The untargeted proteomic study design allowed us to assess age and sex effects across a wide range of cellular functions and specific IR-associated responses. The high-throughput screening clearly demonstrated that the quality and extent of age and sex effects is endpoint-dependent and should not be generalized. In addition, our pipeline identified analytical bias when using data enrichment. When GO term enrichment was used to identify DNA damage and repair associated proteins, responses were moderate in black male mice but undetected in nude male mice. Manual pathway analysis, on the other hand, yielded similar results for both groups. This finding advises caution and advocates cross-validation of data enrichment with other analytical methods. Total body irradiation was chosen to irradiate all tissues homogeneously and allow for multiple sampling of irradiated tissues in agreement with the 3Rs Reduction principle (36). The moderate absorbed dose of 0.5 Gy was chosen to exclude potential low-dose effects that may be exclusive to that regimen (typically up to 0.1 Gy (37)), while being low enough to be of relevance for a wide range of risk exposure scenarios. The proteomic response was analyzed 24 hours after irradiation to match early monitoring applications. Blood (plasma) was chosen in context with diagnostics and minimally-invasive molecular biomarkers. Blood plasma contains a complex protein mixture mainly synthesized by the liver; but also bone marrow, the spleen, and general tissue cells can contribute to its formation and composition (38). As such, the observed differential abundance changes may result in large part from ionizing radiation-induced damage to the hematopoietic system, but potentially also reflect responses in other tissues. Nevertheless, interpretation bias may result from overrepresentation of immune-associated proteins upon IR exposure. This may not necessarily mean a strong response in immune processes at this dose level, but rather reflect the specific makeup of plasma protein, which (excluding albumin) consists in large part of globulins and other immune-related proteins.
Pooling was found highly comparable to individual sample analysis, although a minor artefact on dynamic range was observed. Our analytical approach included immunodepletion of seven highly abundant plasma proteins in order to increase the depth of the proteomic analysis. Immunodepletion allows to achieve broad coverage in the LC-MS-based analysis of plasma (39); however, it is an additional sample preparation step and a potential source of additional analytical bias. The parallel analysis of pooled and individual samples showed the reproducibility of the immunodepletion as a crucial part of the workflow in agreement with literature (40). We achieved good correlation between the abundance patterns from pooled and individual samples, with the Pearson correlation coefficients reaching 0.75-0.81. Accordingly, we conclude that the extra cost of including both sexes or additional age groups in study designs can be mitigated by pooled analysis in biomarker screening studies.
Regarding the observed strain effects, BALB/c nude mice are immunodeficient since they lack a thymus and are unable to produce T-cells. Immune-related IR responses were thus expected to be impaired and were indeed found to be largely suppressed in nude mice. Interestingly, responses in DNA damage & repair pathways (manual analysis) were comparable between black and nude mice at this dose level, although the BALB/c background is known to be DNA repair deficient. It should also be noted that sex as a variable did not appear to contribute to the strain effects per se, which implied that additional (convoluted) sex bias between strains may be negligible.
In the bulk response, sex effects were marginal compared with the pronounced age impact across the proteome. Although hierarchical clustering indicated a separation by sex, this was mainly due to a single cluster of overabundant proteins shared among all female groups. This cluster consisted of low-abundance change proteins which can be assumed to have less impact on cellular responses. (Accordingly, low-abundance change proteins are filtered in functional analyses and this sex-specific cluster would not carry regulatory relevance.) Combining age and sex into one screening cohort, the analysis yielded only two differentially abundant proteins in all groups. Alpha hemoglobin stabilizing protein (AHSP) showed stable IR response across age, but inverted abundance change (over vs. under) between the sexes. With prior knowledge of expected abundance change, it may qualify as a unisex biomarker candidate. AHSP acts as a chaperone during erythroid cell development (41). In both Mus musculus and Homo sapiens, AHSP is expressed in (mainly) bone marrow and blood (41, 42). As such, the biomarker candidate could reflect radiation injury to the hematopoietic system. Serum amyloid A-2 protein (SAA2), a major acute phase reactant (43), showed large FC range across age and sex to the same stressor and its applicability as a pan-group biomarker was rejected. The very low yield of uniage (or unisex) biomarker candidates strongly supports that screening for age-specific and sex-specific biomarkers is the more feasible approach. Our data suggests that previously identified radiation biomarker candidates may not be translatable across age or sex and we reason that age- and sex-specific validation is paramount.
Overall, the study clearly showed that age-based differences in IR-induced responses outweigh sex-based differences for most analytical endpoints–at least in the investigated age binning.
In theory, a stronger IR response may indicate increased radiosensitivity. Conversely, a stronger (early) IR response might increase e.g. DNA damage recognition and repair and thus reduce risk of malignancies. The increased responses in juveniles supported the former reasoning (juveniles are more radiosensitive than adults according to established knowledge), whereas increased responses in males supported the latter (in general, males are perceived as less radiosensitive than females). Follow-up studies are needed to correlate long-term outcome to this early regulatory response. Regarding sex, the difference in functional regulation between males and females strongly suggests the feasibility of sex-specific immune- or apoptosis-based targets for radioprotectors and mitigators. Concerning age, recent work on age-dependent regulation of mitochondrial apoptosis showed that many tissues (e.g. the heart and kidneys) in post-natal individuals are primed for apoptosis, whereas adult tissues are apoptosis refractory with low expression of the apoptotic machinery (44). Assuming a similar apoptosis-primed trend for the juvenile mice studied here, reduced DNA repair might be an effect of damaged cells instead undergoing apoptosis due to priming and high expression of the apoptotic machinery. However, studies show that the hematopoietic system (including peripheral blood mononuclear cells) is still primed for apoptosis in adults (44). Accordingly, age effects from apoptotic priming may be low in blood but could be a significant contributor to IR-responses in other non-hematopoietic tissues.
Proteins for cell cycle regulation, immune responses, or DNA damage & repair that were shared between age, sex, or strain pairs were much less frequent than unique proteins for these processes. Among the shared proteins, only few were identified that may serve as uniage biomarkers of IR-induced responses in females (GMNN and REG3B) and males (SNCA). GMNN (geminin DNA replication inhibitor) is a negative regulator of cell cycle and proliferation; during S-phase, it is involved in suppressing DNA replication and inhibits assembly of the pre-replicative complex until late mitosis. (45, 46). A common cellular outcome in cell cycle regulation, however, was not supported by DNA damage & repair pathway analysis. REG3B (regenerating islet-derived protein 3-beta) is a bactericidal C-type lectin involved in the defense responses of the innate immune system (47). SNCA (alpha-synuclein) is mainly abundant in the presynaptic terminals of brain neurons interacting with phospholipids and proteins (48, 49). Hence, the detection in blood may have come from cranial radiation exposure and SNCA crossing the brain-blood barrier. SNCA may be a potential tissue-specific blood-based biomarker. Nevertheless, since small concentrations are found in other tissues as well, the matter of tissue-specificity needs to be validated. While GMNN, REG3B, and SNCA may be understood as candidates for uniage exposure biomarkers, their dynamic range in dose-response indicated low sensitivity and challenges their applicability. Moreover, the overwhelming dissimilarity of specific protein regulation made a strong case for separate panels of age- and sex-specific radiation biomarkers. It is interesting to note that more proteins with quite similar FC values were identified across strains in females: the multi-ligand integral membrane protein CD36 (cluster of differentiation 36) (50), the protein-conjugating ISG15 (Interferon-stimulated gene 15) (51), and the anti-oxidant enzyme PRDX2 (Peroxiredoxin-2) (52). This finding underlined that strain effects can be negligible for specific endpoints (if only for a given sex).
The qualitative differences in IR-induced responses between age and sex were further highlighted by inferring cellular outcome from DNA damage & repair-associated proteins. As opposed to a mere increase or decrease in response intensity of the same mechanism, juvenile females and males appeared to initiate different signaling cascades. Although both shared suppression of cell cycle progression, females showed activated DNA repair and pro-survival mechanisms whereas males shifted towards pro-apoptosis with mitochondrial stress and reduced DNA repair. In addition, inflammatory regulators, which were not detected in females, appeared to compete over activation and suppression of immune responses. At adult age, sex effects were diminished and, overall, understood as low-to-negligible at this dose level. To the best of our knowledge, these results are the first demonstration of age-dependent sex effects of IR-induced DNA damage and repair. Across strains, the inferred cellular outcome appeared to be rather similar and additional convoluted sex effects were not apparent. Notably, similar outcome was achieved via different mechanisms, as no differential protein levels were shared across strains for either sex. This further strengthened the finding that strain bias in IR-induced responses is a complex matter and should not be generalized across pathways without critical assessment.