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

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. As secondary consumers in both aquatic and terrestrial environments, amphibians play a pivotal role in many ecosystems and are relevant to radioecology. Biological mechanisms operating at low-dose regions relevant to environmental levels of radiation contamination are largely attributed to cellular responses mediated by non-targeted effects (NTEs) such as radiation-induced bystander effect (RIBE) and radioadaptive response (RAR). The natural background radiation level in Chalk River was estimated to be 2.4 mGy/year. The elevated radiation level in DS contributed to less than 1 mGy/year to the natural background radiation. It was found that DS frogs radio-adapted and were more radioresistant than TL frogs; thus, elevated environmental radiation exposure invoked RAR in frogs. The clonogenic reporter assay and calcium flux measurement proved to be useful reporter assays to assess RIBEs in the environment. Bcl-2 and c-myc proved to be reliable biomarkers for chronic low-dose radiation exposures in wild populations of amphibians.