Differential formation of O6-ethylguanine in the DNA of rat brain chromatin fibers of different folding levels exposed to N-ethyl-N-nitrosourea in vitro.

Extended (histone H1-depleted), 11-nm-thick chromatin fibers and condensed 25- to 35-nm-thick chromatin fibers, representing the first and second level of DNA folding in chromatin, respectively, as well as nucleosome core particles, were isolated from fetal rat brain cells and briefly exposed to N-ethyl-N-nitrosourea (EtNU) in vitro. The O6-ethyl-2'-deoxyguanosine (O6-EtdGuo):2'-deoxyguanosine (dGuo) molar ration in DNA enzymatically hydrolyzed to 2'-deoxynucleosides was determined by competitive radioimmunoassay using an anti-O6-EtdGuo monoclonal antibody with an affinity constant for O6-EtdGuo of approximately 2 X 10(10) liters/mol. In comparison to naked DNA (O6-EtdGuo:dGuo relative value, 1.0) the O6 atom of guanine proved to be increasingly protected from ethylation by EtNU in the DNA of the histone H1-depleted chromatin fibers (relative value, approximately 0.6) and of the condensed chromatin fibers (relative value, approximately 0.4). From the O6-EtdGuo:dGuo relative values obtained for the DNA of nucleosome core particles (approximately 0.5) and of H1-depleted chromatin fibers (approximately 0.6), it follows that the accessibility of the O6 atom of guanine to the electrophilic ethyldiazonium ion generated from EtNU in internucleosomal DNA (protein free) is about twice that found in nucleosomal DNA. The overall O6-EtdGuo:dGuo molar ratio in the DNA of H5 rat hepatoma cells exposed to EtNU in vitro was similar to that of the DNA of the condensed 25- to 35-nm chromatin fibers. Since the bulk of genomic DNA is organized in the form of these chromatin fibers, the overall degree of intercellular O6-EtdGuo formation appears to be mainly determined by this folding level of DNA, and not, or only to a low degree, by the DNA folding levels of higher-order chromatin structures such as those found in large loops or domains of chromatin fibers within the cell nucleus.

N-methyl-N-nitrosourea-induced mutations in human cells. Effects of the transcriptional activity of the target gene.

In this study we addressed the question as to whether the mutagenesis by methylating agents is affected by the transcriptional activity of the damaged gene. An Epstein-Barr virus (EBV)-derived shuttle vector system was developed where the genetic target for mutation analysis, the bacterial gpt gene, is under the control of an eukaryotic inducible promoter in plasmid pF1-EBV and lacks the eukaryotic promoter in plasmid pF2-EBV. Two human cell lines that episomically maintain these shuttle vectors were established. In clone 6NT cells, which contain pF1-EBV plasmid, the gpt gene is actively transcribed and the transcription rate is regulated by zinc ions. In clone 3 cells, which harbor pF2-EBV plasmid, the gpt gene is not transcribed. Following treatment of both cell lines with the potent alkylating carcinogen N-methyl-N-nitrosourea (MNU), G.C to A.T transitions were the major mutagenic event, consistent with the miscoding potential of O6-methylguanine. The mutations were predominantly generated in the non-transcribed DNA strand of the active gpt gene. The same strand-bias was observed when the gpt gene was transcriptionally inactive, indicating that MNU-induced strand-specific formation of mutations is not due to transcription. Our data identify as major determinants of this phenomenon the sequence-specificity of MNU mutagenesis and the conformational properties of the target protein. Differences in mutation distribution were observed between the transcriptionally active and inactive gpt gene. This finding suggests that the organization of active genes in chromatin might modulate DNA alkylation and/or DNA repair.

Neutrophils cause oxidative DNA damage in alveolar epithelial cells.

Inflammation has been recognized as a contributing factor in the pathogenesis of some cancers. In the lung, inflammation is characterized by an influx of polymorphonuclear leukocytes (PMN) that release a variety of reactive oxygen species (ROS). The aim of the present study was to investigate the direct effect of PMN on oxidative DNA damage in lung target cells. Therefore, rat alveolar epithelial cells (RLE) were coincubated with PMN or hydrogen peroxide. Known to be correlated with the incidence of cancer, 7-hydro-8-oxo-2'deoxyguanosine (8-oxodG) was used as an effect marker for oxidative damage. Viability of the RLE, when coincubated with PMN, decreased to 43%, dependent on the ratio between PMN and RLE. After washing off PMN, 8-oxodG levels were significantly increased in RLE, but the highest levels were observed in the washed off PMN fraction. In addition, to avoid washing off procedures, immunohistochemical analysis was used to measure the 8-oxodG levels specifically in the RLE and similar results were obtained. In addition, inhibitor experiments showed that antioxidants ameliorated oxidative DNA damage. Our data provide evidence that ROS released by PMN as well as H2O2, cause oxidative DNA damage in epithelial cells.

Human endothelial culture supernatant selectively promotes IgM-producing hybridomas.

We compared the effect of human endothelial cell culture supernatant (HECS), ESG (Ewing sarcoma growth factor), IL-6 (interleukin-6) and peritoneal macrophages on the recovery of hybridoma cells after fusion with respect to growth, stability and distribution of isotype variants. A selective growth of murine IgM-producing hybridoma cells was observed in the presence of HECS after cell fusion.

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)

Formation and persistence of 8-oxoguanine in rat lung cells as an important determinant for tumor formation following particle exposure.

Exposure of rats to quartz (or various other particles) can lead to the development of lung tumors. At the moment, the mechanisms involved in particle-induced tumor formation are not clarified. However, it is suggested that inflammation, in conjunction with the production of reactive oxygen species (ROS) and an enhancement of epithelial cell proliferation, may play a key role in the development of lung tumors. ROS induces 8-oxoguanine (8-oxoGua) and other mutagenic DNA oxidation products, which can be converted to mutations in proliferating cells. Mutation formation in cancer-related genes is a critical event with respect to tumor formation. In this study we investigated the effects of quartz (DQ12) and of the nontumorigenic dust corundum on the induction of 8-oxoGua in the DNA of rat lung cells, as well as on cell proliferation and pulmonary inflammation. Wistar rats were exposed by intratracheal instillation to quartz (2.5 mg/rat) or corundum (2.5 mg/rat) suspended in physiological saline; control animals exposed to physiological saline or left untreated. Measurements were carried out 7, 21, and 90 days after the exposures. 8-oxoGua levels were determined in lung tissue sections at the single cell level by immunocytological assay using a rabbit anti-8-oxoGua antibody. After exposure to quartz, 8-oxoGua levels were significantly increased at all time points of investigation. Additionally, we observed inflammation and an enhanced cell proliferation. Exposure to corundum had no adverse effects on the lung; neither increased 8-oxoGua levels nor enhanced cell proliferation or inflammation were detected. These observations support the suggestion that inflammation associated with increased 8-oxoGua levels in lung cells and increased cell proliferation is an important determinant for particle-induced development of lung tumors in the rat.

Immuno-slot-blot: a highly sensitive immunoassay for the quantitation of carcinogen-modified nucleosides in DNA.

We have established a highly sensitive immuno-slot-blot (ISB) procedure that can be routinely applied for detection and quantitation of any heat- or alkali-stable structural DNA modification (caused by carcinogens or mutagens, for example) for which a specific (monoclonal) antibody (MAB) is available. The essential step in this assay is the immobilization on nitrocellulose filters of the structurally modified DNA in its single-stranded form. The immobilized DNA is first reacted with an MAB specifically directed against a particular modified DNA component (e.g., an alkyldeoxynucleoside), and thereafter with a second antibody directed against the first one. The second antibody can be either labeled with 125I or linked to an enzyme complex capable of eliciting a color reaction with a suitable substrate. The sensitivity of the ISB is demonstrated for two different alkyldeoxynucleosides, O6-ethyldeoxyguanosine (O6-EtdGuo) and O4-ethyldeoxythymidine (O4-EtdThd), both of which are produced in cellular DNA exposed to the alkylating N-nitroso carcinogen N-ethyl-N-nitrosourea and both of which represent DNA lesions miscoding during DNA replication and transcription. Using anti-(O6-EtdGuo) and anti-(O4-EtdThd) MABs, respectively, O6-EtdGuo and O4-EtdThd are detected at levels as low as greater than or equal to 0.3 X 10(-15) mol O6-EtdGuo/3 micrograms DNA (O6-EtdGuo/deoxyguanosine molar ratio in DNA, greater than or equal to 2 X 10(-7) ) and greater than or equal to 0.1 X 10(-15) mol of O4-EtdThd/3 micrograms DNA (O4-EtdThd/deoxythymidine molar ratio in DNA, greater than or equal to 4 X 10(-8) ).

Increased cytotoxicity of 1-(2-chloroethyl)-1-nitroso-3(4-methyl)-cyclohexylurea by pretreatment with O6-methylguanine in resistant but not in sensitive human melanoma cells.

Cells from a resistant ("Gr II") and a sensitive ("Str") human melanoma xenograft were incubated in vitro with O6-methylguanine for 2 h, subsequently treated with 1-(2-chloroethyl)-1-nitroso-3(4-methyl)-cyclohexylurea (MeCCNU) for 1 h and then plated in soft agar. In the resistant cells the O6-methylguanine pretreatment (2 mM) yielded an increase in sensitivity towards MeCCNU by a factor of 7.5. In the sensitive melanoma cells pretreatment with O6-methylguanine did not increase cytotoxicity. Human bone marrow cells from three normal donors were similarly pretreated with O6-methylguanine and MeCCNU. There was a clear increase in MeCCNU-induced cytotoxicity. We conclude, that while O6-methylguanine does potentiate the action of MeCCNU in resistant melanoma cells, the therapeutic usefulness of this treatment strategy may be limited.

Removal of O6-methylguanine from plant DNA in vivo is accelerated under conditions of clastogenic adaptation.

Previously it was shown that the clastogenic efficiency of high doses of alkylating agents in plant root meristems can be reduced significantly by conditioning pretreatment with either a low dose of the same agents, a sublethal heat shock, or heavy metal salts. The molecular mechanisms responsible for these protective effects are still unclear. Here we report on the quantification of O6-methylguanine [O6-MeG] by immuno-slot-blot analysis in DNA of root tip meristems of field bean (Vicia faba) seedlings under conditions of clastogenic adaptation. When root tips were pretreated with a low, conditioning dose of N-methyl-N-nitrosourea (MNU, 10(-4) M, 1 hour) 2 hours before exposure to a high dose of the same clastogen (10(-3) M, 1 hour), the frequency of chromatid aberrations was reduced by more than 50% at a recovery time of 1 B hours, as compared to treatment with the high dose alone. The same was observed when conditioning pretreatment was by a sublethal heat shock [10 minutes, 40 degrees C] or a heavy metal salt (Cd(NO)3, 10(-7) M, 1 hour). The frequency of O6-MeG immediately after exposure to a conditioning and a subsequent challenge treatment was reduced by 43% as compared to treatment with only the high dose. At a recovery time of 18 hours the corresponding frequency of adducts was reduced by 68.3% (related to the initial level) after treatment with the high dose alone, and by 81.3% under adaptive conditions. Sublethal heat shock or heavy metal salt used as conditioning pretreatments also resulted in a decrease of adducts immediately after treatment with the challenge dose. From these data and from prevention of the effects by pretreatment with cycloheximide or O6-benzylguanine we conclude that under conditions of clastogenic adaptation O6-MeG is more efficiently removed from the DNA, presumably by induction of an alkyl acceptor protein such as O6-methylguanine-DNA methyltransferase [MGMT]. This could explain the observed protective effects (clastogenic adaptation.

Formation and repair of O6-methylguanine in recombination hot spots of plant chromosomes.

Mutagen-induced chromatid aberrations are not randomly distributed along the metaphase chromosomes. In the field bean (Vicia faba), defined late-replicating and transcriptionally inactive heterochromatic regions are preferentially involved. After exposure to the alkylating agent N-methyl-N-nitrosourea (MNU) (10(-3) M, 1 hour), 70% of all aberrations are clustered within 6 segments containing tandemly repeated FokI elements of 59 bp, which comprise approximately 10% of the genome. Using immuno-slot-blot analyses, we have studied the frequency of O6-methylguanine (O6-MeG), a mutagenic lesion important for aberration induction, in total genomic DNA as well as in FokI sequences of the field bean after exposure to MNU. In either case, similar numbers of adducts per nucleotide were found immediately after treatment as well as after 18 hours of recovery, when most adducts were removed and significant amounts of chromatid aberrations were detectable. Peculiarities of long FokI element arrays (e.g., formation of specific tertiary structures), resulting in error-prone recombination repair, rather than preferential formation or delayed repair of O6-MeG are apparently responsible for aberration clustering in these hot spot regions.

Ethylation of nucleophilic sites in DNA by N-ethyl-N-nitrosourea depends on chromatin structure and ionic strength.

Depending on ionic strength, chromatin can assume either a condensed, supranucleosomal conformation or the form of an extended nucleosomal fiber. Using sedimentation velocity analysis, both types of structures could be identified in chromatin prepared from cell nuclei of fetal rat brain. When the ionic strength was reduced from 60 to 10 mM NaCl, the average S-value of a defined chromatin fiber fraction (12-15 nucleosomes in size) decreased dramatically from approximately 72 S to approximately 55 S, reflecting the unfolding of condensed chromatin to an extended conformation. Correspondingly, the average S-value of histone H1-depleted chromatin (Ch-) was 54 S at 60 mM NaCl and did not change significantly at lower NaCl concentrations. Ch- contains only the core histones and is, therefore, relaxed into an extended form. Using a monoclonal antibody (ER-6) specific for O6-ethyldeoxyguanosine, we studied the influence of chromatin conformation on the formation of O6-ethylguanine (O6-EtGua) in the DNA of chromatin exposed to the carcinogen N-ethyl-N-nitrosourea (EtNU; 1 mg/ml, 37 degrees C, 20 min) in vitro. When the NaCl concentration during incubations with EtNU was varied between 0 and 100 mM, the amount of O6-EtGua formed in the DNA of complete chromatin (Ch+) was highest at 0 mM NaCl, then decreased exponentially with increasing ionic strength, and remained approximately constant at values greater than or equal to 50 mM NaCl. A similar dependence on ionic strength was found for the formation of O6-EtGua in the DNA of Ch-, and in native DNA. The frequency of O6-EtGua was highest in native DNA, followed by the DNA of Ch-, and lowest in the DNA of Ch+. At each salt concentration, the O6-EtGua content of Ch+ DNA relative to the corresponding values for Ch- DNA and native DNA, remained unchanged (0.70 +/- 0.03 S.D. and 0.42 +/- 0.03 S.D., respectively). In addition to O6-EtGua, the formation of 7-ethylguanine (7-EtGua; major groove of the DNA double helix) and 3-ethyladenine (3-EtAde; minor groove) was analysed after exposure to [1-14C]EtNU. 7-EtGua was the most frequently formed ethylation product, followed by O6-EtGua and 3-EtAde.(ABSTRACT TRUNCATED AT 400 WORDS)

Distribution of O6-ethylguanine in DNA exposed to ethylnitrosourea in vitro as visualized by electron microscopy using a monoclonal antibody.

Application of a monoclonal antibody (Mab ER-6; Rajewsky et al., 1980) specific for O6-ethyl-2'-deoxyguanosine (O6-EtdGuo), in conjunction with a protein-free spreading procedure for double-stranded DNA molecules and transmission electron microscopy, permits the visualization of antibody molecules complexed to O6-EtdGuo residues formed in DNA upon reaction with the carcinogen N-ethyl-N-nitrosourea (EtNU) (Nehls et al., 1984). To obtain information on the distribution of O6-EtdGuo in native DNA exposed to EtNU in vitro, samples of purified rat brain DNA were briefly incubated with EtNU at concentrations differing by a factor of 8 (0.5 and 4 mg of EtNU/ml, respectively). As determined in DNA hydrolysates by competitive radioimmunoassay, the resulting DNA preparations contained O6-EtdGuo at O6-EtdGuo/2'-deoxyguanosine molar ratios of 15.1 X 10(-5) and 116 X 10(-5), respectively. Interspace distances between Mab-binding sites in both sets of ethylated DNA were determined by electron microscopy both in individual DNA fragments of different size, and in computer-generated, long-thread DNA constructs. Comparative statistical analyses by a newly developed MOLRANDO computer program show a non-random distribution pattern of Mab-binding O6-EtdGuo residues.

Monoclonal antibodies to thymidine glycol generated by different immunization techniques.

Monoclonal antibodies specific for thymidine glycol in oxidized DNA were generated by immunization with thymidine glycol monophosphate (TMP-OH) or thymidine glycol (T-OH), respectively, conjugated to keyhole limpet hemocyanin (KLH) or thyreoglobulin (TG). Forty-five clones (TMP-OH) and 70 clones (T-OH) were examined upon antibody production in ELISA. Four clones secreting IgG1, kappa, were characterized further. In several studies the antibodies derived from the immunization with TMP-OH were inhibited by various inhibitors. In descending order of effectiveness, they were thymidine glycol monophosphate (TMP-OH), thymidine glycol (T-OH), thymidine monophosphate (TMP), and thymidine (Thn). After immunization with T-OH, antibodies were inhibited in following order: T-OH > TMP-OH > TMP > Thn. Inhibition by the bases thymine, adenine, cytosine, and guanine were negligible. In ISB (Immuno Slot Blot) performed with OsO4-treated DNA (Poly-[dA-dT]) and amount of 70 fmol thymidine glycol was detectable. DNA had to be irradiated at a level of at least 20 Gy to detect any damage in ELISA but at a lower level of irradiation (10 Gy) in ISB by one of these antibodies, TPS-1. The antibodies obtained after immunization with hapten-protein are therefore capable of the detection of low frequency lesions in DNA generated by free radicals after radiation or oxidative stress.

Monoclonal antibody-based immunoassay for the determination of cellular enzymatic activity for repair of specific carcinogen-DNA adducts (O6-alkylguanine).

We describe a rapid and sensitive, monoclonal antibody (mAb)-based immunoassay that permits the quantitation of cellular enzymatic activity for repair of specific carcinogen-DNA adducts. The assay was established on the basis of the observation that complexes between an anti-(O6-ethyl-2'-deoxyguanosine) mAb (mAb ER-6) and O6-ethylguanine (O6-EtGua) residues in double-stranded DNA can be immobilized on nitrocellulose filters. Circular pSV2gpt plasmid DNA was linearized by digestion with EcoRI and reacted with N-ethyl-N-nitrosourea in vitro to a level of about one O6-EtGua residue per DNA molecule, labeled with 32P, and subsequently incubated with extracts prepared from L929 mouse fibroblasts and with mAb ER-6. The amount of repaired O6-EtGua is proportional to the decrease in 32P activity retained on the filters. Maximum O6-alkyl-guanine repair activity requires preparation of the cell extracts in the presence of high (approximately 0.5 M) NaCl concentrations, whereas the repair reaction per se is markedly inhibited by NaCl. Under the conditions used, the detection limit of the assay is approximately 1 x 10(-16) mol of repaired O6-alkylGua. The repair reaction followed second-order kinetics, indicating that O6-EtGua is predominantly repaired by an enzyme activity inactivated by the repair reaction (e.g. via an alkyl group transfer mechanism analogous to that exhibited by the alkyl group accepting cysteine residues of the 'suicidal' bacterial ogt and ada proteins). The data obtained from the assay can be used for computation of the relative enzymatic repair activity of a given cell extract and of the rate constant K (1 x mol-1 x s-1) of the repair reaction.

Involvement of histone H1 in the organization of the chromosome fiber.

At high ionic strength (e.g., physiological salt concentrations) chromosome fibers are 200 A in diameter and composed of discrete globular structures that are held together by histone H1. At low ionic strength the fibers unfold and appear as the familiar chains of nucleosomes (80 A in diameter). The unfolding of chromosome fibers occurs within a narrow salt range. It results from a change in the mode of the interaction between histone H1 and the chromosome fiber and is very likely the consequence of a change from cooperative binding between histone H1 and DNA to a noncooperative binding. In the noncooperative binding state histone H1 molecules are randomly redistributed along the chromosome fiber.

The chromosome fiber: evidence for an ordered superstructure of nucleosomes.

Chromosome fibers isolated from lymphocyte nuclei and prepared for electron microscopy by techniques designed to preserve their native structure have a distinctly knobby appearance, suggesting that DNA and protein are not distributed evenly along the fiber axis. Individual knobs (superbeads) are arranged in tandem and have an average diameter of about 200 A. Mild nuclease digestion of isolated nuclei releases apparent monomer superbeads that are composed of nucleohistone particles with the properties of nucleosomes. The kinetics of digestion indicate that the superbead is a discrete structural unit containing, on the average, about eight nucleosomes.

After X-irradiation a transient arrest of L929 cells in G2-phase coincides with a rapid elevation of the level of O6-alkylguanine-DNA alkyltransferase.

Following X-irradiation of exponentially growing L929 cells two major phenomena have been observed. First, there was a delay in cell division which can be ascribed to the arrest of cells in the G2-phase (G2-block), and, second, the cellular content of the O6-alkylguanine-DNA alkyltransferase (AGT) was markedly increased. Flow cytometrical DNA-measurements revealed that cells began to accumulate in the G2-phase 4 h after irradiation (p.r.) irrespective of the X-ray dose, while both the fraction of cells blocked in G2 and the time period the cells persisted in G2 increased with the radiation dose. About 24 h past release from the G2-block the distribution of cells in the cell cycle was similar to that of untreated control cells. In comparison with control cells the AGT content in irradiated cells (4 Gy) was highest at about 48 h p.r. (3.4-fold increase). The highest ratio of increase in AGT was, however, observed to occur between about 4 and 13 h p.r. (2.6-fold increase). As shown by flow cytometrical measurements using a BrdUrd/DNA double labeling technique, this rapid primary increase in AGT coincides very well with the entrance of cells into the G2-phase. This indicates that the cellular AGT content in X-irradiated (parental) cells started to exceed the basal level at the beginning of the G2-phase, but not before or during the S-phase. Once the AGT level was elevated it continued to increase for 2 to 3 cell doubling times.

Immunological methods for detection of carcinogen-DNA adducts.

Considerable advances have been made during recent years, with regard to the detection and quantification of carcinogen- or mutagen-induced, structural modifications in the DNA of mammalian cells, by the introduction of immunoanalytical methods, in particular in conjunction with monoclonal antibodies (Mab). Antibodies are characterized by an outstanding capacity for the specific recognition of subtle alterations of molecular structure. They can, therefore, be used as sensitive detection probes in assays for DNA modifications caused by low levels of DNA-reactive (e.g., environmental) agents. Depending on the purpose of analysis, various types of immunoassays can be performed. The competitive radioimmunoassay (RIA) represents a routinely applicable, reproducible and sensitive assay for the detection of defined carcinogen-DNA adducts in hydrolysates of cellular DNA, in body fluids or in urine. Depending on their particular design, enzyme immunoassay (EIA) may have exceptionally low detection limits, due to the enzymatic amplification of the measured radioactivity or colour intensity. Similarly, recently established immuno-slot-blot (ISB) techniques are also characterized by very high sensitivity. Immunocytological assays (ICA) use Mab in conjunction with electronically intensified immunofluorescence for detection of modified DNA components in the nuclei of individual cells. Finally, single modified deoxynucleosides can be detected and localized in individual DNA molecules by immuno-electron microscopy (IEM).