Carmel Mothersill

Radiation-induced bystander effects: Past history and future directions

Abstract Mothersill, C. and Seymour, C. Radiation-Induced Bystander Effects: Past History and Future Directions. Radiat. Res. 155, 757–765 (2001). There has been a recent upsurge of interest in the phenomenon now known as radiation-induced bystander effects. This is largely due to the increased awareness of the contribution of indirect and delayed effects, such as genomic instability, to cellular outcomes after low-dose exposures. It is also due to the availability of tools such as the microbeam and advanced cell culture systems and to the ability to study end points such as gene or protein expression at low doses which were previously difficult to study. This review looks at the history of bystander effects in the earlier literature, in which the clastogenic effect of plasma from irradiated patients was well known. The effect was known to persist for several years and to cause transgenerational effects, making it similar to what we now call genomic instability. The review then examines the current data and controversies which are now beginning to resolve the questions concerning the mechanisms underlying the induction and transmission of both bystander effects and genomic instability. Finally, the possible impact of data concerning radiation-induced bystander effects on radiotherapy and radiation protection is discussed.

Cell-cell contact during gamma irradiation is not required to induce a bystander effect in normal human keratinocytes : Evidence for release during irradiation of a signal controlling survival into the medium

Killing of unirradiated cells by medium from cultures of irradiated cells implies the release of a cytotoxic substance by the irradiated cells. The finding of the gamma-ray-induced cytotoxic effect exclusively in epithelial cells and not in fibroblasts suggested that tissue architecture or cell communication might be important. Normal human keratinocytes and fibroblasts and radiosensitive carcinoma cells were irradiated as single cells, microcolonies of three or four cells, or confluent monolayers. The medium was removed and filtered, and cultures which had never been irradiated were seeded at cloning densities and treated with the medium from the irradiated cells. It was found that the degree of cell-cell contact had no effect on the ability of medium from irradiated epithelial cell cultures to reduce the clonogenic survival of unirradiated cells. Cell density was the only important factor. Inhibition of gap junction intercellular communication using the tumor promoter phorbol myristate acid (PMA), which closes gap junctions, increased killing by the bystander effect when the PMA was added to epithelial cells prior to irradiation. Rescue of epithelial cells exposed to the medium from the irradiated cells was not possible even after only 30 min exposure. This suggests that a signal transduction mechanism may control death or survival by the bystander effect rather than by release of a factor which is directly cytotoxic.

High yields of lethal mutations in somatic mammalian cells that survive ionizing radiation

When mammalian cells are irradiated in vitro, the component cells of a normal-appearing survivor colony or clone are commonly thought to have proliferative capacity equivalent to that of the unirradiated cells. We have found, however, that cells appearing in survivor colonies may carry heritable lethal defects which come to light, perhaps only after numerous successful divisions, in the form of plating efficiencies that are reduced below those of unirradiated cells in a dose-dependent manner. We regard these heritable defects as signs of the induction of lethal mutations, which, like non-lethal mutations, may require many generations before they are expressed. This effect has been noted in two very dissimilar mammalian cell lines, one a primary culture from adult tissue, the other an immortal cell line. We suggest that induction of lethal mutations may occur also in somatic cells in vivo; this would account for the well-known observation that previously irradiated but apparently healed tissue is subsequently proved to be extraordinarily sensitive to subsequent exposure to irradiation or cytotoxic drugs. The results of our experiments in vitro suggest that current methods of estimating mutation or transformation yields may yield underestimates. If lethal mutations are induced also in vivo, interpretations of the results of fractionation experiments on normal tissues may have to be reconsidered.

Relative Contribution of Bystander and Targeted Cell Killing to the Low-Dose Region of the Radiation Dose–Response Curve

Abstract Seymour, C. B. and Mothersill, C. Relative Contribution of Bystander and Targeted Cell Killing to the Low-Dose Region of the Radiation Dose–Response Curve. Human keratinocytes show a bystander effect when exposed to low doses of low-LET radiation. In this paper, data are presented showing a method of correcting the overall survival curve to enable analysis of the relative contributions of the bystander effect and the effect attributable to direct interaction of the radiation with the target cell. The technique used is to obtain a standard clonogenic survival curve using the assay of Puck and Marcus and, with a different set of flasks containing cloning densities of unirradiated cells, to assay the cell killing caused by medium harvested from 2 × 105 cells irradiated with the same doses. The data show that for this human epithelial cell line, doses of 0.01–0.5 Gy show clonogenic death by the bystander effect only, if maximum potential bystander killing is assumed. The magnitude of the effect is relatively constant, and it appears to saturate at doses in the range of 0.03–0.05 Gy. After doses greater than 0.5 Gy, the curves for clonogenic death are the result of a dose-dependent non-bystander effect and a dose-independent bystander effect. If these particular dose–response effects occur in epithelial cells in vivo, they may have important consequences for therapy and studies of low-dose risk.

Production of a signal by irradiated cells which leads to a response in unirradiated cells characteristic of initiation of apoptosis.

This study investigated the ability of medium from irradiated cells to induce early events in the apoptotic cascade, such as mobilization of intracellular calcium, loss of mitochondrial membrane potential and increase in reactive oxygen species, in cells which were never exposed to radiation. Medium from irradiated human keratinocytes was harvested and transferred to unirradiated keratinocytes. Endpoints characteristic of the initiation of apoptosis were monitored for a period of 24 h following medium transfer. Clonogenic survival was also measured. Rapid calcium fluxes (within 30 s), loss of mitochondrial membrane potential, increases in reactive oxygen species (from 6 h after medium transfer), an increase in the number of apoptotic cells (48 hours after medium transfer) and a marked reduction in clonogenic survival (after 9 days) were observed. There was no significant difference between medium generated by cells irradiated at 0.5 Gy or 5 Gy. The data suggest that initiating events in the apoptotic cascade were induced in unexposed cells by a signal produced by irradiated cells.

Initiation of Apoptosis in Cells Exposed to Medium from the Progeny of Irradiated Cells: A Possible Mechanism for Bystander-Induced Genomic Instability?

Abstract Lyng, F. M., Seymour, C. B. and Mothersill, C. Initiation of Apoptosis in Cells Exposed to Medium from the Progeny of Irradiated Cells: A Possible Mechanism for Bystander-Induced Genomic Instability? Radiat. Res. 157, 365–370 (2002). Genomic instability and bystander effects have recently been linked experimentally both in vivo and in vitro. The aim of the present study was to determine if medium from irradiated cells several passages distant from the original exposure could initiate apoptosis in unirradiated cells. Human keratinocytes (from the HPV-G cell line) were irradiated with 0.5 Gy or 5 Gy γ rays. Medium was harvested at each passage up to the 7th passage (approximately 35 population doublings) postirradiation and transferred to unirradiated keratinocytes. Intracellular calcium levels, mitochondrial membrane potential, and the level of reactive oxygen species were all monitored for 24 h after medium transfer. Rapid calcium fluxes (within 30 s), loss of mitochondrial membrane potential, and increases in reactive oxygen species (from 6 h after medium transfer) were observed in the recipient cells. There was no significant difference between medium conditioned by cells irradiated with 0.5 or 5 Gy. The effect of medium from progeny was the same as the initial effect reported previously and did not diminish with increasing passage number. The data suggest that initiating events in the cascade that leads to apoptosis are induced in unirradiated cells by a signal produced by irradiated cells and that this signal can still be produced by the progeny of irradiated cells for several generations.

Relationship between Radiation-Induced Low-Dose Hypersensitivity and the Bystander Effect

Abstract Mothersill, C., Seymour, C. B. and Joiner, M. C. Relationship between Radiation-Induced Low-Dose Hypersensitivity and the Bystander Effect. Radiat. Res. 157, 526–532 (2002). Recent advances in our knowledge of the biological effects of low doses of ionizing radiation have shown two unexpected phenomena: a “bystander effect” that can be demonstrated at low doses as a transferable factor(s) causing radiobiological effects in unexposed cells, and low-dose hyper-radiosensitivity and increased radioresistance that can be demonstrated collectively as a change in the dose–effect relationship, occurring around 0.5–1 Gy of low-LET radiation. In both cases, the effect of very low doses is greater than would be predicted by conventional DNA strand break/repair-based radiobiology. This paper addresses the question of whether the two phenomena have similar or exclusive mechanisms. Cells of 13 cell lines were tested using established protocols for expression of both hyper-radiosensitivity/increased radioresistance and a bystander response. Both were measured using clonogenicity as an end point. The results showed considerable variation in the expression of both phenomena and suggested that cell lines with a large bystander effect do not show hyper-radiosensitivity. The reverse was also true. This inverse relationship was not clearly related to the TP53 status or malignancy of the cell line. There was an indication that cell lines that have a radiation dose–response curve with a wide shoulder show hyper-radiosensitivity/increased radioresistance and no bystander effect. The results may suggest new approaches to understanding the factors that control cell death or the sectoring of survival at low radiation doses.

A Dose Threshold for a Medium Transfer Bystander Effect for a Human Skin Cell Line

Abstract Liu, Z., Mothersill, C. E., McNeill, F. E., Lyng, F. M., Byun, S. H., Seymour, C. B. and Prestwich, W. V. A Dose Threshold for a Medium Transfer Bystander Effect for a Human Skin Cell Line. Radiat. Res. 166, 19–23 (2006). The existence of radiation-induced bystander effects mediated by diffusible factors is now accepted, but the mechanisms and precise behavior at low doses remain unclear. We exposed cells to γ-ray doses in the range 0.04 mGy–5 Gy, harvested the culture medium, and transferred it to unirradiated reporter cells. Calcium fluxes and clonogenic survival were measured in the recipients. We show evidence for a dose threshold around 2 mGy for the human skin cell line used with a suggestion of increased survival below that dose. Similar experiments using direct γ irradiation showed no reduction in survival until the dose exceeded 7 mGy. Preliminary data for neutrons where the γ-ray dose was kept below the bystander threshold do not show a significant bystander effect in the dose range 1–33 mGy. A lack of a bystander response with neutrons occurred at around 1 Gy, where significant cell killing from direct irradiation was observed. The result may have implications for understanding the role of bystander effects at low doses.

Radiation-induced bystander effects, carcinogenesis and models

Implications for carcinogenesis of radiation-induced bystander effects are both mechanistic and practical. They include induction of second cancers, perturbations to tissue social control and induction of genomic instability and delayed or immediate mutations in areas not receiving a direct deposition of energy. Bystander effects have consequences for DNA damage-mutation-cancer initiation paradigms of radiation carcinogenesis that provide the mechanistic justification for low-dose risk estimates. If carcinogenesis does not result from directly induced DNA mutations, then the carcinogenic initiation process may not simply relate to radiation dose. Modification of the preclonal state through genetic and epigenetic mechanisms may occur. To deal with the complexity of these interactions, a ‘chaotic’ or ‘bifurcation’ model invoking autopoietic theory is proposed that could accommodate both beneficial (hormetic) and harmful effects of radiation at comparable doses. Carcinogenesis may then be thought of as the result of a disturbance of the genetic/epigenetic balance occurring within the organ. Ultimate clonality may reflect domination due to selection processes rather than the initiating damage.

Bystander and delayed effects after fractionated radiation exposure.

Abstract Mothersill, C. and Seymour, C. B. Bystander and Delayed Effects after Fractionated Radiation Exposure. Radiat. Res. 158, 626–633 (2002). Human immortalized keratinocytes were exposed to a range of single or fractionated doses of γ rays from 60Co, to medium harvested from donor cells exposed to these protocols, or to a combination of radiation and irradiated cell conditioned medium (ICCM). The surviving fractions after direct irradiation or exposure to ICCM were determined using a clonogenic assay. The results show that medium harvested from cultures receiving fractionated irradiation gave lower “recovery factors” than direct fractionated irradiation, where normal split-dose recovery occurred. The recovery factor is defined here as the surviving fraction of the cells receiving two doses (direct or ICCM) separated by an interval of 2 h divided by the surviving fraction of cells receiving the same dose in one exposure. After treatment with ICCM, the recovery factors were less than 1 over a range of total doses from 5 mGy–5 Gy. Varying the time between doses from 10 min to 180 min did not alter the effect of ICCM, suggesting that two exposures to ICCM are more toxic than one irrespective of the dose used to generate the response. In certain protocols using mixtures of direct irradiation and ICCM, it was possible to eliminate the bystander effect. If bystander factors are produced in vivo, then they may reduce the sparing effect of the dose fractionation.