Molecular Mechanisms of Radiation-Induced Damage to Nucleic Acids1

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The integrity of DNA is essential for the well-being of a cell. Damage to this molecule, whether caused by reaction with chemical mutagens, by irradiation with ultraviolet (UV) light, or by ionizing radiation, has dire consequences. Thus, much effort has been expended in attempting to understand the effects of these agents on DNA. This chapter describes some of the chemical mechanisms whereby DNA is damaged by ionizing radiation. The major advances in understanding of molecular mechanisms have come from the studies of simple model systems. The chapter explains the three basic model systems that have been designed to examine radiation-induced damage to DNA. Each type of system yields useful information, but the applicability of this information to the in vivo situation is limited in all cases. The chapter also discusses the indirect effects of the free radicals produced by the radiolysis of water, in terms of both initial species and final products.

Introduction

The integrity of DNA is essential for the well-being of a cell. Damage to this molecule, whether caused by reaction with chemical mutagens, by irradiation with ultraviolet (UV) light, or by ionizing radiation, has dire consequences. Thus, much effort has been expended in attempting to understand the effects of these agents on DNA.

The intent of this review is to describe some of the chemical mechanisms whereby DNA is damaged by ionizing radiation. Cellular radiobiology has progressed far from the state described by Butler (1956): “We are at the moment in the position of a man who tries to elucidate the mechanism of a telephone exchange by throwing bricks into it and observing some of the results.” The major advances in understanding of molecular mechanisms have come from studies of simple model systems, and it is these systems that will be considered in this review. To place the various model systems in perspective, the general mechanisms of the conversion of energy to damage in a cellular system are discussed in Section II. Against this background, all the model systems discussed in the review are judged as to their validity to the in vivo situation.

The major part of the review is devoted to data derived from aqueous systems. The goal of the discussion is to describe at a molecular level radiation-induced reactions which would account for the DNA damage observed after in vivo irradiation: base damage and strand breaks.

Section snippets

Production of Radiation Damage in a Heterogeneous System

The processes involved in the deposition of energy from ionizing radiation are comparatively well understood (for a review, see Klots, 1968); they result in the formation of ionized species and excited molecules. These initial processes do not occur preferentially with any one molecular species in a mixture; the fraction of the energy deposited in any molecular species in a mixture of species is approximately proportional to the fraction of that species (by mass) present.

Traditionally,

Simulation of in Vivo Radiation Damage Using in Vitro Model Systems

In Sections IV, V, and VI the three basic model systems which have been designed to examine radiation-induced damage to DNA are discussed. Each type of system yields useful information, but the applicability of this information to the in vivo situation is limited in all cases.

Systems described in Sections IV and V provide information on damage which could result from “direct effects.” The data are essentially limited to a description of the radicals produced. Little is known of the final

Irradiation of Solid Materials

The effects produced by deposition of energy in single crystals, mixed crystals, and powders of DNA constituents and in DNA fibers have been widely examined. Two major techniques have been used to examine the initial species produced.

Frozen Solutions

Another method which has been used to stabilize free radicals for observation by EPR spectroscopy is matrix isolation. The molecules of the parent compound are separated from each other in a frozen glass, thus reducing the ability of radicals formed on them to interact.

Unfortunately, the aqueous matrices which can be used to accomplish this require extremes of pH. The pH extremes cause the solute molecules to become ionized; as a consequence they are frozen isolated in the matrix. In neutral

Aqueous Solutions

On irradiation of aqueous solutions of DNA and its analogs, most of the energy is deposited in water. Consequently any effects observed on the solute are the result of reactions with the species produced by water radiolysis. These species are well known and consist of hydrogen atoms (H•), hydrated electrons (eaq−), and hydroxyl radicals (OH•) [reaction (13)]. Extensive investigations of their reactions with a wide variety of compounds have been carried out.

Radical Identification by EPR

Radicals produced in pulse radiolysis are not easily identified from their optical absorption spectra, which are, in general, broad and featureless. The EPR spectra of radicals is of much greater use in determining structure. Limited use has been made of this technique, probably owing to the problems of instrument design. The pioneering work in this area was carried out by Smaller et al., They overcame the limiting time resolution of normal EPR spectrometers by using a higher modulation

Thymine in the Presence of Oxygen

The earliest studies of radiation damage to DNA constituents describe products formed by fragmentation of the molecules (Scholes et al., 1949). Later, hydroperoxides were detected in irradiated DNA by Scholes et al., (1956). Daniels et al., (1957) showed that the peroxides were formed on the pyrimidine bases by saturation of their 5,6 double bond. The hydroxyhydroperoxide formed from thymine has received a great deal of attention. This compound has recently been shown to be formed by

Strand Breaks

Strand break measurement is the major method currently used to detect radiation damage to DNA in vivo. Much less work has been done on model systems for this type of damage than for damage produced in the base moieties. The production of a strand break is probably caused by breakage of a sugar phosphate bond between two nucleoside residues in DNA (Fig. 6A). Thus deoxynucleotides (Fig. 6B) can be used as simple model systems where a strand break-producing reaction would break the sugar phosphate

Modifications of Radiation Damage Caused by Macromolecular Structure

It is appropriate to consider how the radiation chemistry of the monomers could be affected by their involvement in the macromolecular and in vivo situations.

Roots and Okada have shown that OH• radicals are responsible for lethal and strand break damage in vivo. However, high concentrations of OH• scavengers were required to reduce this damage. From the concentration data Roots (1970) has calculated that OH• radicals move only 20–40 Å from their site of production to their site of reaction.

Effects of Other Free Radicals on DNA Constituents

Radiation-induced destruction of DNA constituents by free radicals produced from other solutes has been reported. Brown et al., (1966) reported the radiation-induced binding of ethanol to thymine; this was recently confirmed by Zarebska and Shugar (1972). De Jong et al., (1972) showed that phenylalanyl radicals react with φX DNA to reduce its biological activity. Ward and Kuo (1968) and Patterson et al., (1972) showed that Cl2− ion radicals react rapidly with some DNA constituents. However, Cl2−

Methods of Examining Direct Effects in Aqueous Systems

Several attempts have been made to measure direct effects of radiation in aqueous systems; however, the conditions chosen were not adequate to screen out a significant proportion of the indirect effects.

Some authors carried out irradiation at 77°K at which temperature the free radicals produced from water are essentially immobile. Any damage to the solute molecules measured at 77°K (i.e., by EPR spectra) result from direct effects (or from radicals produced in close proximity to the molecule).

Radiation Modifiers

The response of cells to the effects of radiation can be affected by the presence of various chemical compounds during irradiation. These compounds can be postulated to act either during the period of radiation damage production or during the enzymatic repair of the damage. In this review only the former type of effect will be considered.

If a reaction scheme for the production of damage in DNA in vivo is postulated:

Direct effects

.

Indirect effects DNA+X•→DNA*• DNA*+P→DNA DNA*+

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    The studies carried out in the author's laboratory were supported by Contract AT(04-1) GEN-12 between the U.S. Atomic Energy Commission and the University of California and by a grant from the U.S. Public Health Service.

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