The identification and validation of proteomic biomarkers for detection and/or prediction of radiation injury currently represents an unmet medical need. Interrogating longitudinally collected biospecimens pre- and post-irradiation for downstream molecular phenotyping analyses allows for the identification of several potential proteomic biomarkers. These biomarkers are indicators of overall health or decline thereof, and can be leveraged for early interventions and/or to manage ARS in exposed populations. Additionally, once validated, these biomarkers also have tremendous translational ability and many applications including drug development, understanding the effects of radiation on biological systems, and assessing absorbed radiation doses in exposed populations after a nuclear event.
Extensive research evaluating the changes within proteomic profiles incited by lethal doses of ionizing radiation has been conducted in our laboratory 23. Serum samples of irradiated NHPs 24,25, tissue (jejunum) and biofluids (serum) of irradiated mice 26,27, in addition to irradiated CD34+ cell culture supernatants 28 have been thoroughly evaluated. The radiation sources utilized in our studies contain high level cobalt-60 gamma radiation and various radiation countermeasures under development including tocopherol succinate 27, gamma-tocotrienol 26,27, BIO 300 24, and Ex-Rad 25. Tocopherol succinate and gamma-tocotrienol have been evaluated in murine models 26,27 (tocopherol succinate was also investigated using CD34+ cells in vitro 28), while BIO 300 and Ex-Rad have been investigated using NHP models 24,25. To assess these proteomic changes, methods including NanoUPLC-MS/MS 24,25, two-dimensional differential in-gel electrophoresis (2D-DIGE) 26,27, and a high throughput antibody microarray platform 28 have been used.
In this study, we aimed to characterize the proteomic changes induced by 7.2 Gy total-body irradiation by comparing samples collected before irradiation to samples collected post-irradiation at pre-selected time points (days 1, 13, and 25 post-irradiation). Plasma samples were also collected from moribund animals immediately prior to humane euthanasia; in this study, we have termed these samples “preterminal.” These preterminal samples were compared to the pre-irradiation and post-irradiation time points, and offer insight into the complex changes that are occurring on a cellular level in animals that are experiencing significant health decline and are on the verge of death.
As expected, a lesser degree of significance was noted when comparing preterminal samples to the post-irradiation time points, and these significant differences were more pronounced in the later study days (days 13 and 25). Ultimately, although there was a clear delineation between the pre-irradiation and immediate post-irradiation (day 1) groups, the subsequent time points (day 13 and day 25) demonstrated a trajectory of proteomic alterations, possibly reflecting a biological adaptation or progression of radiation-induced effects.
A deeper analysis revealed that radiation induced significant changes in inflammatory, hemostatic, and cellular structural proteins, suggesting these classes of proteins are detrimentally affected by radiation exposure, confirming previous research in which these radiation-induced changes are well-documented. Radiation induces acute damage in both immune and hematopoietic cells, contributing to the development of ARS. However, the long-term immunological effects of radiation on the immune and hematopoietic systems are lesser known29,30. Additionally, it has also been established that radiation has detrimental effects on the cell membrane, and this damage in turn initiates cellular apoptosis via signaling events31. However, heterogeneity in protein responses underscores the complexity of the NHP plasma proteome's reaction to radiation and the influence of individual physiological variability. In other words, a few proteins displayed consistent patterns in intensities post-irradiation, while others followed more unique trends in irradiated animals. Inter-alpha-trypsin inhibitor heavy chain H4, for example, plays an important role in inflammatory responses32,33. The trajectory of expression in this protein showed a strong positive correlation with proteomic changes in the preterminal phase, in a time dependent manner, suggesting heightened biological stress or damage responses.
The effect of radiation on protein expression varied greatly in terms of patterns in up and downregulation, which further underscores the complex and varied response to radiation, and suggests a cascade of biological events leading to a unique proteomic signature associated with the preterminal state. This disparity not only confirms the immediate effects of radiation but also indicates a progressive and compounded proteomic alteration over time, culminating in a distinct preterminal proteomic signature. These insights provide a valuable framework for understanding the progression of radiation effects on a proteomic level and aid in identifying potential biomarkers that could signal the beginning of the transition to critical health stages in irradiated organisms.
We have also performed metabolomics analysis on plasma samples collected throughout the course of this study at the same time points, which also demonstrated that radiation induced significant time-dependent metabolic perturbations when compared to pre-irradiation profiles. A distinguishable preterminal phenotype was observed, with notable dysregulation in metabolites related to the glycerophospholipid metabolism and steroid hormone biosynthesis and metabolism pathways 21. Notably, metabolomic and proteomic preterminal signatures were demonstrated in both of these studies. Although our results provide a strong proof of concept for delineation of protein biomarkers of the pre-terminal state, ultimately, continued research into the preterminal state of moribund NHPs is needed to further identify and validate proteins and pathways that can be targeted for the development of various therapeutic strategies to treat ARS. To this end, an ongoing study in our laboratory using similar preterminal samples from a large number of NHPs irradiated with two separate doses of cobalt-60 gamma-radiation, will allow for the validation of this study’s results.