40.Luton D, de Lagausie P, Guibourdenche J, Peuchmaur M, Sibony O, Aigrain Y, Oury IF, Blot P. Influence of amnioinfusion in a model of in utero created gastroschisis in the pregnant ewe. Fetal Diagn Ther 2000;15:224–228.

41.Luton D. Etude de L’inflammation, dans le Laparoschisis, Humain et dans un Modele de Laparoschisis de Bredis. These; 2001.

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48.Beydoun S, Yasin S. Premature rupture of the membranes before 28 weeks: Conservative management. Am J Obstet Gynecol 1986;155:471–479.

49.Rotschild A, Ling E, Puterman M. Neonatal outcome after prolonged preterm rupture of the membranes. Am J Obstet Gynecol 1990;162:46–52.

50.Sebire NJ, Snijders RJ, Hughes K, et al. The hidden mortality of monochorionic twin pregnancies. Br J Obstet Gynecol 1997;104(10):1203–1207.

51.Patten RM, et al. Disparity of amniotic fluid volume and fetal size: Problem of the stuck twin-US studies. Radiology 1989;172:153–157.

52.Danskin FH, Neilson JP. Twin-to-Twin transfusion syndrome: What are appropriate diagnostic criteria? Am J Obstet Gynecol 1989;161:365–369.

53.Senat MV, Deprest J, Boulvain M, Paupe A, Winer N, Ville Y. Endoscopic laser surgery versus serial amnioreduction for severe twin-to-twin transfusion syndrome. N Engl J Med 2004;351:136–144.

54.Benirschke K, Driscoll S. The Pathology of the Human Placenta. New York: Springer-Verlag; 1967.

55.Deprest JA, Audibert F, Van Schoubroeck D, Hecher K, Mahieu-Caputo D. Bipolar coagulation of the umbilical cord in complicated monochorionic twin pregnancy. Am J Obstet Gynecol 2000;182:340–345.

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See also FETAL MONITORING; HYDROCEPHALUS, TOOLS FOR DIAGNOSIS AND TREATMENT OF; MONITORING, UMBILICAL ARTERY AND VEIN.

OIL. See LENSES, INTRAOCULAR.

ION-EXCHANGE CHROMATOGRAPHY. See

CHROMATOGRAPHY.

IONIZING RADIATION, BIOLOGICAL EFFECTS OF

ZELENNA GOLDBERG

Department of Radiation

Oncology

Davis, California

INTRODUCTION

Radiation biology refers to all biologic responses induced in cells and tissues by ionizing radiation, a term that encompasses the study of all action of ionizing radiation on living things. Ionizing radiation (IR) is radiation that has sufficient energy to cause the ejection of an orbital electron from its path around the nucleus, which indicates that the photon or charged particle can release large amounts of energy in a very small space. IR is separated into electromagnetic radiation and particulate radiation. Electromagnetic radiation consists of X rays or g rays, which differ only in their mode of production, not in their physical effects in interacting with biologic tissue. X rays are produced by machines that accelerate electrons and then focus them to hit a target usually made of tungsten or gold. The kinetic energy is transferred from the electrons to the target and then is released as photons. These are the most common medical exposures through diagnostic or therapeutic radiation. In contrast, g-radiation is produced within the nucleus from radioactive isotopes. The g rays result from the unstable nuclear configuration decaying to a more stable state and releasing the ‘‘extra’’ energy in this transition in the form of the g ray. Natural background radiation is of this type. Particulate radiation is those radiation sources that are not photons and consist of electrons, protons, a-particles, neutrons, and heavy charged particles such as the nuclei of carbon, neon, argon, or iron. These particles are positively charged because the orbiting electrons have been stripped from them.

As noted, IR is defined by its causing the ejection of an orbital electron when the radiation interacts with tissue. These ionizing events can be further subdivided by being directly ionizing or indirectly ionizing. Directly ionizing events are those in which a charged particle with sufficient kinetic energy interacts with the tissue, directly resulting in the ejection of the orbital electron. These events happen frequently within an exposed tissue, so the charged particle rapidly transfers its energy to the tissue, a concept codified as linear energy transfer (LET). Charged particles have a high LET. In contrast, indirectly ionizing radiation such as g- or X-radiation is first absorbed in the material and secondary, energetic charged particles (electrons) are released. Because this latter process only occurs when the photon is close enough to an atom to interact, it is referred to as sparsely ionizing radiation and has a low linear energy transfer (LET).

182 IONIZING RADIATION, BIOLOGICAL

A longstanding paradigm in radiation biology has been that many effects induced by IR, including its carcinogenic effects and ability to kill cancer cells, are the result of DNA damage arising from the actions of IR in cell nuclei, especially interactions of IR and its products with nuclear DNA (1–3). When a charged particle or secondary electron produced by the interaction of a photon with an orbital electron damages DNA itself, it is known as the direct action of IR. Yet, DNA represents a small fraction of the actual size of the cell. Therefore, it was recognized that most of the interaction of IR within a cell would be with more abundant biomolecules, specifically water. If the action is mediated through the intracellular production of radiolytic reactive products, e.g., OH , H , O2, and H2O2, that are generated in aqueous fluid surrounding DNA, then the DNA damage is called indirect action. These processes unquestionably can result in a variety of types of DNA damage, including DNA singleand double-strand breaks, modifications of deoxyribose rings and bases, intraand inter-strand DNA–DNA cross-links, and DNA–protein cross-links (1,4,5). About a third of all DNA damage is caused by the direct effects of sparsely ionizing g and X rays, with the remaining balance being attributable to the indirect actions of IR. With highLET radiation such as the more densely ionizing a-parti- cles that are emitted by radon and radon progeny, the direct actions of IR on DNA become more predominant and the nature of the DNA modifications become much more complex. Regardless of the type of IR, all of the above forms of DNA damage can lead to untoward effects in cells if unrepaired or misrepaired. With specific regard to carcinogenesis, genomic mutations caused by IR are widely thought to arise from DNA damage that is subsequently converted into a mutation as a result of misprocessing by DNA repair mechanisms or that is converted into a heritable mutation when DNA undergoes replication.

This classic view of radiation biology is giving way to a more complex and complete understanding that the cell membrane and other intracellular compartments, as well as the tissue vasculature, are important targets of IR. Furthermore, as detailed below, although intracellular effects are better defined, we now recognize that the extracellular environment and cell–cell contact play important roles in modulating the effects of IR at the cellular and tissue levels.

MOLECULAR RADIATION BIOLOGY/BYSTANDER EFFECTS

IR is a cellular toxin that is sensed at the cellular level through the ATM-p53-p21 pathway (6,7). Although the upstream sensor of ATM remains to be definitively elucidated, the initial radiation sensing protein likely has a redox sensor and undergoes a conformational change, or possibly is phosphorylated, resulting in the phosphorylation of ATM (8). This, in turn, activates the p53 pathway leading to cell cycle arrest, nuclear translocation of transcription factors such as NF-kB, and either cellular repair or apoptosis. How an individual cell commits to a given fate (repair or apoptosis) remains unclear, but undoubtedly it represents the final integrated response to many simultaneous intracellular events. Several excellent reviews on

this topic have been written (9,10). It should be noted that IR also affects the 26s proteosome, which is another level of non-nuclear intracellular response affecting cell survival, presumably through alterations in the removal of activated (phosphorlyated) proteins, or proteins active in apoptotic processes such as Bcl2 (11,12).

Radiation effects in cells not directly hit by a radiation ionizing event are called bystander effects. Although initial interest in this effect can be found in the medical literature as far back as into the 1950s, the current interest in the area was stimulated by a study published in 1992 in which Nagasawa and Little (13) observed increases in the frequency of sister chromatid exchanges in 30% of immortalized Chinese hamster ovary cells that received low-dose exposure to a-particles, even though less than 1% of the cells’ nuclei were estimated to actually receive direct nuclear hits by an a-particle. That a relatively low percentage of the cells experienced one or more direct ‘‘hits’’ by the a-particles, be they in the cytoplasm or in the nuclei, suggested the possibility that some mechanism was conveying a radiation-associated response to unirradiated cells. Other groups went on to confirm and extend on this finding. It is now well recognized that cells do not require a direct nuclear traversal to result in radiation changes. There are extracellular responses, predominantly TGF-b mediated through media (cell culture experiments) or extracellular fluids (tissues), but other factors cannot be excluded (14). Furthermore, it is recognized that cell–cell contact and gap junctional communications are critical in transmitting the signal from the directly irradiated cell to the neighboring, bystander cells (15).

TIME, DOSE, AND FRACTIONATION

The biologic effects of IR in tissue relate to the size of the dose delivered, time between radiation exposures and the total dose of IR given. Although environmental exposures are chronic and (usually) low level, medical exposures are acute and can be repetitive when given for the treatment of cancer. Radiation therapy for the treatment of malignancy remains the most effective anti-cancer agent discovered, and treatment schedules are predicated on the ‘‘4 R’s of radiobiology’’: repair, repopulation, redistribution, and reoxygenation.

Repair of radiation-induced damage underlies the intrinsic radiation sensitivity of the cell to radiation cell killing. Cells can be broadly grouped into those that can repair significant amounts of damage and those with more limited repair capacity. The former descriptive category corresponds to tissue types where there is limited normal cell turnover, such as lung, kidney, or brain, and these are tissues that display damage after a more prolonged time and are therefore known as late responding tissues. The cells with more limited intrinsic repair capacity are known as acute responding tissues and are typified by skin or gut. These cell types have a limited life span and are constantly being replaced within normal physiologic functioning.

Repopulation refers to the generation of new cells to replace those killed by the IR exposure. Although therapeutically beneficial for containing normal tissue toxicity,

repopulation is also active in malignant tissue and thereby allows greater numbers of tumor clonogens to develop after treatment. For some types of tumors, an acceleration of repopulation begins after 4 weeks of radiation therapy, which can compromise clinical outcome if prolonged therapeutic IR fractionation schemes are used.

Redistribution refers to the changes in cell assortment across the cell cycle. It has long been established that cells vary in their sensitivity to the cytotoxic effects of IR depending on where in the cell cycle they are at the time of irradiation (16,17). Cells are most sensitive to IR effects in the G2/M phase and are most resistant during the S- phase. G1 is intermediate between these two. Thus, clinical radiation therapy schedules use multiple fractions to overcome the relative resistance of the cells in S-phase of the cell cycle on a given treatment day. This difference in radioresistance across stages in the cell cycle also underlies one mechanism of the synergy between radiation therapy and many chemotherapeutic agents in the treatment of malignant disease. Although S-phase cells resist killing from the IR, they are more sensitive to some chemotherapeutics that are active during S-phase when the DNA is most exposed and is replicating. Furthermore, this alteration of cell cycle sensitivity to IR cytotoxicity also has been an area of research for IR-biologic response modifiers. If one can increase the percentage of cells in G2/M at the time of IR, then the relative cell kill per fraction of IR will increase.

Reoxygenation refers to the presence or absence of molecular oxygen within the cell at the time of IR delivery. As detailed at the beginning, most of the cellular damage from IR is mediated through the production of free radicals within the cell. If molecular oxygen is present, these free radicals can be transformed into more complex peroxide radicals, which are more difficult for the cell to repair. This is classically known as ‘‘fixing the radiation damage’’ in the British use of the term ‘‘fix’’ (make more solid), not the American (repair). The increase in cell killing from IR in the presence of oxygen versus under hypoxic conditions is known as the oxygen enhancement ratio, and it is approximately a factor of 2–3, dependent on where in the cell cycle the irradiated cell sits. Although normal tissues have a well-maintained vascular supply so that oxygenation is essentially constant, tumors have a tortuous and unstable vasculature, so that the vessels can open and close erratically. This type of hypoxia is referred to as ‘‘acute hypoxia.’’ Chronic hypoxia occurs when the tumor outgrows its blood supply and the tumor cells are simply beyond the diffusion capability of molecular oxygen. Attempts to exploit this differential, either by sensitizing the hypoxic cells or by specifically targeting them, have been explored and remain active areas of research. Furthermore, identification of the presence of hypoxia remains a significant clinical strategy (18).

The four R’s of radiobiology reflect intracellular controls of overall radiation response. The tissue level effects from IR in the treatment of cancer are dependent on several parameters: volume, dose per fraction, total dose, and time between fractions. The volume of tissue irradiated is critical for determining the long-term repair by repopulation by normal cells to fill in for those irreparably damaged by the IR. Each cell type has its own intrinsic radiation

sensitivity, and the tissue organization (serial or parallel cellular arrangement) as well as the tissue vasculature play critical roles in tissue repair and thus radiosensitivity. Tissues are subdivided into two categories of radiosensitivity based on when they display their damage. Tissues that display their radiation induced damage during a standard course of medical radiation therapy are known as ‘‘acute responding tissues.’’ These tissues naturally have a large amount of cellular turnover, such as skin or gut, and at a cellular level, this corresponds to lesser ability to repair sublethal DNA damage (i.e., a larger proportion of DNA damage is lethal and nonrepairable). In contrast, tissues where there is little or no normal cellular turnover, such as brain, kidney, lung, or spinal cord, there is a substantial ability to repair sublethal damage, and tissue toxicity from radiation therapy is displayed late, long after therapy has finished. These tissues are therefore labeled ‘‘late responding tissues.’’ Therapeutic strategies are designed to separate these two types of responses as malignant tumors are models of acute responding tissues, whereas the dose limiting side effects from radiation therapy are secondary to late responding tissue effects (19,20).

THE LINEAR NO-THRESHOLD MODEL OF RADIATION EFFECTS

Based on the data collected from the victims of the bomb detonations at Hiroshima and Nagasaki, a linear nothreshold model of radiation effects was developed and adopted for public policy applications. In essence, the model states that (1) all radiation exposures are biologically active, (2) the response in the cells/tissue is linear with dose, and (3) there is no threshold below which there is no or negligible effects. The model was developed from the moderately low-dose exposure ranges of 0.5–2 Gy and the medically significant sequelae of increased mortality (carcinogenic and noncarcinogenic, predominantly cardiovascular) (21–23). Hiroshima and Nagasaki data represent the effects of a single acute dose exposure delivered to the whole body with the nutritional deprivation from WW2; as such, they are subject to criticism and questions of their applicability to modern healthy populations where lowdose radiation exposure is often of a more chronic nature. Nevertheless, they remain the best available populationbased data, and they have been painstakingly collected and analyzed. As detailed below, however, the shape of the response curve at the lowest doses remains controversial.

LOW-DOSE EXPOSURES

Low-dose ionizing radiation (LDIR) in the 1–10 cGy range has largely unknown biological activity in the human. Current modeling for health and safety regulations, as well as prediction of carcinogenesis, presupposes a linear, no-threshold model of radiation effects based on the nuclear bomb explosion data, which estimates the effect and risk at low dose by extrapolation from measured effects at high doses. Yet the scientific literature presents a more complex picture, and few data clearly support a linear

184 IONIZING RADIATION, BIOLOGICAL

dose-response model and none in humans. Numerous studies suggest some effects of LDIR may be benign or even beneficial under some circumstances (24,25). Reported benefits include stimulated growth rates in animals, increased rates of wound healing, reductions in cell apoptosis, enhancements in the repair of damaged DNA, and increases in radioresistance via the induction of an adaptive response, among others (26–28). Other lines of evidence, however, suggest LDIR can be hazardous, and if a threshold for detrimental responses does exist, it is operational only at very low dose levels, e.g., 1 cGy. Schiestl et al. (29) found that 1–100 cGy doses of X rays could cause genetic deletions in mice in a linear dose-response manner. This remains an active area of research (30).

Little is known regarding individual variability in sensitivity to radiation exposure. Studies are actively ongoing to develop methods to best assess interindividual variability. This understanding will have both a health risk assessment and medical applications (31).

GENOMIC INSTABILITY

Radiation-induced genomic instability encompasses a range of measurable endpoints such as chromosome destabilization, sister chromatid exchanges, gene mutation and amplification, late cell death, and aneuploidy, all of which may be causative factors in the development of clinical disease, including carcinoma.

Kadhim et. al. identified the persistence of radiationinduced chromosomal instability following a-particle irradiation in clonal populations of murine bone marrow cells (32). The same group followed up this seminal work with an examination of four human bone marrow samples, subjected to an ex vivo a-particle IR (33). Further research then demonstrated that the genomic instability phenotype could be transmitted in vivo when murine hemopoietic cells that had been irradiated in vitro were transplanted into mice that had previously had their native bone marrow purged (34). However, when Whitehouse and Tawn examined radiation workers in Sellafield, England, who had bone marrow plutonium deposition evidence for genomic instability was not found (35). Whether g-IR could induce genomic instability was then examined. The original reports from Kadhim et al. were negative (32,33), but further research examining hprt locus mutations convincingly demonstrated that genomic instability was inducible by g-irradiation (36). Thus, although genomic instability can clearly be demonstrated in the laboratory, whether it occurs in humans after IR exposure remains uncertain (37).

Our understanding of the biologic effects of IR is evolving and ever growing. Research has been active in this field for over 100 years, and it remains a vibrant research area with the new molecular tools now available. The target of interest and concern is as small as the individual DNA base or as large as the whole organism. Mechanistic studies of the subcellular targets of IR and the cellular response signaling cascades must be matched with more complex system evaluations so that the summative effect of these pathways becomes known. Cell signaling exists within and between cells, and this cross-talk affects the

ultimate response to IR exposure. Improving our understanding of radiation response at the cellular and tissue levels will undoubtedly yield advances for medical/therapeutic radiation as well as general cellular biology.

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