Effect of dose-rate and dose fractionation on radiation-induced hemolysis of human erythrocytes.

Human erythrocytes suspended in an isotonic Na-phosphate buffer, pH 7.4 (hematocrit 2%) were exposed under air to gamma radiation at a dose rates of 2.2 kGy.h-1 and 4.2 kGy.h-1. The dose-response curves for hemolysis of erythrocytes indicated that the process of hemolysis is inversely related to the dose-rate. At both dose-rates we observed a reduced level of hemolysis, when erythrocytes were irradiated with a split dose (0.4 kGy + 2.3 kGy with an interval time between the subsequent exposures from 1 to 4 h) in comparison with the same single dose (2.7 kGy). The maximal effect of fractionation was observed when the interfraction time was equal to 3.5 h. The influence of the interfraction temperature on this effect was observed. The results obtained indicate that enucleated human erythrocytes under suitable radiation conditions are capable of repairing radiation damage which leads to hemolysis.     

Quantitative regularities and peculiarities of the development of the cerebral form of radiation sickness at fractionated irradiation

Several peculiarities in manifestations of cerebral form of radiation sickness have been revealed at a fractionated double irradiation with equal and unequal doses per fraction and different intervals between the fractions. A reliable increase in average lifespan of rats irradiated with (100 + 100 Gy) equal doses at 10 and 60 min intervals between two fractions compared to the single radiation exposure to 200 Gy has been obtained. Lifespan of rats irradiated with a total dose greater than 200 Gy in most cases of double exposures with 10 min interval was reliably less than that for animals after a single exposure. The influence of the first dose on the reduction of animal average lifespan increased with fraction dose increasing from 150 to 300 Gy and was most pronounced at the total exposure dose of 400 Gy. Reaction of rats on the repeated irradiation was significantly weakened in comparison with the reaction on the first exposure. At a study of capacitation the interval of 30 min appeared to be more favorable compared to 10 min interval. Importance of a dose value in the first fraction has been demonstrated: the higher this value the worse the capacity of the rats 3 hours after the repeated exposure.     

Activity of radiation degradation products of vitamins A and E to haemolyse erythrocyte

Exposure of vitamin A acetate in freely dissolved state to γ-radiationin vitro caused a dose dependent degradation accompanied by the formation of new products. The radiation degradation products were separated by chromatography using step gradient elution. The parent molecule, vitamin A acetate, induced negligible haemolysis of erythrocytes. In contrast, the polar products formed by irradiation were found to be potent haemolysing agents. A highly polar product, eluted with methanol revealed maximum haemolytic activity. Acetylation of these products resulted in loss of their haemolytic properties. Similarly, vitamin E acetate, a known stabilizer of the biomembranes, after irradiation yielded products which caused haemolysis of erythrocytes. It was demonstrated that irradiation introduces hydroxyl groups which impart haemolytic properties to the radiation degradation products of vitamin A     

Studies of structural modifications induced by gamma-irradiation in distearoylphosphatidylcholine liposomes

An investigation of the structural and thermodynamical modifications induced by gamma-irradiation in model membranes is reported. Differential scanning calorimetry and X-ray diffraction were used to study the different phases and associated transitions of distearoylphosphatidylcholine multilamellar liposomes after 60Co gamma-irradiation. Changes were observed in the shape of the calorimetric peaks and in the corresponding phase transition temperatures. In particular a shoulder was observed at about 20 kGy. The three phases characteristic of lecithins with identical acyl chains were detected also for the highest radiation dose. The formation of lysolecithin and stearic acid upon phospholipid degradation was observed. The lysolecithin concentration increases as a function of irradiation dose, until a saturation value is reached at 40 kGy. These results correlate quite well with those obtained for interlayer and interchain distances and for the width of the main phase transition calorimetric peak. At the highest dose (approximately equal to 80 kGy) cross-linked adjacent radicals and other molecular species are also formed. Appreciable differences, and some similarities, in the behaviour of DSPC and DPPC liposomes under gamma-irradiation were observed.     

Changes in the resistance of mice to whole-body lethal irradiation after local irradiation of the abdominal area

The resistance of mice to whole-body irradiation with lethal doses, after preirradiation of part of the abdomen, was studied with a reference to radiation dose, the volume of local exposure, and the interval between exposures. The radioresistance was found to increase when the preirradiated zone corresponded to the spleen projection, the interval between exposures was 3-7 days, and the dose of local exposure, 2 Gy.     

Action of some hydroxyl radical scavengers on radiation-induced haemolysis

Human and bovine erythrocytes (RBCs) from peripheral blood were gamma-irradiated in vitro to a dose of 500 Gy in the presence of three efficient hydroxyl radical (OH) scavengers: ethanol, ethylene glycol and dimethyl sulphoxide (DMSO). Bovine erythrocytes were strongly protected from radiation induced haemolysis by each of the three scavengers over a concentration range from 10(-4) to 10(-2) molar, presumably as a result of OH scavenging. Human cells were protected as efficiently as bovine RBCs by ethanol and ethylene glycol over the same concentration range, however DMSO failed to protect human cells from haemolysis over a six-decade concentration range up to one molar. Exogenously supplied vitamin E (alpha-tocopherol) protected human RBCs from haemolytic effects of 500 Gy radiation in a dose-dependent fashion; however, bovine cells were not protected over the same concentration range. These preliminary results support evidence from model membrane systems suggesting that secondary radicals of DMSO generated during radiation may be of sufficient reactivity to initiate lipid peroxidation and are suggestive of species differences in the protection of biological membranes from oxidative stress.     

Dielectric breakdown measurements of human and bovine erythrocyte membranes using benzyl alcohol as a probe molecule

Dielectric breakdown of intact erythrocytes and subsequent haemolysis in the presence of increasing concentrations of benzyl alcohol were investigated by means of an electrolytical discharge chamber and a hydrodynamic focusing Coulter Counter.Low concentrations of the drug stabilized human and bovine erythrocytes against haemolysis induced by dielectric breakdown of the cell membrane in isotonic solutions, while high concentrations caused lysis similar to hypotonic and mechanical haemolysis. The stabilizing effect of the drug on electrically induced haemolysis depends on the pulse length of the applied electric field. The critical dielectric breakdown voltage of the membranes of intact cells decreases progressively with increasing benzyl alcohol concentrations, at which the membrane is also more stabilized against electrical and osmotic haemolysis. Occasionally, an increase in the dielectric breakdown voltage is observed at drug concentrations at which lysis occurs. A similar dependence of the breakdown voltage on drug concentration was found for human erythrocyte ghost cells prepared by dielectric breakdown.The results are consistent with the electromechanical model suggested for the dielectric breakdown mechanism and with the assumption of Metcalfe, using NMR and ESR techniques, that the fluidity of the membrane increases with increasing benzyl alcohol concentration.     

Epidemiological studies of cataract risk at low to moderate radiation doses: (not) seeing is believing

The prevailing belief for some decades has been that human radiation-related cataract occurs only after relatively high doses; for instance, the ICRP estimates that brief exposures of at least 0.5-2 Sv are required to cause detectable lens opacities and 5 Sv for vision-impairing cataracts. For protracted exposures, the ICRP estimates the corresponding dose thresholds as 5 Sv and 8 Sv, respectively. However, several studies, especially in the last decade, indicate that radiation-associated opacities occur at much lower doses. Several studies suggest that medical or environmental radiation exposure to the lens confers risk of opacities at doses well under 1 Sv. Among Japanese A-bomb survivors, risks for cataracts necessitating lens surgery were seen at doses under 1 Gy. The confidence interval on the A-bomb dose threshold for cataract surgery prevalence indicated that the data are compatible with a dose threshold ranging from none up to only 0.8 Gy, similar to the dose threshold for minor opacities seen among Chernobyl clean-up workers with primarily protracted exposures. Findings from various studies indicate that radiation risk estimates are probably not due to confounding by other cataract risk factors and that risk is seen after both childhood and adult exposures. The recent data are instigating reassessments of guidelines by various radiation protection bodies regarding permissible levels of radiation to the eye. Among the future epidemiological research directions, the most important research need is for adequate studies of vision-impairing cataract after protracted radiation exposure.     

Assessment of electrophoretic mobility changes of human erythrocytes by free flow electrophoresis after in vitro and in vivo irradiation

Single cell measurements of electrophoretic mobility showed a linear dose-response of human erythrocytes after exposure to ionizing radiation. To establish a biological indicator- or dosimeter-system for application to dose-estimations after accidental exposures, we tried to measure dose dependent EPM-changes by the free flow electrophoresis technique, a rapid and efficient method which supplies the capacity needed for application. The in vitro irradiated erythrocytes of most donors showed an oscillating dose-response but erythrocytes from several other individuals responded in a contradictory manner whereby the EPM of erythrocytes from radiotherapy patients increased linearly with the amount of administered dose. However, the wide interindividual EPM-range of unirradiated erythrocytes inhibits the application of this technique in biological dosimetry.     

Helium-neon laser preirradiation induces protection against UVC radiation in wild-type E. coli strain K12AB1157

We have observed that preirradiation with a helium-neon laser (632.8 nm) induces protection against UVC radiation in wild-type E. coli strain K12AB1157. The magnitude of protection was found to depend on the helium-neon laser irradiance, exposure time, and period of incubation between helium-neon laser exposure and subsequent UVC irradiation. The optimum values for dose, irradiance and interval between the two exposures were found to be 7 kJ/m(2), 100 W/m(2) and 1 h, respectively. The possible involvement of singlet oxygen in the helium-neon laser-induced protection is also discussed.     

Use of the γ-H2AX Assay to Investigate DNA Repair Dynamics Following Multiple Radiation Exposures

Radiation therapy is one of the most common and effective strategies used to treat cancer. The irradiation is usually performed with a fractionated scheme, where the dose required to kill tumour cells is given in several sessions, spaced by specific time intervals, to allow healthy tissue recovery. In this work, we examined the DNA repair dynamics of cells exposed to radiation delivered in fractions, by assessing the response of histone-2AX (H2AX) phosphorylation (γ-H2AX), a marker of DNA double strand breaks. γ-H2AX foci induction and disappearance were monitored following split dose irradiation experiments in which time interval between exposure and dose were varied. Experimental data have been coupled to an analytical theoretical model, in order to quantify key parameters involved in the foci induction process. Induction of γ-H2AX foci was found to be affected by the initial radiation exposure with a smaller number of foci induced by subsequent exposures. This was compared to chromatin relaxation and cell survival. The time needed for full recovery of γ-H2AX foci induction was quantified (12 hours) and the 1:1 relationship between radiation induced DNA double strand breaks and foci numbers was critically assessed in the multiple irradiation scenarios.     

The in vivo dose rate effect of chronic gamma radiation in mice: translocation and micronucleus analyses

The in vivo effects of chronic, ultra low dose rates of gamma radiation in mice were evaluated using fluorescence in situ hybridization and the in vivo micronucleus test. SWR×C57BL/6 mice were divided into nine exposure groups and continuously exposed to 0.5, 2.0 or 4.0 cGy ¹³⁷Cs per day for 30, 60 or 90 days; unexposed control mice were also included. Following exposure, blood samples were taken from each animal and the frequencies of micronucleated polychromatic erythrocytes (MPCE) and micronucleated normochromatic erythrocytes (MNCE) were determined using flow cytometry. Peripheral blood lymphocytes were cultured and analyzed by chromosome painting to determine translocation frequencies. A significant dose rate response was seen in translocations and both MPCE and MNCE. Comparisons were made between the three chronic dose rates and it was determined that there was no significant difference among translocation frequencies for each rate. However, a significant difference was found between the chronic exposures reported here and the fractionated daily exposures reported previously. Dose rate reduction effects, ranging from 3 at low doses to 14 at high doses, were found for chronic versus acute exposures. The possibility of gender effects was investigated in both micronucleus and translocation data. No gender effect was found in translocation induction, but a slight effect was suggested in micronucleus induction.     

Development of a qualitative dose indicator for gamma radiation using lyophilized Deinococcus

The feasibility of using Deinococcus showing strong resistance to both desiccation and ionizing radiation as a dose indicator of gamma radiation exposure was evaluated. Three Deinococcus strains having different levels of radiation resistance, Deinococcus radiodurans (DRD), Deinococcus radiopugnans (DRP), and the DRD pprI mutant (DRM), were selected to develop an appropriate dose indicator for a broad range of exposures. DRD, DRP, and DRM cultures with different numbers of cells [~10(7) to 10(3) colony forming units (CFU)/100 microliter] were lyophilized and subjected to various doses of gamma radiation to determine a critical dose that inhibited bacterial growth completely. Finally, a combination of DRD at ~10(7) and ~10(6) CFU, DRP at ~10(5) CFU, and DRM at ~10(4) CFU successfully indicated exposure to 5, 10, 20, and 30 kGy of gamma radiation, respectively. This study shows the possibility of developing a qualitative indicator of radiation exposure using Deinococcus.     

Effect of dose rate on the induction of experimental lung cancer in hamsters by alpha radiation

The effect of dose rate on the induction of lung cancer in Syrian hamsters by 5.3 MeV alpha particles was examined by varying the number of weekly intratracheal instillations of carrier-free 210Po. By this technique, most of the radiation dose to the lungs was delivered over intervals ranging from 10 to 120 days. Protraction of exposure over 120 days was slightly more carcinogenic at lower total lung doses (24 rad), but slightly less carcinogenic at higher doses (240 rad), than exposure limited to a 10-day interval. No synergism was observed between very low radiation exposures (2.4 rad) and simultaneously administered benzo[a]pyrene. The carcinogenic effect of a single intratracheal instillation of 210Po in isotonic saline was markedly enhanced by subsequent weekly instillations of 0.2 ml of saline alone, emphasizing the importance of noncarcinogenic secondary factors in the expression of radiation-induced lung cancer.     

Improvement of human skin cell growth by radiation-induced modifications of a Ge/Ch/Ha scaffold

Gelatin-/chitosan-/hyaluronan-based biomaterials are used in tissue engineering as cell scaffolds. Three gamma radiation doses (1, 10 and 25 kGy) were applied to scaffolds for sterilization. Microstructural changes of the irradiated polymers were evaluated by using scanning electron microscopy (SEM) and differential scanning calorimetry (DSC). A dose of 25 kGy produced a rough microstructure with a reduction of the porosity (from 99 to 96 %) and pore size (from 160 to 123 μm). Radiation also modified the glass transition temperature between 31.2 and 42.1 °C (1 and 25 kGy respectively). Human skin cells cultivated on scaffolds irradiated with 10 and 25 kGy proliferated at 48 h and secreted transforming growth factor β3 (TGF-β3). Doses of 0 kGy (non-irradiated) or 1 kGy did not stimulate TGF-β3 secretion or cell proliferation. The specific growth rate and lactate production increased proportionally to radiation dose. The use of an appropriate radiation dose improves the cell scaffold properties of biomaterials.     

Delayed haemolytic action of palytoxin. General characteristics

1. Palytoxin is a haemolysin. The erythrocytes from various species can be classified into a sensitive and a hardly sensitive group. The former contain potassium as their main inside cation and are arranged according to their sensitivity as hog greater than or equal to rat, mouse greater than rabbit greater than guinea-pig greater than man. The latter, comprising those from sheep and cattle, have sodium as their main inside cation. In addition, chicken erythrocytes are relatively insensitive. 2. Haemolysis of rat erythrocytes is preceded by a lag period of 1--2 h. With increasing temperature the haemolysis proceeds more quickly but reaches the same final range between 25 and 42 degrees C. The pH optimum in Britton-Robinson buffer supplemented with saline is between 7 and 8. Washing off palytoxin during the prelytic period reduces the haemolytic power. 3. The sensitivity of rat erythrocytes decreases with increase of osmolarity between 235 and 415 mosM. Accordingly, their osmotic resistance is lowered by palytoxin in a concentration-dependent manner. 4. With both rat and sheep erythrocytes, potassium loss by far precedes the haemolysis due to palytoxin. Potassium loss is measurable already after 1 min and increases with time. After 2 hours the quotient between the ED50 of haemolysis and that of potassium loss is around 200. Thus palytoxin is an unusually strong but slow haemolysin of the osmotic type. The extreme prelytic potassium loss and the correlation between susceptibility and potassium content of erythrocytes points towards the relevance of ionic fluxes.     

Radiation Resistance of the Microflora Associated with Amniotic Membranes

Summary Amniotic membrane is widely used in the treatment of burn wounds and ulcers of various etiology. As it comes into contact with open wounds, it needs to be perfectly sterile to avoid the transmission of any disease. Accordingly, amniotic membrane needs to bear a high sterility assurance level (SAL). Conventionally, a radiation dose of 25 kGy is the generally accepted dose for sterilization. But to keep intact the biomechanical and other properties, it is sometimes proposed to use a lower dose without compromising an SAL of 10⁻⁶. The initial microbial contamination level and the radiation resistance of the contaminants determine the dose required for sterilization. The microbial species associated with the amniotic membrane from about 70 different batches were isolated. Twenty-two representative bacterial isolates were characterized and tested for survival in an incremental series of radiation doses from 0.5 to 5.0 kGy. The radiation decimal reduction dose (D₁₀) values for the strains were determined. Relatively higher D₁₀ values were recorded for the gram-positive isolates. The D₁₀ values of microbial isolates ranged from 0.16 to 1.3 kGy, and most resistant Bacillus strain had a D₁₀ value of 2.1 kGy. The radiation dose necessary to achieve an SAL of 10⁻⁶ was calculated based on the D₁₀values of the isolated strains. For a bioburden of 1000 Bacillus organism, the sterilization dose of 18.9 kGy is obtained. However, based on the experimental determination of D₁₀ of the radiation-resistant reference strain Bacillus pumilus, the adequate dose for radiation sterilization is found to be 19.8 kGy if bioburden level of 1000 is granted. The results substantiate that radiation dose of 25 kGy assures sterilization of amniotic membranes with bioburden level of 1000 colony forming units.     

The influence of the irradiation regime upon mycotoxins production under experimental conditions

The paper handles the problem of the inactivation of the toxinogenic strain Aspergillus flavus following the application of gamma radiation to wheat. The amount of the applied dose and of the absorbed dose of ionizing radiation upon the inhibition of mycelium growth and toxin production were defined. The aflatoxin B1 was determined by extracting in chloroform and developed on Silufol R within the choroform; aceton system. The applied doses of gamma radiation (3-30 kGy) have show that the absorbed dose does not inhibit aflatoxin production. By combining the action of gamma radiation with humidity of the wheat (humidity 13-15%; 25% irradiation 6 kGy) an inactivation was reached. With the help of toxicologico-genetical tests (the Dominant Lethal Mutations Test, the Three Generations Test) the influence was traced of contaminated, irradiated substrates upon the health of experimental animals. It follows from the results obtained that in long-term feeding with contaminated wheat irradiated by gamma rays no positive mutagenic activity has been recorded. It allows to presume that wheat of humidity of 25% contaminated by a weakly toxigenic strain Aspergillus flavus irradiated by a dose of 6 kGy, and wheat of a humidity of 13-15%, contaminated by a strongly toxinogenic strain of Aspergillus flavus, irradiated by a dose of 6 kGy, are no genetic risk for white rats.     

Trends in childhood leukaemia in the Nordic countries in relation to fallout from atmospheric nuclear weapons testing.

OBJECTIVE--To obtain further information about the risks of childhood leukaemia after exposure to ionising radiation at low doses and low dose rates before or after birth or to the father''s testes shortly before conception. DESIGN--Observational study of trends in incidence of childhood leukaemia in relation to estimated radiation exposures due to fallout from atmospheric nuclear weapons testing during the 1950s and 1960s. SETTING--Nordic countries. SUBJECTS--Children aged under 15 years. MAIN OUTCOME MEASURES--Incidence rates of leukaemia by age at diagnosis, sex, country, and calendar year of diagnosis or year of birth; exposure category; relation between leukaemia and exposure for children aged 0-14 and 0-4 separately. RESULTS--During the high fallout period the average estimated dose equivalent to the fetal red bone marrow was around 140 mu Sv and the average annual testicular dose 140 mu Sv. There was little evidence of increased incidence of leukaemia among children born in these years. Doses to the red bone marrow of a child after birth were higher, and during the high exposure period children would have been subjected to an additional dose equivalent of around 1500 mu Sv, similar to doses received by children in several parts of central and eastern Europe owing to the Chernobyl accident and about 50% greater than the annual dose equivalent to the red bone marrow of a child from natural radiation. leukaemia incidence and red marrow dose was not related overall, but rates of leukaemia in the high exposure period were slightly higher than in the surrounding medium exposure period (relative risk for ages 0-14: 1.07, 95% confidence interval 1.00 to 1.14; for ages 0-4: 1.11, 1.00 to 1.24). CONCLUSIONS--Current predicted risks of childhood leukaemia after exposure to radiation are not greatly underestimated for low dose rate exposures.     

Chain length-dependent interaction of free fatty acids with the erythrocyte membrane

Free fatty acids protect erythrocytes against hypotonic haemolysis in a certain low concentration range and become haemolytic at higher concentrations. The chain length dependence of this biphasic behaviour was investigated using human erythrocytes. The results can be summarized as follows: (i) A critical minimum chain length is required for both effects. Octanoic acid (C8) and fatty acids with a shorter chain length do not have any effect on the osmotic resistance of erythrocytes. (ii) Decanoic acid (C10) decreases the extent of hypo-osmotic haemolysis and does not become haemolytic at higher concentrations. (iii) Dodecanoic acid (C12) represents the minimum chain length for the typical concentration-dependent biphasic behaviour with protection against hypo-osmotic haemolysis at a certain low concentration range and subsequent haemolysis at higher concentrations. (iv) Tetradecanoic acid (C14) exhibits two concentration ranges of protection against hypo-osmotic haemolysis, each followed by haemolytic concentrations. (v) The observed effects are not correlated with the critical micellar concentrations of the investigated fatty acids.