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PubMed1983

Differences in the DNA of the inactive X chromosomes of fetal and extraembryonic tissues of mice.

We have examined the role of DNA modification in X chromosome inactivation of fetal tissues of the mouse using DNA-mediated gene transfer for the gene hypoxanthine phosphoribosyltransferase (HPRT). Two types of tissues have been examined with respect to randomness of inactivation in 14-day mouse conceptuses: 1) fetal tissue, which undergoes random inactivation of either the maternal or paternal X; and 2) yolk sac endoderm tissue, an extraembryonic membrane, which normally undergoes nonrandom inactivation of the paternal X. Exploiting an electrophoretic variant of HPRT as a means to mark the active and inactive HPRT alleles we provide evidence that: 1) inactive X DNA of the fetus at 14 days behaves like that of both adult tissue and cell lines in that the inactive X DNA is not efficient in gene transfer; and 2) in contrast, inactive X DNA from yolk sac endoderm is functional in gene transfer. Thus, despite the similarity in single active X chromosome expression in yolk sac endoderm and somatic tissues, there appears to be a difference at the level of DNA modification between these two tissues.

Animals↗
PubMed2000

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.

Animals↗
PubMed1997

Determination of steady-state levels of oxidative DNA base modifications in mammalian cells by means of repair endonucleases.

The alkaline elution technique in combination with various repair endonucleases (Fpg protein, endonuclease III, exonuclease III, T4 endonuclease V) was used to quantify steady-state (background) levels of oxidative base modifications in various types of mammalian cells. In human lymphocytes the number of base modifications sensitive to Fpg protein, which include 8-hydroxyguanine, was 0.25 +/- 0.05 per 10(6) base pairs. Even lower levels (0.07 +/- 0.02 per 10(6) bp) were observed in HeLa cells. The numbers of sites sensitive to the other repair endonucleases were below the detection limit (0.05 per 10(6) bp). In a direct comparison, the background level of Fpg-sensitive modifications determined by alkaline elution was much lower than the background level of 8-hydroxydesoxyguanosine (8-oxodG) determined after enzymatic DNA hydrolysis by HPLC and electrochemical detection. However, the number of additional Fpg-sensitive modifications induced by a photosensitizer plus light was similar to the additional number of 8-oxodG residues determined by HPLC with electrochemical detection. This indicates that the enzyme assay does not systematically underestimate the number of lesions and points to an artefactual generation of 8-oxodG during DNA isolation and hydrolysis.

Chromatography, High Pressure Liquid↗
PubMed1988

Release of 7-alkylguanines from haloethylnitrosourea-treated DNA by E. coli 3-methyladenine-DNA glycosylase II.

Previous studies have related DNA modification by the haloethylnitrosoureas to their antitumor activity. Repair of this damage, particularly by O6-alkylguanine-DNA alkyltransferase, has been linked to tumor resistance by several previous investigations. We report here that E. coli 3-methyladenine-DNA glycosylase II can also remove several of the DNA modifications caused by the haloethylnitrosoureas. 7-Chloroethylguanine, 7-hydroxyethylguanine, and diguan-7-ylethane are all released into the supernatant from DNA modified by N-[2-chloroethyl-1,2-14C]-N'-cyclohexyl-N-nitrosourea. Release of diguan-7-ylethane is of particular interest since this entity evidently represents a DNA intrastrand cross-link. If a similar activity is present in mammalian cells, it might be an important source of resistance to the therapeutic action of the haloethylnitrosoureas.

Chromatography, High Pressure Liquid↗
PubMed1998

Oxidative DNA damage in vivo: relationship to age, plasma antioxidants, drug metabolism, glutathione-S-transferase activity and urinary creatinine excretion.

Oxidative DNA modification has been implicated in development of certain cancers and 8-oxodG, the most abundant and mutagenic DNA modification, has for some time been considered a biomarker of this activity. Urinary excretion of 8-oxodG over 24h has been used to estimate the rate of damage to DNA, and animal studies have supported this rationale. Reported determinants include tobacco smoking, heavy exercise, environmental pollution and individual oxygen consumption. Samples from three published studies were used to determine the association of urinary 8-oxodG excretion with age, plasma antioxidants, the glutathione-S-transferase phenotype and the activity of the xenobiotic metabolising enzyme CYP1A2. In the age range 35-65 years, age was not related to urinary 8-oxodG excretion, and there were no relations to either the glutathione-S-transferase phenotype or to the plasma antioxidants: vitamin C, alpha-tocopherol, beta-carotene, lycopene or coenzyme Q10. The activity of CYP1A2 showed a significant correlation in two of the three studies, as well as a significant correlation of 0.26 (p < 0.05) in the pooled data set. Regression analysis of CYP1A2 activity on 8-oxodG indicated that 33% increase in CYP1A2 activity would correspond to a doubling of 8-oxodG excretion. This finding needs to be confirmed in independent experiments. Spot morning urine samples can under certain circumstances be used to estimate 8-oxodG excretion rate provided that creatinine excretion is unchanged (in paired experiments) or comparable (in un-paired experiments), as evaluated from the correlation between 8-oxodG excretion in 24 h urine samples and in morning spot urine samples corrected for creatinine excretion (r = 0.50, p < 0.05). We conclude that 8-oxodG excretion is determined by factors like oxygen consumption and CYP1A2 activity rather than by factors like plasma antioxidant concentrations.

8-Hydroxy-2'-Deoxyguanosine↗
PubMed1999

Monoclonal antibody against DNA adducts with osmium structural probes.

Osmium tetroxide complexes with nitrogen ligands (Os,L) have been widely used as probes of the DNA structure. A monoclonal antibody OsBP7H8 against DNA adducts with Os,L was produced in mice. OsBP7H8 does not bind to proteins or total yeast RNA modified with Os,2,2'-bipyridine (bipy) nor to the unmodified nucleic acids and proteins. The antibody recognizes DNA modified with Os,bipy (DNA-Os,bipy) or with OsO4,1,10-phenanthroline (DNA-Os,phen) but it does not cross-react with oxidized DNA and with DNA adducts of osmium tetroxide complexes with other ligands (such as pyridine, TEMED and bathophenanthroline disulfonic acid). The affinity of OsBP7H8 to DNA-Os,phen is about five-fold higher as compared to DNA-Os,bipy. The antibody can be thus applied either for recognition of single-stranded and distorted regions in DNA (after DNA modification with Os,bipy) or for detection of both single-stranded and double-stranded DNAs (after DNA modification with Os,phen). A new simplified procedure for the dot-blot analysis is proposed, not requiring the purification of DNA-osmium adduct prior to its application to the membrane.

Animals↗
PubMed1988

Inhibition of estrogen-induced renal carcinogenesis in male Syrian hamsters by tamoxifen without decrease in DNA adduct levels.

Estrogens have previously been shown to induce covalent DNA modifications specifically in the hamster kidney, the target organ of estrogen-inducible and -dependent renal carcinoma. The DNA adducts, formed by yet unknown mechanisms, have been postulated to mediate hormonal carcinogenesis in this animal model. In an attempt to study a possible involvement of estrogen receptor mechanisms in the formation of DNA adducts, 17 beta-estradiol and the antihormone tamoxifen were concomitantly administered as s.c. implants to male Syrian hamsters. 17 beta-Estradiol-treated and tamoxifen-treated animals served as positive and negative controls, respectively. The tumor incidence decreased from 100% in 17 beta-estradiol-treated controls to 25% in the group receiving tamoxifen in addition to hormone. Tamoxifen-treated animals did not develop kidney tumors and did not show any detectable DNA damage. DNA adduct levels were comparable in hamsters treated with 17 beta-estradiol and 17 beta-estradiol plus tamoxifen for 5 or 7 months. In hamsters inoculated with H-301 cells, which are derived from the estrogen-induced hamster renal carcinoma and are estrogen dependent for growth, tamoxifen decreased estrogen-dependent H-301 tumor growth. However, in cell culture, neither 17 beta-estradiol nor tamoxifen influenced H-301 cell division. It was concluded that tamoxifen inhibited the growth of estrogen-induced renal carcinoma but did not interfere with tumor initiation since it did not inhibit the formation of DNA adducts. Moreover, receptor mechanisms were most probably not involved in the induction of DNA modifications by estrogens.

Animals↗
PubMed2001

Age-related and tissue-specific accumulation of oxidative DNA base damage in 7,8-dihydro-8-oxoguanine-DNA glycosylase (Ogg1) deficient mice.

Mutations that influence the repair of oxidative DNA modifications are expected to increase the steady-state (background) levels of these modifications and thus create a mutator phenotype that predisposes to malignant transformation. We have analysed the steady-state levels and repair kinetics of oxidative DNA modifications in cells of homozygous ogg1(-/-) null mice, which are deficient in Ogg1 protein, a DNA repair glycosylase that removes the miscoding base 8-hydroxyguanine (8-oxoG) from the genome. Oxidative purine modifications including 8-oxoG were quantified by means of an alkaline elution assay in combination with Fpg protein, the bacterial functional analogue of Ogg1 protein. In primary hepatocytes of adult ogg1(-/-) mice aged 9-12 months, the steady-state level of the lesions was 2.8-fold higher than in wild-type control mice. In contrast, no difference between ogg1(-/-) and wild-type mice was observed in splenocytes, spermatocytes and kidney cells. In hepatocytes of ogg1(-/-) mice, but not in wild-type controls, the steady-state levels increased continuously over the whole lifespan. No significant accumulation of the oxidative base modifications was observed in ogg1(-/-) fibroblasts in culture when they were kept confluent for 8 days. Both in confluent and proliferating ogg1(-/-) fibroblasts, the global repair of additional oxidative base modifications induced by photosensitization was 4-fold slower than in wild-type cells. The results suggest that the consequences of an Ogg1 defect are restricted to slowly proliferating tissues with high oxygen metabolism such as liver, because of a back-up mechanism for the repair of 8-oxoG residues that is independent of transcription and replication.

Age Factors↗
PubMed1987

Guanine modification during chemical DNA synthesis.

Base modification during solid-phase phosphoramidite synthesis of oligodeoxynucleotides has been investigated. We have discovered chemical modification that converts dG and dG-containing oligomers to a fluorescent form. This modification has been linked to N,N-dimethylaminopyridine (DMAP), an acylation catalyst, which can displace phosphate triester adducts at the 6-position of guanine. Further, we have found that this fluorescent intermediate can be converted in ammonium hydroxide solution to 2,6 diaminopurine deoxyribonucleoside (2,6 DAP), a potentially mutagenic nucleoside analog. We have shown that N-methylimidazole (NMI) in place of DMAP eliminates the fluorescent species and reduces 2,6 DAP contamination.

Chromatography, High Pressure Liquid↗
PubMed1980

Uptake and binding of 1-methyl-1-nitrosourea (MNU) and 1-methyl-3-nitro-1-nitrosoguanidine (MNNG) by the isolated guinea pig pancreas.

1-Methyl-1-nitrosourea (MNU) and 1-methyl-3-nitro-1-nitrosoguanidine (MNNG) are carcinogens which methylate nucleic acids and proteins and covalently modify proteins by carbamoylation (MNU) or guanidination (MNNG). Using MNU and MNNG labeled with carbon-14 in the individual carbon positions, the above reactions were quantitated in the isolated guinea pig pancreas, an organ susceptible to tumorigenesis by MNU. Freshly prepared pancreatic lobules were incubated with the labeled drugs (0.03, 0.3 and 1.0 mM) for one hour at 37 degrees C. Alkylated purines from hydrolyzed DNA were separated on Sephadex G10 and acid-soluble nuclear proteins were extracted and separated on polyacrylamide gels. Total uptake of all four labels into lobules was linear with concentration. Acid insoluble radioactivity also increased linearly except for MNNG methylation which plateaued between 0.3 and 1.0 mM. 7-Methylguanine formation by both compounds was approximately ten fold greater than 06-methylation. However, DNA modification by MNU exceeded that by MNNG, especially at the higher drug concentrations. No carbamoylation or guanidination of DNA was detected. Total binding (methylation plus carbamoylation/guanidination) to acid-extractable chromatin proteins was equivalent to DNA modification on a molar basis (approximately 0.35 and 0.08 pmol/microg for exposure to 1.0 mM MNU and MNNG, respectively). All histones were labeled by all drug preparations, with H2A being the principal site of methylation and H2B, H3 and H1 being the major targets of carbamoylation and guanidination. H4 was the least modified histone. Drug binding to cytoplasmic organelles also occurred. These results show a broad spectrum of nuclear and cytoplasmic modification of pancreatic cells by MNU and, to a smaller extent, MNNG.

Animals↗
PubMed1990

[Reactivity of oligonucleotide derivatives, containing methylphosphonate groups. VI. Increase in the effectiveness of directed action of alkylating derivatives of oligonucleotide methylphosphonate analogs on nucleic acids in the presence of effectors--3',5'-bis-N-(2-hydroxyethyl)-phenazine derivatives of oligonucleotides].

Effectors for increasing the efficiency of DNA modification with the alkylating methylphosphonate analogues of oligodeoxyribonucleotides (MFAO) were suggested. Oligodeoxyribonucleotide d(pC5A8ACAATG) used as a target DNA treated with alkylating derivatives of octathymidylate having alternating methylphosphonate and phosphodiester internucleotide bonds (both Rp- and Sp-individual diastereoisomers of MFAO were used) and bearing alkylating 4-(N-methyl-N-2-chloroethylamino)benzyl phosphoramide residue at the 3'-end. The reactions were carried out in the presence of an effector, hexadeoxyribonucleotide derivative PhnNH(CH2)2NHpCATTGTpNH(CH2)2NHPhn bearing two N-(2-hydroxyethyl)phenazinium (Phn) residues at the 3'- and 5'-ends and being complementary to the part of the target DNA neighbouring with octaadenylate. It was shown that Tm of the duplex formed by the target DNA, octathymidylate and effector is by 7-13 degrees C higher than in the absence of the effector, thus considerably increasing the efficiency of the intracomplex alkylation of the target (e.g., at 40 degrees C, the increase for the reagent based on the Rp-isomer is sixfold). Specificity of the target DNA modification by the MFAO alkylating derivatives in the presence of effector is same as with reagents based on oligodeoxyribonucleotides with natural internucleotide bonds.

Alkylating Agents↗
PubMed1990

Unwinding of duplex DNA during transcription initiation at the Escherichia coli galactose operon overlapping promoters.

We have used potassium permanganate as a probe to detect DNA duplex unwinding in vitro, in open complexes between E. coli RNA polymerase and DNa fragments carrying the E. coli galactose operon regulatory region. This zone contains 3 overlapping promoters which specify transcription initiation at 3 distinct startpoints. We have used mutant gal derivatives carrying different single point mutations, each of which allows initiation from only one of the 3 start sites. This has allowed us to compare duplex unwinding in open complexes at the 3 different promoters, and to show that the extent of the unwinding is similar in each case. Further, the pattern of DNA modification by potassium permanganate suggests a model for discrimination between the upper and lower strands. Finally, we show that DNA modification by potassium permanganate at the gal promoters is the same in vivo as in vitro.

Base Sequence↗
PubMed1977

Inhibition of transcription of supercoiled PM2 DNA by carbodiimide modification.

PM2 superhelican DNA (form I), which as been reacted with the single strand specific reagent, N-cyclohexyl-N'-beta-(methylmorpholinium)ethyl carbodiimide (CMC) is more than 95% inhibited in its ability to support transcription with E. coli B RNA polymerase in vitro. Almost complete inhibition of transcription was achieved after 2 hours of reaction with FI when only 1% of the bases were modified. A large increase in S20,* (from 26.8 S to 33.6 S) of FI DNA was observed during the course of reaction. Rifampicin resistant transcription is more susceptible to inhibition by CMC than total transcription, suggesting that the CMC is preferentially binding at promoter sites. These results clearly are in accord with the observation that supercoiled DNA contains localized regions of unpaired bases. The promotor sites for E. coli RNA polymerase in FI PM2 DNA appear to be located at or near these unpaired sites.

Carbodiimides↗
PubMed1985

Quantitation and visualization of alkyl deoxynucleosides in the DNA of mammalian cells by monoclonal antibodies.

Conventional radiochromatographic procedures for the quantitation of carcinogen/mutagen-induced structural DNA modifications have a number of limitations. Thus, these techniques for the most part require application of radioactively labeled carcinogens and the use of relatively large amounts of DNA for analysis at low levels of DNA modification. Radiochromatographic methods also preclude analyses at the level of single cells and DNA molecules. Recently developed immunoanalytical methods have improved this situation considerably. Monoclonal antibodies (Mab) characterized by a high substrate specificity and affinity, in combination with radio- and enzyme-immunoassays, or with "immuno-slot-blot" techniques, now permit the detection of femtomole to subfemtomole amounts of, e.g., alkyldeoxynucleosides in small samples of DNA isolated from tissues or cultured cells previously exposed to nonradioactive N-nitroso compounds. Furthermore, selected Mab can be used to quantitate by direct immunofluorescence (with the aid of computer-based image analysis of electronically intensified fluorescence signals), specific alkyldeoxynucleosides in the nuclear DNA of single cells. With this method, the detection limit for the alkylation product O6-ethyldeoxyguanosine (O6-EtdGuo) is presently of the order of 10(2) -10(3) O6-EtdGuo residues per diploid mammalian genome. Individual cells can thus be monitored for the presence of specific carcinogen-DNA adducts, and with respect to their capacity for enzymatic removal of such modified structures from DNA (as exemplified here by the kinetics of the enzymatic elimination of O6-EtdGuo from the DNA of malignant neurogenic rat cell lines). In combination with transmission electron microscopy, Mab also permit direct visualization (via Mab binding sites) of specific carcinogen-modified structures in individual DNA molecules.(ABSTRACT TRUNCATED AT 250 WORDS)

Alkylating Agents↗
PubMed2006

Diverse roles for histone H2A modifications in DNA damage response pathways in yeast.

There are many types of DNA damage that are repaired by a multiplicity of different repair pathways. All damage and repair occur in the context of chromatin, and histone modifications are involved in many repair processes. We have analyzed the roles of H2A and its modifications in repair by mutagenizing modifiable residues in the N- and C-terminal tails of yeast H2A and by testing strains containing these mutations in multiple DNA repair assays. We show that residues in both tails are important for homologous recombination and nonhomologous end-joining pathways of double-strand break repair, as well as for survival of UV irradiation and oxidative damage. We show that H2A serine 122 is important for repair and/or survival in each of these assays. We also observe a complex pattern of H2A phosphorylation at residues S122, T126, and S129 in response to different damage conditions. We find that overlapping but nonidentical groups of H2A residues in both tails are involved in different pathways of repair. These data suggest the presence of a set of H2A "damage codes" in which distinct patterns of modifications on both tails of H2A may be used to identify specific types of damage or to promote specific repair pathways.

Amino Acid Sequence↗
PubMed1996

Generation of DNA base modification following treatment of cultured murine keratinocytes with benzoyl peroxide.

Benzoyl peroxide (BzPO) is a free radical generating compound that acts as a tumor promoter and progressor in mouse skin. BzPO is cleaved in the presence of copper to produce benzoyloxyl and phenyl radicals. Treatment of mutation reporter plasmids with BzPO and copper yields predominantly single-strand breaks and G-->T transversion mutations. To explore the role of base modifications in the possible mammalian mutagenicity of BzPO the formation of 8-hydroxy-2'-deoxyguanosine (8-OHdG) within the DNA of cultured murine keratinocytes was investigated. Treatment with 10 microM BzPO produced a maximum 3-fold increase in levels of 8-OHdG versus vehicle controls within 1-2 h, with significant levels of 8-OHdG persisting 6 h after initial exposure to BzPO. Pretreatment with the copper chelator bathocuproine disulfonic acid reduced the levels of 8-OHdG generated by BzPO to near background. However, treatment with the iron chelator desferal did not. The stable metabolic product of BzPO benzoic acid was ineffective in producing 8-OHdG. Depletion of cellular glutathione with L-buthionine-(S,R)-sulfoximine increased the amount of BzPO-generated 8-OHdG, while supplementation with glutathione monoethyl ester reduced the number of 8-OHdG molecules formed. Collectively, these results suggest that BzPO at non-cytotoxic concentrations undergoes copper-dependent activation to a reactive product to generate 8-OHdG within cultured murine keratinocytes.

8-Hydroxy-2'-Deoxyguanosine↗
PubMed2003

Influence of substituent modifications on DNA binding energetics of acridine-based anticancer agents.

The DNA binding energetics of a series of analogues derived from the anticancer agent N-[2-(dimethylamino)ethyl]-9-aminoacridine-4-carboxamide (AAC) are investigated. The effects of substituent modification at the C5 position of the acridine chromophore on the interaction of AAC with DNA are determined using spectrophotometry and isothermal titration calorimetry (ITC). The binding affinity and binding free energy associated with the interaction of AAC with DNA are significantly enhanced upon substitution at the C5 position. Energetic profiles describing ligand-DNA complex formation obtained from ITC indicate that C5 substitution significantly enhances binding enthalpy relative to the parent AAC. In many cases, the enhanced binding enthalpies of the C5-substituted analogues correlate with anticancer activity. Because of the cationic character of AAC and its analogues, the DNA binding properties of these compounds are dependent on ionic strength. To quantitate the ionic contributions to complex formation, the observed binding free energy of each compound is parsed into its polyelectrolyte and nonelectrostatic components. Enhanced nonelectrostatic contributions to the overall binding free energies observed with C5-substituted analogues relative to the parent AAC suggest that C5 substituents play a critical role in directing both thermodynamic mechanisms associated with complex formation and molecular interactions between the ligand and its DNA binding site. These studies have demonstrated that substitution of AAC at the C5 position results in enhanced DNA binding affinity and energetics.

Acridines↗