A pilot study of radiation-induced bystander effect in radio-adapting frogs at a radiologically contaminated site located on the chalk river laboratories property

Abstract

Purpose

To measure medium borne bystander effects, to study the influence of radioadaptive response (RAR) on bystander response, and to discover reliable radioresponsive biomarkers in radio-adapting frogs from Duke Swamp contaminated with an above-background radiation level and in naïve frogs from Twin Lake as the background control site.

Materials and methods

Frogs were captured at Duke Swamp and Twin Lake and brought to the lab at the Canadian Nuclear Laboratories facility. Half of the frogs from each site were irradiated with 4 Gy while the other half of the frogs were left with no further radiation treatment. Frog bladders were removed and placed in sterile culture media. Upon arrival at McMaster University, the bladders were processed for tissue cultures. After 48 h, the culture media conditioned by the bladder explants were harvested for clonogenic reporter survival assay and calcium flux measurements for assessing bystander effects. HPV-G cells were used as bystander reporter cells in all radiation-induced bystander effect (RIBE) assays. The frog bladder cultures were incubated for another 10–12 days followed by immunochemical staining for bcl-2 and c-myc expressions to analyze cellular anti-apoptotic (pro-survival) and pro-apoptotic (pro-death) responses, respectively.

Results

Only culture media conditioned by bladders from 4-Gy-irradiated naïve frogs from Twin Lake induced bystander effects (reduction of HPV-G reporter cells’ clonogenic survival and presence of strong calcium flux activities). The 4 Gy irradiation dose increased pro-apoptotic c-myc expression in naïve frogs’ bladder explants. Culture media conditioned by bladders from radio-adapting frogs from Duke Swamp enhanced HPV-G’s clonogenic survival and a 4 Gy irradiation challenge did not change the enhanced clonogenic survival nature nor induce calcium flux. In bladder explants from both control and 4-Gy-irradiated radio-adapting frogs, anti-apoptotic bcl-2 expression for pro-survival responses was ubiquitous while c-myc expression for pro-death responses was limited to a small fraction of cells.

Conclusion

The clonogenic RIBE reporter assay using HPV-G and calcium flux measurements are useful diagnostic tools for RIBE assessment of field biological samples, specifically those from frogs. RAR induced by environmentally relevant low-dose radiation induces protective bystander response. Bcl-2 and c-myc are reliable biomarkers for evaluating low dose radiation responses in wild populations of amphibians. Overall, this pilot study emphasizes the importance of looking at non-targeted effects (NTEs) in natural populations of non-human biota that could be vulnerable to chronic low-dose radiation exposures.

Keywords:

• Frog
• amphibian
• low dose
• tritium
• bystander effect
• non-targeted effect
• adaptive response
• bcl-2
• c-myc

Disclosure statement

The authors report no conflicts of interest.

Additional information

Funding

The research was supported by the Canada Research Chairs Program, the NSERC Industrial Chairs Program, the NSERC Discovery grant program, the NOTE EU Integrated Project, The CANDU Owners Group, and Atomic Energy Canada Ltd. Canada Research Chair in Sexual and Gender Minority Health; Natural Sciences and Engineering Research Council of Canada; Sixth Framework Programme.

Notes on contributors

Nguyen T. K. Vo

Nguyen T. K. Vo, PhD, is a Research Associate in the Department of Biology at McMaster University. His current research interest is low-dose radiation mechanisms in humans and non-human biota.

Harleen Singh

Harleen Singh, MD, holds an MSc degree in Radiation Biology at McMaster University. She is currently practicing medicine in Buffalo, NY.

Marilyne Stuart

Marilyne Stuart, PhD, is a Research Scientist at Canadian Nuclear Laboratories (CNL) in Chalk River, Ontario, Canada. Her research Interest is the long-term effects of low dose radiation in non-human species.

Colin B. Seymour

Colin B. Seymour, PhD, is a Professor of Radiobiology in the Department of Biology at McMaster University. His research interest is low-dose radiation mechanisms including bystander effects and lethal mutations.

Carmel E. Mothersill

Carmel E. Mothersill, PhD, is a Professor and Canada Research Chair in Environmental Radiobiology at McMaster University. Her research interests include low-dose radiation effects on plants and animals with a focus on non-targeted effects.