Levels Of DNA Modifications Research Articles

Dynamic changes in genomic 5-hydroxymethyluracil and N6-methyladenine levels in the Drosophila melanogaster life cycle and in response to different temperature conditions

In this study, the level of DNA modifications was investigated in three developmental stages of Drosophila melanogaster (larvae, pupae, imago) and in an in vitro model (Schneider 2 cells). Analysis was carried out using two-dimensional ultra-performance liquid chromatography with tandem mass spectrometry. Our method made it possible, for the first time, to analyze a broad spectrum of DNA modifications in the three stages of Drosophila. Each stage was characterized by a specific modification pattern, and the levels of these compounds fluctuated throughout the D. melanogaster life cycle. The level of DNA modification was also compared between insects bred at 25 °C (optimal temperature) and at 18 °C, and the groups differed significantly. The profound changes in N6-methyladenine and 5-hydroxymethyluracil levels during the Drosophila life cycle and as a result of breeding temperature changes indicate that these DNA modifications can play important regulatory roles in response to environmental changes and/or biological conditions. Moreover, the supplementation of Schneider 2 cells with 1 mM L-ascorbic acid caused a time-dependent increase in the level of 5-(hydroxymethyl)-2′-deoxyuridine. These data suggest that a certain pool of this compound may arise from the enzymatic activity of the dTET protein.

Distinct and stage-specific contributions of TET1 and TET2 to stepwise cytosine oxidation in the transition from naive to primed pluripotency

Cytosine DNA bases can be methylated by DNA methyltransferases and subsequently oxidized by TET proteins. The resulting 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxylcytosine (5caC) are considered demethylation intermediates as well as stable epigenetic marks. To dissect the contributions of these cytosine modifying enzymes, we generated combinations of Tet knockout (KO) embryonic stem cells (ESCs) and systematically measured protein and DNA modification levels at the transition from naive to primed pluripotency. Whereas the increase of genomic 5-methylcytosine (5mC) levels during exit from pluripotency correlated with an upregulation of the de novo DNA methyltransferases DNMT3A and DNMT3B, the subsequent oxidation steps turned out to be far more complex. The strong increase of oxidized cytosine bases (5hmC, 5fC, and 5caC) was accompanied by a drop in TET2 levels, yet the analysis of KO cells suggested that TET2 is responsible for most 5fC formation. The comparison of modified cytosine and enzyme levels in Tet KO cells revealed distinct and differentiation-dependent contributions of TET1 and TET2 to 5hmC and 5fC formation arguing against a processive mechanism of 5mC oxidation. The apparent independent steps of 5hmC and 5fC formation suggest yet to be identified mechanisms regulating TET activity that may constitute another layer of epigenetic regulation.

Epigenetic Modulators of Monocytic Function: Implication for Steady State and Disease in the CNS.

Epigenetic alterations are necessary for the establishment of functional and phenotypic diversity in the populations of immune cells of the monocytic lineage. The epigenetic status of individual genes at different time points defines their transcriptional responses throughout development and in response to environmental stimuli. Epigenetic states are defined at the level of DNA modifications, chromatin modifications, as well as at the level of RNA base changes through RNA editing. Drawing from lessons regarding the epigenome and epitranscriptome of cells of the monocytic lineage in the periphery, and from recently published RNAseq data deriving from brain-resident monocytes, we discuss the impact of modulation of these epigenetic states and how they affect processes important for the development of a healthy brain, as well as mechanisms of neurodegenerative disease and aging. An understanding of the varied brain responses and pathologies in light of these novel gene regulatory systems in monocytes will lead to important new insights in the understanding of the aging process and the treatment and diagnosis of neurodegenerative disease.

Epigenetic regulation of human adipose-derived stem cells differentiation.

Adult stem cells have more restricted differentiation potential than embryonic stem cells (ESCs), but upon appropriate stimulation can differentiate into cells of different germ layers. Epigenetic factors, including DNA modifications, take a significant part in regulation of pluripotency and differentiation of ESCs. Less is known about the epigenetic regulation of these processes in adult stem cells. Gene expression profile and location of DNA modifications in adipose-derived stem cells (ADSCs) and their osteogenically differentiated lineages were analyzed using Agilent microarrays. Methylation-specific PCR and restriction-based quantitative PCR were applied for 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) detection in selected loci. The level of DNA modifications in the POU5F1 locus was quantified with deep sequencing. Expression levels of selected genes were assayed by real-time PCR. ADSCs differentiation into osteogenic lineages involved marked changes in both 5mC and 5hmC profiles, but 5hmC changes were more abundant. 5mC losses and 5hmC gains were the main events observed during ADSCs differentiation, and were accompanied by increased expression of TET1 (P = 0.009). In ADSCs, POU5F1 was better expressed than NANOG or SOX2 (P ≤ 0.001). Both 5mC and 5hmC marks were present in the POU5F1 locus, but only hydroxymethylation of specific cytosine showed significant effect on the gene expression. In summary, the data of our study suggest significant involvement of changes in 5hmC profile during the differentiation of human adult stem cells.

Alkylation and Cleavage of a DNA Fragment with Trioligonucleotide Reagent

AbstractFor Abstract see ChemInform Abstract in Full Text.

The amazing complexity of transcription factories

Multicellular eukaryotes orchestrate immensely complex patterns of gene expression in order to define numerous specialised cell types. Patterns of expression are regulated at the level of DNA modification, chromatin structure, genome architecture and nuclear organisation. Each aspect of these features of regulation is controlled by complex nuclear systems, and each of the systems must interact to define a coherent regulatory network. In proliferating cells, and particularly during development, patterns of gene expression must be preserved in cells that have active DNA repair mechanisms and efficiently duplicate both DNA and the associated chromatin status--the histone code. The systems that regulate DNA synthesis and genome stability are integrated with those that regulate the efficacy of RNA synthesis, such that patterns of gene expression are maintained. This review analyses how different experimental approaches are revealing the amazing complexity of the transcriptionally active compartment of mammalian cells. The functional implications of this complexity will be discussed.

Alkylation and Cleavage of a DNA Fragment with Trioligonucleotide Reagent

A possibility of site‐directed chemical modification of a ssDNA fragment with “trioligonucleotide reagent” (TOR), consisting of a central oligonucleotide derivative carrying N‐(2‐chloroethyl)‐N‐(p‐formylphenyl)‐N‐propyl‐N‐3‐ydeneamino groups at both 5′‐ and 3′‐thiophosphate ends and two border derivatives with 4‐carbohydrazidephenyl groups at their 3′‐ and 5′‐phosphate ends, respectively, is shown. Products of site‐directed fragment cleavage, more abundant than the alkylation products, were found at 50°C. The overall level of DNA modification by TOR reached 30% at a small excess of the oligonucleotide derivatives.

The level of DNA modification by (+)-syn-(11S,12R,13S,14R)- and (-)-anti-(11R,12S,13S,14R)-dihydrodiol epoxides of dibenzo[a,l]pyrene determined the effect on the proteins p53 and p21WAF1 in the human mammary carcinoma cell line MCF-7.

The polycyclic aromatic hydrocarbon (PAH) dibenzo[a,l]pyrene (DB[a,l]P), the most carcinogenic PAH tested in rodent bioassays, exerts its pathobiological activity via metabolic formation of electrophilically reactive DNA-binding fjord region (+)-syn-(11S,12R,13S,14R)- or (-)-anti-(11R,12S,13S,14R)-DB[a,l]P-dihydrodiol epoxides (DB[a,l]-PDEs). DB[a,l]P is metabolized to these DB[a,l]PDEs which bind to DNA in human mammary carcinoma MCF-7 cells. The molecular response of MCF-7 cells to DNA damage caused by DB[a,l]PDEs was investigated by analyzing effects on the expression of the tumor suppressor protein p53 and one of its target gene products, the cyclin-dependent kinase inhibitor p21WAF1. Treatment of MCF-7 cells with (+)-syn- and (-)-anti-DB[a,l]PDE at a concentration range of 0.001-0.1 microM resulted in DB[a,l]PDE-DNA adduct levels between 2 and 30, and 3 and 80 pmol/mg DNA, respectively, 8 h after exposure. (-)-anti-DB[a,l]PDE exhibited a higher binding efficiency that correlated with a significantly stronger p53 response at low concentrations of the dihydrodiol epoxides. The level of p53 increased by 6-8 h after treatment. The p21WAF1 protein amount exceeded control levels by 12 h and remained elevated for 96 h. At a dose of 0.01 microM (+)-syn-DB[a,l]PDE, an increase in p21WAF1 was observed in the absence of a detectable change in p53 levels. The results indicate that the increase in p53 induced by DB[a,l]PDEs in MCF-7 cells requires an adduct level of approximately 15 pmot/mg DNA and suggest that the level of adducts rather than the specific structure of the DB[a,l]PDE-DNA adduct formed triggers the p53 response. The PAH-DNA adduct level formed may determine whether p53 and p21VAF1 pathways respond, resulting in cell-cycle arrest, or fail to respond and increase the risk of mutation induction by these DNA lesions.

A 32P-postlabeling method for the detection of adducts in the DNA of human fibroblasts exposed to sulfur mustard

Since the toxicities of sulfur mustard are attributed to DNA alkylation, levels of DNA modification in exposed cells should correlate with the intensity of exposure. We have found that 32P-postlabeling can be used successfully to detect the major adduct, 7-hydroxyethylthioethyldeoxyguanosine 5'-phosphate (HETEpdG), that is formed in DNA by sulfur mustard. This method has been used to establish a correlation between exposure and adduct formation in human fibroblasts grown in cell culture and exposed to sulfur mustard concentrations between 2.5 and 15 μM. DNA was recovered from these cells using a salt precipitation method to remove proteins and was found to have an HETEpdG content which increased linearly with SM concentration. This relationship shows that one HETEpdG per 10 6 nucleotides is produced at a SM concentration of 2.3 μM. Growth of fibroblast cells, assayed by trypan-blue exclusion, is somewhat inhibited by 2 μM SM, indicating that 32P-postlabeling has the requisite sensitivity to detect adducts at levels of SM that are minimally toxic.

Enhancement of benzo[a]pyrene diol epoxide mutagenicity by sulfite in a mammalian test system.

Sulfur dioxide, a ubiquitous air pollutant, is a co-carcinogen for benzo[a]pyrene (BP). We have demonstrated previously that the interaction between sulfite, the physiological form of sulfur dioxide, and (+/-) -7r,8t-dihydroxy-9t,10t-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (anti-BPDE), the ultimate carcinogenic form of BP, results in an enhanced mutagenic effect in Salmonella typhimurium strains TA98 and TA100. We report here that this same co-mutagenic effect of sulfite occurs in a mammalian cell line. Treatment of Chinese hamster V79 cells with 50 nM anti-BPDE, a concentration on the linear portion of the dose-response, resulted in a four-fold increase in mutations at the hprt locus relative to the spontaneous rate. When V79 cells were exposed to 1 or 10 mM sulfite immediately prior to the addition of anti-BPDE, the mutation rate increased by 73% and 210%, respectively, over that elicited by anti-BPDE alone. Sulfite itself was moderately cytotoxic, but caused no increase in mutation over the spontaneous rate. Characterization of the dose- and time-dependance of this enhancement of diol epoxide mutagenicity by sulfite closely resembled the effects seen previously in the bacterial system. In particular, enhancement by sulfite was evident when sulfite was added to the cells between 60 min and 1 min prior to the addition of the diol epoxide. Concurrent addition of sulfite and the diol epoxide attenuated the enhancement, and the effect was lost altogether when sulfite was added 10 min after the diol epoxide. The specificity of this effect of sulfite was shown by comparison with sulfate, which at concentrations of either 1 or 10 mM exhibited modest cytotoxicity, but neither was directly mutagenic nor able to enhance the mutagenic effect of anti-BPDE. Binding studies with labeled anti-BPDE showed that the addition of 10 mM sulfite increased binding of anti-BPDE to DNA by over 43%, corresponding to the observed increase in mutant frequency. Interestingly, this difference in level of DNA modification was not apparent after 30 min to 2 h exposures, but only emerged at the 4 h time point. The 4 h point was routinely used for all mutagenicity studies. Binding of anti-BPDE-derived materials to cellular RNA was not altered by 10 mM sulfite. The emergence of increased DNA modification at the latest time point suggests either a more prolonged period of active DNA binding than would occur with diol epoxide, or a difference in the ability to recognize and clear specific DNA adducts. Both possibilities are discussed in regard to the observed formation of 7r,8t,9t-trihydroxy-7,8,9,10-tetrahydrobenzo[a] pyrene-10c-sulfonate (BPT-10-sulfonate) in those incubations. BPT-10-sulfonate is a relatively stable BP derivative which retains the ability to covalently modify DNA. The role of this derivative in the enhancement of diol epoxide mutagenicity by sulfite is strongly suggested by these data.

A new approach to enhancing the efficiency and specificity of interaction in duplexes by the use of tandem structure

Abstract

Benzo[c]phenanthrene-DNA adducts in mouse epidermis in relation to the tumorigenicities of four configurationally isomeric 3,4-dihydrodiol 1,2-epoxides.

P-Postlabeling assays were used to monitor the binding to epidermal DNA that resulted from the application of each of the four configurational isomers of benzo[c]phenanthrene 3,4-dihydrodiol 1,2-epoxide to mouse skin in vivo. For three of these configurational isomers, there was a reasonable correlation between the relative level of binding to epidermal DNA and the known tumorigenic effects of these compounds. However, for the 4(S),3(R)-dihydrodiol 2(S),1(R)-epoxide, the tumorigenic response was considerably greater in relation to the level of DNA modification than was the case for the other isomers. This greater tumorigenic response was consistent with previous observations indicating that this isomer was more mutagenic, at equivalent levels of DNA modification, than the other two tumorigenic dihydrodiol epoxides. Additionally, the 4(S),3(R)-dihydrodiol 2(S),1(R)-epoxide reacts with DNA to generate predominantly (approximately 80%) adducts on the amino group of adenine residues. These findings might imply a greater intrinsic biological effect of such adenine adducts with respect to the other major adduct formed on the amino group of guanine residues.

32P-postlabelling analysis of adducts formed by mitoxantrone and ametantrone with DNA and homopolydeoxyribonucleotides after enzymatic activation

DNA adduct formation by enzymatically activated mitoxantrone and ametantrone has been studied by 32P-postlabelling method. Both drugs were activated by peroxidase/hydrogen peroxide system and formed several DNA adducts when reacted with calf thymus DNA. Mitoxantrone gave 3 and ametantrone 4 different DNA adducts with apparently similar Chromatographic features suggesting that DNA adducts formed by both compounds do not differ significantly in their chemical structure. Despite this similarity, the level of DNA modification is 10 times higher for mitoxantrone compared to ametantrone. We did not observe DNA adducts in control samples where both drugs were incubated with DNA in the absence of the activating system. It indicates the importance of oxidative activation of mitoxantrone and ametantrone for their ability to bind covalently DNA. In order to identify nucleobases involved in the formation of DNA adducts by anthracenediones, polydeoxyadenosine, polydeoxythymidine, deoxyguanosine 3′-monophosphate and deoxycytosine 3′-monophosphate were modified by mitoxantrone and ametantrone activated in the above mentioned oxidating system. We proved that the only nucleobase modified by both drugs is guanine with no alkylation observed at other DNA bases. The pattern of adducts formed with deoxyguanosine 3′-monophosphate is reminiscent of that obtained with calf thymus DNA. In addition, mitoxantrone was found to be phosphorylated during the postlabelling procedure, most probably at the 1,4-hydroxyl groups of the chromophore. Ametantrone which does not possess hydroxyl groups attached to the chromophore core was resistant to phosphorylation by T4 polynucleotide kinase and γ-[ 32P]ATP. These results for the first time provide direct evidence that mitoxantrone and ametantrone form DNA adducts when activated by oxidation in vitro.

Unusual chromosome structure of fission yeast DNA in mouse cells.

Chromosomes from the fission yeast Schizosaccharomyces pombe have been introduced into mouse cells by protoplast fusion. In most cell lines the yeast DNA integrates into a single site within a mouse chromosome and results in striking chromosome morphology at metaphase. Both light and electron microscopy show that the yeast chromosome region is narrower than the flanking mouse DNA. Regions of the yeast insert stain less intensely with propidium iodide than surrounding DNA and bear a morphological resemblance to fragile sites. We investigate the composition of the yeast transgenomes and the modification and chromatin structure of this yeast DNA in mouse cells. We suggest that the underlying basis for the structure we see lies above the level of DNA modification and nucleosome assembly, and may reflect the attachment of the yeast DNA to the rodent cell nucleoskeleton. The yeast integrant replicates late in S phase at a time when G bands of the mouse chromosomes are being replicated, and participates in sister chromatid exchanges at a high frequency. We discuss the implications of these studies to the understanding of how chromatin folding relates to metaphase chromosome morphology and how large stretches of foreign DNA behave when introduced into mammalian cells.

Detection of the major DNA adducts of benzo[j]fluoranthene in mouse skin: nonclassical dihydrodiol epoxides.

Studies have demonstrated that the metabolic activation of benzo[j]fluoranthene (B[j]F) to a genotoxic agent proceeds through the formation of either the trans-4,5-dihydrodiol (B[j]F-4,5-diol) or the trans-9,10-dihydrodiol (B[j]F-9,10-diol) metabolite of B[j]F. Using 32P-postlabeling analysis, the profiles of DNA adducts formed in vivo in mouse skin from B[j]F-4,5-diol and B[j]F-9,10-diol were obtained to establish the contribution of each of these dihydrodiols to the formation of B[j]F-DNA adducts in vivo. B[j]F, B[j]F-4,5-diol and B[j]F-9,10-diol were applied to the shaved backs of mice (100 micrograms/mouse), and DNA adducts were isolated and separated using multidimensional TLC and reverse-phase HPLC. The greatest level of adducts was observed with B[j]F-4,5-diol, which resulted in the formation of 383 pmol of DNA adducts/mg of DNA. This level of DNA modification was more than 2 orders of magnitude greater than that observed with B[j]F or B[j]F-9,10-diol. The major adducts detected from the application of B[j]F-4,5-diol to mouse skin in vivo had chromatographic properties similar to those of the major adducts detected with B[j]F. In contrast, the major DNA adducts detected with B[j]F-9,10-diol had chromatographic properties distinctly different than the adducts formed from either B[j]F or B[j]F-4,5-diol. DNA adducts formed from the syn and anti isomers of the 4,5-dihydrodiol 6,6a-epoxide and the 9,10-dihydrodiol 11,12-epoxide of B[j]F were also evaluated. Each dihydrodiol epoxide derivative was reacted with calf thymus DNA in vitro and applied to mouse skin in vivo.(ABSTRACT TRUNCATED AT 250 WORDS)

DNA adducts in model systems and humans.

The etiology of chemically induced cancer is thought to involve the covalent binding of carcinogens to DNA (adducts) leading to mutations in oncogenes or tumor suppressor genes, and ultimately to tumors. Thus, the DNA-carcinogen adduct has been used as a measurable biochemical endpoint in laboratory studies designed to assess carcinogen exposure, carcinogen metabolism, mutagenesis, and tumorigenesis. Unfortunately, the significance of adducts in the etiology of human cancer is still unclear. This is partially due to the difficulty detecting adducts at carcinogen exposures relevant to humans, which are often orders of magnitude lower than animal model exposures. The relationship between adducts and higher biological effects is also not known at low doses. We have been assessing the DNA damage caused by exposure to heterocyclic amine carcinogens in the diet. Using the technique of 32P-postlabeling in combination with accelerator mass spectrometry, we have determined that DNA adduction in rodents decreases linearly with decreasing dose from the high doses used in typical cancer bioassays to the low doses relevant to human exposures. For a given tissue, adduct levels are correlated with dose, but the level of DNA modification by carcinogens is tissue-specific and does not completely correlate with tumor site. This lack of correlation may be due to differences in adduct formation and repair rates among tissues. Comparison of carcinogen metabolism routes between rodents and humans also indicates that species differences could influence the amount and type of damage resulting from exposure to these carcinogens. The use of model systems to study dosimetry, species differences in adduction, and role of adducts in mutation will ultimately lead to a better understanding of the significance of adducts in human disease. This should eventually allow the use of adducts as biomarkers for estimating carcinogen exposure and individual susceptibility.

Metabolism and binding of benzo[a]pyrene in randomly-proliferating, confluent and S-phase human skin fibroblasts.

The metabolism of benzo[a]pyrene in randomly proliferating and confluent cultures of human skin fibroblast cells was compared with cell cultures in early S phase of the cell cycle after a G1 block. When each cell population was exposed to [G-3H]benzo[a]pyrene for 24 hours and the organic soluble metabolites in the extracellular medium and intracellular components were analyzed by HPLC, a quantitative increase in metabolism was observed in the confluent cell populations. The amount of organic soluble metabolites in the extracellular medium of the confluent dense cultures was 2.7 times the amount found in randomly proliferating cultures and 1.5 times that of the synchronized cultures. The trans-7,8- and 9,10 dihydrodiols and 3-hydroxy benzo[a]pyrene were the major metabolites formed. Small amounts of the sulphate conjugate, 9-hydroxy-benzo[a]pyrene and the tetrols were also detected. Cytoplasmic as well as nuclear extracts from the confluent cell cultures also contained higher amounts of metabolites compared to those from the randomly proliferating and S-phase cells. The levels of DNA modification by metabolically activated benzo[a]pyrene did not differ among the randomly proliferating, confluent and S-phase cells. However, the S-phase cells exhibited approximately 50-fold increase in the frequency of transformation compared to the randomly proliferating cells. Confluent cells were not transformed by benzo[a]pyrene. These data suggest that factors other than random modification of DNA by the carcinogen might have a significant role in the expression of a transformed phenotype and that metabolism and transformation are not directly related. Furthermore, confluent dense cultures with a heightened capability for metabolism of benzo[a]pyrene were more active in the detoxification of benzo[a]pyrene than in the production of the metabolites associated with cellular transformation.

Nitropyrenes are inducers of polyoma viral DNA synthesis

The biological activity of a series of nitopyrenes was assayed by measuring their ability to induce the asynchronous replication of viral DNA in rat fibroblasts transformed by a st-a mutant of polyoma virus. Concentrations of 10–30 μg/ml of 1-nitropyrene (1-NP) induced viral replications, and this effect was enhanced by addition of rat-liver S9 microsomal fraction (300 μ/ml) to the culture medium. The response was less than that obtained with 0.1 μg/ml of the activated metabolite of benzo[ a]pyrene (BP), BP trans-7,8-dihydrodiol-9,10 epoxide (anti) (BPDE). A series of di-, tri-, and tetra-nitropyrenes were also found to induce polyoma DNA replicatin, in the absence of exogenous microsomal activation, displaying strongly positive effects at 0.5–2.0 μg/ml. Dose-response curves with 1,6-dinitropyrene (1,6-DNP) from 0.01 to 0.5 μg/ml inidcated that this compound was approximately equipotent with BPDE for induction of polyoma DNA synthesis. Studies of drug metabolism, DNA binding and DNA adduct formation indicate that 1,6-DNP is metabolized in this cell line, binds to DNA, and forms stable adducts. The level of DNA modifications seen with, 1,6-DNP is higher than that observed under comparable conditions with an equivalent dose of BPDE. These findings provide additional evidence that the nitropyrene class of compounds can exert biological effects in mammalian cells, and that the dinitropyrenes are more potent than 1-NP.

DNA methylation in mammalian nuclei.

A novel system to study the methylation of newly synthesized DNA in isolated nuclei was developed. Approximately 2.5% of cytosine residues incorporated into nascent DNA became methylated by endogenous methylase(s), and the level of DNA modification was reduced by methylation inhibitors. DNA synthesis and methylation were dependent on separate cytosol factors. The cytosol factor or factors required for DNA methylation were sensitive to trypsin digestion and were precipitable by (NH4)2SO4, suggesting that they were proteinaceous. Time-course experiments revealed a short lag of approximately 20 s between synthesis and methylation in nuclei. The DNAs produced in these nuclei were a mixed population of low molecular weight fragments and higher molecular weight fragments shown to be short extension of existing replicons. The methylation level found in low molecular weight DNA was lower than that found in bulk L1210 DNA, indicating that further methylation events might take place after ligation of small fragments. These data suggest that newly synthesized DNA is a good substrate for methylase enzymes and that nuclear cytoplasmic interactions may be important in controlling inheritance of methylation patterns.

Detection of DNA adducts in N-acetoxy-2-acetylaminofluorene-treated human fibroblasts by means of immunofluorescence microscopy and quantitative immunoautoradiography.

An immunohistochemical procedure was developed for the detection of adducts in DNA of cultured cells exposed to N-acetoxy-2-acetylaminofluorene (N-AcO-AAF) with the use of antibodies raised in rabbits against N-(guanosin-8-yl)-2-acetyl-aminofluorene (Guo-8-AAF) conjugated to bovine serum albumin. Binding of these antibodies to the nuclear DNA was visualized by means of immunofluorescence microscopy with fluorescein-conjugated anti-rabbit-Ig antibodies, or by means of immunoautoradiography with 125I-labeled protein A. The dose-response curve obtained when the number of autoradiographic grains developed over the nuclei was plotted as a function of the concentration of N-AcO-AAF used to treat the cells, indicated that the extent of specific antibody-binding is determined by the amount of adducts in the cells. DNA modification levels allowing for 20% survival of the cells could be detected with the immunofluorescence technique, while cells exposed to concentrations of N-AcO-AAF resulting in 60% survival were still positive with the immunoautoradiographic method.