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MOLECULAR AND CELLULAR EFFECTS OF PROTONS, DEUTERONS AND ALPHA-PARTICLES

Objective

Objectives of the project are to:
measure the effectiveness of low doses of protons in inducing HGPRT mutations, as a function of LET;
measure the initial production of DNA double strand breaks in cells irradiated with protons, as a function of LET;
establish a beam line for irradiation of cell monolayers with deuterons to extend to higher LETs that is practically possible with protons;
measure the effectiveness of deuterons for cell inactivation or mutation, as a function of LET;
start comparative experiments with low energy alphaAparticle radionuclide sources at different dose rates;
analyze the microscopic track structure of the radiations to seek features which correlate with their observed biological effectiveness.
Previous studies on cell inactivation of V79 cells caused by low energy protons have shown that the relative biological effectiveness (RBE) linear energy transfer (LET) relationship is in poor agreement with values predicted by the currently assumed curve based on alpha particle data. To extend these studies to end points other than cell inactivation, the mutation induction at the hypozanthine phosphoribosyltransferase (HPRT) locus in V79-753B Chinese hamster cells following irradiation with protons having incident energies of 3.36 1.70 and 1.16 MeV in the dose range 0.5 to 4.0 Gy was analyzed. The mutation curve obtained with 200 kV X-rays was used for comparison. The proton effectiveness increased with the LET and comparison between protons and X-rays showed a substantial difference in the number of induced mutants at the same dose. In the considered LET range low energy protons appear to have a higher effectiveness in mutation induction than other charged particles with the same LET. A similar finding was observed for cell inactivation.

To perform cell irradiations in air with deutron beams the Van de Graft accelerator was fitted with a remote controlled multisample holder for 20 petri dishes to allow irradiation at a prefixed temperature. Preliminary results have been obtained for inactivation of V79 cells irradiated in air with 17.4 and 24.0 keV per um deutrons in the dose range 0.5-4.0 Gy.

Raw experimental data was analyzed for a direct comparison of the biological effectiveness of protons and alpha particles of the same linear energy transfer (LET). From this analysis it is concluded that, overall, protons are more effective at cell inactivation than alpha particles. For mutations at the hypozanthine phosphoribosyltransferase (HPRT) locus in V79-4 cells it was also found that protons were more effective than alpha particles of the same LET.

By contrast, the initial yield of deoxyribonucleic acid (DNA) double strand breaks in V79-4 cells as measured by neutral sucrose sedimentation was smaller for protons than alpha particles. By the Olive method of DNA precipitation the relative yield of breaks was only slightly higher with proton than with alpha particles. Hence it would appear that the residual damage which leads to cell inactivation or mutation is not a random sample from the initial double strand breaks.

The differences in biological effectiveness of protons and alpha particles of the same LET must originate from their differences in track structure. The experimental data provides a useful new constraint on the biologically critical microscopic properties of radiations. To quantify differences between the tracks over dimensions similar to DNA, nucleosomes and chromatin fibre, statistically representative tracks of protons and alpha particles were simulated by the Monte Carlo computer code MOCA14. These were scored for energy deposition in small cylindrical volumes positioned randomly with respect to the tracks. From this absolute frequency distributions of energy deposition in target volumes have been generated for given conditions.

Experimental data on the inactivation, hypoxanthine phosphoribosyltransferase (HPRT) mutations and ribonucleic acid (DNA) double strand breakage in V79 cells irradiated by protons and alpha particles at 20 and 23 keV um-1 has been analyzed. The analysis concludes that protons are more effective at cell inactivation and mutation induction than alpha particles of the same linear energy transfer (LET). By contrast, the initial yield of DNA double strand breakage did not reproduce the differences observed for the cellular effect suggesting that the residual damage responsible for cell inactivation or mutation is not a random sample from the initial double strand breakage.

The frequency of HPRT mutations in V79-793B cells by proton beams having incident energies of 3.36 1.70 and 1.16 MeV were studied. The mutation curve obtained with 2200 kV X-rays was used for comparison. The mutation frequency induced by all the proton beams was higher than that induced by the same dose of X-rays and was linearly related to the dose. The relative biological effectiveness (RBE) for protons increased with the LET and had values higher than those reported in the literature for ions of comparable LET. This parallels previous work found for cell inactivation and indicates that for mutation the RBE-LET relationship may also depend on the type or irradiation.

The initial yields of DNA double strand breakage in V79 cells irradiated with protons of 3.36 1.70 and 1.16 MeV in the dose range 10 to 120 Gy was studied. The sucrose gradient sedimentation technique was used for determinations and irradiation with X-rays was used for comparison. Linear dose response curves were determined. Preliminary data analysis indicated only small differences in the radiation qualities. This finding is consistent with the results of others studying the direct comparison between protons and alpha particles. The experimental data provides useful new constraints on the biologically critical microscopic proper ties of radiations. The observed dependence of biological effectiveness on the LET andradiation type should origniate from changes in the radiation track structure.
It has been well established experimentally in many biological systems that radiations of high linear energy transfer (LET), such as alphaAparticles and neutrons, have greater biological effectiveness than the same absorbed dose of low LET radiations, such as gamma rays or XArays.

It has been difficult to carry out systematic investigations of the quantitative dependence of relative biological effectiveness (RBE) with LET of such protons and alphaAparticles because of the very short ranges of these charged particles. Similar LETs can be obtained with much higher velocity heavier ions which have much larger ranges, but the track structures of these differ greatly from those of protons and alphaAparticles so RBEs obtained from them are less directly relevant.

It is therefore important to investigate the generality of this phenomenon, its extension to deuterons of yet higher LET, its possible dependence on the dose rate and its causes in terms of the microscopic track structure.

The contribution of the Legnaro research group in this project will be the following:
study, design and setup of a new apparatus installed at the CN accelerator for cell irradiation with deuterons, with remote controlled multisample holder and temperature;
study, design and construction of many small size systems for cell irradiation with alpha sources during incubation. These systems will be used to study low dose rate effects; .SP 0 maintenance of cultured mammalian cells and their irradiation using both the already existing proton beam line facility and the ones under development;
cooperation with ISS for cell irradiation and survival and mutation experiments.

The MRC will generate, by Monte Carlo methods, simulated tracks of the radiations at the level of the individual atomic interactions along the path of the particle and all its secondaries.

The ISS group will carry out, at Legnaro, in collaboration with the local group, a more detailed study on the RBE for mutation at low doses as a function of the proton's LET.

Work at the Gray Laboratory will extend these studies to higher LET's using deuterons in place of protons because of their greater range but almost equivalent track structure, also to include studies at low doses and low dose rates.

Funding Scheme

CSC - Cost-sharing contracts

Coordinator

ISTITUTO NAZIONALE DI FISICA NUCLEARE
Address
Via Romea 4
35020 Legnaro
Italy

Participants (3)

GRAY LABORATORY CANCER RESEARCH TRUST
United Kingdom
Address
Mount Vernon Hospital
HA6 2JR Northwood
ISTITUTO SUPERIORE DI SANITA
Italy
Address
Viale Regina Elena 299
00161 Roma
MRC Cell and Molecular Biology Division
United Kingdom
Address
20 Park Crescent
W1N 4AL London