Filter elution assays for DNA damage: practical and mechanistic significance of the DNA in the filter support wash.

The alkaline and neutral (or nondenaturing) filter elution assays are popular methods for the measurement of DNA strand breakage and its repair in eukaryotic cells. In both alkaline and neutral elution, it is recommended practice to wash the filter support after removal of the filter and to analyze the DNA recovered by this procedure together with that remaining on the filter as uneluted DNA, although it is not obvious why the DNA in the filter support wash should be so interpreted. We have observed that the sum of the DNA on the filter and that recovered in the filter support wash is approximately constant when the pH of the alkaline filter elution assay for total strand breaks is increased from 12.1 to 12.6, whereas the fraction on the filter itself is markedly smaller at the higher pH. This behavior characterized DNA elution from undamaged cells, as well as from cells treated with various DNA-damaging agents. These findings are consistent with the "tug-of-war" mechanism that has been proposed for alkaline elution, but are inconsistent with the simplest mechanism of the "sieve" class. In the neutral filter elution assay for double-strand breaks, by contrast, the distribution of DNA between the filter and the filter support wash is pH-independent. This suggests that single- and double-stranded DNA segments traverse a filter by different physical mechanisms. Our observations underscore the importance of carrying out the filter support wash and the analysis of the DNA it contains as uneluted DNA in alkaline elution, while indicating that a different analysis of this DNA might be appropriate for neutral elution.     

Fractionation of DNA from marine invertebrate (Maja crispata, Mytilus galloprovincialis) haemolymph by alkaline elution.

1. An alkaline elution procedure for the detection of DNA damage in marine invertebrate haemolymph has been developed. 2. Provided that three criteria are optimized, such as buffer composition, small filter pores (0.22 microns GVWP 025 00, Millipore), and optimal amounts of haemolymph applied, flow rates may be changed within the range of 0.2 ml/min to 0.05 ml/min without adverse back-pressure on the filter and without blocking filter pores. 3. Under optimal conditions, 70% of mussel haemolymph DNA, and 80% of crab haemolymph DNA will be retained on the filter after 6 hr of elution, indicating shorter DNA in mussel haemolymph. 4. The technique is applicable for testing the in vivo effects of different compounds on DNA in marine invertebrates, and to measurements of DNA damage in naturally exposed mussels. 5. This argues an important case for the use of alkaline elution technique for assessment of environmental genotoxicity, and especially for investigation of DNA damage in different marine organisms which cover a broad range in their DNA molecular weights.     

Automated determination of DNA using the fluorochrome Hoechst 33258

An automated method for the determination of DNA content in fractions from the alkaline filter elution assay of DNA damage has been developed. DNA-containing fractions are mixed with a fluorochrome (Hoechst 33258) and the DNA concentration is measured fluorometrically in a continuous-flow system. The lower limit of detection is 0.05 micrograms DNA/ml, and the linearity range under the conditions used is 0-8 micrograms DNA/ml. The standard deviation (n = 10) was found to be +/- 0.83%. The results are compared with the manual method.     

Filter elution analysis of the effect of alpha-difluoromethylornithine on X-ray-induced DNA damage in 9L cells

We used the filter elution technique to study DNA single- and double-strand scission under denaturing alkaline and nondenaturing conditions in X-irradiated 9L rat brain tumor cells. The amount of DNA damage determined by the alkaline elution assay was similar for different lysis conditions (sodium dodecyl sulfate and sarkosyl) and DNA fluorometric assays (Hoechst 33258 and 3,5-diaminobenzoic acid dyes). Therefore, results of the filter elution assay obtained with the various methods can be compared directly. Using these assays, we found that there was no significant change in the susceptibility to X-ray-induced DNA damage, measured either as single- or double-strand breaks, in 9L cells depleted of polyamines by treatment with alpha-difluoromethylornithine. Results obtained by filter elution are different from results obtained with viscoelastometry, which suggests that the two methods may resolve the effects of changes in DNA structure in different ways.     

Conformation of NAD+ bound to yeast and horse liver alcohol dehydrogenase in solution. The use of the proton-proton transferred nuclear Overhauser enhancement

Changes in reduced viscosity of nuclear lysates from rat liver cells have been studied, in conditions of very low shear stress by the use of an oscillating viscometer, as a function of incubation time in alkaline (pH 12.5) and neutral (pH 8.0) solutions. In non-denaturing conditions, nuclear DNA showed a stepwise, time-dependent increase of reduced viscosity, which suggests that it behaves as a single hydrodynamic unit that progressively changes its radius and viscoelastic properties because of a very slow unfolding, through discrete successive transitions, from a highly superpacked structure toward a linear relaxed B-form fiber. Experimental conditions shown to reduce chromatin-DNA superpacking without changing DNA length (e.g. G₁ cycling versus G₀ non-cycling liver cells, or young versus old rat liver cells) dramatically increased the initial value of reduced viscosity and its time-dependent increment. Conversely, in denaturing conditions, reduced viscosity increased in the initial phase (probably because DNA unfolding prevails on DNA unwinding), then exhibited a plateau level (when unfolding balances unwinding), and subsequently decreased progressively to the value of sheared DNA (when unwinding becomes more rapid due to the progressive breakage of phosphodiester bridges in alkali). Experimental conditions known to induce DNA single- or double-strand breaks (i.e. the use of liver cells from rats treated with dimethylnitrosamine or 2-acetylaminofluorene, or of liver cells exposed to X-rays) caused in both neutral and alkaline solution an increment in the initial reduced viscosity and in the slope of its time-dependent increase, which may be related to a reduction of chromatin-DNA superpacking. Moreover, it became evident in denaturing conditions that a decrease of the maximum viscosity and of the time taken to reach it both related to a reduced DNA length. These viscoelastic properties are constantly correlated with independent DNA structural measurements on the same nuclear lysates, to discriminate the effect due to mere aggregation and disaggregation.     

Measurement of radiation-induced DNA damage using gel electrophoresis or neutral filter elution shows an increased frequency of DNA strand breaks after exposure to pH 9.6

The filter elution technique using nondenaturing conditions is widely used to assay DNA double-strand break (DSB) induction and repair. It has been reported that in the measurement of strand breaks higher rates of elution and of initial rejoining are obtained at pH 9.6 compared to pH 7.2. In the present experiments neutral elution at pH 7.2 and 9.6 were compared in the assay of damage to DNA induced by X rays, 125I decay, and restriction enzyme digestion, in an effort to explain this discrepancy and to determine whether the higher rate of elution observed at pH 9.6 corresponds to a greater number of DSBs. X-ray damage to cellular DNA resulted in significantly different elution profiles at the two pH values. In contrast the elution profiles of the DSB induced by intragenomic 125I decays or restriction endonuclease were independent of the pH of the elution buffer. When gamma-irradiated SV40 DNA was exposed to pH 7.2 or 9.6 elution buffer prior to analysis by gel electrophoresis, a significantly greater number of DNA DSBs were detected in the DNA exposed to pH 9.6. We conclude that X and gamma radiation produce lesions (pH 9.6-labile lesions), in proportion to dose, that have the potential of becoming measurable DSBs following incubation under the mildly alkaline condition of pH 9.6. The data suggest that these lesions may result from single-hit events.     

Comparison of DNA Strand Break Induction in CHO Cells Measured by Alkaline Elution and by Fluorometric Analysis of DNA Unwinding (FADU)

DNA damage in X-irradiated CHO cells was measured by alkaline filter elution and compared to fluorometric analysis of DNA unwinding (FADU). The FADU method proved to be as sensitive as the alkaline filter elution technique in detecting X-ray induced DNA breaks. Strand break induction was also measured after treatment with four radical generating chemicals (hydrogen peroxide, bleomycin, mitomycin C and methyl viologen) using the FADU technique.     

Comparison of DNA Strand Break Induction in CHO Cells Measured by Alkaline Elution and by Fluorometric Analysis of DNA Unwinding (FADU)

DNA damage in X-irradiated CHO cells was measured by alkaline filter elution and compared to fluorometric analysis of DNA unwinding (FADU). The FADU method proved to be as sensitive as the alkaline filter elution technique in detecting X-ray induced DNA breaks. Strand break induction was also measured after treatment with four radical generating chemicals (hydrogen peroxide, bleomycin, mitomycin C and methyl viologen) using the FADU technique.     

Alternating current voltammetric determination of DNA damage

The conditions for alternating current (a.c.) voltammetric DNA determinations have been investigated with respect to its use with alkaline filter elution techniques at low DNA concentrations. In inorganic electrolyte solutions three current peaks can be distinguished: peak I around -1.1 V caused by the reorientation or desorption of DNA segments; peak II around -1.2 V caused by the native DNA (nDNA) form; peak III caused by denatured DNA (dDNA) at -1.4 V. Sonication of nDNA increases the peak current, however not with dDNA. Both dDNA and nDNA give linear peak current increments with DNA increments, their regression lines cutting the concentration axis at the origin. In filter elution techniques organic bases are often used. Adding ethanolamine (EA) elution buffer decreases the peak amplitude of DNA. It turns out that an unknown substance, perhaps a protein or RNA, elutes from the filters and gives rise to a current peak at about -1.3 V. This substance can interfere with the dDNA by competing for electrode surface area, since it diffuses much faster than the large molecules of the DNA. Since however, dDNA has a higher affinity for the electrode surface, after enough time, usually few minutes, the dDNA increasingly displaces the substance and occupies the surface. The same is valid for other organic molecules and thus also for EA. It is therefore remarkable that the unknown substance can be altered by ultrasonication, so that it will no longer interfere with dDNA, in contrast to EA. EA, on the other hand, can be "titrated". When EA is present at short accumulation times it prevents dDNA adsorption. By adding dDNA, the EA can be scavanged and further addition will adsorb and thus increase peak current in proportion to the concentration of the DNA present. The conditions for voltammetric DNA determination have been investigated obeying the recognized interactions. Avoiding organic bases and using inorganic ones would simplify the determination procedure. The reproducibility of the procedure in the range of 50-60 ng DNA/ml has been found to be +/- 6%.     

A technique for the measurement of breakage and repair of DNA alkylated in vivo.

The alkaline elution technique has been adapted for use in the assessment of DNA damage induced in the livers and lungs of mice after administration of an alkylating agent, methylemthanesulfonat (MMS). At 4 h after administration of MMS, damage ot DNA was readily demonstrable; the damage was repaired in liver by 24 h. The lung, particularly of the A/J mouse, exhibited an increased alkaline elution rate when compared to C57BL/6J, and repair was not entirely complete (as judged from the rate of alkaline elution of DNA) by 24 h. The rate of elution was dependent upon temperature. It is believed that this adaptation should have great utility in examining DNA repair in vivo.     

Isolation and purification of large quantities of DNA replication intermediates by pH step alkaline elution

The alkaline elution technique has been modified to be used in the isolation of DNA replication intermediates and in the study of the process of DNA replication. In this procedure pulse labeled CHO cells are layered onto a membrane filter, lysed with detergent, and the nascent DNA eluted in step-wise fashion with tetrapropylammonium hydroxide at pH 11.0, 11.3, 11.5 and 12.1. Alkaline sucrose sedimentation of the eluted DNA shows that the pH 11.0 material consists of < 9S fragments consistant with those described by Okazaki and others. DNA eluting at pH 11.3 has a molecular weight of 8–12 million daltons, DNA which elutes at pH 11.5 sediments with a molecular weight of 20–30 million daltons. Two independent lines of evidence suggest that the pH 11.3 material includes DNA sequences synthesized at replicon origins. (1) Exposure of cells to low doses of X-ray prior to pulse labeling reduces the pH 11.3 fraction by 40–50% while there is little change in the other fractions. (2) Synchronization of cells by inhibiting DNA synthesis with FdU, followed by a 2 min pulse label, yields approximately 50% of the incorporated ³H-thymidine in the pH 11.3 fraction. The pH step elution technique has the following advantages: 1. Intermediates of high specific activity can be isolated from 10⁶ cells per filter; 2. By lysing cells on a filter, proteins, nucleases, and other cellular materials are eliminated; 3. DNA in the lysate is never handled, thus eliminating shearing; 4. Eluted DNA may be instantaneously neutralized by collecting into a buffer to protect it from alkaline degradation.     

An automated alkaline elution system: DNA damage induced by 1,2-dibromo-3-chloropropane in vivo and in vitro

An automated alkaline elution system for the detection of DNA damage has been developed. After manual application of samples, which is completed within 5 min, the subsequent supply of liquids, changes in flow rates, and temperature are controlled automatically. The system operates 16 filters and may easily be expanded. The sensitivity of the fluorometric DNA determinations with the Hoechst 33258 dye is increased by using an elution buffer (20 mM Na2EDTA, pH 12.50) with low background fluorescence. DNA is determined using an automated setup similar to the one recently presented by Sterzel et al. (1985, Anal. Biochem. 147, 462-467). The most significant modification is the use of a neutralization buffer which allows variations in the pH of eluted fractions. This change increases the sensitivity of the DNA measurements. The automated alkaline elution system was evaluated using the nematocide 1,2-dibromo-3-chloropropane (DBCP) in a study of its genotoxic effects in the testes and the kidneys. Significant DNA damage was induced in testicular cells by 2.5 microM DBCP (1 h) in vitro and 85 mumol/kg DBCP ip (3 h) in vivo. The damage appeared after short treatment times (10 min in vivo). Variations in the observed DBCP response in vivo were largely due to interanimal variations. The automated alkaline elution system proved to be a sensitive assay also for the detection of DNA damage in kidney nuclei prepared from rats exposed to DBCP. Provided that kidney nuclei from untreated rats, mice, or hamster were kept ice-cold until lysing, 85-100% of their DNA was retained after 16 h of elution, indicating highly intact DNA. Under the same conditions, guinea pig DNA was rapidly degraded unless the nuclei were prepared in a buffer with a higher concentration of Na2EDTA (20 mM).     

DNA damage and repair in embryonic mouse limbs exposed to teratogenic doses of methylnitrosourea

The alkaline elution assay was used to monitor DNA single-strand breaks in embryonic tissue following exposure to the DNA-damaging teratogen N-methyl-N-nitrosourea (MNU, CAS No. 694-93-5). An animal model was developed in which nearly every fetus exposed to the highest dose of MNU had malformations of the hindlimbs while the fetuses exposed to the lowest dose of MNU had none. Hindlimbs pooled within litters were analyzed for DNA single-strand breaks by alkaline elution conducted at rapid (0.35 ml/min) and slow (0.35 ml/min) speeds. Breaks in the DNA of hindlimbs exposed to teratogenic doses of MNU were readily detected by alkaline elution only if slower speeds were used in the assay. Using the more sensitive procedure, DNA breakage was monitored over a 24-h period. DNA breakage peaked in the MNU-exposed hindlimbs in a dose-dependent manner 4 h after injection. While the elution profiles of hindlimbs exposed to the lower doses of MNU returned to control levels 8 h after injection, single-strand breaks persisted in the hindlimbs exposed to the highest dose of MNU for at least 20 h. These latter data suggest that the highly teratogenic dose of MNU induced DNA damage that was more slowly repaired than that produced at lower doses, possibly by saturation of DNA repair systems. Although some necrosis did occur in hindlimbs exposed at teratogenic dose levels, it was not severe and it did not appear to influence the alkaline elution results. These experiments show that alkaline elution is a sensitive assay for the detection of DNA damage in embryonic tissues.     

Measurement of DNA single-strand breaks by alkaline elution and fluorometric DNA quantification

The method presented is based on the alkaline elution procedure for the determination of DNA single-stand (ss) breaks developed by Kohn and on the principles of DNA quantification after binding with the dye Hoechst 33258. In the present study, modification of the alkaline elution procedure with regard to the elution solution volume was performed. The influences of the DNA strandedness, the ethylenediaminetetraacetate/tetraethylammonium hydroxide denaturation and elution solution presence, the DNA solution pH, the dye amount, and the incubation time for the formation of the dye-ssDNA complex on the DNA fluorometric quantification were also studied. The modified DNA alkaline elution procedure followed by the optimized fluorometric determination of the ssDNA was applied on liver tissue from both untreated and treated (N-nitroso-N-methylurea- administered) Wistar rats. The criteria for the selection of the appropriate estimator and statistical analysis of the obtained results are also presented. The method of the DNA alkaline elution followed by fluorometric determination of ssDNA as modified and evaluated is an accurate and reliable approach for the determination of in vivo induced ssDNA strand breaks.     

Induction and repair of psoralen cross-links in DNA of normal human and xeroderma pigmentosum fibroblasts

Skin fibroblasts from normal human subjects were exposed in vitro to long-wave ultraviolet radiation (UVA, 320–400 nm) alone, or in combination with 8-methoxypsoralen (8-MOP). DNA damage was analysed with the alkaline elution technique before and after post-treatment incubation of the cells at 37°C for various times.Cells treated with UVA at 1.1 J/cm² showed an increased DNA elution rate, which returned to the normal level within 30 min of post-treatment incubation. In cells treated with PUVA (8-MOP at 20 μg/ml plus UVA at 0.04 J/cm²), the alkaline elution rate was not different from untreated control cells, either before or after post-treatment incubation for times up to 7 days.When the PUVA treatment was followed first by a washing, to remove any unbound 8-MOP, and then by UVA (PUVA + UVA) at 1.1 J/cm², the alkaline elution rate decreased below the control level. During the post-treatment incubation of the PUVA + UVA-treated cells there was a gradual increase of the alkaline elution rate to a level significantly above that in control cells. This increase was observed after 30 min. It reached a miaximum after 24 h and remained after 7 days of post-treatment incubation. Cells from a patient with xeroderma pigmentosum of complementation group A, which were given the same PUVA + UVA treatment, did not show any change in the alkaline elution rate during the post-treatment incubation.If, as seems likely, an increased alkaline elution rate indicates an increase of DNA breaks, and a decreased alkaline elution rate indicates the sealing of breaks and/or the formation of cross-links, the results would suggest the following: (1) UVA irradiation in itself is capable of inducing DNA breaks, which are rapidly sealed during post-treatment incubation; (2) PUVA treatment induces mono-adducts, some of which appear to remain in the DNA for at least 7 days of post-treatment incubation and can be activated to form DNA cross-links by a second dose of UVA; (3) DNA cross-links induced by PUVA + UVA can be recognized by a repair process that involves the formation of DNA breaks. This process is not observed in xeroderma pigmentosum cells of group A.     

Fluid Mechanics of DNA Double-Strand Filter Elution

Measurement of infrequent DNA double-strand breaks (DSB) in mammalian cells is essential for the understanding of cell damage by ionizing radiation and many DNA-reactive drugs. One of the most important assays for measuring DSB in cellular DNA is filter elution. This study is an attempt to determine whether standard concepts of fluid mechanics can yield a self-consistent model of this process. Major assumptions of the analysis are reptation through a channel formed by surrounding strands, with only strand ends captured by filter pores. Both viscosity and entanglement with surrounding strands are considered to determine the resistance to this motion. One important result is that the average elution time of a strand depends not only on its length, but also on the size distribution of the surrounding strands. This model is consistent with experimental observations, such as the dependence of elution kinetics upon radiation dose, but independence from the size of the DNA sample up to a critical filter loading, and possible overlap of elution times for strands of different length. It indicates how the dependence of elution time on the flow rate could reveal the relative importance of viscous and entanglement resistance, and also predicts the consequences of using different filters.     

DNA-protein crosslinking by trans-platinum(II)diamminedichloride in mammalian cells, a new method of analysis.

DNA-protien crosslinks produced in mouse leukemia L1210 cells by trans-Pt(II)diamminedichloride were quantitated using the technique of DNA alkaline elution. DNA single-strand segments that were or were not linked to protein were separable into distinct components by alkaline elution after exposure of the cells to 2--15 kR of X-ray. Protein-linked DNA strands were separated on the basis of their retention of filters at pH 12 while free DNA strands of the size generated by 2--15 kR of X-ray passed rapidly through the filters. The retention of protein-linked DNA strands was attributable to adsorption of protein to the filter under the conditions of alkaline elution. The results obeyed a simple quantitative model according to which the frequency of DNA-protein crosslinks could be calculated.     

Interspecies differences in DNA single strand breaks caused by benzo(a)pyrene and marine environment

The presence of DNA single strand breaks in untreated specimens of selected species, mosquito fish Gambusia affinis, painted comber Serranus scriba, blue mussel Mytilus galloprovincialis, spiny crab Maja crispata and sea cucumber Holothuria tubulosa as well as in 10 microg/g benzo(a)pyrene (BaP) treated mosquito fish, blue mussel and spiny crab was measured, using alkaline filter elution. Interspecies differences in alkaline elution profiles were observed and attributed to different lengths of DNA from different sources and to differences in the number of strand breaks present during normal cellular events in different phyla. Spiny crab hemocytes are more sensitive to action of BaP then blue mussel hemocytes and mosquito fish hepatocytes that could be explained by differences in the rates of distinct metabolic reactions and DNA repair among the investigated species. In field study, DNA single strand breaks were measured in hepatocytes of painted comber and in hemocytes of blue mussel and spiny crab from natural population specimens collected at eight sampling sites along Istrian coast, Croatia. Spatial variations in DNA integrity for each species were detected and revealed for the first time that spiny crab is responsive to different environmental conditions. Interspecies variations in the DNA integrity due to environmental conditions, confirmed species specific susceptibility to genotoxicity of certain environment that in long-term may modify the structure of marine communities. The multi-species approach in designing biomonitoring studies was suggested.     

Incomplete rejoining of DNA double-strand breaks in unstimulated normal human lymphocytes

We investigated the repair kinetics of DNA single-strand breaks (SSBs) and double-strand breaks (DSBs) in unstimulated normal human peripheral blood lymphocytes (HPBL). SSBs and DSBs induced by gamma-irradiation (at 0 degree C) were assayed without radiolabel by alkaline and neutral filter elution, respectively. Incubation of irradiated cells at 37 degrees C for various lengths of time demonstrated that the percent DNA rejoined increased until it reached a plateau at approximately 60 min; this repair plateau underwent no substantial change when incubation continued for 20-24 h. The level of the plateau indicated how closely the elution profile of DNA from cells irradiated and incubated (experimental) resembled the elution profile of DNA from unirradiated cells (control). After 6 Gy and 60 min incubation, the alkaline elution profile of DNA from experimental cells from 5 donors was indistinguishable from that seen in DNA from control cells, suggesting that rejoining of SSBs was complete. In contrast after 100 Gy and 60 min incubation the neutral elution profile of DNA from cells from the same donors demonstrated that, compared to DNA from control cells, rejoining of DSBs was approximately two-thirds complete. In the range of 2-8 Gy, 85-104% of SSBs were rejoined after 60 min incubation; in the range of 30-120 Gy, 46-80% of DSBs were rejoined after 60 min incubation. These unexpected results stand in contrast to our previous studies with confluent normal human diploid fibroblasts (HDF), in which rejoining of both SSBs and DSBs was greater than 90% complete by 60 min repair incubation and 100% complete after 18-24 h.     

Quiescent human peripheral blood lymphocytes do not contain a sizable amount of preexistent DNA single-strand breaks

Sedimentation of nucleoids through neutral sucrose density gradients has shown that nucleoids isolated from phytohemagglutinin (PHA)-stimulated human peripheral blood lymphocytes (PBL) sediment faster than nucleoids derived from quiescent lymphocytes, which was attributed to rejoining of DNA single-strand breaks (SSB) present in the resting cells (A.P. Johnstone, and G.T. Williams (1982) Nature (London) 300, 368). We isolated PBL from donors and determined the amount of SSB in nonradiolabeled, untreated resting and PHA-stimulated cells by applying the alkaline filter elution technique. Calibration was based on dose-dependent induction of SSB by 60Co-gamma-radiation. Quiescent cells did not contain a sizable amount of SSB. Mitogen-stimulated cells showed equally low amounts of SSB per cell. The present study indicates that the interpretation of the results obtained with the nucleoid sedimentation technique concerning the supposed rejoining of SSB in PHA-stimulated human lymphocytes is incorrect. Other, equally sensitive, techniques such as alkaline filter elution appear to be preferable for studies on DNA damage and repair.