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.     

Cohesin promotes the repair of ionizing radiation-induced DNA double-strand breaks in replicated chromatin

The cohesin protein complex holds sister chromatids together after synthesis until mitosis. It also contributes to post-replicative DNA repair in yeast and higher eukaryotes and accumulates at sites of laser-induced damage in human cells. Our goal was to determine whether the cohesin subunits SMC1 and Rad21 contribute to DNA double-strand break repair in X-irradiated human cells in the G2 phase of the cell cycle. RNA interference-mediated depletion of SMC1 sensitized HeLa cells to X-rays. Repair of radiation-induced DNA double-strand breaks, measured by γH2AX/53BP1 foci analysis, was slower in SMC1- or Rad21-depleted cells than in controls in G2 but not in G1. Inhibition of the DNA damage kinase DNA-PK, but not ATM, further inhibited foci loss in cohesin-depleted cells in G2. SMC1 depletion had no effect on DNA single-strand break repair in either G1 or late S/G2. Rad21 and SMC1 were recruited to sites of X-ray-induced DNA damage in G2-phase cells, but not in G1, and only when DNA damage was concentrated in subnuclear stripes, generated by partially shielded ultrasoft X-rays. Our results suggest that the cohesin complex contributes to cell survival by promoting the repair of radiation-induced DNA double-strand breaks in G2-phase cells in an ATM-dependent pathway.     

Modulation of restriction enzyme-induced damage by chemicals that interfere with cellular responses to DNA damage: a cytogenetic and pulsed-field gel analysis

The electroporation of restriction enzymes into mammalian cells results in DNA double-strand breaks that can lead to chromosome aberrations. Four chemicals known to interfere with cellular responses to DNA damage were investigated for their effects on chromosome aberrations induced by AluI and Sau3AI; in addition, the number of DNA double-strand breaks at various times after enzyme treatment was determined by pulsed-field gel electrophoresis (PFGE). The poly(ADP-ribose) polymerase inhibitor 3-aminobenzamide (3AB) dramatically increased the yield of exchanges and deletions and caused a small but transitory increase in the yield of double-strand breaks induced by the enzymes. 1-beta-D-Arabinofuranosylcytosine, which can inhibit DNA repair either by direct action on DNA polymerases alpha and delta or by incorporation into DNA, potentiated aberration induction but to a lesser extent than 3AB and did not affect the amount of DNA double-strand breakage. Aphidicolin, which inhibits polymerases alpha and delta, had no effect on AluI-induced aberrations but did increase the aberration yield induced by Sau3AI. The postreplication repair inhibitor caffeine had no effect on aberration yields induced by either enzyme. Neither aphidicolin nor caffeine modulated the amount of DNA double-strand breakage as measured by PFGE. These data implicate poly(ADP-ribosyl)ation and polymerases alpha and delta as important components of the cellular processes required for the normal repair of DNA double-strand breaks with blunt or cohesive ends. Comparison of these data with the effect of inhibitors on the frequency of X-ray-induced aberrations leads us to the conclusion that X-ray-induced aberrations can result from the misjoining or nonrejoining of double-strand breaks, particularly breaks with cohesive ends, but that this process accounts for only a portion of the induced aberrations.     

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.     

Cytogenetical characterization of Chinese hamster ovary X-ray-sensitive mutant cells xrs 5 and xrs 6. VII. Complementation analysis of X-irradiated wild-type CHO-K1 and xrs mutant cells using the premature chromosome condensation technique

The complementation effect of wild-type CHO-K1 and xrs mutants after fusion, as judged by the frequencies of X-ray-induced G1 and G2 premature chromosome condensation (PCC), was studied. For induction of PCC, X-irradiated interphase cells (G1 and G2) were fused immediately with untreated mitotic cells of the same cell line or with mitotic cells of another line. The frequencies of breaks in G1-PCC, or breaks and chromatid exchanges in G2-PCC were determined and the latter parameter was compared with the frequency of chromosomal aberrations in mitotic cells following G2 irradiation. CHO-K1 cells were capable of complementing the X-ray sensitivity of both xrs 5 and xrs 6 cells. However, full restoration of the repair defect in xrs cells could never be accomplished. The mutants failed to complement each other. In CHO-K1 cells, the incidence of chromosomal aberrations was significantly higher in G2-PCC (2.5-fold) than that observed in mitotic cells at 2.5 h after irradiation. The ratio of the induced frequency of aberrations in G2-PCC to that in mitotic cells was correlated with the degree of repair of DNA double-strand breaks (dsb) and reached almost 1 in xrs 5 cells indicating no repair. In addition the data indicated that, during the period of recovery of CHO-K1 cells, X-ray-induced breaks decreased but exchanges remained at the same level. In contrast, due to a deficiency in rejoining of dsb in xrs mutants, breaks remained open for a long period of time, allowing the formation of additional chromatid exchanges during recovery time.     

Initial chromosome damage but not DNA damage is greater in ataxia telangiectasia cells.

Cells derived from individuals with ataxia telangiectasia (AT) exhibit increased sensitivity to ionizing radiation and certain drugs (e.g., bleomycin, neocarzinostatin, and etoposide) as evidenced by decreased survival and increased chromosome aberrations at mitosis when compared with normal cell lines. To understand better the basis of this sensitivity, three AT and two normal lymphoblastoid cell lines were fractionated into cell cycle phase-enriched populations by centrifugal elutriation and then examined for their survival and their relative initial levels of DNA damage (neutral DNA filter elution) and chromosome damage (premature chromosome condensation). AT cells exhibited decreased levels of survival in all phases of the cell cycle; however, AT cells in early G1 phase were especially sensitive compared with normal cells in G1 phase. While AT and normal cells exhibited similar levels of initial DNA double-strand breaks in exponential populations as well as throughout the cell cycle, AT cells showed nearly twofold higher initial levels of chromosome damage than normal control cells in G1 and G2 phase. These results suggest that there is a higher rate of conversion of DNA double-strand breaks into chromosome breaks in AT cells, perhaps due to a difference in chromatin organization or stability. Thus one determining component of cellular radiosensitivity might include chromatin structure.     

Suppressing effect of antimutagenic flavorings on chromosome aberrations induced by UV-light or X-rays in cultured Chinese hamster cells

Chromosome aberrations induced by UV-light or X-rays were suppressed by the post-treatment with antimutagenic flavorings, such as anisaldehyde, cinnamaldehyde, coumarin, and vanillin. UV- or X-ray-irradiated surviving cells increased in the presence of each flavoring. X-ray-induced breakage-type and exchange-type chromosome aberrations were suppressed by the vanillin treatment in the G1 phase of the cell cycle and a greater decrease in the number of X-ray-induced chromosome aberrations during G1 holding was observed in the presence of vanillin. Furthermore, a greater decrease in the number of X-ray-induced DNA single-strand breaks was observed in the presence of vanillin. Treatment with vanillin in the G2 phase suppressed UV- and X-ray-induced breakage-type but not exchange-type chromosome aberrations. The suppression of breakage-type aberrations was assumed to be due to a modification of the capability of the post-replicational repair of DNA double-strand breaks. These G1- and G2-dependent anticlastogenic effects were not observed in the presence of 2',3'-dideoxythymidine, an inhibitor of DNA polymerase beta. Based on these results, the anticlastogenic effect of vanillin was considered to be due to the promotion of the DNA rejoining process in which DNA polymerase beta acts.     

Induced repair of DNA double-strand breaks at the G1/S-phase border

Exposure of human cells to ionizing radiation at the G1/S-phase border of the cell cycle leads to the production of repair patches of 3 nucleotides, representing the constitutive repair response, and very long repair patches (VLRP) of at least 150 nucleotides, representing an induced response. We examined the type of DNA damage that may signal this induced repair response using two chemicals that produce subsets of the damage induced by ionizing radiation. Treatment of cells at the G1/S-phase border with bleomycin, which produces a high proportion of DNA double-strand breaks, also leads to the production of VLRP of at least 130 nucleotides. In contrast, when cells were treated with hydrogen peroxide, which produces base modifications and single-strand breaks, no VLRP were observed. Thus it would appear that DNA double-strand breaks are the signal that leads to the induction of the VLRP. We also examined the relationship between the induced repair response and DNA replication. When cells are treated with hydroxyurea, under conditions that inhibit more than 98% of the DNA synthesis, prior to exposure to 5 Gy, repair patches of 3 and 150 nucleotides are found. This indicates that the longer repair patches are not a result of aberrant DNA replication. However, when cells are treated with the DNA polymerase inhibitor aphidicolin in combination with hydroxyurea and cytosine arabinoside, no induced long patches are found. These results indicate that DNA polymerase alpha, delta or epsilon is required for the synthesis of the VLRP.     

Enhanced induction by X-irradiation of DNA double-strand breakage in mitotic as compared with S-phase V79 cells

A marked enhancement of X-ray-induced DNA double-strand breakage was demonstrated for mitotic Chinese hamster (V79) cells compared with S-phase cells or asynchronous cultures, which can explain the high radiosensitivity of mitotic cells. However, mitotic cells required twice the level of DNA double-strand breakage to produce a lethal lesion of that required for S-phase or asynchronous cells, suggesting that repair in mitotic cells is less error prone.     

Defective repair of DNA single- and double-strand breaks in the bleomycin- and X-ray-sensitive Chinese hamster ovary cell mutant, BLM-2

Using filter elution techniques, we have measured the level of induced single- and double-strand DNA breaks and the rate of strand break rejoining following exposure of two Chinese hamster ovary (CHO) cell mutants to bleomycin or neocarzinostatin. These mutants, designated BLM-1 and BLM-2, were isolated on the basis of hypersensitivity to bleomycin and are cross-sensitive to a range of other free radical-generating agents, but exhibit enhanced resistance to neocarzinostatin. A 1-h exposure to equimolar doses of bleomycin induces a similar level of DNA strand breaks in parental CHO-K1 and mutant BLM-1 cells, but a consistently higher level is accumulated by BLM-2 cells. The rate of rejoining of bleomycin-induced single- and double-strand DNA breaks is slower in BLM-2 cells than in CHO-K1 cells. BLM-1 cells show normal strand break repair kinetics. The level of single- and double-strand breaks induced by neocarzinostatin is lower in both BLM-1 and BLM-2 cells than in CHO-K1 cells. The rate of repair of neocarzinostatin-induced strand breaks is normal in BLM-1 cells but retarded somewhat in BLM-2 cells. Thus, there is a correlation between the level of drug-induced DNA damage in BLM-2 cells and the bleomycin-sensitive, neocarzinostatin resistant phenotype of this mutant. Strand breaks induced by both of these agents are also repaired with reduced efficiency by BLM-2 cells. The neocarzinostatin resistance of BLM-1 cells appears to be a consequence of a reduced accumulation of DNA damage. However, the bleomycin-sensitive phenotype of BLM-1 cells does not apparently correlate with any alteration in DNA strand break induction or repair, as analysed by filter elution techniques, suggesting an alternative mechanism of cell killing.     

Cohesin and DNA damage repair

Replicated DNA molecules are physically connected by cohesin complexes from the time of their synthesis in S-phase until they are segregated during anaphase of the subsequent mitosis or meiosis. This sister chromatid cohesion is essential for the biorientation of chromosomes on the mitotic or meiotic spindle. In addition, cohesion is also essential during G2-phase of the cell cycle to allow repair of DNA double-strand breaks by homologous recombination. Although cohesion can normally only be established during S-phase, recent work in yeast has shown that DNA double-strand breaks induce the recruitment of cohesin to the damage site and lead to the de novo formation of cohesion at this site. It is unknown if similar mechanisms operate in higher eukaryotes, but in mammalian cells phosphorylation of the cohesin subunit Smc1 by the protein kinase Atm has been shown to be important for DNA repair. We discuss how cohesin and sister chromatid cohesion might facilitate the repair of damaged DNA.     

Ionizing radiation damage to DNA: molecular aspects

Radioresistant tumor cells are found in tumor specimens from patients in whom radiotherapy has failed or whose tumors have recurred after therapy. This suggests that inherent cellular radioresistance may in part underlie the failure of radiotherapy, and therefore determination of the presence of resistant cells within a tumor might be a useful predictor of response to radiation therapy. Most standard clonogenic assays of radiation response are time-consuming, and alternative assays of radiation response are being sought. In an earlier publication (J. L. Schwartz et al., Int. J. Radiat. Oncol. Biol. Phys. 15, 907-912, 1988), we reported that radioresistant human tumor cells rejoin DNA double-strand breaks, as measured by DNA neutral filter elution (pH 9.6), faster than more sensitive cell lines. To determine whether DNA elution might have potential as a rapid predictive assay, we examined the relationship between the rate of DNA double-strand break rejoining and radiosensitivity in nine first-passage-after-explant squamous cell carcinomas under conditions that minimized the influence of nontumor and nonclonogenic cells. The frequency of DNA double-strand breaks measured 1 h after irradiation with 100 Gy 60Co gamma rays was used as an estimate of relative rejoining rate. This number is a reflection of both the initial DNA double-strand break frequency and the amount of repair that occurs in 1 h. The relative break frequency was compared to radiosensitivity as measured by standard clonogenic survival assays in later passages (p3-p14) of these same cells. A significant relationship (r = 0.61, P less than 0.01) was found between break frequency measured in first-passage cells and radiosensitivity measured in later passages, suggesting that the neutral elution assay as described here has some promise as a relatively rapid assay of the radiosensitivity of human tumor cells.     

Studies on mutagen-sensitive strains of Drosophila melanogaster. X. Repair of radiation-induced DNA damage in primary cell cultures after irradiation with X-rays

The repair of X-ray-induced DNA lesions in repair-deficient mutant strains was studied as a way of investigating the mechanism of the induction of genetic damage. Genetic effects on the recovery of X-ray-induced damage by the repair-deficient strains ebony (photoreactivation repair-deficient) and mus(1)101D1 (post-replication repair-deficient) were interpreted as impaired repair of single- and double-strand DNA breaks. We investigated the repair of X-ray-induced DNA breaks and alkaline-labile sites in primary cell cultures of ebony and mus(1)101D1 and in cultures of their control strains. No significant differences were found between the repair rates in the mutants and control strains. This indicates that the genetic effects of these mutants are not due to an impaired rate of repair of DNA breaks.     

Biochemical and cytogenetical characterization of Chinese hamster ovary X-ray-sensitive mutant cells xrs 5 and xrs 6. VI. The correlation between UV-induced DNA lesions and chromosomal aberrations, and their modulations with inhibitors of DNA repair synthesis

The role of UV-induced DNA lesions and their repair in the formation of chromosomal aberrations in the xrs mutant cell lines xrs 5 and xrs 6 and their wild-type counterpart, CHO-K1 cells, were studied. The extent of induction of DNA single-strand breaks (SSBs) and DNA double-strand breaks (DSBs) due to UV irradiation in the presence or absence of 1-beta-D-arabinofuranosylcytosine (ara-C) and hydroxyurea (HU) was determined using the alkaline and neutral elution methods. Results of these experiments were compared with the frequencies of induced chromosomal aberrations in UV-irradiated G1 cells treated under similar conditions. Xrs 6 cells showed a defect in their ability to perform the incision step of nucleotide repair after UV irradiation. Accumulation of breaks 2 h after UV irradiation in xrs 6 cells in the presence of HU and ara-C remained at the level of incision breaks estimated after 20 min, which was about 35% of that found in wild-type CHO-K1 cells. In UV-irradiated CHO-K1 and xrs 5 cells, more incision breaks were present after 2 h compared with 20 min post-treatment with ara-C, a further increase was evident when HU was added to the combined treatment. The level of incision breaks induced under these conditions in xrs 5 was about 80% of that observed in CHO-K1 cells. UV irradiation itself did not induce any detectable DNA strand breaks. Accumulation of SSBs in UV-irradiated cells post-treated with ara-C and HU coincides with the increase in the frequency of chromosomal aberrations. These data suggest that accumulated SSBs when converted to DSBs in G1 give rise to chromosome-type aberrations, whereas strand breaks persisting until S-phase result in chromatid-type aberrations. Xrs 6 appeared to be the first ionizing-radiation-sensitive mutant with a partial defect in the incision step of DNA repair of UV-induced damage.     

A calmodulin antagonist has no effect on the repair of X-ray-induced damage in a murine mammary carcinoma cell line

The effects of the calmodulin antagonist W13 were determined on potentially lethal damage repair, sublethal damage repair, and X-ray-induced DNA damage repair following X irradiation of 67 murine mammary carcinoma cells in the proliferative and quiescent states. Studies with W13 (20 micrograms/ml) on proliferating cells showed that the cells rounded up within 2 h but stayed attached to the dishes and there was a slight transient G2 block by 6 h. Also, the proportion of S-phase cells at 12 h was reduced to 65% of control with the concurrent [3H]thymidine incorporation reduced to 62% of control. There was no detectable effect from this pharmacological dose of W13 either on PLDR in proliferating cells at 400 and 800 rad or on quiescent cells at 200 and 400 rad. Likewise, there was no measurable effect on SLDR in either proliferating or quiescent cells at equally split doses of 800 and 600 rad, respectively. In addition, for control vs W13-treated proliferating cells, no difference was detected either in the induction of DNA damage by X irradiation or in the initial rate of repair (T 1/2 approximately equal to 7 min), as measured by the alkaline filter elution assay. In contrast to uv and bleomycin-induced damage, these data suggest that calmodulin may have no major role in either the molecular or cellular recovery from X-ray-induced damage in mammalian cells.     

Effects of trypsin on X-ray-induced cell killing, chromosome abnormalities and kinetics of DNA repair in mammalian cells

When cells are trypsinized before irradiation a potentiation of X-ray damage may occur. This is known as the 'trypsin effect'. Potentiation of X-ray damage on cell killing was seen in V79 Chinese hamster cells but was marginal in Chinese hamster ovary (CHO K1) cells and not evident in murine Ehrlich ascites tumour (EAT) cells. Trypsinization did however increase the number of X-ray-induced chromosomal abnormalities in all 3 lines. To investigate the possibility that trypsin acts by digestion of proteins in chromatin, further experiments were performed to monitor DNA damage and repair. Induction of DNA breaks by X-rays was unaffected by trypsin but trypsinized EAT (suspension) cells repaired single-strand breaks (ssb) less rapidly than controls indicating an inhibitory effect of trypsin on ssb repair. However double-strand break (dsb) repair was unaffected by trypsin. It was also found that the EDTA solution in which the trypsin was dissolved also contributes to the inhibition of dsb repair. The results show that trypsinization can enhance X-ray-induced cell killing, chromosomal damage and DNA repair, the effect varying between cell lines.     

Radiation-induced cytotoxicity,DNA damage and DNA repair: Implications for cell survival theory

Summary The radiosensitivities and the kinetics for removal of radiation-induced DNA damage were compared for proliferative (P) and quiescent (Q) cells of the lines 66 and 67 derived from a mouse mammary adenocarcinoma. As determined from cell survival assays, the 66 and 67 Q cells were more radiosensitive than their 66 and 67 P counterparts. The rank order of their radiosensitivity was: 67 Q > 66 Q 67 P > 66 P. Induction of radiation damage in the DNA of these cells, as measured by the alkaline elution technique, was identical for 66 and 67 P and Q cells. The repair of this DNA damage was biphasic for 66 and 67 P and Q cells. The half-times for the fast and slow repair phases in 66 Q cells were identical to those previously measured in 67 Q cells. The half-times of the fast and slow repair phases in 66 P cells were also identical to those previously measured in 67 P cells. However, the half-times for the fast and slow repair phases in 66 and 67 Q cells were longer than those measured in their 66 and 67 P counterparts. The 66 cell data are consistent with our previously published hypothesis that Q cells are more radiosensitive than their corresponding P cells because they repair their radiation-induced DNA damage slower. However, our results are not consistent with hypotheses that attempt to explain the radiosensitivity differences between lines 66 and 67 solely on the basis of measurable induction and repair of DNA damage.     

A novel cell-cycle-regulated interaction of the Bloom syndrome helicase BLM with Mcm6 controls replication-linked processes

The Bloom syndrome DNA helicase BLM contributes to chromosome stability through its roles in double-strand break repair by homologous recombination and DNA replication fork restart during the replication stress response. Loss of BLM activity leads to Bloom syndrome, which is characterized by extraordinary cancer risk and small stature. Here, we have analyzed the composition of the BLM complex during unperturbed S-phase and identified a direct physical interaction with the Mcm6 subunit of the minichromosome maintenance (MCM) complex. Using distinct binding sites, BLM interacts with the N-terminal domain of Mcm6 in G1 phase and switches to the C-terminal Cdt1-binding domain of Mcm6 in S-phase, with a third site playing a role for Mcm6 binding after DNA damage. Disruption of Mcm6-binding to BLM in S-phase leads to supra-normal DNA replication speed in unperturbed cells, and the helicase activity of BLM is required for this increased replication speed. Upon disruption of BLM/Mcm6 interaction, repair of replication-dependent DNA double-strand breaks is delayed and cells become hypersensitive to DNA damage and replication stress. Our findings reveal that BLM not only plays a role in the response to DNA damage and replication stress, but that its physical interaction with Mcm6 is required in unperturbed cells, most notably in S-phase as a negative regulator of replication speed.     

TNFalpha induces chromosomal abnormalities independent of ROS through IKK, JNK, p38 and caspase pathways

A role for pro-inflammatory cytokines in inflammation-related cancers has been suggested, but mechanisms are not defined. Here, we demonstrate that treatment of HeLa cells with TNFalpha increases chromosomal aberration. In contrast, IL-1beta did not increase, but rather decreased chromosomal aberration. TNFalpha and IL-1beta increased the production of H2O2 to similar levels in cells, suggesting that increased production of reactive oxygen species might not be the premier factor involved. Reducing H2O2 through overexpression of catalase or treatment of cells with NAC or BHA did not have an effect on TNF-induced chromosomal aberration. TNFalpha-induced NO production has been implicated in DNA damage. Inhibiting NO did not reduce TNF-induced chromosomal aberration. Inhibiting IKK, JNK, and p38 kinase as well as caspases decreased TNF-induced chromosomal aberration, and a correlation between TNF-induced apoptosis and CA generation was not found. Single-strand DNA breaks give rise to double-strand breaks, which then results in chromosomal breaks, when replication forks reach the single-strand breaks during S-phase. In cells progressing through S-phase, TNFalpha activation of IKK, JNK, and p38 is significantly reduced. However, these kinases were activated by IL-1beta in S-phase. The possibility that these pathways, in a TNF-specific manner, may regulate either the generation of single- and double-strand breaks or their repair, thereby resulting in increased chromosomal aberration, is discussed.     

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.