By Thomas G. Brock, Ph.D.

Radiation can be divided into two primary categories: ionizing and non-ionizing. The latter includes microwaves, which are relatively low energy and evoke effects primarily through heating. Ionizing radiation, on the other hand, has sufficient energy to displace electrons from atoms or molecules, producing ions. Ultraviolet light, X-rays, and gamma rays, as were released from the Fukushima Daiichi nuclear power plant disaster in Japan recently, are examples of ionizing radiation. Other interesting considerations include collateral effects arising during radiation treatment for cancer and exposure in the laboratory resulting from radiation used for research purposes.

While there are many types of ionizing radiation (e.g., α, β, and γ radiation), the key effect is the ejection of electrons. Atoms or molecules with unpaired electrons are referred to as ‘radicals', often termed ‘free radicals'. The unpaired electrons generally cause radicals to be highly chemically reactive. In biological systems, this means that ionizing radiation can directly damage all types of molecules (e.g., DNA, proteins, lipids) or the effect may be secondary to the generation of free radicals, which react with nearby molecules. As described below, this may range from a good, or even necessary, action to a deleterious (i.e., lethal) event. Radicals tend to attack double bonds, favoring carbonyl, vinyl, and phenolic groups that are common on antioxidants. While radicals are typically highly reactive, some are more stable or persistent. For example, the radical derived from -tocopherol (vitamin E) is long-lived. As a result, vitamin E, a well known antioxidant because of its ability to react with free radicals, may instead be converted by ionizing radiation to a radical form.

Reactive Oxygen Species

Many cells synthesize the reactive oxygen species (ROS) superoxide, O₂^-, enzymatically by an NADPH oxidase complex. Superoxide is also generated as a by-product of mitochondrial respiration. Superoxide is efficiently metabolized by a family of superoxide dismutases (SOD) to produce oxygen and hydrogen peroxide (Figure 1). Hydrogen peroxide, in turn, is converted by catalase to water and oxygen via hydroxyl radical. In humans, there are three distinct SOD genes and gene products (soluble (cytoplasmic) SOD1, mitochondrial SOD2, and extracellular SOD3); the ubiquitous catalase occurs as a single form. The formation and elimination of ROS by this pathway involves the sequential addition of electrons. Ionizing radiation, on the other hand, involves the removal of an electron from water, producing the highly reactive hydroxyl radical. This can be followed, to a lesser extent, by further electron ejection to give H₂O₂.

Figure 1. Formation and elimination of ROS

In concert with SOD, catalase, and other enzymes, antioxidants react with ROS to take them out of play. One of the most important natural antioxidants is glutathione, a tripeptide composed of glycine, cysteine, and glutamine. Normally, glutathione is maintained in a reduced form through the activity of glutathione reductase, which is constitutively active. As a result, the thiol group of the cysteine within glutathione is protonated, with reduced glutathione being abbreviated as GSH. This thiol group is able to donate a reducing equivalent to reactive molecules, including ROS. Upon donating an electron, glutathione itself becomes reactive, joining similarly oxidized molecules to produce the glutathione disulfide (GSSG). Cayman carries a Glutathione Assay Kit as well as a variety of assay kits for enzymes which process glutathione.

DNA Damage

The effects of ionizing radiation can be divided into direct and indirect. Each has distinct ramifications. Radiation can directly disrupt DNA, introducing isolated nucleotide damage, double-strand breaks, or clustered DNA damage. Each type of damage induces its own type of repair pathway. For example, double-strand breaks are mended by homologous recombination if the damage is minimal, but nonhomologous end-joining may occur if the radiation damage produces large or multiple strand breaks. The steps involved in repairing DNA that has been directly damaged by ionizing radiation are complicated, although the basic processes are well-understood.

ROS, produced by ionizing radiation, also damage DNA. The most vulnerable site for oxidative damage on DNA is on guanosine and, specifically on carbon-8 (Figure 2). Note that this site is not normally involved in bonding between guanosine and cytosine. As a result, it can be attacked in both single- and double-stranded DNA. The abstraction of a proton from carbon 8 leads to the production of 8-OH-dG. Tautomerization with nitrogen-7 produces 8-oxo-2-deoxyguanosine (8-oxo-dG), in reference to the carbonyl group at C8; the term 8-oxo-dG is used interchangeably with 8-OH-dG. Other bases can undergo oxidative damage as well. Interestingly, the other purine, adenosine, can be oxidized on either carbon-2 or -8. The pyrimidines typically are hydroxylated on carbon-5.

Figure 2. Oxidative damage to nucleotides: focus on guanosine

Consequent to this DNA damage is base excision repair, which involves removal and replacement of the oxidized base from the sugar-phosphate backbone.¹ The result is the generation of free 8-hydroxy guanine (referred to as either 8-OH-G or 8-OH-Gua). This product is uncharged and thought to be readily secreted from intact cells; it is unclear whether this requires endosomal packaging. Like DNA, RNA can be damaged by reactive oxygen and reactive nitrogen species. A common product of RNA oxidative damage is 8-hydroxy guanosine (8-OH-Guo). Similarly, individual nucleotides can be oxidized: GTP can become 8-OH GTP. Cayman offers an DNA/RNA Oxidative Damage ELISA Kit for evaluating oxidative damage of DNA.

Lipid Damage

The direct effects of ionizing radiation on lipids are less significant, compared to those on DNA: a damaged lipid molecule is easily replaced, whereas damaged DNA must be repaired. The indirect damage of lipids by radiation-induced ROS, however, can be devastating. In membranes, nature has created the ideal setting for a remarkable chain reaction. One important attribute of the membrane is its localized chemistry. Of course, membrane phospholipids commonly have a PUFA in the sn-2 position. These may be any of the medium to long chain fatty acids, with variable numbers or positions of the sites of unsaturation. Significantly, pairs of double bonds on PUFAs are always separated by an intervening methylene group (Figure 3). This configuration makes a hydrogen atom on the methylene group very reactive, so it is readily abstracted by a free radical. The removal of this hydrogen is the initiating step in lipid peroxidation, and the product itself is a fatty acid radical. Molecular oxygen can then react with the lipid radical to produce an unstable peroxyl fatty acid.

Figure 3. The lipid peroxidation chain reaction

This brings to bear the second important attribute of membranes: the abundance and proximity of PUFAs. These fatty acids contribute to membrane fluidity. However, adjacent PUFAs make very nice hydrogen atom donors from the reactive methylene groups to stabilize peroxyl-fatty acids. While this stabilizes the peroxyl by formation of a peroxide on the first lipid, the adjacent fatty acid now contains a radical that can react with oxygen, propagating the chain reaction. In this way, regions of membranes can be rapidly oxidized unless something terminates the cycle. Chemicals that act as antioxidants can effectively terminate the peroxidase chain reaction.

Hormesis

An important concept in toxicology, which also applies to radiation biology as well as physiology in general, is hormesis. Hormesis is defined by Merriam-Webster as "a theoretical phenomenon of dose-response relationships in which something (as a heavy metal or ionizing radiation) that produces harmful biological effects at moderate to high doses may produce beneficial effects at low doses". In fact, hormesis has strong scientific proponents. Over 20 years ago, a group of scientists, representing federal, industrial, and academic interests, formed the Biological Effects of Low Level Exposures (BELLE) Advisory Committee. From the beginning it was clear that ‘biological systems have an impressive array of adaptations that may be turned on in response to various stresses, including physiological stress, as well as exposure to radiation, toxic chemicals, and dietary alterations (belleonline.com). The key concept centers on the adaptive response to an initial, low level cue, which leads to tolerance to subsequent stimuli. For example, ischemic preconditioning (defined, generally, as producing resistance to the loss of oxygen in tissues) is so effective in reducing ischemia/reperfusion injury following surgery that the current question centers on the best of many methods.² In fact, the initial cue can be the same as (homologous) or different from (heterologous) the subsequent stimulus.³ Thus, ischemic preconditioning can be achieved by ischemia, by antioxidants, or by trimetazidine, a fatty acid oxidation inhibitor. With respect to radiation, there is conflicting evidence as to whether low-level, whole body irradiation can be protective or is uniformly deleterious.⁴ Recent reports from BELLE summarize the extensive literature demonstrating the beneficial health effects of low-level exposures to ionizing radiation, as well as reasons why these studies are poorly appreciated.^5-7 Clearly, additional research is necessary.

References

1. David, S.S., O'Shea, V., and Kundu, S. Nature447, 941-950 (2007).

2. Theodoraki, K., Tympa, A., Karmaniolou, I., et al.Surg. Today41, 620-629 (2011).

3. Wiegant, F.A.C., Prins, H.A.B., and Wijk, R.V. Dose Response9, 209-224 (2011).

4. Vaiserman, A.M. Dose Response8, 172-191 (2010).

5. Calabrese, E.J. Hum. Exp. Toxicol.29, 249-261 (2010).

6. Jaworowski, Z. Hum. Exp. Toxicol.29, 263-270 (2010).

7. Liu, S.-Z. Hum. Exp. Toxicol.29, 275-281 (2010).