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T H Zastawny

Publications and source records attributed to T H Zastawny.

At least 19 recordsLinked to original sources
PubMed2001

Repair of gamma-ray-induced base damage in L5178Y sublines is damage type-dependent and unrelated to radiation sensitivity.

The L5178Y (LY) murine lymphoma sublines LY-R and LY-S are differentially sensitive to ionizing radiation. The high radiation sensitivity of LY-S cells is related to impaired rejoining of DNA double strand breaks. We found previously that the gamma-ray-induced base damage is higher in the more radiosensitive LY-S subline. Here, we examine the role of the repair of ionizing radiation induced base damage in relation to the radiosensitivity difference of these sublines. We used the GS/MS technique to estimate the repair rates of six types of base damage in gamma-irradiated LY cells. All modified DNA bases identified in the course of this study were typical for irradiated chromatin. The total amount of initial base damage was higher in the radiation sensitive LY-S subline than in the radiation resistant LY-R subline. The repair rates of 5-OHMeUra, 5-OHCyt, 8-OHAde were similar in both cell lines, the repair rates of FapyAde and 8-OHGua were higher in the radiosensitive LY-S cell line, whereas the repair of 5-OHUra was faster in its radioresistant counter, the LY-R. Altogether, the repair rates of the y-ray-induced DNA base damage in LY sublines are related neither to the initial amounts of the damaged bases nor to the differential lethal or mutagenic effects of ionizing radiation in these sublines.

Animals↗
PubMed2000

Fapyadenine is a moderately efficient chain terminator for prokaryotic DNA polymerases.

Hypoxanthine¿xanthine oxidase¿Fe3+¿ethylenediaminetetraacetate (EDTA) was used to modify ss M13 mp18 phage DNA. The dominant base modifications found by GC/IDMS-SIM were FapyGua, FapyAde, 8-hydroxyguanine, and thymine glycol. Analysis of in vitro DNA synthesis on oxidatively modified template by three DNA polymerases revealed that T7 DNA polymerase and Klenow fragment of polymerase I from Escherichia coli were blocked mainly by oxidized pyrimidines in the template whereas some purines that were easily bypassed by the prokaryotic polymerases constituted a block for DNA polymerase beta from calf thymus. DNA synthesis by T7 polymerase on poly(dA) template, where FapyAde content increased 16-fold on oxidation, yielded a final product with a discrete ladder of premature termination bands. When DNA synthesis was performed on template from which FapyAde, FapyGua, and 8OHGua were excised by the Fpg protein new chain terminations at adenine and guanine sites appeared or existing ones were enhanced. This suggests that FapyAde, when present in DNA, is a moderately toxic lesion. Its ability to arrest DNA synthesis depends on the sequence context and DNA polymerase. FapyGua might possess similar properties.

Animals↗
PubMed1999

SOS-dependent A-->G transitions induced by hydroxyl radical generating system hypoxanthine/xanthine oxidase/Fe3+/EDTA are accompanied by the increase of Fapy-adenine content in M13 mp18 phage DNA.

Gas chromatography/isotope dilution-mass spectrometry with selected ion monitoring (GC/IDMS-SIM) was used to measure oxidised bases in hypoxanthine/xanthine oxidase/Fe3+/EDTA modified ss M13 mp18 phage DNA. A dose-dependent increase of oxidised bases content in DNA was observed with the biggest augmentation of FapyGua, thymine glycol and FapyAde. The amount of 8-OH-Gua was relatively high both in non-oxidised and oxidised DNA, and increased to the same extent as FapyAde and ThyGly. DNA oxidation caused a dramatic decrease in phage survival after transfection to E. coli. Survival was improved 2.8-fold after induction of the SOS system by UV irradiation of bacteria and mutation frequency of the lacZ gene in SOS conditions increased 7-fold over that in non-irradiated bacteria. Spectrum of mutations was different from those reported previously and mutations were distributed rather randomly within M13 lacZ sequence, which was in contrast to previous findings, where with non-chelated metal ions other types of mutations were found in several clusters. Thus, conditions of DNA oxidation and accessibility of metal ions for DNA bases might be important factors for generating different DNA damages and mutations. Major base substitutions found both in SOS-induced and non-induced E. coli but with higher mutation frequency in SOS-induced cells were C-->A (approximately 20-fold increase in SOS-conditions), G-->A (9-fold increase) and G-->C (4.5-fold increase). Very few G-->T transitions were found. A particularly large group of A-->G transitions appeared only in SOS-induced bacteria and was accompanied by augmentation of FapyAde content in the phage DNA with undetectable 2-OH-Ade. It is then possible that imidazole ring-opened adenine mimics guanine during DNA replication and pairs with cytosine yielding A-->G transitions in SOS-induced bacteria.

Bacteriophage M13↗
PubMed1999

Mitochondrial disease in superoxide dismutase 2 mutant mice.

Oxidative stress has been implicated in many diseases. The chief source of reactive oxygen species within the cell is the mitochondrion. We have characterized a variety of the biochemical and metabolic effects of inactivation of the mouse gene for the mitochondrial superoxide dismutase (CD1-Sod2(tm1Cje)). The Sod2 mutant mice exhibit a tissue-specific inhibition of the respiratory chain enzymes NADH-dehydrogenase (complex I) and succinate dehydrogenase (complex II), inactivation of the tricarboxylic acid cycle enzyme aconitase, development of a urine organic aciduria in conjunction with a partial defect in 3-hydroxy-3-methylglutaryl-CoA lyase, and accumulation of oxidative DNA damage. These results indicate that the increase in mitochondrial reactive oxygen species can result in biochemical aberrations with features reminiscent of mitochondrial myopathy, Friedreich ataxia, and 3-hydroxy-3-methylglutaryl-CoA lyase deficiency.

Aconitate Hydratase↗
PubMed1999

Mutagenic specificity of imidazole ring-opened 7-methylpurines in M13mp18 phage DNA.

The most abundant lesion formed in DNA upon modification with methylating agents 7-methylguanine, under alkaline conditions is converted into 2,6-diamino-4-hydroxy-5N-methyl-formamidopyrimidine (Fapy-7MeGua). We have previously shown that treatment of dimethylsulfate methylated DNA with NaOH creates mutagenic base derivatives leading to a 60-fold increase in the frequency of A-->G transitions and a 2-3-fold increase of G-->T and G-->C transversions. We have analyzed which lesions lead to these mutations. We compared mutagenic spectra in the lacZ gene of M13mp18 phage DNA modified with dimethylsulfate and NaOH after selective elimination of damaged bases from molecules used for transfection into SOS-induced E. coli. Partial elimination of Fapy-7MeGua from phage DNA performed by its digestion with formamidopyrimidine-DNA glycosylase resulted in a 2-3-fold decrease of G-->T and G-->C transversions. Selective depurination of methylated bases (9 h, 37 degrees C, pH 7.0) resulting in almost complete loss of 7MeAde as demonstrated by HPLC analysis of [3H]MNU alkylated phage DNA used as a probe, caused a dramatic, 9-fold decrease of A-->G transitions. Alkali-catalysed rearrangement of 7MeAde was followed by HPLC analysis of [3H]MNU alkylated poly(A) and poly(dA). After incubation of these oligonucleotides in NaOH, 7MeAde disappeared from both chromatograms, but only in polyA, 2 new peaks migrating with retention time different from that of 1MeAde, 3MeAde or 7MeAde were detected, suggesting formation of two rotameric forms of Fapy-7MeAde as observed for Fapy-7MeGua. Thus the miscoding lesion, giving rise to A-->G transitions derived from 7MeAde was Fapy-7MeAde. Fapy-7MeGua was at least an order of magnitude less mutagenic, but in SOS-induced cells it gave rise to G-->T and G-->C transversions.

Adenine↗
PubMed1998

Comparison of oxidative base damage in mitochondrial and nuclear DNA.

The levels of endogenous pig liver cells mitochondrial DNA oxidative base damage have been investigated using isotope dilution gas chromatography mass spectrometry (GC/MS). Higher levels of five measured bases were found in mtDNA in relation to nuclear DNA. We have also detected large differences in the modified base ratios of mitochondrial versus nuclear DNA. These ratios for the bases with promutagenic properties as 8OHGua and 5OHCyt are much lower than for other bases (5OHHyd, 5OHMeHyd, 5OHMeUra).

Animals↗
PubMed1998

Ionizing radiation and hydrogen peroxide induced oxidative DNA base damage in two L5178Y cell lines.

Seven oxidized DNA bases were quantified, by gas GC/MS-SIM, in chromatin from gamma-rays and H2O2 treated mouse lymphoma L5178Y (LY) cells, inversely cross-sensitive to these agents. In H2O2 treated cells (2 mM, 1 h, 37 degrees C) we found more damage in LY-R cells than in LY-S cells. On the contrary, in gamma-rays (400 Gy) treated cells we found more damaged DNA bases in LY-S cells. The yield of damaged bases in control cells was similar in both cell lines, with the exception of 8OHAde and FapyGua that were found at a much higher level in LY-S cells. The yields of damaged bases were related to cellular sensitivity to damaging agent; this observation points to a relationship between DNA base damage induction, antioxidant defense system in the intracellular milieu and cell sensitivity.

Animals↗
PubMed1998

Oxidative DNA base modifications as factors in carcinogenesis.

Reactive oxygen species can cause extensive DNA modifications including modified bases. Some of the DNA base damage has been found to possess premutagenic properties. Therefore, if not repaired, it can contribute to carcinogenesis. We have found elevated amounts of modified bases in cancerous and precancerous tissues as compared with normal tissues. Most of the agents used in anticancer therapy are paradoxically responsible for induction of secondary malignancies and some of them may generate free radicals. The results of our experiments provide evidence that exposure of cancer patients to therapeutic doses of ionizing radiation and anticancer drugs causes base modifications in genomic DNA of lymphocytes. Some of these base damages could lead to mutagenesis in critical genes and ultimately to secondary cancers such as leukemias. This may point to an important role of oxidative base damage in cancer initiation. Alternatively, the increased level of the modified base products may contribute to genetic instability and metastatic potential of tumor cells.

Animals↗
PubMed1997

Characterization and mechanism of action of Drosophila ribosomal protein S3 DNA glycosylase activity for the removal of oxidatively damaged DNA bases.

We recently demonstrated that Drosophila ribosomal protein S3 specifically cleaved duplex oligodeoxynucleotides at sites of 7,8-dihydro-8-oxoguanine (8-oxoGua), presumably due to S3 protein possessing an N-glycosylase activity that is associated with its known apurinic/apyrimidinic (AP) lyase activity. Here we show, using DNA substrates prepared by gamma-irradiation under N2O and analyzed by gas chromatography/isotope-dilution mass spectrometry, that S3 protein efficiently liberates 8-oxoGua as a free base from the damaged DNA substrate. Of the 15 additional modified bases present in the DNA substrate, the only other one acted on by S3 protein was 2,6-diamino-4-hydroxy-5-formamidopyrimidine (FapyGua). Specificity constants measured for the removal of 8-oxoGua and FapyGua indicate that S3 protein has a similar preference for both of these modified purines. Having established that S3 protein contains an N-glycosylase activity, we next examined the repair of duplex oligonucleotides containing 8-oxoGua (8-oxoGua-37-mer) positioned opposite Cyt, Gua, Thy, or Ade. Significant cleavage of the 8-oxoGua-37-mer was only detected for an opposing Cyt. Moreover, we show that an imino covalent enzyme-substrate intermediate is formed between S3 protein and 8-oxoGua-37-mer, a result similar to other DNA repair enzymes that catalyze N-glycosylase/AP lyase-type reactions at sites of DNA damage.

Animals↗
PubMed1997

Radiation-induced oxidative DNA base damage and its repair in nuclear matrix-associated DNA and in bulk DNA in hepatic chromatin of rat upon whole-body gamma-irradiation.

In the present work, we examined the formation and repair of DNA base damages induced by gamma-irradiation in different fractions of rat hepatic chromatin. Animals were exposed to radiation with the dose of 10 Gy. Nuclear matrix DNA and whole chromatin were isolated from the liver of rats killed before and in different time after irradiation. In those samples the pyrimidine-derived and the purine-derived modified DNA bases were identified and quantitated by gas chromatography/isotope-dilution mass spectrometry with selected-ion monitoring. We found elevated levels of modified DNA bases over control values after whole body irradiation in both matrix DNA and bulk chromatin samples. Our results suggest that modified bases are preferentially removed from matrix DNA then bulk chromatin.

Animals↗
PubMed1997

Oxidative DNA base damage and its repair in kidneys and livers of nickel(II)-treated male F344 rats.

DNA base damage was assayed using gas chromatography/ mass spectrometry with selected ion monitoring (GC/MS-SIM) in renal and hepatic chromatin of male F344 rats up to 14 days after a single i.p. injection of 90 micromol Ni(II) acetate/kg body wt. Ten different damaged bases were quantified. No damage was found in either organ 12 h after Ni(II) treatment. The damage became significant only from day 1, with magnitude and persistence depending on the organ and base. In livers, levels of five DNA base products were significantly elevated over those in control rats. They were: 8-oxoguanine (by 46% at day 1 postinjection); 2,6-diamino-4-hydroxy-5-formamidopyrimidine (by 107% at day 1); 5-(hydroxymethyl)uracil (by 94% at day 1); 5,6-dihydroxyuracil (by 128% at day 1); and 5-hydroxyhydantoin (by 39% in terms of the overall adjusted means for days 1-14 post-injection). The elevation was highest at day 1 post-injection followed by a decrease at later days, except for 5-hydroxyhydantoin. In kidneys, the levels of only three damaged bases, 8-oxoguanine, 5-hydroxyhydantoin and 5,6-dihydroxyuracil were increased significantly (by 31, 73 and 60%, respectively) and one base, 8-oxoadenine, was increased by 26%, just below significance, all in terms of overall adjusted means for days 1-14 post-injection. Hence, unlike those in the liver, the renal increases persisted for 14 days. The results reveal a tissue specific response to Ni(II)-mediated oxidative DNA base damage with apparently faster DNA repair in liver than in kidney, the main target of Ni(II) carcinogenicity.

Acetates↗
PubMed1996

DNA base damage in lymphocytes of cancer patients undergoing radiation therapy.

We investigated DNA base damage in genomic DNA of lymphocytes of cancer patients undergoing radiation therapy. Lymphocyte chromatin samples were analyzed by gas chromatography/isotope-dilution mass spectrometry for DNA base damage. The results provided evidence for formation of typical hydroxyl radical-induced base modifications in genomic DNA of lymphocytes. Different levels of DNA products in individuals were observed and, in the case of some patients, there was no significant product formation, possibly resulting from differences between individuals and between the types of radiation exposures. Decreases in product levels after an initial increase by radiation exposure were observed. This may indicate the removal of modified bases from lymphocyte DNA by cellular repair.

Adult↗
PubMed1996

DNA damage and DNA sequence retrieval from ancient tissues.

Gas chromatography/mass spectrometry (GC/MS) was used to determine the amounts of eight oxidative base modifications in DNA extracted from 11 specimens of bones and soft tissues, ranging in age from 40 to >50 000 years. Among the compounds assayed hydantoin derivatives of pyrimidines were quantitatively dominant. From five of the specimens endogenous ancient DNA sequences could be amplified by PCR. The DNA from these specimens contained substantially lower amounts of hydantoins than the six specimens from which no DNA could be amplified. Other types of damage, e.g. oxidation products of purines, did not correlate with the inability to retrieve DNA sequences. Furthermore, all samples with low amounts of damage and from which DNA could be amplified stemmed from regions where low temperatures have prevailed throughout the burial period of the specimens.

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

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↗