 3-hydroxytyrosines. These identified radiolytic products were characterized by capillary gas chromatography-mass spectrometry as their trimethylsilyl derivatives after HCl-hydrolysis of irradiated lysozyme. Their initial G-values were also determined using gas chromatography. The possible use of these radiolytic products as monitors of radiation-induced damage to proteins and the sites of attack are also discussed.

Amino Acids↗