Non-targeted bystander effects induced by ionizing radiation

Radiation-induced bystander effects refer to those responses occurring in cells that were not subject to energy deposition events following ionizing radiation. These bystander cells may have been neighbors of irradiated cells, or physically separated but subject to soluble secreted signals from irradiated cells. Bystander effects have been observed in vitro and in vivo and for various radiation qualities. In tribute to an old friend and colleague, Anthony V. Carrano, who would have said "well what are the critical questions that should be addressed, and so what?", we review the evidence for non-targeted radiation-induced bystander effects with emphasis on prevailing questions in this rapidly developing research field, and the potential significance of bystander effects in evaluating the detrimental health effects of radiation exposure.     

Iodine-125 induced micronuclei and sperm head abnormalities in mice

Swiss albino mice were injected with 0 (controls) 5, 10 or 15 microCi of iodine-125 (125I) and a high incidence of micronuclei in young bone marrow erythrocytes and of sperm head abnormalities was observed. The percentage of polychromatic erythrocytes containing micronuclei in mice treated with 0, 2.5, 5 and 7.5 microCi of 125I administered twice (at 30 and 6 h before sampling) was 0.23, 0.44, 0.56 and 0.72, respectively. The percentage (mean) of abnormal sperm recorded in controls during 1-3, 4-5 and 6-8 weeks was 3.3, 3.7 and 3.7 respectively, whereas the values for the same periods in the 5 microCi (5.5, 6.4 and 7.3), 10 microCi (7.7, 8.4 and 10.0) and 15 microCi (10.3, 9.9 and 10.2) groups were significantly higher. These results suggest that 125I can induce genetic damage in both somatic and germ cells.     

DNA double-strand breaks induced by very low X-ray doses are largely due to bystander effects

Ojima, M., Ban, N. and Kai, M. DNA Double-Strand Breaks Induced by Very Low X-Ray Doses are Largely due to Bystander Effects. Radiat. Res. 170, 365-371 (2008).Phosphorylated ATM immunofluorescence staining was used to investigate the dose-response relationship for the number of DNA double-strand breaks (DSBs) induced in primary normal human fibroblasts irradiated with doses from 1.2 to 200 mGy. The induction of DSBs showed a supralinear dose-response relationship. Radiation-induced bystander effects may explain these findings. To test this hypothesis, the number of DSBs in cells treated with lindane, an inhibitor of radiation-induced bystander effects, prior to X irradiation was assessed; a supralinear dose-response relationship was not observed. Moreover, the number of DSBs obtained by subtracting the number of phosphorylated ATM foci in lindane-treated cells from the number of phosphorylated ATM foci in untreated cells was proportional to the dose at low doses (1.2-5 mGy) and was saturated at doses from 10-200 mGy. Thus the increase in the number of DSBs in the range of 1.2-5 mGy was largely due to radiation-induced bystander effects, while at doses >10 mGy, the DSBs may be induced mainly by dose-dependent direct radiation effects and partly by dose-independent radiation-induced bystander effects. The findings in our present study provide direct evidence of the dose-response relationship for radiation-induced bystander effects from broad-beam X rays.     

Radiation-induced bystander effects in vivo are sex specific

Ionizing radiation (IR) effects span beyond the area of direct exposure and can be observed in neighboring and distant na?ve cells and organs. This phenomenon is termed a 'bystander effect'. IR effects in directly exposed tissue in vivo are epigenetically mediated and distinct in males and females. Yet, IR-induced bystander effects have never been explored in a sex-specificity domain. We used an in vivo mouse model, whereby the bystander effects are studied in spleen of male and female animals subjected to head exposure when the rest of the body is protected by a medical-grade lead shield. We analyzed the induction of DNA damage and alterations in global DNA methylation. Molecular parameters were correlated with cellular proliferation and apoptosis levels. The changes observed in bystander organs are compared to the changes in unexposed animals and animals exposed to predicted and measured scatter doses. We have found the selective induction of DNA damage levels, global DNA methylation, cell proliferation and apoptosis in exposed and bystander spleen tissue of male and female mice. Sex differences were significantly diminished in animals subjected to a surgical removal of gonads. These data constitute the first evidence of sex differences in radiation-induced bystander effects in mouse spleen in vivo. We show the role of sex hormones in spleen bystander responses and discuss implications of the observed changes.     

Inhibitory and stimulatory effects of limb ectoderm on in vitro chondrogenesis

Previous studies have indicated possible dual effects of the limb ectoderm in cartilage differentiation. On one hand, explants from early (stage 15) wing buds are dependent on contact with the limb ectoderm for cartilage differentiation (Gumpel-Pinot, J. Embryol. Exp. Morph. 59:157-173, 1980). On the other hand, limb ectoderm from stage 23/24 wing buds inhibits cartilage differentiation by cultured limb mesenchyme cells even without direct contact (Solursh et al., Dev. Biol. 86:471-482, 1981). In the present study, ectoderms from both stage 15/16 and stage 23/24 wings are cultured under the same conditions, and ectoderms from each source are shown to have two effects. Each stimulates chondrogenesis in stage 15 wing bud mesenchyme, and each inhibits chondrogenesis in older wing mesenchyme. The results suggest that the limb ectoderm has at least dual effects on cartilage differentiation, depending on the stage of the mesenchyme. One effect involves an early mesenchymal dependence on the ectoderm. This effect requires contact between the ectoderm and mesoderm (Gumpel-Pinot, J. Embryol. Exp. Morphol. 59:157-173, 1980) but also can be observed at a distance from the ectoderm. Later, the ectoderm can act without direct contact between the ectoderm and mesoderm to inhibit chondrogenesis over some distance.     

Inhibitory and stimulatory effects of amiloride analogues on sodium transport in frog skin

Summary Effects of amiloride analogues on Na transport were studied in isolated skins of the frogRana ridibunda. The pattern of structure-activity relationship of these compounds showed that both the –NH₂ group at position 5 and Cl at position 6 of the pyrazine ring of the amiloride molecule were important for their biological activity. The paramount role of the groups at position 5 was further demonstrated by the striking properties of an analogue resulting from dimethylation of that –NH₂ group. A stimulation of Na transport, opposite to the effect of amiloride itself, was observed in this instance. The increase in Na transport could already be seen at 10^–6 m and was equivalent to the measured increase in Na influx, reversible, dose-dependent, and additive to the natriferic action of oxytocin. Such characteristics resemble those reported with external agents like propranolol and La³⁺. Furthermore, mutual inhibition was observed between the stimulatory effects of this analogue and those of propranolol or La³⁺. These results suggest that the analogue may be considered as another external agent acting at sites of the external membrane distinct from those activated by cAMP but similar to the Ca sites described by Herrera and Curran (Herrera, F.C., Curran, P.F. 1963.J. Gen. Physiol. 46:999).     

Elevated sodium chloride concentrations enhance the bystander effects induced by low dose alpha-particle irradiation

Previous studies have shown that high NaCl can be genotoxic, either alone or combined with irradiation. However, little is known about the relationship between environmental NaCl at elevated conditions and radiation-induced bystander effects (RIBE). RIBE, which has been considered as non-targeted bystander responses, has been demonstrated to occur widely in various cell lines. In the present study, RIBE under the elevated NaCl culture condition was assessed in AG 1522 cells by both the induction of gamma-H2AX, a reliable marker of DNA double-strand break (DSB) for the early process (<1h post irradiation), and the generation of micronuclei (MN), a sensitive marker for relative long process of RIBE. Our results showed that in the absence of irradiation, NaCl at elevated concentration such as 8.0, 9.0 and 10.0g/L did not significantly increase the frequency of gamma-H2AX foci-positive cells and the number of foci per positive cell comparing with that NaCl at a normal concentration (6.8g/L). However, with 0.2cGy alpha-particle irradiation, the induced fraction of gamma-H2AX foci-positive cells and the number of induced gamma-H2AX foci per positive cell were significantly increased in both irradiated and adjacent non-irradiated regions. Similarly, the induction of MN by 0.2cGy alpha-particle irradiation also increased with the elevated NaCl concentrations. With N(G)-methyl-l-arginine, an inhibitor of nitric oxide synthase, the induced fraction of foci-positive cells was effectively inhibited both in 0.2cGy alpha-particle irradiated and adjacent non-irradiated regions under either normal or elevated NaCl conditions. These results suggested that the cultures with elevated NaCl medium magnified the damage effects induced by the low dose alpha-particle irradiation and nitric oxide generated by irradiation was also very important in this process.     

T-cell induced pathogenesis in HIV: bystander effects and latent infection.

The progress of HIV is accompanied by the infection and decline of the population of CD4+ cells. This reduction in cells results from both cytolytic influences of the virus and virus-specific cytotoxic T-cell (CTL) responses. We seek to characterize the extent of CD4+ reduction caused by HIV-specific CTLs at equilibrium. Here we show that intermediate levels of cytotoxic killing of infected cells can be inferior to both strong and weak or absent immune responses. We further show that the deleterious effects of the CTL response are made worse by a slow immune response. Bystander effects in which uninfected cells are thought to be eliminated by non-specific CTL activation lead to small or negligible reductions in uninfected CD4+ cells. Latently infected cells containing pro-viral DNA and which become activated at a constant rate ensure that the immune response is more effective for a larger range of CTL activities and reduces T-cell associated pathology.     

Direct and bystander effects induced by scattered radiation generated during penetration of radiation inside a water-phantom

In this study, the dose distribution of photon (6 MV) and electron (22 MeV) radiation in a water-phantom was compared with the frequency of apoptotic and micronucleated cells of two human cell lines (BEAS-2B normal bronchial epithelial cells and A549 lung cancer epithelial cells). Formation of micronuclei and apoptotic-like bodies was evaluated by the cytokinesis-block micronucleus test. Measurements were performed for five different phantom depths (3-20 cm). Irradiated cells were placed in a water-phantom in three variants: directly on the axis in the beam, under shielding (only in photon radiation) and outside the beam field. The results reveal a discrepancy between the distribution of physical dose at different depths of the water-phantom and biological effects. This discrepancy is of special significance in case of cells irradiated at a greater depth or placed outside the field and under shield during the exposure to radiation. The frequency of cytogenetic damage was higher than the expected value based on the physical dose received at different depths. Cells placed outside the beam axis were exposed to scattered radiation at very low doses, so we tested if bystander effects could have had a role in the observed discrepancy between physical radiation dose and biological response. We explored this question by use of a medium-transfer technique in which medium (ICM-irradiation conditioned medium) from irradiated cells was transferred to non-irradiated (bystander) cells. The results indicate that when cells were incubated in ICM transferred from cells irradiated at bigger depths or from cells exposed outside the radiation field, the number of apoptotic and micronucleated cells was similar to that after direct irradiation. This suggests that these damages are caused by factors released by irradiated cells into the medium rather than being induced directly in DNA by X-rays. Evaluation of biological effects of scattered radiation appears useful for clinical practice.     

Biological damage in testis by Iodine-125 in partially blocked thyroid of rats

Summary Degeneration of testis has been observed after administration of Iodine-125 in potassiumperchlorate treated rats. Histological damage is associated with loss of DNA, RNA, acid phosphatase, total adenosine triphosphatase (ATPase) and Na/K dependent ATPase. Iodine-125 induced atrophic testis shows higher content of sodium and lower levels of potassium as compared to control testis. Damage of testis by Iodine-125 has been compared with atrophied testis, following gamma irradiation earlier reported. Auger effect due to Iodine-125 decay and transmutation at the sites of nuclei and plasma membrane of germinal cells seems to be the possible explanation for testicular damage caused by Iodine-125.     

Inhibitory effects of d-mannose on trypanosomatid lysis induced by Serratia marcescens

Studies were carried out on the effects of different carbohydrates on the lysis of Trypanosoma cruzi, Trypanosoma rangeli and erythocytes caused by the bacteria Serratia marcescens variants SM 365 and RPH. High concentrations of d-mannose were found to protect T. cruzi and T. rangeli markedly diminishing the lysis caused by S. marcescens. However, this carbohydrate is unable to interfere with the hemolysis induced by SM 365 and RPH variants. These results showed that the trypanolytic effect induced by S. marcescens SM 365 and RPH variants is dependent on d-mannose and distinct from the hemolytic activity, strongly suggesting that bacterial fimbriae are relevant to S. marcescens in lysis of parasites.     

Inhibitory and stimulatory effects of fluoride on the calcium pump of cardiac sarcoplasmic reticulum.

While studying the effects of membrane phosphorylation on active Ca2+ transport in cardiac sarcoplasmic reticulum (SR) we used NaF (a conventional phosphatase inhibitor) in the Ca2+ transport assay medium to suppress protein dephosphorylation by endogenous phosphatases. Unexpectedly, depending on the experimental conditions employed, NaF was found to cause a strong inhibitory or stimulatory effect on ATP-dependent, oxalate-facilitated Ca2+ uptake (Ca2+ pump) activity of SR. Investigation of this phenomenon using canine cardiac SR revealed the following. Exposure of SR to NaF in the absence of Ca2+ or ATP in the Ca2+ transport assay medium (prior to initiating Ca2+ transport by the addition of Ca2+ or ATP) promoted a striking concentration-dependent inhibitory effect of NaF (50% and 90% inhibition with approx. 4 and 10 mM NaF, respectively) on Ca2+ uptake by SR; the magnitude of inhibition did not differ appreciably with varying oxalate concentrations. In contrast, exposure of SR to NaF in the presence of both Ca2+ and ATP resulted in a concentration-dependent stimulatory effect of NaF (half-maximal stimulation at approx. 2.5 mM NaF with 2.5 mM oxalate in assay) on Ca2+ uptake; the magnitude of stimulation decreased with increasing oxalate concentration (greater than 2-fold at 1 mM oxalate, 10% at 5 mM oxalate). The inhibitory effect prevailed when SR was exposed to NaF in the presence of Ca2+ alone (without ATP) or ATP alone (without Ca2+). Both the inhibitory and stimulatory effects of NaF were specific to fluoride ion, as NaCl (1-10 mM) showed no effect on Ca2+ uptake by SR under identical assay conditions. A persistently less active state of the Ca2+ pump (evidenced by decreased Ca2+ transport rates) resulted upon pretreatment of SR with NaF in the absence of Ca2+ or ATP; presence of Ca2+ and ATP during pretreatment prevented this transition. The inhibitory action of NaF on the Ca2+ pump was accompanied by a two-fold increase in K0.5 for Ca2+ and decrements in Hill coefficient (nH) and Ca(2+)-stimulated ATP hydrolysis, as well as steady-state level of Ca(2+)-induced phosphoenzyme. The stimulatory effect of NaF, on the other hand, was associated with an increase in the ratio of Ca2+ transported/ATP hydrolysed with only minor changes, if any, in the above parameters. These findings imply that the divergent effects of fluoride are dependent on specific conformational states of the Ca(2+)-ATPase which evolve during the catalytic and ion transport cycle.(ABSTRACT TRUNCATED AT 400 WORDS)     

Radiation-induced bystander effects in malignant trophoblast cells are independent from gap junctional communication

It is controversially discussed that irradiation induces bystander effects via gap junction channels and/or diffusible cellular factors such as nitric oxide or cytokines excreted from the cells into the environment. But up to now the molecular mechanism leading to a bystander response is not well understood. To discriminate between both mechanisms of bystander response, (i) mediated by gap junctional communication and/or (ii) mediated by diffusible molecules, we used non-communicating Jeg3 malignant trophoblast cells transfected with inducible gap junction proteins, connexin43 and connexin26, respectively, based on the Tet-On system. We co-cultivated X-ray irradiated and non-irradiated bystander Jeg3 cells for 4 h, separated both cell populations by flow cytometry and evaluated the expression of activated p53 by Western blot analysis. The experimental design was proven with communicating versus non-communicating Jeg3 cells. Interestingly, our results revealed a bystander effect which was independent from gap junctional communication properties and the connexin isoform expressed. Therefore, it seems more likely that the bystander effect is not mediated via gap junction channels but rather by paracrine mechanisms via excreted molecules in Jeg3 cells.     

Engaged and bystander T cell receptors are down-modulated by different endocytotic pathways

T cell antigen receptor (TCR) engagement by stimulatory antibodies or its major histocompatibility complex-antigen ligand results in its down-modulation from the cell surface, a phenomenon that is thought to play a role in T cell desensitization. However, TCR engagement results in the down-modulation not only of the engaged receptors but also of non-engaged bystander TCRs. We have investigated the mechanisms that mediate the down-modulation of engaged and bystander receptors and show that co-modulation of the bystander TCRs requires protein-tyrosine kinase activity and is mediated by clathrin-coated pits. In contrast, the down-modulation of engaged TCRs is independent of protein-tyrosine kinases and clathrin pits, suggesting that this process is mediated by an alternate mechanism. Indeed, down-modulation of engaged TCRs appears to depend upon lipid rafts, because cholesterol depletion with methyl-beta-cyclodextrin completely blocks this process. Thus, two independent pathways of internalization are involved in TCR down-modulation and act differentially on directly engaged and bystander receptors. Finally, we propose that although both mechanisms coexist, the predominance of one or the other mechanisms will depend on the dose of ligand.