Structural, electronic and energetic consequences of epigenetic cytosine modifications.

The hydrogen bonding patterns of cytosine and its seven C5-modifed analogues paired with canonical guanine were studied using the first principle approach. Both global minima and biologically relevant conformations were studied. The former resulted from full gradient geometry optimizations of hydrogen bonded pairs, while the latter were obtained based on 125 d(GpC) dinucleotides found in the PDB database. The obtained energetic, electronic and structural data lead to the conclusion that the epigenetically relevant modification of cytosine may have serious consequences on hydrogen bonding with guanine. First of all, the significant substituent effects were observed for such trends as charges on sites involved in hydrogen bonding, the total intermolecular interaction energy or electron densities at bond critical points. Moreover, the molecular orbital polarization contribution resulting from energy decomposition expressed in terms of absolutely localized molecular orbitals exhibited an inverse linear correlation with frozen density contributions. A substituent effect on the amount of charge transfer from pyrimidine toward guanine was also observed. The increase of intermolecular interactions of guanine with modified cytosine is associated with the increase of the electro-donating character of the C5-substituent. However, only pairs involving 5-methylcytosine are more stable than those formed by canonical cytosine. Furthermore, the energy differences observed for global minima also remain important for a broad range of displacement and angular parameters defining pair conformations in model d(GpC) dinucleotides. Due to the sensitivities of intermolecular interactions to mutual arrangements of monomers the modification of cytosine at the C5 site can significantly alter the actual energy profiles. Consequently, it may be anticipated that the modified dinucleotides will adopt different conformations than a standard G-C pair in a B-DNA double helix.

Endogenous oxidative DNA base modifications analysed with repair enzymes and GC/MS technique.

GC/MS technique was used to identify endogenous levels of oxidatively modified DNA bases. To avoid possible artefact formation we used Fpg and Endo III endonucleases instead of acid hydrolysis to liberate the base products from unmodified DNA samples. Several different DNA preparations were used: (i) commercial calf thymus DNA, (ii) DNA isolated from rat liver, (iii) DNA isolated from human lymphocytes and (iv) nuclei isolated from rat liver. In all DNA samples used in our assays the most efficiently removed bases by Fpg protein are FapyG and FapyA although 8-oxoG was also detected in all preparations. The amount of 8-oxoG in human lymphocytes and in rat liver DNA was 3 and 2 per 10(7)bases, respectively. It is reasonable to postulate that the presented method is one of the techniques which should be used to reveal the enigma of endogenous, oxidative DNA damage.

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.

In vivo DNA-protein cross-linking by cis- and trans-diamminedichloroplatinum(II).

When Novikoff hepatoma-bearing rats were given injections of a therapeutic dose of cis-diamminedichloroplatinum(II) (cis-DDP) (7 mg/kg), DNA-protein cross-links could be detected by using antisera to dehistonized chromatin, nuclear matrix, or Novikoff hepatoma cytoskeletal preparation. The extent of cross-linking increased in time up to 24 h after the injection, after which time the DNA-protein cross-links were gradually repaired, with no cross-links detectable at 72 h. trans-DDP in equitoxic (40 mg/kg) dose was very efficient in forming DNA-protein cross-links. Although formed more rapidly, these trans-DDP-mediated cross-links were repaired faster, within 48 h after the injection. The repair of cross-links at equimolar trans-DDP dose (7 mg/kg) was even more rapid. The principal proteins cross-linked to the DNA by both cis- or trans-DDP were Novikoff hepatoma cytokeratins (Mr 39,000, 49,000, 56,000, and an additional protein band reacting with the antiserum to Novikoff hepatoma cytoskeletal preparation at Mr approximately 68,000).

cis-diamminedichloroplatinum(II) cross-linking of the human myeloid cell nuclear differentiation antigen to DNA in HL-60 cells following 1,25-dihydroxy vitamin D3-induced monocyte differentiation.

Protein-DNA interactions of the human myeloid cell nuclear differentiation antigen (MNDA) were examined in vivo in proliferating HL-60 promyelocytic leukemia cells and following induction of differentiation by 1,25-dehydroxyvitamin D3. Intact cells were treated with the reversible cross-linking agent cis-diamminedichloroplatinum(II) and the MNDA levels in the isolated protein-DNA complexes were determined. Less than 1% of the total intracellular level of MNDA was cross-linked to DNA in the noninduced proliferating HL-60 cells. Once the cells were induced to differentiate into monocytes, the amount of antigen cross-linked to the DNA increased to over 5% of the total intracellular level. The increased efficiency of cross-linking the MNDA to DNA was specific for monocyte-induced HL-60 differentiation, achieved with three inducers, and was not observed in association with granulocyte-induced differentiation. On a molar basis the phorbol ester (12-O-tetradecanoylphorbol-13-acetate) was the most effective inducer of monocyte differentiation, followed by 1,25-dihydroxy-16-ene-23-ynecholecalciferol which was more effective than 1,25-dihydroxycholecalciferol. A cesium chloride gradient analysis of the nucleic acid-protein fraction isolation from cis-diamminedichloroplatinum(II)-treated, monocyte-induced HL-60 cells documented the authenticity of the association between the MNDA and DNA. The results indicate that a significant level of chromatin reorganization may accompany monocyte-induced differentiation that leads to much higher levels of MNDA-DNA cross-linking to DNA. The expression of the MNDA is restricted to human myeloid cells and the present results indicate that a fraction of this low abundance nuclear protein is specifically located near the DNA [within cis-diamminedichloroplatinum(II) cross-linking distance] and that this association may be modulated specifically during monocyte differentiation.

The level of typical biomarker of oxidative stress 8-hydroxy-2'-deoxyguanosine is higher in uterine myomas than in control tissues and correlates with the size of the tumor.

The results of this work show a higher level of 8-hydroxy-2'-deoxyguanosine (8-OH-dG), a typical biomarker of oxidative stress, in uterine myoma tissues than in their respective tumor-free tissues. The level of this modified base was elevated in uterine tissues of premenopausal women when compared with postmenopausal ones. We have also found the correlation between the size of the tumor and the amount of 8-OH-dG. These results suggest that estrogen-produced 8-OH-dG may be one of the factors responsible for the formation of the myoma, and it may contribute to malignant transformation of myoma cells.

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.

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.

Further evidence that oxidative stress may be a risk factor responsible for the development of atherosclerosis.

There are numerous data suggesting that oxidative stress may be involved in the development of atherosclerosis. Therefore, in the present study we measured the amount of 8-hydroxy-2'-deoxyguanosine (8-OH-dG), one of the typical biomarkers of oxidative stress, in DNA isolated from lymphocytes of the patients and in the control group. Levels of antioxidant vitamins (A, C, and E) and intracellular labile iron pool (LIP), which can influence oxidative stress, were also determined. Blood samples were obtained from a control group of 55 healthy persons and from 43 atherosclerotic patients. 8-OH-dG and the vitamin levels were measured by high-performance liquid chromatography. Labile iron pool in lymphocytes was analyzed by fluorescent assay. The levels of 8-OH-dG and LIP were significantly higher and vitamin C concentration was significantly lower in the patient group than in the control group. The rest of the analyzed parameters do not significantly differ between the groups. A lower concentration of vitamin C and higher levels of labile iron pool in a group of atherosclerotic patients when compared with the control group may lead to oxidative stress, which is manifested by a higher level of 8-OH-dG in blood lymphocytes. All these factors may create an environment that promotes the development of atherosclerosis.

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.

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.

DNA base modifications in chromatin of human cancerous tissues.

Free radical-induced damage to DNA in vivo is implicated to play a role in carcinogenesis. Evidence exists that DNA damage by endogenous free radicals occurs in vivo, and there is a steady-state level of free radical-modified bases in cellular DNA. We have investigated endogenous levels of typical free radical-induced DNA base modifications in chromatin of various human cancerous tissues and their cancer-free surrounding tissues. Five different types of surgically removed tissues were used, namely colon, stomach, ovary, brain and lung tissues. In chromatin samples isolated from these tissues, five pyrimidine-derived and six purine-derived modified DNA bases were identified and quantitated by gas chromatography/mass spectrometry with selected-ion monitoring. These were 5-hydroxy-5-methylhydantoin, 5-hydroxyhydantoin, 5-(hydroxymethyl)uracil, 5-hydroxycytosine, 5,6-dihydroxycytosine, 4,6-diamino-5-formamidopyrimidine, 8-hydroxyadenine, xanthine, 2-hydroxyadenine, 2,6-diamino-4-hydroxy-5-formamidopyrimidine, and 8-hydroxyguanine. These compounds are known to be formed typically by hydroxyl radical attack on DNA bases. In all cases, elevated amounts over control levels of modified DNA bases were found in cancerous tissues. The amounts of modified bases depended on the tissue type. Lung tissues removed from smokers had the highest increases of modified bases above the control levels, and the highest overall amounts. Colon cancer tissue samples had the lowest increases of modified bases over the control levels. The results clearly indicate higher steady-state levels of modified DNA bases in cancerous tissues than in their cancer-free surrounding tissues. Some of these lesions are known to be promutagenic, although others have not been investigated for their mutagenicity. Identified DNA lesions may play a causative role in carcinogenesis.

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.

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).

8-Oxo-2'-deoxyguanosine level in lymphocytes DNA of cancer patients undergoing radiotherapy.

We analyzed the level of 8-oxo-2'-deoxyguanosine in lymphocytes DNA of cancer patients undergoing radiotherapy. The results of this work indicate that exposure of cancer patients to therapeutic doses of ionizing radiation causes significant increase of the amount of 8-oxo-dG in DNA isolated from their lymphocytes.

Oxidative DNA base damage in cancerous tissues of patients undergoing brachytherapy.

This aim of this study was to measure the typical free radical-induced products of DNA bases in cellular DNA of cervical cancer tissues directly irradiated by applying brachytherapy to the patients. Significant increases in the amounts of modified bases over the control level were observed in the samples isolated after irradiation for all patients. These increases differed among patients and among products. The repair capacity and/or the amount of hypoxic cells inside the tumor may account for the different levels of modified bases. It is possible that the observed variabilities may account for the differences in clinical responses to brachytherapy.

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.

Oxidative DNA base damage in lymphocytes of HIV-infected drug users.

In the present study, we have studied the level of oxidative DNA base damage in lymphocytes of HIV-infected intravenous drug users (IDUs) and a seronegative control group. Chromatin was isolated from the lymphocytes and then analyzed by gas chromatography/isotope-dilution mass spectrometry with selected-ion monitoring (GC/IDMS-SIM). Significantly greater levels of four oxidatively modified DNA bases were observed in chromatin samples from the symptomatic HIV-infected patients than in those from the seronegative patients. These were 5-hydroxyuracil, 5-hydroxycytosine, 8-hydroxyadenine and 8-hydroxyguanine. In the case of 5-hydroxyuracil and 8-hydroxyguanine, a statistically significant difference was also found between the control group and the asymptomatic HIV-positive patients. These results suggest that oxidative stress may play an important role in the pathogenesis of acquired immune deficiency syndrome (AIDS), and that administration of antioxidant drugs to HIV-infected patients may offer protection against AIDS-related carcinogenesis.

8-Oxo-7,8-dihydroguanine and 8-oxo-7,8-dihydro-2'-deoxyguanosine levels in human urine do not depend on diet.

In the present study, we used the method involving HPLC pre-purification followed by gas chromatography with isotope dilution mass spectrometric detection for the determination of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodGuo) and 8-oxo-7,8-dihydroguanine (8-oxoGua) in human urine. The mean levels of 8-oxoGua and 8-oxodGuo in the urine samples of the subjects on unrestricted diet were respectively 1.87 nmol/kg 24 h (+/-0.90) and 0.83 nmol/kg 24 h (+/-0.49), and in the case of the groups studied, they did not depend on the applied diet. The sum of the amounts of both compounds in urine can give information about the formation rate of 8-oxoGua in cellular DNA. It is also likely that the levels of modified nucleo-base/side in urine sample are reflective of the involvement of different repair pathways responsible for the removal of 8-oxodGuo from DNA, namely base excision repair (BER) and nucleotide excision repair (NER).

Oxidative damage to plant DNA in relation to growth conditions.

In this study we investigated the level of 8-oxo-2'-deoxyguanosine (8-oxodG) in DNA of Cardamine pratensis plants subjected to different growth conditions trying to answer the question whether factors like light and water accessibility or low temperature may have an impact on the total DNA oxidative damage. The level of this modified nucleoside was determined using HPLC coupled to UV absorbance and electrochemical detection (HPLC-UV-EC). We did not observe any statistically significant differences in 8-oxodG level between DNA of etiolated and light exposed plants as well as between DNA of regularly watered and drought-subjected plants. In contrast, we have shown that chilling (1 degree C for 28 h) brings about the increase of 8-oxodG level in DNA.