J.T. Lett

Effects of Heavy Ions on Rabbit Tissues: Cataractogenesis

As part of an investigation of the responses of optic and proximate tissues to heavy-ion irradiation, the lenses of New Zealand white rabbits were exposed to the Bragg plateau regions of 530 MeV/amu Ar ions and 365 MeV/amu Ne ions and also to 60Co gamma-photons. The linear energy transfers (LET infinity s) for the radiations were 90 +/- 5, 35 +/- 3, and 0.3 keV/micrometer, respectively. After irradiation, lenticular opacities were monitored through their incipient and/or clinical stages (less than or equal to 5 years) by slit-lamp biomicroscopy and scored with subjective, but well-defined, indices. Cataractogenesis, which progressed according to the model proposed by Rubin and Casarett (1968), was modified by radiation quality in the following ways. (1) The rate of development of the early (acute) stage increased with the LET infinity of the incident radiation; (2) at the intermediate (plateau) stage, the values for the relative biological effectiveness (r.b.e.) of the heavy ions were similar to those reported for proliferating cells in culture; (3) for a given intermediate level, the onset of late cataractogenesis occurred earlier the higher the LET affinity of the radiation involved. As with alopecia, the r.b.e.s for cataractogenesis varied with post-irradiation time.

Late cataractogenesis in rhesus monkeys irradiated with protons and radiogenic cataract in other species

Rhesus monkeys (Macaca mulatta) which were irradiated at ca. 2 years of age with acute doses (less than or equal to 5 Gy) of protons (32-2300 MeV) are exhibiting the late progressive phase of radiation cataractogenesis 20-24 years after exposure, the period during which we have been monitoring the sequelae of irradiation of the lens. The median life span of the primate is approximately 24 years. Analogous late ocular changes also occur in a similar period of the lifetimes of New Zealand White (NZW) rabbits (Oryctolagus cuniculus) exposed at 8-10 weeks of age to 460-MeV 56Fe ions. In this experiment, which has been in progress for ca. 6 years, we are following the development of radiation-induced lenticular opacification (cataractogenic profiles) throughout the life span. The median life span of the lagomorph is 5-7 years. Cataractogenic profiles for NZW rabbits irradiated with 20Ne and 40Ar ions and 60Co gamma photons were obtained previously. Reference is also made to measurements of the cataractogenic profiles of a short-lived rodent, the Fischer 344 rat (Rattus norvegicus) during the first year after exposure at 8-10 weeks of age to spread-Bragg-peak protons of 55 MeV nominal energy. The median life span of the rodent is reported to be 2-3 years.

Cellular and tissue responses to heavy ions: Basic considerations

SummaryResponses of the S/S variant of the L5178Y murine leukemic lymphoblast, the photoreceptor cell of the rabbit retina and the lenticular epithelium of the rabbit to heavy ions (20Ne,28Si,40Ar and56Fe) are described and discussed primarily from the standpoint of the need for a comprehensive theory of cellular radiosensitivity from which a general theory of tissue radiosensitivity can be constructed.The radiation responses of the very radiosensitive, repair-deficient S/S variant during the G1- and early S phases of the cell cycle were found to be unlike those of normally radioresistant cells in culture: the relative biological effectiveness (RBE) did not increase with the linear energy transfer (LET∞) of the incident radiation. Such behavior could be anticipated for a cell which is lacking the repair system that operates in other (normal) cells when they are exposed to ionizing radiations in the G1 phase of the cell cycle. The S/S variant does exhibit a peak of radioresistance to X-photons mid-G1 + 8 h into the cell cycle, however, and as the LET∞ was increased, the repair capacity responsible for that radioresistance was reduced progressively.Sensory cells (photoreceptors) in the retina of the New Zealand white (NZW) rabbit are very radioresistant to ionizing radiations, and several years elapsed after localized exposure (e.g., 5–10 Gy) to heavy ions (20Ne,40Ar) before photoreceptor cells were lost from the retina. During the first few weeks after such irradiations, damage to DNA in the photoreceptor cells was repaired to a point where it could not be demonstrated by reorienting gradient sedimentation under alkaline conditions, a technique that can detect DNA damage produced by <0.1 Gy of X-photons. Restitution of DNA structure was not permanent, however, and months or years later, butbefore loss of photoreceptor cells from the retina could be detected, progressive deterioration of the DNA structure began.

Cataractogenesis from high-LET radiation and the Casarett model.

Space radiations, especially heavy ions, constitute significant hazards to astronauts. These hazards will increase as space missions lengthen. Moreover, the dangers to astronauts will be enhanced by the persistence, or even the progression, of biological damage throughout their subsequent life spans. To assist in the assessment of risks to astronauts, we are investigating the long-term effects of heavy ions on specific animal tissues. In one study, the eyes of rabbits of various ages were exposed to a single dose of Bragg plateau 20Ne ions (LET infinity approximately equals 30 keV/micrometer). The development of cataracts has shown a pronounced age-related response during the first year after irradiation, and will be followed for two more years. In other studies, mice were exposed to single or fractionated doses of 12C ions (4-cm spread-out Bragg peak; dose-averaged LET infinity = 70-80 keV/micrometer) or 60Co gamma-photons (LET infinity = 0.3 keV/micrometer). Measurements of the frequency of posterior lens opacification have shown that the tissue sparing observed with dose fractionation of gamma-photons was absent when 12C-ion doses were fractionated. Development of posterior lens cataracts was also followed for long periods (up to 21 months) in mice exposed to single doses of Bragg plateau HZE particles (40Ar, 20Ne and 12C ions: LET infinity approximately equals 100, 30 and 10 keV/micrometer, respectively) or 225 kVp X-rays. Based on average cataract levels at the different observation times, the RBEs (RBE = relative biological effectiveness) for the ions were circa 5, 3 and 1-2, respectively, over the range of doses used (0.05-0.9 Gy). Investigations of cataractogenesis are useful for exploring the model of radiation damage proposed by Casarett and by Rubin and Casarett with a tissue not connected directly to the vasculature.

Deficient repair and degradation of DNA in X-irradiated L5178Y S/S cells: cell-cycle and temperature dependence

The rejoining of DNA strand breaks induced by X rays in the radiosensitive S/S variant of the L5178Y murine leukemic lymphoblast has been studied by alkaline-EDTA-sucrose sedimentation using swinging-bucket and zonal rotors. After irradiation, incubation resulted in an increase in DNA size, but the DNA structures were not restored in all cells, even when the x-ray dose was only 50 rad. Subsequently, 10 to 20 h after irradiation, heavily degraded DNA began to appear. When cells were irradiated at different parts of the cycle, the extent of DNA degradation varied in a fashion similar to survival: Least DNA degradation was found after irradiation at the most radioresistant stage (G/sub 1/ + 8 h), and most DNA degradation occurred after irradiation at the radiosensitive stage (G/sub 1/). Changes in cell survival caused by postirradiation hypothermia were also reflected in the extent of DNA degradation. Populations of G/sub 1/ cells, which show marked increases in survival after postirradiation hypothermic exposure, exhibited a lower level of DNA degradation, whereas populations of G/sub 1/ + 8 h cells, whose survival is affected little by postirradiation hypothermia, showed limited changes in DNA degradation. The onset of degradation was delayed by hypothermia in all cases.

Correlation of survival with the restoration of DNA structure in x-irradiated L5178Y S/S cells

GOLDIN, E. M., Cox, A. B., AND LETT, J. T. Correlation of Survival with the Restoration of DNA Structure in X-Irradiated L5178Y S/S Cells. Radiat. Res. 83, 668-676 (1980). After exponentially growing populations of the radiosensitive S/S variant of the L5178Y murine leukemic lymphoblast were exposed to low doses of X rays (-100 rad), the majority of the radiation-induced strand breaks in the DNA appeared to be rejoined within 5-10 hr. Subsequently, the DNA resolved slowly into a component that finally degraded into small pieces and a component that regained the size of the DNA in unirradiated cells long after irradiation (days). The fraction of the latter component correlated directly with the fraction of surviving cells. A similar correlation was found when semisynchronous populations of the S/S variant were X-irradiated (?200 rad) during the radioresistant phase of the cell cycle.

Cataractogenic potential of ionizing radiations in animal models that simulate man

Aspects of experiments on radiation-induced lenticular opacification during the life spans of two animal models, the New Zealand white rabbit and the rhesus monkey, are compared and contrasted with published results from a life span study of another animal model, the beagle dog, and the most recent data from the ongoing study of the survivors from radiation exposure at Hiroshima and Nagasaki. An important connection among the three animal studies is that all the measurements of cataract indices were made by one of the authors (A.C.L.), so variation form personal subjectivity was reduced to a minimum. The primary objective of the rabbit experiments (radiations involved: 56Fe, 40Ar and 20Ne ions and 60Co gamma photons) is an evaluation of hazards to astronauts from galactic particulate radiations. An analogous evaluation of hazards from solar flares during space flight is being made with monkeys exposed to 32, 55, 138 and 400 MeV protons. Conclusions are drawn about the proper use of animal models to simulate radiation responses in man and the levels of radiation-induced lenticular opacification that pose risks to man in space.

Effects of heavy ions on rabbit tissues: Induction of DNA strand breaks in retinal photoreceptor cells by high doses of radiation

Excised retinas from New Zealand white (NZW) rabbits were irradiated at 0° C with 9–260 Gy (depending on the type of radiation) of 300 kVp X-rays, or the first 5 cm (range: ∼ 14 cm in water) of 365 MeV/u Ne ions or 530 MeV/u Ar ions (LET∞s : ∼ 1, 35 ± 3 and 90 ± 5 keV/µm, respectively). Other positions (LET∞s) in the Ne-ion beam (Bragg curve) were employed in more limited experiments. The retinas were frozen and stored in liquid nitrogen until analysis.Total strand breakage in the DNA of retinal photoreceptor (sensory) cells was determined from sedimentation profiles obtained by velocity sedimentation through reoriented alkaline sucrose gradients under conditions free from anomalies related to rotor speed. For the radiation doses employed: the reciprocal of the number average molecular weight,Mn, was related linearly to dose for each radiation quality and extrapolation to zero dose in each case gave positive intercepts for which the mean unitradiated molecular weight,M0, was 6.1 ± 1.0 × 108 daltons; the efficiencies of total strand breakage for the different radiations were 50 ± 3, 110 ± 2 and 240±6 eV/strand break, respectively. For the heavy ions, accurate analogous calculations for other positions in the Bragg curves were precluded by beam degeneration due to fragmentation of the primary particles, etc.Overall, the experimental results support the concept that ionizing radiations damage cellular DNA by two general processes. One process causes localized damage, which under our experimental conditions is revealed as strand breaks and/or alkali-labile bonds in regions between molecules of sizecirca 109 daltons (subunits); the other causes essentially random damage. Base damage caused by either process would not have been delineated in our experiments.

Late effects from particulate radiations in primate and rabbit tissues

Optic tissues in groups of New Zealand white rabbits were irradiated locally at different stages throughout the median life span of the species with a single dose (9 Gy) of 425 MeV/amu Ne ions (LET infinity approximately 30 keV/micrometer) and then inspected routinely for the progression of radiation cataracts. The level of early cataracts was found to be highest in the youngest group of animals irradiated (8 weeks old), but both the onset of late cataracts and loss of vision occurred earlier when animals were irradiated during the second half of the median life span. This age response can have serious implications in terms of space radiation hazards to man. Rhesus monkeys that had been subjected to whole-body skin irradiation (2.8 and 5.6 Gy) by 32 MeV protons (range in tissue approximately 1 cm) some twenty years previously were analysed for radiation damage by the propagation of skin fibroblasts in primary cultures. Such propagation from skin biopsies in MEM-alpha medium (serial cultivation) or in supplemented Hams F-10 medium (cultivation without dilution) revealed late damage in the stem (precursor) cells of the skins of the animals. The proton fluxes employed in this experiment are representative of those occurring in major solar flares.

Responses of Synchronous L5178Y S/S Cells to Heavy Ions and Their Significance for Radiobiological Theory

Synchronous suspensions of the radiosensitive S/S variant of the L5178Y murine leukaemic lymphoblast at different positions in the cell cycle were exposed aerobically to segments of heavy-ion beams (20Ne, 28Si, 40Ar, 56Fe and 93Nb) in the Bragg plateau regions of energy deposition. The incident energies of the ion beams were in the range of 460 ± 95 MeV u-1, and the calculated values of linear energy transfer (LET∞) for the primary nuclei in the irradiated samples were 33 ± 3, 60 ± 3, 95 ± 5, 213 ± 21 and 478 ± 36 keV μm-1, respectively; 280 kVp X-rays were used as the baseline radiation. Generally, the maxima or inflections in relations between relative biological effectiveness (RBE) and LET∞ were dependent upon the cycle position at which the cells were irradiated. Certain of those relations were influenced by post-irradiation hypothermia. Irradiation in the cell cycle at mid -G1 to mid-G1 +3 h, henceforth called G1 to G1 + 3 h, resulted in survival curves that were close approximations to simple exponential functions. As the LET∞ was increased, the RBE did not exceed 1.0, and by 478 keV μm-1 it had fallen to 0.39. Although similar behaviour has been reported for inactivation of proteins and certain viruses by ionizing radiations, so far the response of the S/S variant is unique for mammalian cells. The slope of the survival curve for X-photons (D0:0.27 Gy) is reduced in G1 to G1 + 3 h by post-irradiation incubation at hypothermic temperatures and reaches a minimum (D0: 0.51 Gy) at 25 °C. As the LET∞ was increased, however, the extent of hypothermic recovery was reduced progressively and essentially was eliminated at 478 keV μm-1. At the cycle position where the peak of radioresistance to X-photons occurs for S/S cells, G1 + Sh, increases in LET∞ elicited only small increases in RBE (at 10% survival), until a maximum was reached around 200 keV μm-1. At 478 keV μm-1, what little remained of the variation in response through the cell cycle could be attributed to secondary radiations (δ rays) and smaller nuclei produced by fragmentation of the primary ions. Definitions 1. Linear energy transfer (LET∞) is the energy deposited per unit length of track by an ionizing particle and usually is measured in kiloelectron volts per micrometer (in water). 2. Penumbra. Atomic interactions along the track of a heavy ion result in the ejection of electrons with energies sufficient to move beyond the region of dense ionization which constitutes the track core, and so may be considered to form a penumbra of sparsely ionizing radiations around the track core. 3. RBE. The effectiveness of a densely ionizing radiation (heavy ion) compared to a sparsely ionizing radiation, e. g. X- or γ -photons, is measured by the inverse ratio of the doses of each radiation needed to produce a given radiobiological effect, and is known as the relative biological effectiveness (RBE): the usual reference radiation is 250 kVp X-rays. 4. D0 is a measure of the radiosensitivity of a cell as determined from the (limiting) linear slope of the survival curve, and is the dose in Gray (1 Gy ≡ 1 Joule kg-1) required to reduce the survival at a point anywhere in that region of the survival curve to 37% of its value at that point.