H. Loman

Radiation-induced Strand Breaks in ϕX174 Replicative Form DNA: An Improved Experimental and Theoretical Approach

To determine the yield of radiation-induced single-strand, double-strand and potential breaks (breaks which are converted into actual breaks by alkali or heat treatment) oxygenated aqueous solutions of phi X174 supercoiled circular double-stranded (RFI) DNA were irradiated with increasing doses of gamma-irradiation and subjected to electrophoresis on agarose gels both before and after heat treatment. A complete separation was obtained of RFI, RFII (relaxed circle due to one or more single-strand breaks) and RFIII (linear DNA due to one double-strand break). A computer-assisted spectrophotometric procedure was developed, which enabled us to measure very accurately the amount of DNA present in the three DNA fractions. The quantitative changes of each fraction of DNA with dose could be fitted to a straightforward statistical model, which described the dose-dependent formation of the different types of breaks and from which the D37-values of single-strand, potential single-strand and double-strand breaks could be calculated to be 0.42 +/- 0.02, 1.40 +/- 0.25 and 57 +/- 36 Gy respectively. Potential double-strand breaks were not formed significantly under our conditions. In addition the maximum distance between two independently introduced single-strand breaks in opposite strands resulting in a double-strand break could be determined. The values before and after heat treatment are shown to be 29 +/- 6 and 102 +/- 13 nucleotides, respectively.

Reaction rate of OH radicals with phi X174 DNA: influence of salt and scavenger.

A derivation is given for the dependence of the rate constant of the reaction of OH radicals with a spherical macromolecule on the rate by which such radicals are scavenged by the medium. Experiments were carried out with oxygenated solutions of dilute single-stranded phi X174 DNA at 10(-4)M NaCl (large reaction radius of DNA) or at 10(-4)M NaCl + MgCl2 (small reaction radius) with t-butanol as a scavenger. The results of these experiments cannot be described by simple second-order competition, but can be explained by the predicted dependence of the rate constant of the reaction OH + DNA on the concentration of t-butanol. Furthermore, the results show that only part of the reactions of OH radicals with phi X174 DNA leads to DNA inactivation, and that even at zero scavenger concentration OH radicals are scavenged by other molecules than DNA, presumably impurities remaining even after careful purification of the DNA.

Inactivation of Biologically-active DNA by Radiation-induced Phenylalanine Radicals

SummaryTo investigate the possibility of the involvement of organic radicals, e.g. those derived from amino acids, in the inactivation of DNA in living cells by ionizing radiation, we studied, as a model system, dilute solutions of the biologically-active DNA of the bacteriophage ΦX174 in the presence of relatively high concentrations of phenylalanine. The solutions were saturated with N2, O2 or N2O and irradiated with 60Co γ-rays. Under these conditions a negligible fraction of the primary water radicals reacts with DNA. Nevertheless inactivation of DNA occurs, due to reactions of amino-acid radicals. These reactions probably do not produce DNA chain breaks, but another type of nucleotide damage, which has not yet been identified. In anoxic solutions the phenylalanine radicals can be reduced or oxidized by small amounts of cysteamine and paranitroacetophenone, respectively, which prevents their attack on DNA, resulting in an enormous protection. Moreover, it was found that the ratio of breaks and other d...

DNA damage induced by reduced nitroimidazole drugs

Five nitroimidazole drugs were reduced electrolytically and by gamma-radiolysis at fast (300 mumoles or 100% per hr) and slow (3-9 mumoles or 1-3% per hr) reduction rates in the presence of Escherichia coli DNA and single stranded or double stranded DNA from the bacteriophage phi X174. The degree of DNA damage depends upon the rate of drug reduction, where slow reduction produces more damage than fast reduction. The efficiency of damage produced is in the order metronidazole greater than ornidazole greater than azomycin greater than misonidazole greater than benznidazole which reveals a linear correlation between the one-electron reduction potential (E17) and the negative logarithm of the concentration of reduced drug at which 37% of the original DNA activity remains. Damage is not influenced by the presence of O2 at least between about 1-100 ppm. We suggest the protonated one-electron nitro radical anion as a possible candidate for the active damaging species and explain the basis of the relative cytotoxicity of these drugs under conditions of hypoxia.

Inactivation of Biologically Active DNA by Isopropanol and Formate Radicals

If OH and H radicals, produced by absorption of ionizing radiation in aqueous solutions, are scavenged with isopropanol or sodium formate, secondary radicals are formed which can inactivate phi X174 DNA. From experiments at various DNA concentrations and dose rates we were able to determine the rate constant and the inactivation efficiency of the reaction of these organic radicals with single stranded DNA.

Effects of sulphydryl compounds on the radiation damage in biologically active DNA.

The effect of sulphydryl compounds on the induction of alkali-labile sites and on the contribution of such sites to the inactivation of single-stranded phi X174 DNA was studied. Cysteamine is capable of reacting with DNA radicals, thereby modifying the radiation damage in such a way that the induction of immediate and latent breaks is reduced. This depends on the pH of the solution. With cysteine only, a pH dependent protection, against lethal alkali-labile potential breaks could be observed. The damage other than breaks is not influenced by the presence of sulphydryl compounds.

Alkali-labile sites and post-irradiation effects in gamma-irradiated biologically active double-stranded DNA in aqueous solution.

Gamma-irradiation of double-stranded RF-DNA of bacteriophage phi X174 in aqueous solution in the presence of oxygen produces at least one type of alkali-labile site. It is lethal and gives rise to breaks by alkali and is identical with the damage which becomes manifest by post-irradiation heat treatment. The effect of alkali is dependent on temperature. Furthermore, the excision repair system is not involved in eliminating lethal nucleotide damage in RFI-DNA.

Alkali-labile Sites in Biologically Active DNA: Comparison of Radiation Induced Potential Breaks and Apurinic Sites

Gamma-ray induced alkali-labile sites, which are converted into breaks by alkali or heat, are compared with apurinic sites in both single-stranded and phi X174RF-DNA. It is shown that the kinetics of the conversion into breaks are completely different for both sites. The conclusion is: the gamma-ray induced alkali-labile sites are not identical with apurinic or/and apyrimidinic sites.

Inactivation of Biologically Active DNA by Hydrated Electrons

SummaryFrom a comparison of the inactivation yields of gamma-irradiated ϕX174 DNA with and without an electron scavenger, we conclude that hydrated electrons inactivate DNA. However the number of inactivated molecules is only small compared with the number of hydrated electrons available, which is attributed to a low inactivation efficiency (about 8 per cent) and to the presence of impurities. A significant inactivation of ϕX174 DNA by hydrated electrons could be measured, only when the contribution of OH and H radicals was largely suppressed by specific scavengers for these primary radicals.

Influence of Anoxic Sensitizers on the Radiation Damage in Biologically Active DNA in Aqueous Solution

The competition between biologically active single-stranded phiX174 DNA and the anoxic radiosensitizers metronidazole, misonidazole, paranitroacetophenone or nifuroxime for OH radicals is studied. The results are compared with experiments in which the protection of the DNA by t-butanol is determined. Also the effects of the sensitizers on the chemical nature of the damage (immediate and potential break, immediate and potential base damage) is studied. It is found that in diluted aqueous solutions of DNA these radiosensitizers do not sensitize with respect to the biological inactivation. The only effect observed is a shift from potential to immediate breaks with misonidazole and also nifuroxime.