Production Of Single-strand Breaks Research Articles

UVA-photoprotective potential of silymarin and silybin.

Exposure to solar radiation is a major cause of environmental human skin damage. The main constituent of solar UV light is UVA radiation (320-400nm); however, the need for protection against UVA has been marginalized for a long time. As a result, there is still a lack of useful agents for UVA protection. In this study, the effect of silymarin (SM) and its main constituent silybin (SB) pre-treatment on UVA-stimulated damage to primary human dermal fibroblasts were carried out. The cells were pre-treated for 1h with SB or SM and then were exposed to UVA light, using a solar simulator. The effect of SB and SM on reactive oxygen species (ROS) and glutathione (GSH) level, caspase-3 activity, single-strand breaks (SSB) formation and protein level of matrix metalloproteinase-1 (MMP-1), hemeoxygenase-1 (HO-1), and heat shock protein (HSP70) was evaluated. Treatment with both SM and SB resulted in a reduction in UVA-stimulated ROS generation and SSB production, as well as in the prevention of GSH depletion, a decrease in the activation of caspase-3 and protein level of MMP-1. They also moderately increased HO-1 level and reduced HSP70 level. Our data showed that both SM and SB are non-phototoxic and have UVA-photoprotective potential and could be useful agents for UV-protective dermatological preparations.

In vitro DNA damage characterisation studies on plasmid pBR322 after exposure to γ radiation by 60Co

When a biological system is either accidentally or intentionally exposed to radiation, the energy absorbed triggers a number of successive events including damage to living tissues. Major radiation damage is due to the aqueous free radicals generated by the radiolysis of water. These free radicals act as molecular marauders and in turn damage DNA, mitochondrial membrane, lipid, cellular protein, resulting in cellular dysfunction and mortality. In view of the above mentioned facts an experiment was conducted to study the genotoxic effects of γ radiation and its dose effectiveness. The present experiment was conducted on samples of plasmid pBR322 DNA as the in vitro experimental model devoid of any DNA repair and replication machinery. The samples were exposed to different doses of gamma radiations from 1 to 200 Gy. Exposure of plasmid pBR322 DNA to γ radiation resulted in production of single strand breaks as a result of which, the supercoiled (SC) form was converted to relaxed form (RL). Exposure of radiation, even at very low dose of 1 Gy, exhibited a significant damage to DNA resulting in about 70% SC form and 30% RL form of DNA. At a dose of 10 Gy the SC form was reduced to about 37% and further 5% at a dose of 50 Gy with about 88.5 and 6.5% RL and linear (L) forms of DNA respectively. Thus, the disappearance of supercoiled form of plasmid pBR322 DNA was found to be directly related to radiation dose and exhibited a radiation dose dependent pattern.

Lead contamination results in late and slowly repairable DNA double-strand breaks and impacts upon the ATM-dependent signaling pathways

Despite a considerable amount of data, evaluation of the potential genotoxicity and cancer proneness of lead compounds remains unclear, probably due to the plethora of experimental procedures, biological endpoints and cellular models used. In parallel, the understanding in DNA damage formation, repair and signaling has considerably progressed all along these last years, notably for DNA double-strand breaks (DSBs). Here, were examined DNA damage formation and repair in human cells exposed to lead nitrate (Pb(NO 3) 2) and their consequences upon the ATM-dependent stress signaling, cell cycle progression and cell death. As observed with anti-pH2AX immunofluorescence, exposure to Pb(NO 3) 2 results in formation of late DSBs, that would not originate from conversion of nucleotide damage but likely by a direct production of single-strand breaks. Lead contamination inhibits non-homologous end-joining repair process by preventing the DNA-PK kinase activity whereas the MRE11-dependent repair pathway is exacerbated. Lead contamination triggers successive synchronization of cells in G2/M phase in which the RAD51-dependent homologous recombination was found to be activated. Altogether, our findings support that lead contamination generates late unrepairable DSBs that impact upon the ATM-dependent stress signaling pathway by favoring propagation of errors. Such findings should help to consider more carefully the biological action of lead compounds in the frame of public and occupational exposures.

Effective Cross Sections for Production of Single-Strand Breaks in Plasmid DNA by 0.1 to 4.7 eV Electrons

We determined effective cross sections for production of single-strand breaks (SSBs) in plasmid DNA [pGEM 3Zf(-)] by electrons of 10 eV and energies between 0.1 and 4.7 eV. After purification and lyophilization on a chemically clean tantalum foil, dry plasmid DNA samples were transferred into a high-vacuum chamber and bombarded by a monoenergetic electron beam. The amount of the circular relaxed DNA in the samples was separated from undamaged molecules and quantified using agarose gel electrophoresis. The effective cross sections were derived from the slope of the yield as a function of exposure and had values in the range of 10(-15)- 10(-14) cm2, giving an effective cross section of the order of 10(-18) cm2 per nucleotide. Their strong variation with incident electron energy and the resonant enhancement at 1 eV suggest that considerable damage is inflicted by very low-energy electrons to DNA, and it indicates the important role of pi* shape resonances in the bond-breaking process. Furthermore, the fact that the energy threshold for SSB production is practically zero implies that the sensitivity of DNA to electron impact is universal and is not limited to any particular energy range.

Causes of DNA single-strand breaks during reduction of chromate by glutathione in vitro and in cells

Carcinogenic chromates induce DNA single-strand breaks (SSB) that are detectable by conventional alkali-based assays. However, the extent of direct breakage has been uncertain because excision repair and hydrolysis of Cr–DNA adducts at alkaline pH also generate SSB. We examined mechanisms of SSB production during chromate reduction by glutathione (GSH) and assessed the significance of these lesions in cells using genetic approaches. Cr(VI) reduction was biphasic and the formation of SSB occurred exclusively during the slow reaction phase. Catalase or iron chelators completely blocked DNA breakage, as did the use of GSH purified by a modified Chelex procedure. Thus, the direct intermediates of GSH–chromate reactions were unable to cause SSB unless activated by H 2O 2. SSB repair-deficient XRCC1 −/− and proficient XRCC1 + EM9 cells had identical survival at doses causing up to 60% clonogenic death and accumulation of 1 mM Cr(VI). However, XRCC1 −/− cells displayed higher lethality in the more toxic range and the depletion of GSH made them hypersensitive even to moderate doses. Elevation of cellular catalase or GSH levels eliminated survival differences between XRCC1 −/− and XRCC1 + cells. In summary, formation of toxic SSB in cells occurs at relatively high chromate doses, requires H 2O 2, and is suppressed by high GSH concentrations.

Induction of apoptosis by photoexcited tetracyclic compounds derivatives of benzo[ b]thiophenes and pyridines

The antiproliferative activity, upon UVA irradiation, of two tetracyclic derivatives of benzo[ b]thiophenes and pyridines, a benzo[ b]thienopyridopyrimidone ( 1) and a thienocarboline ( 2), has been investigated in a panel of human tumor cell lines. The two compounds present a remarkable cytotoxicity after UVA irradiation (365 nm), reaching an IC 50 of 0.1 μM in the leukaemia cell lines and 0.3–0.5 μM in the solid tumour cell lines. Their effect on the cell cycle was measured by flow cytometry in Jurkat cells. The compounds induce cell cycle perturbations and trigger a massive apoptosis as revealed by the externalisation of Annexin V-targeted residues at the outer plasmatic membrane. Furthermore the drugs induce, upon UVA irradiation significant variations of the mitochondrial potential (Δ ψ mt) measured by flow cytometry using the fluorochrome JC-1. In addition we characterized the mitochondrial production of reactive oxygen species (ROS) using the probe dihydroethidine (HE) and the oxidations of the mitochondrial phospholipid cardiolipin using the interacting probe nonyl acridine orange (NAO). Both compounds stimulate the production of ROS, and remarkably induce oxidation of cardiolipin. We have investigated the DNA-binding properties of these two compounds by means of UV–Vis spectroscopy and fluorescence. The two compounds exhibit a low affinity toward the macromolecule. The mode of binding was also investigated by means of flow linear dichroism (LD) which has revealed that the two compounds do not efficiently intercalate into DNA. Finally, the DNA-photocleavaging properties of the test compounds were studied on pBR322 plasmid DNA as a model. Only compound 1 is able to induce a significant production of single strand breaks only after digestion with the base excision repair enzyme Endo III. Altogether these data suggest that DNA is not a preferential target of these molecules and other subcellular structures may be responsible for their high phototoxic activity.

The modulatory effects of ellagic acid and vitamin E succinate on TCDD-induced oxidative stress in different brain regions of rats after subchronic exposure.

The effects of ellagic acid (EA) and vitamin E succinate (VES) on 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-induced oxidative stress in different brain regions of rats have been studied after subchronic exposure to the compounds. TCDD was administered to groups of rats at a dose of 46 ng/kg/day for 90 days. EA and VES were administered to groups of rats, either separately or simultaneously with TCDD, every other day for 90 days. At the end of the treatment period, animals were sacrificed and brains were dissected to cerebral cortex (Cc), hippocampus (H), cerebellum (C), and brain stem (Bs), and were assayed for production of superoxide anion (SA), lipid peroxidation (LP), and DNA single-strand breaks (SSBs). While TCDD administration to rats resulted in significant production of SA, LP, and DNA SSBs in Cc and H, simultaneous administration of VES or EA with the xenobiotics resulted in significant protection against those effects. The results also indicate that VES provided a better protyection against TCDD-induced effects in brains when compared with EA.

Iron chelators modulate the production of DNA strand breaks and 8-hydroxy-2′-deoxyguanosine

The interaction of chelators and reducing agents is of particular importance in understanding iron-associated pathology since catalytic iron undergoes cyclic reduction and oxidation in vivo. Therefore, we treated plasmid DNA with free or chelated Fe(III) in the presence of biological reductants, and simultaneously measured the number of single strand breaks (SSBs) and oxidative base modification (8-hydroxy-2′-deoxy-guanosine; 8-OHdG) by quantitative gel electro-phoresis and HPLC with electrochemical detection, respectively. Production of SSBs and 8-OHdG was linearly correlated suggesting that these two different lesions share a common chemical mechanism. The levels of both lesions were enhanced when Fe(III) was chelated to citrate or nitrilotriacetic acid. Reducing agents showed different potency in inducing DNA damage catalyzed by chelated iron (L-ascorbate > L-cysteine > H2O2). Chelation increased SSB formation by ∼ 8-fold and 8-OHdG production by ∼ 4-fold. The ratio of SSB/8-OHdG catalyzed by chelated iron, which is twice as high as by unchelated iron, indicates that chelation affects iron-catalyzed oxidative DNA damage in a specific way favoring strand breakage over base modification. Since iron is mostly chelated in biological systems, the production of genomic and mitochondrial DNA damage, particularly strand breaks, in diseases involving iron overload is likely to be higher than previously predicted from studies using unchelated iron.

Response of V79 cells to low doses of X-rays and negative pi-mesons: clonogenic survival and DNA strand breaks.

Mammalian cells are hypersensitive to very low doses of X-rays (< 0.2 Gy), a response which is followed by increased radioresistance up to 1 Gy. Increased radioresistance is postulated to be a response to DNA damage, possibly single-strand breaks, and it appears to be a characteristic of low linear energy transfer (LET) radiation. Here we demonstrate a correspondence between the extent of the increased radioresistance and linear energy transfer of 250 kVp X-rays and plateau and Bragg peak negative pi-mesons. The results support our hypothesis since the size of the increased radioresistant response appears to correspond to the number of radiation induced single-strand breaks. Furthermore, since survival prior to the increased radioresistant response (< 0.2 Gy) was LET-independent, these data support the notion that the increased radioresistant response may dictate the overall survival response to higher doses. However, while these data provide further circumstantial evidence for the involvement of DNA strand breaks in the triggering of increased radioresistance, more direct conclusions cannot be made. The data are not accurate enough to detect structure in the single-strand break profiles, the production of single-strand breaks being apparently linear with dose.

DNA Breakage upon K-Shell Excitation of Phosphorus as a Model for Direct Effects in Radiation Biology

Single-strand breaks (SSBs) and double-strand breaks (DSBs) induced in DNA under phosphorus K-shell resonant absorption have been studied using supercoiled plasmids. The kinetics of the production of SSBs and DSBs exhibits a linear and a quadratic dependence, respectively, on photon fluence. Cross sections and quantum yields have been measured. The resonant photoexcitation of the phosphorus atoms was found to increase the DSB/SSB ratio compared to the off-resonance excitation. This enhancement factor can be related to the measured enhancement of the rate of cellular death and gene mutation in yeast under similar experimental conditions reported previously in the literature. Such resonant excitation of a specific atom belonging to DNA turns out to be an elegant method to investigate pure direct effects.

Association between 8-hydroxy-2′-deoxyguanosine formation and DNA strand breaks mediated by copper and iron

Oxidative DNA damage is involved in diverse biological phenomena and consists of several kinds of lesions, mainly, strand breaks, base modifications, and DNA-protein crosslinking. However, little is known about the existence of a chemical relationship among them or the ratio by which these different types of lesions are produced. In the present study we investigated whether a relationship exists between DNA strand breakage and base modification. We selected cupric [Cu(II)] and ferric [Fe(III)] ions for this study because these transition metals are active catalysts of DNA damage in vivo. Supercoiled plasmid DNA pZ189 was treated with Cu(II) or Fe(III) in the presence of different reducing agents. We measured in each sample both the number of DNA single-strand breaks (SSB) by quantitative electrophoresis and the amount of a modified DNA base, 8-hydroxy-2′-deoxyguanosine (8-OHdG) by HPLC with simultaneous electrochemical (EC) and spectrophotometric detection. The number of DNA SSBs produced was linearly related to the number of 8-OHdG present. The regression of the number of SSBs as a function of the number of 8-OHdG is expressed by the equation [SSBs] = b × [8-OHdG], where b = 1.7, 2.0, 2,7, 1.7, and 9.4, for Cu(II) in the presence of H 2O 2, l-cysteine and l-ascorbate, and for Fe(III) in the presence of H 2O 2 and l-ascorbate, respectively. The linear correlation we observed between the production of SSB and 8-OHdG mediated by Fe(III) and by Cu(II) suggests that these products may arise via a common chemical mechanism and could allow an easier and more precise estimation of DNA breakage.

Carboplatin Enhances the Production and Persistence of Radiation-Induced DNA Single-Strand Breaks

Fluorometric analysis of DNA unwinding and alkaline elution were used to investigate the production and persistence of DNA single-strand breaks (SSBs) in Chinese hamster V79 and xrs-5 cells treated with the chemotherapeutic agent carboplatin in combination with radiation. Carboplatin was administered to cells before irradiation in hypoxic conditions, or the drug was added immediately after irradiation during the postirradiation recovery period in air. The results of DNA unwinding studies suggest that carboplatin enhances the production of radiation-induced SSBs in hypoxic V79 cells and xrs-5 cells by a factor of 1.86 and 1.83, respectively, when combined with radiation compared to the SSBs produced by irradiation alone. Carboplatin alone did not produce a measurable number of SSBs. Alkaline elution profiles also indicated that the rate of elution of SSBs was higher in cells treated with the carboplatin-radiation combination in hypoxia when compared to irradiation alone, resulting in an increased yield of radiation-induced SSBs by a factor of 1.46 in V79 cells with 20 Gy irradiation and by a factor of 2.02 in xrs-5 cells with 20 Gy irradiation. When carboplatin is present after irradiation and during the postirradiation recovery period, the rejoining of radiation-induced SSBs is inhibited during this postirradiation incubation period (radiopotentiation) with a relative inhibition factor at 1 h postirradiation of 1.25 in V79 cells and 1.15 in xrs-5 cells. An increased production and persistence of SSBs resulting from the interaction of carboplatin with radiation may be an important step in the mechanism responsible for the potentiated cell killing reported previously from studies in animal tumors and in cultured cells.

DNA Damage Produced by Exposure of Supercoiled Plasmid DNA to High- and Low-LET Ionizing Radiation: Effects of Hydroxyl Radical Quenchers

Production of DNA damage by exposure to ionizing radiation was measured in two in vitro systems. A supercoiled plasmid of 7.3 kbp was isolated and exposed in an aqueous environment to 60Co gamma rays and JANUS 0.85 MeV fission-spectrum neutrons. Dose responses for the production of single-strand breaks (ssbs) and double-strand breaks (dsbs) were computed from the conversion of the supercoil to its relaxed and linear forms. The relative effectiveness (neutrons:gamma-rays) for destruction of genetic transforming activity of M13 viral DNA was 0.23, close to that for ssb production, in contrast with the situation for biological effects such as lethality, mutagenesis and cellular transformation measured in mammalian cells, where RBEs are > 1. The role of hydroxyl (OH) radicals in DNA damage induction by neutrons was investigated by exposure of plasmid in the presence of known quenchers of this species. Of four quenchers tested, all were able to reduce the yields of both ssbs and dsbs. These findings are consistent with a model for ssb and dsb induction by high linear energy transfer radiation that involves OH radical medication.

DNA strand breakage during physiological apoptosis of the embryonic chick lens: free 3' OH end single strand breaks do not accumulate even in the presence of a cation-independent deoxyribonuclease.

Epithelial cells from the lens equator differentiate into elongated fiber cells. In the final steps of differentiation, the chromatin appears quite condensed and chromatin breakdown into nucleosomes occurs. DNA breaks due to an endodeoxyribonuclease activity corresponding to at least two polypeptides of 30 and 40 kDa have been identified. To identify the nature and the developmental appearance of initial breaks, nick translation reaction was followed both biochemically and in situ in fiber and epithelial cells from chick embryonic lenses. There is no accumulation of single-strand breaks (SSB) with 3'OH ends in lens fiber cells during embryonic development. Such damage can be increased in these cells by treatment with DNAase I indicating the absence of an inhibitor of the nick translation reaction in fiber cells. However, there are indications of the presence of DNA breaks with blocked termini when the phosphatase activity of nuclease P1 is used. The presence of breaks is also indicated by the large amounts of (ADP-ribose)n found in lens fibers particularly at 11 days of embryonic development (E11) as ADP-ribosyl transferase binds to and is activated by DNA strand breaks. Incubation of lens cells in vitro, which causes nucleosomal fragmentation only in fiber cells, produces SSB with 3'OH ends in both epithelia and fibers. Incubation for short periods, observed in experiments in situ, induces SSB first in the central fiber nuclei, which are late in differentiation. This may indicate that these SSB play a physiological role. Long incubations produce larger numbers of SSB in epithelia than fibers. The SSB in the fibers may have been converted into double-strand breaks (D SB), seen as nucleosomal fragments, and therefore no longer act as substrates for nick translation. The nuclease activity responsible for SSB production is independent of divalent cations and could be implicated in lens terminal differentiation.

Effects of treatment with methyl methanesulfonate during meiotic and postmeiotic stages and maturation of spermatozoa in mice

DNA damage and its related effects were quantitatively measured in germ cells at various meiotic and postmeiotic stages in male mice treated with methyl methanesulfonate (MMS). Unscheduled DNA synthesis (UDS) took place most efficiently in germ cell stages from middle to late spermatocytes at the time of MMS treatment, and essentially no UDS was detected in the stages from late spermatid to maturing spermatozoa, suggesting the absence of DNA repair in late spermatids and maturing spermatozoa. Late spermatids and immature spermatozoa were, on the other hand, most sensitive to MMS in producing DNA single-strand breaks (SSB) as well as chromosome aberrations in eggs fertilized by the treated males. The storage of the spermatozoa in the reproductive tract appeared to enhance the production of SSB. DNA damages induced by MMS should cause various genetic effects during the postmeiotic stages and maturation of spermatozoa through mechanisms not related to excision repair.

Effects of metal ion chelators on DNA strand breaks and inactivation produced by hydrogen peroxide in Escherichia coli: detection of iron-independent lesions

In order to study the role of metallic ions in the H2O2 inactivation of Escherichia coli cells, H2O2-sensitive mutants were treated with metal ion chelators and then submitted to H2O2 treatment. o-Phenanthroline, dipyridyl, desferrioxamine, and neocuproine were used as metal chelators. Cell sensitivity to H2O2 treatment was not modified by neocuproine, suggesting that copper has a minor role in OH production in E. coli. On the other hand, prior treatment with iron chelators protected the cells against the H2O2 lethal effect, indicating that iron participates in the production of OH. However, analysis of DNA sedimentation profiles and DNA degradation studies indicated that these chelators did not completely block the formation of DNA single-strand breaks by H2O2 treatment. Thiourea, a scavenger of OH, caused a reduction in both H2O2 sensitivity and DNA single-strand break production. The breaks observed after treatment with metal chelators and H2O2 were repaired 60 min after H2O2 elimination in xthA but not polA mutant cells. Therefore, we propose that there are at least two pathways for H2O2-induced DNA lesions: one produced by H2O2 through iron oxidation and OH production, in which lesions are repaired by the products of the xthA and polA genes, and the other produced by an iron-independent pathway in which DNA repair requires polA gene products but not those of the xthA gene.

10,11-Methylenedioxycamptothecin, a Topoisomerase I Inhibitor of Increased Potency: DNA Damage and Correlation to Cytotoxicity in Human Colon Carcinoma (HT-29) Cells

We had previously shown that 10,11-methylenedioxy-20-(RS)-camptothecin (MDO-CPT) is a more potent inhibitor of purified DNA topoisomerase I than 20-(S)-camptothecin (CPT). The current studies compared the cytotoxicity and DNA damage induced by MDO-CPT and CPT in the human colon carcinoma cell line, HT-29. MDO-CPT was 7- to 10-fold more potent than CPT both for cytotoxicity (ID50 = 25 vs. 180 nM) and production of DNA single-strand breaks (SSB). Kinetics of SSB formation and reversal were similar for MDO-CPT and CPT. DNA-protein crosslinks (DPC) were also produced by both drugs with a SSB/DPC ratio of 1/1. Moreover, no SSB were detected under non-deproteinizing conditions, indicating that both CPT and MDO-CPT produced protein-linked DNA single-strand breaks. A good correlation between cytotoxic potency and protein-linked DNA single-strand break production was observed for CPT and MDO-CPT, implying a causal relationship between drug-induced cytotoxicity and topoisomerase I inhibition. The sensitivity of human colon HT-29 cancer cells to camptothecins may be a selective phenomenon since these cells normally express natural resistance to current chemotherapeutic drugs, including topoisomerase II inhibitors.

Reverse gyrase binding to DNA alters the double helix structure and produces single-strand cleavage in the absence of ATP.

Stoichiometric amounts of pure reverse gyrase, a type I topoisomerase from the archaebacterium Sulfolobus acidocaldarius were incubated at 75 degrees C with circular DNA containing a single-chain scission. After covalent closure by a thermophilic ligase and removal of bound protein molecules, negatively supercoiled DNA was produced. This finding, obtained in the absence of ATP, contrasts with the ATP-dependent positive supercoiling catalyzed by reverse gyrase and is interpreted as the result of enzyme binding to DNA at high temperature. Another consequence of reverse gyrase stoichiometric binding to DNA is the formation of a cleavable complex which results in the production of single-strand breaks in the presence of detergent. Like eubacterial type I topoisomerase (protein omega), reverse gyrase is tightly attached to the 5' termini of the cleaved DNA. In the light of these results, a comparison is tentatively made between reverse gyrase and the eubacterial type I (omega) and type II (gyrase) topoisomerases.

Production of DNA single-strand breaks in unstimulated splenocytes by dimethylnitrosamine

In an attempt to elucidate the mechanism whereby primary hepatocytes, but not liver S9 homogenates, generate immunosuppressive metabolites of dimethylnitrosamine (DMN), the production of DNA single-strand breaks (SSB) in unstimulated splenocytes was investigated with alkaline-elution analysis. Both hepatocytes and S9 hormogenates induced SSB in cultured splenocytes by DMN — minimum detectable doses with the two metabolic activation systems (MAS) were 1 μM and 5 mM, respectively. DNA elution profiles were linear in splenocytes co-cultured with DMN and hypatocytes and convex in splenocytes incubated with DMN and S9 homogenates. Aminoacetonitrile (AAN; 10 mM), a DMN demethylase inhibitor, reversed SSB in splenocytes when incubated with either MAS. Addition of exogenous calf-thymus DNA to the hepatocyte co-culture medium did not affect the production of SSB. Rocking the hepatocyte-splenocyte cultures changed the elution profile from linear to convex. All of these treatments have been previously shown to block the immunosuppressive by DMN in the hepatocyte co-culture system. These results indicate that the immunosuppressive by DMN is not related to DNA damage, as measured by the production of SSB, and suggest that the metabolism of DMN to intermediates capable of producing genotoxicity and immunotoxicity may be qualitatively and/or quantitatively different.

Calculation of Initial Yields of Single- and Double-strand Breaks in Cell Nuclei from Electrons, Protons and Alpha Particles

A model of the DNA and electron and ion track structure computer codes are used to model damage in the DNA by direct action. This damage is converted into single-strand breaks using the method described by Charlton and Humm (1988) in which a minimum energy deposited in a critical volume of the DNA is correlated with the production of single-strand breaks. It is then assumed that if these single-strand breaks lie on opposite strands and are separated by less than a few base pairs they produce double-strand breaks. Absolute yields of both single- and double-strand breaks expressed in breaks/Gy-dalton are calculated and compared to measured yields. Good agreement is obtained for single-strand breaks while the calculated yields for double-strand breaks are greater than those measured.