Dilution of irradiated cell conditioned medium and the bystander effect

While nontargeted and low-dose effects such as the bystander effect are now accepted, the mechanisms underlying the response have yet to be elucidated. It has been shown that the transfer of irradiated cell conditioned medium (ICCM) can kill cells that are not directly irradiated; however, to date the effect of ICCM concentration on cell killing has not been reported. The occurrence of a bystander effect was determined by measuring cell survival after exposure to various ICCM dilutions, using the colony-forming assay, in cells of six human cell lines with varied bystander responses and tumor/ p53 status. Autologous ICCM transfer for these cell lines induced a bystander effect as reported previously. ICCM from these cell lines was transferred to cells of a common reporter cell line (HPV-G) to investigate whether the lack of an induced bystander effect was due to their inability to generate or to respond to a bystander signal(s). ICCM from cells of four cell lines induced a bystander effect in HPV-G reporter cells, confirming that signal production is a critical factor. A saturation response was observed when ICCM was diluted. Survival was found to increase linearly until a plateau was reached and the bystander effect was abolished at 2x dilution. The effect of ICCM from the different cell lines reached a plateau at different dilutions, which were found to correlate with the cell line's radiosensitivity.     

Increased mitochondrial mass in cells with functionally compromised mitochondria after exposure to both direct gamma radiation and bystander factors

The bystander effect describes radiation-like damage in unirradiated cells either in the vicinity of irradiated cells or exposed to medium from irradiated cells. This study aimed to further characterize the poorly understood mitochondrial response to both direct irradiation and bystander factor(s) in human keratinocytes (HPV-G) and Chinese hamster ovarian cells (CHO-K1). Oxygen consumption rates were determined during periods of state 4, state 3 and uncoupled respiration. Mitochondrial mass was determined using MitoTracker FM. CHO-K1 cells showed significantly reduced oxygen consumption rates 4 h after exposure to 5 Gy direct radiation and irradiated cell conditioned medium (ICCM) and an apparent recovery 12-24 h later. The apparent recovery was likely due to the substantial increase in mitochondrial mass observed in these cells as soon as 4 h after exposure. HPV-G cells, on the other hand, showed a sustained increase in oxygen consumption rates after ICCM exposure and a transient increase 4 h after exposure to 5 Gy direct radiation. A significant increase in mitochondrial mass per HPV-G cell was observed after exposure to both direct radiation and ICCM. These findings are indicative of a stress response to mitochondrial dysfunction that increases the number of mitochondria per cell.     

Effective target size for the induction of bystander effects in medium transfer experiments

Although radiation-induced bystander effects are frequently observed biological phenomena, the mechanism for these effects has not been fully determined. The target-hit theory and related concepts from microdosimetry provide a convenient formalism to help identify the nature of the targets responsible for initiating the emission of diffusible factors in medium transfer experiments. We used the microdosimetric models proposed by Stewart et al. (Radiat. Res. 165, 460-469, 2006) to analyze the results of published medium transfer experiments for gamma-ray doses in the range of 0.04 mGy to 5 Gy. The analysis suggests that the effective size of the target responsible for initiating signal emission in HPV-G human keratinocyte donor cells is approximately 2 microm.     

The molecular and cellular consequences of the chernobyl accident

In this paper the results of research at 5–10 and 24 years after the Chernobyl accident are summarized. These results include the investigation of genomic instability, formation of the adaptive response, genome damage, and oxidative status. The studies were performed on cells in culture, mice, children and adults who lived in the contaminated areas, and liquidators of the consequences of the Chernobyl accident. Inhibition of cell proliferative activity, late cell death, and the increase in micronucleus and giant cell frequency were observed after the exposure of cells in culture in the accident zone followed by their culturing in laboratory conditions. In the progeny of the exposed cells, the effect of enhanced radiosensitivity was detected. Thus, it can be assumed that exposure of parental cells in culture in the area of the accident induced genomic instability that resulted in the development of various abnormalities in progeny cells. At the organism level, the Chernobyl zone exposure of mice caused an increase in radiosensitivity; as well, a decrease in the endotheliocyte density in the cerebral cortex and other brain tissues was observed. In the blood lymphocytes of children stimulated by PHA, a more than two times increase in micronucleus cell frequency was detected. A reduced number of individuals with significant adaptive response was found in both the juvenile and adult groups. In all investigated populations, an increased number of individuals with enhanced radiosensitivity were observed in response to low-dose radiation exposure. At 24 years after the accident liquidators were subjected to examinations, which revealed an increased frequency of cells with micronuclei and chromosome-type aberrations in blood lymphocytes, an elevated level of DNA double strand breaks, and a reduced level of reactive oxygen species compared to those of the control group. This means that the genomic instability that was accumulated by the residents of the contaminated regions and liquidators as a result of the accident leads to damage of the genetic apparatus, an increase in radiosensitivity, and hypoxia as late consequences that all are risk factors and increase the probability of the development of tumor and non-tumor diseases. The development of the above-mentioned pathological processes may occur in the distant future.

     

The effect of melanin on the bystander effect in human keratinocytes

The influence of melanin on radiation-induced bystander effects has been studied. Melanin is known to be a natural substance with proved radioprotective properties in different organisms and cell lines. It is non-toxic and is effective against acute and chronic irradiation. The lower the radiation dose, the higher the relative impact of melanin protection. In this study influence of melanin on human keratinocytes (HPV-G cells) has been studied using the colony-forming assay. We have shown that bystander donor medium from 0.5 Gy irradiated cells when transferred to unirradiated cells, caused almost the same effect as direct irradiation. Melanin increased the colony-forming ability of bystander recipient cells when it was added into culture medium before irradiation. The effect of melanin added after irradiation was to produce less protection in both the directly irradiated and bystander medium treated groups. The absorption spectrum of the filtered medium is identical to one of the intact culture medium showing that melanin was not present in filtered medium. Thus, it cannot protect recipient cells but reduces the amount of the bystander effect. It is concluded that melanin added before irradiation effectively decreased the radiation dose. The reduction of the impact of the bystander signal on recipient cells when melanin was added to the donor medium after harvest but before filtration, may mean that the bystander signal has a physical component as melanin can absorb all types of physical energy.     

The involvement of calcium and MAP kinase signaling pathways in the production of radiation-induced bystander effects

Much evidence now exists regarding radiation-induced bystander effects, but the mechanisms involved in the transduction of the signal are still unclear. The mitogen-activated protein kinase (MAPK) pathways have been linked to growth factor-mediated regulation of cellular events such as proliferation, senescence, differentiation and apoptosis. Activation of multiple MAPK pathways such as the ERK, JNK and p38 pathways have been shown to occur after exposure of cells to radiation and a variety of other toxic stresses. Previous studies have shown oxidative stress and calcium signaling to be important in radiation-induced bystander effects. The aim of the present study was to investigate MAPK signaling pathways in bystander cells exposed to irradiated cell conditioned medium (ICCM) and the role of oxidative metabolism and calcium signaling in the induction of bystander responses. Human keratinocytes (HPV-G cell line) were irradiated (0.005-5 Gy) using a cobalt-60 teletherapy unit. The medium was harvested 1 h postirradiation and transferred to recipient HPV-G cells. Phosphorylated forms of p38, JNK and ERK were studied by immunofluorescence 30 min-24 h after exposure to ICCM. Inhibitors of the ERK pathway (PD98059 and U0126), the JNK pathway (SP600125), and the p38 pathway (SB203580) were used to investigate whether bystander-induced cell death could be blocked. Cells were also incubated with ICCM in the presence of superoxide dismutase, catalase, EGTA, verapamil, nifedipine and thapsigargin to investigate whether bystander effects could be inhibited because of the known effects on calcium homeostasis. Activated forms of JNK and ERK proteins were observed after exposure to ICCM. Inhibition of the ERK pathway appeared to increase bystander-induced apoptosis, while inhibition of the JNK pathway appeared to decrease apoptosis. In addition, reactive oxygen species, such as superoxide and hydrogen peroxide, and calcium signaling were found to be important modulators of bystander responses. Further investigations of these signaling pathways may aid in the identification of novel therapeutic targets.     

20 years after Chernobyl accident--is it a lot or not for the estimation its characteristics ?

The results of long standing researches in the zones, which suffered from Chernobyl accident, on cultured cells, mice, blood lymphocytes of inhabitants of polluted regions (adults and children) are brought in the paper. The conclusion is that residence in polluted territories results in genomic instability, that is manifested in many effects on cytogenetic, cellular and organisms levels. It was shown, that in late descendants of cells after exposition in Chernobyl zone the increase of cell death, the micronuclei level, the frequency of giant cells, the enhancement of radiosensitivity and the absence of the adaptive response was observed. In the culture of embryonic fibroblasts, that was obtained from the exposed mice, the increase of cells with aberrations of chromosome and the frequency of multiaberrant cells was noticed. In mice exposed in Chernobyl zone the decrease of amounts of the endothelial cells in the different parts of the brain, the enhancement of mice radiosensitivity was observed. All effects were discovered in the late descendants of cells and can be the result of genomic instability induced by low level irradiation in polluted by Chernobyl accident regions.     

Molecular and cellular consequences of the Chernobyl accident

In this paper the results of the Chernobyl accident investigation 5-10 and 24 years after are summarized. The genomic instability, adaptive response formation, genome damage and oxidative status have been investigated. The studies were performed on cells in culture, mice, children and adults living in contaminated areas and liquidators. On cells in culture after exposition in the accident zone and culturing thereafter in laboratory conditions the cell proliferative activity decrease; the late cell death, the frequency of cells with micronuclei and giant cells increasing have been observed. In the progeny of exposed cells the enhancement of radiosensitivity has been noticed. So we can suppose that in cultured cells exposition in the zone of the accident the genomic instability is induced which results in many disturbances. At the organism level in mice exposed in the Chernobyl zone the radiosensitivity increase and the decrease of endotheliocytes density in brain tissue has been observed. On the stimulated by PHA blood lymphocytes of children the increase of the frequency of cells with micronuclei more than 2 time have been noticed. In all groups investigated, the decrease of individuals with significant adaptive response was observed. In children and adults inhabitants the increase of radiosensitivity after low dose of irradiation has been noticed. 24-year after the accident it was discovered that in liquidators lymphocytes the frequency of cells with micronuclei, with chromosome type aberrations, with DNA double strand breaks have been increased; the reactive oxygen species (ROS) were decreased in comparison with the control population. We can suppose that genomic instability induced in residents of contaminated regions and liquidators long after the accident results in the genetic apparatus damage, radiosensitivity enhancement, hypoxia that represent risk factors and increase the probability of tumour and non-tumour diseases. The development of these pathological processes may happen in much more remote periods.     

The natural anti-Pseudomonas aeruginosa and anti-Proteus immunity of the suitable donor population]

The natural anti-pyocyanic and anti-Proteus immunity of donorable population of Kiev before the accident at Chernobyl Atomic Power Plant has been studied. Blood serums of 3024 deliberately non-immunized persons have been studied. It is determined that there are natural anti-pyocyanic and anti-Proteus antibodies in blood serum of people with different group belonging during the all periods of a year (by ABO system). The anti-pyocyanic and anti-Proteus antibodies of high titers in blood serum are found in summer most frequently.     

The state of immunity in the liquidators of consequences of the Chernobyl accident with cardio-vascular diseases

The purpose of the work was research and functional reserves immunity in participants in the Chernobyl with cardiovascular disease. A Clinical Lab 49 men aged 44 to 52 years with diseases of the cardiovascular system, participated in the Chernobyl nuclear power plant in 1986 to 1988. As control used data 33 patients with similar pathology, and 16 healthy men. Researched the total number of white blood cells, lymphocytes and transfusions, the absolute number and relative CD4+ and CD8+, CD 16, CD20+, CD95+ lymphocytes peripheral blood, number mononuclear, synthesizing IL-1beta, TNF-alpha, INF-alpha, IL-4, the content of lipids and proteins, the activity of alkaline phosphatase and mieloperoksidazy in neutrophils. To assess the functional reserve immune system blood samples studied people subjected to radiation doses 0.25, 50, 1.0 Gy of in vitro and studied the reaction cytochemical indicators neutrophils before and after the radiative forcing. The liquidators not detected significant changes in the absolute number of leucocytes, but compared with control groups noted significant reduction in the absolute number of CD8+ and CD20+ lymphocytes, increased the number of cells, expression of FAS-antigen, change the number of mononuclear spontaneously synthesizing and produce cytokines, decreased maintenance of cation proteins in neutrophils. Radiation samples peripheral blood liquidators caused the same reaction cytochemical indicators of neutrophils and control groups, the compensatory and adaptive nature of the changes in the immune system, developed in response to complex factors radiation accident.     

Cambial history of Scots pine trees (Pinus Sylvestris) prior and after the Chernobyl accident as encoded in the xylem

Studies were carried out on wood samples collected in October 1997 from breast height of Scots pine trees (Pinus sylvestris) from site located 5 km south from the Chernobyl nuclear power plant. The radioactive contamination at the site was 3.7x10(5) kBq m(-2). These samples of secondary wood were used as an archive of information about the dynamics of a meristematic tissue cambium affected by ionising radiation from the Chernobyl reactor accident. The results show that frequency of the cambial cells events like anticlinal divisions, intrusive growth and cells elimination, was after the Chernobyl accident, about three times higher in comparison to preceding years. The most interesting finding was that after irradiation the length of tracheids increased. This increase is interpreted as an effect of intracambial competition among cells in the initial layer.     

Late consequences of action of low doses of ionizing radiation on human enzymatic antioxidant system

Age-related changes in the activity of superoxide dismutase and glutathione peroxidase in the blood of participants of the Chernobyl accident liquidation were studied. According to our findings the people under 30 years old are the most sensitive to irratiation.     

Analysis of blood lymphocyte subsets in children living around Chernobyl exposed long-term to low doses of cesium-137 and various doses of iodine-131

Epidemiological studies have found that children living around Chernobyl have rates of respiratory tract illness that are higher than those seen in the area before the Chernobyl accident. The present study investigates the possible effects of radiation exposure on the composition of peripheral blood lymphocyte subsets in children living around Chernobyl. Two hundred nineteen healthy children and children suffering from recurrent respiratory diseases aged 6-14 years who received both low doses of radiation to the whole body from (137)Cs and various doses of radiation to the thyroid from (131)I as fallout from the accident were assessed 5 (1991) and 8-10 years (1994-1996) after the accident. A total of 148 healthy children and children suffering from recurrent respiratory diseases living in noncontaminated areas were also evaluated as controls. Children with recurrent respiratory diseases who lived around Chernobyl had a significantly lower percentage of T cells and a higher percentage of NK cells compared to control children with recurrent respiratory diseases during the study period. In contrast to the findings in 1991, a significant decrease in the percentage of helper-inducer cells was observed in children with recurrent respiratory diseases in 1994-1996. In contrast to 1991, there is a positive correlation between the percentage of helper-inducer cells, the helper-inducer/cytotoxic-suppressor cell ratio, and the dose of radiation to the thyroid of healthy children from (131)I in 1994-1996. There was a positive correlation between the dose of radiation to the thyroid from (131)I and the percentage of helper-inducer cells in children with recurrent respiratory diseases 5 years (1991) after the accident. Further, the dose of radiation to the thyroid from (131)I correlated negatively with the percentage of T and B cells and positively with the percentage of NK cells in children with recurrent respiratory diseases 8-10 years (1994-1996) after the accident. These results raise the possibility that long-term exposure to low doses of (137)Cs may have altered the composition of the T-cell subsets and NK cells in children with recurrent respiratory diseases. The differences in the composition of the peripheral blood lymphocyte subsets between healthy children and those with recurrent respiratory diseases may be attributed to long-term low-dose exposure of the whole body to radiation from (137)Cs and exposure of the thyroid to radiation from (131)I subsequent to the Chernobyl accident.     

Assessment of lipid peroxidation and plasma membrane permeability for Ca2+ in the red blood cells of cattle after the long term grazing on radioactively contaminated territory

Ten years after the Chernobyl accident a physiological condition of cows was examined on radioactivy contaminated territory of the Novozibkov district of the Bryansk region. The long grazing of cattle on radioactivly contaminated territory revealed the increase in permeability of plasmatic membrane of the red blood cells to Ca2+ and the activation of process of lipid peroxidation. The sensitivity of the red blood cells of cows to incubations in hypertonic conditions was demonstrated.     

Chromosome alterations in cleanup workers sampled years after the Chernobyl accident

Cytogenetic analysis performed 4-13 years after the Chernobyl accident showed an elevated frequency of acentrics, chromatid exchanges, dicentrics and rings in Chernobyl cleanup workers compared to the control group. Cytogenetic data were analyzed according to the information on exposure to radiation (the year when the cleanup workers worked at the Chernobyl station, doses rates, time elapsed since exposure to radiation, and cytogenetic examination) and some lifestyle factors. The data obtained suggested that some types of chromosome aberrations could be influenced by the action of different environmental factors or lifestyle factors. The frequency of acentrics was correlated with the age of the cleanup workers, and the increased chromatid exchange frequency was attributed to smoking. The numbers of dicentrics and rings suggested a genotoxic effect of ionizing radiation that is still present over 13 years after the exposure.     

Results of the cytogenetic examination of the people taking part in the clean-up of the accident at the Chernobyl AES]

Chromosome preparations from peripheral blood cells of 117 subjects who took part in liquidation of Chernobyl NPP accident consequences in 1986 have been investigated. The number of chromosome aberrations has been shown to increase considerably. A short-term exposure has been found to be 5-7 times more effective than long-term one.     

Increased radiosensitivity in cells of two human cell lines treated with bystander medium from irradiated repair-deficient cells

Radiation-induced bystander factors have been shown to be more toxic if they are from medium harvested from irradiated repair-deficient cells. The aim of this study was to test the hypothesis that the radiosensitivity of repair-proficient cells can be increased by exposing them to medium-borne factors harvested from sensitive cells and vice versa. Cells from a mismatch repair (MMR)-deficient cell line (Raji 10) with a sensitive response to radiation or the wild-type parent cell line were irradiated to 0.5 Gy gamma rays and then monitored for growth rate in their own medium or in the alternative conditioned medium. In other experiments, cells or conditioned medium were added to reporter cells (HPV-G, which are relatively sensitive keratinocytes, or highly radioresistant HT29 cells). The subsequent responses of the two cell lines to a 0.5-Gy dose of (60)Co gamma rays were measured. The results show that prior exposure of resistant cells to medium from irradiated sensitive cells reduced the clonogenic survival of the subsequently irradiated resistant cells. The reverse is also true. Measurement of the apoptosis index and BCL2 expression confirmed that the harvested medium was capable of modulating apoptosis after irradiation. This may have important applications in tumor therapy and also in the understanding of mechanisms involved in induction of adaptive responses.     

Modulation of radiation responses by pre-exposure to irradiated cell conditioned medium

The aim of this study was to investigate whether exposure of HPV-G cells to irradiated cell conditioned medium (ICCM) could induce an adaptive response if the cells were subsequently challenged with a higher ICCM dose. Clonogenic survival and major steps in the cascade leading to apoptosis, such as calcium influx and loss of mitochondrial membrane potential, were examined to determine whether these events could be modified by giving a priming dose of ICCM before the challenge dose. Clonogenic survival data indicated an ICCM-induced adaptive response in HPV-G cells "primed" with 5 mGy or 0.5 Gy ICCM for 24 h and then exposed to 0.5 Gy or 5 Gy ICCM. Reactive oxygen species (ROS) were found to be involved in the bystander-induced cell death. Calcium fluxes varied in magnitude across the exposed cell population, and a significant number of the primed HPV-G cells did not respond to the challenge ICCM dose. No significant loss of mitochondrial membrane potential was observed when HPV-G cells were exposed to 0.5 Gy ICCM for 24 h followed by exposure to 5 Gy ICCM for 6 h. Exposure of HPV-G cells to 5 mGy ICCM for 24 h followed by exposure to 0.5 Gy ICCM for 18 h caused a significant increase in mitochondrial mass and a change in mitochondrial location, events associated with the perpetuation of genomic instability. This study has shown that a priming dose of ICCM has the ability to induce an adaptive response in HPV-G cells subsequently exposed to a challenge dose of ICCM.     

Leukemia incidence in the Russian cohort of Chernobyl emergency workers

Of all potentially radiogenic cancers, leukemia, a type of cancer of the blood, has the highest risk attributable to ionizing radiation. Despite this, the quantitative estimation of radiation risk of a leukemia demands studying very large exposed cohorts, because of the very low level of this disease in unexposed populations and because of the tendency for its radiation risk to decrease with time. At present, the Japanese cohort of atomic bomb survivors is still the primary source of data that allows analysis of radiation-induced leukemia and the underlying dose–response relationship. The second large cohort that would allow to study radiation-induced leukemia is comprised of individuals who were exposed due to the accident of the Chernobyl nuclear power plant in 1986. The objective of the present study was to estimate radiation risks of leukemia incidence among the Russian cohort of Chernobyl emergency workers, for different time periods after the accident. Twenty-five years after the Chernobyl accident and based on the results of the present study, one can conclude that the radiation risk of leukemia incidence derived from the Russian cohort of Chernobyl emergency workers is similar to that derived from the cohort of atomic bomb survivors: The time-averaged excess relative risk per Gray (ERR Gy⁻¹) equals 4.98 for the Russian cohort and 3.9 for the life span study (LSS) cohort; excess absolute risk decreases with time after exposure at an annual rate of 9% for the Russian cohort, and of 6.5% for the LSS cohort. Thus, the excess in risk of leukemia incidence in a population due to a single exposure is restricted in time after exposure by the period of about 15 years.     

Trends in infant leukaemia in West Germany in relation to in utero exposure due to the Chernobyl accident

A temporary increase in the incidence of infant leukaemia in Greece was reported by Petridou et al., which was attributed to in utero exposure to ionising radiation resulting from the Chernobyl accident. We performed a similar analysis based on the data of the German Childhood Cancer Registry in order to check whether the observation could be confirmed by means of independent data. Applying the same definitions as Petridou et al., we also observed an increased incidence of infant leukaemia in a cohort of children born after the Chernobyl accident. More detailed analyses, regarding areas with different contamination levels and dose rate gradients over time after the accident, showed, however, no clear trend with regard to exposure. It would therefore appear less likely that the observed effect was caused by exposure to ionising radiation due to the Chernobyl accident. Received: 30 January 1998 / Accepted in revised form: 23 April 1998