Physics 561, Lecture 7

Modifiers of Response to Radiation

Types of Modifiers.

The fundamental question we ask ourselves about radiation's effect on biological tissue is: how much damage will a given amount of radiation cause? The answer, glibly, is: it depends. This unit is about what the response depends on.

There are, broadly, two kinds of modifiers: biological modifiers and physicochemical modifiers. We will treat the biological modifiers first. This constitutes a reversal of our normal practice: for the most part we've covered physics and chemistry first and moved on to biology only afterward.

Biological modifiers

There are a variety of ways that a biological system introduces its own set of modifiers to its response to radiation. An obvious way is the variation associated with cell type and tissue type. Cells that divide frequently are more radiation-sensitive than those that divide rarely. Tissues that are made up of rapidly dividing cells are similarly radiation-sensitive. It is fairly easy to understand this: the more frequently cells divide, the more likely it is that radiation-induced damage to DNA will manifest itself in phenotypic alterations at mitosis rather than being repaired somewhere along the cell cycle. Cells that are fully oxygenated tend to be more sensitive than those that are less well-oxygenated. The reasons for this will be discussed later in the lecture.

Probably the most evident biological mechanism that introduces variability in response to radiation is the cell cycle. This is a large subject, and a quick review of what constitutes the cell cycle is in order.

Cells that go through periodic cell division, i.e. all cells except terminally differentiated cells that are destined never to divide again, are considered to pass through four phases within a single life cycle, i.e. in the progression from one cell division to the next. These four phases are known as: the mitotic (M), presynthetic (G1), synthetic (S), and postsynthetic (G2) phases. Cells are more sensitive to radiation damage at certain phases than at others, and they are more prone to delays in the cell-cycle velocity at some stages than others. The properties of these phases relevant to radiation biophysics are as follows.
Phase Symbol Timespan Defining
Activity		 Radiation
Sensitivity		 Radiation-induced
Cycle Delay
Mitotic M 5-10% Cell
Division		 Sensitive  
Presynthetic G1 10-50% Preparation for
DNA synthesis		 Moderate Minimal
Synthetic S 30-50% DNA Synthesis Insensitive Substantial
Postsynthetic G2 15-25% Preparation
for mitosis		 Sensitive Substantial

Thus the least sensitive cells are those in the synthetic phase, particularly the later part of the S phase. The most sensitive are those in the G2 and M phases. It is likely that the radio-resistance is strong in the S phase because the DNA repair enzymes that allow radiation-induced damage to appear in DNA are at full activity during S phase and less so at other stages. During G2 and M the DNA is packaged in chromosomes, where the repair enzymes would have difficulty operating. The mechanisms by which cell progression is delayed by radiation are less well understood. There are several protein kinases involved in regulating the cell cycle, and it may be that radiation-induced degradation of the these kinases slows the cycle.

Physico-chemical Effectors


Water

The first physical or chemical effector of the sensitivity of tissue to radiation that we will discuss is water. We recall that much of the damage from radiation is mediated through free radicals derived from water (OH., H., O2-.) and energetic ions produced in water. The radicals and high-energy ions are responsible for damage to DNA and other biomolecules, so the less water we have in a system, the less damage will occur-- all other things being equal, which they rarely are.

This dependence on water availability, i.e. on the concentration of water, is almost irrelevant to biology. The molarity of pure water is ρM, where ρ is the density of water and M is its molecular weight. Thus water is
(1 kg/L) / [ (18 g/mole) * (0.001 kg / g) ] = 55.5M. Even in biological systems that contain high concentrations of salts and other solutes the water activity will be only slightly lower than this maximum value of 55.5M, so variations based on differences in water availability matter little.
Temperature

Another physical effector is temperature. Most chemical reactions proceed more rapidly at high temperature than at low; typically the log of the reaction rate is linearly related to the inverse of the Kelvin temperature, with a negative slope. The slope is equal to -ΔG‡ / R, i.e. the activation energy of the reaction divided by the gas-law constant:

Thus any chemical reaction will display a temperature dependence that is relatively easy to characterize. Among the reactions that constitute the interaction between radiation and biological systems are covalent bond breakages, restitutions of radicals, and enzymatic repair of damages. Each of these processes proceeds through a different mechanism, and there is no reason to expect any two of them to have the same activation energy. So the overall temperature dependence of some chemical endpoint associated with irradiation of biological samples will be complex.

The reality is that temperature will rarely matter. Activation energies associated with biological reactions are small enough that the difference in rate between, say, 25°C = 298K and 37°C = 310K will be small. Alpen characterizes various categories of radiation-related reactions in three ranges of temperatures--T <. 100K, 100K < T < 170K, and 170 < T < 420K. Studies in these first two ranges have little relevance to real biological systems, and the variability in temperature in the last range is small. One could use thermal kinetics--that is, the temperature dependence of reaction rates-- as a way of sorting out the differences between direct action of radiation, viz.
R-H + hν -> R-H.+ + e-
and indirect action, viz.
R-H + .OH -> R. + H2O
since the thermal implications of these two approaches will be different. But in reality temperature effects are of minor influence on the biological response to radiation.
Oxygen

A more significant chemical effector is oxygen, based on the following facts:

Higher organisms have developed elaborate mechanisms for delivering oxygen to outlying tissues. Typically they have some version of an oxygen-delivery molecule, hemoglobin, that carries oxygen from the site where it is available to the site where it is needed. They also typically have a storage molecule, myoglobin, which hangs on to an oxygen molecule until the cell needs it for a reaction. Other oxygen-related control systems occur as well. In spite of all these control systems, the partial pressure of oxygen does vary substantially from one tissue to another. Many reactions proceed differently in the presence of high concentrations of oxygen as compared with the rate at low or zero P(O2). These differences will influence the radiation sensitivity of tissues.

One of the mechanisms by which oxygen influences radiation response is damage fixation by oxygen. In this instance a macromolecular or small-molecule reactant reacts with molecular oxygen, and a peroxyl radical results:
R. + O2 -> R-O2.
which is a semi-stable free radical (T1/2 = 10-7 to 10-3), capable of remaining in the neighborhood of the tissue where it was produced and causing further damage over the long haul. This oxygen-related mechanism is called "damage fixation by oxygen". We say that the free radical becomes "fixed", i.e. stabilized enough so that further damage may arise. Note that this definition of "fixed" differs almost 180° from the conventional usage, where "fixed" means "repaired." Reactions like the one just mentioned can compete with the organism's built-in mechanisms for scavenging free radicals. An example of these scavenger mechanisms are the reactions of radicals with sulfhydryl reagents:
R. + R'-SH -> R-H + R'S.
R'S. + R'S. -> R'-S-S-R'
where we show that the most common restitution event with the resulting organic sulfur free radicals is dimerization.

A series of cell-culture experiments on the influence of oxygen were conducted in the 1950's. It was found that survival was diminished in oxygen relative to the results obtained in nitrogen. Thus the survival curves look like this:

In fact, it is possible to plot the relative sensitivity of a set of cells to oxygen as a function of the oxygen partial pressure, as shown:

From these considerations we seek a quantitative characterization for the influence of oxygen on a cell culture. The definition we use is of the oxygen enhancement ratio or OER, as such:
OER = (dose in N2 for surviving fraction, S / S0) /
        (dose in O2 for surviving fraction, S / S0)
That is, we choose a particular survival fraction, say S = S0 / 10, and determine the radiation dose necessary to reach S both under oxic and anoxic conditions. Since the cells are more sensitive to radiation under oxic than oxic conditions, the dose in nitrogen necessary to bring the survival fraction down to S0 / 10 will be larger than it is in oxygen. Thus in this figure, we can define OER = DN2 / DO2:


We wish to fit the data from the plot of relative sensitivity against partial pressure. Over a reasonable range of oxygen concentrations a good match to data is obtained from the Howard-Flanders & Alper equation:
S/SN = (m[O2] + K) / ([O2] + K)
where we note that K is in concentration units, i.e. the same units as [O2], and m must be unitless. The value m is known as the maximum relative sensitivity, because if [O2] >> K, (S / SN) = m[O2] / [O2] = m.
We can also examine plots of S vs. [O2] to obtain K. We do this by saying that if K == [O2], then at that particular S value, SK, we find that
SK / SN = (mK + K) / (K + K) = (m+1) / 2.
Some actual values for m and K for three organisms are given below:
Organism m K,µM
Shigella 2.9 4.0
E.coli 3.1 4.7
S.cerevisiae 2.4 5.8

i.e., the most striking differences in behavior is between the bacterial cell systems (Shigella and E. coli) and the eukaryotic cells (Saccharomyeces cerevisiae, i.e., yeast).

Thiols

We have already mentioned the role of thiols in protecting cells from free-radical damage from radiation. The most important type of thiol present is reduced glutathione,

which is reasonably plentiful in tissues. It can react with macromolecular free radicals,
[macro]-R. + R'-SH -> [macro]-R-H + R'-S.,
i.e. a hydrogen atom is transferred from the thiol (glutathione or its equivalent) to the macromolecule so that the thiol becomes the free-radical species and the macromolecule becomes unreactive. The other mechanism by which glutathione exerts its protecive action is through interactions with small-molecule radicals that might otherwise react with macromolecules to cause damage. Formally this is almost an identical reaction:
[small]-. + R'-SH -> [small]-H + R'-S..
In either case the resulting quasi-stable glutathione (or other sulfhyrdryl) radical finds a mate and dimerizes:
R'-S. + R'-S. -> R'-S-S-R'

Radiation Sensitizers

As a final topic for this week we consider whether there might be ways to increase the radiation-sensivity of cells in a tumor. If we are using radiation to kill a tumor, we would like it (the tumor) to be as sensitive as possible to the tumor, so that we will be able kill it with minimal damage to the surrounding non-tumorous tissue. Since tumors are sometimes poorly supplied with oxygen, they are often less radiation-sensitive than are neighboring non-tumor cells that are dividing as fast as they are. The challenge, then, is to devise a way to make the cells in the rapidly growing tumor as radiation-sensitive at it would be if the partial pressure of oxygen around the tumor were as high as in healthy tissue. It is found that certain nitroaromatic compounds, including metronidazole,
,
increase the radiation sensitivity of cells to levels similar to that of fully oxygenated tissue--provided that large doses (several millimolar) are used. Toxicity limits the utility of this method, but if the metronidazole can be administered in the tumor mass without acute toxicity, it could become an important adjoint to radiation therapy.