John F. Ward

The Complexity of DNA Damage: Relevance to Biological Consequences

Ionizing radiation causes both singly and multiply damaged sites in DNA when the range of radical migration is limited by the presence of hydroxyl radical scavengers (e.g. within cells). Multiply damaged sites are considered to be more biologically relevant because of the challenges they present to cellular repair mechanisms. These sites occur in the form of DNA double-strand breaks (dsb) but also as other multiple damages that can be converted to dsb during attempted repair. The presence of a dsb can lead to loss of base sequence information and/or can permit the two ends of a break to separate and rejoin with the wrong partner. (Multiply damaged sites may also be the biologically relevant type of damage caused by other agents, such as UVA, B and/or C light, and some antitumour antibiotics.) The quantitative data available from radiation studies of DNA are shown to support the proposed mechanisms for the production of complex damage in cellular DNA, i.e. via scavengable and non-scavengable mechanisms. The yields of complex damages can in turn be used to support the conclusion that cellular mutations are a consequence of the presence of these damages within a gene. Literature data are used to support these statements and to develop overall mechanisms connecting the production of primary species to the production of biologically relevant damages. The consequences of the LET of the radiation on multiplicity of damage are discussed and suggestions made for the cause of the decrease of the oxygen enhancement ratio as the LET increases.

Biochemistry of DNA lesions.

Ionizing radiation produces a range of damage types in cellular DNA. All damage types do not have the same biological significance. Here arguments are presented supporting the view that lesions in which damage is present on both strands in a local region of the DNA (locally multiply damaged sites--LMDS) will present problems for cellular repair processes. We have previously shown that lesions produced in DNA by individual OH radicals, i.e., single OH species acting alone, are ineffective in mammalian cell killing [J.F. Ward, W.F. Blakely, and E.I. Joner, Radiat. Res. 103, 383-392 (1985)]. We have similar evidence in mutagenesis studies (Ward and Calabro-Jones, unpublished data). Thus the formation of such damage by individual OH radicals formed by ionizing radiation would be similarly ineffectual. Earlier [J.F. Ward, Radiat. Res. 86, 185-195 (1981)] we suggested that OH-radical scavenging studies were consistent with the scavenging of OH radicals in volumes of high radical density, spurs, etc., i.e., in volumes which, when they overlap the DNA, will cause the production of LMDS. The individual constituent lesions of LMDS will be formed as a result of direct ionization or as a result of an OH-radical attack. Both mechanisms can lead to base damage or strand breakage. It is clear that damage in both bases of a deoxyribonucleotide pair leads to loss of base sequence information and can be repaired correctly only by accident or in a recombinational process.(ABSTRACT TRUNCATED AT 250 WORDS)

The Yield of DNA Double-strand Breaks Produced Intracellularly by Ionizing Radiation: A Review

The mechanisms of radiation damage production are used to examine the following premises: (1) the number of DNA double-strand breaks per unit dose increases with dose; (2) cell type to cell type variations in yield of DNA dsb per dose occur. Two stages of damage production are identified as possible sources of damage yield modulation; numbers of OH. free radicals reacting with the target, and amount of chemical repair occurring on the target radicals. These factors are discussed in the light of the structures within which cellular DNA is packaged and the known rate constants for the reactions involved. It is concluded from our current knowledge that, in the presence of oxygen: (a) the number of DNA dsb is linearly related to dose, and (b) the yields of DNA damage per dose among cell types are constant. There is a caveat to the latter conclusion: the chromatin structure may be different in radiosensitive cell lines. In the absence of such a difference, variations in radiosensitivity with dose or with cell type are assigned to differences in repair speed and/or accuracy.

Constraints on energy deposition and target size of multiply damaged sites associated with DNA double-strand breaks

We suggest that the observed experimental data on relative double-strand break (dsb) yield as a function of radiation quality can act as valuable constraints in defining the type of energy deposition which causes this basic lesion in radiation biology. Both heavy-ion and alpha-particle data show sufficient trends for quantitative comparisons with calculation to be made. We use the technique of track-structure simulation and search for energy-deposition clusters (containing at least a given number of ionizations in a given diameter) whose relative frequencies (compared to sparsely ionizing radiation) correlate with the relative biological effects (RBEs) for dsb induction. We conclude that locally multiply damaged sites (LMDS) which cause dsb are probably energy depositions of at least two to five ionizations localized, respectively, in sites of diameters of 1-4 nm. Although our derived cluster sizes should be viewed in light of the quality of the experimental data and uncertainties in the computer simulations at the nanometre level, it is unlikely that these estimates of cluster sizes would change greatly.

Alkaline Phosphatase Promotes Radioprotection and Accumulation of WR-1065 in V79-171 Cells Incubated in Medium Containing WR-2721

Addition of alkaline phosphatase and WR-2721 to culture medium containing V79-171 cells leads to production of WR-1065 and its disulphide forms in the medium, to cellular accumulation of WR-1065, and to radioprotection which correlates with cellular WR-1065 level.

URETERAL STENTING AFTER URETEROSCOPY FOR DISTAL URETERAL CALCULI: A MULTI-INSTITUTIONAL PROSPECTIVE RANDOMIZED CONTROLLED STUDY ASSESSING PAIN, OUTCOMES AND COMPLICATIONS

PURPOSE We compare postoperative pain, stone-free rates and complications after ureteroscopic treatment of distal ureteral calculi with or without the use of ureteral stents. MATERIALS AND METHODS A total of 113 patients with distal ureteral calculi amenable to ureteroscopic treatment were prospectively randomized into stented (53) and unstented (60) groups. Stones were managed with semirigid ureteroscopes with or without distal ureteral dilation and/or intracorporeal lithotripsy. Preoperative and postoperative pain questionnaires were obtained from each patient. Patients with stents had them removed 3 to 10 days postoperatively. Radiographic followup was performed postoperatively to assess stone-free rates and evidence of obstruction. RESULTS Six patients randomized to the unstented group were withdrawn from the study after significant intraoperative ureteral trauma was recognized, including 3 ureteral perforations, that required ureteral stent placement, leaving 53 with stents and 54 without for analysis. Patients with stents had statistically significantly more postoperative flank pain (p = 0.005), bladder pain (p <0.001), urinary symptoms (p = 0.002), overall pain (p <0.001) and total narcotic use (p <0.001) compared to the unstented group. Intraoperative ureteral dilation or intracorporeal lithotripsy did not statistically significantly affect postoperative pain or narcotic use in either group (p >0.05 in all cases). Overall mean stone size in our study was 6.6 mm. There were 4 (7.4%) patients without stents who required postoperative readmission to the hospital secondary to flank pain. All patients (85%) who underwent imaging postoperatively were without evidence of obstruction or ureteral stricture on followup imaging (mean followup plus or minus standard deviation 1.8 +/- 1.5 months), and the stone-free rate was 99.1%. CONCLUSIONS Uncomplicated ureteroscopy for distal ureteral calculi with or without intraoperative ureteral dilation can safely be performed without placement of a ureteral stent. Patients without stents had significantly less pain, fewer urinary symptoms and decreased narcotic use postoperatively.

Mechanisms of DNA repair and their potential modification for radiotherapy

The potentiation of radiation damage, which can be accomplished by the inhibition of repair, is estimated from published studies of repair deficient mutants. Sensitization factors as high as 10 have been achieved. Because it has previously been suggested that the most probable lethal lesion is a DNA double strand break (DSB), it is not surprising that cells deficient in repairing this type of damage are the most radiosensitive. The structures of DNA DSBs and other Locally Multiply Damaged Sites (LMDS) (involving both single strand breaks (SSB) and base damage sites) are reviewed, together with the processes by which cells may attempt to repair these lesions. Repair processes occur in competition with damage fixation, again, mechanisms of damage fixation are predicted from studies in model systems. A strategy for inhibiting the repair processes is devised that consists of holding the first SSB constituent of the LMDS open by repairing in the presence of deoxynucleoside analogues, such as ara-C, so that there is a higher probability of the formation of a DSB upon cleavage of the second site (on the other strand) by hydrolysis of a labile bond or by endonuclease cleavage of a base damaged site. To achieve preferential sensitization of tumor vs. normal tissue it may be possible to take advantage of the deficiency in alkaline phosphatase in tumor vs. normal vasculature, that is, in analogy with treatment with WR-2721. The deoxynucleoside analogue would be delivered together with the phosphate ester (deoxynucleotide) of the correct deoxynucleoside, for example, ara-C, in the presence of deoxycytidine monophosphate (dCMP). Higher alkaline phosphatase levels in normal tissue capillaries would hydrolyse the dCMP to deoxycytidine, which competes effectively with ara-C in repair replication.

Nature of Lesions Formed by Ionizing Radiation

A major goal of those studying ionizing radiation-induced damage in DNA is the identification of the alterations that (when produced within a cell) are the source of higher-level damage (killing, mutation, transformation). Of course, the damage that is produced initially by radiation is subject to enzymatic repair, and hence, to assess the ability of a cell to repair the change, it is important to know the identities of the types of damage. Product identification in the DNA of irradiated cells is not straightforward. Yields of specific base damages have been measured after high doses, but damage to the deoxyribose moieties is less studied, except as the general consequence of such damage, i.e., strand breakage. The majority of studies in which products have been identified and characterized have been carried out in model systems. In these systems, a variety of routes to damage production are known, and an understanding of these routes in the context of the intracellular environment of the DNA makes possible valid extrapolation to the types of damage produced in the cell and, hence, to the identification of the important lesions.

Radioimmunoassay of a thymine glycol.

A sensitive and specific radioimmunoassay for 5,6-dihydroxy-5,6-dihydrothymine (thymine glycol) was developed. As little as 4 femtomoles of thymine glycol can be detected in

Radioimmunoassay of 7,8-dihydro-8-oxoadenine (8-hydroxyadenine).

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