Osmotically driven radial diffusion of single-stranded DNA fragments on an agarose bed as a convenient measure of DNA strand scission

The present study describes the development and characterization of a novel technique, the alkaline-halo assay, for the assessment of DNA single strand breakage in mammalian cells. This technique allows the measurement of DNA lesions at the single cell level and presents the additional advantages of being rapid, sensitive, virtually costless and environmentally friendly, because it does not require the use of isotopes. The alkaline halo assay involves a series of sequential steps in which the cells are first treated, then embedded in melted agarose and spread onto microscope slides that are incubated for 2 min at ice-bath temperature to allow complete geling. The slides are then incubated for 20 min in a high salt alkaline lysis solution, for an additional 15 min in a hypotonic alkaline solution and, finally, for 10 min in ethidium bromide. Under these conditions, single-stranded DNA fragments spread radially from the nuclear cage and generate a fluorescent image that resembles a halo concentric to the nucleus remnants. The area of the halos increased at increasing levels of DNA fragmentation and this process was associated with a progressive reduction of areas of the nuclear remnants. These events were conveniently monitored with a fluorescence microscope and quantified by image processing analysis. The sensitivity of the alkaline-halo assay, which is based on the osmotically driven radial diffusion of single-stranded DNA fragments through agarose pores, is remarkably similar to that of the widely used alkaline elution and comet assays.     

Characterization of DNA damage in yeast apoptosis induced by hydrogen peroxide, acetic acid, and hyperosmotic shock

Saccharomyces cerevisiae has been reported to die, under certain conditions, from programmed cell death with apoptotic markers. One of the most important markers is chromosomal DNA fragmentation as indicated by terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) staining. We found TUNEL staining in S. cerevisiae to be a consequence of both single- and double-strand DNA breaks, whereas in situ ligation specifically stained double-strand DNA breaks. Cells treated with hydrogen peroxide or acetic acid staining positively for TUNEL assay stained negatively for in situ ligation, indicating that DNA damage in both cases mainly consists of single-strand DNA breaks. Pulsed field gel electrophoresis of chromosomal DNA from cells dying from hydrogen peroxide, acetic acid, or hyperosmotic shock revealed DNA breakdown into fragments of several hundred kilobases, consistent with the higher order chromatin degradation preceding DNA laddering in apoptotic mammalian cells. DNA fragmentation was associated with death by treatment with 10 mM hydrogen peroxide but not 150 mM and was absent if cells were fixed with formaldehyde to eliminate enzyme activity before hydrogen peroxide treatment. These observations are consistent with a process that, like mammalian apoptosis, is enzyme dependent, degrades chromosomal DNA, and is activated only at low intensity of death stimuli.     

Analysis of DNA double- and single-strand breaks by two dimensional electrophoresis: action of micrococcal nuclease on chromatin and DNA, and degradation in vivo of lens fiber chromatin.

We describe a novel system for two dimensional electrophoresis at neutral and alkaline pH for determining the double-stranded and single-stranded lengths of DNA. With this system we analysed the mode of micrococcal nuclease digestion of DNA in cellular and SV40 viral chromatin and of supercoiled SV40 DNA. The enzyme reaction occurred in two steps : the enzyme first introduced single-strand breaks, then converted these to double-strand breaks by an adjacent cleavage on the opposite strand. Digestion of cellular chromatin DNA occurred by a similar mechanism. Chromatin fragments produced by limited micrococcal nuclease action contained many single-strand breaks, which may be important when this method is used to prepare chromatin fragments for biochemical and biophysical studies. Nucleosome monomer to tetramer produced at later stages of digestion contained few if any single-strand breaks.     

Application of alkaline unwinding assay for detection of mutagen-induced DNA strand breaks

The frequency of single-strand breaks in parental DNA and gaps in nascent DNA in various cells exposed to methyl methanesulfonate (MMS) or methylnitrosourea (MNU) was investigated by alkaline unwinding assay using two types of alkaline lysis conditions, 22°C lysis versus 0°C lysis. The DNA damage induced by MMS and MNU is considered to be characteristic of lesions produced in DNA by alkylating agents. The aim of our research project was to adjust this method to be able to detect the greatest number of DNA lesions induced by alkylating agents in parental DNA of different mammalian cells. In our experiments we used human cell lines EUE, GM637 and XP12, Chinese hamster V79 cells, and Syrian hamster embryo cells. The higher level of strand interruptions was detected under conditions of lysis of cells at 22°C. Probably the level of strand interruptions found after the lysis of cells at 22°C correlates with the increased number of disrupted alkali-labile sites of DNA. It is remarkable that the different lysis conditions did not influence the number of gaps detected in nascent DNA of alkylated cells. Comparing induction of breaks and gaps in radiolabelled strands of parental and daughter DNA under different lysis conditions, we succeeded in defining the optimum conditions for detection of alkali-labile sites of parental DNA.     

Length distributions of single-stranded DNA in Chinese hamster ovary cells.

The distribution of lengths of single-strand DNA in Chinese hamster ovary cells in the G₁ phase of the cell cycle has been observed for various conditions of cell lysis and incubation of the lysates. The method of analysis was band sedimentation through a self-generating density gradient, a technique developed originally for the analytical ultracentrifuge, but modified here for the preparative ultracentrifuge so that measurements of sedimentation coefficients could be made under conditions that minimize shearing of the single-stranded DNA. The effect of rotor speed dependence of the sedimentation coefficient is considered in developing the relation between the sedimentation coefficient and molecular weight for this technique.Special precautions were taken to ensure that complete separation of long single strands took place upon alkaline denaturation, to preclude the possibility of anomalous sedimentation due to interstrand entanglement. Bromodeoxyuridine was incorporated into the DNA in the last round of replication. Advantage was taken of the increased sensitivity to ultraviolet irradiation for the production of single-strand breaks in DNA strands substituted with bromodeoxyuridine. After irradiation the bromodeoxyuridine-substituted strand could be completely separated from the complementary strand in alkaline sedimentation profiles without any apparent breakage in the unsubstituted strand.The conditions of lysis, chosen to minimize the degradation of DNA in the lysates, included lysis at pH 9.3 with Pronase and lysis at high pH (10.8 and 12.0). Sedimentation analysis was performed at various time intervals after incubation at 4 °C or 37 °C. Lysis and incubation at pH 12.0 produced a continuous single-strand breakdown of the DNA in the lysate. Analysis of the sedimentation profiles indicates that these alkaline-induced breaks are randomly distributed. However, lysis and incubation at pH 10.8 and at pH 9.3 with Pronase produced stable sedimentation profiles with number-average molecular weights of 1.7 × 10⁸ and 6.0 × 10⁷, respectively. Analysis of the single-strand DNA sedimentation profiles for these lysates indicates that the distribution of lengths of single-stranded DNA is non-random, i.e. that the distributions may represent regular subunits of chromosomal DNA structure. Suggestive evidence is presented that the approximately 60-μm units are structurally alternated in the two chains. The possible origin of the discontinuities between the subunits is also discussed.     

Chromatin Collapse during Caspase-dependent Apoptotic Cell Death Requires DNA Fragmentation Factor, 40-kDa Subunit-/Caspase-activated Deoxyribonuclease-mediated 3′-OH Single-strand DNA Breaks

Apoptotic nuclear morphology and oligonucleosomal double-strand DNA fragments (also known as DNA ladder) are considered the hallmarks of apoptotic cell death. From a classic point of view, these two processes occur concomitantly. Once activated, DNA fragmentation factor, 40-kDa subunit (DFF40)/caspase-activated DNase (CAD) endonuclease hydrolyzes the DNA into oligonucleosomal-size pieces, facilitating the chromatin package. However, the dogma that the apoptotic nuclear morphology depends on DNA fragmentation has been questioned. Here, we use different cellular models, including MEF CAD^−/− cells, to unravel the mechanism by which DFF40/CAD influences chromatin condensation and nuclear collapse during apoptosis. Upon apoptotic insult, SK-N-AS cells display caspase-dependent apoptotic nuclear alterations in the absence of internucleosomal DNA degradation. The overexpression of a wild-type form of DFF40/CAD endonuclease, but not of different catalytic-null mutants, restores the cellular ability to degrade the chromatin into oligonucleosomal-length fragments. We show that apoptotic nuclear collapse requires a 3′-OH endonucleolytic activity even though the internucleosomal DNA degradation is impaired. Moreover, alkaline unwinding electrophoresis and In Situ End-Labeling (ISEL)/In Situ Nick Translation (ISNT) assays reveal that the apoptotic DNA damage observed in the DNA ladder-deficient SK-N-AS cells is characterized by the presence of single-strand nicks/breaks. Apoptotic single-strand breaks can be impaired by DFF40/CAD knockdown, abrogating nuclear collapse and disassembly. In conclusion, the highest order of chromatin compaction observed in the later steps of caspase-dependent apoptosis relies on DFF40/CAD-mediated DNA damage by generating 3′-OH ends in single-strand rather than double-strand DNA nicks/breaks.     

DNA damage induced by three major metabolites of 1,3-butadiene in human hepatocyte L02 cells

1,3-Butadiene (BD) is a carcinogenic air pollutant. Its bioactivation produces four major metabolites, i.e., 3,4-epoxy-1-butene (EB), 3,4-epoxy-1,2-butanediol (EBD), 1,2,3,4-diepoxybutane (DEB), and 3-butene-1,2-diol (BDD). Studies have been mostly focused on DEB due to its strong mutagenicity/carcinogenicity. In contrast, studies of genotoxicity of EB, EBD, and BDD have been limited. In particular, genotoxicity of EBD and BDD using strand breaks as the endpoint has not been investigated. To obtain a more complete understanding of BD toxicity, in the present study, we used comet assay to investigate DNA damage induced by EB, EBD, and BDD in human hepatocyte L02 cells, with the aim to determine their relative potencies, the types of DNA damage, and the possible pathway to form strand breaks. Using alkaline comet assay (pH>13), it was observed that EB and EBD caused similar concentration-dependent increases in DNA migration from 50 to 1000μM. However, BDD induced a statistically significant increase only at 1000μM, and the increase itself was very small. EBD was as potent as EB at lower concentrations (≤200μM), and was slightly less potent than EB at higher concentrations. The results indicated that these metabolites could generate strand breaks in cells with the rank order of the potencies being EB>≈EBD?BDD. All three compounds failed to cause statistically significant increases in DNA migration in pre-lysed cells, suggesting that they did not produce strand breaks through chemical pathways under our experimental conditions. By using comet assays at pH 11.9 and pH 9, it was demonstrated that EB and EBD generated both single-strand breaks (SSB) and alkali-labile sites, but BDD produced only SSB. To our knowledge, this is the first report to investigate EBD- and BDD-induced strand breaks in cells. The results implied that EBD could play an important role in toxicity of BD.     

DNA double-strand breaks and alkali-labile bonds produced by bleomycin.

Both in linear T2 DNA, analyyzed by velocity sedimentation, and in supercoiled Col EL DNA, analyzed by gel electrophoresis, the number of double-strand breaks produced by bleomycin was directly propotional to the number of single-strand breaks and was far greater than the number expected from random coincidence of single-strand breaks, suggesting that the bleomycin-induced double-strand breaks occur as an independent event. In Col EL DNA, at least twice as many single-strand breaks were found under alkaline assay conditions as were found under neutral conditions, showing the production of alkaline-labile bonds by bleomycin.     

Overnight lysis improves the efficiency of detection of DNA damage in the alkaline comet assay

The ability to detect DNA damage using the alkaline comet assay depends on pH, lysis time and temperature during lysis. However, it is not known whether different lysis conditions identify different types of DNA damage or simply measure the same damage with different efficiencies. Results support the latter interpretation for radiation, but not for the alkylating agent MNNG. For X-ray-induced damage, cells showed the same amount of damage, regardless of lysis pH (12.3 compared to >13). However, increasing the duration of lysis at 5 degrees C from 1 h to more than 6 h increased the amount of DNA damage detected by almost twofold. Another twofold increase in apparent damage was observed by conducting lysis at room temperature (22 degrees C) for 6 h, but at the expense of a higher background level of DNA damage. The oxygen enhancement ratio and the rate of rejoining of single-strand breaks after irradiation were similar regardless of pH and lysis time, consistent with more efficient detection of strand breaks rather than detection of damage to the DNA bases. Conversely, after MNNG treatment, DNA damage was dependent on both lysis time and pH. With the higher-pH lysis, there was a reduction in the ratio of oxidative base damage to strand breaks as revealed using treatment with endonuclease III and formamidopyrimidine glycosylase. Therefore, our current results support the hypothesis that the increased sensitivity of longer lysis at higher pH for detecting radiation-induced DNA damage is due primarily to an increase in efficiency for detecting strand breaks, probably by allowing more time for DNA unwinding and diffusion before electrophoresis.     

Identification of apoptotic cells by formamide-induced dna denaturation in condensed chromatin.

In this article we describe a novel effect of formamide on DNA of apoptotic nuclei and present a method for specific detection of apoptotic cells based on this effect. Our observations show that formamide induces DNA denaturation in apoptotic nuclei but has no such effect on DNA of non-apoptotic cells. Formamide-induced DNA denaturation combined with detection of denatured DNA with a monoclonal antibody (MAb) against single-stranded DNA made it possible to specifically identify the apoptotic cells. This procedure produced intense staining of the condensed chromatin in the apoptotic nuclei. In contrast, necrotic cells from cultures treated with sodium azide, saponin, or hyperthermia did not bind this antibody, demonstrating the specificity of the formamide-MAb assay for the apoptotic cells. However, TUNEL stained 90-100% of necrotic cells in all three models of necrosis. Because the MAb did not stain cells with single- or double-stranded DNA breaks in the absence of apoptosis, we conclude that staining of the apoptotic nuclei is not influenced by DNA breaks and is induced by specific changes in condensed chromatin, such as damage to the DNA-histone interactions. Importantly, the formamide-MAb technique identified apoptotic cells in frozen sections and in histological sections of formalin-fixed, paraffin-embedded tissues.     

The relationship between molecular and cellular repair from potentially lethal damage induced by N-methyl-N'-nitro-N-nitrosoguanidine in Chinese hamster V79 cells

The relationship between molecular and cellular repair from potentially lethal damage (PLD) induced by N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) was investigated in exponentially growing V79 Chinese hamster cells. We compared the repair processes by an alkaline sucrose sedimentation analysis and a colony formation assay. MNNG-treated cells were exposed to the conditioned medium (CM) from density-inhibited plateau-phase V79 cell cultures, as a post-treatment for the induction of PLD repair. When MNNG-treated cells were postincubated in CM, cell survival continuously increased for 18 h, and during this period, DNA replication was substantially suppressed. CM did not inhibit the rejoining of the single-strand breaks of parental DNA. Rather, parental DNA fragments sedimented more rapidly when postincubated in CM than in fresh medium. These data indicate that cellular recovery from MNNG-induced PLD increases in proportion to the resealing of MNNG-induced single-strand breaks of DNA during the suppression of DNA replication, suggesting that excision repair is involved in the PLD repair process.     

Quantitation of chemically induced DNA strand breaks in human cells via an alkaline unwinding assay

DNA strand breaks induced in human CCRF-CEM cells by electrophilic chemicals (carcinogens/mutagens) can be readily quantitated via a facile alkaline unwinding assay. This procedure estimates the number of chemically induced DNA strand breaks on the basis of the percentage DNA converted from double-stranded to single-stranded form during an exposure to the alkaline unwinding conditions. The assay is based on the assumption that each strand break serves as a strand unwinding point during the alkaline denaturation. The extent of strand separation can be standardized with respect to the initial level of induced strand breaks by the use of X-rays, which produce known levels of DNA strand breaks per rad in mammalian cells. Subsequent to the alkaline exposure, the single- and double-stranded DNA were separated by use of thermostated hydroxylapatite columns (60 degrees C), and the DNA was quantitated via a fluorescence assay (Hoechst 33258 compound). A correlation was shown between mammalian DNA strand-breaking potential (as measured in this procedure) and the propensity of these chemicals to revert Salmonella typhimurium TA100.     

Explanation for excessive DNA single-strand breaks and endogenous repair foci in pluripotent mouse embryonic stem cells

Pluripotent mouse embryonic stem cells (mES cells) exhibit ∼ 100 large γH2AX repair foci in the absence of measurable numbers of DNA double-strand breaks. Many of these cells also show excessive numbers of DNA single-strand breaks (> 10,000 per cell) when analyzed using the alkaline comet assay. To understand the reasons for these unexpected observations, various methods for detecting DNA strand breaks were applied to wild-type mES cells and to mES cells lacking H2AX, ATM, or DNA-PK_cs. H2AX phosphorylation and expression of other repair complexes were measured using flow and image analysis of antibody-stained cells. Results indicate that high numbers of endogenous γH2AX foci and single-strand breaks in pluripotent mES cells do not require ATM or DNA-PK kinase activity and appear to be associated with global chromatin decondensation rather than pre-existing DNA damage. This will limit applications of γH2AX foci analysis in mES cells to relatively high levels of initial or residual DNA damage. Excessive numbers of single-strand breaks in the alkaline comet assay can be explained by the vulnerability of replicating chromatin in mES cells to osmotic shock. This suggests that caution is needed in interpreting results with the alkaline comet assay when applied to certain cell types or after treatment with agents that make chromatin vulnerable to osmotic changes. Differentiation of mES cells caused a reduction in histone acetylation, γH2AX foci intensity, and DNA single-strand breakage, providing a link between chromatin structural organization, excessive γH2AX foci, and sensitivity of replicating mES cell chromatin to osmotic shock.     

Viscoelastic characterization of single-stranded DNA from Escherichia coli.

Single-stranded DNA released from E. coli wild type and mutant cells by alkaline-EDTA-detergent was analyzed using the recently developed biophysical technique of viscoelastometry. Under the lysis conditions used, it was possible to detect single strands of molecular weight approximately 2 times 10-9 daltons. Little difference was detected in the size of single-stranded DNA from log phase vs. stationary phase cultures, or from cells treated with chloramphenicol to allow completion of replicating chromosomes. The largest single strands from ligase overproducing, endonuclease minus, and pol A1 mutants were likewise of approximately the same size as wild type, but were present in smaller yields. The reduction in single-strand molecular weight as a result of heating intact cells was investigated as a function of time and temperature. Heating at 37 degrees C for up to 20 min produced no additional single-strand breaks, but temperatures from 45 to 65 degrees introduced breaks. Solutions maintained at pH 12.5 were not stable indefinitely, and the relative viscosity of such solutions was found to decrease over a period of several hours.     

Rejoining kinetics of DNA single- and double-strand breaks in normal and DNA ligase-deficient cells after exposure to ultraviolet C and gamma radiation: an evaluation of ligating activities involved in different DNA repair processes

The repair kinetics for rejoining of DNA single- and double-strand breaks after exposure to UVC or gamma radiation was measured in cells with deficiencies in DNA ligase activities and in their normal counterparts. Human 46BR cells were deficient in DNA ligase I. Hamster EM9 and EM-C11 cells were deficient in DNA ligase III activity as a consequence of mutations in the XRCC1 gene. Hamster XR-1 cells had mutation in the XRCC4 gene, whose product stimulates DNA ligase IV activity. DNA single- and double-strand breaks were assessed by the comet assay in alkaline conditions and by the technique of graded-field gel electrophoresis in neutral conditions, respectively. 46BR cells, which are known to re-ligate at a reduced rate the DNA single-strand breaks incurred during processing of damage induced by UVC but not gamma radiation, were shown to have a normal repair of radiation-induced DNA double-strand breaks. EM9 cells exhibited a reduced rate of rejoining of DNA single-strand breaks after exposure to ionizing radiation, as reported previously, as well as UVC radiation. EM-C11 cells were deficient in the repair of radiation-induced-DNA single-strand breaks but, in contrast to EM9 cells, demonstrated the same kinetics as the parental cell line in the resealing of DNA breaks resulting from exposure to UVC radiation. Both EM9 and EM-C11 cells displayed a significant defect in rejoining of radiation-induced-DNA double-strand breaks. XR-1 cells were confirmed to be highly deficient in the repair of radiation-induced DNA double-strand breaks but appeared to rejoin DNA single-strand breaks after UVC and gamma irradiation at rates close to normal. Taken together these results indicate that: (1) DNA ligase I is involved only in nucleotide excision repair; (2) DNA ligase IV plays an important role only in repair of DNA double-strand breaks; and (3) DNA ligase III is implicated in base excision repair and in repair of DNA double-strand breaks, but probably not in nucleotide excision repair.     

DNA damage induced by nitropyrenes in primary mouse hepatocytes and in rat H4-II-E hepatoma cells

The capacity of nitropyrenes to cause DNA damage in primary mouse hepatocytes (C57BL/6N mice) and rat H4-II-E hepatoma cells was studied by estimating single-strand breaks using the alkaline elution technique. 1-Nitropyrene (10-200 microM) caused clear dose-dependent increases in DNA strand breaks in both cell types, whereas no increase in DNA strand breaks was observed in hepatocytes treated with 1.3-, 1,6-, 1,8-dinitropyrene, 1,3,6-trinitropyrene and 1,3,6,8-tetranitropyrene under standard assay conditions (5-20 microM 30-min incubation). However, 1,8-dinitropyrene (1,8-DNP) caused dose-dependent increases in DNA strand breaks when incubated with the H4-II-E cells for 48 h, while no single-strand breaks were observed following treatment with 1,6-dinitropyrene (1,6-DNP) under the same conditions. Neither 1,6-DNP nor 1,8-DNAP induced DNA crosslinks in the H4-II-E cells. These data indicate that substrate specificity exists in the metabolic activation of nitropyrenes in murine liver.     

Measurement of DNA damage induced by irradiation with gamma-rays from a TRIGA Mark II research reactor in human cells using Fast Micromethod.

The Fast Micromethod is a novel quick and convenient microplate assay for determination of DNA single-strand breaks. This method measures the rate of unwinding of cellular DNA upon exposure to alkaline conditions using a fluorescent dye which preferentially binds to double-stranded DNA. Here we applied this method to determine the levels of DNA single-strand breaks in HeLa cells induced by y-irradiation deriving from fission isotopes and activation products at the TRIGA Mark II research reactor in Mainz. An increased strand scission factor (SSF) value, which is indicative for DNA damage, was found at doses of 1 Gy and higher. A similar increase in SSF value, which further increased in a dose-dependent manner, was found in human peripheral blood mononuclear cells after irradiation with 6 MV X-rays from a linear accelerator to give a total exposure of 0.5 to 10 Gy.     

Repair of DNA single-strand breaks in radiation-sensitive mutants of mouse cells

The radiation-sensitive mutant M10 of mouse lymphoma L5178Y cells was examined for its ability to rejoin DNA single-strand breaks induced by gamma-rays. The alkaline sucrose gradient sedimentation analysis revealed that M10 cells repaired single-strand breaks but simultaneously produced increasing amounts of small DNA fragments with time of postirradiation incubation, something which was not observed in L5178Y cells. Since small fragments did not appear in M10 cells irradiated at room temperature, DNA fragmentation may result from cold treatment during irradiation followed by incubation at 37 degrees C. This indicates that the cold susceptibility is characteristic of M10 cells and is not related to radiation sensitivity of this mutant. This conclusion is supported by the finding that no DNA degradation takes place after cold treatment with a subsequent incubation in the other radiosensitive mutant LX830 that belongs to the same complementation group as M10.     

Abundant alkali-sensitive sites in DNA of human and mouse sperm

The DNA of human and mouse sperm cells was analyzed by single-cell microgel electrophoresis, by agarose gel electrophoresis, and by alkaline elution--three techniques that can detect single-strand DNA breaks and/or labile sites. Under these conditions a surprisingly large number of single-strand DNA breaks, approximately 10(6) to 10(7) per genome, were detected in human and mouse sperm but not in human lymphocytes or in mouse bone marrow cells. These breaks were also present in chicken erythrocyte DNA, which is also highly condensed. These breaks were not observed under neutral pH conditions nor under denaturing conditions not involving alkali, suggesting that these sites are alkali-sensitive and do not represent preexisting single-strand breaks. The high frequency of such sites in sperm from healthy mouse and human donors suggests that they represent a functional characteristic of condensed chromatin rather than DNA damage.     

Differential staining of DNA strand breaks in dried comet assay slides.

The comet assay involves embedding cells in agarose on microscope slides. After lysis and electrophoresis, staining is usually performed with a fluorescent DNA-binding dye and observation is carried out on fresh wet slides through an epifluorescence microscope. We present here a simple alternative for preservation of the agarose comet slides and a fluorescent staining that allows fine differential analysis of DNA strand breaks under confocal microscopy. Lymphocytes were processed according to previous published methods. Slides were quickly dehydrated in a hot oven at 50C for 20 min. Once the agarose layer was dried and reduced to a thin film, slides were treated with RNase. Image analysis showed higher tail length, total area, and tail moment. Using confocal microscopic optical sectioning, a thickness of approximately 180 microm for wet slides and 12 microm for dehydrated gels was calculated. Acridine orange, used for DNA differential staining, allowed quantitation of metachromasia and orthochromasia with confocal scanning microscopy. Differences between alkaline and neutral comet assay with AO were clear-cut and, in principle, a metachromatic index can be calculated. (J Histochem Cytochem 49:921-922, 2001)