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.     

Absence of a dose-rate effect in the transformation of C3H 10T1/2 cells by alpha-particles

The findings of Hill et al. (1984) on the greatly enhanced transformation frequencies at very low dose rates of fission neutrons induced us to perform an analogous study with alpha-particles at comparable dose rates. Transformation frequencies were determined with gamma-rays at high dose rate (0.5 Gy/min), and with alpha-particles at high (0.2 Gy/min) and at low dose rates (0.83-2.5 mGy/min) in the C3H 10T1/2 cell system. alpha-particles were substantially more effective than gamma-rays, both for cell inactivation and for neoplastic transformation at high and low dose rates. The relative biological effectiveness (RBE) for cell inactivation and for neoplastic transformation was of similar magnitude, and ranged from about 3 at an alpha-particle dose of 2 Gy to values of the order of 10 at 0.25 Gy. In contrast to the experiments of Hill et al. (1984) with fission neutrons, no increased transformation frequencies were observed when the alpha-particle dose was protracted over several hours.     

Evidence for reduced capacity for damage accumulation and repair in plateau-phase C3H 10T1/2 cells following multiple-dose irradiation with gamma rays

The effects of multiple-dose gamma irradiation on the shape of survival curves were studied with mouse C3H 10T1/2 cells maintained in contact-inhibited plateau phase. The dose-fractionation intervals included 3, 6, and 24 h. Following three fractionated doses (5 Gy per dose) of exposures, cells responded to further irradiation by displaying a survival curve with a much reduced shoulder width (Dq) compared to that of the survival curve measured in cells irradiated with single-graded doses alone. The effect on the mean lethal dose (D0) was small and appeared to be significant. The effect on reduction of Dq could not be completely overcome by lengthening the fractionation intervals from 3 to 6 h or 24 h, times in which repair of sublethal damage (SLD) measured by simple split-dose scheme and potentially lethal damage (PLD) measured by postirradiation incubation was completed. Other experiments showed that pretreatments of cells with fractionated irradiation appeared to slow down the cellular repair processes of SLD and PLD. Therefore, the observed change in the shape of survival curves after fractionation treatments may be attributed to a reduction of the cells' capacity for damage accumulation by an enhancement of the lethal expression of SLD and PLD. Although the molecular mechanism(s) is not known, the results of this study indicate that the acute graded dose-survival curve cannot be used a priori to extrapolate and reliably predict results of hyperfractionation. It is probable that for a nondividing or slowly dividing cell population, such an extrapolation may lead to an underestimation of cell killing. Furthermore, the findings of this investigation appear to support an interpretation, alternative to the high-linear energy transfer (LET) track-end postulate, for the effects on cell survival seen at low doses or low dose rates.     

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.     

Relative sensitivities of repair-deficient mammalian cells for clonogenic survival after alpha-particle irradiation

The clonogenic survival of cells of the radiation-sensitive hamster cell lines irs1, irs2, irs3 and xrs5, representing different DNA repair pathways, was compared to that of their parent lines after alpha-particle irradiation. Measurements of nuclear area were made to calculate the probability of surviving a single alpha-particle traversal, the average number of lethal lesions per track and per unit dose, along with the "intrinsic radiosensitivity" of these cells, allowing for the potential of multiple lethal lesions per traversal. For all cell lines studied, alpha particles were found to be more biologically effective per unit absorbed dose than X rays at inducing cell inactivation. The repair-deficient cells showed an enhanced sensitivity to alpha particles compared to their parent line, but the degree of enhancement was less than for X rays. The reduction in additional sensitivity for alpha-particle irradiation was shown not to be due predominantly to differences in cell geometry limiting the probability of a cell nucleus being traversed. The results suggest that both the nonhomologous end-joining pathway and to a lesser extent the homologous recombination repair pathway play a role in successful repair of alpha-particle-induced damage, although a large proportion of damage is not repaired by either pathway.     

The in vitro radiobiology of astatine-211 decay

Chinese hamster V79 cells in culture were exposed to astatine-211, an alpha-particle-emitting radiohalogen. The dose-log survival response was linear with no detectable shoulder. Cells in monolayers had a D0 of 1.0 microCi/ml. Suspended cells had a D0 of 0.60 microCi/ml with a cellular uptake of 2.5 fCi/cell; this is equal to approximately 1.5 alpha-particle traversals per cell nucleus. The frequencies of chromosome and chromatid breaks were linear with dose, but the number declined rapidly with time. These data are discussed in relation to published alpha-particle beam studies and the potential use of 211At in radionuclide therapy.     

Characterization of hydroxyl radical-induced damage after sparsely and densely ionizing irradiation

The extent of hydroxyl radical mediated cell inactivation was measured for a variety of particle beams ranging from 8.5 Me V/u neon ions to 570 Me V/u argon ions. In general, the fraction of the total radiosensitivity caused by OH decreases from close to 60 per cent at low ionization density or low linear energy transfer (low LET) to close to 25 per cent at high LET for aerobically irradiated mammalian cells. The extent of OH induced cell lethality can be explained in terms of LET infinity only for low energy or low atomic number particles where fragmentations and complicated track structures do not contaminate the characteristic particle LET. For example, at a calculated LET infinity of 100 ke V/micron, the OH mediated fraction of the total radiation damage is about 25 per cent for low energy carbon but close to 40 per cent for high energy carbon ions. For low energy charged nuclei of approximately the same energy, as the 5.4-13.4 MeV/u He, Li, C and Ne ions in this report, there is a predictable diminution of the OH mediated effect with increasing LET infinity; however, the biological effect cannot be predicted accurately from calculated LET infinity values for high energy particle irradiation, nor indeed from a variety of low energy charged particles of quite different energies (incident velocities). This illustrates the unsuitability of using LET as a unifying parameter, except under specific circumstances. As more is learned about the energy deposition for energized charged particles in terms of track structure (core and penumbra), it may be possible to characterize the radiobiological data with a better physical parameter than LET infinity.     

The relationship between radiation-induced DNA double-strand breaks and cell kill in hamster V79 fibroblasts irradiated with 250 kVp X-rays, 2.3 MeV neutrons or 238Pu alpha-particles

Using the neutral filter elution technique, the induction of DNA double-strand breaks (dsb) has been measured in 250 kVp X-irradiated V79-379A Chinese hamster cells irradiated under air or nitrogen. The dose-effect curves for induced dsb were curvilinear, mirroring cell survival curves, such that there was an approximately linear relationship between induced dsb and lethal lesions (-In (cell survival)) which was independent of oxygen. With cells irradiated with 2.3 MeV neutrons or 238Pu alpha-particles the correlations between lethal events and dsb, although also approximately linear, do not match those for X-rays. With neutrons there is approximately a 2.5-fold reduction in the level of dsb induction per lethal event. Thus either the apparently linear relationships found are spurious, and there is no general correlation between induced dsb and lethal effect, or there are qualitative differences between neutron, alpha-particle and X-ray induced dsb that give them differing probabilities of cell kill.     

Caffeine-mediated release of alpha-radiation-induced G2 arrest increases the yield of chromosome aberrations

Severe and partly irreversible G2 arrest caused by americium-241 alpha-particles in Chinese hamster V79 cells acted as a competing process to the yield of detectable aberrant mitoses at metaphase. With increasing dose of alpha-radiation an increasing fraction of cells was irreversibly arrested in G2 with the consequence of interphase death before the first post-irradiation mitosis. This irreversible G2 arrest (demonstrated by flow cytofluorometry and mitotic indices) could be overcome by adding caffeine 8 hours after irradiation, the time point of maximum G2 arrest (80-90 per cent of all cells). Within 3.5 hours the number of aberrant mitoses increased by this treatment from 54 to 96 per cent and from 65 to 99.9 per cent for doses of 1.75 and 4.38 Gy of alpha-particles, respectively. The aberration frequency per mitotic cell, scored as chromatid and isochromatid breaks, rings, interchanges and dicentrics increased by a factor of about 3 after releasing G2 arrested cells. The frequency distribution of aberrations per cell revealed that, after 4.38 Gy, 58 per cent of the formerly G2-arrested cells had more than five aberrations per cell compared to only 8 per cent without the interaction of caffeine.     

Biophysical analysis of the dose-dependent overdispersion and the restricted linear energy transfer dependence expressed in dicentric chromosome data from alpha-irradiated human lymphocytes

Experimental data for the induction of dicentric chromosomes in phytohemagglutinin (PHA)-stimulated human T lymphocytes by ²⁴¹Am alpha-particles obtained by Schmid et al. have been analyzed in the light of biophysical theory. As usual in experiments with alpha-particles, the relative variance of the intercellular distribution of the number of aberrations per cell exceeds unity, and the multiplicity of the aberrations per particle traversal through the cell is understood as the basic effect causing this overdispersion. However, the clearly expressed dose dependence of the relative variance differs from the dose-independent relative variance predicted by the multiplicity effect alone. Since such dose dependence is often observed in experiments with alpha-particles, protons, and high-energy neutrons, the interpretation of the overdispersion needs to be supplemented. In a new, more general statistical model, the distribution function of the number of aberrations is interpreted as resulting from the convolution of a Poisson distribution for the spontaneous aberrations with the overdispersed distributions for the aberrations caused by intratrack or intertrack lesion interaction, and the fluctuation of the cross-sectional area of the cellular chromatin must also be considered. Using a suitable mathematical formulation of the resulting dose-dependent overdispersion, the mean number λ ₁ of the aberrations produced by a single particle traversal through the cell nucleus and the mean number λ ₂ of the aberrations per pairwise approach between two alpha-particle tracks could be estimated. Coefficient α of the dose-proportional yield component, when compared between ²⁴¹Am alpha-particle irradiation and ¹³⁷Cs gamma-ray exposure, is found to increase approximately in proportion to dose-mean restricted linear energy transfer, which indicates an underlying pairwise molecular lesion interaction on the nanometer scale. Received: 17 December 1996 / Accepted in revised form: 20 April 1997     

Lethal and mutagenic effect of the XeCl laser on Escherichia coli

The survival rate and reversions to tryptophan-independence of Escherichia coli after XeCl laser irradiation (lambda = 308 nm) within the dose range from 10(3) to 10(5) J/m2 have been studied to show that LD37 is 10(4) J/m2, the survival rate at a maximum dose of 10(5)J/m2 is 1 per cent, and the number of mutants per 10(6) cells survived is 100.     

Dose-rate effects of 8-methoxypsoralen plus 365-NM irradiation on cell killing in Saccharomyces cerevisiae.

Tow types of dose-rate effect that alter the survival response of haploid yeast cells to 8-methoxypsoralen (8-MOP) plus treatment with irradiation at 365 nm were studied. (1) When the concentration of 8-MOP was varied between 9.2 X 10(-5) and 2.3 X 10(-8) M and the dose rate of 365-nm irradiation kept constant, the efficiency of the irradiation for killing increased relatively to that of 8-MOP whe the concentration of 8-MOP decreased. This indicated that there was no strict reciprocity between radiation dose and concentration of drug. (2) When the dose rate of radiation was varied between 0.66 X 10(3) and 108 X 10(3) J m-2 h-1 and the concentration of 8-MOP was kept constant, the survival of wild-type cells increased strikingly at low dose rates of radiation as compared with high dose rates. Cells responded more to changes at low dose rates than to equal changes a high dose rates. The high resistance of wild-type cells to 8-MOP plus radiation delivered at low dose rates absent from rad 1-3 cells defective in excision-repair. This suggests that the dose-rate effect seen in wild-type cells depended at least in part on an active excision-repair function. At low dose rates of radiation, the shoulder of the survival curve for rad1-3 cells, i.e. the ability to accumulate sub-lethal damage, was increased by a factor of about 2 when compared with that seen at a high dose rate. Thus it is likely that at low dose rates a repair function other than excision-resynthesis may operate in rad1-3 cells.     

Kinetics of uptake, retention, and radiotoxicity of 125IUdR in mammalian cells: implications of localized energy deposition by Auger processes

The kinetics of uptake, retention, and radiotoxicity of 125IUdR have been studied in proliferating mammalian cells in culture. The radioactivity incorporated into the DNA is directly proportional to the duration of incubation and to the extracellular concentration of 125I. The rate of proliferation of cells is related to the intracellular radioactive concentration and is markedly reduced at medium concentrations greater than or equal to 0.1 mu Ci/ml. At 37% survival the high LET type cell survival curve is characterized by an uptake of 0.035 pCi/cell, and the cumulated mean lethal dose to the cell nucleus is about 80 rad compared to 580 rad of X-ray dose for this cell line. The strong cytocidal effects of the decay of 125I correlate with localized irradiation of the DNA by the low energy Auger electrons.     

The RBE-LET relationship for rodent intestinal crypt cell survival, testes weight loss, and multicellular spheroid cell survival after heavy-ion irradiation.

This report presents data for survival of mouse intestinal crypt cells, mouse testes weight loss as an indicator of survival of spermatogonial stem cells, and survival of rat 9L spheroid cells after irradiation in the plateau region of unmodified particle beams ranging in mass from 4He to 139La. The LET values range from 1.6 to 953 keV/microns. These studies examine the RBE-LET relationship for two normal tissues and for an in vitro tissue model, multicellular spheroids. When the RBE values are plotted as a function of LET, the resulting curve is characterized by a region in which RBE increases with LET, a peak RBE at an LET value of 100 keV/microns, and a region of decreasing RBE at LETs greater than 100 keV/microns. Inactivation cross sections (sigma) for these three biological systems have been calculated from the exponential terminal slope of the dose-response relationship for each ion. For this determination the dose is expressed as particle fluence and the parameter sigma indicates effect per particle. A plot of sigma versus LET shows that the curve for testes weight loss is shifted to the left, indicating greater radiosensitivity at lower LETs than for crypt cell and spheroid cell survival. The curves for cross section versus LET for all three model systems show similar characteristics with a relatively linear portion below 100 keV/microns and a region of lessened slope in the LET range above 100 keV/microns for testes and spheroids. The data indicate that the effectiveness per particle increases as a function of LET and, to a limited extent, Z, at LET values greater than 100 keV/microns. Previously published results for spread Bragg peaks are also summarized, and they suggest that RBE is dependent on both the LET and the Z of the particle.     

Immediate localized CDKN1A (p21) radiation response after damage produced by heavy-ion tracks

Using confocal microscopy on immunofluorescence-stained cells, we have investigated the response of CDKN1A (p21), one of the key proteins involved in the DNA damage response pathway, after irradiation with accelerated lead or chromium ions. Each traversal of an accelerated ion leads to the formation of a single, bright focus of the CDKN1A protein in the nuclei of human fibroblasts within 2 min after irradiation at 4 degrees C. This immediate, localized CDKN1A response is specific for particle irradiation with a high linear energy transfer (LET), whereas X irradiation, after a period of induction, yields a diffusely spread pattern, in line with the differences in the microscopic dose deposition pattern of both radiation types. The particle-induced CDKN1A foci persist for several hours until they become diffuse and vanish. These findings suggest that CDKN1A accumulates at the sites of primary DNA damage, possibly mediated by the interaction with proteins involved in DNA repair. Here, for the first time, an immediate biological response confined to the radial extension of low-energy particle tracks ( approximately 1 micrometer) is directly visualized and correlated to ion traversals. This indicates that particle irradiation represents an ideal tool to study the processing of biological damage induced in defined subnuclear regions.     

Calculation of boron neutron capture cell inactivation in vitro based on particle track structure and x-ray sensitivity

The Monte-Carlo technique was used to perform quantitative microdosimetric model calculations of cell survival after boron neutron capture irradiations in vitro. The high energy ⁷Li and alpha-particles resulting from the neutron capture reaction ¹⁰B (n,α)⁷Li are of short range and are highly damaging to cells. The biophysical model of the Monte-Carlo calculations is based on the track structure of these α-particles and ⁷Li-ions and the x-ray sensitivity of the irradiated cells. The biological effect of these particles can be determined if the lethal effect of local doses deposited in very small fractional volumes of the cell nucleus is known. This lethal effect can be deduced from experimental data of cell survival after x-ray irradiation assuming a Poisson distribution for lethal events. The input data used in a PC-based computer program are the radial dose distribution inside the track of the released particles, cell survival after x-ray irradiation, geometry of the tumor cells, subcellular ¹⁰B concentration, and thermal neutron fluence. The basic concept of this Monte-Carlo computer model is demonstrated. Validations of computer calculations are presented by comparing them with experimental data on cell survival.     

Evaluation of the expression of damage of the DNA-structural complex of rat thymocytes based on kinetics of alkaline lysis following irradiation

Rotary viscosimeters were used to study the postirradiation destruction of the DNA-structural complex (DSC) of rat thymocyte nuclei exhibited by a change in alkaline denaturation of DSC upon lysis. The S area, limited by the characteristic viscosity values obtained during alkaline lysis of thymocyte nuclei, was used as a characteristic of DSC. Immediately after irradiation the S area changed up to 81-84 per cent at 0.5-1.5 Gy and up to 56-44 per cent at 2-10 Gy. 6 to 24 h following irradiation a change in the profile of alkaline denaturation of DSC was a function of dose and dropped from 100 down to 11 per cent at doses of 0 to 10 Gy. After 2-3 days, the changes in S were also observed but they were not a strict function of dose and were the same with the values obtained immediately after irradiation.     

Enhanced retention of the alpha-particle-emitting daughters of Actinium-225 by liposome carriers

Targeted alpha-particle emitters hold great promise as therapeutics for micrometastatic disease. Because of their high energy deposition and short range, tumor targeted alpha-particles can result in high cancer-cell killing with minimal normal-tissue irradiation. Actinium-225 is a potential generator for alpha-particle therapy: it decays with a 10-day half-life and generates three alpha-particle-emitting daughters. Retention of (225)Ac daughters at the target increases efficacy; escape and distribution throughout the body increases toxicity. During circulation, molecular carriers conjugated to (225)Ac cannot retain any of the daughters. We previously proposed liposomal encapsulation of (225)Ac to retain the daughters, whose retention was shown to be liposome-size dependent. However, daughter retention was lower than expected: 22% of theoretical maximum decreasing to 14%, partially due to the binding of (225)Ac to the phospholipid membrane. In this study, Multivesicular liposomes (MUVELs) composed of different phospholipids were developed to increase daughter retention. MUVELs are large liposomes with entrapped smaller lipid-vesicles containing (225)Ac. PEGylated MUVELs stably retained over time 98% of encapsulated (225)Ac. Retention of (213)Bi, the last daughter, was 31% of the theoretical maximum retention of (213)Bi for the liposome sizes studied. MUVELs were conjugated to an anti-HER2/neu antibody (immunolabeled MUVELs) and were evaluated in vitro with SKOV3-NMP2 ovarian cancer cells, exhibiting significant cellular internalization (83%). This work demonstrates that immunolabeled MUVELs might be able to deliver higher fractions of generated alpha-particles per targeted (225)Ac compared to the relative fractions of alpha-particles delivered by (225)Ac-labeled molecular carriers.     

Elevated sodium chloride concentrations enhance the bystander effects induced by low dose alpha-particle irradiation

Previous studies have shown that high NaCl can be genotoxic, either alone or combined with irradiation. However, little is known about the relationship between environmental NaCl at elevated conditions and radiation-induced bystander effects (RIBE). RIBE, which has been considered as non-targeted bystander responses, has been demonstrated to occur widely in various cell lines. In the present study, RIBE under the elevated NaCl culture condition was assessed in AG 1522 cells by both the induction of gamma-H2AX, a reliable marker of DNA double-strand break (DSB) for the early process (<1h post irradiation), and the generation of micronuclei (MN), a sensitive marker for relative long process of RIBE. Our results showed that in the absence of irradiation, NaCl at elevated concentration such as 8.0, 9.0 and 10.0g/L did not significantly increase the frequency of gamma-H2AX foci-positive cells and the number of foci per positive cell comparing with that NaCl at a normal concentration (6.8g/L). However, with 0.2cGy alpha-particle irradiation, the induced fraction of gamma-H2AX foci-positive cells and the number of induced gamma-H2AX foci per positive cell were significantly increased in both irradiated and adjacent non-irradiated regions. Similarly, the induction of MN by 0.2cGy alpha-particle irradiation also increased with the elevated NaCl concentrations. With N(G)-methyl-l-arginine, an inhibitor of nitric oxide synthase, the induced fraction of foci-positive cells was effectively inhibited both in 0.2cGy alpha-particle irradiated and adjacent non-irradiated regions under either normal or elevated NaCl conditions. These results suggested that the cultures with elevated NaCl medium magnified the damage effects induced by the low dose alpha-particle irradiation and nitric oxide generated by irradiation was also very important in this process.