Results from several laboratories, including ours, have suggested that measurements of radiation-induced DNA strand breaks and DNA-protein crosslinks (DPCs) may be used to estimate the hypoxic fraction or fractional hypoxic volume of tumors and normal tissues. This suggestion has been predicated on both published and unpublished information that (1) the oxygen dependence of the formation of strand breaks in irradiated mammalian cells is similar to the oxygen dependence of radiation-induced cell killing, and (2) the oxygen dependence of the formation of DPCs in irradiated mammalian cells is the mirror image of the oxygen dependence of radiation-induced cell killing. However, the published studies that attempted to determine the relationship between the oxygen dependence of the formation of strand breaks and the radiation sensitivity of mammalian cells were not performed at 37°C, the exact oxygen concentrations were not always known, and the results were conflicting. In addition, most of the data on the oxygen dependence of the formation of DPCs are unpublished. Consequently, we have undertaken a comprehensive investigation of one cell line, 9L/Ro rat brain tumor cells, to determine if the shape of the oxygen dependence curve and the K m value for radiation-induced strand breaks and DPCs were similar when 9L cells were irradiated under both ideal gas-liquid equilibrium conditions at 4°C and nonideal gas-liquid equilibrium conditions at 37°C. At 4°C under ideal gas-liquid equilibrium conditions, the K m for the formation of strand breaks was approximately 0.0045 mM, and the K m for radiation sensitivity was approximately 0.005 mM. A similar comparison for the formation of DPCs at 4°C could not be made, because the efficiency of the formation of DPCs was much lower at 4°C than at 37°C. At 37°C under nonideal gas-liquid equilibrium conditions, the apparent K m for the formation of strand breaks and radiation sensitivity was approximately 0.032 mM, and the K m for the formation of DPCs was approximately 0.02 mM. The data for strand breaks are in agreement with the published data of Chapman et al. (Int. J. Radiat. Biol. 26, 383-389, 1974), and the data for DPCs are in agreement with the unpublished data of Meyn (personal communication). These results support the suggestion that measurements of radiation-induced strand breaks and/or DPCs may be used to detect hypoxic cells and estimate the hypoxic fraction or fractional hypoxic volume of tumors and normal tissues.

One important goal of radiobiology is to describe the response to radiation damage in quantitative terms. Because the dose response is nonlinear, this typically involves the comparison of several dose-dependent parameters. Past attempts at simplifying this process have often involved manipulations of these dose-dependent parameters to derive a single comparative number. Unfortunately, the advantages of a single comparative number, often a ratio or even a ratio of ratios, can be outweighed by the loss of significant biological information. Examples are given in four areas of research: (1) definition of radiation response at clinically relevant radiation doses, (2) modification of radiation sensitivity by oxygen, (3) effects of combined radiation modifiers (e.g., sensitizers and protectors), and (4) comparisons of radiation modifiers in different dose/response regions. In each area, to define and compare dose response, we propose the use of consistent, simple, and absolute radiation response parameters: the inverse dose (or dose) required to produce a given effect. The use of absolute sensitivity (or resistance) avoids the use of many other parameters, ratios, and definitions and permits a uniform and unambiguous description of "radiation response."

The formation of (R)- and (S)-8,5′-cycloadenosine 5′-monophosphate (8,5′-cycloAMP), 8-hydroxyadenosine 5′-monophosphate (8-hydroxyAMP), and radiolytic adenine release from irradiated solutions of adenosine 5′-monophosphate (5′-AMP) was measured as a function of increasing liquid-phase oxygen concentration. Three classes of specific molecular damage were identified on the basis of the oxygen dependence for product formation. Major changes in product yield occurred near the range of oxygen concentrations associated with the radiobiological oxygen effect. In addition to these data, systematic increases in the concentration of hydrogen peroxide at the time of irradiation resulted in an increase in the yield of 8-hydroxyAMP and a component of radiolytic adenine release in nitrogen-saturated solutions of 5′-AMP. However, no changes in the yield of the 8,5′-cyclonucleotides were observed under these conditions.

The presence of low levels of oxygen may have profound effects on the cytotoxic activity of radiation, radiosensitizers, and bioreductive alkylating agents. As others have shown, low oxygen tensions may significantly alter rates of cellular and chemical oxygen consumption. When experiments are performed at very low oxygen concentrations, the opposing effects of oxygen leakage into and cellular/chemical oxygen consumption from the system can lead to unpredictable results. Use of a newly designed, highly sensitive Clark-type oxygen sensor has permitted accurate and reproducible measurement of low levels of oxygen. Cellular depletion of oxygen at various cell densities has been monitored for a series of oxygen tensions in solution and the corresponding respiration rates have been calculated. Although oxygen depletion was found to be quite significant at low oxygen tensions, not all oxygen present could be removed by cellular respiration. Respiration rate decreased as oxygen tension decreased and approached zero at low oxygen tensions. This result was independent of cell density. A model is presented to account for the observed effect of oxygen tension on cellular oxygen utilization.

Pretreatment of V79-WNRE cells with 150 μM diethylmaleate for 1 hr at 37°C caused a decrease in intracellular glutathione levels to approximately 10-15% of control levels (0.5 vs <tex-math>$5.0\ {\rm nmol}/10^{6}\ \text{cells}$</tex-math>). The cells could be washed free of diethylmaleate and held at 0°C for several hours without toxicity and with no increase in glutathione concentration, although the glutathione concentration rapidly increased to normal levels at higher temperatures. Survival curves were determined as a function of oxygen or misonidazole concentration (the latter in the absence of oxygen). A new "thin-film" technique was used to avoid changes in oxygen concentration because of radiochemical or cellular oxygen consumption. Glutathione depletion itself caused a small but consistent radiosensitization of hypoxic cells (dose enhancement ratio of 1.2). However, glutathione depletion caused a profound change in the radiosensitizing efficiency of misonidazole, with a decrease in K m of about sevenfold from 0.6 to 0.09 mM. In contrast, only a 2.5-fold decrease was found in the K m for radiosensitization by oxygen with diethylmaleate pretreatment. These results suggest a fundamental problem with the conventional theory of radiosensitivity whereby one considers a first-order competition for reaction with target radicals between radical-fixing versus radical-repairing species. It also suggests difficulties in the interpretation of glutathione as the only endogenous protective species.

Radiation survival curves of EMT6/Ed spheroids have been obtained under conditions which eliminate changes in oxygen concentration between growth and irradiation. These curves show a high-dose, resistant component which is nearly parallel to the curves obtained when spheroids were irradiated under nitrogen. Thus EMT6 spheroids appear to model accurately the radiation responses of EMT6 tumors. In contrast, when spheroids were grown to relatively high density (300-400 spheroids per 250-ml spinner flask), then separated into several flasks for irradiation, an increase in oxygen concentration in the medium occurred which fully oxygenated the previously hypoxic cells. The two causes for the oxygen depletion in sealed growth flasks were quantitated. Depletion of total oxygen in the flask occurred, and, more importantly, oxygen consumption kept the growth medium well below equilibrium with the oxygen in the gas phase. Smaller but similar effects on oxygen concentration were found in flasks containing V79 spheroids.

Diamide [diazenedicarboxylic acid bis (N,N′-dimethylamide)] radiosensitizes hypoxic Chinese hamster cells by decreasing the shoulder of the survival curve (at low concentrations) and increasing the slope (at high concentrations). We studied the mechanisms responsible for these two phenomena by irradiating cells under different conditions of temperature and substrate availability and measuring either their survival or DNA damage (chromosome aberrations and strand breaks). The results confirm that different mechanisms are responsible for the two effects. The effect on the shoulder appeared to be due to oxidation of endogenous nonprotein sulfhydryls and reduced pyridine nucleotides, compounds that would normally effect rapid chemical repair of certain (single-hit type?) lesions. The slope effect, on the other hand, may have been due to reactions with DNA similar to those that have been described for the electron-affinic compounds. These results, together with the finding that hypoxic cells remained sensitized after the removal of diamide when they were irradiated in the cold, provide a useful model and a new experimental approach to the question of chemical radiosensitization.

Diamide sensitizes hypoxic mammalian cells to X-radiation in a complex manner. It sensitizes CHO cells more effectively than V79-S171 cells and is far more efficient at 0°C than at 18°C in the presence of glucose. In the cold, diamide is more efficient than either nifuroxime or metronidazole (Flagyl), but this advantage is lost at 18°C. Low concentrations of diamide (40-100 μM, depending on cell line) decrease the extrapolation number to or close to 1 while affecting the D 0 only slightly, but higher concentrations decrease the D 0 to the euoxic level. The single-dose survival of V79 cells irradiated in the presence of increasing diamide concentrations follows a biphasic curve, with a break in the region of 200-300 μM. Since diamide is not inactivated by X-radiation and since there is no evidence of differential sensitization of cells in M, G 1 late S, or plateauphase $G_{1}/G_{0}$ , we interpret these data to mean that diamide acts by at least two mechanisms, one operating mainly at low concentrations to reduce the shoulder and the other predominating at high concentrations to decrease the D 0 .

Para-nitroacetophenone (PNAP) selectively sensitizes hypoxic mammalian cells to ionizing radiation. The growth of Chinese hamster V79 cells as multicellular spheroids provides a convenient tumor model to test this drug. When irradiated, cells from these spheroids exhibit a multicomponent survival curve. The resistant portion of the curve is primarily due to hypoxic, noncycling cells near the center of the spheroid. However, radiation survival data indicates that these cells are as resistant as the most resistant cycling cells when the latter are made hypoxic. Addition of 500 μM PNAP prior to and during irradiation under oxic conditions decreases the survival on the resistant portion of the spheroid survival curve although the dose-modification is not as large as in hypoxic, monolayer systems. The analog, m-nitroacetophenone, acts in a similar fashion in spheroids.

Growth curves for Chinese hamster fibroblasts are determined for various partial pressures of oxygen. It is found that for oxygen tensions greater than 500 parts per million, growth is only minimally affected. At less than 500 parts per million, the number of cells increases exponentially for only a limited time and then remains constant. Viability, as determined by colony forming ability, remains high during this plateau phase even after 4 days under extremely hypoxic conditions.

Complete radiation survival curves for mammalian cells as a function of partial pressure of oxygen have been obtained with considerable medium present. The results show an apparent decrease in extrapolation number (n) when irradiation is carried on during conditions of moderate hypoxia (5000 ppm oxygen) with the n value returning to the air level when the hypoxia becomes extreme. This phenomenon can be explained by radiochemical depletion of oxygen. At less than 25 ppm oxygen, the cells are not capable of sublethal repair, while at 200 ppm oxygen repair proceeds at almost the aerobic rate.