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Purpose: Occupational exposure to neutrons typically includes significant doses of low-energy neutrons, with energies below 100 keV. In addition, the normal-tissue dose from boron neutron capture therapy will largely be from low-energy neutrons. Microdosimetric theory predicts decreasing biological effectiveness for neutrons with energies below about 350 keV compared with that for higher-energy neutrons; based on such considerations, and limited biological data, the current radiation weighting factor (quality factor) for neutrons with energies from 10 keV to 100 keV is less than that for higher-energy neutrons. By contrast, some reports have suggested that the biological effectiveness of low-energy neutrons is similar to that of fast neutrons. The purpose of the current work is to assess the relative biological effectiveness of low-energy neutrons for an endpoint of relevance to carcinogenesis: in vitro oncogenic transformation., Methods: Oncogenic transformation induction frequencies were determined for C3H10T1/2 cells exposed to two low-energy neutron beams, respectively, with dose-averaged energies of 40 and 70 keV, and the results were compared with those for higher-energy neutrons and X-rays., Results: These results for oncogenic transformation provide evidence for a significant decrease in biological effectiveness for 40 keV neutrons compared with 350 keV neutrons. The 70 keV neutrons were intermediate in effectiveness between the 70 and 350 keV beams., Conclusions: A decrease in biological effectiveness for low-energy neutrons is in agreement with most (but not all) earlier biological studies, as well as microdosimetric considerations. The results for oncogenic transformation were consistent with the currently recommended decreased values for low-energy neutron radiation weighting factors compared with fast neutrons.

Both cell lethality and neoplastic transformation were assessed for C3H10T1/2 cells exposed to neutrons with energies from 0.040 to 13.7 MeV. Monoenergetic neutrons with energies from 0.23 to 13.7 MeV and two neutron energy spectra with average energies of 0.040 and 0.070 MeV were produced with a Van de Graaff accelerator at the Radiological Research Accelerator Facility (RARAF) in the Center for Radiological Research of Columbia University. For determination of relative biological effectiveness (RBE), cells were exposed to 250 kVp X rays. With exposures to 250 kVp X rays, both cell survival and radiation-induced oncogenic transformation were curvilinear. Irradiation of cells with neutrons at all energies resulted in linear responses as a function of dose for both biological endpoints. Results indicate a complex relationship between RBEm and neutron energy. For both survival and transformation, RBEm was greatest for cells exposed to 0.35 MeV neutrons. RBEm was significantly less at energies above or below 0.35 MeV. These results are consistent with microdosimetric expectation. These results are also compatible with current assessments of neutron radiation weighting factors for radiation protection purposes. Based on calculations of dose-averaged LET, 0.35 MeV neutrons have the greatest LET and therefore would be expected to be more biologically effective than neutrons of greater or lesser energies.

Domestic, low-level exposure to radon gas is considered a major environmental lung-cancer hazard involving DNA damage to bronchial cells by alpha particles from radon progeny. At domestic exposure levels, the relevant bronchial cells are very rarely traversed by more than one alpha particle, whereas at higher radon levels-at which epidemiological studies in uranium miners allow lung-cancer risks to be quantified with reasonable precision-these bronchial cells are frequently exposed to multiple alpha-particle traversals. Measuring the oncogenic transforming effects of exactly one alpha particle without the confounding effects of multiple traversals has hitherto been unfeasible, resulting in uncertainty in extrapolations of risk from high to domestic radon levels. A technique to assess the effects of single alpha particles uses a charged-particle microbeam, which irradiates individual cells or cell nuclei with predefined exact numbers of particles. Although previously too slow to assess the relevant small oncogenic risks, recent improvements in throughput now permit microbeam irradiation of large cell numbers, allowing the first oncogenic risk measurements for the traversal of exactly one alpha particle through a cell nucleus. Given positive controls to ensure that the dosimetry and biological controls were comparable, the measured oncogenicity from exactly one alpha particle was significantly lower than for a Poisson-distributed mean of one alpha particle, implying that cells traversed by multiple alpha particles contribute most of the risk. If this result applies generally, extrapolation from high-level radon risks (involving cellular traversal by multiple alpha particles) may overestimate low-level (involving only single alpha particles) radon risks.

The oncogenic potential of high-energy 56Fe particles (1 GeV/nucleon) accelerated with the Alternating Gradient Synchrotron at the Brookhaven National Laboratory was examined utilizing the mouse C3H 10T1/2 cell model. The dose-averaged LET for high-energy 56Fe is estimated to be 143 keV/micrometer with the exposure conditions used in this study. For 56Fe ions, the maximum relative biological effectiveness (RBEmax) values for cell survival and oncogenic transformation were 7.71 and 16.5 respectively. Compared to 150 keV/micrometer 4He nuclei, high-energy 56Fe nuclei were significantly less effective in cell killing and oncogenic induction. The prostaglandin E1 analog misoprostol, an effective oncoprotector of C3H 10T1/2 cells exposed to X rays, was evaluated for its potential as a radioprotector of oncogenic transformation with high-energy 56Fe. Exposure of cells to misoprostol did not alter 56Fe cytotoxicity or the rate of 56Fe-induced oncogenic transformation.

About 25% of human tumors contain a mutated member of the ras gene family. Neutron exposure is an occupational risk in several work places and while we know that cells exposed to neutrons can become transformed, the molecular basis of this process is not understood. To determine whether neutron-induced cellular transformation involves ras mutation, C3H10T1/2 cells were exposed to a single dose of 5.9 MeV neutrons. Type II and type III foci were isolated and established as cell lines. A total of 34 foci were selected and expanded for analysis of tumorigenicity, chromosomal aberrations and mutations in members of the ras gene family. The presence of mutations in genomic DNA in N-ras or K-ras of each focus was examined by either single-strand conformational polymorphism (SSCP) analysis or by asymmetric PCR coupled cell cycle sequence analysis. Although chromosomal aberrations were detected at metaphase, no alterations in either ras gene were detected. We conclude that in vitro neutron-induced transformation must occur through a mechanism other than ras mutation.

Generation of estimates of risk caused by exposure to radon in the home, either from miner data or from A-bomb data, requires several scaling factors such as for dose, dose rate and radiation quality, and possible synergisms. Such scaling factors are best developed from laboratory-based studies. Two possible sources of alpha particles for such studies are (1) a polyenergetic spectrum, generated directly by radon and its progeny, or (2) a series of monoenergetic alpha particles. We compare here the results of oncogenic transformation from studies using both systems. At the Columbia University Radiological Research Accelerator Facility (RARAF), C3H 10T1/2 cells were irradiated with alpha particles of various energies, with defined LETs from 70 to 200 keV/mum. At Pacific Northwest Laboratory, cells from the same stock were exposed to alpha particles from radon gas and its progeny, which were in equilibrium with the culture medium. There was good agreement between the results of oncogenic transformation experiments using the two different exposure systems. Apart from the experimental transformation frequencies themselves, such a comparison requires (1) reliable dosimetry at both facilities and (2) estimated LET distributions for the polyenergetic alpha-particle irradiator. Thus this good agreement gives some confirmation to the technique which is used to fold together oncogenic transformation rates from monoenergetic alpha particles to yield a predicted rate for a spectrum of alpha particles.

Exposure of synchronized populations of mouse C3H 10T1/2 cells to a single dose (0.6 Gy) of 5.9 MeV neutrons at intervals after mitotic shake-off results in a distinctive variation in the oncogenic transformation frequency through the cell cycle. Previous findings show a sensitive window for X-ray-induced oncogenic transformants at late times after mitotic shake-off (14-16 h). Optimal sensitivity to neutrons was observed for cell populations irradiated soon after mitotic shake-off (4-6 h), where the majority of cells would be in the G1 phase of the cell cycle. Additionally, enhanced sensitivity was also found for that period after shake-off (14-16 h) which was maximally sensitive to X rays corresponding to cell populations with a high proportion of G2-phase cells. That is, low-LET radiation (250 kVp X rays) largely appears to produce oncogenic transformants in G2-phase cells, while intermediate-LET radiation (5.9 MeV neutrons) is effective principally on G1- and, to a somewhat lesser extent, G2-phase cells. Cells irradiated with neutrons showed less variation for lethality through the cell cycle than those irradiated with X rays, in agreement with previous findings. The mechanistic basis for the difference in the response of cells in the different phases of the cell cycle to radiations of different quality is unknown but is suggestive of distinct ("signature") molecular changes leading to the observed oncogenic transformation response.

Domestic radon risk estimates are typically based either on data for uranium miners or on data derived from A-bomb survivors; comparison of domestic radon risk estimates derived from these two disparate sources represents an important test of their reliability. There is currently a significant discrepancy of about a factor of three between domestic radon risk estimates generated with these two independent methods. To base such risk estimates on the data for A-bomb survivors, who were exposed mainly to low-LET radiation, requires a quality factor for alpha particles from random progeny; the final risk estimate is then directly proportional to this quality factor. We have used the most extensive quantitative in vitro data set currently available at high LET for an oncogenic end point, to make the best estimate we can that could be used as a basis for a quality factor. Our best estimates of values appropriate for the quality factor for radom progeny are significantly lower than those currently used (20-25) in estimating lung cancer mortality due to randon. Specifically, our best estimate for home dwellers is around 10. In addition, because of the different geometry in the bronchial epithelia of nonsmokers compared to smokers, our best estimate of an appropriate quality factor for home dwellers is about 18% greater than that for miners; thus our best estimate of the "effective K factor" to convert to effective dose/WLM in home dwellers from effective dose/WLM in miners would be increased by this factor. Based on a quality factor of approximately 10, the dosimetrically based estimate of radon-induced mortality would be approximately 35,000 per year in the U.S. rather than the value of approximately 70,000 obtained using a quality factor of 20. The value of 35,000, while larger than the values based on data for miners (approximately 20,000), is much smaller than previous estimates of approximately 70,000 based on dosimetric methods; thus risk estimates based on the two approaches, dosimetric and epidemiological, may be partially reconciled. Finally, a quality factor of 10 would reduce the proportion of the collective effective dose caused by radon progeny from the currently accepted value of 55% down to about 38%.

Primary explants of Syrian hamster embryo (SHE) cells were exposed to either low-LET 250 kVp X rays or graded single doses of defined high-LET alpha particles (90, 100, 120, 150, 180 and 200 keV/microns), simulating those produced by radon progeny, and monitored for cell inactivation and oncogenic transformation. For the alpha particles the doses delivered ranged from 1 cGy to 1 Gy with an emphasis on doses less than 20 cGy, while for the X rays the doses ranged from 20 cGy to 4 Gy. The dose-response curves for cell killing by alpha particles approximated an exponential function of dose, whereas the X rays produced a curve with a shoulder characteristic of linear-quadratic relationships seen for low-LET radiations. The RBE at 10% survival varied between 3.6-7.0 depending on the LET of the alpha particles, with the RBEm ranging between 7-12. The most effective alpha particles were those with an LET of 120 keV/microns. All radiations produced initial increases in the frequency of morphological transformants, as a function of dose, with a rise to a maximum followed by a plateau in the response which was relatively constant at approximately 2-6 x 10(-3) transformants frequency, expressed per initial cell at risk, had a tendency to decline to parallel the cell survival response. Both the dose at which the maximum frequency of transformants was expressed and the initial slope of the dose-response relationship differed substantially between the different radiation qualities. Maximal transformation per initial cell at risk occurred at doses as low as 1-4 cGy for the 90 and 100 keV/microns particles with the maximum occurring at higher doses (to 16 cGy) as the LET increased toward 200 keV/microns. In contrast, the maximal transformation for 250 kVp X rays was at 50 cGy. The 90 and 100 keV/microns particles, with an RBEm of 60 and 37, respectively, based on the ratios of the initial slopes of the dose-response curves, were the most effective LETs in terms of the ability to induce morphological transformation of SHE cells. When expressed in terms of particle fluence, it appears that in the LET range of radon progeny approximately two to four particle traversals per nucleus are required per killing event, whereas it is at doses corresponding to less than one particle per nucleus that maximal oncogenic transformation is expressed.(ABSTRACT TRUNCATED AT 400 WORDS)

Epidemiological studies have established an association between exposure to radon and carcinoma of the lung. However, based on data for either lung cancer in uranium miners exposed to radon or bronchial epithelial carcinomas in Japanese A-bomb survivors, it has not been possible to assign estimates of risk of lung cancer for the general population exposed to radon in their homes. Based on past success with the excellent quantitative properties of the C3H 10T1/2 in vitro oncogenic transformation assay system, the relative biological effectiveness (RBE) for radiation-induced transformation for charged particles of defined LET has been determined. As the LET of the radiation was increased, the rate of induction of oncogenic transformation increased and the RBEm approached 20. At higher LETs, RBE dropped precipitously. The rapid drop in effectiveness for alpha particles with LETs between 120 and 265 keV/microns implies a lower quality factor than the 20-25 currently considered appropriate when estimating lung cancer mortality.

Misoprostol, a PGE1 analog, is an effective radioprotector of murine intestine and hematopoietic and hair cell renewal systems. The radioprotective nature of misoprostol was extended to examine its ability to influence clonogenic cell survival and induction of oncogenic transformation in Syrian hamster embryo cells exposed to X rays in utero and assayed in vitro. Hamsters in their 12th day of pregnancy were injected subcutaneously with misoprostol, and 2 h later the pregnant hamsters were exposed to graded doses of X rays. Immediately after irradiation, hamsters were euthanized and embryonic tissue was explanted into culture dishes containing complete growth medium. After a 2-week incubation period, clonogenic cell survival and morphologically transformed foci were determined. Survival of misoprostol-treated SHE cells was increased and yielded a dose reduction factor of 1.5 compared to SHE cells treated with X rays alone. In contrast, radiation-induced oncogenic transformation of misoprostol-treated cells was reduced by a factor of 20 compared to cells treated with X rays alone. These studies suggest that misoprostol not only protects normal tissues in vivo from acute radiation injury, but also protects cells, to a large extent, from injury leading to transforming events.

Purpose: Prostaglandins are associated with a variety of both pathologic and normal physiological effects in mammals. Among this broad array of effects, prostaglandins have been shown to provide protection to tissues from a variety of injurious agents including ionizing radiation. Of the prostaglandins tested to date, an analogue of prostaglandin E1, misoprostol (cytotec) was found to be a very effective radioprotector. The purpose of this study was to assess the ability of misoprostol to protect cells from the cytotoxic and oncogenic effects of ionizing radiation., Methods and Materials: Pregnant Syrian hamsters were injected subcutaneously with 125 micrograms misoprostol/100 g body weight 2 h before being exposed to graded doses of X rays. Embryos were excised immediately after irradiation and cells were explanted into culture dishes. Following 14 days of incubation, cells were fixed in formalin and stained with giemsa for examination of cell clonogenicity and morphological transformation., Results: First, misoprostol protected cells from some degree of radiation toxicity. A reduction in cell killing by a factor of 1.5 was seen at 10% cell survival. Second, based on transformation studies, a higher frequency of oncogenic transformation is seen for cells exposed in utero to graded doses of X rays alone than for cells exposed to the combination of misoprostol followed by radiation. In the presence of misoprostol, transformation is reduced by a factor of 20 at the level of 10(-3) transformants per surviving cell., Conclusion: Misoprostol may have clinical utility, not only in protecting selected normal tissues during cancer therapy, but it may also be useful in protecting cells from secondary tumors caused by ionizing radiation.

Synchronised populations of mouse C3H/10T-1/2 cells were obtained by a stringent mitotic dislodgment procedure. Mitotic cells rapidly attach and progress sequentially through the cell cycle. Irradiation (3 Gy of X rays) was carried out at intervals from 0 to 18 h after initiating cell cycle progression of the mitotic cells. Oncogenic transformation was enhanced 10-fold over cells irradiated soon after replating (G1 and S phases) for cells in a near 2 h period corresponding to cells in G2 phase but not in mitosis. The cell surviving fraction had a 2-1/2-fold variation with resistant peaks corresponding to the late G1 and late S phases. These findings provide experimental support for the hypothesis initiated by Rossi and Kellerer and developed by Brenner and Hall to explain the LET dependent inverse dose rate effect for oncogenic transformation.

Hyperthermia was combined with bleomycin, melphalan and cis-platinum in order to examine cell lethality and oncogenic transformation in C3H10T1/2 cells from the adjuvant use of heat with chemotherapy agents. When cells were exposed concurrently to 42.5 degrees C and each of the three chemotherapy agents, heat enhanced both the cytotoxic and oncogenic potential of the drugs. Hyperthermia-enhanced ratios were largest for bleomycin-treated cells. Examination of transformation incidences expressed as a function of surviving fraction, i.e. the cytotoxicity of treatment and therefore drug-heat efficacy, showed that for a given level of cell killing the combination of heat and cis-platinum resulted in fewer transformants per surviving cell than for cis-platinum alone. Hyperthermia appears to reduce the oncogenic potential of low concentrations of melphalan but has no effect on bleomycin-induced oncogenic transformation.

Mouse C3H 10T1/2 cells were exposed to single or fractionated doses of charged particles of defined linear energy transfer (LET) from 25 to 200 keV/microns. Dose fractionation with prolonged time intervals enhanced the yield of transformed foci compared with a single acute dose for a range of LET values between 40 and 120 keV/microns. Radiations of lower or higher LET did not show the enhancement that is commonly referred to as the inverse dose-rate effect. The fractionation scheme that was used consisted of three dose fractions; the maximum enhancement of transformation occurred with an interval of 150 min between dose fractions. This inverse dose-rate effect, demonstrated for cycling cells in log phase, was not seen for cells in plateau phase.

When exposure to densely ionizing radiation is protracted, the resulting biological effect is sometimes, but not always, enhanced for transformational end points, relative to acute exposure. A pattern has emerged as to the dependence of this effect on dose, dose rate, and radiation quality. Previous calculations indicated that the dose and dose-rate trends can be predicted by a model in which there is a period within the cell cycle of very high sensitivity to oncogenesis. Recent experiments indicate that the inverse dose-rate effect is significant over a very limited range of LETs--from about 30 to 130 keV/microns. We discuss such LET effects in the context of cell cycle-dependent models, and suggest that the effects are understandable on the basis of such models. In essence, the inverse dose-rate effect disappears at high LET because of a reduction in the number of cells being hit, and disappears at LETs below about 30 keV/microns because most of the dose is deposited at low specific energies, insufficient to produce the saturation effect which is central to the phenomenon. At even lower LETs, damage repair yields the familiar sparing associated with protraction of X- or gamma-ray doses.

C3H 10T1/2 cells were synchronized by a modified mitotic shake-off procedure. X irradiation of cells at various intervals after mitotic harvest indicated a single narrow window (about 2 h) of sensitivity to the induction of oncogenic transformation. It is not possible to delineate precisely the time in the cycle at which this sensitivity is expressed. The most likely candidate is G2 phase, though we cannot eliminate the possibility that the sensitive period begins in late S phase. In the same synchronized cells, cell lethality showed the conventional pattern, i.e., sensitivity in mitosis and resistance in late S and in G1 phase.

An in vitro assay was used to assess cell killing and induction of neoplastic transformation in C3H 10T1/2 cells exposed to X rays and a range of monoenergetic neutrons administered at various dose rates. Curves for cell survival and induction of neoplastic transformation showed nonlinearity for cells exposed to acute graded doses of X rays, while irradiation of cells with 0.05 to 1.5 Gy of 0.23-, 0.35-, 0.45-, 0.70-, 0.96-, 5.90-, and 13.7-MeV neutrons resulted in a linear response as a function of dose for both neoplastic transformation and killing. When compared to results obtained with 250-kVp X rays, all neutron energies were more effective at both cell killing and induction of neoplastic transformation. When expressed as maximum biological effectiveness (RBEM), both cell survival and induction of neoplastic transformation showed an initial increase with neutron energy (maximal at 0.35 MeV), followed by a decrease in effectiveness with further increases in energy. These responses are consistent with microdosimetric predictions in that recoil protons from neutron interaction are shifted to lower lineal energies as neutron energies increase. To examine the effects of temporal distribution of dose on neutron-induced neoplastic transformation, cells were exposed to either a single dose or five equal dose fractions spread over 8 h. As a function of dose for single or fractionated exposures to 0.5 Gy or 0.23-, 0.35-, 0.45-, 5.9-, or 13.7-MeV neutrons, neither a sparing nor an enhancing effect was seen with survival. Similarly, the frequency of induction of neoplastic transformation was independent of dose fractionation for all but 5.9-MeV neutrons. The enhancing effects of exposure to fractionated doses of 5.9-MeV neutrons were further studied by comparing exposures for a range of doses given singly, in five fractions over 8 h, or continuously for 8 h. Results reaffirm the enhancing effects of dose fractionation on the induction of oncogenic transformation for 5.9-MeV neutrons. Within the limits of the data, there is modest enhancement of neutron-induced neoplastic transformation when the exposure dose is extended from a few minutes to several hours, with the enhancement being dependent on neutron energy.

Considerable evidence has recently accumulated, both in vivo and in vitro, to suggest that neutrons at low dose rates, or in multiple fractions, have an enhanced effectiveness for inducing neoplastic transformation. A consistent pattern has emerged as to the dependence of the effect on dose, dose rate, and radiation type. A consistent model of the phenomenon as a function of dose, dose rate, and radiation type is presented. The approach, first suggested by Rossi, is that cells are extra sensitive in part of their cycle. Then an acute exposure of cycling cells to high-LET radiation results in some fraction of the sensitive cells receiving large depositions of energy--greater than required for the effect. If the exposure is protracted, a larger proportion of sensitive cells will be exposed, but to smaller numbers of energy depositions--still enough, however, to produce the effect. The model produces results consistent with all available data on enhancement of neoplastic transformation in the C3H 10T1/2 system by protraction or fractionation at medium or high LET. Implications for radiation protection and the radon problem are discussed.

Mouse C3H 10T1/2 cells were exposed sequentially to low doses (0.1 and 0.3 Gy) of monoenergetic neutrons (0.35, 0.45, 5.9, and 13.7 MeV) and 250-kVp X rays (1 and 3 Gy). The incidences of oncogenic transformation in the cells exposed to neutrons followed by X rays indicated that the effects of the individual radiations were simply additive. This supports the contention that risks associated with the two different radiation modalities may be considered to be additive.

The effects of dose rate and dose fractionation on high-LET radiation-induced oncogenic transformation of C3H 10T1/2 cells were examined. Cells were irradiated with graded doses of 5.9-MeV monoenergetic neutrons administered either in single acute exposures (30 mGy/min) or extended over an 8-h period at low dose rates (from 0.21 to 1 mGy/min). Although cell survival studies showed no difference in effect with a change in radiation delivery rate, enhancement of oncogenic transformation occurred when the dose rate was reduced. When the neutron dose was divided into three fractions over 8 h, the biological effect was intermediate between that for the acute and that for the low-dose-rate exposures. Further irradiations were made using deuterons with an LET of 40 keV/microns. The dose-mean lineal energy was comparable to that measured for the 5.9-MeV monoenergetic neutrons. An inverse dose-rate/fractionation effect for the induction of transformation by high-LET deuterons was observed when the time between each of three fractions for a 0.3-Gy total dose was at least 45 min. No further enhancement was seen for longer dose fractionations, suggesting that very long protracted exposures of high-LET radiation would produce no additional enhancement.

Oncogenic transformation was assayed after C3H 10T1/2 cells were irradiated with monoenergetic neutrons; cells were exposed to 0.23-, 0.35-, 0.45-, 5.9-, and 13.7-MeV neutrons given singly or in five equal fractions over 8 h. At the biologically effective neutron energy of 0.45 MeV, enhancement of transformation was evident with some small fractionated doses (below 1 Gy). When transformation was examined as a function of neutron energy at 0.5 Gy, enhancement was seen for cells exposed to three of the five energies (0.35, 0.45, and 5.9 MeV). Enhancement was greatest for cells irradiated with 5.9-MeV neutrons. Of the neutron energies examined, 5.9-MeV neutrons had the lowest dose-averaged lineal energy and linear energy transfer. This suggests that enhancement of transformation by fractionated low doses of neutrons may be radiation-quality dependent.

The hypoxic cell sensitizer Misonidazole (Ro-07-0582) induces oncogenic transformations in the C3H/10T1/2 mouse embryo cell line cultured in vitro. A drug concentration of 0.4 mM applied to aerated cells for 3 or 6 days results in a transformation rate comparable to that observed following an X-ray dose of 1 Gray. A higher drug concentration of 6.0 mM is equivalent to 4 Gy. The combination of Misonidazole and X-rays produces a significant increase in the frequency of transformation over either drug or radiation alone, but the data are equivocal on the question of additivity vs synergism.

An established line of mouse fibroblasts (10T1/2 cells) cultured in vitro was used to compare the incidence of oncogenic transformation produced by x rays, the hypoxic cell radiosensitizer misonidazole, and a range of commonly used chemotherapy agents. A 3-day exposure to misonidazole at a concentration obtainable during treatment produced an incidence of transformation similar to that of about 50 rad. When chemotherapy agents were tested at concentrations comparable to those used clinically and matched to produce similar cell killing, the incidence of transformation varied widely: some agents, such as vincristine, did not produce transformation at a level detectable above background, while others, such as cis-platinum, appear to be potent carcinogens and produce transformation at a rate orders of magnitude higher than that achieved with x rays.

The potential of hyperthermia for producing oncogenic transformation in vitro was assessed using C3H/10T 1/2 mouse embryo cells. Temperatures in the range of 40 degrees to 45 degrees C caused no transformations, whether or not the heat treatment was associated with significant cell killing. In addition modest hyperthermia appreciably reduced the transformation frequencies associated with X-ray doses. The findings suggest an additional benefit in combining ionizing radiations with hyperthermia.

The modulating effect of 43 degrees C hyperthermia on the induction of oncogenic transformation by the antineoplastic agents, actinomycin D, mitomycin C, and 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) was examined using the C3H 10T1/2 cell line. For any given level of cytotoxicity, cells exposed to the three chemotherapy agents at 37 degrees C showed similar frequencies of transformation. Transformation frequencies induced by all three drugs were reduced by hyperthermia. The reduction was most pronounced for cells exposed to BCNU, and to a lesser extent, by cells exposed to actinomycin D and mitomycin C. The modulating effects of heat on drug-induced transformation incidence appeared to be independent of whether application of heat and drug was concurrent or sequential.

An established line of mouse fibroblasts, C3H/10T1/2 cells, was used for the assessment in vitro of oncogenic transformations caused by single and split doses of x-rays. The shape of the dose-response relationship was determined over the range from 0.1 to 10 Gy. It was found that splitting the x-ray dose into two equal fractions, separated by 5 hr, led to a reduction in transformation frequency at doses above 1.5-2 Gy but to an enhancement of transformation at lower doses. The observations reported cast doubt on the assessment of human cancer risk at lo dose levels by a linear extrapolation from available high-dose data from the Japanese atomic bomb survivors or from persons exposed for medical purposes.

There is increasing concern for the oncogenic potential of agents used to treat cancer. Hyperthermia is one of the few modalities that does not, of itself, produce transformed foci in vitro. The adjuvant use of heat with X-rays or chemotherapy agents is an interesting approach to increasing the cell-killing potential while decreasing the oncogenicity of combined-modality therapy. Following a priming heat dose in C3H 10T1/2 cells, resistance to cell killing by a second heat dose develops and is maximal by 10 h. This is known as thermotolerance, and can be monitored by the appearance of proteins of specific molecular weight known as heat-shock proteins. By contrast, a priming heat dose does not confer resistance to killing by cis-platinum (cis-DDP). Indeed, heat potentiates the cytotoxicity due to cis-DDP. The interaction is greatest if heat and drug are applied simultaneously, but is still substantial if the drug is applied many hours after heating. The loss of interaction between heat and cis-DDP occurs slowly, but by 48 h, heat and drug act independently. Thermotolerant cells are less sensitive to the induction of transformation by X-rays than previously unheated cells. On the other hand, 48 h after a heat exposure, when cells have regained their normal sensitivity to killing by cis-DDP, their sensitivity to the induction of transformation by cis-DDP has also returned to normal.

The relative biological effectiveness (RBE) of a range of neutron energies relative to 250-kVp X rays has been determined for oncogenic transformation and cell survival in the mouse C3H 10T 1/2 cell line. Monoenergetic neutrons at 0.23, 0.35, 0.45, 0.70, 0.96, 1.96, 5.90, and 13.7 MeV were generated at the Radiological Research Accelerator Facility of the Radiological Research Laboratories, Columbia University, and were used to irradiate asynchronous cells at low absorbed doses from 0.05 to 1.47 Gy. X irradiations covered the range 0.5 to 8 Gy. Over the more than 2-year period of this study, the 31 experiments provided comprehensive information, indicating minimal variability in control material, assuring the validity of comparisons over time. For both survival and transformation, a curvilinear dose response for X rays was contrasted with linear or nearly linear dose responses for the various neutron energies. RBE increased as dose decreased for both end points. Maximal RBE values for transformation ranged from 13 for cells exposed to 5.9-MeV neutrons to 35 for 0.35-MeV neutrons. This study clearly shows that over the range of neutron energies typically seen by nuclear power plant workers and individuals exposed to the atomic bombs in Japan, a wide range of RBE values needs to be considered when evaluating the neutron component of the effective dose. These results are in concordance with the recent proposals in ICRU 40 both to change upward and to vary the quality factor for neutron irradiations.

Modification of sister chromatid exchanges and radiation-induced transformation in mouse C3H/10T 1/2 and Syrian hamster embryo cells by the tumor promoter 12-O-tetradecanoylphorbol-13-acetate and two retinoids, the trimethylmethoxyphenyl analog of N-ethyl retinamide and beta-all-trans-retinoic acid, has been studied. 12-O-tetradecanoylphorbol-13-acetate alone enhances, and retinoids alone reduce radiation-induced transformation. When both compounds were present, the retinoids not only reduced the oncogenic effects of radiation but completely eliminated the promoting effects of 12-O-tetradecanoylphorbol-13-acetate. These results were not paralleled by changes in sister chromatid exchange frequencies, indicating that, while sister chromatid exchanges may be useful as indicators of primary carcinogen mutagens, they may have little utility when secondary agents after the response of cells to a primary initiator.

The C3H/10T 1/2 cell line, cultured in vitro, was used to study the induction of oncogenic transformations produced by electron affinic compounds which function as hypoxic cell radiosensitizers. The relationship between the incidence of oncogenic transformations and drug concentration was obtained for 3-day exposures to misonidazole. At a concentration of 1 mM, a comparison was made of the carcinogenic potential of misonidazole, desmethylmisonidazole, Ro-07-0741, and SR 2508. Misonidazole and desmethylmisonidazole induced similar frequencies of transformation, while the frequency was doubled for Ro-07-0741 and increased by a factor of 5 for SR 2508. The relative carcinogenicity of the various alternatives to misonidazole is clearly a factor in choosing the next compound to be tested in the clinic.

Depending on its time of addition to Syrian hamster embryo or mouse C3H 10T1/2 cells the protease inhibitor antipain (AP) can enhance, or reduce, radiation induced-oncogenic transformations. These opposing influences are not paralleled by changes in sister chromatid exchanges in either cell system. A 24 h treatment with 10 microM AP prior to, and during irradiation, with removal 10 min after irradiation results in greater than a two-fold increase in transformants. Conversely, a 24 h treatment beginning 10 min after irradiation results in about a two-fold decrease in transformants. The utility of AP as an agent for the reduction of tumorigenesis is brought into question, since quite short temporal differences in application can result in near five-fold differences in the frequencies of oncogenic transformations.