The 32P-postlabeling assay for DNA adducts

32P-postlabeling analysis is an ultrasensitive method for the detection and quantitation of carcinogen-DNA adducts. It consists of four principal steps: (i) enzymatic digestion of DNA to nucleoside 3'-monophosphates; (ii) enrichment of the adduct fraction of the DNA digest; (iii) 5'-labeling of the adducts by transfer of 32P-orthophosphate from [gamma-32P]ATP mediated by polynucleotide kinase (PNK); (iv) chromatographic or electrophoretic separation of the labeled adducts or modified nucleotides and quantitation by measurement of their radioactive decay. The assay requires only microgram quantities of DNA and is capable of detecting adducts at frequencies as low as 1 in 10(10) nt, making it applicable to the detection of events resulting from environmental exposures, or experiments using physiological concentrations of agents. It has a wide range of applications in human, animal and in vitro studies, and can be used for a wide variety of classes of compound and for the detection of adducts formed by complex mixtures. This protocol can be completed in 3 d.     

Reliability of bulky DNA adducts measurement by the nuclease P1 32P-post-labelling technique.

The aim was to assess the reliability of bulky DNA adducts measurement by means of the 32P-post-labelling assay. The research design consisted of an intramethod reliability study. Buffy coats from 41 subjects were used to obtain two aliquots of 1-5 microg DNA for each subject; bulky DNA adducts were measured using the nuclease P1 32P-post-labelling technique. The reliability of the measurement was assessed by means of the intraclass correlation coefficient (ICC), the distribution of the differences between the two measurements and the limits of agreement. The estimated ICC was 0.977, with a 95% confidence interval between 0.921 and 0.977. The limits of agreement were +/- 0.44 (DNA adducts per 10(8) nucleotides). Only three subjects had differences lying out of such limits. Bulky DNA adduct levels measured by the 32P-post-labelling technique showed good reliability. Only one measurement is needed to use DNA adducts as a biomarker of exposure and, possibly, cancer risk. Besides, as a validation analysis, 32P-post-labelling measurements can be repeated in only 20-30% of samples.     

Reliability of bulky DNA adducts measurement by the nuclease P1 32P-post-labelling technique

The aim was to assess the reliability of bulky DNA adducts measurement by means of the ³²P-post-labelling assay. The research design consisted of an intramethod reliability study. Buffy coats from 41 subjects were used to obtain two aliquots of 1–5 μg DNA for each subject; bulky DNA adducts were measured using the nuclease P1 ³²P-post-labelling technique. The reliability of the measurement was assessed by means of the intraclass correlation coefficient (ICC), the distribution of the differences between the two measurements and the limits of agreement. The estimated ICC was 0.977, with a 95% confidence interval between 0.921 and 0.977. The limits of agreement were ±0.44 (DNA adducts per 10⁸ nucleotides). Only three subjects had differences lying out of such limits. Bulky DNA adduct levels measured by the ³²P-post-labelling technique showed good reliability. Only one measurement is needed to use DNA adducts as a biomarker of exposure and, possibly, cancer risk. Besides, as a validation analysis, ³²P-post-labelling measurements can be repeated in only 20–30% of samples.     

Heterocyclic amine-DNA adducts analyzed by 32P-postlabeling method

DNA adducts formed by 12 heterocyclic amines were analyzed by 32P-postlabeling method. Several DNA adducts were detected in rat liver by administration of each heterocyclic amine. Total adduct levels ranged from 0.5 for 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) to more than 250 for 3-amino-1,4-dimethyl-5H-pyrido[4,3-b]indole (Trp-P-1) per 10(7) nucleotides 24 hr after intragastric administration of these compounds. The N-hydroxy derivative of 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) was reactive toward DNA in vitro to form adducts. Addition of acetic anhydride to N-OH-MeIQx greatly enhanced its reactivity to DNA. 32P-Postlabeling analysis revealed that the MeIQx-DNA adducts formed in vivo and in vitro were identical. Thus, MeIQx would be metabolized in vivo to N-hydroxy form and further esterified to produce more reactive species, such as N-acetoxy form, which modify DNA to form adducts.     

32P-postlabeling assay for the quantification of the major platinum-DNA adducts.

To allow more sensitive, selective, and routine analyses of platinum(Pt)-GG and -AG intrastrand cross-links we have significantly improved our quantitative (32)P-postlabeling assay (M. J. P. Welters et al. Carcinogenesis 18, 1767-1774, 1997). Instead of off-line scintillation counting we introduced an on-line flow radioisotope detector into the HPLC system. Furthermore, the isolation protocol for the adducts was significantly modified and optimized to reduce interfering background peaks that prevented quantification of low levels of the cisplatin-DNA adducts in white blood cells obtained from patients. Reduction of background signals was obtained by boiling the samples, followed by phenol/chloroform/isoamylethanol extraction after the DNA digestion step. The labeling efficiency for the adducts was increased by 40% by using Na-formate instead of NH(4)-formate for elution of the adducts from the strong cation-exchange columns. Finally, a calibration curve and quality controls were implemented. The labeling efficiencies were not different between the dinucleotides. The between- and within-run precision for the Pt-GG and Pt-AG adducts measured at the lower limit of quantification of 87 and 53 amol/microg DNA, respectively, was less than 20% CV. The adducts were stable in DNA stored for a 2-month time period at -80 degrees C. The assay is now routinely used for high-precision analyses of patient and cell line samples containing very low adduct levels.     

Caffeine enhancement of digestion of DNA by nuclease S1.

The activity of Aspergillus orzae nuclease S1 on DNA has been investigated under varying pH and metal ion conditions. Nuclease S1 was found to preferentially digest denatured DNA. With native DNA as substrate the enzyme could only digest the DNA when caffeine was added to the reaction mixture. The enzyme was more active in sodium acetate buffer (pH 4.5), than in either standard saline citrate (PH 7.0) or sodium phosphate buffer (pH 6.8). Caffeine was also found to affect the thermal stability of DNA, resulting in a melting profile characterized by two transitions. The first transition (poorly defined) was below the normal melting temperature of the DNA, while the next transition was at the normal melting temperature of the DNA, while the next transition was at the normal melting temperature of the DNA. The susceptibility of caffeine-treated DNA to nuclease digestion seems to be a result of the local unwinding that caffeine causes in the regions of DNA that melt in the first transition. This selective destabilization presumably sensitizes the unwound regions to nuclease hydrolysis. The hydrolysates of the DNA digested by nuclease S1 were subjected first to ion exchange chromatography followed by paper chromatography. The results from this partial characterization of the digestion products showed that they contain mononucleotides as well as oligonucleotides of varying lengths. The base composition of the mononucleotide digests suggests that caffeine has greater preference for interacting with A-T base-pairs in DNA.     

Analysis of tamoxifen-DNA adducts in endometrial explants by MS and 32P-postlabeling

The nonsteroidal antiestrogen tamoxifen increases the risk of endometrial cancer; however, the mechanism for the induction of these tumors is not known. Recently, Sharma et al. [Biochem. Biophys. Res. Commun. 307 (2003) 157], using high performance liquid chromatography (HPLC) with online postcolumn photochemical activation and fluorescence detection, reported the presence of (E)-alpha-(deoxyguanosin- N2-yl)tamoxifen in DNA from human endometrial explants incubated with tamoxifen. Inasmuch as the methodology used by these investigators does not allow unambiguous characterization of tamoxifen-DNA adducts, we have used two additional techniques (HPLC coupled with electrospray ionization tandem mass spectrometry and 32P-postlabeling analyses) to assay for the presence of tamoxifen-DNA adducts in the human endometrial explant DNA. Tamoxifen-DNA adducts were not detected by either method.     

Isolation of foldback DNA utilizing nuclease S1 digestion in aqueous dioxane.

An improved method for the isolation of the inverted repetitive (foldback) sequences present in mammalian DNAs is described. It makes use of the new observation that nuclease S1 digestion of denatured DNA occurred at a faster rate and was more extensive in medium containing dioxane. The temperature-absorbance characteristics of nuclease S1-resistant DNA were systematically studied as a function of the temperature employed during the step of enzymic hydrolysis. Specimens of human placental and calf thymus DNA which had been denatured and renatured to C₀t ≤ 10^?3 mol s liter^?1 were used as substrates. Foldback DNA was isolated from the enzymic digests by means of hydroxylapatite chromatography. Temperature-absorbance studies showed the enzyme-resistant DNA had a high degree of thermal stability; the hyperchromic rises equaled those obtained in the native speciments. The amount of foldback DNA which could be obtained was not influenced by the fragment size of the starting material, above a certain molecular weight range. Foldback DNA represented about 4% of the human genome and at least 5% of the bovine genome. The size distributions of these strands were studied by means of polyacrylamide gel electrophoresis.     

Flow cytometric evaluation of DNA digestion with S1 nuclease

The digestion of DNA with the S1 nuclease has been investigated by flow cytometry on two cultured cell lines and on splenic lymphocytes. The nucleolytic activity of the enzyme has been determined by measuring the decrease in fluorescence intensity of DNA stained with Propidium Iodide (PI). The reaction kinetics could be estimated through the shift of the G1 peak, since the shape of the DNA histograms obtained from cell submitted to S1 digestion did not show any particular variation as compared with the histograms of undigested control cells. DNA digestion was found to be more pronounced when cells were previously damaged with agents known to induce strand breaks, such as gamma-rays or Adriamycin. The assessment of DNA sensitivity to S1 nuclease with flow cytometry may thus provide a fast and reliable procedure for evaluating chromatin structural changes, both in physiological and pathological conditions.     

Statistical calculation of detection limits for DNA adducts using the 32P-postlabeling assay with a standard addition procedure

The 32P-postlabeling assay is widely used for the analysis of DNA adducts. Some adducts can be detected with very high sensitivity but quantification can be unreliable, particularly if it is based only on comparison with unmodified nucleotides (relative adduct labeling, RAL values). Furthermore, guidelines to calculate detection limits for adduct concentrations are lacking. This is particularly important for human biomonitoring studies of environmental exposures, where a low adduct level can remain undetected. Reports of null results of toxicity studies should always include a limit of detection, indicating the effect magnitude that would have produced, with a given probability of false negative (type II error), a statistically significant increase (type I error). Here, we report on a procedure based on t-statistics to calculate two types of detection limits, the "critical level (CL)" and the "detection level (DL)". The first is the size of the difference between exposed and controls required to achieve statistical significance. The second is the size of the difference that will be detected with a chosen probability of a false negative. For the degrees of freedom (d.f.) to be used for the t-values, a general formula is given so that different standard deviations and group sizes of control and exposed groups can be handled. A sample calculation of the whole procedure is shown, using the null data for the formation of a particular adduct in lung DNA of styrene-treated mice, analyzed by 32P-postlabeling. The procedure takes into account: (i) TLC-specific background radioactivity; (ii) variability within the control and exposed groups; and (iii) confidence limits for the factor to convert 32P-radioactivity to amounts of adduct. The latter step incorporates the variance of the differences between the samples and replicates spiked with adduct standard. A statement such as follows is the result: the concentration of the alpha-isomer adduct of styrene 7,8-oxide at the O(6)-position of guanine in mouse lung DNA would have to be at least 12 adducts per 10(8) nucleotides to be detected in the given experiment on a 5% level (type I error), with a probability of 5% to miss an existing effect (type II error).     

Micrococcal nuclease digestion study of spermidine-condensed DNA

Spermidine-condensed lambda DNA tertiary structures have been studied by micrococcal nuclease digestion. Broad but discrete DNA bands were observed in gel electrophoresis experiments of digests at sizes of: 1003 +/- 115 bp, 1972 +/- 190 bp and 3100 +/- 350 bp. These bands comprise an arithmetic series, similar to, but larger than, arithmetic DNA band series sizes we have observed previously in calf thymus and phi x-174 DNA condensates. The 1003bp monomer lambda DNA band size corresponds to wrapping B DNA once circumferentially about the toroidal-shaped tertiary structures, the predominant condensed structures present in these preparations, and is consistent with the measured electron microscopic dimensions for hydrated lambda DNA toruses previously presented. DNA fragment length stability was determined by release from the digested condensates. Fragments of 80-85bp and sizes below are thermodynamically unstable in the lambda DNA condensates. This fragment size agrees well with a recent determination of the cooperativity size in DNA condensates.     

Selective hydrolysis of damaged DNA by nuclease P1

The turnover rates for hydrolysis by nuclease P1 of the 16 unmodified dideoxynucleoside monophosphates were measured. In addition, the turnover rates were measured in a variety of dideoxynucleoside monophosphates containing free radical-induced base modifications. The modified bases included cis-5,6-dihydroxy-5,6-dihydrothymine (thymine glycol), 5,6-dihydrothymine, 5-hydroxymethyuracil, 8-hydroxyguanine, 5-hydroxy-5-methylhydantoin and the formamido remnant which can be derived from either a thymine or a cytosine base. The turnover rate for dinucleoside monophosphates containing 4,8-dihydro-4-hydroxy-8-oxo-guanine modifications, which are induced by singlet oxygen, were also measured. A model was devised for the hydrolysis of DNA by nuclease P1 which uses the observed turnover rates as parameters. The model predicts the abundance of monomers and dimers as hydrolysis proceeds. Whereas the level of monomers increases monotonically, the level of each dimer first increases and then falls off. There are advantages to phosphorylating dimers, as compared with monomers, using polynucleotide kinase. Consequently this model may be of interest in connection with ³²P-postlabeling applied to the measurement of DNA damage in nuclease P1 partial hydrolysates of DNA.     

Identification of new DNA adducts in human bladder epithelia exposed to the proximate metabolite of 4-aminobiphenyl using 32P-postlabeling method

The DNA adducts were analyzed by 32P-postlabeling method following exposure of human uroepithelial cells (HUC) to N-hydroxy-4-aminobiphenyl (N-OH-ABP), the proximate metabolite of the human bladder carcinogen 4-aminobiphenyl (ABP). TLC of the postlabeled products on the first dimension revealed several products, the majority of which stayed close to the origin and were earlier identified as the 3',5' -bisphospho derivatives of N-(deoxyguanosin-8-yl)-4-aminobiphenyl and N-(deoxyadenosin-8-yl)-4-aminobiphenyl (Carcinogenesis 13 (1993) 955; Carcinogenesis 16 (1995) 295). Here we report characterization of two additional adducts that amounted to less than 5% of the total adducts. Autoradiography of D1 chromatogram of the postlabeled products of calf thymus DNA chemically interacted with N-OH-ABP under acidic conditions revealed two adducts, #1 and #2, with R(f) values of about 0.2 and 0.3, respectively. Two adducts with D1 thin layer chromatographic properties similar to those of adducts #1 and #2 were obtained on postlabeling analyses of products generated by chemical interaction of N-acetoxy-4-aminobiphenyl (N-OAc-ABP) with deoxyguanosine-3' -monophosphate (dGp). Based on proton NMR and mass spectroscopic analyses of the synthetic products derived from N-OAc-ABP, the chemical structures of adducts #1 and #2 have been identified as 3-(deoxyguanosin-N(2)-yl)-4-aminobiphenyl, and N-(deoxyguanosin-N(2)-yl)-4-aminobiphenyl, respectively. Both of these adducts were insensitive to digestion with nuclease P1. 32P-Postlabeling analysis of the nuclease P1 enriched DNA hydrolysate of HUC cells treated with N-OH-ABP showed the presence of adduct #2 but not adduct #1. Adduct #2 was also detected in calf thymus DNA incubated with HUC cytosol and N-OH-ABP in the presence of acetyl CoA. These results suggest that in the target cells for ABP carcinogenesis in vivo, N-OH-ABP is bioactivated by acetyl CoA-dependent acyltransferases to reactive arylnitrenium ions that covalently interact at N(2)-position of deoxyguanosine in DNA.     

Induction of double-strand breaks by S1 nuclease, mung bean nuclease and nuclease P1 in DNA containing abasic sites and nicks.

Defined DNA substrates containing discrete abasic sites or paired abasic sites set 1, 3, 5 and 7 bases apart on opposite strands were constructed to examine the reactivity of S1, mung bean and P1 nucleases towards abasic sites. None of the enzymes acted on the substrate containing discrete abasic sites. Under conditions where little or no non-specific DNA degradation was observed, all three nucleases were able to generate double-strand breaks when the bistranded abasic sites were 1 and 3 base pairs apart. However, when the abasic sites were further apart, the enzymes again failed to cleave the DNA. These results indicate that single abasic sites do not cause sufficient denaturation of the DNA to allow incision by these single-strand specific endonucleases. The reactivity of these enzymes was also investigated on DNA substrates that were nicked by DNasel or more site-specifically by endonuclease III incision at the discrete abasic sites. The three nucleases readily induced a strand break opposite such nicks.     

Protection of discrete DNA fragments by the complex H1-octamerhistones or H5-octamerhistones after micrococcal nuclease digestion.

Several authors, including ourselves, have reported the existence of chromatosomes with DNA size larger than 166 bp in bird erythrocyte chromatin. It was tempting to correlate this increased DNA size with the presence of histone H5. In order to substantiate this hypothesis, we performed a micrococcal nuclease digestion kinetic on: chicken erythrocyte chromatin, either native, selectively depleted from H1, or from H1 and H5; and rat liver chromatin, either native or partially H1 depleted. The comparative analysis of the lengths of DNA in the chromatosome size region led to the following conclusions: - denaturing gels clearly reveal a first discrete pause at 178 nucleotides in H1 depleted chicken erythrocyte chromatin as well as in partially H1-depleted rat liver chromatin, before the material accumulates at the next intermediate 166 nucleotide chromatosome pause. - the generation of all discrete chromatosome bands is critically dependent on low ionic strength conditions and low Ca++ concentrations during the digestion, suggesting it may result from the protection of DNA cleavage sites by histone H5 or H1, C or N terminal domains.     

Nucleosome assembly capacity of nuclear lysate after nuclease digestion

Limited digestion of lymphocyte nuclei with micrococcal nuclease degrades the nuclear DNA and results in a resistant plateau of about 50% of the original DNA. During the course of the nuclease cleavage as more and more DNA becomes acid-soluble an increasing amount of core histone is released from the disintegrated chromatin indicating that a part of nucleosomal protected DNA is degraded. These free histones appeared not to be different from those arising from resistant chromatin fragments. The released histones are in a native state which allows the exogenous DNA to be converted into nucleoprotein complexes which appear to exhibit a typical nucleosomal structure as tested by several criteria.