Dietary modulation of DNA damage in human.

Manipulation of human diet can modulate urinary biomarkers of oxidative DNA base damage (UBODBD), reflecting changes in levels of DNA damage. When dietary composition is maintained but caloric intake is decreased (caloric restriction), UBODBD excretion is suppressed. At isocaloric dietary intake the level of damage depends on diet composition. For diets consisting of foods containing carbohydrates, proteins, and fats but lacking fruits and vegetables, the level of damage is higher than for diets including fruits and vegetables, which are rich in natural antioxidants. Assay of urinary biomarkers is suggested as a potential test for quantitative assessment of the carcinogenic or anticarcinogenic properties of foods, food components, and diets and for individual responses to nutritional regimens.

Biomarkers of OH radical damage in vivo.

Mechanisms of formation of o-tyrosine (o-Tyr) and thymine glycol (TG), the two possible markers of OH radical generation in biosystems and in vivo are described. The o-Tyr measurements require invasive approaches, while TG detection may be accomplished by noninvasive analysis in the urine.

The 8-hydroxyguanine content of isolated mitochondria increases with lipid peroxidation.

When lipid peroxidation was induced in isolated mitochondria there was a marked increase in the 8-hydroxyguanine content of the nucleic acids extracted from these mitochondria. The elevation of 8-hydroxyguanine levels was associated with an extensive alteration of normal electrophoretic mobility of mitochondrial DNA. However, suppression of lipid peroxidation with alpha-tocopherol proportionally attenuated 8-hydroxyguanine production and limited the electrophoretic mobility change of mitochondrial DNA.

Generation of oxy radicals in biosystems.

Many recent lines of evidence indicate that endogenous free radicals contribute to spontaneous mutagenesis through the direct induction of DNA damage. However, the mechanisms underlying this process are not yet fully understood. A brief overview of the knowledge that is currently available is provided here, with emphasis on the generation of oxy radicals in biosystems, the reactions of those radicals with biomolecules, and the induction of oxidative DNA base damage that might lead to mutation.

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 degrees C with 9-260 Gy (depending on the type of radiation) of 300 kVp X-rays, or the first 5 cm (range: approximately 14 cm in water) of 365 MeV/u Ne ions or 530 MeV/u Ar ions (LET infinity's: approximately 1, 35 +/- 3 and 90 +/- 5 keV/micron, respectively). Other positions (LET infinity'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 unirradiated molecular weight, M0, was 6.1 +/- 1.0 X 10(8) 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 size circa 10(9) daltons (subunits); the other causes essentially random damage. Base damage caused by either process would not have been delineated in our experiments.

Characterization of free radical-induced base damage in DNA at biologically relevant levels.

DNA damage induced by oxygen radicals, e.g., hydroxyl radicals generated in living cells either by cellular metabolism or external agents such as ionizing radiations, appears to play an important role in mutagenesis, carcinogenesis, and aging. Elucidation of the chemical nature of such DNA lesions at biologically significant quantities is required for the assessment of their biological consequences and repair. For this purpose, a sensitive method using gas chromatography-mass spectrometry with the selected-ion-monitoring technique (GC-MS/SIM) was developed in the present work. DNA was exposed to hydroxyl radicals and hydrogen atoms produced by ionizing radiation in N2O-saturated aqueous solution. DNA samples were subsequently hydrolyzed with formic acid, trimethylsilylated, and analyzed by GC-MS/SIM. Characteristic ions from previously known mass spectra of DNA base products as their trimethylsilyl derivatives were recorded and the area counts of each ion were integrated. From these acquired data, a partial mass spectrum of each product was generated and then compared with those of authentic materials. This technique permitted the detection and characterization of a large number of free radical-induced based products of DNA, i.e., 5,6-dihydrothymine, 5-hydroxy-5,6-dihydrothymine, 5-hydroxymethyluracil, 5-hydroxyuracil, 5-hydroxycytosine, thymine glycol, 4,6-diamino-5-formamidopyrimidine, 8-hydroxyadenine, 2,6-diamino-4-hydroxy-5-formamidopyrimidine, and 8-hydroxyguanine, simultaneously in a single sample after radiation doses from 0.1 to 10 Gy. Detectable amounts of the base products were found to be as low as approximately 10 fmol per injection.(ABSTRACT TRUNCATED AT 250 WORDS)

Cellular and tissue responses to heavy ions: basic considerations.

Responses 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 and 56Fe) 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 infinity) 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 infinity 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 less than 0.1 Gy of X-photons. Restitution of DNA structure was not permanent, however, and months or years later, but before loss of photoreceptor cells from the retina could be detected, progressive deterioration of the DNA structure began. Age dependencies of late sequelae from densely ionizing radiations are matters of concern both for the therapeutic uses of radiation and the risk/benefit considerations of environmental exposure, especially in outer space. A pilot experiment with a single acute exposure to 20Ne ions has illustrated the need for careful examination of the role of animal age at the time of irradiation in subsequent tissue responses.(ABSTRACT TRUNCATED AT 400 WORDS)

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 Ham's 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.

Further evidence that the survival of irradiated mammalian cells is controlled by temporal processes.

Theories of mammalian cellular radiosensitivity that exclude metabolic modification of radiation damage are untenable (Nagasawa et al., 1980). Evidence supporting that conclusion has been obtained from experiments with a radiosensitive, proliferating cell, the S/S variant of the L5178Y murine leukaemic lymphoblast, and a radioresistant, nondividing cell, the retinal photoreceptor (rod) of the rabbit. When S/S cells (mid-) G1 + 8 h in the cycle, at the peak of radioresistance, were X-irradiated at 37 degrees C and then treated hyperthermically (12 h, 38.7-40.3 degrees C), the survival curve, which has a shoulder at 37 degrees C, changed progressively to the simple exponential obtained for G1 cells. Under conditions where Ne ions (LET = 35 keV microns-1) have a relative biological effectiveness (RBE) of approximately 2 for normally radioresistant cells in vitro and in situ, the RBE for G1 S/S cells was approximately 1. Neon ions (1-50 Gy) caused similar amounts of DNA damage in S/S cells and photoreceptors, but the cellular responses were very different. After 5 Gy, the surviving fraction of asynchronous S/S cells was 10(-5), DNA structures were not restored and by 8 h post-irradiation extensive DNA degradation was evident; in the retina, however, the photoreceptor complement was unchanged for greater than 1 year, DNA structures appeared to be restored and remained so (many months) until late DNA degradation began. These phenomena can be explained satisfactorily only if temporal processes play a significant role in cellular radiosensitivity.

Effects of heavy ions on rabbit tissues: analysis of low levels of DNA damage in retinal photoreceptor cells.

When suitable precautions are taken, sedimentation of DNA through reoriented alkaline sucrose gradients in zonal rotors can be used to determine small amounts of DNA damage in mammalian cells without resorting to radioactive precursors. Hence, the method is especially useful for studying the efficacies of DNA repair mechanisms in the neurons of the central nervous system (CNS) and the accumulation of DNA damage in the ageing CNS. Here we describe the technique as it has been used to examine the DNA damage occurring in the photoreceptor cells of the retina of the New Zealand white rabbit during the course of natural ageing or after exposure to heavy ions. This article is an integral part of a series of reports of the latter studies (Lett et al. 1980, Keng and Lett 1981, Cox et al. 1981, 1982, Keng et al. 1982). With the same analytical technique, very low levels of radioactive DNA precursors can be used to advantage in investigations of proliferating cells.

Late skin damage in rabbits and monkeys after exposure to particulate radiations.

Skin biopsies were taken from the central regions of the ears of New Zealand white rabbits following localized exposure of one ear of each rabbit to 530 MeV/amu Ar or 365 MeV/amu Ne ions. The unirradiated ears served as controls. Biopsies were taken also from the chests and inner thighs of rhesus monkeys after whole-body exposure to 32 MeV protons and from unirradiated control animals. The linear energy transfers (LET infinity's) for the radiations were 90 +/- 5, 35 +/- 3, and approximately 1.2 keV/micrometer, respectively. In the rabbit studies, explants were removed with a 2 mm diameter dermal punch at post-irradiation times up to five years after exposure. Similar volumes of monkey tissue were taken from skin samples excised surgically 16-18 years following proton irradiation. Fibroblast cultures were initiated from the explants and were propagated in vitro until terminal senescence (cessation of cell division) occurred. Cultures from irradiated tissue exhibited decreases in doubling potential that were dependent on radiation dose and LET infinity and seemed to reflect damage to stem cell populations. The implications of these results for astronauts exposed to heavy ions and/or protons in space include possible manifestations of residual effects in the skin many years after exposure (e.g. unsatisfactory responses to trauma or surgery).

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 degeneration in rabbit tissues after irradiation by heavy ions.

In 1974, using the rabbit as a model, we began long-term experiments designed to help in the evaluation of the hazards to man from extended exposure to heavy ions in space. Such exposure would occur, for example, during the construction of solar power stations in stationary orbits or on round trips to Mars. Our experiments with 400 MeV/nucleon Ne ions and 570 MeV/nucleon Ar ions have shown that true late effects of a degenerative nature are manifested only years after irradiation. At the appropriate doses (the high end of the experimental dose range), the magnitudes of the late effects are comparable with those encountered in human patients given radiation therapy with neutrons. Such comparisons show that the rabbit experiments are applicable to man. Given that basis, the results from the low end of the experimental dose range lead to the conclusion that astronauts subjected to the radiation fluxes anticipated during flights of the above duration could experience late radiation effects one or more decades after exposure. Late degenerative changes will occur in tissues of the central nervous system, terminally differentiating systems and stem cell populations. The studies also indicate that individual tissues may be "prematurely aged" by radiation in the sense that the "life spans" of those tissues can be decreased without the appearance of malignancies.