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Radiobiology of alpha particles. III. Cell inactivation by alpha-particle traversals of the cell nucleus.

Cell inactivation after exposure to collimated 3.5-MeV alpha particles in three hamster cell lines, V79, CHO-10B, and HS-23, one mouse cell line, C3H 10T1/2, and a human skin fibroblast cell line were studied. Several parameters were investigated for each cell line. Theoretical calculations were performed to find the distribution of energy deposited in the nuclear volume for each cell line. The mean number of alpha-particle traversals required to induce a lethal lesion varied between two for HS-23 cells and six for C3H 10T1/2 cells. The number of traversals per unit area and the total track length of alpha particles that inactivated a cell were found to be nearly constant for the hamster and mouse cell lines. These quantities were found to be lower for the human skin fibroblast cell line. The RBE values for all cell lines were found to be about 3.8 at 10% survival. Thus cell lines that are more sensitive to alpha radiation are also more sensitive to gamma radiation. The average number of alpha-particle traversals producing a single lethal lesion is greater than one. The passages of alpha particles through the cell nucleus that do not kill the cell may lead to carcinogenic effects.

Alpha Particles

Radiobiology of alpha particles. II. Dosimetry of low-energy alpha particles using parallel-plate ionization chambers and a surface barrier detector.

Three small parallel-plate ionization chambers were developed for measuring dose rates, of primarily low-energy alpha particles in the energy range 0.4-3.5 MeV, at a defined cell-Mylar interface. Spectral energy distributions of these alpha particles were also measured at the same position using a specially designed small-area silicon surface barrier detector. Dose rates were derived from the spectral distributions and compared with those derived from the ionization chambers. Different alpha-particle energies were obtained using a 144-MBq 238Pu collimated source and a variety of Mylar moderator foils of different thicknesses. These measurements, extended to mean alpha-particle energies as low as 0.4 MeV, will enable us to correlate radiobiological data with effects of alpha particles terminating in different regions of cell nuclei.

Alpha Particles

A device for in vitro irradiation with alpha-particles using an alpha-emitting radioactive source.

A device to irradiate a monolayer of cultured cells with alpha-particles using an Am-241 alpha-source (33.4 MBq) was designed to investigate RBEs of alpha-particles in cell killing, induction of chromosome aberration, mutagenic changes and transformation. This device can be used conveniently in a common laboratory by a small number of researchers without any limitation of machine time. The device performs as follows: (1) The energy of alpha-particles at the entrance of the cell layer is 3.20 MeV with a standard deviation of 0.25 MeV, (2) the incident angle to the cell layer is 82.8 degrees with a standard deviation of 3.2 degrees, (3) the fluence rate is 4.7 x 10(5) cm-2.min-1, (4) the average LET infinity for a cell layer 5 microns thick is 138 keV/micron, (5) the average dose rate for a cell layer 5 microns thick is 0.10 Gy/min., (6) a temperature and CO2 concentration conducive to cell cultivation are maintained during irradiation.

Alpha Particles

Radioimmunotherapy with alpha-particle-emitting immunoconjugates.

Alpha particles are energetic short-range ions whose higher linear energy transfer produces extreme cytotoxicity. An alpha-particle-emitting radioimmunoconjugate consisting of a bismuth-212-labeled monoclonal immunoglobulin M specific for the murine T cell/neuroectodermal surface antigen Thy 1.2 was prepared. Analysis in vitro showed that the radioimmunoconjugate was selectively cytotoxic to a Thy 1.2+ EL-4 murine tumor cell line. Approximately three bismuth-212-labeled immunoconjugates per target cell reduced the uptake of [3H]thymidine by the EL-4 target cells to background levels. Mice inoculated intraperitoneally with EL-4 cells were cured of their ascites after intraperitoneal injection of 150 microcuries of the antigen-specific radioimmunoconjugate, suggesting a possible role for such conjugates in intracavitary cancer therapy.

Alpha Particles

Peroxidation of the dried thin film of lipid by high-energy alpha particles from a cyclotron.

High-energy alpha particles produced a dose-dependent linear increase in different lipid peroxidation products (e.g., malondialdehyde (MDA), conjugated dienes, and hydroperoxides) in the dried thin film state. An inverse dose-rate effect was observed when the dose rate was varied by changing either the alpha-particle fluence rate or the alpha-particle energy. The antioxidants alpha-tocopherol and butylated hydroxytoluene (BHT) suppressed the alpha-particle-induced lipid peroxidation in the dried thin film state, and in this respect alpha-tocopherol was found superior to BHT. It was found that alpha-tocopherol was equally efficient in inhibiting lipid peroxidations by alpha particles and ultraviolet light.

Alpha Particles

Alpha-particle autoradiography in CR-39: a technique for quantitative assessment of alpha-emitters in biological tissue.

The techniques for alpha-particle autoradiography based on the plastic nuclear track detector CR-39, previously reported, have been developed considerably. The techniques are applied to alpha-autoradiography of human lung tissue in particular but are applicable to any biological tissue. The most important developments are: (i) Improvements in the manufacture and pre-etching of the plastic. These allow activities as low as approximately 10(-15) Ci g-1 to be determined. (ii) High resolution alpha-particle spectroscopy in CR-39 plastic based on the analysis of the structure of the etched track. This enables the energy of individual alpha-particles to be determined to approximately 35 keV. (iii) Calculation of the effective thickness of tissue sampled by the plastic. This relates the tissue activity to the track density on the plastic. (iv) A deconvolution analysis which takes the distributions of track length and dip angle in the plastic and determines the alpha-particle range spectrum and distribution of tissue activity with height above the plastic surface. This enables both the absolute abundance and the microdistribution of alpha-active nuclides present to be determined. (v) The analysis of radon diffusion in tissue to determine the mean radon diffusion distance in tissue and plastic.

Adult

Interaction between X-ray and alpha-particle damage in V79 cells.

V79 cells have been exposed to X-rays or 238Pu alpha-particles or to X-rays following priming alpha-particle doses of 0.5, 2 or 2.5 Gy. The survival curve for exposure to alpha-particles was exponential with a D0 of 0.89 Gy. Following exposure to priming alpha-particle doses the resulting X-ray survival curves had the same slope as the single dose X-ray curve, but a reduced shoulder. For alpha-particle priming doses of 0.5 and 2 Gy this reduction was the same as for the same X-ray doses. 2.5 Gy alpha-particles reduced the subsequent X-ray curve Dq to almost zero. alpha-particles do cause damage capable of interacting with X-ray damage.

Alpha Particles

Response of X-ray-sensitive CHO mutant cells (xrs-6c) to radiation. II. Relationship between cell survival and the induction of chromosomal damage with low doses of alpha particles.

The induction of cytotoxicity, chromosomal aberrations, and sister chromatid exchanges (SCEs) was measured in CHO K-1c cells and in isogenic X-ray-sensitive mutant xrs-6c cells that had been irradiated with X rays and alpha particles in isoleucine-deficient alpha-minimal essential medium in G1 phase of the cell cycle. There was a noticeable shoulder region on the survival curve for CHO K-1c cells irradiated with very low doses of alpha particles, whereas this feature was absent for xrs-6c cells with alpha-particle doses as low as 0.5 cGy. Higher frequencies of chromatid-type aberrations were induced in G1-phase xrs-6c cells than in G1-phase CHO K-1c cells by both gamma- and alpha-particle irradiation. Induction of nonlethal chromosomal aberrations was observed following exposure to 2-6 cGy of alpha particles, doses yielding 97-100% cell survival. Irradiation with 0.5 cGy of alpha particles induced SCE; nearly 60% of irradiated cells contained significantly increased levels of SCE. However, only 3% of the nuclei of cells exposed to 0.5 cGy of alpha-particle radiation were actually traversed by an alpha particle. The observation that a large fraction of cells apparently survive exposure to very low doses of alpha-particle radiation with persistent genetic damage manifested by both chromosomal aberrations and SCEs may have important implications for the carcinogenic hazards of high-LET radiation.

Alpha Particles

Cell killing and division delay in asynchronous and synchronized HeLa cells irradiated with alpha particles or X rays.

HeLa cells irradiated with a single or two split doses of alpha particles or X rays were observed with time-lapse photography or examined for their colony-forming ability. The cell cycle-dependent variation of cell killing and division delay were compared in synchronous and asynchronous cell populations. Cellular damage by alpha particles was manifested in the form of cessation of division, or death, rather than partial division which was predominant for X irradiation. Furthermore, an increased number of interrupted divisions leading to death following split doses correlated with the lack of production of reparable damage from alpha particles. As to the cell cycle-dependent variation of radiosensitivity, a somewhat different pattern was noted with alpha particles, especially for division delay. The pattern of cell killing with alpha particles was similar to that found with X rays, in that high sensitivity was noted at or close to mitosis, while a resistant peak remained at late S but not in early G1. The pattern of division delay was similar for X rays and alpha particles during G2-M, with a maximum delay at mid G2 and no delay past the transition point, but differed during G1-S. During this period, division delay increased with cell age, whereas it showed a broad peak at G1-S boundary and a trough at late S for X rays. There was a reverse correlation between division delay and cell killing except for G2-M in the case of X rays. However, such was not the case for alpha particles.

Alpha Particles

Response of cultured human airway epithelial cells to X-rays and energetic alpha-particles.

Radon and its progeny, which emit alpha-particles during decay, may play an important role in inducing human lung cancer. To gain a better understanding of the biological effects of alpha-particles in human lung we studied the response of cultured human airway epithelial cells to X-rays and monoenergetic helium ions. Our experimental results indicated that the radiation response of primary cultures was similar to that for airway epithelial cells that were transformed with a plasmid containing an origin-defective SV40 virus. The RBE for cell inactivation determined by the ratio of D0 for X-rays to that for 8 MeV helium ions was 1.8-2.2. The cross-section for helium ions, calculated from the D0 value, was about 24 microm 2 for cells of the primary culture. This cross-section is significantly smaller than the average geometric nuclear area (approximately 180 microms 2), suggesting that an average of 7.5 alpha-particles (8 MeV helium ions) per cell nucleus are needed to induce a lethal lesion.

Alpha Particles

A comparative study of rejoining of DNA double-strand breaks in yeast irradiated with 3.5 MeV alpha-particles or with 30 MeV electrons.

Yeast cells were irradiated with 3.5 MeV alpha-particles and 30 MeV electrons, as reference radiation. The kinetics of DNA double-strand break (dsb) rejoining during incubation of cells under non-growth conditions (PLDR conditions) were measured using the neutral sedimentation technique. A monophasic kinetic was found after irradiation of cells with alpha-particles, with a dose-independent t1/2 value of about 13 h. The kinetics of rejoining of dsb induced by 30 MeV electrons was found to be biphasic, with dose-independent t1/2 values of 3.8 h for the initial and of about 11 h for the slow component. The fraction of the slow component was, however, dose-dependent. These kinetics were measured for both types of radiation at doses yielding high surviving fractions (5% up to 100%). Dsb are induced linearly with dose of both radiations. The RBE value of alpha-particles was found to be 2.5 for initial dsb. The RBE of alpha-particles increased as a consequence of dsb rejoining. This increase in RBE value suggests that DSB may be primary lesions for chromosome aberrations, cellular inactivation and oncogenic transformation of mammalian cells which all exhibit high RBE values of alpha-particles.

Alpha Particles

The combined effects of alpha-particles and X-rays on cell killing and micronuclei induction in lung epithelial cells.

Understanding how cellular damage produced by high-linear energy transfer (LET) radiation interacts with that produced by low-LET is important both in radiation therapy and in evaluating risk. To study such interactions, rat lung epithelial cells (LEC) were grown on Mylar films and exposed to both X-rays and alpha-particles, separately or simultaneously. Cell killing, and the numbers of binucleated cells and micronuclei, were measured as indicators of damage. X-rays and alpha-particles given separately caused dose-related increases in cell cycle time, with alpha-particles producing greater mitotic delay than X-rays. Damage from alpha-particles and X-rays given simultaneously did not interact to alter further the cell cycle. Cell survival data following exposure to X-rays and alpha-particles, combined or individually, were fitted by linear-quadratic models. Survival curves following exposure to alpha-particles only, or to 1.0 Gy alpha-particles plus graded X-ray doses, were adequately described using only the linear (alpha) term of a linear-quadratic model with alpha coefficients of 0.9 +/- 0.04 and 1.03 +/- 0.18 Gy-1, respectively. Survival following exposure to X-rays only or to 0.06 Gy alpha-particles combined with X-rays was best fitted using both alpha and beta terms of the linear-quadratic model (0.12 +/- 0.03)D + (0.007 +/- 0.002)D2 and (0.57 +/- 0.08)D + (0.3 +/- 0.02)D2, respectively. The numbers of micronuclei produced by exposure to alpha-particles or X-rays alone increased linearly with dose, with slopes of 0.48 +/- 0.07 and 0.19 +/- 0.05 micronuclei/binucleated cell per Gy for alpha and X-rays, respectively. Simultaneous exposure to graded levels of X-rays and a constant alpha dose of either 1.0 or 0.06 Gy increased micronuclei frequency, with a slope of 0.74 +/- 0.05 or 0.58 +/- 0.04 micronuclei/binucleated cell per Gy, respectively. These slopes are similar to that produced by alpha-particles alone. These studies demonstrated that both cell killing and the induction of micronuclei were increased by combined exposures compared with that predicted for separate exposures.

Alpha Particles

Induction of sister chromatid exchanges (SCE) in G0 lymphocytes by plutonium-238 alpha-particles.

Irradiation of human G0 lymphocytes with plutonium-238 alpha-particles and X-rays was performed to investigate the production of sister chromatid exchanges (SCE). Alpha-particles produce a significant increase in SCE and this elevation is more significant when separated lymphocytes are irradiated. X-ray irradiation did not induce any significant increase in SCE. Therefore the relative biological effectiveness (RBE) for the induction of SCE by alpha-particles in this system is undefined and effectively infinite.

Alpha Particles

Inactivation of C3H 10T1/2 cells by monoenergetic high LET alpha-particles.

Inactivation of mouse C3H 10T1/2 cells in plateau-phase (7.8 x 10(4) cells/cm2) was studied by using alpha-particles from the irradiation facility installed for radiobiological experiments at the 3 MV Tandem accelerator, University of Naples. Silicon detectors and CR39 plastic track detectors were employed for dosimetric purposes. The cells were exposed to high LET monoenergetic alpha-particles (energy of 1.8 MeV at the centre of the cell nucleus, track-averaged LET of 177 keV/micron and dose-rate of 1.1 Gy/min) and low-LET 80 kVp X-rays. The X-ray survival curve showed a significant shoulder (alpha/beta = 9 Gy) while the survival curve for alpha-particles was close to exponential. The mean lethal dose of alpha-particles was 0.77 +/- 0.02 Gy and the RBE was 5.2 at 80% survival and 3.0 at 5% survival. Survival of exponentially growing cells (2 x 10(4) cells/cm2) following irradiation with the alpha-particle beam is also reported. The nuclear areas of 10T1/2 cells were measured as 299 +/- 9 micron 2 and 250 +/- 8 micron 2 for cells in log phase and plateau phase, respectively. The inactivation cross-section, obtained from the mean lethal dose, was 34 micron 2 and 37 micron 2 for cells in log phase and plateau phase, respectively. These values appear to be the maximum measured values for the inactivation cross-section of 10T1/2 cells as a function of the alpha-particle LET. This saturation cross-section is very similar to the saturation values reported in the literature for other mammalian cell lines.

Alpha Particles

Experimental examination of cavity ionisation theory applied to alpha-particle fields.

The form of a general theory of cavity ionisation is outlined and its applications to alpha-particle fields considered. The attenuation of the alpha particles within the cavity, which gives rise to the weighting factor in general cavity theory, is considered in detail and an appropriate treatment for alpha-particle fields developed. Apparatus, consisting of an ionisation chamber composed of electrodes emitting alpha rays, was constructed in order to impose a rigorous test on the theory in the most critical region of cavity size. The general theory of cavity ionisation as applied to alpha-particle fields is shown to be in close agreement with the experimental results. As in the case of photon and electron fields, it is possible to characterise a cavity by a single chord length (path length) but the charged particle energy spectrum and the variation of stopping power with energy must be considered in detail.

Alpha Particles

Elemental analysis of bone mineral by backscattering of alpha particles.

Backscattering of 2 MeV alpha particles has been applied for the first time to studies of the inorganic substances of human bone. Bone samples taken from the femoral shaft (dense bone) and iliac crest (spongy bone) of 18 cadavers were analysed for the calcium, phosphorus and oxygen content, after lyophilisation and heating for 1.5 h at 500 degrees C to remove water and organic material. The backscattering method produces a spectrum in which all the elements present in the sample except hydrogen can be seen at once. The reproducibility of the measurements was less than 2%. No previous studies were found in the literature of the measurement of elemental oxygen in bone samples. Ca/P ratios varied from 1.97 to 2.47, Ca/O ratios from 0.76 to 0.91 and P/O ratios from 0.34 to 0.41, the latter two being about 10--15% lower than expected if bone mineral were hydroxyapatite (Ca/O = 0.96 and P/O = 0.44). The results are consistent with previous work on Ca/P ratios but the low Ca/O and P/O ratios could not be explained without postulation of crystal or hydroxyl water in bone mineral. Support for the explanation was gained by differential gravimetric and thermal analysis.

Alpha Particles

A versatile plutonium-238 irradiator for radiobiological studies with alpha-particles.

A versatile irradiator has been constructed for in vitro irradiation of mammalian cells with alpha-particles of well-defined energy, LET, direction, dose and dose rate. It is based on approximately 1.2 x 10(9) Bq of 238Pu (on a platinum disc) contained in a He-filled chamber. In a standard configuration, monolayers of cells grown in 10 Hostaphan-based dishes are irradiated with 3.26 +/- 0.22 MeV alpha-particles (LET 121 keV microns-1) at selectable dose rates from approximately 2 Gy min-1 down to less than 10(-4) Gy min-1 (i.e. fluence rates of 1 x 10(7) cm-2 min-1 to 3 x 10(2) cm-2 min-1). Single dishes can be irradiated at dose rates up to 24 Gy min-1 (fluence rate 1 x 10(8) cm-2 min-1). Incident energy and LET can be varied from 0.8 to 4.2 MeV and 266 to 102 keV microns-1, respectively. The irradiator has full incubation and gassing facilities for protracted irradiations. The irradiator is particularly suitable for in vitro analytical studies of the biological effects of alpha-particles of energies and LETs similar to those which cells may receive in vivo from radionuclides such as radon and the actinides. It has been used successfully for investigations of a variety of alpha-particle-induced effects in different cell types irradiated either as attached monolayers or as very thin suspensions.

Alpha Particles

Mutations induced by ionizing radiation in a plasmid replicated in human cells. II. Sequence analysis of alpha-particle-induced point mutations.

The human shuttle plasmid pZ189, containing the Escherichia coli supF gene as the mutational target, was irradiated in vitro with 210Po alpha particles and transfected into human lymphoblastoid cells. Plasmids which were replicated in human cells were recovered and those containing mutant supF genes were isolated by phenotypic screening in E. coli. The mutations were characterized by sequencing the tRNA gene. The mutant frequency increased linearly with the alpha-particle dose and, at 259 Gy, it was 16 times (0.29%) that observed in unirradiated controls (0.018%). The distribution of alpha-particle-induced point mutations was highly nonrandom and similar to that observed in the unirradiated or X-irradiated plasmid DNAs. The majority of the mutations were G.C----A.T transitions and occurred selectively at most 5'-TC (3'-AG) and 5'-CC (3'-GG) sequences. For the unirradiated control DNA, these mutations at C's (G's) were preferentially located in the nontranscribed strand, similar to the observation previously made for mutations in X-irradiated DNA. Such a strand bias was not observed for mutations in the alpha-particle-irradiated DNA. The data suggest that, although similar types of point mutations are induced in unirradiated, X-irradiated, and alpha-particle-irradiated DNAs, the mechanisms of their induction and the exact nature of the lesions involved may be quite different.

Alpha Particles