Measurements of relative biological effectiveness (RBE) have been made on the range-modulated 70 MeV proton beam at TRI-UMF using a precise cell sorting survival assay. In this study, Chinese hamster V79-WNRE cells were suspended in medium containing liquid gelatin at 37°C in irradiation tubes and the gel was allowed to solidify by cooling to 4°C. Complete cell survival responses were measured at 11 positions with 2 mm spacing within a proton stopping peak width of approximately 2 cm. Survival responses after proton irradiation were compared with responses to 60 Co γ rays measured at the same time, and RBE values were determined as a function of both dose and depth. Above doses of 4 Gy, the average RBE for these cells throughout the modulated proton stopping distribution was 1.21 ± 0.05, measured at a survival of 1%. However, we also observed that, within the spread-out Bragg peak, the RBE increased with increasing depth, from ∼1.2 at the proximal part to >1.3 at the distal part of the peak. At the distal edge of the stopping distribution, the RBE value increased significantly, to an extent that may be of concern when this region of the treatment volume is close to sensitive tissues. Below 4 Gy, the RBE value was also dependent on radiation dose, increasing significantly to values of approximately 1.37 and 1.56 at 2 and 1 Gy, respectively. Our results illustrate that the use of a single RBE value in different irradiation protocols can be an oversimplification, and argues for the use of "proton gray doses" rather than "γ-ray equivalent grays."

We have observed that when a single linear-quadratic (LQ) function is used to fit the radiation survival response of an asynchronously dividing population of V79 cells, a consistent misfit occurs at low doses. The data can be better described by fitting the low-dose and high-dose ranges separately, and there is evidence of a two-component response. The most obvious explanation is that we may simply be seeing the response of subpopulations of cells of different radiosensitivity: sensitive ${\rm G}_{1}\text{-}$ , ${\rm G}_{2}\text{-}$ and M-phase cells and resistant S-phase cells. The cell sorting assay for cell survival which we have used in these studies may thus be providing sufficient accuracy to resolve these subpopulations, not previously seen in conventional survival measurements. An alternative explanation is that the linear-quadratic function may be inappropriate for accurate description of the radiation survival response at low dose, at least for these cells. To test this hypothesis we have used three other models to fit the data: the single-hit plus multi-target (SHMT) model and the two-parameter repair-misrepair (RMR) model both yielded inferior fits to the asynchronous survival data; the three-parameter RMR model provided an improved fit to the data. The best fit, however, was obtained using a two-population LQ model, which suggested approximately equal numbers of sensitive and resistant cells. When the survival response of tightly synchronized G 1 / S-phase cells was measured using the cell sorting assay, no substructure was observed. This offers strong support to the hypothesis that the substructure observed in the asynchronous survival response is due to subpopulations of cells of different, cycle-dependent radiosensitivity.

The survival of asynchronous, exponentially growing DU-145 human tumor cells was measured after single doses of X rays in the dose range of 0.05-4 Gy using the cell sorting assay. When the response was modeled with the linear-quadratic (LQ) equation, a good fit to the data was observed for dose levels above 1 Gy; however, a region of enhanced sensitivity was observed at doses less than this. One possible explanation of this low-dose substructure is that a small, sensitive subpopulation of cells is selectively killed at low doses. Modeling of the radiation response with a two-population LQ model suggests that for these data this explanation is unlikely. Another possibility is that the whole cell population is initially hypersensitive, becoming radioresistant as damage is sustained by the cell. Conceivably this radioprotective mechanism could act in one of two ways. The cell could move from a radiation-sensitive to a radiation-resistant state by a continuous function of dose, or alternatively, only after a sufficient accumulation of damage, i.e. a "triggering dose." Both of these possibilities have been explored in the results of fitting two "induced resistance" models.

The dose dependence of the oxygen enhancement ratio (OER) has been examined through multiple measurements of the response of Chinese hamster V79-171 cells to low and high doses of radiation under aerobic and hypoxic conditions. In this series of experiments the cells were maintained at 37°C throughout the gassing and irradiation periods, to simulate normal physiological conditions. Flow cytometry and cell sorting techniques were used to facilitate accurate measurement of cell survival throughout the dose range, but particularly at low dose. The OER was found to decrease significantly at low dose, qualitatively confirming earlier reports from this laboratory, though the decrease was somewhat smaller in the present series. This difference may be a temperature effect since in the earlier experiments irradiation was at 0°C. This report shows that the OER decreases from a value of 2.87 ± 0.16 (standard deviation of mean) at S = 0.01 to 2.36 ± 0.19 at S = 0.80. Both α and β are altered by the presence of oxygen. The OER is presented as a function of dose in nitrogen.

Patient treatments at TRIUMF (Tri-University Meson Facility, Vancouver, B. C.) use a moving spot raster scan technique where the pion range is modulated in depth for each position of the moving spot. The spot scans in a stepwise fashion and can produce any desired field shape. This approach provides very good dose uniformity across the treatment field and allows maximum flexibility in shaping the treatment volume. Survival of cultured cells has been used as a biological dosimeter to test the isoeffectiveness of the pion dose distributions, which must be shaped in depth to compensate for the depth-dependent LET distribution. Isoeffectiveness across the treatment field has also been verified using this system, which involves irradiating cells supported in a gelatin matrix. The response of pig skin to pion irradiation at TRIUMF has provided a check on the in vivo RBE for acute effects derived from our earlier studies with mouse foot. In addition, the pig skin reactions have been followed for several months to assess the later dermal response. The RBE of our pion beam relative to 270 kVp X rays is approximately 1.5 for both the acute epidermal and the later dermal responses.

A decreased oxygen enhancement ratio (OER) at lower radiation doses has been previously reported (B. Palcic, J. W. Brosing, and L. D. Skarsgard, Br. J. Cancer 46, 980-984 (1984)). The question remained whether or not this effect is due to a possible oxygen contamination at low doses, which was not the case at high doses. To ensure a sufficient degree of hypoxia prior to the start of irradiation, Chinese hamster cells (CHO) were made hypoxic by gas exchange combined with metabolic consumption of oxygen at 37°C. At the same time oxygen levels in cell suspension were measured using a Clark electrode. It was found that under experimental conditions used in this laboratory for hypoxic irradiations, the oxygen levels before the start of irradiation are always below the levels which could give any significant enhancement to radiation inactivation by X rays. Full survival curves were determined in the dose range 0-30 Gy using the conventional survival assay and in the dose range 0-3 Gy using the low dose survival assay. The results confirmed the earlier finding that the OER decreases at low doses. It is therefore believed that the dose-dependent OER is a true radiobiological phenomenon and not an artifact of the experimental method used in the low dose survival assay.

Using an automated low dose survival assay, the radiosensitizing effectiveness of misonidazole at low radiation dose (0-6 Gy) was measured in cultured mammalian cells. Also measured was its effectiveness at high doses of radiation (0-35 Gy) using the conventional survival assay. In both cases, several concentrations of the drug from 0 to 5 mM were used. The data at low doses were analyzed by a two-parameter mathematical equation with linear and quadratic dose terms, $S=e^{-\alpha D-\beta D^{2}}$ , which proved to be a good fit to the experimental data at all misonidazole concentrations. It is shown that whereas the coefficient of the quadratic dose term, β, increases significantly with increasing misonidazole concentration, the drug does not significantly affect the coefficient of the linear term, α. The enhancement ratio (ER) of misonidazole is shown to be decreased at lower doses. The clinical implications of this result are discussed.

It has been shown previously that TAN does not enhance production of single-strand breaks (SSB) in DNA of Chinese hamster cells irradiated under hypoxia. In contrast, misonidazole does enhance the yield of SSB, but this SSB enhancement by misonidazole is reduced if TAN is present with misonidazole during irradiation. It was also shown that each of these sensitizers enhances base/sugar damage, measured with the aid of bacterial enzymes (MLS). The results presented here for MLS damage in mammalian cells irradiated in the presence of the misonidazole-TAN combination indicate that the two drugs act independently in enhancing MLS damage, and hence they interact with different types of lesions to produce base/sugar damage. It is proposed that the protection by TAN in mammalian cells observed at the survival level is due to "repair" of some of that type of damage which would otherwise become a misonidazole-enhanced SSB.

The effect of ionizing radiation on the toxicity of misonidazole to hypoxic mammalian cells was examined. Cell toxicity response (log surviving fraction vs time of exposure to misonidazole in hypoxia) can be approximated by a biphasic curve: an initial period of approximately zero-slope shoulder, followed by exponential decrease in survival. Radiation reduced the zero-slope shoulder of toxicity response in a dose-dependent manner and at a given dose, the shoulder totally disappeared. The slope of the exponential region of the toxic response was unaffected. The same final survival level was achieved regardless of the sequence in which radiation and misonidazole exposure were applied to cells; in fact, there was no detectable repair of that radiation-induced damage which interacts with misonidazole toxicity (up to 24 hr). A mechanism for this interaction is proposed. Clinical implications are considered assuming that similar interaction between the two modalities takes place in vivo. Since the shoulder of toxic response is eliminated at high radiation doses, repeated administration of radiation and misonidazole could lead to additional kill of chronically and acutely hypoxic cells, if indeed both types are present in human tumors.

When misonidazole is present during irradiation of hypoxic mammalian cells, an enhancement of single-strand breaks (SSB) in DNA is observed. Oxygen also enhances SSB, presumably in a manner similar to that of misonidazole. The dose-modifying factor (DMF) for 15 m M misonidazole was found to be 3.4, compared to an oxygen enhancement ratio (OER) of 3.5. Another class of DNA damage, namely, sites exposed by an extract of Micrococcus luteus , was examined. Radiation-induced M. luteus extract-sensitive sites (MLS) were also found to be enhanced by the presence of misonidazole or molecular oxygen. The DMF for this damage by 15 m M misonidazole was 1.6 while the OER was 2.5. The ratio of MLS to SSB is approximately 1.25 under hypoxia, 0.9 in the presence of oxygen, and 0.6 in the presence of 15 m M misonidazole under hypoxic conditions. Incubation with misonidazole under conditions which are toxic to mammalian cells (37°C, hypoxia), and which result in many SSB, produces no detectable lesions sensitive to the M. luteus extract.

The properties of the electron-affinic 2-nitroimidazole Ro-07-0582 have been examined in vitro in two Chinese hamster cell lines, CHO and ${\rm CH}2{\rm B}_{2}$ . The drug was found to sensitize hypoxic cells to radiation damage selectively with a high efficiency. At concentrations greater than 10 mM the drug has a dose-modifying factor of approximately 3.0 in hypoxic cultures. It does not alter the response of aerobic cells. Pre- or postirradiation treatment of cells with Ro-07-0582 has little, if any, effect. At high cell concentrations, where many sensitizers are ineffective, this compound retains most of its radiosensitizing capability. Ro-07-0582 is also shown to have a chemotherapeutic potential in that it demonstrates a very selective toxicity for hypoxic cells after a few hours of exposure. It is much less toxic to aerobic cells. This property, while it may serve as a useful complement to the radiosensitizing effect of the drug, suggests that care must be exercised in the clinical application of Ro-07-0582 to avoid possible injury to normal hypoxic tissues in the body.

The effect of several electron affinic compounds on the radiation response of Chinese hamster cells irradiated in both dilute cell suspension and centrifugation pellets was investigated. p-Nitroacetophenone (500 μM), 175 μM nitrofurazone, and 200 μM dimethylfurmarate, when present during the irradiation of anoxic, dilute cell suspensions, produced dose-modifying factors of 1.5, 1.8, and 1.8, respectively. Anoxic cells irradiated at high cell concentrations and in the presence of a sensitizer experienced a two-stage loss of much of the sensitization normally seen in dilute cell suspension. A large component of sensitization was lost upon formation of the cell pellet, reducing the dose modifying factors to values approximately 70% of those obtained in dilute cell suspensions. This was followed by a slow disappearance of the remaining component of sensitization. Some possible mechanisms for this cell concentration effect are discussed. It is suggested that the cell pellet experiment may prove to be a useful indicator of the effectiveness of radiosensitizers in vivo.

It has been shown that exposure to triacetoneamine-N-oxyl (TAN) can enhance the damage produced in Chinese hamster cells by ionizing radiation even if the drug is not present during irradiation. Preirradiation exposure to TAN gives a dose modifying factor (DMF) of 1.3 while postirradiation treatment with TAN can be nearly as effective as TAN present at the time of irradiation (DMF = 1.5). Postirradiation sensitization is strongly dependent on temperature and is only observed if TAN is added within a few minutes after irradiation. The mode of action of TAN in mammalian cells is therefore not the same as in bacterial cells where no postirradiation sensitization occurs. Electron spin resonance (ESR) measurements indicate that TAN is entering the cells but that at high cell concentrations there is a gradual decay of free spins which is accompanied by a small loss in sensitization. A much larger loss in sensitization, however, occurs very rapidly at these high cell concentrations (centrifugation pellets) without any apparent loss in spins.