Horst Jung

The Oxygen Effect

The term “oxygen effect” refers to the observation that the radiation sensitivity of macromolecules and biological systems irradiated in the presence of oxygen or air is generally higher than when they are irradiated under vacuum or in an inert atmosphere. This only applies, however, with ionizing radiations; in UV irradiation experiments, an oxygen effect is only rarely observed. As with the temperature effect, justice is not done to the oxygen effect by treating it merely as a troublesome side-effect of radiation action. It is actually a phenomenon of great heuristic importance for the elucidation of the molecular nature of radiation damage. It is a pity that here, as in many other aspects of radiation biology, relevant experiments are scarce and the many facets of the oxygen effect tend in general to produce confusion rather than understanding. It is therefore not surprising that there is no satisfactory interpretation of the oxygen effect as yet. Nevertheless, an attempt will be made to describe the oxygen effect quantitatively, with the aid of known physico-chemical data and taking specific aspects of the inactivation of microorganisms into account. The chemical mechanisms underlying the oxygen effect will be studied, in the light of experiments on the radiation inactivation of biological macromolecules.

The Temperature Effect

In Chapter 5, target molecular weights for various enzymes were calculated from the 37%-doses measured at room temperature, and the results compared with the true molecular weights of the irradiated biomolecules. As there is no rational reason to attach any special significance to the results obtained at room temperature, the dependence of inactivation rate on the temperature during exposure will now be examined. In some ways, this chapter could be considered as an extension of the target theory; furthermore, some additional features of the indirect effect (see Chapter 6) will become apparent, the significance of which is enhanced by the observation of a remarkable uniformity in the temperature-dependence of many biological systems under irradiation.

The Action of Radiation on Viruses

Viruses are biological objects which have no metabolism of their own and can replicate only with the aid of the genetic and metabolic systems of a host cell, i. e. they are “parasites at the molecular level”. If the host cell is a bacterium they are referred to as bacteriophages, or phages. Since the effects of radiation have been investigated more thoroughly in bacterial viruses than in other systems, a large proportion of the experimental material discussed in this chapter is derived from investigations on bacteriophages.

Radiation Sensitivity and Biological Complexity

With the investigation of the action of radiation on bacteria, the initially declared aims of this book have been achieved: to discuss the most important molecular mechanisms causing damage, and to describe their action on elementary biological systems. A certain basic idea has been repeatedly confirmed during the discussion of radiation sensitivity; this is the concept of the target theory, which basically states that the radiation sensitivity of a biological system increases with the size of its sensitive target. The target in enzymes was shown to be the whole molecule (see Fig. 28), and in viruses and bacteria the total DNA (see Chapters 12 and 13). However, when the examples of single and double strand viruses are considered, the relationship between radiation sensitivity (1/D 37) and the DNA molecular weight, requires the use of different proportionality constants, which are referred to as inactivation probabilities (killing-efficiency, MW T /MW, Tables 15 and 16). The fact that different inactivation probabilities are obtained is due to the specific biological factors which decide whether or not a system can “survive” a specific lesion in the DNA, e. g. whether or not it can repair the lesion. It also, however, depends on the specific type of nucleic acid (single strand or double strand) and ultimately, if higher cells are included, on the specific arrangement of DNA in the chromosomes, which may even be present in multiples of the normal quantity (polyploidy).

The Action of Radiation on Enzymes: The Example of Ribonuclease

The aim of the previous chapters has been to emphasize the basic features as well as the general “laws” of the action of radiation. This forms a sound basis for the understanding of the “biomolecular” portion of this treatise. The study begins with radiation effects on enzymes, although this should not be taken to imply that these phenomena are particularly simple. The structure of enzymes is in some ways much more complicated than that of nucleic acids, and their three-dimensional structure reflects the highly specific catalytic properties. Interest in the action of radiation on enzymes results from the fact that they are essential for the maintenance of vital processes. The aim of this chapter is to derive an approximate picture of the action of radiation on enzymes from the multitude of different observations; but the discussion will be mainly confined to a particularly well-examined enzyme, ribonuclease (RNase), since the quantity and variety of experimental data involved does not allow a collective treatment of the various enzymes.

Direct and Indirect Action of Radiation

In the previous chapters, an attempt has been made to draw conclusions as to the nature of the radiation lesions by applying formal physical and mathematical procedures to the interpretation of dose-response curves, and by investigating the LET-dependence of radiation sensitivity. It was assumed that such a thing as a well defined target really exists. Basically, this assumption seems to be justified, since the formal definition of target used in the hit theory was so general that difficulties are only encountered when attempts are made to identify the target with sensitive biological structures. However, as this is the main theme of the target theory, it is necessary to consider the extent to which realistic targets can be obtained from dose-response curves. The concept of a target does not make any allowance for damage from the “outside”, which is the rule rather than the exception. If the concept is to be retained, then the fraction of the energy contributed from outside to the target must be determined. This leads to the concepts of direct and indirect effect as outlined in Chapter 1.3. Such a classification is meaningful only at the molecular level, where the chances of distinguishing between these two effects are greatest. If the absorption of radiation occurs in the molecule in which the lesion appears, then this is the direct action of radiation,while with indirect action the absorption of the radiation energy and the response to this energy occur in different molecules. This definition is considerably more rigorous than the one used in the past, in which the irradiation of dry systems was considered as direct, while the indirect effect was considered to occur predominantly in the presence of water.

Primary Processes of Energy Absorption

Up to this point, the aim has been to describe the form of dose-response curves schematically, using the principles of hit theory, kinetic theory or general stochastics. A knowledge of the primary processes of absorption is necessary before this discussion can be extended. There are two ways to approach this problem: either by looking at the loss of radiation energy (the slowing down) of a charged particle, or alternatively by looking at the uptake of energy by molecules of the irradiated material. Both approaches will be used, commencing with a consideration of the processes of interaction of radiation with matter. This will include electromagnetic radiation, as well as charged and uncharged particles. The section is subdivided according to the different interaction processes.

Physico-Chemical Changes in Irradiated Nucleic Acids

The nucleic acids have a fundamental role in the maintenance of vital processes. While deoxyribonucleic acid (DNA) carries genetic information, the various ribonucleic acids fulfil important functions in the realization of this information (see Chapter 11.1). The key position in biological processes occupied by nucleic acids made the investigation of the action of radiation on DNA and RNA the central theme of molecular radiation biology. As in the case of enzymes, the main problem is to correlate loss of biological function with the occurrence of physical and chemical changes, and thereby to gain an understanding of the inactivation mechanism. There are numerous biological functions of the nucleic acids that are accessible to measurement, as well as physico-chemical changes induced by irradiation, so that the simultaneous discussion of both of these facets would affect the clarity of this presentation. The physico-chemical and chemical changes occurring in irradiated nucleic acids will, therefore, be considered first, and in the three succeeding chapters an attempt will be made to correlate these changes with the inhibition of certain biological functions. This will not always be simple, as in most cases only one of these effects has been examined by a particular author. The attempts to relate functional with physico-chemical changes have only developed during recent years, especially in the work with bacteriophages.

Inactivation of Nucleic Acid Functions

The nucleic acids fulfil important functions within the complex biochemical processes which are the basis of what we know as life. For example, the total genetic information of an individual is contained in its nucleic acids; this information is carried in most organisms by double-stranded DNA, but in some viruses the genome consists of a single strand of DNA or RNA, and in rare cases of double-stranded RNA. Duplication of the genetic material must occur to enable the genetic information to be transferred from one generation to the next (for example, in cell division). According to the Watson-Crick mechanism for the semi-conservative replication of double-stranded DNA, the original strands separate while the complementary strands are synthesized. This process can also occur in vitro; such a system must contain DNA polymerase and deoxyribonucleoside-triphosphate, as well as a DNA template, also known as “primer” DNA (cf. Kornberg, 1961).

The Hit Theory

The hit theory is the oldest and at the same time the most illustrative of the theories that have been developed to interpret radiation dose-response curves. From the comparison of the effects of poison and radiation carried out in the preceding chapter it is evident that the form of radiation dose-response curves cannot be explained in terms of biological variability alone. The consideration of this initially unexplainable fact led to an entirely new approach: the application of quantum-physical ideas to biological problems. This laid the foundation for an interpretation of dose-response curves in terms of the hit theory, based on two physical observations and one postulate: 1. Ionizing radiation transfers its energy in discrete packets. 2. The interactions (hits) are independent of each other and follow a Poisson distribution. 3. The response under investigation occurs if a specified target has received a defined number (n)of hits.