Plasmid DNA breakage by decay of DNA-associated Auger electron emitters: approaches to analysis of experimental data

Plasmid DNA is a popular substrate for the assay of DNA strand breakage by a variety of agents. The use of the plasmid assay relies on the assumption that individual damaging events occur at random, which allows the application of Poisson statistics. This assumption is not valid in the case of damage arising from decay of DNA-associated Auger electron emitters, since a single decay event can generate a few breaks in the same DNA strand, which is indistinguishable from a single break in the assay. The consequent analytical difficulties are overcome by considering relaxation events rather than single-strand breaks, and linearization events rather than double-strand breaks. A further consideration is that apart from damage at the site of DNA-associated decay, which is the principal interest of the analysis, some DNA damage also arises from the radiation field created by all decay events. These two components of damage are referred to as internal and external breakage, respectively, and they can be separated in the analysis since their contribution depends on the experimental conditions. The DNA-binding ligand Hoechst 33258 labeled with 125I was used in our experiments to study breakage in pBR322 plasmid DNA arising from the decay of this Auger electron emitter. The values obtained for the efficiency (per decay) of plasmid relaxation and linearization by the 125I-labeled ligand were 0.090 +/- 0.035 and 0.82 +/- 0.04, respectively. When dimethylsulfoxide was included as a radical scavenger, the efficiency values for relaxation and linearization were 0.15 +/- 0.02 and 0.65 +/- 0.05, respectively.     

Auger electron-induced double-strand breaks depend on DNA topology

From a structural perspective, the factors controlling and the mechanisms underlying the toxic effects of ionizing radiation remain elusive. We have studied the consequences of superhelical/torsional stress on the magnitude and mechanism of DSBs induced by low-energy, short-range, high-LET Auger electrons emitted by (125)I, targeted to plasmid DNA by m-[(125)I]iodo-p-ethoxyHoechst 33342 ((125)IEH). DSB yields per (125)I decay for torsionally relaxed nicked (relaxed circular) and linear DNA (1.74+/-0.11 and 1.62+/-0.07, respectively) are approximately threefold higher than that for torsionally strained supercoiled DNA (0.52+/-0.02), despite the same affinity of all forms for (125)IEH. In the presence of DMSO, the DSB yield for the supercoiled form remains unchanged, whereas that for nicked and linear forms decreases to 1.05+/-0.07 and 0.76+/-0.03 per (125)I decay, respectively. DSBs in supercoiled DNA therefore result exclusively from direct mechanisms, and those in nicked and linear DNA, additionally, from hydroxyl radical-mediated indirect effects. Iodine-125 decays produce hydroxyl radicals along the tracks of Auger electrons in small isolated pockets around the decay site. We propose that relaxation of superhelical stress after radical attack could move a single-strand break lesion away from these pockets, thereby preventing further breaks in the complementary strand that could lead to DSBs.     

Biological damage from the Auger effect,possible benefits

Summary Decay of radioactive isotopes by K-capture leads to the Auger effect and results in the loss of several orbital electrons and the emission of X-rays. Whereas radiation effects are produced from the emitted electrons, the consequences of the Auger effect are strictly localized to the site of the decaying nuclide.The paper reviews the biological consequences of the decay of¹²⁵I which produces the Auger effect. Nearly all data were obtained from DNA labeled with¹²⁵I-5-iodo-2-deoxyuridine (IUdR) in bacteria and mammalian cells. Parameters of effects were cell death, DNA strand breaks, and mutation induction. In order to recognize in a cell the contribution from the Auger effect and that of absorbed radiation, experimental data are analysed in terms of the specific energy for the nuclear volume which contains the isotope.The data indicate that decay of¹²⁵I is far more toxic than is expected on the basis of absorbed dose to the labeled nucleus. Moreover, it is emphasized that the toxicity of the¹²⁵I decay is largely determined by events immediately localized to the site of decay.Because the consequences of the Auger effect are strictly localized to the molecular site of the decay,¹²⁵I and perhaps other nuclides decaying by K-capture promise to be interesting tools in cell biology and molecular biology. First data on the Auger effect as a tool are summarized.It appears that recognizable biological damage is only observed when the Auger effect takes place in vitally important molecules, an example of which is DNA.Dedicated to Prof. Dr. H. Muth on the occasion of his 60th birthday.     

Iodine-125 decay in a synthetic oligodeoxynucleotide. II. The role of auger electron irradiation compared to charge neutralization in DNA breakage

Lobachevsky, P. N. and Martin, R. F. Iodine-125 Decay in a Synthetic Oligodeoxynucleotide. II. The Role of Auger Electron Irradiation Compared to Charge Neutralization in DNA Breakage. The dramatic chemical and biological effects of the decay of DNA-incorporated (125)I stem from two consequences of the Auger electron cascades associated with the decay of the isotope: high local deposition of radiation energy from short-range Auger electrons, and neutralization of the multiply charged tellurium atom. We have analyzed the extensive data reported in the companion paper (Radiat. Res. 153, 000-000, 2000), in which DNA breakage was measured after (125)I decay in a 41-bp oligoDNA. The experimental data collected under scavenging conditions (2 M dimethylsulfoxide) were deconvoluted into two components denoted as radiation and nonradiation, the former being attributed to energy deposition by Auger electrons. The contribution of the components was estimated by adopting various assumptions, the principal one being that DNA breakage due to the radiation mechanism is dependent on the distance between the decaying (125)I atom and the cleaved deoxyribosyl unit, while the nonradiation mechanism, associated with neutralization of the multiply charged tellurium atom, contributes equally at corresponding nucleotides starting from the (125)I-incorporating nucleotide. Comparison of the experimental data sets collected under scavenging and nonscavenging (without dimethylsulfoxide) conditions was used to estimate the radiation-scavengeable component. Our analysis showed that the nonradiation component plays the major role in causing breakage within 4-5 nucleotides from the site of (125)I incorporation and produces about 50% of all single-stranded breaks. This overall result is consistent with the relative amounts of energy associated with Auger electrons and the charged tellurium atom. However, the nonradiation component accounts for almost four times more breaks in the top strand, to which the (125)I is bound covalently, than in the bottom strand, thus suggesting an important role of covalent bonds in the energy transfer from the charged tellurium atom. The radiation component dominates at the distances beyond 8-9 nucleotides, and 36% of the radiation-induced breaks are scavengeable.     

Inhibitory and stimulatory bystander effects are differentially induced by Iodine-125 and Iodine-123

The bystander effect, originating from cells irradiated in vitro, describes responses of surrounding cells not targeted by the radiation. Previously we demonstrated that the subcutaneous injection into nude mice of human adenocarcinoma LS174T cells lethally irradiated by Auger electrons from the decay of DNA-incorporated (125)I inhibits growth of co-injected LS174T cells (inhibitory bystander effect; Proc. Natl. Acad. Sci. USA 99, 13765-13770, 2002). We have repeated these studies using cells exposed to lethal doses of (123)I, an Auger electron emitter whose emission spectrum is identical to that of (125)I, and report herein that the decay of (123)I within tumor cell DNA stimulates the proliferation of neighboring unlabeled tumor cells growing subcutaneously in nude mice (stimulatory bystander effect). Similar inhibitory bystander effects ((125)I) and stimulatory bystander effects ((123)I) are obtained in vitro. Moreover, supernatants from cultures with (125)I-labeled cells are positive for tissue inhibitors of metalloproteinases (TIMP1 and TIMP2), and those from cultures with (123)I-labeled cells are positive for angiogenin. These findings call for the re-evaluation of current dosimetric approaches for the estimation of dose-response relationships in individuals after radiopharmaceutical administration or radiocontamination and demonstrate a need to adjust all "calculated" dose estimates by a dose modification factor (DMF), a radionuclide-specific constant that factors in hitherto not-so-well recognized biophysical processes.     

Strand breaks in plasmid DNA after positional changes of Auger electron-emitting iodine-125: direct compared to indirect effects.

To elucidate the nature and kinetics of DNA strand breaks caused by low-energy Auger electron emitters, we compared the yields of DNA breaks in supercoiled pUC19 DNA in the presence of the (.)OH scavenger dimethyl sulfoxide (DMSO) after the decay of (125)I (1) in proximity to DNA after minor-groove binding ((125)I-iodoHoechst 33342, (125)IH) and (2) at a distance from DNA ((125)I-iodoantipyrine, (125)IAP). DMSO is efficient at protecting supercoiled plasmid DNA from the decay of (125)I free in solution (dose modification factor, DMF = 59 +/- 4) and less effective when the (125)I decays occur close to DNA (DMF = 3.8 +/- 0.3). This difference is due mainly to the inability of DMSO to protect DNA from the double-strand breaks produced by groove-bound (125)I (DMF = 1.0 +/- 0.2). Additionally, the fragmentation of plasmid DNA beyond the production of single-strand and double-strand breaks that is seen after the decay of (125)IH and not (125)IAP (Kassis et al., Radiat. Res. 151, 167-176, 1999) cannot be modified by DMSO. These results demonstrate that the mechanisms underlying double-strand breaks caused by the decay of (125)IH differ in nature from those caused by the decay of (125)IAP.     

Comparison of cell inactivation by Auger electrons using the two reagents 4-[123I]iodoantipyrine and [123I]NaI

The Auger electron emitter ¹²³I was examined in the form of 4-[¹²³I]iodoantipyrine and as [¹²³I]NaI for its effectiveness in killing cells of different sensitivity to photon irradiation. Micronucleus assays showed that 4-[¹²³I]iodoantipyrine is 2–3 times more effective in cell inactivation than [¹²³I]NaI. This can be attributed to the fact that antipyrine, for reason of its lipid solubility, can enter cells and can reach the nucleus, whereas [¹²³I]NaI is excluded from the cytoplasm. In the nucleus Auger decay is conceivably located on the DNA where it may invoke high-LET irradiation damage. Irradiation damage by [¹²³I]NaI is by long range Auger and internal conversion electrons and hence less densely ionising. Results of the present study demonstrate, however, that the enhancement of micronuclei frequency (MNF) seen with 4-[¹²³I]iodoantipyrine as compared to [¹²³I]NaI is similar for all cell lines and that the ratio of 4-[¹²³I]iodoantipyrine/[¹²³I]NaI MN response remains the same. Experiments with the free radical scavenger DMSO, indicated nearly identical dose reduction factors for both ¹²³I carriers. These two observations strongly suggest that the cell inactivation by 4-[¹²³I]iodoantipyrine is not by direct high-LET ionisation of DNA, but is due to an indirect effect. The indirect radiation effect of Auger decay in the nucleus could arise because 4-[¹²³I]iodoantipyrine is not incorporated into the DNA, but is only associated with chromatin where the DNA is shielded by histones. Received: 24 May 2000 / Accepted: 1 November 2000     

Repair of sequence-specific 125I-induced double-strand breaks by nonhomologous DNA end joining in mammalian cell-free extracts

In mammalian cells, nonhomologous DNA end joining (NHEJ) is considered the major pathway of double-strand break (DSB) repair. Rejoining of DSB produced by decay of (125)I positioned against a specific target site in plasmid DNA via a triplex-forming oligonucleotide (TFO) was investigated in cell-free extracts from Chinese hamster ovary cells. The efficiency and quality of NHEJ of the "complex" DSB induced by the (125)I-TFO was compared with that of "simple" DSB induced by restriction enzymes. We demonstrate that the extracts are indeed able to rejoin (125)I-TFO-induced DSB, although at approximately 10-fold decreased efficiency compared with restriction enzyme-induced DSB. The resulting spectrum of junctions is highly heterogeneous exhibiting deletions (1-30 bp), base pair substitutions, and insertions and reflects the heterogeneity of DSB induced by the (125)I-TFO within its target site. We show that NHEJ of (125)I-TFO-induced DSB is not a random process that solely depends on the position of the DSB but is driven by the availability of microhomology patches in the target sequence. The similarity of the junctions obtained with the ones found in vivo after (125)I-TFO-mediated radiodamage indicates that our in vitro system may be a useful tool to elucidate the mechanisms of ionizing radiation-induced mutagenesis and repair.     

Low-energy electrons inside active DNA models: A tool to elucidate the radiation action mechanisms

To postulate radiation action mechanisms and to test them by Monte Carlo simulation, a complex computer model was developed consisting of major components for the generation of a radiation spectrum, biomolecular structures, and electron track structures in liquid water. As the radiation source¹²⁵I is employed here; it is an excellent test radiation due to its exactly localized position in the DNA molecule and high biological toxicity as a consequence of the emission of short-ranging Auger electrons. A linear DNA plasmid model (Pomplun 1991) which can actively respond to radical attack (Terrissol and Pomplun 1994) has been modified into a nucleosome model representing the double-helix of DNA with 146 basepairs and more than 9000 atoms surrounding the histones. The introduction of this new target structure allows a more realistic simulation of cellular conditions. Using the model's decay accumulation aspect, the situation of many break and survival experiments can be approximated and the influence of several cellular parameters tested. As a first step, a correlation between the size of energy depositions and strand-break patterns was sought.     

Efficient mutation induction by 125I and 131I decays in DNA of human cells

To examine the role of radiation energy deposition in DNA on cellular effects, we investigated the ability of 125IdUrd and 131IdUrd to kill cells and induce mutations at the hprt locus. We employed human lymphoblastoid cells proficient (TK6) or deficient (SE30) in the ability to incorporate a thymidine analog into DNA by way of the thymidine kinase (TK) scavenger pathway. Iodine-125 releases a shower of low-energy Auger electrons upon decay which deposit most of their energy within 20 nm of the decay site, whereas 131I is a high-energy beta/gamma emitter that is generally considered to emit sparsely ionizing radiation. Although 125IdUrd incorporated into cellular DNA was very effective at producing toxic and mutagenic effects in TK6 cells, virtually no effect was seen in TK-deficient cells incubated with similar levels of 125IdUrd in the extracellular medium. In response to 131IdUrd treatment, 0.45 X 10(-6) mutants were induced per centigray dose deposited within the nucleus in TK-proficient cells, whereas few mutations were induced in TK-deficient cells at doses up to 38 cGy from 131I decays occurring in the medium. The differences in biological response between TK6 and SE30 cells cannot be explained by differential radiosensitivity or IdUrd sensitization of the cell lines involved. We conclude that both 125I and 131I decays occurring while incorporated into DNA are more effective at inducing cell killing and mutations in human cells than either nonincorporated decays or low-LET radiations. These results suggest that localized energy deposition is an important factor in producing biologically important damage by both of these isotopes, and that residual lesions following the decay of DNA-incorporated radioisotopes may contribute to the toxic and mutagenic effects observed in TK-proficient cells. Furthermore, they emphasize that certain beta/gamma-emitting isotopes such as 131I may be particularly hazardous when incorporated into DNA.     

Chemical and biological consequences of the radioactive decay of iodine-125 in plasmid DNA

Doubly labeled [U-14C, 5-125I]iododeoxycytidine (IdC) triphosphate was synthesized and incorporated enzymatically into defined positions of the plasmid pBR322. After storage under various conditions, the stable end products were analyzed using radio-GC, radio-HPLC, and electron microscopy. In addition, solutions of 14C-IdC-labeled DNA containing Na125I as an internal radiation source were studied to investigate the influence of internal radiolysis. Transmutation of the covalently bound 125I leads to complete destruction of the labeled nucleotide, giving rise to 14CO2 and 14CO as major products. Fragmentation of the pyrimidine base is independent of solvent and DNA configuration. Internal radiolysis caused by Na125I leads to only minor damage. Electron microscopy studies reveal that decay-induced double strand breaks (dsb) occur both at the site of decay and in areas as far as hundreds of base pairs apart from that site. Number and distribution of the breaks is strongly dependent on solvent and DNA configuration. A direct correlation exists between the extent of fragmentation of the nucleotide and the mean number of dsb.     

A Monte Carlo simulation of Auger cascades

The energy imparted to biological tissue after the decay of incorporated Auger emitters stems from two sources: (a) energy deposition by the Auger and Coster-Kronig electrons and (b) the charge potential which remains on the multiple ionized atom after the end of the cascade. For the numerical assessment of both the kinetic energy of the released electrons and the charge potential, a new and--for purposes of microdosimetry--precise method is presented. Based on relativistic Dirac-Fock calculations and a rigorous bookkeeping, this method provides a perfect energy balance of the considered atomic system when applied to Monte Carlo simulations of Auger cascades. By comparing the results for charge distribution for krypton and iodine with experimental data and the electron spectrum of 125I with theoretical data, it can be shown that the approach followed in this work is reasonable and appropriate for the determination of the energy deposited by incorporated Auger emitters in small volumes of condensed matter. The total energy deposited by 125I in a volume of 20-nm diameter is 2.03 keV which is made up by multiple ionization (1.07 keV) and energy deposition by the emitted Auger electrons (0.96 keV).     

Mechanisms underlying production of double-strand breaks in plasmid DNA after decay of 125I-Hoechst

Previously, the kinetics of strand break production by (125)I-labeled m-iodo-p-ethoxyHoechst 33342 ((125)IEH) in supercoiled (SC) plasmid DNA had demonstrated that approximately 1 DSB is produced per (125)I decay both in the presence and absence of the hydroxyl radical scavenger DMSO. In these experiments, an (125)IEH:DNA molar ratio of 42:1 was used. We now hypothesize that this DSB yield (but not the SSB yield) may be an overestimate due to subsequent decays occurring in any of the 41 (125)IEH molecules still bound to nicked (N) DNA. To test our hypothesis, (125)IEH was incubated with SC pUC19 plasmids ((125)IEH:DNA ratio of approximately 3:1) and the SSB and DSB yields were quantified after the decay of (125)I. As predicted, the number of DSBs produced per (125)I decay is one-half that reported previously ( approximately 0.5 compared to approximately 1, +/- DMSO) whereas the number of SSBs ( approximately 3/(125)I decay) is similar to that obtained previously ( approximately 90% are generated by OH radicals). Direct visualization by atomic force microscopy confirms formation of L and N DNA after (125)IEH decays in SC DNA and supports the strand break yields reported. These findings indicate that although SSB production is independent of the number of (125)IEH bound to DNA, the DSB yield can be augmented erroneously by (125)I decays occurring in N DNA. Further analysis indicates that 17% of SSBs and 100% of DSBs take place within the plasmid molecule in which an (125)IEH molecule decays, whereas 83% of SSBs are formed in neighboring plasmid DNA molecules.     

Strand breaks in whole plasmid dna produced by the decay of (125)I in a triplex-forming oligonucleotide.

DNA strand breaks produced by the decay of (125)I positioned against a specific site in plasmid DNA via a triplex-forming oligonucleotide were studied both in the immediate vicinity of the site of the decay with a single nucleotide resolution and in the whole plasmid by measuring the percentages of supercoiled, open-circular and linear forms. The localized breaks are distributed within 10 bp in each direction from the decay site with maxima in both strands just opposite the (125)I-dC residue in the triplex-forming oligonucleotide. The distributions of breaks in the two DNA strands are almost symmetrical, in agreement with the geometry of the pyrimidine motif triplex. We found that about 25% of the double-strand breaks were located outside the 90-bp fragment containing the triplex-forming oligonucleotide binding sequence. The ratio of single- to double-strand breaks in the whole plasmid was 11 for bound triplex-forming oligonucleotide compared to 26 when the triplex-forming oligonucleotide was free in solution. The number of double-strand breaks per decay of (125)I was 0.46 for bound triplex-forming oligonucleotide and 0.17 for free triplex-forming oligonucleotide. Comparing the data on the localized damage and those for the whole plasmid, we concluded that, in addition to DNA breaks that are confined to a helical turn around the (125)I atom, the decay can produce breaks hundreds of base pairs away in the plasmid molecule. This linear plasmid molecule containing radiation-induced damage at a specific DNA site should be useful in studies of the molecular mechanisms of DNA repair.     

Site-specific strand breaks in RNA produced by (125)I radiodecay

Decay of ¹²⁵I produces a shower of low energy electrons (Auger electrons) that cause strand breaks in DNA in a distance-dependent manner with 90% of the breaks located within 10 bp from the decay site. We studied strand breaks in RNA molecules produced by decay of ¹²⁵I incorporated into complementary DNA oligonucleotides forming RNA/DNA duplexes with the target RNA. The frequencies and distribution of the breaks were unaffected by the presence of the free radical scavenger dimethyl sulfoxide (DMSO) or by freezing of the samples. Therefore, as was the case with DNA, most of the breaks in RNA were direct rather than caused by diffusible free radicals produced in water. The distribution of break frequencies at individual bases in RNA molecules is narrower, with a maximum shifted to the 3′-end with respect to the distribution of breaks in DNA molecules of the same sequence. This correlates with the distances from the radioiodine to the sugars of the corresponding bases in A-form (RNA/DNA duplex) and B-form (DNA/DNA duplex) DNA. Interestingly, when ¹²⁵I was located close to the end of the antisense DNA oligonucleotide, we observed breaks in RNA beyond the RNA/DNA duplex region. This was not the case for a control DNA/DNA hybrid of the same sequence. We assume that for the RNA there is an interaction between the RNA/DNA duplex region and the single-stranded RNA tail, and we propose a model for such an interaction. This report demonstrates that ¹²⁵I radioprobing of RNA could be a powerful method to study both local conformation and global folding of RNA molecules.     

DNA-incorporated 125I induces more than one double-strand break per decay in mammalian cells

The Auger-electron emitter 125I releases cascades of 20 electrons per decay that deposit a great amount of local energy, and for DNA-incorporated 125I, approximately one DNA double-strand break (DSB) is produced close to the decay site. To investigate the potential of 125I to induce additional DSBs within adjacent chromatin structures in mammalian cells, we applied DNA fragment-size analysis based on pulsed-field gel electrophoresis (PFGE) of hamster V79-379A cells exposed to DNA-incorporated 125IdU. After accumulation of decays at -70 degrees C in the presence of 10% DMSO, there was a non-random distribution of DNA fragments with an excess of fragments <0.5 Mbp and the measured yield was 1.6 DSBs/decay. However, since these experiments were performed under high scavenging conditions (DMSO) that reduce indirect effects, the yield in cells exposed to 125IdU under physiological conditions would most likely be even higher. In contrast, using a conventional low-resolution assay without measurement of smaller DNA fragments, the yield was close to one DSB/decay. We conclude that a large fraction of the DSBs induced by DNA-incorporated 125I are nonrandomly distributed and that significantly more than one DSB/decay is induced in an intact cell. Thus, in addition to DSBs produced close to the decay site, DSBs may also be induced within neighboring chromatin fibers, releasing smaller DNA fragments that are not detected by conventional DSB assays.     

Induction of DNA double-strand breaks by 157Gd neutron capture

The rationale of boron (10B) neutron capture therapy (BNCT) is based on the high thermal neutron capture cross section of 10B and the limited maximum range (about one cell diameter) of the high LET fission products of the boron neutron capture (NC) reaction. The resulting radiochemical damage is confined to the cell containing the BNC reaction. Although other nuclides have higher thermal neutron capture cross sections than 10B, NC by such nuclides results in the emission of highly penetrating gamma rays. However, gadolinium-157 (157Gd) n-gamma reaction is also accompanied by some internal conversion and, by implication, Auger electron emission. Irradiation of Gd3+-DNA complexes with thermal neutrons results in the induction of DNA double-strand (ds) breaks, but the effect is largely abrogated in the presence of EDTA. Thus, by analogy with the effects of decay of Auger electron-emitting isotopes such as 125I, the Gd NC event must take place in the close proximity of DNA in order to induce a DNA ds break. It is proposed that 157Gd-DNA ligands therefore have potential in NCT. The thermal neutron capture cross section of 157Gd, a nonradioactive isotope, is more than 50 times that of 10B.     

Induction and repair of double- and single-strand DNA breaks in bacteriophage lambda superinfecting Escherichia coli

Induction and repair of double- and single-strand DNA breaks have been measured after decays of 125I and 3H incorporated into the DNA and after external irradiation with 4 MeV electrons. For the decay experiments, cells of wild type Escherichia coli K-12 were superinfected with bacteriophage lambda DNA labelled with 5'-(125I)iodo-2'-deoxyuridine or with (methyl-3H)thymidine and frozen in liquid nitrogen. Aliquots were thawed at intervals and lysed at neutral pH, and the phage DNA was assayed for double- and single-strand breakage by neutral sucrose gradient centrifugation. The gradients used allowed measurements of both kinds of breaks in the same gradient. Decays of 125I induced 0.39 single-strand breaks per double-strand break. No repair of either break type could be detected. Each 3H disintegration caused 0.20 single-strand breaks and very few double-strand breaks. The single-strand breaks were rapidly rejoined after the cells were thawed. For irradiation with 4 MeV electrons, cells of wild type E. coli K-12 were superinfected with phage lambda and suspended in growth medium. Irradiation induced 42 single-strand breaks per double-strand break. The rates of break induction were 6.75 x 10(-14) (double-strand breaks) and 2.82 x 10(-12) (single-strand breaks) per rad and per dalton. The single-strand breaks were rapidly repaired upon incubation whereas the double-strand breaks seemed to remain unrepaired. It is concluded that double-strand breaks in superinfecting bacteriophage lambda DNA are repaired to a very small extent, if at all.     

A method of calculating initial DNA strand breakage following the decay of incorporated 125I

Two sources of individual Auger electron spectra and an electron track code were used with a simple model of the DNA to successfully simulate the single-strand DNA breakage measured by Martin and Haseltine (1981). The conditions of the calculation were then extended to examine patterns of single-strand breaks in both strands of the DNA duplex to score double-strand breaks. The occurrences of five types of break were scored. The total number of double-strand breaks (dsb) per decay at the site of the decay was 0.90 and 0.65 for the different Auger electron spectra. It was shown that for mammalian cells an additional source of double-strand breaks from low LET radiation added approximately 0.17 dsb/decay to each, giving a final total of 1.07 and 0.85 dsb/decay for mammalian cells depending on the electron spectrum. Further is is shown that the energy deposition in the DNA from the iodine decay is very complex, with a broad range of energy depositions and products. Even for a particular energy deposited in the DNA different types of strand break are produced. These are identified and their probabilities calculated.     

Kinetics of uptake, retention, and radiotoxicity of 125IUdR in mammalian cells: implications of localized energy deposition by Auger processes

The kinetics of uptake, retention, and radiotoxicity of 125IUdR have been studied in proliferating mammalian cells in culture. The radioactivity incorporated into the DNA is directly proportional to the duration of incubation and to the extracellular concentration of 125I. The rate of proliferation of cells is related to the intracellular radioactive concentration and is markedly reduced at medium concentrations greater than or equal to 0.1 mu Ci/ml. At 37% survival the high LET type cell survival curve is characterized by an uptake of 0.035 pCi/cell, and the cumulated mean lethal dose to the cell nucleus is about 80 rad compared to 580 rad of X-ray dose for this cell line. The strong cytocidal effects of the decay of 125I correlate with localized irradiation of the DNA by the low energy Auger electrons.