Women’s contributions to radiobiology in Ireland; from small beginnings….

Abstract

Objectives

To describe the contribution of women radiobiologists in Ireland to the development of the discipline internationally and at home and to discuss the history of radiobiology in Ireland to date. This parallels the history of the evolution of a small radiobiology group in Kevin Street, Dublin Institute of Technology (DIT) which was formerly part of the City of Dublin Vocational Education Committee. There followed years of development first as a radiobiological research center which evolved in the FOCAS Research Institute now embedded within Technological University Dublin (TU Dublin).

Conclusions

Over the last 45 years, the women of the Radiation and Environmental Science Centre (RESC) contributed to the major paradigm shift in low dose radiobiology contributing exciting new research concerning non-targeted effects, including discovery of lethal mutations, medium transfer bystander mechanisms, and signaling pathways. They also developed translational research using human explant culture systems with unique immunocytochemical methods and more recently evolved to molecular and spectroscopic analysis of clinical samples. The RESC also developed unique in vitro research methods into effects of radiation on non-human species of concern in ecosystems.

Keywords:

• Radiation biology
• low dose radiation
• non-targeted effects
• bystander effects
• lethal mutations
• genomic instability

Early days

The evolution of radiobiology in Ireland as a discipline distinct from radiotherapy research or medical physics developed almost in parallel with the evolution of Technological University Dublin (TU Dublin) from the Kevin Street College of the City of Dublin Vocational Education Committee (CDVEC) through the Dublin Institute of Technology (DIT). Along the way was a strategic alliance with the Irish Radiotherapy Hospital (St Luke’s) where Cobalt 60 radiation facilities were available. This narrative traces the work of Irish women who contributed to this evolution. Figure 1 depicts the parallel evolution of the structures in Dublin (Figure 1(a)) and the science (Figure 1(b)).

Figure 1. (A) The major milestones of RESC from its initial development in St. Luke’s Radiotherapy Hospital in 1978 through Dublin Institute of Technology and now Technological University Dublin with major International funding awards included. (B) The main radiobiological discoveries by RESC spanning 50 years (1970s–present).

St Luke’s Hospital and Kevin Street VEC

Radiobiology started in Ireland in the mid-1970s with a European grant obtained by Dr Jim Malone on which he employed Carmel Mothersill (CM) as a post-doctoral fellow who had no experience of radiation and was a muscle biochemist. The grant was to look for potential natural substances such as vitamins A, C and E which could alter the shoulder of the radiation survival curve and which might have potential as radioprotective or sensitizing agents. Nowadays, we recognize that agents that modulate the shoulder are affecting mechanisms operating in the low dose region of the dose response curve but back in the seventies, shoulders were thought to describe sub-lethal lesions that could be repaired or could recover from the damage to the DNA target. Alternative but largely ignored alternatives suggested the shoulder was due to a limited pool of repair substance which once depleted, caused the cell death to become exponential. The history of this period and evolution of low dose radiobiology was recently reviewed by CM (Mothersill and Seymour 2019). CM came from a background of studying post mortem glycolysis in muscle, so started to manipulate energy metabolism in cells to see what the effects were on the shoulder. Results suggested energy for repair was what determined the width of the shoulder, supporting pool model theories although as this tended to vary from cell line to cell line there was obviously a genetic component involved.

The need for access to radiation facilities in St Luke’s radiotherapy hospital led to a small room being made available for tissue culture in the basement of the hospital. It was here that CM in association with Colin Seymour, discovered that progeny of irradiated cells which had the ability to form a colony in the Puck and Marcus assay (Puck and Marcus 1956) were not fully recovered and did not behave in subsequent experiments as if never irradiated. These results were met with incredulity when presented at a meeting of the UK Association for Radiation Research (ARR) in 1984 and it was only thanks to the mentorship of Tikvah Alper that the now classic paper was published in 1986 describing ‘High Yields of Lethal Mutations in the progeny of cells which survived irradiation’ (Seymour et al. 1986). By this time, CM had been appointed as a lecturer in the Physics Department in DIT Kevin Street seconded half time to do research as St Luke’s Hospital. Grants from the Irish Cancer Society and a ready supply of students from Kevin Street allowed the group to grow.

Work on this phenomenon kept CM occupied for several years along with postgraduate supervision of several women radiobiologists including Sandra O’Reilly, Margaret Lehane, Stephanie Mulgrew and Anne Cusack who also graduated during this period. The main studies focused on the lethal mutation effect, seeking to characterize the relationship to dose and fractionation. It became apparent that lethal mutations occurred predominantly in the low dose region and when plotted against dose a saturated dose response was seen with no increase in frequency with increasing dose above about 2 Gy. This is now a recognized response pattern in most non-targeted effects (Seymour et al. 1986; Alper et al. 1988; Seymour and Mothersill 2000). Alongside this work, the group also developed a human tissue explant culture method (Mothersill, Cusack, MacDonnell, et al. 1988; Mothersill, Cusack, Seymour 1988) and working with surgeons from several Dublin hospitals, looked at the growth post irradiation of normal and tumor explants, treated with various chemotherapy agents. These studies used growth as a major endpoint but also looked at expression of proteins such as p53, Bcl2 and cmyc. Studies using bladder explants were extended to look at patterns of protein expression in smokers and nonsmokers and how these changed after exposure to radiation or other carcinogens. This is described in more detail below in the translational research section. The work on lethal mutations continued and Fiona Lyng (FL) joined the group in 1991 as an undergraduate thesis student and later as a PhD student. At that time, the field of radiobiology was on the cusp of exploding. Radiation-induced genomic instability (RIGI) became the new and very exciting area of research (reviewed in Mothersill and Seymour 2012) worldwide with our lethal mutation discovery acknowledged as among the first descriptions of the delayed non-clonal and high frequency emergence of abnormalities in distant progeny of apparently normal irradiated surviving cells.

Establishment of Radiation Research Centre 1992-RIGI becomes trendy but not fundable in Europe necessitating a sideways move to environmental toxicology and fish disease research

While RIGI was creating worldwide excitement among radiobiologists, those involved in radiation protection were skeptical because RIGI made it very difficult to support the linear-no-threshold (LNT) dose response relationship which was a DNA centric model that had at its core the assumption that direct energy deposition in cells caused DNA breaks directly in proportion to the dose. RIGI suggested something else was going on and that low dose radiation exposure caused an ‘instability’ of unknown cause which meant that non clonal lethal mutations could occur which could not have been induced directly by the radiation as they were lethal for the cell. As a result of the skepticism, CM lost funding from Europe and moved into DIT Kevin St. full time. She re-invented herself using expertise in human cell culture and explant culture to develop a niche in fish research and fish toxicology based on the culture of fish cells, gaining EU grants as a partner with the ability to culture tissues from a variety of organisms. This widening remit brought many new International collaborators to work with, and allowed diversification which ultimately led to major funded collaborations with University College Cork and Heriot Watt University in Scotland. Large EU grants were awarded and CM then led an EU concerted action to coordinate research into culture methods for invertebrate species. Maria Lyons-Alcantara (MLA) joined the group as a post-doctoral fellow specializing in fish research and Maria Davoren also worked on ecotoxicology projects along with Sharon Ni Shuilleabhan and Alma Mulford. The major milestones in this environmental research area are shown in Figure 2.

Figure 2. Timeline of the development of environmental radiation research in the RESC from 1992 until 2008.

DIT makes RSC a center that is later adapted to RESC

In 1992, DIT obtained autonomy as a degree-awarding institution and then in 1995 decided to establish designated research centers within the institute to foster applied research and aid its efforts ultimately to become a university. CM bid for one and the radiation science center (RSC) was established as an official funded research center within the Institute. To reflect the increasing emphasis on environmental work the center name was changed to the Radiation and Environmental Science Centre (RESC) in 1998. Radiation research had never stopped but the center was now poised to become a leading group in the efforts to study impacts of radiation on non-human species in the environment. This was part of a worldwide effort to include impacts on the environment as a consideration in radiation protection planning. The work of CM, FL and MLA in particular established the RESC as a leader in this field and participated in the EU ERICA and PROTECT projects aimed at developing suitable tools and frameworks for an integrated approach to radiation protection of humans and non-humans.

Alongside the environmental development, the RESC never stopped doing radiation research. In fact, the Irish Radiation Research Society (IRRS) was established and launched in 1995 at the same ceremony celebrating RSC as a DIT center. This expanded the scope for collaboration and dissemination of radiobiological research on the island of Ireland. In 1997, we published that culture medium from irradiated cells could reduce the survival of unirradiated cells. Although Nagasawa and Little (1992) had described a bystander effect using a very low fluence of alpha particles, ours was the first demonstration that ‘soluble factors’ could carry information which made unirradiated cells behave as if they had been irradiated (Mothersill and Seymour 1997).

The other major branch of radiobiology the RESC pursues is translational research for biomarkers of radio-sensitivity or radioresistance. Early work in this area described above involved collaborations with surgeons at local hospitals to obtain patient tumor samples then subjected to the explant culture system. This culture system was established at RSC by CM first on human esophageal normal and malignant tissue (Seymour et al. 1988) and then urothelium (Mothersill et al. 1991) and other carcinomas such as the bladder (Mothersill et al. 1992). Biomarkers such as Ki-67 were applied to explant studies, first to monitor explant cell growth from the different tissues but then as tumor and mechanistic markers including c-Myc and P53 (Mothersill, Harney, et al. 1994; Mothersill, Seymour, et al. 1994) and BCL2 (Harney et al. 1995), p21, Ras and EGFR with both acute and low doses of ionizing radiation (Cobalt-60) (Mothersill et al. 1995, 1997). Sheridan used this explant technique at RSC to study human esophageal endoscopic biopsies (Sheridan et al. 1995) and she also established primary cultures from head and neck (H&N) tumors taken at surgery, and immunohistochemical expression of Ki67, EGFR and c-Myc biomarkers for the radiosensitive and radioresistant groups was shown (Sheridan et al. 1997). During this time in RSC, Lambkin applied immunohistochemical expression of P53 in cervical carcinoma paraffin wax sections (Lambkin et al. 1994) and she also extensively studied the role of HPV in cervical carcinomas. She later joined the School of Biological Sciences (in DIT) and continued to significantly contribute to HPV research for cervical carcinomas (Keegan et al. 2005; Oberg et al. 2005; Zhao et al. 2005, 2007; Fay et al. 2009). At the same in RSC, Creane performed radiobiological studies showing the drug docetaxel (Taxotere) to be a weak radiosensitizer in three cell lines (Creane, Seymour, Colucci, et al. 1999) and furthermore when used in combination with radiation could prevent the induction of lethal mutations (Creane, Seymour, Mothersill 1999). Dunne later studied the cell cycle and apoptotic mechanisms of docetaxel in both radioresistant and radiosensitive colorectal cancer cell lines showing accumulation of cells exposed in the radiosensitive phase G2/M and a large 60–80% induction of apoptosis (Dunne 2004).

Much of this radiobiological work continued in RESC to the present day with major mechanistic contributions from a number of female researchers and funded by a Science Foundation Ireland award to CM and by EU FP5 RADINSTAB and EU FP6 Integrated Project, NOTE. FL returned to RESC as a postdoc in 1999 and brought new skills and expertise in live cell imaging. She focused on trying to define the early signaling events occurring in bystander cells. At this time, DIT had limited microscopy facilities so the initial experiments were carried out in University College Cork where FL had previously carried out postdoctoral research involving confocal imaging. Initiating events in the apoptotic cascade were shown to be induced in unirradiated bystander cells by a signal produced in irradiated cells (Lyng et al. 2000). Rapid transient calcium fluxes were observed within 30 seconds after addition of irradiated cell conditioned media (ICCM) and induction of ROS and loss of mitochondrial membrane potential (MMP) was observed up to 24 hours after ICCM treatment. A follow on study (Lyng et al. 2002) showed that the bystander signal could be produced by the progeny of irradiated cells for several generations. Human keratinocytes were irradiated and ICCM was harvested at each passage post irradiation up to approximately 35 population doublings. Calcium fluxes, loss of MMP and ROS induction were observed following transfer of ICCM to unirradiated cells. Similar effects were observed for ICCM from initially irradiated cells and from the progeny of these irradiated cells.

Orla Howe (OH) joined the RSC in 1998 as a research assistant, later PhD student with TCD (2000–2004), DIT post-doctoral fellow and Arnold Graves Scholar (2004–2009), then joining the School of Biological Sciences at DIT in 2009 to present day (currently School of Biological and Health Sciences in TUDublin). As a research assistant in RSC, she worked primarily on cell and explant culture with immunohistochemistry techniques and she then developed her own translational research interests. She established collaborations with the Paterson Institute of Medical Research in Manchester UK, St. James Hospital (breast cancer) and St. Vincents Hospital (benign and malignant prostate tissue and colorectal cancer tissue) in Dublin. This initial collaborative work involved culturing of whole blood samples obtained from donor and patient cohorts to determine their intrinsic G2 chromosomal radiosensitivity by cytogenetic techniques where elevated G2 radiosensitivity was reported in cancer cohorts compared to normal donors (Bryant et al. 2002; Howe, Daly, et al. 2005; Howe, O'Malley, et al. 2005; Howe et al. 2009). Between 2000 and 2004, OH expanded this translational work to molecular focused studies at the Queensland Institute of Medical Research, Brisbane, Australia, to further understand the mechanisms of radiation-induced radiosensitivity in cells obtained from ataxia-telangiectasia (AT) and ataxia-ocular motor apraxia 1 (AOA1) patients with elevated cellular radiosensitivity (Gueven et al. 2004, 2007).

In these early days of RESC, the center was growing rapidly and gaining exposure nationally and internationally. CM won a bid to host the International Congress of Radiation Research (ICRR) in 1999, which attracted over 1000 delegates, and FL, OH, MLA and Natasha Coen (NC) all assisted in the organization of this conference. See Figure 3 for a snapshot of the opening ceremony by then Irish President Mary McAleese. Many other conferences/meetings were organized by the RESC which included the National IRRS meetings, ARR UK meetings, European Radiation Research (ERR) meetings, LowRad and Low level Radiation and Health society meetings, EU concerted action workshops and LH Gray workshops which brought international attention to Irish radiobiology and to DIT research.

Figure 3. A press release photo taken at the opening ceremony of the International Congress of Radiation Research (ICRR) July 1999 with the primary host CM (1) and the President of Ireland Mary McAleese (2) along with FL (3), TL (4), OH (5), NC (6) and MLA (7).

Scientifically, at this time radiobiological concepts were further broadened to integrate research on environmental toxicants and different radiation types (UV). The work of NC in collaboration with the Medical Research Council in the UK demonstrated genomic-instability in cells exposed to heavy metals cadmium and nickel persisting for generations after exposure (Coen et al. 2001) and also in particulate titanium debris from a hip implant in collaboration with Avon Orthopaedic Centre, Southmead Hospital in the UK (Coen et al. 2003). Around this time, a subgroup of female postgraduate researchers led by Dr. James Murphy shifted their efforts from the classic radiobiological concept that genomic DNA is the critical target to mitochondrial direct and bystander DNA damage effects in cells exposed to low-LET radiation (Murphy et al. 2005; Nugent et al. 2007, 2010) and UV exposure (Zanchetta et al. 2010, 2011). CM moved to McMaster University in Canada in 2003 and FL was appointed RESC manager who then continued to grow and expand radiobiological research. A strong link was maintained between RESC and CM, and collaborative radiobiological work continued with Fiona McNeill, Lorna Ryan, Lisa Liu and Michelle Le in Canada and Hayley Furlong from Ireland who worked partly in Canada making significant contributions to the understanding of bystander signaling mechanisms. In fact, CM then became an adjunct professor of the new TUDublin (formerly DIT) late in 2019 and gave her inaugural adjunct professor lecture on campus in January 2020.

DIT establishes the FOCAS Research Institute and RESC re-location

DIT researchers from the Schools of Physics and Chemistry, including CM, MLA and FL, were involved in obtaining large scale funding from the Higher Education Authority Programme for Research in Third Level Institutions (PRTLI), Cycle 1 (1999–2001), co-funded by the EU. The aim was to establish a facility for optical characterization and spectroscopy (FOCAS) and to consolidate and develop established expertise while nurturing developing research activities in DIT. In 2004, RESC moved into a purpose built 200 m² research laboratory in the 3200 m² FOCAS Research Institute to the rear of the DIT Kevin Street site. The new FOCAS microscopy facilities and funding for postgraduate students allowed us to continue our bystander signaling research. Maguire et al. (2005) showed a dose dependence of bystander responses where exposure to <0.5 Gy ICCM resulted in no significant bcl-2 expression and cell death through a caspase-dependent pathway, whereas exposure to 5 Gy ICCM resulted in significant bcl-2 expression and cell death through a caspase-independent pathway. In addition, cell death could be blocked by N-acetylcysteine (NAC) for all ICCM doses indicating a role for ROS. Similar responses, including MMP depolarization, induction of ROS, expression of bcl-2 and release of cytochrome c, were shown for bystander cells following microbeam irradiation with protons (Lyng, Maguire, Kilmurray, et al. 2006). Further evidence of the role of mitochondria in bystander responses was shown by Nugent et al. as an increase in mitochondrial mass (2007) and mitochondrial DNA damage (2010) following exposure to ICCM suggesting a stress response to mitochondrial dysfunction. MAPK signaling pathways were also shown to be triggered in cells exposed to ICCM and again calcium and ROS were key players in these pathways (Lyng, Maguire, McClean, et al. 2006).

Further development of the FOCAS Research Institute

The facilities of the FOCAS Research Institute were further developed under PRTLI Cycle 4 (2007–2013), co-funded by the EU Regional Development Fund, as DIT became a partner in the Integrated NanoScience Platform for Ireland and the National Biophotonics and Imaging Platform Ireland. This saw the further development of RESC research into biophotonics/imaging and nanotoxicology but our radiation research remained a core focus and our efforts on bystander signaling pathways continued.

Maguire et al. (2007) showed that bystander signals produced by irradiated cells induce an adaptive response in unirradiated cells to a further exposure to bystander signals. Interestingly, Vines et al. (2008) showed that different cell lines showed varying responses to ICCM generated from different cell lines indicating that the signal produced by the irradiated cell determines the overall bystander effect rather than the individual response of the recipient cell. In 2011, the sequence of early bystander signaling events was elucidated (Lyng et al. 2011). Membrane signaling was found to be the first response to ICCM followed by calcium influx, a rapid increase in ROS levels and a subsequent increase in nitric oxide (NO) levels. Calcium and ROS were found to be involved in the production of the bystander signal while calcium, ROS and NO were all found to be important signaling molecules involved in bystander responses.

Furlong et al. (2013) reported significant molecular expression of apoptotic genes in bystander reporter cells exposed to 0.5 and 0.05 Gy IR compared to their direct irradiated counterpart and provided further molecular mechanistic insight to the apoptotic cascade for both direct and indirect IR. Later, in CMs laboratory in Canada, HF then demonstrated the expression of proteins found in bystander reporter HaCaT cells exposed to 0.5 Gy media obtained from skin cultures of rainbow trout (Oncorhynchus mykiss). Annexin A2 and cingulin (upregulated) and Rho-GDI2, F-actin-capping protein subunit beta, microtubule-associated protein RP/EB family member, and 14-3-3 proteins (downregulated) were reported providing further molecular mechanistic insights of radiation-induced bystander effects (Furlong et al. 2015).

Research into extracellular vesicles and exosomes was taking off around this time and their role in bystander induced signaling was shown by Jella et al. (2014). Two different types of vesicles were identified in ICCM, exosomes (30–100 nm) and microvesicles (>100 nm) and the concentration of these extracellular vesicles was found to increase in a dose dependent manner. Jella et al. (2018) also showed a persistent production of ROS and NO in bystander cells following exposure to ICCM and a link between ROS production and cell survival pathways. See Figure 4 for these proposed bystander signaling pathways.

Figure 4. Schematic showing an overview of the signaling pathways induced in bystander cells with calcium signaling playing a central role as discovered by RESC researchers.

Designation as Ireland’s first Technological University January 2019 and the move to a new campus at Grangegorman March 2021 (delayed due to the pandemic)

Ireland’s first Technological University, TU Dublin, was formally established on 1 January 2019 as a result of a partnership between DIT, Institute of Technology Blanchardstown and Institute of Technology Tallaght. TU Dublin will build on its more than 130 year history of providing technological higher education in Dublin (the first Technical School was established at Kevin St in 1887 by Dublin Corporation). In addition to the new designation, a new city campus is currently being developed on a 73 acre site at Grangegorman in Dublin city, the largest higher education development project in Europe.

During these new developments, RESC has continued to focus on bystander and non-targeted effects and recent work by Isabel Vega-Carrascal and Jane Bryant involved out of field effects of radiation (Shields et al. 2014; Bryant et al. 2019). The role of exosomes as molecular signaling mediators of out of field effects was investigated as part of the Horizon 2020 EJP-CONCERT funded SEPARATE project, in collaboration with colleagues in ENEA, HMGU and Oxford Brookes University (Cagatay et al. 2020). Induction of calcium, ROS and NO was observed in mouse embryonic fibroblasts exposed to exosomes derived from organs of whole body irradiated (WBI) or partial body irradiated (PBI) mice.

Translational research has also continued on predictive assays for individual radiosensitivity and normal tissue effects/late radiation toxicity in prostate cancer patient blood samples in collaboration with our clinical and medical physics colleagues in the St Luke’s Radiation Oncology Network. This research with our RESC colleague Dr Aidan Meade, funded by Science Foundation Ireland and the EU FP7 Network of Excellence DoReMi, focused on both radiobiological assays applied to patient samples such as the G2 radiosensitivity assay and the γH2AX assay, and the development of novel spectroscopic assays based on Raman and Infrared spectroscopy (Maguire 2015; Medipally et al. 2017, 2019; Meade 2019; Cullen et al. 2020). A recent study by White et al. (2020) compared G2 chromosomal radiosensitivity in prostate cancer samples exposed to different low LET radiation types and doses. The quest to find radiosensitivity biomarkers has continued and we have recently reported PTEN and CCDN1 from MicroRNA analysis of AT cells (Bryant et al. 2020) as potential radiosensitive biomarkers along with several others not yet published.

In conclusion, this short narrative provides a history of the significant contributions of Irish women to radiobiology (as displayed in Figure 5 for the RESC) and to the development of the discipline as a major research focus in TU Dublin.

Figure 5. The Venn diagram representing >40 women radiobiologists/environmental scientists and supporting staff who contributed to the field through the RESC over the last 50 years.

Acknowledgements

The authors sincerely thank Technological University Dublin (formerly Dublin Institute of Technology) for their continuing support of RESC and the Clinical Trials Unit along with our Radiation Oncology and Medical Physics colleagues in St Luke’s Radiation Oncology Network and Cancer Trials Ireland for their ongoing support and excellent collaboration.

Disclosure statement

The authors report no conflict of interest for this manuscript.

Additional information

Funding

We are very grateful to the funding agencies who have supported our research including Science Foundation Ireland [PI award, 11/RFP.1/BMT/3317, 15/TIDA/2883], the Higher Education Authority Programme for Research in Third Level Institutions, Cycle 1, Cycle 2 and Cycle 4, the Health Research Board [ILP-POR-2019-114], the Cancer Research Advancement Board, St Luke’s Institute for Cancer Research, the European Commission [funding since 1980 including RADINSTAB Grant agreement ID: FIGH-CT-1999-00003, NOTE Integrated Project Grant agreement ID: 36465, DoReMi Network of Excellence Grant agreement ID: 249689, EJP-CONCERT SEPARATE, Grant no. 004/2017 662287].

Notes on contributors

Orla Howe

Orla Howe is a professor and senior researcher at the School of Biological & Health Sciences at Technological University Dublin. Her research interests include low dose radiation effects, translational radiobiology and molecular biomarkers.

Fiona M. Lyng

Fiona M. Lyng is a professor and Head of the Radiation and Environmental Science Center at Technological University Dublin. Her research interests include low dose radiation effects and translational radiobiology.

Carmel Mothersill

Carmel Mothersill is a professor and Canada Research Chair in Environmental Radiobiology at McMaster University in Canada and adjunct professor at Technological University Dublin. Her interests include low dose radiation effects in the non-human biota.