Interactions between radiation and matter nuclear reactions and the production of radioisotopes instrumentation - an introduction to alpha, beta and gamma radiation detectors the radiation from nuclear decay applications of radioisotopes in medicine and biology neutrons in biology and clinical medicine other accelerator-based applications in medicine and biology principles of radiation protection.

Dimethyl fumarate (DMF) depletes intracellular glutathione (GSH) by covalent bond formation in a reaction mediated by GSH-S-transferase. Treatment of hypoxic Chinese hamster V79 cells with 5 mM DMF before irradiation radiosensitizes the cells, resulting in an enhancement ratio (ER) of about 2.7 with minimal toxicity, when the end point is clonogenic cell survival. Under the same conditions aerobic cells are sensitized, and ER of about 1.3 is found, and GSH is reduced to about 3% of control. Very similar results were obtained previously with Chinese hamster ovary (CHO) cells. In addition, new data presented here show that DMF treatment of V79 or CHO cells immediately after irradiation under hypoxic conditions sensitizes the cells, resulting in an ER of about 1.5, DMF treatment after irradiation under aerobic conditions results in an ER of 1.3, and this DMF treatment reduces protein thiols (PSH) to about 70% of control. When induction of DNA damage is measured using the neutral elution assay, treatment of V79 or CHO cells with DMF prior to irradiation under hypoxic conditions results in an ER of 1.9-2.0, but there is no enhancement of DNA damage when DMF is added after irradiation under hypoxic conditions or when cells are treated with DMF before or after irradiation under aerobic conditions. Based on these data we postulate that DMF radiosensitizes killing of hypoxic cells by two actions: depletion of GSH interferes with the chemical competition between damage fixation and repair, and depletion of PSH causes an inhibition of enzymatic repair processes. We also suggest that DMF sensitizes aerobic cells only by inhibition of enzymatic repair processes.

From analytical expressions derived for the radical-repair (competition) model describing the relationship between cellular radiosensitivity and oxygen concentration, "K-curve" behavior has been quantified as a function of the concentration of the species S which restitutes the radiation-induced radicals to their original molecular configuration. If these species are identified with thiols, K-curves modified by fractionally depleting [S] through calculation can be compared with experimental data where cells have their thiols depleted using various means, for example, by chemical agents or by the use of cells with decreased thiols because of genetic deficiency. Families of curves have been calculated related both to the S-depleted and the non-S-depleted hypoxic control, the latter of which is used to calculate enhancement ratios. Comparison of the model with experimental data is made.

We will review the relationships between glutathione (GSH), protein thiols, and cellular responses to radiation, peroxides, and peroxide-producing drugs. Our primary interest involves the behavior of sulfhydryls as electron and hydrogen carriers, and their capacity to protect various target molecules against radiation and peroxidative damage. We used reagents such as L-buthionine sulfoximine (LBSO), alone and in combination with N-ethyl maleimide (NEM), diamide, and dimethylfumarate, to decrease GSH so that it could no longer participate in the electron transfer reactions. Our results indicate that aerobic sensitization produced by GSH depletion can be further enhanced if electron-accepting agents, such as tertiary butyl hydroperoxide (t-BOOH), are present during irradiation. Hydroperoxide is a substrate for glutathione peroxidase and diverts electrons and hydrogen away from target molecules during its reduction. Sensitivity to radiation seems to be due to the inhibition of the mitochondria's capacity to reduce hydroperoxide. We will also report the mitochondria's ability to reduce the oxygen radicals produced by radiation and drugs. Data also indicate that t-BOOH oxidizes protein thiols which are enzymatically involved in repair of DNA damage.

Dimethylfumarate (DMF) depletes intracellular glutathione (GSH) by covalent bond formation in a reaction which may be mediated by GSH-S-transferase. In Chinese hamster ovary cells this depletion is rapid; e.g., 0.5 mM DMF depletes GSH to less than 10% of control in 5 min at room temperature. DMF is a very effective hypoxic cell radiosensitizer, with an enhancement ratio (ER) of about 3 obtained by a 5-min exposure of cells at room temperature to 5 mM DMF, without significant toxicity. At this same concentration of drug, there is a small enhancement of aerobic cells (ER = 1.3), but the 5 mM DMF in hypoxia results in nearly a complete collapse of the hypoxic dose-response curve to the same level as seen in air with DMF. It has been suggested previously that DMF sensitizes cells via electron affinic mechanisms. However, this appears not to be the case in this study, as shown by the fact that cells pretreated with DMF and then washed free of the drug remained equally radiosensitive as cells irradiated in the presence of the drug. This large enhancement of radiation sensitivity appears to be related to the drug's ability to deplete thiols; i.e., thiols appear to be a major factor responsible for radioresistance of hypoxic cells.

In summary, there are theoretically several ways by which a cell's content of DNA repair genes or other radioprotective proteins can be increased. At present the most feasible way to do so seems to be to search for repair systems, such as the adaptive repair observed in human lymphocytes, whose activity can be increased by exogenous inducing agents. Now that this effect has been established in human lymphocytes, a search for similar systems in other cells should begin. Transfection of DNA repair genes into bone marrow cells that could be used to make the blood-forming organs more radioresistant is actually very close to being possible; the problem is more a matter of morality than of feasibility. The practicability of amplification of DNA repair genes or genes coding for other radioprotective proteins in the cells of a whole organism is at present close to zero. It is clear, however, that continued research on the basic mechanisms of cellular radiosensitivity could eventually lead to genetically engineered radioresistance in humans, but only if society approves.

Therapeutic gain factors (TGFs) have been determined for three spontaneous tumors of the C3H mouse treated by photons + normobaric oxygen (O2(1) ATA), photons + hyperbaric oxygen (O2 3 ATA), photons + misonidazole, or fast neutrons. The tumors were early generation isotransplants of spontaneous tumors: MCaIV, a mammary carcinoma; FSaII, a fibrosarcoma; and SCCVII, a squamous cell carcinoma. The tumors, transplanted to the right leg, were 6 mm at start of treatment. Normal tissue responses studied were acute reaction of normal skin (all treatment modalities) and LD50 following irradiation of the upper abdomen (in test of photons + O2 at 1 or 3 ATA). Thus both the tumor and normal tissues would be classified as "acute responding." All subject tissues were at congruent to 34.5-35 degrees C at irradiation. Treatments were based on d(25)Be or p(43)Be fast neutron beams, 60Co and 137Cs photon beams. Treatments were given in 5 or 15 equal doses in 5 days. For photon treatments, TGFs (air/O2 3 ATA) were substantially and significantly larger than 1 for all three tumor systems treated at small or large doses per fraction when related to skin or abdominal tissue responses. The TGFs (air/O2 1 ATA) were greater than 1 at small doses per fraction for MCaIV and FSaII for skin as the normal tissue; the TGFs for all three tumors and at all doses per fraction would be greater than 1 when related to upper abdominal tissues. TGFs (O2 1 ATA/O2 3 ATA) for photon irradiation greater than 1 were found only for SCCVII and that obtained for both large and small doses per fraction. Misonidazole achieved impressive TGFs (air/air + miso or air/O2 1 ATA + miso); the drug was tested only at 10-12 Gy/fraction and relative to skin. RBEs(FN) for the three tumors were lower at 1.5-2 Gy(FN)/fraction than at 5-6 Gy(FN)/fraction, i.e. the opposite to that reported for normal tissue (RBE increases with decreasing dose per fraction). A TGF (relative to skin reaction) greater than 1 for fast neutron therapy was found only for SCCVII when treated at large doses/fraction; this was true for air or O2 1 ATA conditions.

The effect of changes in both the intracellular glutathione (GSH) concentration and the concentration of extracellular reducing equivalents on the aerobic radiosensitization was studied in three cell lines: CHO-10B4, V79, and A549. Intracellular GSH was metabolically depleted after the inhibition of GSH synthesis by buthionine sulfoximine (BSO), while the extracellular environment was controlled through the replacement of growth medium with a thiol-free salt solution and in some experiments by the exogenous addition of either GSH or GSSG. Each of the cell lines examined exhibited an enhanced aerobic radioresponse when the intracellular GSH was extensively depleted (GSH less than 1 nmol GSH/10(6) cells after 1.0 mM BSO/24 h treatment) and the complexity of the extracellular milieu decreased. Although the addition of oxidized glutathione (5 mM GSSG/30 min) to cells prior to irradiation was without effect, much or all of the induced radiosensitivity was overcome by the addition of reduced glutathione (5 mM GSH/15 min). However, the observation that the exogenous GSH addition restores the control radioresponse without increasing the intracellular GSH concentration was entirely unexpected. These results suggest that a number of factors exert an influence on the extent of GSH depletion and determine the extent of aerobic radiosensitization. Furthermore, the interaction of exogenous GSH with--but without penetrating--the cell membrane is sufficient to result in radiorecovery.

Chronic aerobic exposure of A549 human lung carcinoma cell cultures to 0.1 mM L-buthionine-SR-sulfoximine and 1 mM misonidazole, or 1 mM SR-2508 results in inhibition of cell growth and decreased clonogenic survival. These patterns are not apparent with the individual drug treatments. Both parameters demonstrate maximum toxicity after 72 hr in culture, which parallels the time required to deplete A549 cells of glutathione with 0.1 mM L-BSO under these growth conditions. Toxicity appears to be related to hydrogen peroxide-produced during 1 electron reduction of the nitro compounds in the presence of oxygen. A549 cells have a lowered capacity to reduce peroxide due to the effect of thiol depletion on the enzymes GSH-peroxidase and GSH-S-transferase, which require the tripeptide as a substrate. The addition of catalase, another important enzyme involved in peroxide reduction, partially reverses the observed toxicity. 4-Hydroxypyrazole, which inhibits endogenous catalase activity, causes an increase in the observed cytotoxicity. Similar effects of L-BSO and 4-hydroxypyrazole are seen for toxicity due to hydrogen peroxide being added directly to cell cultures.

Our data show that A549 cells are increasingly radiosensitive with prolonged exposure to L-BSO. The resulting glutathione and protein thiol depleted cells show both loss of shoulder and slope modification. Furthermore, there is an increase in single strand DNA breaks and irrepairable cross-linking. The aerobic radiation damage in the thiol depleted state appears to be different from that obtained with hypoxic cells. Any postulated role for GSH in reducing or preventing peroxidative radiation damage must also include protection against single strand DNA breaks as well as involvement in repairing DNA-protein cross-links. The latter effect may be related to decreased protein thiol content as reflected in a decreased enzyme capacity to repair DNA damage.

The problem of neutrons produced by many of the high-energy x-ray therapy machines (10 MV and above) is reviewed, and the possible risk their presence poses to radiotherapy patients is estimated. A review of the regulatory background containing a summary of the recommendations of the U.S. Council of State Governments (USCSG), and of the International Electro-Technical Commission (IEC), as well as an indication that recommendations will be forthcoming from the National Council on Radiation Protection (NCRP) and the International Commission of Radiological Protection (ICRP) is presented. The neutrons in question are produced by high-energy photons (x rays) incident on the various materials of the target, flattening filter, collimators, and other essential components of the equipment. The neutron yield (per treatment dose) increases rapidly as the megavoltage is increased from 10 to 20 MV, but remains approximately constant above this. Measurements and calculations of the quantity, quality, and spatial distribution of these neutrons and their concomitant dose are summarized. Values of the neutron dose are presented as entrance dose, midline dose (10-cm depth), and integral dose, both within and outside of the treatment volume. These values are much less than the unavoidable photon doses which are largely responsible for treatment side effects. For typical equipment, the average neutron integral dose from accelerator-produced neutrons is about 4-7 g cGy (per treatment cGy), depending on the treatment plan. This translates into an average dose of neutrons [averaged over the body of a typical 70-kg (154 lb) patient] of 0.06-0.10 cGy for a treatment of 1000 cGy. Using these neutron doses and the best available neutron risk coefficients, it is estimated that 50 X 10(-6) fatal malignancies per year due to the neutrons may follow a typical treatment course of 5000 rads of 25-MV x rays. This is only about 1/60th of the average incidence of malignancies for the general population. Thus, the cancer risk to the radiotherapy patient from accelerator-produced neutrons poses an additional risk to the patient that is negligible in comparison.