Alkaline Unwinding Technique Research Articles

FGF-10 prevents mechanical stretch-induced alveolar epithelial cell DNA damage via MAPK activation.

Cyclic stretch of alveolar epithelial cells (AEC) can alter normal lung barrier function. Fibroblast growth factor-10 (FGF-10), an alveolar type II cell mitogen that is critical for lung development, may have a role in promoting AEC repair. We studied whether cyclic stretch induces AEC DNA damage and whether FGF-10 would be protective. Cyclic stretch (30 min of 30% strain amplitude and 30 cycles/min) caused AEC DNA strand break formation, as assessed by alkaline unwinding technique and DNA nucleosomal fragmentation. Pretreatment of AEC with FGF-10 (10 ng/ml) blocked stretch-induced DNA strand break formation and DNA fragmentation. FGF-10 activated AEC mitogen-activated protein kinase (MAPK), and MAPK inhibitors prevented FGF-10-induced AEC MAPK activation and abolished the protective effects of FGF-10 against stretch-induced DNA damage. In addition, a Grb2-SOS inhibitor (SH(3)b-p peptide), a RAS inhibitor (farnesyl transferase inhibitor 277), and a RAF-1 inhibitor (forskolin) each prevented FGF-10-induced extracellular signal-regulated kinase (ERK) 1/2 phosphorylation in AEC. Moreover, N17-A549 cells that express a RAS dominant/negative protein prevented the FGF-10-induced ERK1/2 phosphorylation and RAS activation in AEC. We conclude that cyclic stretch causes AEC DNA damage and that FGF-10 attenuates these effects by mechanisms involving MAPK activation via the Grb2-SOS/Ras/RAF-1/ERK1/2 pathway.

Relationship between cellular radiosensitivity and DNA damage measured by comet assay in human normal, NBS and AT fibroblasts

Purpose : To study the relationship between cellular radiosensitivity and DNA damage measured by the comet assay. Materials and methods : Experiments were performed with nine human fibroblast lines (six normal, one NBS, and two AT). Cellular radiosensitivity was determined by colony assay and DNA damage was assessed by the comet assay. Results : The cellular radiosensitivity of the fibroblast lines used covered a broad range with SF2 values varying between 1.3% and 53%. The comets analysed immediately after irradiation with doses up to 5 Gy showed marked differences among the cell lines; the relative initial tail moment at a dose of 5 Gy, ITM5, varied from 2.7 ±0.2 to 5.0 ±0.3. This variation was considered not to result from different numbers of DNA strand breaks induced but from differences in chromatin structure. There was an inverse correlation between SF2 and ITM5, i.e. radiosensitive cell lines exhibited a higher initial tail moment than radioresistant cell lines. In contrast, the repair kinetics measured with the comet assay for a dose of 2Gy followed by an incubation of up to 2h showed little variation and were found not to correlate with SF2. Repair kinetics as well as the amount of residual damage measured by this version of the comet assay were fairly similar to those measured by the alkaline unwinding technique and unlike that measured by neutral gel electrophoresis, indicating that this comet assay detects primarily single-strand breaks and alkali-labile sites, not double-strand breaks. Conclusions : The correlation between SF2 and the initial tail moment at 5 Gy found here suggests that the cellular radiosensitivity of human fibroblasts also depends on the chromatin structure.

Repair of Radiation-Induced DNA Strand Breaks Does Not Occur Preferentially in Transcriptionally Active DNA

Certain DNA base lesions induced by ionizing radiation or oxidative stress are repaired faster from the transcribed strand of active genes compared to the genome overall. In this study, it was investigated whether radiation-induced DNA strand breaks are preferentially repaired in active genes compared to the genome as a whole in CHO cells. The alkaline unwinding technique coupled to slot-blot hybridization with specific DNA probes was used to study the induction and repair of DNA strand breaks in defined DNA sequences. Results using this technique showed a linear dose response for the formation of radiation-induced DNA strand breaks in the dihydrofolate reductase (DHFR) gene. Furthermore, the half-life of radiation-induced strand breaks was less than 5 min in the DHFR gene, in the ribosomal genes, and in the genome as a whole. These results suggest that the repair of DNA strand breaks is fast and uniform in the genome of mammalian cells.

Correlation between cellular radiosensitivity and non-repaired double-strand breaks studied in nine mammalian cell lines

Purpose: To test the relationship between cell killing and nonrepaired DNA strand breaks both in repair proficient and deficient cell lines. Materials and methods: Five of the cell lines used are repair competent (CHO, CHO K1, rat rhabdomyosarcoma R1H, mouse balb and normal human fibroblasts), while four display a reduced repair capacity (scid, xrs 1, xrs 5, AT). Cell survival was determined by colony formation assay. The total number of strand breaks was measured by the alkaline unwinding technique and the numbers of double-strand breaks by constant-field gel electrophoresis. Results: The nine cell lines showed a broad spectrum in radiosensitivity with SF values ranging from 0.018 to 0.58. The cell lines 2 did not vary in the number of induced strand breaks, neither for all strand breaks nor for double-strand breaks alone. In contrast, there was a large variation in the number of non-repaired strand breaks measured 24 h after irradiation. Comparison of cell killing with the number of non-repaired breaks measured after a dose of 90 Gy showed no correlation for single-strand breaks (r 2 0.29) but a fairly good correlation for double-strand breaks (r 2 0.87). This correlation was found to hold both for repair proficient and deficient cell lines. Conclusions: The results obtained strongly suggest that the number of non-repaired double-strand breaks measured 24 h after irradiation can be used as an indicator of cellular radiosensitivity.

Disassociation of oxidant-induced ATP depletion and DNA damage from early cytotoxicity in LLC-PK1 cells.

To determine the mechanism(s) of oxidant-mediated cell lysis in renal tubular epithelial cells, we determined ATP depletion, DNA damage, lipid peroxidation, and cytotoxicity in LLC-PK1 cells exposed to 500 microM hydrogen peroxide for 1 h with and without inhibitors of lipid peroxidation including a lazaroid compound, 2-methylaminochroman (2-MAC), and Trolox, a vitamin E analog. ATP levels were determined by luciferin-luciferase, DNA damage by the alkaline unwinding technique, lipid peroxidation by the generation of malondialdehyde, and early cytotoxicity (5 h) by the release of 51Cr, whereas late cytotoxicity (24 h) was determined by release of [3H]leucine from prelabeled cells. Cells exposed to 500 microM hydrogen peroxide demonstrated significant (P < 0.01) ATP depletion, DNA damage, and lipid peroxidation, followed by cell death at 5 h. Concentrations of 0.1-25 microM 2-MAC or 25-500 microM Trolox each markedly and significantly (P < 0.01) inhibited lipid peroxidation and early cytotoxicity but had little to no effect on ATP depletion or DNA damage. Thus oxidant-stressed cells remained intact for several hours despite significant ATP depletion and DNA damage when lipid peroxidation was inhibited with the antioxidant compounds. At 24 h, 2-MAG and Trolox had lost their protective effect, suggesting that mechanisms other than lipid peroxidation play a role in later cytotoxicity. We conclude that ATP depletion and DNA damage are not the primary mediators of early cytotoxicity following oxidant stress, whereas lipid peroxidation plays an central role in mediating early cytotoxicity following oxidant injury.

Cobalt(II) inhibits the incision and the polymerization step of nucleotide excision repair in human fibroblasts

Compounds of cobalt are carcinogenic to experimental animals, but the mutagenicity in mammalian cells in culture is rather weak. In contrast, cobalt(II) has been shown to inhibit the removal of DNA damage induced by UVC light, indicating an interference with cellular DNA repair processes. In the present study it was investigated which step of the nucleotide excision repair is affected by cobalt(II) and which mechanisms are involved. In this context, the effect of non-cytotoxic cobalt(II) concentrations on the induction as well as on the repair of UVC-induced DNA lesions has been examined in human fibroblasts by using the alkaline unwinding technique under various conditions. Cobalt(II) concentrations as low as 50 μM inhibit the incision as well as the polymerization step. In contrast, the ligation of repair patches is not disturbed by this metal. By combining the alkaline unwinding technique with the repair enzyme T4 endonuclease V, it is demonstrated that the incision at the site of cyclobutane pyrimidine dimers is affected at concentrations of 150 μM and higher. As one mode of action, the competition with essential magnesium(II) ions by cobalt(II) ions could be identified.

Sensitive analysis of oxidative DNA damage in mammalian cells: use of the bacterial Fpg protein in combination with alkaline unwinding

The measurement of oxidative DNA base modifications by different methods has received special attention in recent years. Here we describe a procedure to quantify DNA lesions recognized by the bacterial formamido-pyrimidine-DNA glycosylases (Fpg protein). These include 7,8-dihydro-8-oxoguanine (8-hydroxyguanine) as well as some other forms of imidazole ring-opened purines, which are converted into abasic sites and subsequently into DNA single-strand breaks by the associated endonuclease activity. The frequency of DNA strand breaks is determined by the alkaline unwinding technique. The procedure provides a fast and sensitive tool to assess the extent of spontaneous as well as induced oxidative DNA damage in mammalian cells.

Rejoining of DNA double-strand breaks in X-irradiated CHO cells studied by constant- and graded-field gel electrophoresis.

Induction and repair of double-strand breaks (dsb) were measured in exponentially growing CHO-10A cells using the constant- and graded-field gel electrophoresis. Dsb repair was studied after an X-ray dose of 60 Gy. The repair curve obtained was biphasic with the respective half-times of tau 1 = 3.8 +/- 0.9 and tau 2 = 118 +/- 30 min. The number of non-reparable dsb was measured for X-ray doses up to 180 Gy and was found to be only a small fraction (14%) of all non-rejoinable breaks determined previously using the alkaline unwinding technique. The ratio of non-reparable dsb to the number of lethal events calculated from survival curves is 0.14:1. This result indicates that for CHO cells nonreparable dsb represent only a small fraction of lethal damage. This is in line with the cytogenetic observation that cell killing mainly results from mis-rejoined events (i.e. exchange aberrations, translocations, interstitial deletions). The kinetics of dsb rejoining were found to be independent of the size of the fragments involved (between 1 and 10 Mbp). In addition, the rejoining kinetics of DNA fragments < or = 1 Mbp did not show the formation of new DNA fragments with time after irradiation indicating the absence of programmed cell death in irradiated CHO cells.

DNA damage in lung cells in vivo and in vitro by 1,3-butadiene and nitrogen dioxide and their photochemical reaction products

A UV-irradiated mixture of 1,3-butadiene and nitrogen dioxide (NO 2) was tested for its potency to induce DNA damage measured as single-strand breaks (SSB) in lungs of mice. Both gases were also tested separately. After 16 h exposure a UV-irradiated mixture of 40 ppm butadiene + 20 ppm NO 2, but not 20 ppm butadiene + 10 ppm NO 2 + UV, induced a significant increase in SSB as measured by the alkaline unwinding technique. There was no increase in the level of SSB using the alkaline elution technique during the same testing conditions. However, after 5 h exposure to 60 ppm butadiene + 30 ppm NO 2 + UV both methods demonstrated a significant increase in SSB. Mice were also exposed to butadiene at 80 and 200 ppm for 16 h and at 500 ppm for 5 h. DNA damage was demonstrated in both liver and lung after 5 and 16 h (only at 200 ppm) of exposure using the unwinding technique. Using the alkaline elution assay, a significant increase in the level of SSB in lung and liver was found only after 5 h of exposure. When mice were exposed to 30 ppm NO 2 for 16 h or 50 ppm for 5 h, a significant increase in SSB was found with the unwinding technique. Alveolar macrophages from mice were also exposed in vitro to the gas mixture and to butadiene and NO 2 separately. In these experiments, the DNA damage was studied with the unwinding technique. A significant effect was demonstrated with 40 ppm butadiene + 20 ppm NO 2 + UV. NO 2 itself contributed to some extent to the increase. Reasons for the discrepancies between the unwinding and the alkaline elution techniques are discussed.

Correlation between thermal radiosensitization and slowly rejoined DNA strand breaks in CHO cells.

The effect on the repair of slowly rejoined strand breaks was studied in CHO cells using the alkaline unwinding technique. Heat (45 degrees C, 20 min) combined with a X-ray dose of 9 Gy was found to result in an increased half-time of repair but also in an increased number of slowly rejoined strand breaks. When a time interval at 37 degrees C was inserted between irradiation and heat, the half-time of repair was not altered, whereas the number of slowly rejoined strand breaks as measured 300 min after irradiation decreased with increasing time interval between the two treatments. The half-time of 18 +/- 2 min suggested that the additionally formed, slowly rejoined strand breaks arise from a certain type of radiation-induced DNA base lesions with repair of which is modified by heat. The effect of X-irradiation combined with heat was also studied for cell survival. When irradiation and heat were separated by an incubation at 37 degrees C, cell survival increased with a half-time of 20 +/- 2 min, which is similar to that measured for the number of additional, slowly rejoined strand breaks. For a great variety of combined treatments, the reduction in cell survival correlates well with the enhanced number of slowly rejoined strand breaks measured 300 min after irradiation. This positive correlation and the similarity in the half-times mentioned above suggests that thermal radiosensitization results from the number of additional, slowly rejoined strand breaks formed when irradiation was combined with heat.

Relationship between non-reparable DNA strand breaks and cell survival studied in X-irradiated CHO, CHO K1, xrs1 and xrs5 cells.

Non-reparable DNA strand breakage was measured by means of alkaline unwinding technique in CHO, CHO K1, xrs1 and xrs5 cells for X-ray doses ranging from 15 to 180 Gy. For comparison, cell survival was recorded for doses up to 9 Gy using the colony forming assay. DNA strand breaks determined 24 h after irradiation were considered as non-preparable, since it was shown previously that repair processes are completed by 20 h after irradiation and the level reached remained constant for at least another 10 h. The number of non-reparable strand breaks was found to increase with dose. This increase was much steeper for xrs1 and xrs5 cells as compared with that for the parental strain CHO K1. This difference correlates with the respective cellular radiosensitivities. For the three cell lines the ratio of non-reparable strand break/lethal event is about 15:1. For the original CHO cells, which showed the same radiosensitivity as K1 cells, much less non-reparable breaks were found when compared with K1 cells. For CHO cells the ratio of residual strand breaks/lethal event is about 1:1. From these data it is concluded that about one non-reparable DNA strand break is sufficient for cell kill, and that the higher number of non-reparable breaks found for CHO K1 cells and xrs mutants results from unrejoined breaks which do not affect lethality. These breaks might result from DNA degradation.

Non-reparable DNA strand breaks and cell killing studied in CHO cells after X-irradiation at different passage numbers.

Non-reparable DNA strand breaks were measured in X-irradiated CHO cells by means of the alkaline unwinding technique and were compared with cell survival measured by the colony assay. The experiments were performed with cells at passage numbers 10, 50 and 110 after thawing from stock culture. Cellular radiosensitivity was found to be identical for all three passage numbers used. By contrast, the dose-response of non-reparable DNA strand breaks was only the same for passage numbers 10 and 50 but significantly steeper for cells irradiated in passage number 110. The ratio of non-reparable breaks to lethal events, as calculated from the survival curves, was found close to 1:1 for cells irradiated at passage numbers 10 and 50 but increased to 20:1 at passage number 110. These data indicate that the number of non-reparable strand breaks measured after irradiation not only depends on cellular radiosensitivity but also on other parameters such as the age of the cell culture.

Saturated and unsaturated repair of DNA strand breaks in CHO cells after X-irradiation with doses ranging from 3 to 90 Gy.

The kinetics of DNA strand break repair was studied in exponentially-growing CHO cells after X-irradiation with doses of 3, 9, 30, 60 and 90 Gy. DNA strand breaks were measured using the alkaline unwinding technique. For all X-ray doses applied the kinetics of DNA strand break repair consisted of fast, intermediate and slow phases. The latter, which was interpreted as the repair kinetic of DNA double-strand breaks, was best described by an exponential decline. The actual repair half-time of double-strand break repair, tau dsb, was obtained from the slope of the slow component after subtracting the number of non-reparable breaks measured 24 h after irradiation. This half-time was found to be independent of the dose applied with a mean value of tau dsb = 168 +/- 10 min. This result indicated that the repair of double-strand breaks was unsaturated for doses up to 90 Gy. The repair kinetics of the breaks of the fast and intermediate phases were found to be dependent on the dose applied. These kinetics were associated with the induction and repair of primary and secondary single-strand breaks, the latter possibly generated by enzymatic incision at damaged bases. Analysis of these curves using the Michaelis-Menten equation showed that the half-time of enzymatic incision, tau in, and the half-time at which both primary and secondary single-strand breaks were rejoined, tau rep, varied with the amount of damage present in the cell. tau in Increased from a minimum value tau in,min = 13 +/- 2 min proportionally to the number of base damage with a rate of alpha in = 0.138 +/- 0.015 min Gy-1, and tau rep from a minimum value tau rep,min = 1.4 +/- 0.2 proportionally to the number of single-strand breaks with a rate of alpha rep = 0.038 +/- 0.001 min Gy-1. The enzymatic incision was unsaturated for doses up to about 30 Gy, whereas the repair of single-strand breaks was unsaturated only for doses up to about 10 Gy. Up to these doses the increase in the half-time tau in and tau rep was so small that, within the range of experimental errors, the parameters may be approximated by constant values. From the results it is concluded that for CHO cells the continuously-bending dose-response curve obtained for radiation-induced killing cannot be attributed to a saturated repair.

Reparable and non-reparable DNA strand breaks induced by X-irradiation in CHO K1 cells and the radiosensitive mutants xrs1 and xrs5.

Repair kinetics of X-ray-induced DNA lesions was measured for CHO K1, xrs1 and xrs5 cells using the alkaline unwinding technique. Cells were irradiated on ice with X-ray doses of 9, 30 and 90 Gy followed by repair incubation at 37 degrees C. Repair was studied for up to 60 h to cover the kinetics of reparable and non-reparable DNA damage. The kinetics of single-strand break (ssb) repair was found to be the same in CHO K1 and xrs cells. In contrast, the number of double-strand breaks (dsbs) was shown to decline with half-times that were longer for xrs1 and xrs5 as compared with CHO K1 cells (12.2 and 15.0 h versus 4.9 h for 90 Gy). These differences are primarily due to differences in the fraction of non-reparable dsbs which was higher by a factor of 3-4 for xrs1 and xrs5 cells as compared with K1 cells (3.2 and 3.7% versus 1.0% for 90 Gy). However, the kinetics of those dsbs which were actually repaired did not show any significant differences for the three cell lines studied and were rejoined at a mean half-time of 3.8 h. It is suggested that the higher number of non-reparable breaks in xrs cells may not be attributable to a deficient enzymatic repair system. It is suggested that alterations in chromatin conformation might prevent rejoining of a portion of dsbs.

Hydroxyurea-induced cell death in human T lymphoma cells as related to imbalance in DNA/protein cycle and deoxyribonucleotide pools and DNA strand breaks.

The aim of this study was to elucidate the mechanism(s) behind the cellular toxicity of therapeutic concentrations of hydroxyurea (HU). Treatment of human T lymphoma cells (CCRF-CEM) with 60-100 microM of HU for 24 h decreased the growth rate by 90% due to accumulation of cells in early S phase. It induced a marked imbalance in both the DNA/protein cycle (as measured by two-parameter flow cytometry) and the deoxyribonucleotide (dNTP) pools. HU treatment did not enhance the frequency of DNA single-strand breaks (SSBs), as measured by the alkaline unwinding technique. Cell viability was unaffected. However, removal of HU led to 10-15% cell loss during the following 12 h period in parallel with increasing SSBs, and a rapid progression of cells through S and G2 stages. The unbalanced DNA to protein content per cell and the dNTP pools were normalized 6-12 and 24 h after removal of HU, respectively. These results show that marked changes in the DNA to protein ratio and dNTP pools alone are not directly lethal, but when combined with a high replicative DNA synthesis rate, as found after removal of HU, apparently lead to elevated cell death.

Effect of caloric restriction on hepatic nuclear DNA damage in male Fischer 344 rats treated with aflatoxin B 1

Caloric restriction is known to reduce chemically-induced tumor incidence in laboratory animals. The effect is believed to be mediated in part by modification of hepatic drug metabolism, including both phase I and phase II enzymes. Using aflatoxin B 1 (AFB 1) as a model carcinogen, we studied the effect of caloric restriction on the modification of rat liver nuclear DNA by AFB 1 and DNA damage due to the formation of apurinic sites from the AFB 1-DNA adduct removal process. Caloric restriction reduced the metabolic activation of AFB 1 which resulted in a decrease of AFB 1-DNA binding by more than 50%. The results of the study of the effect of caloric restriction on the AFB 1-induced DNA strand breakage assayed by the alkaline unwinding technique showed that caloric restriction protected the formation of apurinic sites from the AFB 1-DNA adducts and reduced the double strand DNA breakages by 1.3– 2.5-fold. Thus, the lower initial AFB 1-DNA binding and less DNA damage, presumably by the less apurinic sites formed during the depurination process of AFB 1-DNA adducts, contributed to the protective effect of caloric restriction.

Faster rates of DNA unwinding under alkaline conditions in xrs-5 cells may reflect chromatin structure alterations

The Chinese hamster ovary (CHO) cell line xrs-5 is a radiation-sensitive mutant isolated from CHO-K1 cells. The radiation sensitivity is associated with a defect in DNA double-strand break rejoining. The DNA alkaline unwinding technique was used to measure the DNA single-strand breakage caused by γ-rays in xrs-5 and CHO-K1 cells. Greater rates of DNA unwinding were found in xrs-5 cells as compared to CHO-K1. Independent measurement of DNA strand breakage by DNA filter elution or pulsed-field gel electrophoresis failed to show any difference between the two cell lines. The greater rate of unwinding in xrs-5 cells may reflect an alteration in chromosome structure.

Effect of heat on induction and repair of DNA strand breaks in X-irradiated CHO cells.

Chinese hamster ovary cells were exposed to various heat treatments followed by X-irradiation, and the induction and repair of DNA strand breaks was studied using the alkaline unwinding technique. Heat treatments alone were found to cause DNA strand breakage only for temperatures greater than or equal to 43 degrees C, whereas the number of radiation-induced strand breaks was unaffected by additional heating. Strand break repair was studied for irradiated cells preheated at temperatures ranging from 42 degrees C to 45 degrees C. The total repair curve could be separated into three phases, a fast (t = 0-15 min), an intermediate (t = 15-120 min) and a slow (t greater than or equal to 120 min) phase. All phases were altered when cells were heated either prior to or after irradiation. The fast and the intermediate phase could be well interpreted by the assumption that irradiation leads to both primary and secondary single-strand breaks, the latter being generated by enzymatic incision at sites of damaged bases. For irradiation alone, the ratio of all secondary strand breaks to all primary breaks was fsec = 1.5 +/- 0.5. This ratio was not altered by preceding heat treatments (mean fsec = 1.7 +/- 0.2). The main effect of heating on the repair kinetics of single-strand breaks was an increase in the repair half-time of primary and secondary breaks (maximum increase by a factor of 3.4), whereas the generation of secondary breaks was only slightly retarded (factor 1.3). The slow repair phase, which is assumed to represent the repair of DNA double-strand breaks, was best described by a single exponential component. The half-time of this component was found to increase from tau slow = 170 +/- 70 min for non-heated cells to tau slow = 345 +/- 80 min for cells heated at 45 degrees C for 20 min, indicating that heat inhibited the repair of double-strand breaks. For irradiation alone, the initial fraction of the slow component was fslow = 0.065 +/- 0.004. This fraction was enhanced by additional heating, with a maximum increase by a factor of 2.7 for cells heated at 45 degrees C for 20 min. This elevation cannot be the result of an enhanced induction of double-strand breaks, but must be associated with an additional formation of slowly repaired strand breaks during repair incubation. These additional strand breaks must arise from strand breaks which in non-heated cells are repaired during the fast or intermediate phase.(ABSTRACT TRUNCATED AT 400 WORDS)

X-ray induced DNA strand break induction and rejoining in cultured bovine lens epithelial cells.

DNA strand break induction and rejoining in cultured bovine lens epithelial cells were measured by the alkaline unwinding technique followed by hydroxyapatite chromatography. DNA damage is described in terms of dose effect curves for unwinding immediately after irradiation (0 min) and for unwinding after a recovery period (30 min). The dose effect curves obtained are purely exponential with D0 values of 29.3 Gy (0 min) and 138 Gy (30 min), respectively. Rejoining data are given in terms of rejoining enhancement curves with half-times for overall rejoining being in the range from 4 to 10 min, independent of the dose. Kinetics of strand breaks are also presented in terms of decay curves, which are best described by the sum of three exponential components. The half-times of these components have been determined as tau(I) approximately 2 min, tau(II) approximately 15 min and tau(III) approximately 500 min. These components of damage decay curves are discussed in relation to cell killing.

Efficient protection against oxidative DNA damage in chromatin.

The role of histones and higher order chromatin structures in protecting against oxidative DNA damage was investigated using an in vitro system consisting of nuclear and nucleoid monolayers as model chromatin substrates. These substrates, derived from human skin fibroblasts, were challenged with hydroxyl radicals produced via a Fenton reaction involving Fe(II)-ethylenediaminetetraacetic acid and ascorbic acid. The resulting DNA strand breaks were measured using the alkaline unwinding technique. The sequential removal of chromosomal proteins from the DNA by pretreating nuclear monolayers with increasing concentrations of salt dramatically increased the frequency of hydroxyl radical-induced DNA strand breaks. Furthermore, the DNA in decondensed chromatin was found to contain 14-fold fewer DNA strand breaks than naked, supercoiled DNA, whereas the DNA of "native" chromatin and "condensed" chromatin contained 100-fold and 300-fold fewer breaks, respectively. We conclude that the binding of histones to the DNA and its organization into higher order chromatin structures dramatically protects the DNA against hydroxyl radical-induced DNA strand breaks and thus should be considered part of the cellular defense against the induction of oxidative DNA damage.