Comparative radiobiology of fast neutrons: Relevance to radiotherapy and basic studies

In:. J. Rad~onan

Oncolo~j

Brol

Phys

1977. Volume

3. pp

187.193

Perpamon Rcss.

Printed m the U.S.A.

0 Neutrons-II

COMPARATIVE RADIOBIOLOGY RELEVANCE TO RADIOTHERAPY

OF FAST NEUTRONS: AND BASIC STUDIES?

FRANK Q. H. NGO, Ph.D., ANTUN HAN, M.D., HIROSHI UTSUMI, Ph.D. and M. M. ELKIND, Ph.D. Division

of Biological

and Medical Research,

Argonne

National

Laboratory,

Argonne.

IL 60439. U.S.A.

Comparative neutron radiobiological properties relevant to radiation therapy are being investigated using V79 neutrons, Chinese hamster cells in culture. Three neutron beams are being used: linear accelerator-produced p+ + Be, at the Cancer Therapy Facility of the Fermi National Accelerator Laboratory (Fermi); cyciotronproduced neutrons, d++Be, at the Franklin McLean Research institute, University of Chicago @MI): and fission-produced neutrons at the JANUS Reactor of the Argonne National Laboratory (JANUS). The mean neutron energies of the Fermi, FM1 and JANUS beams are 25 MeV, 3.6 MeV and 0.85 MeV, respectively. The RBE vplues of cell survival relative to 250 kVp X-rays, at a given surviving fraction, decreased in the order JANUS, FMI, Fermi, a trend opposite to the mean neutron energy. The OER’s measured with Fermi and JANUS neutrons were essentially the same, however. Other radiibiolosicpl studies have been initiated with JANUS neutrons. Results from two-dose fractionation experiments showed that very little repair of neturon-induced subkthai damage occurred for incubation at 37°C for intervals up to 5 hr between exposures. Data from postirradiition growth kinetics indicated that neutron radiation produces cell division delays that increase linearly with dose. An RBE for cell division delay, relative to 55 kV X-rays, was calculated to be approximately 3.5, which is close to the upper limit of the RBE vaifor survivals. Hyperthermic treatment, immediately following neutron irradiation, enhanced cell killing. Fostirradiation treatments of ceils with hype- and hyper-tonicity also resulted in enhanced cell killing. Fast neutrons, Tonicity.

V79 Chinese

hamster

cells, RBE, OER, Sublethal

INTRODUCTION

damage,

Division

delay, Hypertbennia,

In order for fast neutrons to be applied to radiation therapy in a safe and efficient manner, comparative radiobiological studies and more basic research are required. Comparative studies are necessary because most neutron sources in use, or being developed for use in clinical trials, consist of broad and yet different energy spectra and therefore broad and different LET distributions; radiobiological responses to a beam of such characteristics are difficult to predict, and, unfortunately, the kind of physical and biological information needed for such a prediction is thus far lacking. Basic radiobiological research with neutrons is essential because it is the only course by which a

better understanding of radiation effects induced by the neutral particle can be accomplished. Until we have improved our understanding and accumulated sufficient basic information, the clinical application of fast neutrons cannot be developed confidently and correctly. In the Chicago area, two fast neutron beams are being developed for therapeutic use: one at the Cancer Therapy Facility at the Fermi National Accelerator Laboratory and the other at the Franklin McLean Research Institute, University of Chicago. A third fast neutron beam, which was designed primarily for biological research purposes, is available from the JANUS reactor at the Argonne National

*The work was supported by the U.S. Energy Research and Development Administration and Grant number CA 18434 from the U.S. National Cancer Institute. Ackno&?dgments-We wish to thank F. S. Williamson. Dr. T. B. Borak. G. L. Holmblad. and J. E. Trier of our division at the Argonne National Laboratory; Dr. L. Cohen, Dr. M. Awschalom, and A. Jones of the Cancer Therapy Facility.

the Fermi National Accelerator Laboratory; and Dr. F. T. Kuchnir. Dr. F. Watermann, Dr. F. M. Skaggs and Dr. M. L. Greim of the Franklin McLean Research Institute for their dosimetry measurements and their efforts in making the neutron facilities accessible to us. Excelient technical assistance from M. Geroch, C.-M. Chang-Liu, E. Kautzky and M. D. Long is gratefully acknowledged.

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Laboratory. These three neutron beams produce energy spectra which differ considerably. In this report we use mammalian cells in vitro to compare the relative biological effectiveness (RBE) and the oxygen enhancement ratio (OER) for the aforementioned neutron beams. We also include studies using the JANUS neutrons to elucidate dose fractionation effects relative to repair of neutroninduced sublethal damage, growth kinetics following neutron irradiation and modification of neutron-induced damage by hyperthermia and hypo- or hypertonicity. Results will be compared with those from X-rays and their implications will be discussed in terms of clinical applications, and, where possible, in terms of basic mechanisms. broad

! of Chlcago

F

1

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20

NEUTRON

METHODS AND MATERIALS Cell cultures V79-AL162, a subline of Chinese hamster cells, was used in this study. The cells were grown exponentially, with a generation time of 8-9 hr, in plastic pertri dishes (lo-cm dia.) at 37°C in a humid atmosphere containing 2% CO* (pH = 7.3). Growth medium used was a a-modified Eagle’s mediumm supplemented with 15% fetal calf serum. Radiation sources

Three fast neutron beams of different, broad energy spectra were used: a fission-produced neutron beam at the JANUS Reactor at the Argonne National Laboratory (JANUS), a cyclotron-produced neutron beam, d’+Be. at the Franklin McLean Research (FMI), and a linear accelerator-produced neutron beam, p+-,Be, at the Cancer Therapy Facility of the Fermi National Accelerator Laboratory (Fermi). Figure 1 shows the energy spectra of these beams; the average energies of the JANUS, FM1 and Fermi neutrons are 0.85, 3.6 and 25 MeV respectively.6”5’29 Neutron dosimetry was determined by the physicists associated with each facility prior to each experiment. The dose rates used were 37.8 rad/min for 13-15 rad/min for FMI, and JANUS neutrons. - 28 rad/min for Fermi. Two X-ray sources were used: a G. E. Maxitron operated at 250 kVp, 30 mA, HVL = 1.10 mm Cu; and a Picker X-ray unit operated at 50 kVp, 20 mA, HVL = 0.14 mm Al. The absorbed dose rates were 137 rad/min for the 250 kVp X-rays for the 50 kVp. Experimental

and

1615 rad/min

procedure

For experiments other than OER measurements, cells from exponentially growing culture were trypsinized and inoculated into polystyrene T-flasks (25 cm*) or Petri dishes (IO-cm dia.) approximately

FM I (d, Be)

30

40

50

60

ENERGY, MeV

Fig. I. Energy spectra of JANUS, FM1 and Fermi neutron beams produced by the physical processes described in the parentheses. The relative neutron intensities for each beam have been normalized to scale 100 on the abscissa. 36 hr prior to radiation. Before exposure to neutrons, flasks containing attached cells were filled with growth medium and transported in ice to the neutron facilities. With the JANUS neutrons, the flasks were irradiated in air. Flasks were located so that cells were exposed to the direct neutron source through the polystyrene wall of the flask to which they were attached, and to the indirect, reflected source through the medium contained in the flasks. The difference in hydrogen content between the wall (-CH-) and medium (primarily HzO) produces a discontinuity in the dose at the interface. The JANUS neutron doses quoted in this paper reflect a correction factor of 0.86 for this discontinuity. Studies are continuing to improve the accuracy of this factor.5 With the Fermi or FM1 neutron beams, samples were irradiated in water phantoms. Flasks were immersed in a position to allow the neutron flux to pass through the medium before the cells. For X-irradiation with 250-kVp X-rays, cells were irradiated in 10 ml growth medium in flasks: for 50 kVp they were irradiated in Petri dishes with no cover and no medium. Hyperthermic treatments were accomplished by immersing flask samples immediately after radiation into a water bath with temperature preset at 41.7 2 0.2”C. Solutions with varying strengths of tonicity were prepared by adding NaCl into a phosphate buffered saline (PBS), pH = 7.4. initially exclusive of NaCl. For tonicity treatments, cells immediately after radiation were first rinsed once with isotonic PBS (0.14 M NaCI) and treated with the respective tonic solution for 20 min at 37°C. Upon completion of radiation or treatments, cells

Comparative radiobiology of fast neutrons 0 F. Q. H. NW,

in duplicate flasks or dishes were trypsinized, pooled, counted and plated in triplicates in complete growth medium at appropriate dilutions in Petri dishes. Colonies formed in these dishes in 7-8 days under the culture conditions described earlier were considered to represent surviving cells. In the OER experiments, hypoxic cells were prepared by inoculating cells into 2-ml glass ampoules at a high concentration (-2x lo6 cells/ml). Cells in the ampoule were bubbled for 2 min with a gas mixture of 95% nitrogen and 5% CO? at a flow rate approximately 650 cm3/min. The ampoule was then sealed while the flow rate was gradually reduced. To deplete the oxygen in the medium even further. ampoules were then shaken for 45 min at 37°C. Aerobic cells were also prepared in glass ampoules, using only - 1 x IO5cells/ml and no gas bubbling or shaking. During neutron exposures, the ampoules were inserted into a polyethylene holder which was set to vibrate horizontally to improve dose uniformity to the suspended cells, and also to insure hypoxic condition for the hypoxic samples. Upon completion of radiation, cells from ampoules irradiated in triplicates were pooled, without trypsinization, and were plated as described above. RESULTS AND DISCUSSION biological effectiveness and oxygen

Relative hancement

189

et al.

JANUS neutrons. This trend is qualitatively in accord with the theoretical prediction made by Kellerer and Rossi2’ and is consistent with experimental results reported by various investigators, including Hall et al.,” Bewley et a1.,4 Withers et aL3’ and Gragg et al.” One of the main advantages of using fast neutrons in radiotherapy is the reduced dependence on oxygen in cell inactivation. As shown in Fig. 3, using survival curves of aerobic and hypoxic cells, we deduced an OER of 1.8 for the Fermi beam and 1.7 for the JANUS, as compared to 3.4 for 250 kVp X-rays. Using similar techniques, Hall et af.19 have reported an OER of 1.6-1.7 for the Fermi neutrons, a result in fair agreement with the present data. According to these values. the RBE of hypoxic cells would be greater than that of oxic cells and thus a therapeutic gain factor of greater than one for tumors containing hypoxic cells can be expected.” However, the closeness of the OER values of the Fermi and the JANUS neutrons implies that the high energy Fermi beam would not be significantly more effective for sterilization of hypoxic cells.

en-

ratio

Figure 2 shows the survival data obtained with V79 cells exposed to graded doses of JANUS, FM1 and Fermi neutrons and to 250 kVp X-rays. All survival curves were fitted by eye. These data indicate that at a given survival level, the RBE increases as the mean energy decreases, namely from Fermi to FM1 to

0.2

0.4

DOSE,krad 0.6 0.8 1.0

1.2

14

1.6

rJANUS NEUTRONS o OER:17

Fig. 3. Survival

. Ll

0001~

curves

of V79 Chinese hamster cells ex-

(left) and Fermi (right) neutrons under aerobic and hypoxic conditions. The OER’s shown are based upon ratios of Dn’s. Error bars represent standard error of the mean when larger than the symbol.

posed to JANUS

li

Repair of neutron-induced

Fig. 2. Survival curves of V79 Chinese hamster cells exposed to JANUS, FM1 and Fermi neutron beams. a@ 250 kVp X-rays. P.E. represent the plating efficiency. N denotes average. cell multiplicity. Error bars represent standard

error

of the mean

when

larger

than

the symbol.

sublethal damage

both radiotherapy and radiobiology, sublethal damage and its repair are important considerations. It is not known if the neutron-induced sublethal damage is the same in all respects as the sublethal damage caused by low LET radiations, such as X-rays. It is known, however, that in mammalian systems the capacity for cells to accumulate neutron-induced sublethal damage varies only slightly with neutron energy and is much smaller than that with X-rays.“14.~ This is evident in the smaller shoulder on the survival curve after neutron as opposed to X-ray radiation (e.g. Fig. 2). Consequently. the absolute In

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amount of neutron-induced sublethal damage that can be repaired would also be relatively small. Figure 4 shows the results of a split dose experiment using the JANUS neutrons. Little if any repair of sublethal damage was observed under these conditions. It should be noted, however, that these results do not rule out a partial repair of sublethal damage in the intervals studied. The apparent lack of sublethal damage repair is also supported by the two-dose fractionated survival curve, results that we have reported previously.25 Clinically, this implies that the RBE for a given tissue would be greater if multifractionation as opposed to single exposures of neutrons were used.

V79-AL162 JANUS

0

IO

20 HOURS

/

V_79-AL162 t

1

NUI PE =89% JANUS FAST NEUTRONS 176rods+176rods

37°C

4 j

I2

3

HOURS BETWEEN

40

50

-

60

'0

EXPOS~JAE,~'~C

Fig. 5. Growth kinetics of V79 Chinese hamster cells after exposures to various single doses of JANUS neutrons. Open circles trace the growth of unirradiated cells. Lines with no data points were drawn parallel to the line which was fitted to the open circles (unirradiated cells). Tz denotes the population doubling time.18

i _I

227radst227rads

G.0011 0

30 AFTER

NEUTRONS

456 EXPOSURES

Fig. 4. Responses of V79 Chinese hamster cells to two fractionated doses of JANUS neutrons as a function of incubation intervals between the exposures, for doses and

tion of the total population. These data indicate that: (1) neutrons, like X-rays, induce delay of cell division, and the delay increases with dose; and (2) the amount of postirradiation division decreases as the dose increases. It is interesting to note that to reduce the growth of non-surviving cells to about one division requires a dose of approximately 300 rad of neutrons, as compared to approximately 1000 rad of 55 kVp X-rays.9 Division delay of the surviving cells can be examined by plating cells in growth medium and measuring the proportion of colonies formed. Figure 6 shows that: (1) the average division delay of the

incubation temperatures specified.

IOOL

:

V79-AL162 JANUS NEUTRONS: s_

Postirradiation growth kinetics Although considerable information has been accumulated for postirradiation cell kinetics following low LET radiation,‘.‘.‘“.“.” little if any data are available for high LET or neutron radiation. It was found, for example, in previous studies that division delay caused by X-irradiation is a linear function of radiation dose, and the delay for non-surviving or killed cells (cells which fail to form colony) is the same as for surviving cells (cells which form colony).’ The postirradiation growth kinetics has been followed using the JANUS neutrons. Figure 5 shows the growth kinetics of cells immediately after exposure to various single doses of neutrons; zero hour corresponds to the time of exposure. The data reflect to a large extent the non-surviving population, because the surviving moiety occupies only a very small por-

:

P

cellsy

c,34~~--,c--20~-~~----30 HOURS

40

_ __ ~_~_Z 50 60

76

AfTEREXPOSURE,37"C

Fig. 6. Growth kinetics of V79 Chinese hamster cells after an exposure to JANUS neutrons for surviving (0) and total cell (m) populations. Ad denotes division delay time and Tz denotes the popuration doubling time.

Comparative

radiohiology

of fast neutrons

survivivng cells is essentially the same as that of the total population; and (2) after a limited number of divisions, the killed cells stop their proliferation. while survivors keep dividing exponentially at approximately the same rate as unirradiated cells. In Fig. 7. the division delays of the total population and surviving population are plotted as a function of dose. These data show that delay of cell division increases approximately linearly with dose. from 0 to 320 rad. and at a rate of about 3.5 hr per 100rad for both the non-surviving and surviving cells. If we compared the rate of division delay of 3.S hr1100 rad of neutrons to a division delay of about 1 hr/lOO rad of 55 kVp X-rays,’ we obtained an RBE of 3.5 for division delay. In respect to survival. the RBE of 55 kVp X-rays

relative

to 250 kVp X-rays

is 1.1-1.2 as

measured by the ratio of Do’s. If we assume that the RBE for division delay between these two X-ray beams is similar in magnitude as for survival, then the RBE for division delay for the JANUS neutrons relative to 250 kVp X-rays would increase to 3.9-4.2.

,

16

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14:

. 3

,

3.5hr/lOO rod

0 .o o

1 i 1

.

% 56: s

i-

(

JANUS NEUTRONS

IZ2 f IO: 4 x 8-

4?

1

V79-AL162

1

0

F. Q. H. NGO. et al.

Modification thermia

191

of neutron-induced

damage

by hyper-

Enhancement by hyperthermia of the cell killing response to X- or y-irradiation has been reviewed recently by Dewey er al.* The increased understanding of hyperthermic effects in the past few years will undoubtedly help develop their clinical applications. It is of interest to find out if hyperthermia potentially has a role to play in therapy with high LET radiations. Although some information recently became available for hyperthermia and helium ions,16 information about hyperthermia and neutrons has been lacking. We have initiated a study in this area to build upon earlier data obtained with V79 cells and Xrays.2.3 In the experiments to be described, we used postirradiation hyperthermia in order to insure that damage expression. rather than target modification, could be examined. Figure 8 shows the effect of the hyperthermic treatments, 2 hr or 3 hr at 41.7”C, immediately following neutron irradiation. These data demonstrate that hyperthermia used in conjunction with neutrons does enhance cell killing and, furthermore, the killing increases as the heat treatment is lengthened. The relative shoulder width, &/DO. increases slightly with increasing heat treatment, suggesting that the principal effect is a greater expression of potentially lethal damage. Regardless of the mechanism, however, heat treatment after neutron exposure increases cell killing.

4 A

2 0 Q 0 *

TOTAL CELLS j,

SURVlVORS ! ._I I Yt o._100 200 300 400 500 600 C DOSE, rod ??

Modification changes

of neutron-induced

damage

by tonicity

Enhancement of radiation sensitivity by hypertonic treatments has been demonstrated in mammalian DOSE roe

Fig. 7. Delay of cell division as a function of dose of JANUS neutrons for surviving (closed symbols) and total (open symbols) populations of V79 Chinese hamster ceils. (Different symbols represent results from different experiments.)

These values are close to or higher than the upper limit of the RBE values for survival. Recently Rasey et al.” also observed a higher RBE for mitotic delay than for survival in EMT-6 cells irradiated with cyclotron-produced neutrons at the University of Washington with respect to 250 kVp X-rays. These in vitro data with two types of mammalian cells therefore suggest that radiation-induced delay in cell proliferation is an additional factor which will significantly affect the outcome of clinical trials using fast neutrons. It remains to be determined whether the RBE of division delay depends upon neutron energy.

Fig. 8. Survival data of V79 Chinese hamster cells exposed to JANUS neutrons, immediately followed by hyperthermia (41.7”C) for 2 hr (A) and 3 hr fm). Error bars represent standard error of the mean when larger than the symbol.

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ce,ls.i.?3.?4

Recently, Raaphorst and Kruu? examined tonicity effects on V79-Chinese hamster cells using a wide range of NaCl concentrations in distilled water. They found that, by irradiating cells just before the end of the salt treatment. radiation sensitivity increased as the tonicity departed from the isotonic point toward either the hypo- or hyper-tonic regions. We have extended these results with two changes. First, NaCl concentrations were varied in PBS to insure pH control. Second. treatments (20min) with solutions of different tonicities were given after radiation to insure that changes only in damage expression were examined. The 20 min treatments by themselves were not toxic. In Fig. 9, the variation in survival is shown for 50 kVp X-ray and neutron doses that result in close to the same survivals under isotonic conditions. Clearly, the molarity dependence differs qualitatively at high salt concentrations; also the RBE’s of survival are less for both hypo- and hyper-tonic conditions than for isotonic treatment.

SUMMARY AND CONCLUSIONS Comparative radiobiological studies with Chinese hamster cells in vifro are reported using Fermi, FM1 and JANUS neutrons. The RBE values of these neutron beams for cell survival have been measured relative to 250 kVp X-rays. At a given survival level, the JANUS beam yielded the highest RBE, whereas the Fermi beam gave the lowest. The OER values of

179-AL I62

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a07rods 20mlr

-4

;o _~_

d

_- -- .,

X-rays-

NoCI

OSM ..-_...--do~-~--

IO

IC

IO'

iNaCI! MOLAR

Fig. 9. Variations in radiosensitivity of V79 Chinese hamster cells as a function of NaCl concentrations (in PBS) for 20 min treatment immediately following 50 kVp X- or JANUS neutron-irradiation (0 and A. respectively). 0.14 M corresponds to isotonicity. Error bars represent standard error of the mean when larger than the symbols.

these two beams. however, were found to be essentially constant. The OER for the FM1 beam has not yet been determined but, because the energy distribution of its neutrons lies between those of JANUS and Fermi. we do not expect its OER to differ significantly.

When a dose of JANUS neutrons was divided into two equal fractions, there was little repair of neutroninduced sublethal damage. In addition to the advantage associated with the reduced OER, the reduced magnitude of repair with fast neutrons may increase the gain factor because the RBE for a given tissue would be greater if multifractionation compared to single doses of neutrons were used. This requires, of course, that the RBE of the normal tissue involved due to fractionated doses be increased by a smaller amount than that of the malignant tissue. Results from postirradiation growth kinetics indicate that neutron irradiation from the JANUS reactor is quantitatively more effective in producing division delays than X-rays. Qualitatively, however, neutrons are similar to X-rays; the induced delays are a linear function of dose and, moreover, the delay times are the same for surviving and for killed cells. Thus, the RBE for neutron-induced division delay relative to X-rays is independent of dose, at least for the dose range studied. This is very different from the RBE for survival, which decreases with increasing dose. The RBE for division delay was calculated to be approximately 3.5 relative to 55 kVp X-rays and 3.9-4.2 to 250 kVp X-rays. These values are close to or higher than the upper limit of the RBE for survival. While the RBE for division delay may vary depending on neutron energy, cell type, and perhaps other factors, the data presently available suggest that this end point is another significant parameter for consideration in the planning of clinical protocols for neutron therapy. Postirradiation hyperthermic treatments enhanced the expression of potentially lethal damage produced by neutrons. Hypo- and hyper-tonic salt treatments following neutron irradiation also enhanced cell killing. These enhancements, caused by tonicities. appeared to be more pronounced with X-rays than neutrons. The mechanisms involved when such physical and chemical agents are used are under investigation. Nevertheless, our results suggest that hyperthermia. and hypo- and hyper-tonicity can be considered as sensitizing agents in fast neutron therapy.

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synthesis to radiation-induced division delay in Chinese hamster cells. Radiat. Res. 44: 788-806, 1970. 2. Ben-Hur. E., Elkind. M.M.. Bronk, B.V.: Thermally

enhanced radioresponse of cultured Chinese hamster Inhibition of repair of sublethal damage and enhancement of lethal damage. Radiat. Res. 58: 38-5 1, 1974.

cells:

Comparative

radiobiology

of fast neutrons

3. Ben-Hur, E., Elkind, M.M.: Mechanisms for enhanced radiation-induced cell killing in hyperthermic mammalian cells. In Proc. of the Int. Symp. on Cancer Therapy by Hyperthermia and Radiation, Washington, D.C., 1975, pp. 34-40. 4. Bewley, D.K., Cullen, B., Field, S.B., Hornsey, S., Page, B.C.: A comparison for use in radiotherapy of neutron beams generated with 16 and 42 MeV deuterons on beryllium. Br. J. Radial. 49: 36&366, 1976. 5. Borak, T.B.: Division of Biological and Medical Research, Argonne National Laboratory, Private communication. 6. Cohen, L., Awschalom, M.: The cancer therapy facility at the Fermi National Laboratory: A preliminary report. Appl. Radiol. Nov.-Dec.: 51-60, 1976. 7. Dettor, C.M., Dewey, W.C., Winans, L.F., Noel, J.S.: Enhancement of X-ray damage in synchronous Chinese hamster cells by hypertonic treatments. Radiat. Res:52: 352-372, 1972. 8. Dewey, W.C., Hopwood, L.E., Sapareto, S.A., Gerweek, L.E.: Cellular responses to combinations of hyperthermia and radiation. Radiology 123: 46-74, 1977. 9. Elkind, M.M., Han, A., Volz, K.W.: Radiation response of mammalian cells grown in culture-IV. Dose dependence of division delay and postirradiation growth of surviving and nonsurviving Chinese hamster cells. J. Nat1 Cancer Insfit. 30: 705-721, 1%3. 10. Elkind, MM., Whitmore, G.F.: The Radiobiology of Cultured Mammalian Cells. New York, Gordon & Breach, 1967, Chap. 7, pp. 303-382. 11. Elkind, M.M., Withers, H.R., Belli, J.A.: Intracellular repair and the oxygen effect in radiobiology and radiotherapy. Front. Radiar. Ther. Oncol. 3: 55-87, 1%8. 12. Elkind, MM: Damage and repair processes relative to neutron (and charged particle) irradiation. Curr. Topics Radiat. Res. Quant. 7: l-44, 1970. 13. Elkind, MM.: Damage registration and repair following neutron irradiation. Brookhaven Nat1 Lab., A.E.C. Rep. BNL-14116, Fall 1970. 14. Field, S.B.: An historical survey of radiobiology and radiotherapy with fast neutrons. Curr. Topics Radial. Res. @ant. 11: l-86, 1976. 15. Frigerio, N.A.: Neutron spectrometry of the JANUS high tlux room. Division of Biological and Medical Research Annual Report. Argonne Nat1 Lab., Rep. ANL-7870, 8-10, 1971. 16. Gerner, W., Leith, J.T., Boone, M.L.: Mammalian cell survival response following irradiation with 4MeV Xrays or accelerated helium ions combined with hyperthermia. Radiology 119: 715-720, 1976. 17. Gragg, R.L., Humphrey, R.M., Meyn, R.E.: Response of Chinese hamster ovary cells to fast neutron radiotherapy beams-I. Relative biological effectiveness and

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