Near-total glutathione depletion and age-specific cataracts induced by buthionine sulfoximine in mice.

The specific inhibitor of glutathione biosynthesis, L-buthionine sulfoximine (L-BSO), although relatively nontoxic in adult mice, induces severe glutathione depletion and age-specific pathological changes when repeatedly administered to male suckling mice. Dense cataracts developed when mice aged 9 to 12 days were given a series of injections of L-BSO, despite excellent survival and the absence of other significant long-term effects. By contrast, similar treatment of mice aged 14 to 17 days, although slightly less effective in reducing glutathione levels, resulted frequently in death, hind-leg paralysis, or impaired spermatogenesis, but did not produce cataracts. Administration of L-BSO to preweanling mice provides a novel model system for the induction of cataracts by depletion of lens glutathione and may enable the study of critical functions of glutathione in the lens and other growing tissues during early postnatal development.

Dosimetry for a study of low-dose radiation cataracts among Chernobyl clean-up workers.

A cohort of 8,607 Ukrainian Chernobyl clean-up workers during 1986-1987 was formed to study cataract formation after ionizing radiation exposure. Study eligibility required the availability of sufficient exposure information to permit the reconstruction of doses to the lens of the eye. Eligible groups included civilian workers, such as those who built the "sarcophagus" over the reactor, Chernobyl Nuclear Power Plant Workers, and military reservists who were conscripted for clean-up work. Many of the official doses for workers were estimates, because only a minority wore radiation badges. For 106 military workers, electron paramagnetic resonance (EPR) measurements of extracted teeth were compared with the recorded doses as the basis to adjust the recorded gamma-ray doses and provide estimates of uncertainties. Beta-particle doses to the lens were estimated with an algorithm devised to take into account the nature and location of Chernobyl work, time since the accident, and protective measures taken. A Monte Carlo routine generated 500 random estimates for each individual from the uncertainty distributions of the gamma-ray dose and of the ratio of beta-particle to gamma-ray doses. The geometric mean of the 500 combined beta-particle and gamma-ray dose estimates for each individual was used in the data analyses. The median estimated lens dose for the cohort was 123 mGy, while 4.4% received >500 mGy.

Cataracts among Chernobyl clean-up workers: implications regarding permissible eye exposures.

The eyes of a prospective cohort of 8,607 Chernobyl clean-up workers (liquidators) were assessed for cataract at 12 and 14 years after exposure. The prevalence of strictly age-related cataracts was low, as expected (only 3.9% had nuclear cataracts at either examination), since 90% of the cohort was younger than 55 years of age at first examination. However, posterior subcapsular or cortical cataracts characteristic of radiation exposure were present in 25% of the subjects. The data for Stage 1 cataracts, and specifically for posterior subcapsular cataracts, revealed a significant dose response. When various cataract end points were analyzed for dose thresholds, the confidence intervals all excluded values greater than 700 mGy. Linear-quadratic dose-response models yielded mostly linear associations, with weak evidence of upward curvature. The findings do not support the ICRP 60 risk guideline assumption of a 5-Gy threshold for "detectable opacities" from protracted exposures but rather point to a dose-effect threshold of under 1 Gy. Thus, given that cataract is the dose-limiting ocular pathology in current eye risk guidelines, revision of the allowable exposure of the human visual system to ionizing radiation should be considered.

Mice heterozygous for the ATM gene are more sensitive to both X-ray and heavy ion exposure than are wildtypes.

Previous studies have shown that the eyes of ATM heterozygous mice exposed to low-LET radiation (X-rays) are significantly more susceptible to the development of cataracts than are those of wildtype mice. The findings, as well as others, run counter to the assumption underpinning current radiation safety guidelines, that individuals are all equally sensitive to the biological effects of radiation. A question, highly relevant to human space activities is whether or not, in similar fashion there may exist a genetic predisposition to high-LET radiation damage. Mice haplodeficient for the ATM gene and wildtypes were exposed to 325 mGy of 1 GeV/amu 56Fe ions at the AGS facility of Brookhaven National Laboratory. The fluence was equivalent to 1 ion per lens epithelial cell nuclear area. Controls consisted of irradiated wildtype as well as unirradiated wildtype and heterozygous mice. Prevalence analyses for stage 0.5-3.0 cataracts indicated that not only cataract onset but also progression were accelerated in the mice haplo-deficient for the ATM gene. The data show that heterozygosity for the ATM gene predisposes the eye to the cataractogenic influence of heavy ions and suggest that ATM heterozygotes in the human population may also be radiosensitive. This may have to be considered in the selection of individuals who will be exposed to both HZE particles and low-LET radiation as they may be predisposed to increased late normal tissue damage.

Quantitative comparisons of continuous and pulsed low dose rate regimens in a model late-effect system.

PURPOSE: There is increasing interest and usage of pulsed low dose rate (PDR) brachytherapy, in which a single source is shuttled through the catheters of an implant, typically for about 10 min each hour. This study was designed to compare the late effects produced in various PDR regimens with those from the corresponding continuous low dose rate (CLDR) regimens. METHODS AND MATERIALS: A model late-responding system was used, namely, cataract induction in the rat lens. This system has the advantage of being highly quantifiable. The rats eyes were exposed to a total dose of 15 Gy either continuously over 24 h, or with three different PDR regimens, all with the same total dose and overall time. We addressed three questions: (a) are late effects increased when a CLDR regimen is replaced with 10-min pulses repeated every hour? (b) Are late effects increased if hourly 10-min pulses are replaced with 10-min pulses repeated every 4 h? (c) Are late effects increased if 10-min pulses are replaced with 100-s pulses? RESULTS: We found that the four regimens under test, continuous, 10-min pulses each hour, 10-min pulses every 4 h, and 100-s pulses every hour, showed no significant differences in cataractogenic potential, as estimated with the Wilcoxon-Gehan test. Power tests indicated that the experimental design was adequate to detect relatively small differences in cataractogenicity between regimens. CONCLUSIONS: The equality of late effects from CLDR and PDR in these experiments must imply that sublethal damage repair is quite slow in this model late-responding system, in agreement with trends observed in the clinic for sublethal damage repair of late sequelae. Such trends would suggest that PDR is unlikely to produce significantly worse late effects than the corresponding CLDR regimen, which is in agreement with early clinical data using PDR. Caution, however, is strongly recommended.

The effect of endotoxin-induced intraocular inflammation on the rat lens epithelium.

Intraocular inflammation induced by an intravitreal injection of Shigella endotoxin into the rat eye produces early changes in the number of dividing cells of the lens epithelium and affects the organization of the meridional rows. A depression in mitotic activity in the germinative zone is observed during the first 24 hr after injection. At 48 hr, despite the continued mitotic inhibition in the germinative zone, an increase in cell division occurs in the central zone. By 72 hr, the germinative zone mitosis reappears and exceeds control values, whereas the central zone mitotic activity returns to normal. At that time mitotic figures are found in the transitional zone. Disorganization of the meridional rows is seen as early as 12 hr after injection (the first time period observed) and reaches a peak by 48 hr. During the next 5 days, however, the severity of the disorganization diminishes. By the seventh day the rows appear, for the most part, fully recovered, and the mitotic activity reaches normal or near-normal levels in all regions. The details of these observations and their possible relationship to inflammatory cataracta complicata are discussed.

Accelerated heavy particles and the lens II. Cytopathological changes.

To assess more fully the risk to normal tissue exposed to accelerated heavy particles in the space program and during radiotherapy on earth, the cytopathological effects of a variety of doses of accelerated (570 MeV/amu) Argon (40Ar) ions on the rat lens were investigated. Time-course analyses of lenses exposed to a 1 Gy (100 rad)-dose revealed that the effects of the particles on mitotic index, nuclear fragmentation, and meridional row (MR) cytoarchitecture were qualitatively similar to those caused by 185 kVp x-rays. The effects of dose on the lens epithelium was also examined at 67 wk post-irradiation. The mitotic index returned to normal levels by that time; however, the biological effectiveness (RBE) of 40Ar relative to x-rays, in causing MR disorganization, increased with decreasing dose and closely resembled the RBE for cataractogenesis. The RBE data are consonant with the view that radiation cataracts are the result of damage to the lens epithelial population, which is later expressed as aberrant differentiation during fibergenesis.

Accelerated heavy particles and the lens. VII: The cataractogenic potential of 450 MeV/amu iron ions.

PURPOSE: To determine the cataractogenic potential dose of high velocity iron ions as a fixation of dose administered singly or fractionated. The dose is critical to risk assessment and to theories of radiation action and cataractogenesis. METHODS: Twenty-eight-day-old rats were examined by slit-lamp biomicroscopy on a weekly-bi-weekly basis for more than 2 yr after radiation exposure. For the acute exposure study doses of 1, 2, 5, 25, and 50 cGy were evaluated. The fractionated regimens involved total doses of 2, 25, and 50 cGy. The reference radiation consisted of 50, 100, 200, or 700 cGy of 250 kilovolt (peak) x-rays. RESULTS: In accordance with previous findings in the rat using 570 MeV/amu 40Ar ions, the relative biologic effectiveness increased rapidly with decreasing dose, reaching values as high as 100. Unlike 40Ar ions, fractionation of the 56Fe doses did not produce a consistent enhancement at any of the doses examined. CONCLUSIONS: The data support the previous findings of a high cataractogenic potential for high linear energy transfer (LET) radiation. The effectiveness for the production of cataracts increases with decreasing dose relative to x-rays and is independent of dose protraction. Although the present study did not reveal a consistent enhancement of effect when the ions were applied in fractions, the results are consistent with at least one theory of the inverse dose-rate effect observed for high-LET radiation.

Rapid deterioration of lens fibers in GSH-depleted mouse pups.

Lens opacities developed within 48-72 hr in mice that received a series of eight injections of L-buthionine sulfoximine, a specific inhibitor of glutathione (GSH) biosynthesis, on postnatal days 8 and 9. Initial histopathologic features consisted of swollen fibers in the central anterior cortex and displacement of cell nuclei from the bow region to the posterior cortex. These aberrations suggest early fiber cell membrane and/or cytoskeletal dysfunction. A massive wave of fiber cell lysis then engulfed the entire lens cortex and nucleus within 24 hr and left only epithelial cells intact, suggesting a concerted mechanism of cataract generation. The acellular core of the mature cataract seen on postnatal day 16 consisted of a granular matrix in which pycnotic and fragmented cell nuclei were located near the terminus of the lens epithelium. The epithelium displayed increased mitotic activity and meridional row disorganization. During the next two weeks, rapid regeneration of lens fibers, displacement of the acellular necrotic cytoplasm to the center and rear of the lens, and vacuole formation were observed. As new fibers were differentiated, partial regeneration of the bow was seen. However, the cataract was irreversible.

The effects of nuclear magnetic resonance imaging on ocular tissues.

Nuclear magnetic resonance, an imaging technique with great promise for detecting cerebral abnormalities, was studied to determine its possible deleterious effects on the mammalian eye. Young (3.5-week-old) Columbia-Sherman rats were exposed simultaneously to a constant magnetic field of 2.7 tesla and radio frequency pulses of 29 MHz at 800-ms intervals for six hours at field strengths representing the maximum used in a clinical setting. The six-hour exposure is many times greater than the four to six minutes currently employed in most diagnostic protocols. The animals were examined by slit-lamp biomicroscopy and ophthalmoscopy at regular intervals. Autoradiograms of lenses from animals injected with tritiated thymidine prior to exposure did not reveal any disturbances in cell-cycle kinetics. Eyes from rats not previously injected with the isotope were processed for cytopathologic analysis at various intervals. A two-year follow-up has indicated that at both the slit-lamp biomicroscopic and the light microscopic levels, there were no discernable effects on the rat eye.

Lens epithelium and radiation cataract. I. Preliminary studies.

The migration of epithelial cells during fibergenesis in the lenses of young rats was determined by tritiated thymidine autoradiography. Cells initially labeled in the germinative zone appeared in the lens bow within seven days. Irradiation with either 600 or 2,400 rads of x-rays did not greatly alter the migration rate, but it did interfere with normal differentiation and proper fiber deposition, as manifested by a displacement posteriorly of the lens bow nuclei and an accumulation of abnormal nucleated fibers in the posterior part of the cortex. The presence of labeled cells in those regions prior to the onset of opacification is consistent with the possibility of direct epithelial cell involvement in radiation cataractogenesis.

Neutron effects on the lens.

Few in vivo systems have received the investigative attention or have provided the grist for our understanding of basic radiobiological principles as have the lens and the cataract. From Roentgen's time the lens has been recognized as a "biological dosimeter" for gauging radiation response. Its advantages range from its in vivo status to its qualification as an integrated tissue. From the time of the Hiroshima/Nagasaki experience, there has been some urgency in attempting to understand the breadth of neutron-radiation effects on humans. The major obstacle has been our understanding of the doses which were received by the individuals who express the damage. The majority of the work has been derived from experimental animals and findings related to photons: X and gamma rays. Cataractogenesis provides insights in terms of not only ocular radiopathy but also the basic mechanisms of the action of radiation. Often referred to as the "classic" nonstochastic radiation effect, it is becoming increasingly clear that the suggestion of a threshold reflects the limitations imposed on expression by the life span. Thus the primary damage which appears to be a somatic mutation is fully stochastic. This being the case, it is not surprising that, as is the case for simpler systems, the RBEs for cataracts following neutron exposure are significantly higher than for X rays, and that there is evidence for an inverse dose-rate effect in their production. This presentation focuses on these data and on the merits of the lens for the assessment of neutron effects on humans, the existing data for known dose levels in the human population, and the confounding issues associated with extrapolation from experimental work. Data from Western sources as well as those from the USSR are presented.

Effect of accelerated iron ions on the retina.

The eyes of rats were exposed to doses of 0.1 and 2.5 Gy of 450-MeV/amu 56Fe particles (LET approximately 195 keV/microns). The beam axes were oriented perpendicular to the central retina of the animals. Retinas were harvested immediately (less than 10 min), 24 h, 15 days, 136 days, and 186 days after the experiment. The retinas of animals of equivalent ages were sampled at the same intervals. By Day 15, the spatial densities of the pigment epithelial, photoreceptor, and bipolar cells in retinas irradiated with 2.5 Gy were 15 to 20% lower than those of the controls. The cellular density of the pigment epithelium returned to the control level by Day 186 while photoreceptor and bipolar cell numbers remained depressed. One and fifteen days after irradiation, the choroidal vessels showed signs of radiation damage. Exposure to 0.1 Gy did not affect the cellular density within the retina at the interval examined (186 days). None of the retinas showed evidence of track-specific injury that could be interpreted as microlesions or tunnel lesions.

Accelerated heavy particles and the lens. VIII. Comparisons between the effects of acute low doses of iron ions (190 keV/microns) and argon ions (88 keV/microns).

The nature of the RBE-LET relationship for radiation-induced effects in vivo is not well known in the high-LET range above about 100 keV/microns. Here, we compare the cataractogenic effects of acute doses of 190 keV/microns iron ions on the eyes of rats with those of 88 keV/microns argon ions. The RBEs of the two radiations cannot be distinguished statistically, both being between 50 and 200 at our lowest dose of 0.01 Gy and decreasing to between 2 and 14 at our highest dose of 0.5 Gy; these values are consistent with results obtained in vivo, both for cataractogenesis and for oncogenesis. For this end point, therefore, the RBE-LET relationship probably varies very slowly between 88 and 190 keV/microns. On the basis of these studies with acute doses of 88 and 190 keV/microns particles, the detailed distribution in LET of the very high-LET galactic cosmic-ray dose to which astronauts in deep space are exposed may not be critical for the prediction of biological hazard. Such a conclusion might simplify the task of high-LET radiation risk estimation in space.

Accelerated heavy particles and the lens. III. Cataract enhancement by dose fractionation.

For a number of biological end points it has been shown that, in contrast to low linear energy transfer (LET) radiation, dose fractionation of high-LET radiation does not result in a reduction in overall effectiveness. Studies were conducted to determine the effect of fractionating the exposures to heavy ion doses on the development of cataracts. Rat eyes were exposed to single doses of 1, 5, and 25 cGy of 570 MeV/amu40Ar ions and to 2, 4, and 10 Gy of 250 kVp X rays. These were compared to unirradiated controls and eyes which were exposed to the same total dose delivered in four fractions over 12 h. While in all cases fractionation of the exposure to X rays produced significant reduction in cataractogenic potential, fractionating doses of 40Ar ions caused a dose- and stage-dependent enhancement in the development of cataracts.

Accelerated heavy ions and the lens. IV. Biomicroscopic and cytopathological analyses of the lenses of mice irradiated with 600 MeV/amu 56Fe ions.

The lenses of mice exposed to 600 MeV/amu iron ions were evaluated by slit-lamp biomicroscopy and cytopathological analyses. The doses ranged from 0.05 to 1.6 Gy, and the lenses were assessed at several intervals postirradiation. Cataract, the development of which is dependent on both time and dose, is significantly more advanced in all of the exposed mice when compared to the unirradiated controls. The great difference between the severity of the cataracts caused by 0.05 Gy (the lowest dose used) and those that developed spontaneously in the control animals is an indication that 0.05 Gy may far exceed the threshold dose for the production of cataracts by accelerated iron ions. Cytopathologically, a similar dose dependence was observed for a number of end points including micronucleation, interphase death, and meridional row disorganization. In addition the exposure to the 56Fe ions produced a long-term effect on the mitotic population and a pronounced "focal" loss of epithelial cytoarchitecture. The microscopic changes support the view that the mechanism of heavy-ion-induced cataractogenesis is the same as that for cataracts caused by low-LET radiation.

The effect of accelerated argon ions on the retina.

It has been postulated that high energy heavy ions cause a unique form of damage in living tissue, which results from the high linear energy transfer of accelerated single particles. We have searched for these single-particle effects, so-called "microlesions," in composite electron micrographs of retinas of rats which had been irradiated with a dose of 1 Gy of 570 MeV/amu argon ions. The calculated rate of energy deposition of the radiation in the retina was about 100 keV/micron and the influence was four particles per 100 micron 2. Different areas of the irradiated retinas which combined would have been expected to be traversed by approximately 2400 particles were examined. We were unable to detect ultrastructural changes in the irradiated retinas distinct from those of controls. The spatial cellular densities of pigment epithelial and photoreceptor cells remained within the normal range when examined at 24 h and at 6 months after irradiation. These findings suggest that the retina is relatively resistant to heavy-ion irradiation and that under the experimental conditions the passage of high energy argon ions does not cause retinal microlesions that can be detected by ultrastructural analysis.

Accelerated heavy particles and the lens. VI. RBE studies at low doses.

We report on the prevalence, hazard, and relative biological effectiveness (RBE) for various stages of lens opacification in rats induced by very low doses of fast argon ions of LET 88 keV/microns, compared to those for X rays. Doses of argon ions from 0.01 to 0.25 Gy were used and RBEs of these ions relative to X rays estimated using a nonparametric technique. At the end of the follow-up period, which encompasses a significant fraction of the animals' lifetime, 90% confidence intervals for the RBE of the argon ions relative to X rays were 4-8 at 0.25 Gy, 10-40 at 0.05 Gy, and 50-100 at 0.01 Gy. Our results are consistent with the point-estimate neutron RBEs in Japanese A-bomb survivors, though broad confidence bounds are present in the Japanese results. If a reasonable extrapolation to higher doses is used, our results are also consistent with data reported earlier at higher doses for argon-ion cataractogenesis in rats, mice, and rabbits. We conclude from these results that at very low doses the RBE for cataractogenesis from HZE particles in space is considerably more than 20, and use of a quality factor of at least 50 would be prudent.

Quantitative assessment of the cataractogenic potential of very low doses of neutrons.

We report on the prevalence and relative biological effectiveness (RBE) for various stages of lens opacification in rats induced by very low doses (2 to 250 mGy) of medium-energy (440 keV) neutrons, compared to those for X rays. Neutron doses were delivered either in a single fraction or in four separate fractions and the irradiated animals were followed for over 100 weeks. At the highest observed dose (250 mGy) and at early observation times, there was evidence of an inverse dose-rate effect; i.e., a fractionated exposure was more potent than a single exposure. Neutron RBEs relative to X rays were estimated using a non-parametric technique. The results were only weakly dependent on time postirradiation. At 30 weeks, for example, 80% confidence intervals for the RBE of acutely delivered neutrons relative to X rays were 8-16 at 250 mGy, 10-20 at 50 mGy, 50-100 at 10 mGy and 250-500 at 2 mGy. The results are consistent with the estimated neutron RBEs in Japanese A-bomb survivors, though broad confidence bounds are present in the Japanese results. Our findings are also consistent with data reported earlier for cataractogenesis induced by heavy ions in rats, mice, and rabbits. We conclude from these results that, at very low doses (<10 mGy), the RBE for neutron-induced cataractogenesis is considerably larger than the RBE of 20 commonly used, and use of a significantly larger value for calculating equivalent dose would be prudent.

Accelerated heavy particles and the lens. I. Cataractogenic potential.

The effect of varying doses of accelerated (570 MeV/ amu ) argon ions on the rat lens is described with detailed observations on the sequence of development of the cataracts, the time-dose relationship, and the analysis of their cataractogenic potential. The relative biological effectiveness (RBE) of the heavy particles for cataract production, compared to low linear energy transfer (LET) radiation (X rays), has been established. These data indicate that, as with neutrons, the RBE increases with decreasing dose and that at a dose of 0.05 Gy an RBE of about 40 was observed.