Is there a role for the adverse outcome pathway framework to support radiation protection?

PURPOSE: In 2012, the Organization for Economic Cooperation and Development (OECD) formally launched the Adverse Outcome Pathway (AOP) Programme. The AOP framework has the potential for predictive utility in identifying early biological endpoints linked to adverse effects. It uses the weight of correlative evidence to identify a minimal set of measurable key events that link molecular initiating events to an adverse outcome. AOPs have the capability to identify knowledge gaps and priority areas for future research based on relevance to an adverse outcome. In addition, AOPs can identify pathways that are common among multiple stressors, thereby allowing for the possibility of refined risk assessments based on co-exposure considerations. The AOP framework is increasingly being used in chemical and ecological risk assessment; however, its use in the development of radiation-specific pathways has yet to be fully explored. To bring awareness of the AOP framework to the Canadian radiation community, a workshop was held in Canada in June 2018 that brought together radiation experts from Health Canada, the Canadian Nuclear Laboratories, and the Canadian Nuclear Safety Commission. METHODS: The purpose of the workshop was to share knowledge on the AOP framework, specifically (1) to introduce the concept of the AOP framework and its possible utility to Canadian radiation experts; (2) to provide examples on how it has advanced risk assessment; (3) to discuss an illustrative example specific to ionizing radiation; and lastly (4) to identify the broad benefits and challenges of the AOP framework to the radiation community. RESULTS: The participants showed interest in the framework, case examples were described and areas of challenge were identified. Herein, we summarize the outcomes of the workshop. CONCLUSIONS: Overall, participants agreed that by building AOPs in the radiation field, a network of data-sharing initiatives will enhance our interpretation of existing knowledge where current scientific evidence is minimal. They would provide new avenues to understand effects at low-dose and dose-rates and help to quantify the combined effect of multiple stressors on shared mechanistic pathways.

A study on the effect of the internal exposure to (210)Po on the excretion of urinary proteins in rats.

This study was designed to assess the feasibility of a noninvasive urine specimen for the detection of proteins as indicators of internal exposure to ionizing radiation. Three groups of rats (five in each group) were intravenously injected with 1601 ± 376, 10,846 ± 591 and 48,467 ± 2812 Bq of (210)Po in citrate form. A sham-exposed control group of five rats was intravenously injected with sterile physiological saline. Daily urine samples were collected over 4 days following injection. Purification and pre-concentration of urinary proteins were carried out by ultrafiltration using a 3000 Da molecular weight cutoff membrane filter. The concentration of common urinary proteins, namely albumin, alpha-1-acid glycoprotein, immunoglobulins IgA and IgG, was measured by an enzyme-linked immunosorbent assay. Urinary excretion of albumin decreased dose-dependently (p < 0.05) 96 h post-injection relative to the control group. In contrast, no statistically significant effects were observed for other proteins tested. The dose-dependent decrease in urinary excretion of albumin observed in this study underscores the need for further research, which may lead to the discovery of new biomarkers that would reflect the changes in the primary target organs for deposition of (210)Po.

Using tea as an artificial urine in a Canadian performance testing program for fission/activation products.

In recent years, the National Calibration Reference Centre for Bioassay and In Vivo Monitoring (NCRC) at the Radiation Protection Bureau (RPB), Health Canada, has been conducting investigations with black tea to develop a matrix that can be used to replace urine in each of the following performance testing programs (PTP): (1) tritium, (2) carbon-14, (3) the DUAL (i.e., 3H/14C), and (4) fission/activation products (F/AP). A 1% tea solution with thimerosal, which had worked successfully for tritium, carbon-14, and the DUAL, was selected and tested for the F/AP PTP because of its similarity to urine in color and UV-VIS spectra. However, application of this tea to samples of the F/AP program containing 133Ba, 137Cs, 57Co, and 60Co produced precipitates, which was an unexpected result. Further experiments showed that replacement of thimerosal with an alcohol at about 5% eliminated the precipitation problem. The alcohol can be ethanol, methanol, or isopropanol. In the experiments, the 1% tea, preserved with alcohol, remained clear and stable for at least 100 d. The duration of each PTP for the NCRC is limited to 90 d. Application of the CNSC S-106 regulatory standard to the tea produced acceptable accuracy and precision results. It was concluded that a suitable tea matrix for the F/AP program had been found.

Evaluation of tea as a matrix in a dual (3H/14C) performance testing program in Canada.

Urine is the most popular matrix used in performance testing programs (PTP) and inter-comparison programs (ICP) for bioassay. Because it comes from humans, there are concerns regarding its biosafety. For large programs, its collection can take several hours or days to complete. In addition, natural urine has an unpleasant smell, which tends to worsen with increasing storage time. In order to solve some of these problems, the Bioassay Section at the Radiation Protection Bureau in Health Canada has been investigating the use of tea in both PTP and ICP exercises. A method based on diluting tea steeps and scanning them in the UV-VIS range of the light spectrum to select appropriate concentrations as simulated urine for the programs has been published. So far, however, only single H and single C in tea have been studied. The results were found to be compatible and very successful under the S-106 standard of the Canadian Nuclear Safety Commission. This report is an extension of similar investigations and shows that tea samples spiked with both H and C (DUAL) are also compatible and produce excellent PTP results.

Preparation and application of steeps of tea as new simulations of urine for the performance testing programme of 14C.

(14)C is one of the radionuclides for which the Canadian Nuclear Safety Commission has developed performance testing programmes (PTPs). During the PTP exercises, clients receive samples of natural urine containing spiked radionuclides, for testing. In these programmes, urine has disadvantages. These include (1) slow collection times from donors, (2) unpleasant smell and (3) potential to transmit diseases. To assist in solving some of these problems, the Canadian National Calibration Reference Centre for Bioassay and In Vivo Monitoring has conducted research with tea solutions, to find simpler, safer and more readily available alternatives to urine. This paper provides a new technique by which steeps of black tea have been successfully prepared for the (14)C PTP. The results of tea solutions compared well with those of urine. It was concluded that tea steeps, of which the spectroscopic and colour quenching properties have been adjusted, do provide appropriate urine simulations, suitable for use in PTPs.

Preparation and application of tea to a tritium performance testing programme.

A simple, but novel technique, for adjusting steeps of black tea to produce fluids, which are visually and spectroscopically similar to urine, has been developed at the National Calibration Reference Centre for Bioassay and In Vivo Monitoring in Canada. The method uses scans of absorbance versus wavelength, in the UV-VIS range (200-800 nm) to select diluted tea steeps that simulate urine. Tea solutions (1 and 10 %) were spiked with tritium and distributed to laboratories for performance testing (PT). The PT exercise was done as in a regular bioassay programme. The results showed that all samples satisfied the pass/fail conditions of the S-106 standard of the Canadian Nuclear Safety Commission, suggesting that adjusted tea successfully simulated urine for the tritium PT programmes. Also, since unlike urine whose use may increase the probability of contaminating and transmitting diseases (e.g. hepatitis C), tea is a safer alternative. When needed, it can readily be prepared for the laboratories.

Laboratory evaluation of a SpectraMax microplate reader and test strips for field measurement of creatinine in spot urine samples in the event of a radiological accident.

The fear that terrorists might use radiological or nuclear (RN) devices to attack others is a new but growing phenomenon, arising mainly from the events of 11 September 2001. Research on rapid analytical methods that can allow analyses of large numbers of people who may become internally contaminated with radionuclides due to a RN accident is still limited. To contribute to this bioassay capacity for emergency response, the Radiation Protection Bureau of Health Canada has identified and evaluated two new portable SpectraMax plate readers (model 250 and Plus 384) and one brand of dry reagent strips for rapid measurement of creatinine in spot urine samples. Concentrations of creatinine in spot urine samples provide a means of adjusting or normalizing urine collections to 24 h, upon which accurate internal dose assessments due to the radionuclides can be made. Preliminary test results of the devices showed the two SpectraMax plate readers and the TECO dry creatinine reagent strips were portable, rapid and reliable for urinary creatinine measurements in spot samples, suggesting they can be used in rapid dose screening of people.

Requirements for radiation emergency urine bioassay techniques for the public and first responders.

Following a radiation emergency, the affected public and the first responders may need to be quickly assessed for internal contamination by the radionuclides involved. Urine bioassay is one of the most commonly used methods for assessing radionuclide intake and radiation dose. This paper attempts to derive the sensitivity requirements (from inhalation exposure) for the urine bioassay techniques for the top 10 high-risk radionuclides that might be used in a terrorist attack. The requirements are based on a proposed reference dose to adults of 0.1 Sv (CED, committed effective dose). In addition, requirements related to sample turnaround time and field deployability of the assay techniques are also discussed. A review of currently available assay techniques summarized in this paper reveals that method development for ²4¹Am, ²²6Ra, ²³8Pu, and 9°Sr urine bioassay is needed.

The Canadian National Calibration Reference Center for Bioassay and in Vivo Monitoring: an update.

The Canadian National Calibration Reference Center (NCRC) for Bioassay and In Vivo Monitoring is part of the Radiation Protection Bureau, Health Canada. The NCRC operates three performance testing programs that are designed to confirm that workplace monitoring results are accurate and provide the necessary external verification that is part of a comprehensive quality assurance program. The NCRC performance testing programs cover the in vitro, in vivo, and internal dosimetry parts of Canadian facilities' radiation protection programs. The internal dosimetry performance testing is a new addition to the performance testing suite. This summary also describes the recent reorganization of the NCRC.

A study on the effect of degradation media on the physical and mechanical properties of porous PLGA 85/15 scaffolds.

This study investigates the effect of PLGA 85/15 scaffold on the cell growth and viability of a cell line, and the degradation of the scaffold in different media. The cell line used was human promyelocytic leukemia cells (HL-60). Three different media were considered: distilled water, a phosphate buffered saline (PBS) solution, and HL-60 cell line. Porous PLGA 85/15 scaffolds were prepared with an optimized gas foaming/salt leaching technique using a NaCl/polymer mass ratio of five, a saturation pressure of 5.52 MPa and a saturation time of 12 h. The cell growth and viability were not impaired by the presence of the scaffold. The mass change of the scaffold due to degradation over the period was varied only by 4% across all three media. The average macropore size and molecular weight decreased as the degradation time increased in each medium. The scaffolds maintained mechanical and structural integrity throughout the study in all three media over the degradation period studied, and the change of Young's modulus of the scaffold under wet condition was not significant. Overall, PBS solution most strongly affected physical and mechanical properties, followed by dH(2)O and HL-60 cells. The distinct variations of the scaffold's properties using different media, demonstrated the importance of carefully selecting the medium to perform in vitro studies. The medium must replicate the actual environment where the scaffold would be used, to represent accurately the changes in properties that the scaffold would be undergoing.

A laboratory observation of some toxic effects of commercially available implant polyurethanes on HL-60 cancer cells.

We studied the interaction of HL-60 cells with films of commercial polyurethanes (PU). The cast films were dried in an oven at 60 degrees C for forty eight hours, washed in doubly distilled water, and soaked in fresh water for another 48 hours to ensure that all of the leachables including the dimethylacetamide used as casting solvent was removed before they were introduced to the cells. The HL-60 cells were cultured in the RPM1-1640, at 37 degrees C, 100% humidity and 5% CO2. Evaluation of the effects between the cells and the films, was done by counting the live cells using a hemacytometer and a light microscope. Preliminary analysis of our data have shown that while some brands of polyurethane can slow down or suppress the growth of the phagocytic type of cells, others can enhance the cell growth. It was concluded that this observation was new but should be investigated further to better understanding the interactions which may lead to the development of in vitro methods for screening the safety and stability of PU intended for implantation.